Aiping Lü

Emerging evidence indicates that osteoclasts direct osteoblastic bone formation. MicroRNAs (miRNAs) have a crucial role in regulating osteoclast and osteoblast function. However, whether miRNAs mediate osteoclast-directed osteoblastic bone formation is mostly unknown. Here, we show that increased osteoclastic miR-214-3p associates with both elevated serum exosomal miR-214-3p and reduced bone formation in elderly women with fractures and in ovariectomized (OVX) mice. Osteoclast-specific miR-214-3p knock-in mice have elevated serum exosomal miR-214-3p and reduced bone formation that is rescued by osteoclast-targeted antagomir-214-3p treatment. We further demonstrate that osteoclast-derived exosomal miR-214-3p is transferred to osteoblasts to inhibit osteoblast activity in vitro and reduce bone formation in vivo. Moreover, osteoclast-targeted miR-214-3p inhibition promotes bone formation in ageing OVX mice. Collectively, our results suggest that osteoclast-derived exosomal miR-214-3p transfers to osteoblasts to inhibit bone formation. Inhibition of miR-214-3p in osteoclasts may be a strategy for treating skeletal disorders involving a reduction in bone formation.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

Aptamers are oligonucleotide sequences with a length of about 25–80 bases which have abilities to bind to specific target molecules that rival those of monoclonal antibodies. They are attracting great attention in diverse clinical translations on account of their various advantages, including prolonged storage life, little batch-to-batch differences, very low immunogenicity, and feasibility of chemical modifications for enhancing stability, prolonging the half-life in serum, and targeted delivery. In this Review, we demonstrate the emerging aptamer discovery technologies in developing advanced techniques for producing aptamers with high performance consistently and efficiently as well as requiring less cost and resources but offering a great chance of success. Further, the diverse modifications of aptamers for therapeutic applications including therapeutic agents, aptamer–drug conjugates, and targeted delivery materials are comprehensively summarized.

Syndrome differentiation (Bian Zheng) in traditional Chinese medicine (TCM) is the comprehensive analysis of clinical information gained by the four main diagnostic TCM procedures: observation, listening, questioning, and pulse analysis, and it is used to guide the choice of treatment either by acupuncture and/or TCM herbal formulae, that is, Fufang. TCM syndrome differentiation can be used for further stratification of the patients' conditions with certain disease, identified by orthodox medical diagnosis, which could help the improvement of efficacy of the selected intervention. In modern TCM research it is possible to integrate syndrome differentiation with orthodox medical diagnosis leading to new scientific findings in overall medical diagnosis and treatment. In this review, the focus is to screen published evidence on the role of syndrome differentiation in modern TCM research with particular emphasis on basic and clinical research as well as, pharmacological evaluation of TCM herbal formulary for drug discovery.

Currently, major concerns about the safety and efficacy of RNA interference (RNAi)-based bone anabolic strategies still exist because of the lack of direct osteoblast-specific delivery systems for osteogenic siRNAs. Here we screened the aptamer CH6 by cell-SELEX, specifically targeting both rat and human osteoblasts, and then we developed CH6 aptamer-functionalized lipid nanoparticles (LNPs) encapsulating osteogenic pleckstrin homology domain-containing family O member 1 (Plekho1) siRNA (CH6-LNPs-siRNA). Our results showed that CH6 facilitated in vitro osteoblast-selective uptake of Plekho1 siRNA, mainly via macropinocytosis, and boosted in vivo osteoblast-specific Plekho1 gene silencing, which promoted bone formation, improved bone microarchitecture, increased bone mass and enhanced mechanical properties in both osteopenic and healthy rodents. These results indicate that osteoblast-specific aptamer-functionalized LNPs could act as a new RNAi-based bone anabolic strategy, advancing the targeted delivery selectivity of osteogenic siRNAs from the tissue level to the cellular level.

Molecular descriptors and fingerprints have been routinely used in QSAR/SAR analysis, virtual drug screening, compound search/ranking, drug ADME/T prediction and other drug discovery processes. Since the calculation of such quantitative representations of molecules may require substantial computational skills and efforts, several tools have been previously developed to make an attempt to ease the process. However, there are still several hurdles for users to overcome to fully harness the power of these tools. First, most of the tools are distributed as standalone software or packages that require necessary configuration or programming efforts of users. Second, many of the tools can only calculate a subset of molecular descriptors, and the results from multiple tools need to be manually merged to generate a comprehensive set of descriptors. Third, some packages only provide application programming interfaces and are implemented in different computer languages, which pose additional challenges to the integration of these tools. A freely available web-based platform, named ChemDes, is developed in this study. It integrates multiple state-of-the-art packages (i.e., Pybel, CDK, RDKit, BlueDesc, Chemopy, PaDEL and jCompoundMapper) for computing molecular descriptors and fingerprints. ChemDes not only provides friendly web interfaces to relieve users from burdensome programming work, but also offers three useful and convenient auxiliary tools for format converting, MOPAC optimization and fingerprint similarity calculation. Currently, ChemDes has the capability of computing 3679 molecular descriptors and 59 types of molecular fingerprints. ChemDes provides users an integrated and friendly tool to calculate various molecular descriptors and fingerprints. It is freely available at http://www.scbdd.com/chemdes . The source code of the project is also available as a supplementary file.

Nucleic acid aptamers have minimal immunogenicity, high chemical synthesis production, low cost and high chemical stability when compared with antibodies. However, the susceptibility to nuclease degradation, rapid excretion through renal filtration and insufficient binding affinity hindered their development as drug candidates for therapeutic applications. In this review, we will discuss methods to conquer these challenges and highlight recent developments of chemical modifications and technological advances that may enable early aptamers to be translated into clinical therapeutics.

Abstract Purpose: KIT aberrations were described in acral and mucosal melanomas in largely Caucasian populations. Asian populations are more prone to develop acral and mucosal than cutaneous melanomas, and may harbor a high frequency of KIT aberrations. Experimental Design: Melanoma subtypes (n = 502) were analyzed histologically to determine melanoma subtype. Tissue samples were analyzed for mutations in exons 9, 11, 13, 17, and 18 of KIT gene in genomic DNA by PCR amplification and Sanger sequencing. The copy numbers of the KIT gene were analyzed by quantitative PCR, and protein expression levels of KIT (CD117) were determined by immunohistochemistry. Results: The most common melanoma subtypes were acral (38.4%) and mucosal (33.3%) melanomas in this population. The overall incidence of somatic mutations within the KIT gene was 10.8% (54/502), and all subtypes of melanoma contained KIT mutations. Increases in KIT gene copy numbers were correlated to CD117 overexpression. The genetic mutations of KIT were unrelated to the age, gender, stage, thickness, and ulceration of primary melanomas. Importantly, the overall survival of melanoma patients with KIT mutations (P = 0.001) or with KIT aberrations (mutation plus amplification, P = 0.0002) was significantly shorter than that of patients without such alterations. Conclusion: In China, the prevalent melanomas are acral and mucosal melanomas. KIT mutations are detected in all melanoma subtypes. Our study suggests that increases in KIT gene copy numbers, but not KIT mutations, may be correlated to CD117 overexpression. For the first time, our study suggests that genetic KIT aberration is an adverse prognostic factor for melanoma. Clin Cancer Res; 17(7); 1684–91. ©2011 AACR.

Paclitaxel (PTX) is among the most commonly used first-line drugs for cancer chemotherapy. However, its poor water solubility and indiscriminate distribution in normal tissues remain clinical challenges. Here we design and synthesize a highly water-soluble nucleolin aptamer-paclitaxel conjugate (NucA-PTX) that selectively delivers PTX to the tumor site. By connecting a tumor-targeting nucleolin aptamer (NucA) to the active hydroxyl group at 2' position of PTX via a cathepsin B sensitive dipeptide bond, NucA-PTX remains stable and inactive in the circulation. NucA facilitates the uptake of the conjugated PTX specifically in tumor cells. Once inside cells, the dipeptide bond linker of NucA-PTX is cleaved by cathepsin B and then the conjugated PTX is released for action. The NucA modification assists the selective accumulation of the conjugated PTX in ovarian tumor tissue rather than normal tissues, and subsequently resulting in notably improved antitumor activity and reduced toxicity.

With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs, isolated from young wild-type mice, to progeroid mice confer significant lifespan and healthspan extension. The transplanted MDSPCs improve degenerative changes and vascularization in tissues where donor cells are not detected, suggesting that their therapeutic effect may be mediated by secreted factor(s). Indeed, young wild-type-MDSPCs rescue proliferation and differentiation defects of aged MDSPCs when co-cultured. These results establish that adult stem/progenitor cell dysfunction contributes to ageing-related degeneration and suggests a therapeutic potential of post-natal stem cells to extend health.

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of in vivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment.

Effective therapy for Alzheimer’s disease is a major challenge in the pharmaceutical sciences. There are six FDA approved drugs (e.g., donepezil, memantine) that show some effectiveness; however, they only relieve symptoms. Two factors hamper research. First, the cause of Alzheimer’s disease is not fully understood. Second, the blood-brain barrier restricts drug efficacy. This review summarized current knowledge relevant to both of these factors. First, we reviewed the pathophysiology of Alzheimer’s disease. Next, we reviewed the structural and biological properties of the blood-brain barrier. We then described the most promising drug delivery systems that have been developed in recent years; these include polymeric nanoparticles, liposomes, metallic nanoparticles and cyclodextrins. Overall, we aim to provide ideas and clues to design effective drug delivery systems for penetrating the blood-brain barrier to treat Alzheimer’s disease.

Abstract Background Skeletal muscle atrophy induced by either aging (sarcopenia) or mechanical unloading is associated with serious health consequences. Long non‐coding RNAs (lncRNAs) are implicated as important regulators in numerous physiological and pathological processes. Methods Microarray analysis was performed to identify the differentially expressed lncRNAs in skeletal muscle between adult and aged mice. The most decreased lncRNA in aged skeletal muscle was identified. The C2C12 mouse myoblast cells were used to assess the biological function of the lncRNA in vitro . The target microRNA of lncRNA and the target protein of microRNA were predicted by bioinformatics analysis and validated in vitro . Furthermore, the biology function of the lncRNA in vivo was investigated by local overexpression or knockdown the lncRNA in skeletal muscle. The therapeutic effect of the lncRNA overexpression in age‐related or mechanical unloading‐induced muscle atrophy was also evaluated. Results We identified a novel lncRNA (muscle anabolic regulator 1, MAR1) which was highly expressed in mice skeletal muscle and positively correlated with muscle differentiation and growth in vitro and in vivo . We predicted and validated that microRNA‐487b (miR‐487b) was a direct target of MAR1. We also predicted and validated that Wnt5a, an important regulator during myogenesis, was a target of miR‐487b in C2C12 cells. Our findings further demonstrated that enforced MAR1 expression in myoblasts led to derepression of Wnt5a. Moreover, MAR1 promoted skeletal muscle mass/strength and Wnt5a protein level in mice. Enforced MAR1 expression in mice attenuated muscle atrophy induced by either aging or unloading. Conclusions The newly identified lncRNA MAR1 acts as a miR‐487b sponge to regulate Wnt5a protein, resulting in promoting muscle differentiation and regeneration. MAR1 could be a novel therapeutic target for treating muscle atrophy induced by either aging or mechanical unloading.

Several virtual screening models are proposed to screen small molecules only targeting primary miRNAs without selectivity. Few attempts have been made to develop virtual screening strategies for discovering small molecules targeting mature miRNAs. Mature miRNAs and their specific target mRNA can form unique functional loops during argonaute (AGO)-mediated miRNA-mRNA interactions, which may serve as potential targets for small-molecule drug discovery. Thus, a loop-based and AGO-incorporated virtual screening model is constructed for targeting the loops. The previously published studies have found that miR-214 can target ATF4 to inhibit osteoblastic bone formation, whereas miR-214 can target TRAF3 to promote osteoclast activity. By using the virtual model, the top ten candidate small molecules targeting miR-214-ATF4 mRNA interactions and top ten candidate small molecules targeting miR-214-TRAF3 mRNA interactions are selected, respectively. Based on both in vitro and in vivo data, one small molecule can target miR-214-ATF4 mRNA to promote ATF4 protein expression and enhance osteogenic potential, whereas one small molecule can target miR-214-TRAF3 mRNA to promote TRAF3 protein expression and inhibit osteoclast activity. These data indicate that the loop-based and AGO-incorporated virtual screening model can help to obtain small molecules specifically targeting miRNA-mRNA interactions to rescue bone phenotype in genetically modified mice.

Traditional Chinese medicine, including herbal medicine and acupuncture, as one of the most important parts in complementary and alternative medicine (CAM), plays the key role in the formation of integrative medicine. Why do not the modern drugs targeting the specificity of diseases produce theoretical effects in clinical observation? Why does not the traditional Chinese medicine targeting the Zheng (syndrome) produce theoretical effects in clinic? There should have some reasons to combine Western medicine with Chinese herbal medicine so as to form the integrative medicine. During the integration, how to clarify the impact of CAM theory on Western medicine has become an emergent topic. This paper focuses on the exploration of the impact of theory of traditional Chinese medicine on the therapy of diseases in Western medicine.

We have shown that muscle-derived stem cells (MDSCs) transplanted into dystrophic (mdx) mice efficiently regenerate skeletal muscle. However, MDSC populations exhibit heterogeneity in marker profiles and variability in regeneration abilities. We show here that cell sex is a variable that considerably influences MDSCs' regeneration abilities. We found that the female MDSCs (F-MDSCs) regenerated skeletal muscle more efficiently. Despite using additional isolation techniques and cell cloning, we could not obtain a male subfraction with a regeneration capacity similar to that of their female counterparts. Rather than being directly hormonal or caused by host immune response, this difference in MDSCs' regeneration potential may arise from innate sex-related differences in the cells' stress responses. In comparison with F-MDSCs, male MDSCs have increased differentiation after exposure to oxidative stress induced by hydrogen peroxide, which may lead to in vivo donor cell depletion, and a proliferative advantage for F-MDSCs that eventually increases muscle regeneration. These findings should persuade researchers to report cell sex, which is a largely unexplored variable, and consider the implications of relying on cells of one sex.

Studies on traditional Chinese medicine (TCM), like those of other systems of traditional medicine (TM), are very variable in their quality, content and focus, resulting in issues around their acceptability to the global scientific community. In an attempt to address these issues, an European Union funded FP7 consortium, composed of both Chinese and European scientists and named "Good practice in traditional Chinese medicine" (GP-TCM), has devised a series of guidelines and technical notes to facilitate good practice in collecting, assessing and publishing TCM literature as well as highlighting the scope of information that should be in future publications on TMs. This paper summarises these guidelines, together with what has been learned through GP-TCM collaborations, focusing on some common problems and proposing solutions. The recommendations also provide a template for the evaluation of other types of traditional medicine such as Ayurveda, Kampo and Unani.GP-TCM provided a means by which experts in different areas relating to TCM were able to collaborate in forming a literature review good practice panel which operated through e-mail exchanges, teleconferences and focused discussions at annual meetings. The panel involved coordinators and representatives of each GP-TCM work package (WP) with the latter managing the testing and refining of such guidelines within the context of their respective WPs and providing feedback.A Good Practice Handbook for Scientific Publications on TCM was drafted during the three years of the consortium, showing the value of such networks. A "deliverable - central questions - labour division" model had been established to guide the literature evaluation studies of each WP. The model investigated various scoring systems and their ability to provide consistent and reliable semi-quantitative assessments of the literature, notably in respect of the botanical ingredients involved and the scientific quality of the work described. This resulted in the compilation of (i) a robust scoring system and (ii) a set of minimum standards for publishing in the herbal medicines field, based on an analysis of the main problems identified in published TCM literature.Good quality, peer-reviewed literature is crucial in maintaining the integrity and the reputation of the herbal scientific community and promoting good research in TCM. These guidelines provide a clear starting point for this important endeavour. They also provide a platform for adaptation, as appropriate, to other systems of traditional medicine.

Traditional Chinese medicine (TCM) is an integral part of mainstream medicine in China. Due to its worldwide use, potential impact on healthcare and opportunities for new drug development, TCM is also of great international interest. Recently, a new era for modernisation of TCM was launched with the successful completion of the Good Practice in Traditional Chinese Medicine Research in the Post-genomic Era (GP-TCM) project, the European Union’s Seventh Framework Programme (FP7) coordination action on TCM research. This 3.5-year project that involved inputs from over 200 scientists resulted in the production of 20 editorials and in-depth reviews on different aspects of TCM that were published in a special issue of Journal of Ethnopharmacology (2012; volume 140, issue 3). In this narrative review, we aim to summarise the findings of the FP7 GP-TCM project and highlight the relevance of TCM to modern medicine within a historical and international context. Advances in TCM research since the 1950s can be characterised into three phases: Phase I (1950s-1970s) was fundamental for developing TCM higher education, research and hospital networks in China; Phase II (1980s-2000s) was critical for developing legal, economic and scientific foundations and international networks for TCM; and Phase III (2011 onwards) is concentrating on consolidating the scientific basis and clinical practice of TCM through interdisciplinary, interregional and intersectoral collaborations. Taking into account the quality and safety requirements newly imposed by a globalised market, we especially highlight the scientific evidence behind TCM, update the most important milestones and pitfalls, and propose integrity, integration and innovation as key principles for further modernisation of TCM. These principles will serve as foundations for further research and development of TCM, and for its future integration into tomorrow’s medicine.

Network pharmacology is becoming a cutting-edge research field in current drug discovery and drug development thanks to rapid progress in systems biology, network biology, and chemical biology. By integrating reductionist and systems approaches as well as computational and experimental methods, network pharmacology has great potential to act as the next generation mode of drug research. Network pharmacology studies emphasize the paradigm shift from “one target, one drug” to “network target, multicomponent therapeutics,” highlighting a holistic thinking also shared by traditional Chinese medicine (TCM). In TCM, the perspective of holism has long been central to herbal treatments of various diseases. Characterized by holistic theory and rich experience in multicomponent therapeutics, TCM herbal formulae offer bright prospects for the control of complex diseases in a systematic manner. Thus, introducing network pharmacology in TCM will provide novel methodologies and new opportunities for discovering bioactive ingredients and endogenous/exogenous biomarkers, revealing mechanisms of action and exploring scientific evidence of numerous herbs and herbal formulae in TCM on the basis of complex biological systems of human body. Moreover, the integration of TCM and network pharmacology can greatly promote the progress of network pharmacology as well. Here, we have grouped together 27 papers in this burgeoning field, put forward for publication in this special issue on TCM network pharmacology. In the special issue, we have firstly published four concise reviews or perspectives across the two fields between TCM and network pharmacology. The topics range from the research paradigm of network pharmacology based on TCM theory and practice, the available databases and computational tools in TCM network pharmacology, to the applications of network pharmacology in TCM. These papers highlighted some specific themes, such as the concept of network target, mechanisms of TCM herbal formulae, and target identification of herbal active ingredients. For example, a review article provided a perspective regarding TCM-based network pharmacology and its use in multiple compound drug discovery, by following an analysis of the merged networks of differentially expressed genes in rheumatoid arthritis-cold pattern and protein targets related to Fu Zi, Xi Xin and Gui Zhi. Three other review articles comprehensively addressed the origin and development of TCM network pharmacology, the definitions of basic network concepts, the computational tools and data sources regarding TCM network pharmacology, and the significance and approaches of network pharmacology in the TCM field, as well as the target identification methods of herbal active ingredients and the use of ligand-protein networks. A remarkable feature of TCM is the use of herbal formulae. Understanding the mechanisms of action and combinatorial rules of herbal formulae is of great significance in TCM modernization and is also one of the important steps in modern drug discovery. The emerging TCM network pharmacology offers a unique opportunity to explore systematically not only the molecular complexity of an herbal formula, but also the molecular relationships between an herbal formula and complex diseases. A practical strategy of TCM network pharmacology is the combined use of network-based computational predictions and experimental validations. In this special issue, we have published 11 interesting research papers covering 10 classic herbal formulae by employing network-based approaches and omics-based experimental studies. For example, two research papers established an integrative platform of TCM network pharmacology on the basis of the concept of “network target, multicomponent therapeutics,” and then applied this platform in the identification of active compounds and mechanisms of action of an herbal formula Qing-Luo-Yin for the treatment of rheumatoid arthritis, as well as Ge-Gen-Qin-Lian decoction recorded in “Shang-Han-Lun” for the treatment of Type 2 diabetes. Moreover, network analysis and omics technologies including genomic, metabolomic, and proteomic studies are always integrated together to understand the molecular actions exerted by herbal formulae at a systematic level. In the special issue, four papers addressed the metabolomic analysis for determining the effects of Huang-Lian-Jie-Du-Tang in a rat model of collagen-induced arthritis, the metabolomic analysis for She-Xiang-Bao-Xin pill in treating myocardial infarction in rats, the proteomic analysis for determining the possible proteomic network associated with the antiarthritic effects of Qing-Fu-Guan-Jie-Shu in collagen-induced arthritis rats, and the experimental study of the protective effect of Xiao-Xu-Ming decoction in a rat model of cerebral ischemia and reperfusion. It is known that identifying the target proteins and combinatorial rules of active ingredients in herbal formulae remains to be a difficult issue. There are six papers to address such an issue from the network point of view by using bioinformatics analysis and experiments, for example, the mechanism of antirheumatic actions of Huang-Lian-Jie-Du-Tang by network analysis, the molecular mechanism of Shu-Feng-Jie-Du formula by a module analysis, drug-target network of Yuan-Hu-Zhi-Tong prescription, the pharmacological mechanisms of Wu-Tou-Tang, and the mechanism of cell apoptosis induced by Fu-Zheng-Hua-Yu tablet. Herbal active ingredients have long been viewed as a rich source of therapeutic leads in drug discovery. Network pharmacology is expected to be a new strategy and powerful tool to find the bioactive compounds as well as their potential molecular targets from numerous herbs or herbal formulae. For herbal active ingredients, there are eight papers published in this special issue with the efforts to study herbal active ingredients by network pharmacology approaches. For example, a paper predicted the target network of vitexicarpin and experimentally validated the molecular network account for its antiangiogenic activities. A paper proposed a computational method to identify the effective herbal components and mechanisms of action. A paper revealed a pharmacological signaling pathway network of baicalin in the treatment of ischaemia-reperfusion in mice. Three other papers addressed the molecular mechanisms of herbal active ingredients with the help of omics technologies, including the metabolomic analysis of genipin in rats and identification of metabolites by LC/MS, the neuroprotective effects of hydroxysafflor yellow A via the NF-kappaB pathway by NMR-based metabonomics, and expression profiling and proteomic analysis of JIN Chinese herbal formula in lung carcinoma H460 xenografts. Lastly, two experimental studies are published on the epithelial-mesenchymal transition induction of shikonin in skin wound healing and capsaicin-induced cancer cell apoptosis through ER stress. Uncovering the scientific basis of herbal traditional properties especially the molecular association between herbal formulae and TCM syndrome (ZHENG) is absolutely critical to preserve and develop TCM. Four papers in this special issue focus on the traditional properties of TCM. A paper identified the network-based biomarkers for the Cold Coagulation Blood Stasis Syndrome and showed the therapeutic effects of Shao-Fu-Zhu-Yu decoction in rats. To identify potent synergistic combinations in herbal formulae, a paper developed an interesting Feedback System Control Scheme to optimize combinations of flavonoids derived from Astragali Radix. As the potential adverse effects of herbs have limited their clinical applications, a paper proposed a network-based computational model to predict the safety of herbs through a comparative analysis of withdrawn drugs. Based on combining formula network, chemical space, and metabolic space, a paper analyzed the properties of the incompatible herbal pairs associated with the traditional rule termed “eighteen antagonisms and nineteen mutual inhibitors” in herbal formulae. In summary, TCM network pharmacology is a new interdisciplinary frontier in both ancient TCM and modern drug discovery and development fields, which represents valuable interactions and exchanges between traditional Chinese medicine and those of network, pharmacological, biomedical and computational sciences. This special issue provides a high-profile venue for dissemination of significant scientific findings in TCM network pharmacology. It is just the beginning. We encourage researchers in TCM and related fields to support the development of this novel and promising direction. Shao Li Tai-Ping Fan Wei Jia Aiping Lu Weidong Zhang

<h3>Objective</h3> <i>Helicobacter pylori</i> infection and overexpression of cyclo-oxygenase-2 (COX-2) are associated with gastric cancer and its precursors. To evaluate the effect of a selective COX-2 inhibitor alone and combined with <i>H pylori</i> eradication on the evolution of precancerous gastric lesions, a randomised, placebo-controlled trial was conducted in Linqu County, Shandong Province, China. <h3>Methods</h3> A total of 1024 participants aged 35–64 years with <i>H pylori</i> infection and advanced gastric lesions were randomly assigned in a factorial design to two interventions or placebo: anti-<i>H pylori</i> treatment for 7 days, and a COX-2 inhibitor (celecoxib) for 24 months. The effects of the interventions were evaluated by the regression or progression of advanced gastric lesions. <h3>Results</h3> Of the 1024 participants who received anti-<i>H pylori</i> treatment or placebo, 919 completed a subsequent 24-month treatment with celecoxib or placebo. The <i>H pylori</i> eradication rate by per-protocol analysis was 78.2%. Compared with placebo, the proportions of regression of gastric lesions significantly increased in the celecoxib treatment (52.8% vs 41.2%) and anti-<i>H pylori</i> treatment (59.3% vs 41.2%) group, and OR by per-protocol analysis was 1.72 (95% CI 1.07 to 2.76) for celecoxib and 2.19 (95% CI 1.32 to 3.64) for <i>H pylori</i> eradication. No statistically significant effect was found for <i>H pylori</i> eradication followed by celecoxib on the regression of advanced gastric lesions (OR 1.48, 95% CI 0.91 to 2.40). <h3>Conclusion</h3> This population-based intervention trial revealed that celecoxib treatment or <i>H pylori</i> eradication alone had beneficial effects on the regression of advanced gastric lesions. No favourable effects were seen for <i>H pylori</i> eradication followed by celecoxib treatment. <h3>Trial registration</h3> HARECCTR0500053 in accordance with WHO ICTRP requirements.

Traditional Chinese medicine (TCM) is a medical system with over 3000 years of continuous practice experience and refinement through treatment observations. The TCM pattern classification (also defined as Syndrome or Zheng differentiation) and treatment of ill health is the basis and the key concept of the TCM theory. All diagnostic and therapeutic methods in TCM are based on the differentiation of TCM pattern. TCM pattern can be considered as the TCM theoretical interpretation of the symptom profiles. Pattern classification is often used as a guideline in disease classification in TCM practice and has been recently incorporated with biomedical diagnosis, resulting in the increasing research interest of TCM pattern among various disciplines of integrative medicine. This paper describes the historical evolution on the integration of the TCM pattern classification and disease diagnosis in biomedicine, the methodology of pattern classification for diseases, efficacy of TCM practice with integration of TCM pattern classification and biomedical disease diagnosis, and the biological basis of TCM pattern. TCM pattern classification, which may lead to new findings in biological sciences, was also discussed.

Patient-reported outcomes (PROs) play an increasingly important role in clinical practice and research. Modern psychometric methods such as item response theory (IRT) enable the creation of item banks that support fixed-length forms as well as computerized adaptive testing (CAT), often resulting in improved measurement precision and responsiveness. Here we describe and discuss the case for developing an international core set of PROs building from the US PROMIS® network. PROMIS is a U.S.-based cooperative group of research sites and centers of excellence convened to develop and standardize PRO measures across studies and settings. If extended to a global collaboration, PROMIS has the potential to transform PRO measurement by creating a shared, unifying terminology and metric for reporting of common symptoms and functional life domains. Extending a common set of standardized PRO measures to the international community offers great potential for improving patient-centered research, clinical trials reporting, population monitoring, and health care worldwide. Benefits of such standardization include the possibility of: international syntheses (such as meta-analyses) of research findings; international population monitoring and policy development; health services administrators and planners access to relevant information on the populations they serve; better assessment and monitoring of patients by providers; and improved shared decision making. The goal of the current PROMIS International initiative is to ensure that item banks are translated and culturally adapted for use in adults and children in as many countries as possible. The process includes 3 key steps: translation/cultural adaptation, calibration, and validation. A universal translation, an approach focusing on commonalities, rather than differences across versions developed in regions or countries speaking the same language, is proposed to ensure conceptual equivalence for all items. International item calibration using nationally representative samples of adults and children within countries is essential to demonstrate that all items possess expected strong measurement properties. Finally, it is important to demonstrate that the PROMIS measures are valid, reliable and responsive to change when used in an international context. IRT item banking will allow for tailoring within countries and facilitate growth and evolution of PROs through contributions from the international measurement community. A number of opportunities and challenges of international development of PROs item banks are discussed.

Interference of the binding of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has become a new inspiring immunotherapy for resisting cancers.To date, the FDA has approved two PD-1 monoclonal antibody drugs against cancer as well as a monoclonal antibody for PD-L1.More PD-1 and PD-L1 monoclonal antibody drugs are on their way in clinical trials.In this review, we focused on the mechanism of the PD-1/PD-L1 signaling pathway and the monoclonal antibodies (mAbs) against PD-1 and PD-L1, which were approved by the FDA or are still in clinical trials.And also presented is the prospect of the PD-1/PD-L1 immune checkpoint blockade in the next generation of immunotherapy.

Novel vaccines are urgently needed to reduce the burden of severe malaria. Using a differential whole-proteome screening method, we identified Plasmodium falciparum schizont egress antigen-1 (PfSEA-1), a 244-kilodalton parasite antigen expressed in schizont-infected red blood cells (RBCs). Antibodies to PfSEA-1 decreased parasite replication by arresting schizont rupture, and conditional disruption of PfSEA-1 resulted in a profound parasite replication defect. Vaccination of mice with recombinant Plasmodium berghei PbSEA-1 significantly reduced parasitemia and delayed mortality after lethal challenge with the Plasmodium berghei strain ANKA. Tanzanian children with antibodies to recombinant PfSEA-1A (rPfSEA-1A) did not experience severe malaria, and Kenyan adolescents and adults with antibodies to rPfSEA-1A had significantly lower parasite densities than individuals without these antibodies. By blocking schizont egress, PfSEA-1 may synergize with other vaccines targeting hepatocyte and RBC invasion.

Osteoporosis, a bone disease resulting in the loss of bone density and microstructure quality, is often associated with fragility fractures, and the latter imposes a great burden on the patient and society. Although there are several different treatments available for osteoporosis such as hormone replacement therapy, bisphosphonates, Denosumab, and parathyroid hormone, some concerns have been raised regarding the inherent side effects of their long term use. It would be of great relevance to search for alternative natural compounds, which could complementarily overcome the limitations of the currently available therapy. Herein, we review current literature on natural compounds that might have therapeutic values for osteoporosis. Search terms included bone resorption, bone density, osteoporosis, postmenopausal, osteoporosis or bone density conservation agents, and any of the terms related to traditional, herbal, natural therapy, natural health, diet, or phytoestrogens. All the compounds and herbs included in the review are naturally bioactive or are used in folk herbal medicine and have been reported to be capable of attenuating osteopenia or osteoporosis in vivo or in vitro, through various mechanisms - estrogen-like activity, antioxidant and anti-inflammatory properties, or by modulating the key signaling pathways in the pathogenesis of osteoporosis. Through our assessment of the therapeutic potential and outlook of alternative medicine, we aim to provide an appealing perspective for the consideration of the application of a complementary anti-osteoporotic treatment option and prevention strategy for osteoporosis or osteolytic bone disorders.

Recovery from skeletal muscle injury is often incomplete because of the formation of fibrosis and inadequate myofiber regeneration; therefore, injured muscle could benefit significantly from therapies that both stimulate muscle regeneration and inhibit fibrosis. To this end, we focused on blocking myostatin, a member of the transforming growth factor-β superfamily and a negative regulator of muscle regeneration, with the myostatin antagonist follistatin. In vivo, follistatin-overexpressing transgenic mice underwent significantly greater myofiber regeneration and had less fibrosis formation compared with wild-type mice after skeletal muscle injury. Follistatin's mode of action is likely due to its ability to block myostatin and enhance neovacularization. Furthermore, muscle progenitor cells isolated from follistatin-overexpressing mice were significantly superior to muscle progenitors isolated from wild-type mice at regenerating dystrophin-positive myofibers when transplanted into the skeletal muscle of dystrophic mdx/severe combined immunodeficiency mice. In vitro, follistatin stimulated myoblasts to express MyoD, Myf5, and myogenin, which are myogenic transcription factors that promote myogenic differentiation. Moreover, follistatin's ability to enhance muscle differentiation is at least partially due to its ability to block myostatin, activin A, and transforming growth factor-β1, all of which are negative regulators of muscle cell differentiation. The findings of this study suggest that follistatin is a promising agent for improving skeletal muscle healing after injury and muscle diseases, such as the muscular dystrophies.

Drug discovery is a risky, costly and time-consuming process depending on multidisciplinary methods to create safe and effective medicines. Although considerable progress has been made by high-throughput screening methods in drug design, the cost of developing contemporary approved drugs did not match that in the past decade. The major reason is the late-stage clinical failures in Phases II and III because of the complicated interactions between drug-specific, human body and environmental aspects affecting the safety and efficacy of a drug. There is a growing hope that systems-level consideration may provide a new perspective to overcome such current difficulties of drug discovery and development. The systems pharmacology method emerged as a holistic approach and has attracted more and more attention recently. The applications of systems pharmacology not only provide the pharmacodynamic evaluation and target identification of drug molecules, but also give a systems-level of understanding the interaction mechanism between drugs and complex disease. Therefore, the present review is an attempt to introduce how holistic systems pharmacology that integrated in silico ADME/T (i.e., absorption, distribution, metabolism, excretion and toxicity), target fishing and network pharmacology facilitates the discovery of small molecular drugs at the system level.

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

Dysregulated microRNAs in osteoclasts could cause many skeletal diseases. The therapeutic manipulation of these pathogenic microRNAs necessitates novel, efficient delivery systems to facilitate microRNAs modulators targeting osteoclasts with minimal off-target effects. Bone resorption surfaces characterized by highly crystallized hydroxyapatite are dominantly occupied by osteoclasts. Considering that the eight repeating sequences of aspartate (D-Asp8) could preferably bind to highly crystallized hydroxyapatite, we developed a targeting system by conjugating D-Asp8 peptide with liposome for delivering microRNA modulators specifically to bone resorption surfaces and subsequently encapsulated antagomir-148a (a microRNA modulator suppressing the osteoclastogenic miR-148a), i.e. (D-Asp8)-liposome-antagomir-148a. Our results demonstrated that D-Asp8 could facilitate the enrichment of antagomir-148a and the subsequent down-regulation of miR-148a in osteoclasts in vivo, resulting in reduced bone resorption and attenuated deterioration of trabecular architecture in osteoporotic mice. Mechanistically, the osteoclast-targeted delivery depended on the interaction between bone resorption surfaces and D-Asp8. No detectable liver and kidney toxicity was found in mice after single/multiple dose(s) treatment of (D-Asp8)-liposome-antagomir-148a. These results indicated that (D-Asp8)-liposome as a promising osteoclast-targeting delivery system could facilitate clinical translation of microRNA modulators in treating those osteoclast-dysfunction-induced skeletal diseases.

Adult multipotent stem cells have been isolated from a variety of human tissues including human skeletal muscle, which represent an easily accessible source of stem cells. It has been shown that human skeletal muscle-derived stem cells (hMDSCs) are muscle-derived mesenchymal stem cells capable of multipotent differentiation. Although hMDSCs can undergo osteogenic differentiation and form bone when genetically modified to express BMP2; it is still unclear whether hMDSCs are as efficient as human bone marrow mesenchymal stem cells (hBMMSCs) for bone regeneration. The current study aimed to address this question by performing a parallel comparison between hMDSCs and hBMMSCs to evaluate their osteogenic and bone regeneration capacities. Our results demonstrated that hMDSCs and hBMMSCs had similar osteogenic-related gene expression profiles and had similar osteogenic differentiation capacities in vitro when transduced to express BMP2. Both the untransduced hMDSCs and hBMMSCs formed very negligible amounts of bone in the critical sized bone defect model when using a fibrin sealant scaffold; however, when genetically modified with lenti-BMP2, both populations successfully regenerated bone in the defect area. No significant differences were found in the newly formed bone volumes and bone defect coverage between the hMDSC and hBMMSC groups. Although both cell types formed mature bone tissue by 6 weeks post-implantation, the newly formed bone in the hMDSCs group underwent quicker remodelling than the hBMMSCs group. In conclusion, our results demonstrated that hMDSCs are as efficient as hBMMSCs in terms of their bone regeneration capacity; however, both cell types required genetic modification with BMP in order to regenerate bone in vivo.

Triptolide (TP), an active component isolated from Tripterygiumwilfordii Hook F, has therapeutic potential against rheumatoid arthritis (RA). However, the underlying molecular mechanism has not been fully elucidated. The aim of this study is to investigate the mechanisms of TP acting on RA by combining bioinformatics analysis with experiment validation. The human protein targets of TP and the human genes of RA were found in the PubChem database and NCBI, respectively. These two dataset were then imported into Ingenuity Pathway Analysis (IPA) software online, and then the molecular network of TP on RA could be set up and analyzed. After that, both in vitro and in vivo experiments were done to further verify the prediction. The results indicated that the main canonical signal pathways of TP protein targets networks were mainly centered on cytokine and cellular immune signaling, and triggering receptors expressed on myeloid cells (TREM)-1 signaling was searched to be the top one shared signaling pathway and involved in the cytokine and cellular immune signaling. Further in vitro experiments indicated that TP not only remarkably lowered the levels of TREM-1 and DNAX-associated protein (DAP)12, but also significantly suppressed the activation of janus activating kinase (JAK)2 and signal transducers and activators of transcription (STAT)3. The expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in lipopolysaccharides (LPS)-stimulated U937 cells also decreased after treatment with TP. Furthermore, TREM-1 knockdown was able to interfere with the inhibition effects of TP on these cytokines production. In vivo experiments showed that TP not only significantly inhibited the TREM-1 mRNA and DAP12 mRNA expression, and activation of JAK2 and STAT3 in ankle of rats with collagen-induced arthritis (CIA), but also remarkably decreased production of TNF-α, IL-1β and IL-6 in serum and joint. These findings demonstrated that TP could modulate the TREM1 signal pathway to inhibit the inflammatory response in RA.

Triptolide (TP), a major extract of the herb Tripterygium wilfordii Hook F (TWHF), has been shown to exert potent pharmacological effects, especially an immunosuppressive effect in the treatment of rheumatoid arthritis (RA). However, its multiorgan toxicity prevents it from being widely used in clinical practice. Recently, several attempts are being performed to reduce TP toxicity. In this review, recent progress in the use of TP for RA, including its pharmacological effects and toxicity, is summarized. Meanwhile, strategies relying on chemical structural modifications, innovative delivery systems, and drug combinations to alleviate the disadvantages of TP are also reviewed. Furthermore, we also discuss the challenges and perspectives in their clinical translation.

Therapeutic genome editing technology has been widely used as a powerful tool for directly correcting genetic mutations in target pathological tissues and cells to cure of diseases.The modification of specific genomic sequences can be achieved by utilizing programmable nucleases, such as Meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly-interspaced short palindromic repeat-associated nuclease Cas9 (CRISPR/Cas9).However, given the properties, such as large size, negative charge, low membrane penetrating ability, as well as weak tolerance for serum, and low endosomal escape, of these nucleases genome editing cannot be successfully applied unless in vivo delivery of related programmable nucleases into target organisms or cells is achieved.Here, we look back at delivery strategies having been used in the in vivo delivery of three main genome editing nucleases, followed by methodologies currently undergoing testing in clinical trials, and potential delivery strategies provided by analyzing characteristics of nucleases and commonly used vectors.

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1-/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.

Breast cancer remains one of the most life-threatening tumors affecting women. Most patients with advanced breast cancer eventually develop metastatic diseases, which cause significant morbidity and mortality. Approximately two-thirds of patients with advanced breast cancer exhibit osteolytic-type bone metastasis, which seriously reduce the quality of life. Therefore, development of novel therapeutic strategies for treating breast cancer patients with bone metastasis is urgently required. The "seed and soil" theory, which describes the interaction between the circulating breast cancer cells (seeds) and bone microenvironment (soil), is widely accepted as the mechanism underlying metastasis. Disruption of any step in this cycle might have promising anti-metastasis implications. The interaction of receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK is fundamental in this vicious cycle and has been shown to be a novel effective therapeutic target. A series of therapeutic strategies have been developed to intervene in this cross-talk. Therefore, in this review, we have systematically introduced the functions of the RANKL/RANK signaling system in breast cancer and discussed related therapeutic strategies.

NF-κB is a transcription factor activated in response to inflammatory, genotoxic and oxidative stress and important for driving senescence and aging. Ataxia-telangiectasia mutated (ATM) kinase, a core component of DNA damage response signaling, activates NF-κB in response to genotoxic and oxidative stress via post-translational modifications. Here we demonstrate that ATM is activated in senescent cells in culture and murine tissues from Ercc1-deficient mouse models of accelerated aging, as well as naturally aged mice. Genetic and pharmacologic inhibition of ATM reduced activation of NF-κB and markers of senescence and the senescence-associated secretory phenotype (SASP) in senescent Ercc1-/- MEFs. Ercc1-/Δ mice heterozygous for Atm have reduced NF-κB activity and cellular senescence, improved function of muscle-derived stem/progenetor cells (MDSPCs) and extended healthspan with reduced age-related pathology especially age-related bone and intervertebral disc pathologies. In addition, treatment of Ercc1-/∆ mice with the ATM inhibitor KU-55933 suppressed markers of senescence and SASP. Taken together, these results demonstrate that the ATM kinase is a major mediator of DNA damage-induced, NF-κB-mediated cellular senescence, stem cell dysfunction and aging and thus represents a therapeutic target to slow the progression of aging.

Aptamers are short single-stranded DNA, RNA, or synthetic Xeno nucleic acids (XNA) molecules that can interact with corresponding targets with high affinity. Owing to their unique features, including low cost of production, easy chemical modification, high thermal stability, reproducibility, as well as low levels of immunogenicity and toxicity, aptamers can be used as an alternative to antibodies in diagnostics and therapeutics. Systematic evolution of ligands by exponential enrichment (SELEX), an experimental approach for aptamer screening, allows the selection and identification of in vitro aptamers with high affinity and specificity. However, the SELEX process is time consuming and characterization of the representative aptamer candidates from SELEX is rather laborious. Artificial intelligence (AI) could help to rapidly identify the potential aptamer candidates from a vast number of sequences. This review discusses the advancements of AI pipelines/methods, including structure-based and machine/deep learning-based methods, for predicting the binding ability of aptamers to targets. Structure-based methods are the most used in computer-aided drug design. For this part, we review the secondary and tertiary structure prediction methods for aptamers, molecular docking, as well as molecular dynamic simulation methods for aptamer-target binding. We also performed analysis to compare the accuracy of different secondary and tertiary structure prediction methods for aptamers. On the other hand, advanced machine-/deep-learning models have witnessed successes in predicting the binding abilities between targets and ligands in drug discovery and thus potentially offer a robust and accurate approach to predict the binding between aptamers and targets. The research utilizing machine-/deep-learning techniques for prediction of aptamer-target binding is limited currently. Therefore, perspectives for models, algorithms, and implementation strategies of machine/deep learning-based methods are discussed. This review could facilitate the development and application of high-throughput and less laborious in silico methods in aptamer selection and characterization.

Drug-drug interaction (DDI) can trigger many adverse effects in patients and has emerged as a threat to medicine and public health. Despite the continuous information accumulation of clinically significant DDIs, there are few open-access knowledge systems dedicated to the curation of DDI associations. To facilitate the clinicians to screen for dangerous drug combinations and improve health systems, we present DDInter, a curated DDI database with comprehensive data, practical medication guidance, intuitive function interface, and powerful visualization to the scientific community. Currently, DDInter contains about 0.24M DDI associations connecting 1833 approved drugs (1972 entities). Each drug is annotated with basic chemical and pharmacological information and its interaction network. For DDI associations, abundant and professional annotations are provided, including severity, mechanism description, strategies for managing potential side effects, alternative medications, etc. The drug entities and interaction entities are efficiently cross-linked. In addition to basic query and browsing, the prescription checking function is developed to facilitate clinicians to decide whether drugs combinations can be used safely. It can also be used for informatics-based DDI investigation and evaluation of other prediction frameworks. We hope that DDInter will prove useful in improving clinical decision-making and patient safety. DDInter is freely available, without registration, at http://ddinter.scbdd.com/.

Traditional Chinese medicine (TCM) is currently considered a complementary or alternative medical system in most Western countries and has been increasingly accepted worldwide. More and more clinical trials on TCM have been conducted internationally, and scientists worldwide are becoming increasingly interested in the evaluation of clinical efficacy of TCM based on clinical trials. This paper reviews the situation of clinical trials on TCM in the past decade, including systematic reviews about clinical trials either focusing on the treatment of disease with TCM approaches or focusing on one herbal product, conduction of clinical trials on TCM either with randomization and controlled methods or general observation. Some general issues on the conduct of clinical trials on TCM, such as randomization, control, quality of life (QOL), patient reported outcomes (PROs) and biomarkers, quality control, safety evaluation and case studies, are discussed, and accordingly some suggestions are proposed.

In Brief Background: Complex chronic diseases such as rheumatoid arthritis have become a major challenge in medicine and for the pharmaceutical industry. New impulses for drug development are needed. Objective: A systems biology approach is explored to find subtypes of rheumatoid arthritis patients enabling a development towards more personalized medicine. Methods: Blood samples of 33 rheumatoid arthritis (RA) patients and 16 healthy volunteers were collected. The RA patients were diagnosed according to Chinese medicine (CM) theory and divided into 2 groups, the RA Heat and RA Cold group. CD4+ T-cells were used for a total gene expression analysis. Metabolite profiles were measured in plasma using gas chromatography/mass spectrometry. Multivariate statistics was employed to find potential biomarkers for the RA Heat and RA Cold phenotype. A comprehensive biologic interpretation of the results is discussed. Results: The genomics and metabolomics analysis showed statistically relevant different gene expression and metabolite profiles between healthy controls and RA patients as well as between the RA Heat and RA Cold group. Differences were found in the regulation of apoptosis. In the RA Heat group caspase 8 activated apoptosis seems to be stimulated while in the RA Cold group apoptosis seems to be suppressed through the Nrf2 pathway. Conclusions: RA patients could be divided in 2 groups according to CM theory. Molecular differences between the RA Cold and RA Heat groups were found which suggest differences in apoptotic activity. Subgrouping of patients according to CM diagnosis has the potential to provide opportunities for better treatment outcomes by targeting Western or CM treatment to specific groups of patients. This exploratory study suggests that classification by Chinese Medicine (CM) as “RA heat” or “RA cold” may correlate with molecular differences and that CM features might identify subtypes of RA amenable to different therapies.

Objectives: The objective of this study is to explore the role of Traditional Chinese Medicine (TCM) pattern differentiation in identifying a subset of patients with rheumatoid arthritis (RA) who are more likely to respond to biomedical combination therapy. Methods: This study uses data from a previous multicenter randomized-controlled clinical trial. One hundred and ninety-four (194) patients were treated with biomedical combination therapy (diclofenac, methotrexate, and sulfasalazine). ACR20 response at 12 weeks and 24 weeks was used for evaluation of efficacy. Eight (8) symptoms, which are the most important for establishing TCM cold and hot patterns in patients with RA, were analyzed in this study. TCM patterns were obtained using factor analysis of the eight symptoms. Thirst, vexation, hot feeling in the joints, turbid yellow-colored urine, and fever were classified as factor 1. Cold feeling in the whole body, cold feeling in the limbs, and cold feeling in the joints were classified as factor 2. The classification into factor 1 and 2 is similar to TCM hot pattern and cold pattern differentiation, since the symptoms in factor 1 and 2 are the key symptoms in TCM hot and cold patterns, respectively. The effective rates in patients with different TCM patterns were analyzed with the χ2 method. Results: At 12 weeks, ACR20 response in patients treated with the biomedical combination therapy was 36.08%. At 24 weeks, ACR20 response was 69.59%. Based on the eight symptoms used in factor analysis, the effective rates in the patients with cold pattern and hot pattern were 51.67% and 29.09%, respectively, after 12 weeks of treatment; and 88.52% and 55.36%, respectively, after 24 weeks of treatment. Conclusions: TCM pattern differentiation based on symptoms can help identify a subset of patients with RA that will more likely respond to biomedical therapy, consisting of diclofenac, methotrexate, and sulfasalazine.

Peripheral nerve injuries and neuropathies lead to profound functional deficits. Here, we have demonstrated that muscle-derived stem/progenitor cells (MDSPCs) isolated from adult human skeletal muscle (hMDSPCs) can adopt neuronal and glial phenotypes in vitro and ameliorate a critical-sized sciatic nerve injury and its associated defects in a murine model. Transplanted hMDSPCs surrounded the axonal growth cone, while hMDSPCs infiltrating the regenerating nerve differentiated into myelinating Schwann cells. Engraftment of hMDSPCs into the area of the damaged nerve promoted axonal regeneration, which led to functional recovery as measured by sustained gait improvement. Furthermore, no adverse effects were observed in these animals up to 18 months after transplantation. Following hMDSPC therapy, gastrocnemius muscles from mice exhibited substantially less muscle atrophy, an increase in muscle mass after denervation, and reorganization of motor endplates at the postsynaptic sites compared with those from PBS-treated mice. Evaluation of nerve defects in animals transplanted with vehicle-only or myoblast-like cells did not reveal histological or functional recovery. These data demonstrate the efficacy of hMDSPC-based therapy for peripheral nerve injury and suggest that hMDSPC transplantation has potential to be translated for use in human neuropathies.

Rheumatoid arthritis (RA) is the most severe type of chronic inflammatory disease and has always been a research hotspot in different fields. In this study, a non-targeted metabonomics approach was carried out to profile metabolic characteristics of RA and its Chinese medicine subtypes by using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Plasma samples of 57 RA patients and 23 healthy controls were collected. On the basis of the traditional Chinese medicine (TCM), RA patients were classified into two main patterns, the cold pattern and the heat pattern. By using univariate and multivariate data analysis, we found that the RA patients presented diverse dysfunctions in inositol phosphate metabolism, lipid metabolism, amino acid metabolism, glucose metabolism, ascorbate metabolism, glyoxylate and dicarboxylate metabolism. The metabolic phenotypes were different between the RA cold pattern and the RA heat pattern. Compared with the RA cold pattern, the RA heat pattern showed elevated plasma concentrations of glycochenodeoxycholate, proline, saturated and mono-unsaturated phosphatidylcholine (PC) but decreased levels of urea, free fatty acid (FFA) and polyunsaturated PC. Our data show that metabonomics is a valuable tool in disease and TCM subtype research.

Our lab developed and optimized a method, known as the modified pre-plate technique, to isolate stem/progenitor cells from skeletal muscle. This method separates different populations of myogenic cells based on their propensity to adhere to a collagen I-coated surface. Based on their surface markers and stem-like properties, including self-renewal, multi-lineage differentiation, and ability to promote tissue regeneration, the last cell fraction or slowest to adhere to the collagen-coated surface (pre-plate 6; pp6) appears to be early, quiescent progenitor cells termed muscle-derived stem/progenitor cells (MDSPCs). The cell fractions preceding pp6 (pp1–5) are likely populations of more committed (differentiated) cells, including fibroblast- and myoblast-like cells. This technique may be used to isolate MDSPCs from skeletal muscle of humans or mice regardless of age, sex or disease state, although the yield of MDSPCs varies with age and health. MDSPCs can be used for regeneration of a variety of tissues including bone, articular cartilage, skeletal and cardiac muscle, and nerve. MDSPCs are currently being tested in clinical trials for treatment of urinary incontinence and myocardial infarction. MDSPCs from young mice have also been demonstrated to extend life span and healthspan in mouse models of accelerated aging through an apparent paracrine/endocrine mechanism. Here we detail methods for isolation and characterization of MDSPCs.

Current strategies for drug discovery have reached a bottleneck where the paradigm is generally “one gene, one drug, one disease.” However, using holistic and systemic views, network pharmacology may be the next paradigm in drug discovery. Based on network pharmacology, a combinational drug with two or more compounds could offer beneficial synergistic effects for complex diseases. Interestingly, traditional chinese medicine (TCM) has been practicing holistic views for over 3,000 years, and its distinguished feature is using herbal formulas to treat diseases based on the unique pattern classification. Though TCM herbal formulas are acknowledged as a great source for drug discovery, no drug discovery strategies compatible with the multidimensional complexities of TCM herbal formulas have been developed. In this paper, we highlighted some novel paradigms in TCM-based network pharmacology and new drug discovery. A multiple compound drug can be discovered by merging herbal formula-based pharmacological networks with TCM pattern-based disease molecular networks. Herbal formulas would be a source for multiple compound drug candidates, and the TCM pattern in the disease would be an indication for a new drug.

Malignant melanoma is one of the most common malignancies. Its incidence grows rapidly at an annual rate of 3−5%. Although the incidence of melanoma remains low in China, it has increased rapidly, with approximately 20,000 new cases reported each year. The mortality of melanoma also has been increasing rapidly; in contrast, although the incidence of melanoma is also increasing in western countries, its mortality basically remains unchanged and does not rise along with the escalation in prevalence. Thus, there is still a wide gap between China and Western countries in the diagnosis and treatment of melanoma. Melanoma has become one of the diseases that pose a major threat to health of Chinese people. However, compared with other common malignant tumors, there is still a far way to go to achieve the standardized diagnosis and treatment of melanoma. In May 2007, the Chinese Society of Clinical Oncology (CSCO) formally established the CSCO Melanoma Panel with an attempt to promote the development of clinical oncology, facilitate the multidisciplinary standardized treatment for melanoma, advocate the active learning and application of currently available scientific evidences at home and abroad, and explore the development of Chinese guidelines on the clinical practices on melanoma. After consultations with multidisciplinary experts, the first edition of the Chinese Consensus on the Diagnosis and Treatment of Melanoma was released in 2008; in 2009, 2011, and 2013, three revisions of this consensus document were published after many multidisciplinary seminars. The past 5 years have witnessed several breakthroughs in the clinical treatment of melanoma. Melanoma has become one of the malignant tumors whose treatment patterns have changed rapidly. To adapt to the fast advances in melanoma treatment and make the clinical management of melanoma in China more standardized and internationalized, the 2015 edition of the Chinese Guidelines on the Diagnosis and Treatment of Melanoma was finalized after repeated and wide consultations with multidisciplinary experts and updated and added with much new information, with an attempt to provide the up-todated and reliable instructions on clinical practices based recent scientific evidences.

Similar symptoms of the different types of arthritis have continued to confound the clinical diagnosis and represent a clinical dilemma making treatment choices with a more personalized or generalized approach. Here we report a mass spectrometry-based metabolic phenotyping study to identify the global metabolic defects associated with arthritis as well as metabolic signatures of four major types of arthritis--rheumatoid arthritis (n = 27), osteoarthritis (n = 27), ankylosing spondylitis (n = 27), and gout (n = 33)--compared with healthy control subjects (n = 60). A total of 196 metabolites were identified from serum samples using a combined gas chromatography coupled with time-of-flight mass spectrometry (GC-TOF MS) and ultraperformance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-QTOF MS). A global metabolic profile is identified from all arthritic patients, suggesting that there are common metabolic defects resulting from joint inflammation and lesion. Meanwhile, differentially expressed serum metabolites are identified constituting an unique metabolic signature of each type of arthritis that can be used as biomarkers for diagnosis and patient stratification. The results highlight the applicability of metabonomic phenotyping as a novel diagnostic tool for arthritis complementary to existing clinical modalities.

Abstract The role of osteoclastic miRNAs in regulating osteolytic bone metastasis (OBM) of breast cancer is still underexplored. Here, we examined the expression profiles of osteoclastogenic miRNAs in human bone specimens and identified that miR-214-3p was significantly upregulated in breast cancer patients with OBM. Consistently, we found increased miR-214-3p within osteoclasts, which was associated with the elevated bone resorption, during the development of OBM in human breast cancer xenografted nude mice (BCX). Furthermore, genetic ablation of osteoclastic miR-214-3p in nude mice prevent the development of OBM. Conditioned medium from MDA-MB-231 cells dramatically stimulated miR-214-3p expression to promote osteoclast differentiation. Mechanistically, a series of in vitro study showed that miR-214-3p directly targeted Traf3 to promote osteoclast activity and bone-resorbing activity. In addition, osteoclast-specific miR-214-3p knock-in mice showed remarkably increased bone resorption when compared to the littermate controls, which was attenuated after osteoclast-targeted treatment with Traf3 3′UTR-containing plasmid. In BCX nude mice, osteoclast-targeted antagomir-214-3p delivery could recover the TRAF3 protein expression and attenuate the development of OBM, respectively. Collectively, inhibition of osteoclastic miR-214-3p may be a potential therapeutic strategy for breast cancer patients with OBM. Meanwhile, the intraosseous TRAF3 could be a promising biomarker for evaluation of the treatment response of antagomir-214-3p.

Boswellic acids have long been considered the main bioactive components of frankincense, and many studies <i>in vitro</i> and in animals as well as several clinical studies have confirmed their various bioactivities. In particular, a large number of mechanistic studies have confirmed their anti-inflammatory and antitumor activities. However, not every boswellic acid exhibits a satisfactory pharmacological performance, which depends on the chemical structure and functional groups of the acid. To enhance the pharmacological values of boswellic acids, derivatization has been specifically applied with the aim of discovering more active derivatives of BAs. In addition, the preliminary pharmacokinetic studies of these compounds using various standard methods show their poor bioavailability in humans and rodents, which has led to questions of their pharmacological relevance and potentially limits their use in clinical practice and pharmaceutical development. To improve these effects, some approaches have shown some improvements in effectiveness, and the new formula compatibility approach is considered a very reasonable method for improving the bioavailability of boswellic acids.

The STandards for Reporting Interventions in Clinical Trials Of Moxibustion (STRICTOM), in the form of a checklist and descriptions of checklist items, were designed to improve reporting of moxibustion trials, and thereby facilitating their interpretation and replication. The STRICTOM checklist included 7 items and 16 sub-items. These set out reporting guidelines for the moxibustion rationale, details of moxibustion, treatment regimen, other components of treatment, treatment provider background, control and comparator interventions, and precaution measures. In addition, there were descriptions of each item and examples of good reporting. It is intended that the STRICTOM can be used in conjunction with the main CONSORT Statement, extensions for nonpharmacologic treatment and pragmatic trials, and thereby raise the quality of reporting of clinical trials of moxibustion. Further comments will be solicited from the experts of the CONSORT Group, the STRICTA Group, acupuncture and moxibustion societies, and clinical trial authors for optimizing the STRICTOM.

Abstract Introduction Duchenne muscular dystrophy (DMD), caused by a lack of the functional structural protein dystrophin, leads to severe muscle degeneration where the patients are typically wheelchair-bound and die in their mid-twenties from cardiac or respiratory failure or both. The aim of this study was to investigate the potential of human dental pulp stem cells (hDPSCs) and human amniotic fluid stem cells (hAFSCs) to differentiate toward a skeletal myogenic lineage using several different protocols in order to determine the optimal conditions for achieving myogenic commitment and to subsequently evaluate their contribution in the improvement of the pathological features associated with dystrophic skeletal muscle when intramuscularly injected into mdx /SCID mice, an immune-compromised animal model of DMD. Methods Human DPSCs and AFSCs were differentiated toward myogenic lineage in vitro through the direct co-culture with a myogenic cell line (C2C12 cells) and through a preliminary demethylation treatment with 5-Aza-2′-deoxycytidine (5-Aza), respectively. The commitment and differentiation of both hDPSCs and hAFSCs were evaluated by immunofluorescence and Western blot analysis. Subsequently, hDPSCs and hAFSCs, preliminarily demethylated and pre-differentiated toward a myogenic lineage for 2 weeks, were injected into the dystrophic gastrocnemius muscles of mdx /SCID mice. After 1, 2, and 4 weeks, the gastrocnemius muscles were taken for immunofluorescence and histological analyses. Results Both populations of cells engrafted within the host muscle of mdx /SCID mice and through a paracrine effect promoted angiogenesis and reduced fibrosis, which eventually led to an improvement of the histopathology of the dystrophic muscle. Conclusion This study shows that hAFSCs and hDPSCs represent potential sources of stem cells for translational strategies to improve the histopathology and potentially alleviate the muscle weakness in patients with DMD.

Purpose: This study aimed to explore underlying action mechanism of Wu-Tou decoction (WTD) in rheumatoid arthritis (RA) through network pharmacology prediction and experimental verification. Methods: Chemical compounds and human target proteins of WTD as well as RA-related human genes were obtained from TCM Database @ Taiwan, PubChem and GenBank, respectively. Subsequently, molecular networks and canonical pathways presumably involved in the treatment of WTD on RA were generated by ingenuity pathway analysis (IPA) software. Furthermore, experimental validation was carried out with MIP-1β-induced U937 cell model and collagen induced arthritis (CIA) rat model. Results: CCR5 signaling pathway in macrophages was shown to be the top one shared signaling pathway associated with both cell immune response and cytokine signaling. In addition, protein kinase C (PKC) δ and p38 in this pathway were treated as target proteins of WTD in RA. In vitro experiments indicated that WTD inhibited MIP-1β-induced production of TNF-α, MIP-1α, and RANTES as well as phosphorylation of CCR5, PKC δ, and p38 in U937 cells. WTD treatment maintained the inhibitory effects on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. In vivo experiments demonstrated that WTD ameliorated symptoms in CIA rats, decreased the levels of IL-1β, IL-2, IL-6, TNF-α, MIP-1α, MIP-2, RANTES, and IP-10 in serum of CIA rats, as well as mRNA levels of MIP-1α, MIP-2, RANTES, and IP-10 in ankle joints of CIA rats. Furthermore, WTD also lowered the phosphorylation levels of CCR5, PKC δ and p38 in both ankle joints and macrophages in ankle joints from CIA rats. Conclusion: It was demonstrated in this research that WTD played a role in inhibiting inflammatory response in RA which was closely connected with the modulation effect of WTD on CCR5 signaling pathway in macrophages.

Cathepsin K (Cat K), highly expressed in osteoclasts, is a cysteine protease member of the cathepsin lysosomal protease family and has been of increasing interest as a target of medicinal chemistry efforts for its role in bone matrix degradation. Inhibition of the Cat K enzyme reduces bone resorption and thus, has rendered the enzyme as an attractive target for anti-resorptive osteoporosis therapy. Over the past decades, considerable efforts have been made to design and develop highly potent, excellently selective and orally applicable Cat K inhibitors. These inhibitors are derived from synthetic compounds or natural products, some of which have passed preclinical studies and are presently in clinical trials at different stages of advancement. In this review, we briefly summarised the historic development of Cat K inhibitors and discussed the relationship between structures of inhibitors and active sites in Cat K for the purpose of guiding future development of inhibitors.

The Journal of PathologyVolume 239, Issue 1 p. 72-83 Original Paper Clonality analysis of multifocal papillary thyroid carcinoma by using genetic profiles Zheming Lu, Zheming Lu Laboratory of Genetics, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaThese authors contributed equally to this work.Search for more papers by this authorJindong Sheng, Jindong Sheng Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaThese authors contributed equally to this work.Search for more papers by this authorYujie Zhang, Yujie Zhang Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorJianhua Deng, Jianhua Deng Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorYong Li, Yong Li Laboratory of Animal Center of the Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorAiping Lu, Aiping Lu Department of Pathology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorJuan Zhang, Juan Zhang Novogene Bioinformatics Technology Co, Ltd, 38 Xueqing Road, Beijing, ChinaSearch for more papers by this authorHuan Yu, Huan Yu Novogene Bioinformatics Technology Co, Ltd, 38 Xueqing Road, Beijing, ChinaSearch for more papers by this authorMin Zhang, Min Zhang Novogene Bioinformatics Technology Co, Ltd, 38 Xueqing Road, Beijing, ChinaSearch for more papers by this authorZikai Xiong, Zikai Xiong Genetron Health, Inc, 8 Life Science Parkway, Beijing, ChinaSearch for more papers by this authorHai Yan, Hai Yan Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC USASearch for more papers by this authorBill H Diplas, Corresponding Author Bill H Diplas Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC USACorrespondence to: B Liu, Department of Head and Neck Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or Y Lu, MD, PhD, Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or BH Diplas, Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. E-mail: [email protected]Search for more papers by this authorYouyong Lu, Corresponding Author Youyong Lu Laboratory of Molecular Oncology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaCorrespondence to: B Liu, Department of Head and Neck Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or Y Lu, MD, PhD, Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or BH Diplas, Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. E-mail: [email protected]Search for more papers by this authorBaoguo Liu, Corresponding Author Baoguo Liu Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaCorrespondence to: B Liu, Department of Head and Neck Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or Y Lu, MD, PhD, Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or BH Diplas, Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. E-mail: [email protected]Search for more papers by this author Zheming Lu, Zheming Lu Laboratory of Genetics, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaThese authors contributed equally to this work.Search for more papers by this authorJindong Sheng, Jindong Sheng Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaThese authors contributed equally to this work.Search for more papers by this authorYujie Zhang, Yujie Zhang Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorJianhua Deng, Jianhua Deng Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorYong Li, Yong Li Laboratory of Animal Center of the Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorAiping Lu, Aiping Lu Department of Pathology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaSearch for more papers by this authorJuan Zhang, Juan Zhang Novogene Bioinformatics Technology Co, Ltd, 38 Xueqing Road, Beijing, ChinaSearch for more papers by this authorHuan Yu, Huan Yu Novogene Bioinformatics Technology Co, Ltd, 38 Xueqing Road, Beijing, ChinaSearch for more papers by this authorMin Zhang, Min Zhang Novogene Bioinformatics Technology Co, Ltd, 38 Xueqing Road, Beijing, ChinaSearch for more papers by this authorZikai Xiong, Zikai Xiong Genetron Health, Inc, 8 Life Science Parkway, Beijing, ChinaSearch for more papers by this authorHai Yan, Hai Yan Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC USASearch for more papers by this authorBill H Diplas, Corresponding Author Bill H Diplas Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC USACorrespondence to: B Liu, Department of Head and Neck Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or Y Lu, MD, PhD, Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or BH Diplas, Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. E-mail: [email protected]Search for more papers by this authorYouyong Lu, Corresponding Author Youyong Lu Laboratory of Molecular Oncology, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaCorrespondence to: B Liu, Department of Head and Neck Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or Y Lu, MD, PhD, Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or BH Diplas, Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. E-mail: [email protected]Search for more papers by this authorBaoguo Liu, Corresponding Author Baoguo Liu Department of Head and Neck Surgery, Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing, ChinaCorrespondence to: B Liu, Department of Head and Neck Surgery, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or Y Lu, MD, PhD, Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital and Institute, 52 Fucheng Road, Beijing 100142, China. E-mail: [email protected] Or BH Diplas, Department of Pathology, Duke University Medical Center, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. E-mail: [email protected]Search for more papers by this author First published: 01 February 2016 https://doi.org/10.1002/path.4696Citations: 48 No conflicts of interest were declared. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract Papillary thyroid carcinoma (PTC) is the most common adult thyroid malignancy and often presents with multiple anatomically distinct foci within the thyroid, known as multifocal papillary thyroid carcinoma (MPTC). The widespread application of the next-generation sequencing technologies in cancer genomics research provides novel insights into determining the clonal relationship between multiple tumours within the same thyroid gland. For eight MPTC patients, we performed whole-exome sequencing and targeted region sequencing to identify the non-synonymous point mutations and gene rearrangements of distinct and spatially separated tumour foci. Among these eight MPTCs, completely discordant mutational spectra were observed in the distinct cancerous nodules of patients MPTC1 and 5, suggesting that these nodules originated from independent precursors. In another three cases (MPTC2, 6, and 8), the distinct MPTC foci of these patients had no other shared mutations except BRAF V600E, also indicating likely independent origins. Two patients (MPTC3 and 4) shared almost identical mutational spectra amongst their separate tumour nodules, suggesting a common clonal origin. MPTC patient 7 had seven cancer foci, of which two foci shared 66.7% of mutations, while the remaining cancer foci displayed no common non-synonymous mutations, indicating that MPTC7 has multiple independent origins accompanied by intraglandular disease dissemination. In this study, we found that 75% of MPTC cases arose as independent tumours, which supports the field cancerization hypothesis describing multiple malignant lesions. MPTC may also arise from intrathyroidal metastases from a single malignant clone, as well as multiple independent origins accompanied by intrathyroidal metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Supporting Information Filename Description path4696-sup-0001-AppendixS1.docxWord 2007 document , 14 KB Supporting information path4696-sup-0002-AppendixS2.xlsxExcel 2007 spreadsheet , 37.6 KB Table S1 Literature summary of studies on the clonal origin of MPTC tumor foci Table S2 Clinical characteristics and genomic alternations of tumour samples analyzed from eight MPTC patients Table S3 Related genes and rearrangement in thyroid carcinoma panel 244 cancer related genes, oncogenes and tumor suppressor genes Table S4 The single nucleotide variants identified by whole exome sequencing path4696-sup-0003-Figure1.tifTIFF image, 3.2 MB MPTC3 is an example of MPTC with tumour foci of common clonal origins. path4696-sup-0004-FigureS2.tifTIFF image, 1.2 MB MPTC5 is an example of MPTC with tumour foci of independent clonal origins. path4696-sup-0005-FigureS3.tifTIFF image, 1.2 MB MPTC6 is an example of MPTC with tumour foci of independent clonal origins. path4696-sup-0006-FigureS4.tifTIFF image, 1.2 MB MPTC8 is an example of MPTC foci developing from independent clonal origins. 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Osteoporosis is a progressive skeletal disorder characterized by low bone mass and increased risk of fracture in later life. The incidence and costs associated with treating osteoporosis cause heavy socio-economic burden. Currently, the diagnosis of osteoporosis mainly depends on bone mineral density and bone turnover markers. However, these indexes are not sensitive and accurate enough to reflect the osteoporosis progression. Metabolomics offers the potential for a holistic approach for clinical diagnoses and treatment, as well as understanding of the pathological mechanism of osteoporosis. In this review, we firstly describe the study subjects of osteoporosis and bio-sample preparation procedures for different analytic purposes, followed by illustrating the biomarkers with potentially predictive, diagnosis and pharmaceutical values when applied in osteoporosis research. Then, we summarize the published metabolic pathways related to osteoporosis. Furthermore, we discuss the importance of chronological data and combination of multi-omics in fully understanding osteoporosis. The application of metabolomics in osteoporosis could provide researchers the opportunity to gain new insight into the metabolic profiling and pathophysiological mechanisms. However, there is still much to be done to validate the potential biomarkers responsible for the progression of osteoporosis and there are still many details needed to be further elucidated.

Baicalin is an important flavonoid compound THAT is isolated from the Scutellaria baicalensis Georgi Chinese herb and plays a critical role in anti‑oxidative, anti‑inflammatory, anti‑infection and anti‑tumor functions. Although baicalin can suppress the proliferation of tumor cells, the underlying mechanisms of baicalin in bleomycin (BLM)‑induced pulmonary fibrosis remain to be elucidated. Thus, the aim of the present study was to determine the role of baicalin in pulmonary fibrosis and fibroblast proliferation in rats. Hematoxylin and eosin (H&E) and Masson staining were used to measure the morphology of pulmonary fibrosis, ELIASA kits were used to test the ROS and inflammation, and western blotting and TUNEL were performed to study the apoptosis proteins. In vitro, MTT assay, flow cytometry, western blotting and immunofluorescence were performed to investigate the effects of baicalin on proliferation of fibroblasts. The most significantly fibrotic changes were identified in the lungs of model rats at day 28. Baicalin (50 mg/kg) attenuated the degree of pulmonary fibrosis, and the hydroxyproline content of the lung tissues was decreased in the baicalin group, compared with the BLM group. Further investigation revealed that baicalin significantly increased glutathione peroxidase (GSH‑px), total‑superoxide dismutase (T‑SOD) and glutathione (GSH) levels, whilst decreasing that of serum malondialdehyde (MDA). TUNEL‑positive cells were significantly decreased in rats treated with baicalin group, compared with the model group. Furthermore, it was found that BLM promoted fibroblasts viability in a dose‑dependent manner in vivo, which was restricted following treatment with different concentrations of baicalin. Moreover, BLM promoted the expression levels of cyclin A, D and E, proliferating cell nuclear antigen, phosphorylated (p)‑AKT and p‑calcium/calmodulin‑dependent protein kinase type. BLM also promoted the transition of cells from the G0/G1 phase to the G2/M and S phases, and increased the intracellular Ca2+ concentration, which was subsequently suppressed by baicalin. Collectively, the results of the present study suggested that baicalin exerted a suppressive effect on BLM‑induced pulmonary fibrosis and fibroblast proliferation.

The inflammatory response has a complex pathogenesis; thus, it is a critical contributor to the development and complication of many diseases. Zhishi and Zhiqiao are famous Citrus herbal medicines that are rich in bioactive phenolic constituents with multiple anti-inflammatory activities.Establishment of a multi-component-target-pathway network strategy to investigate the usage of Zhishi and Zhiqiao on inflammatory diseases can provide a reference for mechanisms of traditional Chinese medicine (TCM).A multi-component-target-pathway network strategy was constructed to elucidate the various antiinflammatory effects of Zhishi and Zhiqiao by integrating multi-constituent determination, network pharmacology, molecular mechanisms in cells and integrated metabolomics in animals.Based on the quantitatively determined global and characteristic chemical profiles of Zhishi and Zhiqiao, the component-target-pathway network was predicted by network pharmacology coupled with text mining and docking. The potential antiinflammatory mechanism of the various components in Zhishi and Zhiqiao were verified using LPS-induced inflammatory responses in RAW 264.7 cells. The different metabolic regulating effects of Zhishi and Zhiqiao against an LPS-induced inflammation model were investigated using a plasma metabolomics strategy.The molecular mechanism of Zhishi mainly suppressed the MAPK signaling pathway, whereas Zhiqiao emphasized the PPAR-AKT signaling pathways simultaneously to block the inflammatory process. Meanwhile, Zhishi and Zhiqiao both exhibited an anti-inflammatory effect by inhibiting the NF-κB signaling pathway to reduce the production of inflammatory mediators. In the metabolomics study, Zhishi and Zhiqiao exhibited variant corrections of the disordered metabolic pathways through amino acid metabolism, glycometabolism and lipid metabolism.All of these results indicate that Zhishi and Zhiqiao, in a diversified mixture, exert their anti-inflammatory effect through variant pathways. These findings can assist in developing the use of Zhishi and Zhiqiao for inflammatory diseases.

Antibiotics-induceds changes in intestinal flora (dysbiosis) may have various effects on the host. Dysbiosis is associated with numerous metabolites including bile acids, which are produced in the liver from cholesterol and metabolized in the gut by intestinal microbiota. Total phenolic extracts of Citrus aurantium L. (TPE-CA) are rich in dietary flavanones and their glycosyl derivatives, including flavones, flavonols, polymethoxyflavones and coumarins, which exert positive health effects on the microbiota. The aim of this study is to elucidate the interplays between the intestinal microbiota and bile acids metabolism attributed to antibiotics. Mice were exposed to broad-spectrum antibiotics, such as ampicillin, streptomycin and clindamycin, for 14 days. This exposure resulted in reduced bacterial diversity and richness, and destroyed intestinal permeability. The homeostasis of bile acids was also affected. Subsequent TPE-CA administration, counteracted most of the dysbiosis, and reshaped intestinal permeability, these effects occurred via upregulation of zonula occludens 1 and occludin associated proteins and downregulation of serum endotoxin compared to the antibiotics group. TPE-CA maintained the homeostasis of bile acids via modulation of the liver -gut axis related farnesoid X receptor (FXR)/ fibroblast growth factor 15 (FGF15) pathway and FXR-targeted protein. Our findings indicated that TPE-CA exerted a protective effect on the restoration of intestinal microbiota composition, reshaped barrier integrity and maintained bile acid homeostasis via the liver - gut axis with antibiotics-induced dysbiosis.

Aptamers are promising therapeutic and diagnostic agents for various diseases due to their high affinity and specificity against target proteins. Structural determination in combination with multiple biochemical and biophysical methods could help to explore the interacting mechanism between aptamers and their targets. Regrettably, structural studies for aptamer–target interactions are still the bottleneck in this field, which are facing various difficulties. In this review, we first reviewed the methods for resolving structures of aptamer–protein complexes and for analyzing the interactions between aptamers and target proteins. We summarized the general features of the interacting nucleotides and residues involved in the interactions between aptamers and proteins. Challenges and perspectives in current methodologies were discussed. Approaches for determining the binding affinity between aptamers and target proteins as well as modification strategies for stabilizing the binding affinity of aptamers to target proteins were also reviewed. The review could help to understand how aptamers interact with their targets and how alterations such as chemical modifications in the structures affect the affinity and function of aptamers, which could facilitate the optimization and translation of aptamers-based theranostics.

The CRISPR/Cas systems in prokaryotes such as bacteria and archaea are the adaptive immune system to prevent infection from viruses, phages, or other foreign substances. When viruses or phages first invade the bacteria, Cas proteins recognize and cut the DNA from viruses or phages into short fragments that will be integrated into the CRISPR array. Once bacteria are invaded again, the modified CRISPR and Cas proteins react quickly to cut DNA at the specified target location, protecting the host. Due to its high efficiency, versatility, and simplicity, the CRISPR/Cas system has become one of the most popular gene editing technologies. In this review, we briefly introduce the CRISPR/Cas systems, focus on several delivery methods including physical delivery, viral vector delivery, and non-viral vector delivery, and the applications of disease therapy. Finally, some problems in CRISPR/Cas9 technology have been proposed, such as the off-target effects, the efficiency of DNA repair mechanisms, and delivery of CRISPR/Cas system safely and efficiently to the target location.

Endogenous reprogramming of pancreas-derived non-beta cells into insulin-producing cells is a promising approach to treat type 1 diabetes (T1D). One strategy that has yet to be explored is the specific delivery of insulin-producing essential genes, Pdx1 and MafA, to pancreatic alpha cells to reprogram the cells into insulin-producing cells in an adult pancreas. In this study, we used an alpha cell-specific glucagon (GCG) promoter to drive Pdx1 and MafA transcription factors to reprogram alpha cells to insulin-producing cells in chemically induced and autoimmune diabetic mice. Our results showed that a combination of a short glucagon-specific promoter with AAV serotype 8 (AAV8) can be used to successfully deliver Pdx1 and MafA to pancreatic alpha cells in the mouse pancreas. Pdx1 and MafA expression specifically in alpha cells were also able to correct hyperglycemia in both induced and autoimmune diabetic mice. With this technology, targeted gene specificity and reprogramming were accomplished with an alpha-specific promotor combined with an AAV-specific serotype and provide an initial basis to develop a novel therapy for the treatment of T1D.

<ns4:p>Background Acute lymphoblastic leukaemia (ALL) is a common type of cancer in children. General anaesthetics are often used on patients undergoing painful procedures during ALL treatments but their effects on ALL malignancy remain unknown. Herein, we aim to study the effect of propofol and sevoflurane on the migration, homing and chemoresistance of ALL cells. Methods NALM-6 and Reh cells were treated with propofol (5 and 10 μg/ml) or sevoflurane (3.6%) <ns4:italic>in vitro</ns4:italic> for six hours. Then, cells were harvested for adhesion assay and migration assay <ns4:italic>in vitro</ns4:italic>. In <ns4:italic>in vivo</ns4:italic> experiments, GFP-NALM-6 cells were pre-treated with propofol (10 μg/ml) or sevoflurane (3.6%) for six hours. Then, cells were injected intravenously to C57BL/6 female mice followed by intravital microscopy. For chemoresistance study, cells were treated with rising concentrations of Ara-c (0.05-50 nM) plus 10μg/ml of propofol or Ara-C plus 3.6% of sevoflurane for 4 hours, followed by the assessment of cell viability via CCK-8 assay and detection of autophagy via flow cytometry. Results Both anaesthetics reduced <ns4:italic>in vivo</ns4:italic> migration and <ns4:italic>in vivo</ns4:italic> homing as exemplified by 1) the reduction in the number of cells entering the bone marrow and 2) the disturbance in homing location in relation to endosteal surface. Our results indicated that general anaesthetics reduced the surface CXCR4 expression and the adhesion of leukaemia cells to thrombin cleaved osteopontin (OPN) was reduced. Those changes might result in the alterations in migration and homing. In addition, both anaesthetics sensitised ALL cells to Ara-c possibly through CXCR4 mediated mechanisms. Propofol but not sevoflurane enhanced chemo-related cell death via inducing cytotoxic autophagy. Conclusion Together, our data suggest that both propofol and sevoflurane could reduce ALL migration, and homing <ns4:italic>in vivo</ns4:italic> and <ns4:italic>in vitro</ns4:italic> via CXCR4 and OPN mediated mechanisms. Both anaesthetics could sensitise ALL cells to chemotherapy possibly via CXCR4 mediated mechanisms.</ns4:p>

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis that predominantly affects the axial skeleton in adolescent patients. The natural history of the disease remains poorly characterized. In this study, we combined GC–MS and LC–MS techniques to evaluate the major metabolic changes in the plasma of AS patients in view of metabonomics. Univariate and multivariate analysis were employed for altered metabolite comparison and pattern recognition. Application of supervised partial least-squares discrminant analysis to either GC–MS or LC–MS data allowed accurate discrimination of AS patients from normal controls, demonstrating its potential diagnostic utilization. In addition, AS patients presented elevated plasma concentrations of proline, glucose, phosphate, urea, glycerol, phenylalanine and homocysteine but reduced levels of phosphocholines, tryptophan and a bipeptide – phenylalanyl-phenylalanine. In the context of their involved metabolic pathways, the identified metabolites were discussed accordingly. This investigation primarily proved that integrated chromatography–mass spectrometry and integrated uni- and multi-variate statistical analysis facilitated metabonomics to be a more promising tool in disease research.

The research is aimed to explore the distinct molecular signatures in discriminating the rheumatoid arthritis patients with traditional Chinese medicine (TCM) cold pattern and heat pattern. Twenty patients with typical TCM cold pattern and heat pattern were included. Microarray technology was used to reveal gene expression profiles in CD4+ T cells. The signal intensity of each expressed gene was globally normalized using the R statistics program. The ratio of cold pattern to heat pattern in patients with RA at more or less than 1:2 was taken as the differential gene expression criteria. Protein-protein interaction information for these genes from databases was searched, and the highly connected regions were detected by IPCA algorithm. The significant pathways were extracted from these subnetworks by Biological Network Gene Ontology tool. Twenty-nine genes differentially regulated between cold pattern and heat pattern were found. Among them, 7 genes were expressed significantly more in cold pattern. Biological network of protein-protein interaction information for these significant genes were searched and four highly connected regions were detected by IPCA algorithm to infer significant complexes or pathways in the biological network. Particularly, the cold pattern was related to Toll-like receptor signaling pathway. The following related pathways in heat pattern were included: Calcium signaling pathway; cell adhesion molecules; PPAR signaling pathway; fatty acid metabolism. These results suggest that better knowledge of the main biological processes involved at a given pattern in TCM might help to choose the most appropriate treatment.

S100A6 has been implicated in a variety of biological functions as well as tumorigenesis. In this study, we investigated the expression status of S100A6 in relation to the clinicopathological features and prognosis of patients with gastric cancer and further explored a possible association of its expression with epigenetic regulation. S100A6 expression was remarkably increased in 67.5% of gastric cancer tissues as compared with matched noncancerous tissues. Statistical analysis demonstrated a clear correlation between high S100A6 expression and various clinicopathological features, such as depth of wall invasion, positive lymph node involvement, liver metastasis, vascular invasion, and tumor-node metastasis stage (P < 0.05 in all cases), as well as revealed that S100A6 is an independent prognostic predictor (P = 0.026) significantly related to poor prognosis (P = 0.0004). Further exploration found an inverse relationship between S100A6 expression and the methylation status of the seventh and eighth CpG sites in the promoter/first exon and the second to fifth sites in the second exon/second intron. In addition, the level of histone H3 acetylation was found to be significantly higher in S100A6-expressing cancer cells. After 5-azacytidine or trichostatin A treatment, S100A6 expression was clearly increased in S100A6 low-expressing cells. In conclusion, our results suggested that S100A6 plays an important role in the progression of gastric cancer, affecting patient prognosis, and is up-regulated by epigenetic regulation. S100A6 has been implicated in a variety of biological functions as well as tumorigenesis. In this study, we investigated the expression status of S100A6 in relation to the clinicopathological features and prognosis of patients with gastric cancer and further explored a possible association of its expression with epigenetic regulation. S100A6 expression was remarkably increased in 67.5% of gastric cancer tissues as compared with matched noncancerous tissues. Statistical analysis demonstrated a clear correlation between high S100A6 expression and various clinicopathological features, such as depth of wall invasion, positive lymph node involvement, liver metastasis, vascular invasion, and tumor-node metastasis stage (P < 0.05 in all cases), as well as revealed that S100A6 is an independent prognostic predictor (P = 0.026) significantly related to poor prognosis (P = 0.0004). Further exploration found an inverse relationship between S100A6 expression and the methylation status of the seventh and eighth CpG sites in the promoter/first exon and the second to fifth sites in the second exon/second intron. In addition, the level of histone H3 acetylation was found to be significantly higher in S100A6-expressing cancer cells. After 5-azacytidine or trichostatin A treatment, S100A6 expression was clearly increased in S100A6 low-expressing cells. In conclusion, our results suggested that S100A6 plays an important role in the progression of gastric cancer, affecting patient prognosis, and is up-regulated by epigenetic regulation. S100A6, also known as calcyclin, is a low-molecular-weight acidic protein containing 2 EF-hand calcium-binding motifs.1Ferrari S Calabretta B deRiel JK Battini R Ghezzo F Lauret E Griffin C Emanuel BS Gurrieri F Baserga R Structural and functional analysis of a growth-regulated gene, the human calcyclin.J Biol Chem. 1987; 262: 8325-8332Abstract Full Text PDF PubMed Google Scholar, 2Donato R S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles.Int J Biochem Cell Biol. 2001; 33: 637-668Crossref PubMed Scopus (1327) Google Scholar It was discovered on the basis of its cell cycle-dependent expression and is preferentially expressed in G1 phase of the cell cycle after mitogenic stimuli.3Calabretta B Battini R Kaczmarek L de Riel JK Baserga R Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein.J Biol Chem. 1986; 261: 12628-12632Abstract Full Text PDF PubMed Google Scholar, 4Calabretta B Kaczmarek L Mars W Ochoa D Gibson CW Hirschhorn RR Baserga R Cell-cycle-specific genes differentially expressed in human leukemias.Proc Natl Acad Sci USA. 1985; 82: 4463-4467Crossref PubMed Scopus (77) Google Scholar S100A6 is a member of the S100 family, which are found localized to the cytoplasm and/or nucleus in a wide range of cell types.5Cross SS Hamdy FC Deloulme JC Rehman I Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: s100A6. S100A8, S100A9 and S100A11 are all overexpressed in common cancers.Histopathology. 2005; 46: 256-269Crossref PubMed Scopus (223) Google Scholar S100A6 may interact with binding or target proteins, thereby regulating dynamics of cytoskeleton constituents, cell growth and differentiation, and calcium homeostasis.5Cross SS Hamdy FC Deloulme JC Rehman I Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: s100A6. S100A8, S100A9 and S100A11 are all overexpressed in common cancers.Histopathology. 2005; 46: 256-269Crossref PubMed Scopus (223) Google Scholar, 6Emberley ED Murphy LC Watson PH S100 proteins and their influence on pro-survival pathways in cancer.Biochem Cell Biol. 2004; 82: 508-515Crossref PubMed Scopus (119) Google Scholar, 7Golitsina NL Kordowska J Wang CL Lehrer SS Ca2+-dependent binding of calcyclin to muscle tropomyosin.Biochem Biophys Res Commun. 1996; 220: 360-365Crossref PubMed Scopus (49) Google Scholar, 8Mani RS McCubbin WD Kay CM Calcium-dependent regulation of caldesmon by an 11-kDa smooth muscle calcium-binding protein, caltropin.Biochemistry. 1992; 31: 11896-11901Crossref PubMed Scopus (52) Google Scholar, 9Slomnicki LP Nawrot B Lesniak W S100A6 binds p53 and affects its activity.Int J Biochem Cell Biol. 2009; 41: 784-790Crossref PubMed Scopus (96) Google Scholar, 10Tsoporis JN Izhar S Parker TG Expression of S100A6 in cardiac myocytes limits apoptosis induced by tumor necrosis factor-alpha.J Biol Chem. 2008; 283: 30174-30183Crossref PubMed Scopus (38) Google Scholar Subsequent studies showed that S100A6 may also be involved in the regulation of cancer progression.11Filipek A Kuznicki J Calcyclin–from basic research to clinical implications.Acta Biochim Pol. 1993; 40: 321-327PubMed Google Scholar The deregulation of S100A6 expression during malignant transformation has thus far been described in human pancreatic cancer, malignant thyroid neoplasms, malignant melanoma, breast cancer, hepatocellular carcinoma, lung cancer, prostate cancer, and colorectal carcinoma.12Shekouh AR Thompson CC Prime W Campbell F Hamlett J Herrington CS Lemoine NR Crnogorac-Jurcevic T Buechler MW Friess H Neoptolemos JP Pennington SR Costello E Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma.Proteomics. 2003; 3: 1988-2001Crossref PubMed Scopus (156) Google Scholar, 13Brown LM Helmke SM Hunsucker SW Netea-Maier RT Chiang SA Heinz DE Shroyer KR Duncan MW Haugen BR Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue.Mol Carcinog. 2006; 45: 613-626Crossref PubMed Scopus (113) Google Scholar, 14Stulik J Osterreicher J Koupilova K Knizek J Bures J Jandik P Langr F Dedic K Schafer BW Heizmann CW Differential expression of the Ca2+ binding S100A6 protein in normal, preneoplastic and neoplastic colon mucosa.Eur J Cancer. 2000; 36: 1050-1059Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 15Komatsu K Kobune-Fujiwara Y Andoh A Ishiguro S Hunai H Suzuki N Kameyama M Murata K Miyoshi J Akedo H Tatsuta M Nakamura H Increased expression of S100A6 at the invading fronts of the primary lesion and liver metastasis in patients with colorectal adenocarcinoma.Br J Cancer. 2000; 83: 769-774Crossref PubMed Scopus (75) Google Scholar, 16Weterman MA Stoopen GM van Muijen GN Kuznicki J Ruiter DJ Bloemers HP Expression of calcyclin in human melanoma cell lines correlates with metastatic behavior in nude mice.Cancer Res. 1992; 52: 1291-1296PubMed Google Scholar, 17Pedrocchi M Schafer BW Mueller H Eppenberger U Heizmann CW Expression of Ca(2+)-binding proteins of the S100 family in malignant human breast-cancer cell lines and biopsy samples.Int J Cancer. 1994; 57: 684-690Crossref PubMed Scopus (132) Google Scholar, 18Rehman I Cross SS Catto JW Leiblich A Mukherjee A Azzouzi AR Leung HY Hamdy FC Promoter hyper-methylation of calcium binding proteins S100A6 and S100A2 in human prostate cancer.Prostate. 2005; 65: 322-330Crossref PubMed Scopus (44) Google Scholar, 19Rehman I Cross SS Azzouzi AR Catto JW Deloulme JC Larre S Champigneuille J Fromont G Cussenot O Hamdy FC S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors.Br J Cancer. 2004; 91: 739-744Crossref PubMed Scopus (29) Google Scholar, 20Kim J Kim J Yoon S Joo J Lee Y Lee K Chung J Choe I S100A6 protein as a marker for differential diagnosis of cholangiocarcinoma from hepatocellular carcinoma.Hepatol Res. 2002; 23: 274-286Crossref PubMed Scopus (29) Google Scholar, 21De Petris L Orre LM Kanter L Pernemalm M Koyi H Lewensohn R Lehtio J Tumor expression of S100A6 correlates with survival of patients with stage I non-small-cell lung cancer.Lung Cancer. 2009; 63: 410-417Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar S100A6 overexpression has been reported to link with metastasis in colon cancer14Stulik J Osterreicher J Koupilova K Knizek J Bures J Jandik P Langr F Dedic K Schafer BW Heizmann CW Differential expression of the Ca2+ binding S100A6 protein in normal, preneoplastic and neoplastic colon mucosa.Eur J Cancer. 2000; 36: 1050-1059Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 15Komatsu K Kobune-Fujiwara Y Andoh A Ishiguro S Hunai H Suzuki N Kameyama M Murata K Miyoshi J Akedo H Tatsuta M Nakamura H Increased expression of S100A6 at the invading fronts of the primary lesion and liver metastasis in patients with colorectal adenocarcinoma.Br J Cancer. 2000; 83: 769-774Crossref PubMed Scopus (75) Google Scholar and is a well-established marker of melanoma in which its level correlates with tumor invasiveness and poor prognosis. Although the high expression of S100A6 was reported in gastric cancer, its correlation with patient prognosis and clinicopathological features has not been fully investigated.22Yang YQ Zhang LJ Dong H Jiang CL Zhu ZG Wu JX Wu YL Han JS Xiao HS Gao HJ Zhang QH Upregulated expression of S100A6 in human gastric cancer.J Dig Dis. 2007; 8: 186-193Crossref PubMed Scopus (35) Google Scholar Like other S100 proteins, S100A6 may promote cancer progression through specific roles in cell survival and apoptotic pathways,6Emberley ED Murphy LC Watson PH S100 proteins and their influence on pro-survival pathways in cancer.Biochem Cell Biol. 2004; 82: 508-515Crossref PubMed Scopus (119) Google Scholar however the exact mechanism is unclear. In this study, we performed a detailed analysis of S100A6 expression in primary gastric cancer, matched metastatic lymph nodes, and liver metastatic nodules. Then we analyzed the relationship between S100A6 overexpression and clinicopathological features and patients prognosis. And to gain insight into the mechanism of the regulation of S100A6 expression in gastric cancer, we examined DNA methylation and histone modifications along the S100A6 gene, which may affect S100A6 expression in cancer cell lines by previous reports.23Lesniak W Slomnicki LP Kuznicki J Epigenetic control of the S100A6 (calcyclin) gene expression.J Invest Dermatol. 2007; 127: 2307-2314Crossref PubMed Scopus (21) Google Scholar, 24Lesniak W Swart GW Bloemers HP Kuznicki J Regulation of cell specific expression of calcyclin (S100A6) in nerve cells and other tissues.Acta Neurobiol Exp (Wars). 2000; 60: 569-575PubMed Google Scholar One hundred sixty-six patients with gastric cancer were studied, including 114 males and 52 females (mean age, 58 years; range, 27–82 years) diagnosed and surgically treated in Beijing Cancer Hospital between 1995 and 2001. The depth of tumor invasion, histological grade, lymph node metastasis, liver metastasis, and vascular invasion were obtained from histopathological reports. Stage of gastric cancer was classified according to 2002 tumor-node metastasis (TNM) classification recommended by the American Joint Committee on Cancer.25American Joint Committee on Cancer Gastric Cancer. AJCC Cancer Staging. Manual. 6th ed. Springer-Verlag, New York2002: 99-103Google Scholar None of the patients received chemotherapy or radiation therapy preoperatively. All patients were followed up until January 2007. After gastrectomy, resected specimens were processed routinely for macroscopic pathological assessment, and tissues were sampled and snap-frozen in liquid nitrogen. In addition, 28 matched liver metastases and 30 matched metastatic lymph nodes were also collected from these patients. Fresh human tissues were stored at −80°C for Western blot analyses and also fixed with 10% formalin in PBS for immunohistochemistry. This investigation was performed after approval by Ethics Committee of Peking University. Informed consent was obtained from each patient. Gastric cancer cell lines KATO3, RF1, RF48, MKN45, and AGS were obtained from ATCC (American Type Culture Collection, Manassas, VA), and cell lines BGC823 and SGC7901 were established in China and obtained from Cell Research Institute, Shanghai, China.26Ji J Chen X Leung SY Chi JT Chu KM Yuen ST Li R Chan AS Li J Dunphy N So S Comprehensive analysis of the gene expression profiles in human gastric cancer cell lines.Oncogene. 2002; 21: 6549-6556Crossref PubMed Scopus (60) Google Scholar Cancer cells were routinely grown as a monolayer in RPMI-1640 medium (GIBCO BRL, Carlsbad, CA), supplemented with 10% (v/v) fetal calf serum (FCS, GIBCO) and antibiotics at 37°C in a humidified 5% CO2 atmosphere. Cells seeded on glass slides were fixed by 4% formaldehyde for immunocytochemistry. Four-micrometer sections from formalin-fixed paraffin-embedded tissues were mounted on poly-L-lysine-coated slides and then deparaffinized in xylene and rehydrated through alcohol to distilled water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 minutes at room temperature. After pressure cooking the slides in 10 mmol/L EDTA (pH 8.0) for 3 minutes, the sections were incubated overnight at 4°C with mouse anti-S100A6 antibody (1:500, H00006277-M16, Abnova, Taiwan). Primary antibodies were detected using a two-step EnVision System (Dako, Denmark). Positive and negative immunohistochemistry controls were routinely used. For negative controls, the primary antibody was replaced by nonimmune mouse serum to confirm its specificity. Moreover, we used internal positive control in immunohistochemistry for quality assurance.27Packeisen J Buerger H Krech R Boecker W Tissue microarrays: a new approach for quality control in immunohistochemistry.J Clin Pathol. 2002; 55: 613-615Crossref PubMed Scopus (78) Google Scholar Evaluation of S100A6 staining was principally according to the scoring criteria described previously.28Vimalachandran D Greenhalf W Thompson C Luttges J Prime W Campbell F Dodson A Watson R Crnogorac-Jurcevic T Lemoine N Neoptolemos J Costello E High nuclear S100A6 (Calcyclin) is significantly associated with poor survival in pancreatic cancer patients.Cancer Res. 2005; 65: 3218-3225PubMed Google Scholar The information recorded included subcellular location of S100A6 staining (nuclear and/or cytoplasmic), intensity of staining (negative, weak, moderate, and strong), and percentage of cells demonstrating positive immunoreactivity. High expression cases were moderately or strongly stained with >20% of cells demonstrating positive immunoreactivity in nucleus and/or cytoplasm, and the rest (low expression) were negatively or weakly stained. For double staining, mouse anti-human S100A6 antibody (1:500, Abnova) and rabbit anti-human Ki-67 antibody (1:100, RM-9106-S0, LabVision) were used as primary antibodies. The secondary antibodies used for double staining were FITC-conjugated goat anti-mouse antibody and Rhodamine (TRITC)-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA). Confocal images were acquired using Leica TCS SP5 confocal microscope (Leica, Mannheim, Germany). Excitation wavelength for TRITC was 586 nm, and that for FITC 488 nm. In addition, the nuclei of specimens were simultaneously stained with DAPI, excitation at 358 nm. A postembedding immunogold method was applied for the detection of S100A6 proteins. The anti-S100A6 antibody was obtained from Abnova and used at a dilution of 1:40. Cases immunohistochemically positive for S100A6 were selected. Then small samples (1 mm3 in size) of these positive cases were immersed 6 hours in fresh glutaraldehyde fixative solution, washed in 0.2 mol/L sucrose, and then fixed in osmium tetroxide. These samples were then dehydrated through a graded acetone series (−4°C), embedded in epoxy resin, and polymerized at 37°C for 24 hours and 60°C for 48 hours. Ultrathin sections were obtained on a ultramicrotome and collected on nickel grids. All sections were incubated in 10% H2O2 then rinsed with PBS enriched with 1% bovine serum albumin (BSA) and 0.5% powdered skim milk three times for 5 minutes each and incubated in the primary antibodies at 4°C overnight. After being washed three times in TBS (pH 7.6), the sections were incubated in the goat anti-mouse IgG conjugated to 10 nm gold particles antibody (Sigma-Aldrich, St. Louis, MO) at a dilution of 1:100 in TBS (pH 7.6). Sections were rinsed three times for 10 minutes each in enriched TBS and three times for 10 minutes each in distilled water, and stained with uranyl acetate and lead citrate. The sections were observed and photographed in a Zeiss 900 transmission electron microscope (Oberkochen, Germany). For negative controls, the primary antibody was replaced by nonimmune mouse serum. According to manufacturer’s instructions, total cell lysates from 10 paired fresh human tissues (primary gastric cancer tissues and matched adjacent nonneoplastic mucosa) were separated into cytoplasmic and nuclear fractions using a NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce, Rockford, IL). Total proteins were extracted by Trizol according to manufacturer’s instructions. Protein concentration was determined using a BCA Protein Assay Kit (Pierce), and samples were normalized by dilution with appropriate lysis buffer. Samples were denatured in SDS sample loading buffer by heating to 100°C for 3 minutes and resolved by SDS-PAGE (12% minigels), followed by transfer to Immobilon PVDF transfer membranes (Millipore, Watford, UK), and then subjected to Western blot analysis. Blots were blocked by 5% nonfat milk in PBS for 30 minutes and incubated in mouse anti-S100A6 monoclonal antibody (1:2000) and mouse anti-α-tubulin monoclonal antibody (1:5000, T5168, Sigma-Aldrich) for 2 hours at room temperature. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:1000 dilution. Protein–antibody complexes conjugated with peroxidase were visualized with SuperSignal West Pico Chemiluminscent Substrate (Pierce). S100A6 levels were quantified by a computerized densitometer. Quantitative assessment of S100A6 mRNA levels by real-time PCR. Total RNA was extracted from BGC823, SGC7901, KATO3, AGS, MKN45, RF1, and RF48 cells by Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Specific primers for S100A6 and actin were as reported.29Ohuchida K Mizumoto K Ishikawa N Fujii K Konomi H Nagai E Yamaguchi K Tsuneyoshi M Tanaka M The role of S100A6 in pancreatic cancer development and its clinical implication as a diagnostic marker and therapeutic target.Clin Cancer Res. 2005; 11: 7785-7793Crossref PubMed Scopus (132) Google Scholar Quantitative RT-PCR was done with a SYBR Green PCR kit (ABI, USA). Each sample was run thrice. Expression of each gene was given as the ratio of expression of each target gene mRNA to that of β-actin mRNA. DNA was isolated according to the standard phenol/chloroform protocol from MKN45, BGC823, SGC7901, KATO3, AGS, RF1, RF48 cell lines, and 53 pairs fresh human tissues (primary gastric cancer tissues and matched nonneoplastic gastric mucosa). Bisulfite modification was performed in accordance with the standard procedures of the EZ DNA methylation kit (Zymo Research, Orange, CA). The methylation profile of S100A6 gene (accession number: NM_014624, and the base number was according to the UCSC genome browser), comprising 1000 bp upstream of the transcription start site and 1646 bp transcribed sequence comprising three exons and two introns (47 CpG sites, 151775549-151775008 and 151774423-151774103), were detected by nested PCR to amplify the bisulfite-modified DNA. The primers were as reported.23Lesniak W Slomnicki LP Kuznicki J Epigenetic control of the S100A6 (calcyclin) gene expression.J Invest Dermatol. 2007; 127: 2307-2314Crossref PubMed Scopus (21) Google Scholar Then the PCR products of cell lines were subcloned into the pMD18-T vector and sequenced, while the tissue PCR products were sequenced directly. The methylated percentages of the CpG sites in primary gastric cancer tissues and adjacent nonneoplastic gastric mucosa were calculated separately. The base number was according to the UCSC Genome Browser. MKN45, RF1, and RF48 cells were incubated for 4 days in RPMI 10% (v/v) fetal calf serum containing 5, 10 μmol/L 5-azacytidine (Sigma-Aldrich) or 4 days with 1 μg/ml trichostatin A (Sigma-Aldrich), a histone deacetylase inhibitor. Media were changed every 24 hours. Total RNA was extracted by Trizol reagent, and then the mRNA expression of S100A6 was detected by real-time PCR. Ten primary gastric cancer tissues and matched nonneoplastic gastric mucosa were chopped into small pieces, the AGS, SGC7901, RF1, RF48 cell lines were washed with PBS, and the samples were crosslinked by 1% formaldehyde and lysated in the lysis buffer. Chromatin of the samples was sonicated to small fragments, then the sonicated chromatin was immunoprecipitated with the antibodies of anti-acetyl-H3 (06-599, Upstate Biotech), anti-acetyl-H3 (lysine 9) (07-352, Upstate Biotech), anti-trimethyl-H3 (lysine 9) (07-442, Upstate Biotech), and anti-trimethyl-H3 (lysine 27) (07-449, Upstate Biotech). Then the protein A Sepharose Fast Flow (Sigma-Aldrich) was added to each sample. The samples were incubated in the 65°C waterbath to reverse formaldehyde crosslink. DNA was extracted and dissolved in 50 μl TE buffer. PCR was applied to amplify the products above. The subsequent PCR was performed with the forward: 5′-CTGGCTCGAGGGTCA-3′ and backward: 5′-GGCTGTTCCGGGTTAGGA-3′ primers.23Lesniak W Slomnicki LP Kuznicki J Epigenetic control of the S100A6 (calcyclin) gene expression.J Invest Dermatol. 2007; 127: 2307-2314Crossref PubMed Scopus (21) Google Scholar Then the average percentage of input signal of the primary gastric cancer and nonneoplastic mucosa were compared. Clinicopathologic variables were extracted from histopathological reports. To obtain associations between S100A6 expression and clinicopathologic variables, data were cross-tabulated and a χ2 test performed, except for age parameter, which was assessed by Student’s t-test. Cumulative survival was estimated by Kaplan–Meier method, and comparisons between groups were done using a log-rank test. Overall survival was measured from date of initial surgery to date of death, counting death from any cause as the end point, or the last date of information as the end point if no event was documented. A multivariate analysis of Cox proportional hazards regression model (backward, stepwise) was created to assess the influence of each variable on survival. Significance was set at P < 0.05. The variables of the histone H3 modification was assessed by Student’s t-test between the primary gastric cancer tissues and nonneoplastic gastric mucosa. The intensity of S100A6 staining was remarkably higher in primary gastric cancer tissues compared with matched noncancerous mucosa, which had either absent or weak cytoplasmic staining (see supplemental Figure 1 at http://ajp.amjpathol.org). The high expression of S100A6 was detected in 67.5% (112 of 166) of gastric cancer tissues. Immunohistochemical staining of S100A6 in gastric cancer tissues was graded and classified by criteria described in Materials and Methods (see supplemental Figure 2 at http://ajp.amjpathol.org). In primary lesions where tumor invading muscularis propria, S100A6 nuclear staining was frequently found more intense in the invading fronts than in central portions (Figure 1A). Moreover, infiltrating neutrophils and macrophages were also sporadically S100A6-positive. High S100A6 expression was also observed in both lymph node metastases (Figure 1B) and liver metastases (Figure 1C). High S100A6 immunostaining was detected in all lymph node metastases (100%, 30/30), infiltrating into the marginal sinus or diffusing into the lymph nodes. However, only 66.7% (20/30) of matched primary gastric cancer expressed high S100A6. The difference was significant between the two groups (P = 0.001). Twenty-six of 28 (92.9%) liver metastases showed high S100A6 expression, as did 24 of 28 (85.7%) matched primary gastric cancer, without statistical difference (P = 0.388). Table 1 shows the correlation between immunohistochemical expression of S100A6 and clinicopathological parameters. Statistical analysis showed that S100A6 expression was strongly associated with depth of wall invasion (P = 0.030), lymph node metastasis (P = 0.037), liver metastasis (P = 0.024), vascular invasion (P = 0.044), and TNM stage (P = 0.048). The analysis of S100A6 expression and survival was depicted in Figure 2. Kaplan–Meier survival curves showed high S100A6 expression group had significantly lower 5-year survival rate (18.62%) than low S100A6 expression group (49.65%; log-rank = 12.52, P = 0.0004; Figure 2). A multivariate Cox proportional hazards model (Table 2) using variables associated with survival in our study revealed that S100A6 expression was a significant independent prognostic indicator (P = 0.026).Table 1Association of S100A6 Expression with Clinicopathological Parameters in Gastric Cancer PatientsS100A6 expressionVariablesCasesNegative − (n = 54)Positive + (n = 112)PSex Male11440740.298 Female521438Age (y), mean ± SE16659.9 ± 1.457.2 ± 1.10.149Depth of wall invasion T1 + T22814140.03 T3 + T41384098Differentiation Well and moderated4514310.812 Poorly1214081Lymph node metastasis Negative3115160.037 Positive1353996Liver metastasis M013850880.024 M128424Vascular invasion V (−)6025350.044 V (+)992673 Not recorded*Data incomplete.7TNM stages I + II3717200.048 III+ IV1293792* Data incomplete. Open table in a new tab Table 2Multivariate Analysis of Prognostic Factors by Cox Proportional Hazard ModelVariablesPRelative riskCI (95%)Depth of wall invasion T3 + T4 vs. T1 + T20.0052.911.38–6.14Differentiation Poor versus well/moderate0.4370.840.55–1.30Lymph node metastasis Positive vs. negative0.0312.101.07–4.12Vascular invasion Positive vs. negative0.3341.250.80–1.95Liver metastasis Positive vs. negative0.0002.831.70–4.71S100A6 expression High versus low0.0261.671.06–2.61CI indicates confidence interval. Open table in a new tab CI indicates confidence interval. By both immunohistochemistry and immunofluorescence, S100A6 distribution was found throughout the cytoplasm and/or nucleus. In addition, by laser confocal scanning, the result of Ki-67 antigen30Kill IR Localisation of the Ki-67 antigen within the nucleolus. Evidence for a fibrillarin-deficient region of the dense fibrillar component.J Cell Sci. 1996; 109: 1253-1263PubMed Google Scholar and S100A6 double-staining showed S100A6 nuclear distribution was either evenly throughout the whole nucleus or mainly in the nucleolus in gastric caner tissues (Figure 3A). In nonneoplastic mucosa, Ki-67 antigen was weakly expressed in the nucleus (see supplemental Figure 3 at http://ajp.amjpathol.org). Ultrastructural images captured by immunoelectron microscope demonstrated that S100A6 was deposited throughout the cytoplasm staining endoplasmic reticulum, mitochondria, and lysosome, and within the nucleus where it distributed to both nucleoplasmic and nucleolar structures in gastric cancer tissues (Figure 3B). There was no S100A6 deposition in the negative control staining (data not shown). In addition, Western blot was performed to detect the expression of S100A6. It revealed a single band of 10.5 kDa, which was correctly positioned for S100A6. In primary gastric cancer, 96.8% (31/32) was S100A6-positive and 71.9% (23/32) of cancer showed higher expression (average 2.9-fold) than did the nonneoplastic mucosa. Moreover, the localization of S100A6 protein in nuclear and/or cytoplasmic compartments was confirmed by immunoblotting of nuclear and/or cytoplasmic fractions from ten paired gastric cancer samples (Figure 3C). Because previous studies have shown that epigenetic factors may regulate S100A6 gene expression,23Lesniak W Slomnicki LP Kuznicki J Epigenetic control of the S100A6 (calcyclin) gene expression.J Invest Dermatol. 2007; 127: 2307-2314Crossref PubMed Scopus (21) Google Scholar to further explore the mechanism of the up-regulation of S100A6, epigenetic regulation was investigated. First, we chose the S100A6 high- and low-expressing cell lines by measuring S100A6 expression levels in

Inhibition of the inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) pathway enhances muscle regeneration in injured and diseased skeletal muscle, but it is unclear exactly how this pathway contributes to the regeneration process. In this study, we examined the role of NF-κB in regulating the proliferation and differentiation of muscle-derived stem cells (MDSCs). MDSCs isolated from the skeletal muscles of p65(+/-) mice (haploinsufficient for the p65 subunit of NF-κB) had enhanced proliferation and myogenic differentiation compared to MDSCs isolated from wild-type (wt) littermates. In addition, selective pharmacological inhibition of IKKβ, an upstream activator of NF-κB, enhanced wt MDSC differentiation into myotubes in vitro. The p65(+/-) MDSCs also displayed a higher muscle regeneration index than wt MDSCs following implantation into adult mice with muscular dystrophy. Additionally, using a muscle injury model, we observed that p65(+/-) MDSC engraftments were associated with reduced inflammation and necrosis. These results suggest that inhibition of the IKK/NF-κB pathway represents an effective approach to improve the myogenic regenerative potential of MDSCs and possibly other adult stem cell populations. Moreover, our results suggest that the improved muscle regeneration observed following inhibition of IKK/NF-κB, is mediated, at least in part, through enhanced stem cell proliferation and myogenic potential.

Chinese herbal medicines (HMs) is one of the great herbal systems of the world, which play an important role in current health care system in many countries. In the view of tradition Chinese medicine (TCM) theory, Yin-yang and five-elements theory is the central theory, which is used to explain how the world and body work. Under the guidance of such philosophy, TCM considers that HMs have different properties, which are the important factors for prescribing herbal formulae; such prescriptions are based on TCM pattern classification in clinical practice. The cold and hot property are commonly defined for HM property identification; however, the biological activities that are related to the HM property remain a mystery because of a lack of appropriate methods. A bioinformatics approach was applied to identify the distinguishing biological activities of HMs that have these cold and hot properties.Twenty HMs with typical cold and hot properties (10 cold and 10 hot) were selected based on TCM clinical application records and Chinese pharmacopeia. The active target proteins of each HM were searched in the PubChem database and were analyzed in Ingenuity Pathway Analysis (IPA) platform to find out the HM property-related biological activities. In addition, the main compounds of the HMs were fragmented using a fragment-based approach and were analyzed for the purpose of deciphering the properties.The main biological networks of HMs with cold and hot properties include cell cycle, cellular growth, proliferation and development, cancer, cytokine signaling, and intracellular and second messenger signaling; 11 specific pathways are presented to be perturbed only by HMs with the hot property, and the 27 specific target protein molecules include PRKACA, PRKCA, PRKCB, PRKCD, PRKCE, PRKCG, PRKD1, TLR4, TLR7, TLR8, TLR9, HTR4, HTR6, HTR7, HTR2A, HTR1B, HTR2B, GNAO1, GNAI1, TNF, IL8, ROCK2, AKT1, MAPK1, RPS6KA1, RPS6KA3 and JAK2, which are involved in the biological network. One specific pathway is detected to be involved in the biological network of HMs with the cold property, the specific molecules are RAN and KPNB1. Cold propertied HMs show intensive toxicity in the heart, liver and kidney compared with hot HMs, which is likely to be correlated with the specific chemical fragments constructions in the HMs with the cold property, such as long chain alkenes, Benzo heterocycle and azotic heterocycle according to the chemical fragment analysis for the HMs.Inflammation and immunity regulation are more related to HMs with the hot property, and cold propertied HMs possess the tendency to impact cell growth, proliferation and development. Integrative bioinformatics analysis and chemical structure analysis are a promising methods for identifying the biological activity of HM properties.

Puerarin is a major active ingredient extracted from the root of P. lobata, a traditional Chinese herb, and possesses anti-oxidative and anti-inflammatory activities. However, the low oral bioavailability of puerarin limits its further application. Therefore, we synthesized tetraacetyl puerarin (4AC) through acetylation to improve its liposolubility and bioavailability. In the present investigations, we tested the anti-oxidative and TNF-α suppressive activity of 4AC in lipopolysaccharide (LPS)-induced RAW264.7 macrophages and bovine type II collagen-induced arthritic (CIA) rats. The results showed that 4AC retained the bioactivity of puerarin. And 4AC significantly increased the activity of SOD and reduced the level of MDA both in vitro and in vivo. It also improved the level of GSH-PX and the total antioxidant capacity in vivo. Furthermore, it dramatically decreased TNF-α level in the cultured supernatant of RAW264.7 cells treated with LPS and in the serum of CIA rats. These initial results indicated that 4AC had a potential therapeutic effect on CIA rats through an anti-oxidative and TNF-α suppressive activity. In addition, the molecular mechanism of anti-oxidation of 4AC was explored by testing the MAPKs/NF-κB signaling pathway. The results showed that 4AC significantly inhibited NF-κB expression and down-regulated the levels of p-ERK and p-JNK in LPS-activated RAW264.7 cells. These results indicated that 4AC had bioactive anti-oxidative effects and suggest the potential value of 4AC for the treatment of rheumatoid arthritis.

For thousands of years, tonic herbs have been successfully used all around the world to improve health, energy, and vitality. However, their underlying mechanisms of action in molecular/systems levels are still a mystery. In this work, two sets of tonic herbs, so called Qi-enriching herbs (QEH) and Blood-tonifying herbs (BTH) in TCM, were selected to elucidate why they can restore proper balance and harmony inside body, organ and energy system. Firstly, a pattern recognition model based on artificial neural network and discriminant analysis for assessing the molecular difference between QEH and BTH was developed. It is indicated that QEH compounds have high lipophilicity while BTH compounds possess high chemical reactivity. Secondly, a systematic investigation integrating ADME (absorption, distribution, metabolism, and excretion) prediction, target fishing and network analysis was performed and validated on these herbs to obtain the compound-target associations for reconstructing the biologically-meaningful networks. The results suggest QEH enhance physical strength, immune system and normal well-being, acting as adjuvant therapy for chronic disorders while BTH stimulate hematopoiesis function in body. As an emerging approach, the systems pharmacology model might facilitate to understand the mechanisms of action of the tonic herbs, which brings about new development for complementary and alternative medicine.

Zheng, which is also called a syndrome or pattern, is the basic unit and a key concept of traditional Chinese medicine (TCM) theory. Zheng can be considered a further stratification of patients when it is integrated with biomedical diagnoses in clinical practice to achieve higher efficacies. In an era of evidence-based medicine, confronted with the vast and increasing volume of TCM data, there is an urgent need to explore these resources effectively using techniques of knowledge discovery in databases. The application of effective data mining in the analysis of multiple extensively integrated databases can supply new information about TCM Zheng research. In this paper, we screened the published literature on TCM Zheng-related studies in the SinoMed and PubMed databases with a novel data mining approach to obtain an overview of the Zheng research landscape in the hope of contributing to a better understanding of TCM Zheng in the era of evidence-based medicine. In our results, contrast was found in Zheng in different studies, and several determinants of Zheng were identified. The data described in this paper can be used to assess Zheng research studies based on the title and certain characteristics of the abstract. These findings will benefit modern TCM Zheng-related studies and guide future Zheng study efforts.

Focal bone destruction within inflamed joints is the most specific hallmark of rheumatoid arthritis (RA). Our previous study indicated that the therapeutic efficiency of triptolide in RA may be due partially to its chondroprotective and anti-inflammatory effects. However, its roles in bone destruction are still unclear. In this study, our data firstly showed the therapeutic effects of triptolide on severity of arthritis and arthritis progression in collagen-induced arthritis (CIA) mice. Then, by micro-CT quantification, triptolide treatment significantly increased bone mineral density, bone volume fraction, and trabecular thickness and decreased trabecular separation of inflamed joints. Interestingly, triptolide treatment could prevent the bone destruction by reducing the number of osteoclasts in inflamed joints, reducing the expression of receptor activator of NF- κ B (RANK) ligand (RANKL) and RANK, increasing the expression of osteoprotegerin (OPG), at both mRNA and protein levels, and decreasing the ratio of RANKL to OPG in sera and inflamed joints of CIA mice, which were further confirmed in the coculture system of human fibroblast-like synovial and peripheral blood mononuclear cells. These findings offer the convincing evidence for the first time that triptolide may attenuate RA partially by preventing the bone destruction and inhibit osteoclast formation by regulating RANKL/RANK/OPG signal pathway.

Chinese medicine (CM) has a long history of experience and proven successful treatment for chronic diseases and has also played an important role in the provision of health care in China. Patients with chronic diseases are happy to accept CM and physicians are willing to use CM to relieve patients suffering from chronic illnesses. The Chinese health authorities encourage CM development to meet the requirements for the treatment of chronic diseases. CM products are an essential part of medications that have a predominant role in the prevention and treatment of chronic diseases in China. A large number of CM clinical studies, including a substantial number of available randomized controlled trials and systematic reviews, have shown that CM is effective and safe in the treatment of chronic diseases. Although the efficacies of some evaluated CM therapies remain uncertain, it is worth assessing them by using CM pattern (Zheng or syndrome) differentiation to verify treatment outcomes. CM is considered to have a better safety profile compared to pharmaceutical chemicals, but inappropriate applications of CM also makes the safety issues a hot discussed subject. As a medical system, CM should be able to provide worldwide contribution for the patients who are suffering from chronic diseases. The application of CM pattern classification in diagnosis with corresponding prescribed treatment using herbal formulae in the relief of chronic diseases can be linked with modern biomedical parameters (biomarkers) as treatment outcomes. These outcome parameters, together with the patients' reported quality of life assessment, can provide innovative approaches for evidence-based estimation of the efficacy of CM treatment in chronic diseases.

Rheumatoid arthritis (RA) is a heterogeneous disease, and traditional Chinese medicine (TCM) can be used to classify RA into different patterns such as cold and hot based on its clinical manifestations. The aim of this study was to investigate potential network-based biomarkers for RA with either a cold or a hot pattern.Microarray technology was used to reveal gene expression profiles in CD4(+) T cells from 21 RA patients with cold pattern and 12 with hot pattern. A T-test was used to identify significant differences in gene expression among RA patients with either cold or hot pattern. Cytoscape software was used to search the existing literature and databases for protein-protein interaction information for genes of interest that were identified from this analysis. The IPCA algorithm was used to detect highly connected regions for inferring significant complexes or pathways in this protein-protein interaction network. Significant pathways and functions were extracted from these subnetworks by the Biological Network Gene Ontology tool.Four genes were expressed at higher levels in RA patients with cold pattern than in patients with hot pattern, and 21 genes had lower levels of expression. Protein-protein interaction network analysis for these genes showed that there were four highly connected regions. The most relevant functions and pathways extracted from these subnetwork regions were involved in small G protein signaling pathways, oxidation-reduction in fatty acid metabolism and T cell proliferation.Complicated network based pathways appear to play a role in the different pattern manifestations in patients with RA, and our results suggest that network-based pathways might be the scientific basis for TCM pattern classification.

Duchenne muscular dystrophy (DMD) patients lack dystrophin from birth; however, muscle weakness becomes apparent only at 3-5 years of age, which happens to coincide with the depletion of the muscle progenitor cell (MPC) pools. Indeed, MPCs isolated from older DMD patients demonstrate impairments in myogenic potential. To determine whether the progression of muscular dystrophy is a consequence of the decline in functional MPCs, we investigated two animal models of DMD: (i) dystrophin-deficient mdx mice, the most commonly utilized model of DMD, which has a relatively mild dystrophic phenotype and (ii) dystrophin/utrophin double knock-out (dKO) mice, which display a similar histopathologic phenotype to DMD patients. In contrast to age-matched mdx mice, we observed that both the number and regeneration potential of dKO MPCs rapidly declines during disease progression. This occurred in MPCs at both early and late stages of myogenic commitment. In fact, early MPCs isolated from 6-week-old dKO mice have reductions in proliferation, resistance to oxidative stress and multilineage differentiation capacities compared with age-matched mdx MPCs. This effect may potentially be mediated by fibroblast growth factor overexpression and/or a reduction in telomerase activity. Our results demonstrate that the rapid disease progression in the dKO model is associated, at least in part, with MPC depletion. Therefore, alleviating MPC depletion could represent an approach to delay the onset of the histopathologies associated with DMD patients.

Murine muscle-derived stem cells (MDSCs) have been shown capable of regenerating bone in a critical size calvarial defect model when transduced with BMP 2 or 4; however, the contribution of the donor cells and their interactions with the host cells during the bone healing process have not been fully elucidated. To address this question, C57/BL/6J mice were divided into MDSC/BMP4/GFP, MDSC/GFP, and scaffold groups. After transplanting MDSCs into the critical-size calvarial defects created in normal mice, we found that mice transplanted with BMP4GFP-transduced MDSCs healed the bone defect in 4 wk, while the control groups (MDSC-GFP and scaffold) demonstrated no bone healing. The newly formed trabecular bone displayed similar biomechanical properties as the native bone, and the donor cells directly participated in endochondral bone formation via their differentiation into chondrocytes, osteoblasts, and osteocytes via the BMP4-pSMAD5 and COX-2-PGE2 signaling pathways. In contrast to the scaffold group, the MDSC groups attracted more inflammatory cells initially and incurred faster inflammation resolution, enhanced angiogenesis, and suppressed initial immune responses in the host mice. MDSCs were shown to attract macrophages via the secretion of monocyte chemotactic protein 1 and promote endothelial cell proliferation by secreting multiple growth factors. Our findings indicated that BMP4GFP-transduced MDSCs not only regenerated bone by direct differentiation, but also positively influenced the host cells to coordinate and promote bone tissue repair through paracrine effects.—Gao, X., Usas, A., Proto, J. D., Lu, A., Cummins, J. H., Proctor, A., Chen, C.-W., Huard, J. Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects. FASEB J. 28, 3792–3809 (2014). www.fasebj.org

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affects in vivo versus in vitro maturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination using in vivo - or in vitro -matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes), in vitro -matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), and in vitro -matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for both in vivo and in vitro oocyte maturation. In addition, for oocytes matured in vivo , the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes matured in vitro , the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

Abstract Muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle by a modified preplate technique exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. MDSCs retrovirally transduced to express bone morphogenetic proteins (BMPs) can differentiate into osteocytes and chondrocytes and enhance bone and articular cartilage repair in vivo, a feature that is not observed with nontransduced MDSCs. These results emphasize that MDSCs require prolonged exposure to BMPs to undergo osteogenic and chondrogenic differentiation. A sustained BMP protein delivery approach provides a viable and potentially more clinically translatable alternative to genetic manipulation of the cells. A unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD), was used to bind, protect, and sustain the release of bone morphogenetic protein-2 (BMP2) in a temporally and spatially controlled manner. Prolonged exposure to BMP2 released by the PEAD:heparin delivery system promoted the differentiation of MDSCs to an osteogenic lineage in vitro and induced the formation of viable bone at an ectopic site in vivo. This new strategy represents an alternative approach for bone repair mediated by MDSCs while bypassing the need for gene therapy.

Tripterygium wilfordii Hook F. (TwHF) based therapy has been proved as effective in treating rheumatoid arthritis (RA), yet the predictors to its response remains unclear. A two-stage trial was designed to identify and verify the baseline symptomatic predictors of this therapy. 167 patients with active RA were enrolled with a 24-week TwHF based therapy treatment and the symptomatic predictors were identified in an open trial; then in a randomized clinical trial (RCT) for verification, 218 RA patients were enrolled and classified into predictor positive (P+) and predictor negative (P-) group, and were randomly assigned to accept the TwHF based therapy and Methotrexate and Sulfasalazine combination therapy (M) for 24 weeks, respectively. Five predictors were identified (diuresis, excessive sweating, night sweats for positive; and yellow tongue-coating, thermalgia in the joints for negative). In the RCT, The ACR 20 responses were 82.61% in TwHF/P+ group, significantly higher than that in TwHF/P- group (P = 0.0001) and in M&S/P+ group (P < 0.05), but not higher than in M&S/P- group. Similar results were yielded in ACR 50 yet not in ACR 70 response. No significant differences were detected in safety profiles among groups. The identified predictors enable the TwHF based therapy more efficiently in treating RA subpopulations.

A novel neuropeptide spexin was found to be broadly expressed in various endocrine and nervous tissues while little is known about its functions. This study investigated the role of spexin in bowel movement and the underlying mechanisms. In functional constipation (FC) patients, serum spexin levels were significantly decreased. Consistently, in starved mice, the mRNA of spexin was significantly decreased in intestine and colon. Spexin injection increased the velocity of carbon powder propulsion in small intestine and decreased the glass beads expulsion time in distal colon in mice. Further, spexin dose-dependently stimulated the intestinal/colonic smooth muscle contraction. Galanin receptor 2 (GALR2) antagonist M871, but not Galanin receptor 3 (GALR3) antagonist SNAP37899, effectively suppressed the stimulatory effects of spexin on intestinal/colonic smooth muscle contraction, which could be eliminated by extracellular [Ca(2+)] removal and L-type voltage-dependent Ca(2+) channel (VDCC) inhibitor nifedipine. Besides, spexin dramatically increased the [Ca(2+)]i in isolated colonic smooth muscle cells. These data indicate that spexin can act on GALR2 receptor to regulate bowel motility by activating L-type VDCC. Our findings provide evidence for important physiological roles of spexin in GI functions. Selective action on spexin pathway might have therapeutic effects on GI diseases with motility disorders.

This retrospective study aimed to assess the safety of patients with severe cerebral palsy (CP), who received allogeneic umbilical cord blood stem cells (UCBSCs) treatment from August 2009 to December 2012 in Guangdong Provincial Hospital of Chinese Medicine. A total of 47 patients with average age of<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" id="M1"><mml:mn>5.85</mml:mn><mml:mo>±</mml:mo><mml:mn>6.12</mml:mn></mml:math>years were evaluated in this study. There was no significant association with allogeneic UCBSCs treatments found in the data of the laboratory index . No casualties occurred. Some adverse events during treatments were found in 26 (55.3%) patients, including fever (42.6%) and vomiting (21.2%). Intrathecal infusion and the ages at the initiation of treatment (≤10 years old) were risk factors for the occurrence of adverse events by logistic regression analysis. However, all adverse events disappeared after symptomatic treatment. No treatment related serious adverse events were found in follow-up visits within 6 months. In conclusion, allogeneic UCBSCs treatment was relatively safe for severe CP patients.

The Ebola virus is highly pathogenic and destructive to humans and other primates. The Ebola virus encodes viral protein 40 (VP40), which is highly expressed and regulates the assembly and release of viral particles in the host cell. Because VP40 plays a prominent role in the life cycle of the Ebola virus, it is considered as a key target for antiviral treatment. However, there is currently no FDA-approved drug for treating Ebola virus infection, resulting in an urgent need to develop effective antiviral inhibitors that display good safety profiles in a short duration.This study aimed to screen the effective lead candidate against Ebola infection. First, the lead molecules were filtered based on the docking score. Second, Lipinski rule of five and the other drug likeliness properties are predicted to assess the safety profile of the lead candidates. Finally, molecular dynamics simulations was performed to validate the lead compound.Our results revealed that emodin-8-beta-D-glucoside from the Traditional Chinese Medicine Database (TCMD) represents an active lead candidate that targets the Ebola virus by inhibiting the activity of VP40, and displays good pharmacokinetic properties.This report will considerably assist in the development of the competitive and robust antiviral agents against Ebola infection.

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading unnecessary or dysfunctional cellular organelles and proteins in all living cells. In addition to its vital homeostatic role, this degradation pathway also involves in various human disorders, including metabolic conditions, neurodegenerative diseases, cancers and infectious diseases. Therefore, the comprehensive understanding of autophagy process, autophagy-related modulators and corresponding pathway and disease information will be of great help for identifying the new autophagy modulators, potential drug candidates, new diagnostic and therapeutic targets. In recent years, some autophagy databases providing structural and functional information were developed, but the specific databases covering autophagy modulator (proteins, chemicals and microRNAs)-related target, pathway and disease information do not exist. Hence, we developed an online resource, Human Autophagy Modulator Database (HAMdb, http://hamdb.scbdd.com ), to provide researchers related pathway and disease information as many as possible. HAMdb contains 796 proteins, 841 chemicals and 132 microRNAs. Their specific effects on autophagy, physicochemical information, biological information and disease information were manually collected and compiled. Additionally, lots of external links were available for more information covering extensive biomedical knowledge. HAMdb provides a user-friendly interface to query, search, browse autophagy modulators and their comprehensive related information. HAMdb will help researchers understand the whole autophagy process and provide detailed information about related diseases. Furthermore, it can give hints for the identification of new diagnostic and therapeutic targets and the discovery of new autophagy modulators. In a word, we hope that HAMdb has the potential to promote the autophagy research in pharmacological and pathophysiological area.

Lack of pharmacological strategies in clinics restricts the patient prognosis with myocardial ischemia/reperfusion (I/R) injury. The aim of this study was to evaluate the cardioprotection of combined salvianolic acid B (SalB) and ginsenoside Rg1 (Rg1) against myocardial I/R injury and further investigate the underlying mechanism. I/R injury was induced by coronary artery ligation for Wistar male rats and hypoxia/reoxygenation injury was induced on H9c2 cells. Firstly, the best ratio between SalB and Rg1was set as 2:5 based on their effects on heart function detected by hemodynamic measurement. Then SalB-Rg1 (2:5) was found to maintain mitochondrial membrane potential and resist apoptosis and necrosis in H9c2 cell with hypoxia/reoxygenation injury. Companying with same dose of SalB or Rg1 only, SalB-Rg1 showed more significant effects on down-regulation of myocardial infarct size, maintenance of myocardium structure, improvement on cardiac function, decrease of cytokine secretion including TNF-α, IL-1β, RANTES and sVCAM-1. Finally, the SalB-Rg1 improved the viability of cardiac myocytes other than cardiac fibroblasts in rats with I/R injury using flow cytometry. Our results revealed that SalB-Rg1 was a promising strategy to prevent myocardial I/R injury.

Breast cancer is the second leading cause of cancer death among women.Human epidermal receptor 2 (HER2) positive breast cancer (HER2+ BC) is the most aggressive subtype of breast cancer, with poor prognosis and a high rate of recurrence.About one third of breast cancer is HER2+ BC with significantly high expression level of HER2 protein compared to other subtypes.Therefore, HER2 is an important biomarker and an ideal target for developing therapeutic strategies for the treatment HER2+ BC.In this review, HER2 structure and physiological and pathological roles in HER2+ BC are discussed.Two diagnostic tests, immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH), for evaluating HER2 expression levels are briefly introduced.The current mainstay targeted therapies for HER2+ BC include monoclonal antibodies, small molecule tyrosine kinase inhibitors, antibody-drug conjugates (ADC) and other emerging anti-HER2 agents.In clinical practice, combination therapies are commonly adopted in order to achieve synergistic drug response.This review will help to better understand the molecular mechanism of HER2+ BC and further facilitate the development of more effective therapeutic strategies against HER2+ BC.

Yupingfeng San (YPFS) is a representative Traditional Chinese Medicine (TCM) formula with accepted therapeutic effect on Asthma. However, its action mechanism is still obscure. In this study, we used network pharmacology to explore potential mechanism of YPFS on asthma. Nucleotide-bindling oligomerization domain (NOD)-like receptor pathway was shown to be the top one shared signaling pathway associated with both YPFS and asthma. In addition, NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome was treated as target protein in the process of YPFS regulating asthma. Further, experimental validation was done by using LPS-stimulated U937 cells and ovalbumin (OVA)-sensitized BALB/c mice model. In vitro experiments showed that YPFS significantly decreased the production of TNF-α and IL-6, as well as both mRNA and protein levels of IL-1β, NLRP3, Caspase-1 and ASC in LPS-stimulated U937 cells. In vivo experiment indicated that YPFS treatment not only attenuated the clinical symptoms, but also reduced inflammatory cell infiltration, mucus secretion and MUC5AC production in lung tissue of asthmatic mice. Moreover, YPFS treatment remarkably decreased the mRNA and protein levels of IL-1β, NLRP3, Caspase-1 and ASC in lung tissue of asthmatic mice. In conclusion, these results demonstrated that YPFS could inhibit NLRP3 inflammasome components to attenuate the inflammatory response in asthma.

Reversing established muscle atrophy following mechanical unloading is of great clinical challenge. Long noncoding RNAs (lncRNAs) have been demonstrated to play important roles in myogenesis. Here we identified a lncRNA (mechanical unloading-induced muscle atrophy-related lncRNA [lncMUMA]) enriched in muscle, which was the most downregulated lncRNA during muscle atrophy development in hindlimb suspension (HLS) mice. The <i>in vitro</i> and <i>in vivo</i> data demonstrated that the decreased expression levels of lncMUMA closely associated with a reduction of myogenesis during mechanical unloading. Mechanistically, lncMUMA promoted myogenic differentiation by functioning as a miR-762 sponge to regulate the core myogenic regulator MyoD <i>in vitro</i>. The enforced expression of lncMUMA relieved the decreases in MyoD protein and muscle mass in miR-762 knockin mice. Therapeutically, the enforced expression of lncMUMA improved the <i>in vitro</i> myogenic differentiation of myoblasts under microgravity simulation, prevented the muscle atrophy development, and reversed the established muscle atrophy in HLS mice. These findings identify lncMUMA as an anabolic regulator to reverse established muscle atrophy following mechanical unloading.

This manuscript elaborates on the establishment of a chemotaxonomic classification strategy for closely-related Citrus fruits in Traditional Chinese Medicines (TCMs). UPLC-Q-TOF-MS-based metabolomics was applied to depict the variable chemotaxonomic markers and elucidate the metabolic mechanism of Citrus TCMs from different species and at different ripening stages. Metabolomics can capture a comprehensive analysis of small molecule metabolites and can provide a powerful approach to establish metabolic profiling, creating a bridge between genotype and phenotype. To further investigate the different metabolites in four closely-related Citrus TCMs, non-targeted metabolite profiling analysis was employed as an efficient technique to profile the primary and secondary metabolites. The results presented in this manuscript indicate that primary metabolites enable the discrimination of species, whereas secondary metabolites are associated with species and the ripening process. In addition, analysis of the biosynthetic pathway highlighted that the syntheses of flavone and flavone glycosides are deeply affected in Citrus ripening stages. Ultimately, this work might provide a feasible strategy for the authentication of Citrus fruits from different species and ripening stages and facilitate a better understanding of their different medicinal uses.

Abnormalities in the integral components of bone, including bone matrix, bone mineral and bone cells, give rise to complex disturbances of skeletal development, growth and homeostasis. Non-specific drug delivery using high-dose systemic administration may decrease therapeutic efficacy of drugs and increase the risk of toxic effects in non-skeletal tissues, which remain clinical challenges in the treatment of skeletal disorders. Thus, targeted delivery systems are urgently needed to achieve higher drug delivery efficiency, improve therapeutic efficacy in the targeted cells/tissues, and minimize toxicities in non-targeted cells/tissues. In this review, we summarize recent progress in the application of different targeting moieties and nanoparticles for targeted drug delivery in skeletal disorders, and also discuss the advantages, challenges and perspectives in their clinical translation.

Functional dyspepsia (FD) is a widely prevalent gastrointestinal disorder throughout the world, whereas the efficacy of current treatment in the Western countries is limited. As the symptom is equivalent to the traditional Chinese medicine (TCM) term "stuffiness and fullness," FD can be treated with Zhi-zhu Wan (ZZW) which is a kind of Chinese patent medicine. However, the "multi-component" and "multi-target" feature of Chinese patent medicine makes it challenge to elucidate the potential therapeutic mechanisms of ZZW on FD. Presently, a novel system pharmacology model including pharmacokinetic parameters, pharmacological data, and component contribution score (CS) is constructed to decipher the potential therapeutic mechanism of ZZW on FD. Finally, 61 components with favorable pharmacokinetic profiles and biological activities were obtained through ADME (absorption, distribution, metabolism, and excretion) screening in silico. The related targets of these components are identified by component targeting process followed by GO analysis and pathway enrichment analysis. And systematic analysis found that through acting on the target related to inflammation, gastrointestinal peristalsis, and mental disorder, ZZW plays a synergistic and complementary effect on FD at the pathway level. Furthermore, the component CS showed that 29 components contributed 90.18% of the total CS values of ZZW for the FD treatment, which suggested that the effective therapeutic effects of ZZW for FD are derived from all active components, not a few components. This study proposes the system pharmacology method and discovers the potent combination therapeutic mechanisms of ZZW for FD. This strategy will provide a reference method for other TCM mechanism research.

Aging-related loss of adult stem cell function contributes to impaired tissue regeneration. Mice deficient in zinc metalloproteinase STE24 (Zmpste24-/-) exhibit premature age-related musculoskeletal pathologies similar to those observed in children with Hutchinson-Gilford progeria syndrome (HGPS). We have reported that muscle-derived stem/progenitor cells (MDSPCs) isolated from Zmpste24-/- mice are defective in their proliferation and differentiation capabilities in culture and during tissue regeneration. The mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth, and inhibition of the mTORC1 pathway extends the lifespan of several animal species. We therefore hypothesized that inhibition of mTORC1 signaling would rescue the differentiation defects observed in progeroid MDSPCs. MDSPCs were isolated from Zmpste24-/- mice, and the effects of mTORC1 on MDSPC differentiation and function were examined. We found that mTORC1 signaling was increased in senescent Zmpste24-/- MDSPCs, along with impaired chondrogenic, osteogenic, and myogenic differentiation capacity versus wild-type MDSPCs. Interestingly, we observed that mTORC1 inhibition with rapamycin improved myogenic and chondrogenic differentiation and reduced levels of apoptosis and senescence in Zmpste24-/- MDSPCs. Our results demonstrate that age-related adult stem/progenitor cell dysfunction contributes to impaired regenerative capacities and that mTORC1 inhibition may represent a potential therapeutic strategy for improving differentiation capacities of senescent stem and muscle progenitor cells.

Abstract Bone morphogenetic protein (BMP) signaling is essential for osteogenesis. However, recombinant human BMPs (rhBMPs) exhibit large inter-individual variations in local bone formation during clinical spinal fusion. Smurf1 ubiquitinates BMP downstream molecules for degradation. Here, we classify age-related osteoporosis based on distinct intraosseous BMP-2 levels and Smurf1 activity. One major subgroup with a normal BMP-2 level and elevated Smurf1 activity (BMP-2 n /Smurf1 e ) shows poor response to rhBMP-2 during spinal fusion, when compared to another major subgroup with a decreased BMP-2 level and normal Smurf1 activity (BMP-2 d /Smurf1 n ). We screen a chalcone derivative, i.e., 2-(4-cinnamoylphenoxy)acetic acid, which effectively inhibits Smurf1 activity and increases BMP signaling. For BMP-2 n /Smurf1 e mice, the chalcone derivative enhances local bone formation during spinal fusion. After conjugating to an osteoblast-targeting and penetrating oligopeptide (DSS) 6 , the chalcone derivative promotes systemic bone formation in BMP-2 n /Smurf1 e mice. This study demonstrates a precision medicine-based bone anabolic strategy for age-related osteoporosis.

<h3>Background & Aims</h3> The Chinese herbal medicine, MaZiRenWan (MZRW), has been used for more than 2000 years to treat constipation, but it has not been tested in a randomized controlled trial. We performed a trial to evaluate the efficacy and safety of MZRW, compared with the stimulant laxative senna or placebo, for patients with functional constipation (FC). <h3>Methods</h3> We performed a double-blind, double-dummy, trial of 291 patients with FC based on Rome III criteria, seen at 8 clinics in Hong Kong from June 2013 through August 2015. Patients were observed for 2 weeks and then assigned randomly (1:1:1) to groups given MZRW (7.5 g, twice daily), senna (15 mg daily), or placebo for 8 weeks. Patients were then followed for 8 weeks and evaluated at baseline and weeks 4, 8 (end of treatment), and 16 (end of follow up). Participants recorded information on stool form and frequency, feeling of complete evacuation, and research medication taken. Data on individual bowel symptoms, global symptom improvement, and adverse events were collected. A complete response was defined as an increase ≥1 complete spontaneous bowel movement (CSBM)/week from baseline (the primary outcome). Secondary outcomes included response during the follow-up period, colonic transit, individual and global symptom assessments, quality of life measured with 36-item short form Chinese version, and adverse events. <h3>Results</h3> Although there was no statistically significant difference in proportions of patients with a complete response to MZRW (68%) vs. senna (57.7%) (<i>P</i> = .14) at week 8, there was a statistically significant difference vs. placebo (33.0%) (<i>P</i> < .005). At the 16-week timepoint (after the 8-week follow-up period), 47.4% of patients had a complete response to MZRW, 20.6% had a complete response to senna, and 17.5% had a complete response to placebo (<i>P</i> < .005 for MZRW vs. placebo). The group that received MZRW group also had significant increases in colonic transit and reduced severity of constipation, straining, incomplete evacuation, and global constipation symptoms compared with the groups that received placebo or senna in (P < .05 for all comparisons). <h3>Conclusions</h3> In a randomized controlled trial of 291 patients with FC, we found MZRW to be well-tolerated and effective in increasing CSBM/week. MZRW did not appear to be more effective than senna and might be considered as an alternative to this drug. ClincialTrials.gov no: NCT01695850.

Traditional Chinese medicine (TCM) with the characteristics of "multi-component-multi-target-multi-pathway" has obvious advantages in the prevention and treatment of complex diseases, especially in the aspects of "treating the same disease with different treatments". However, there are still some problems such as unclear substance basis and molecular mechanism of the effectiveness of formula. Network pharmacology is a new strategy based on system biology and poly-pharmacology, which could observe the intervention of drugs on disease networks at systematical and comprehensive level, and especially suitable for study of complex TCM systems. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, causing articular and extra articular dysfunctions among patients, it could lead to irreversible joint damage or disability if left untreated. TCM formulas, Danggui-Sini-decoction (DSD), Guizhi-Fuzi-decoction (GFD), and Huangqi-Guizhi-Wuwu-Decoction (HGWD), et al., have been found successful in controlling RA in clinical applications. Here, a network pharmacology-based approach was established. With this model, key gene network motif with significant (KNMS) of three formulas were predicted, and the molecular mechanism of different formula in the treatment of rheumatoid arthritis (RA) was inferred based on these KNMSs. The results show that the KNMSs predicted by the model kept a high consistency with the corresponding C-T network in coverage of RA pathogenic genes, coverage of functional pathways and cumulative contribution of key nodes, which confirmed the reliability and accuracy of our proposed KNMS prediction strategy. All validated KNMSs of each RA therapy-related formula were employed to decode the mechanisms of different formulas treat the same disease. Finally, the key components in KNMSs of each formula were evaluated by in vitro experiments. Our proposed KNMS prediction and validation strategy provides methodological reference for interpreting the optimization of core components group and inference of molecular mechanism of formula in the treatment of complex diseases in TCM.

Since calcium and phosphorus play vital roles in a multitude of physiologic systems, disorders of calcium and phosphorus metabolism always lead to severe consequences such as skeletal-related and cardiovascular morbidity, or even life-threatening. Physiologically, the maintenance of calcium and phosphorus homeostasis is achieved via a variety of concerted actions of hormones such as parathyroid hormone (PTH), vitamin D, and fibroblast growth factor (FGF23), which could be regulated mainly at three organs, the intestine, kidney, and bone. Disruption of any organ or factor might lead to disorders of calcium and phosphorus metabolism. Currently, lacking of accurate diagnostic approaches and unknown molecular basis of pathophysiology will result in patients being unable to receive a precise diagnosis and personalized treatment timely. Therefore, it is urgent to identify early diagnostic biomarkers and develop therapeutic strategies. Fortunately, proteomics and metabolomics offer promising tools to discover novel indicators and further understanding of pathological mechanisms. Therefore, in this review, we will give a systematic introduction on PTH-1,25(OH)2D-FGF23 axis in the disorders of calcium and phosphorus metabolism, diagnostic biomarkers identified, and potential altered metabolic pathways involved.