Cong Xue

PD-L1 expression is a feature of Epstein-Barr virus (EBV) associated malignancies such as nasopharyngeal carcinoma (NPC). Here, we found that EBV-induced latent membrane protein 1 (LMP1) and IFN-γ pathways cooperate to regulate programmed cell death protein 1 ligand (PD-L1). Expression of PD-L1 was higher in EBV positive NPC cell lines compared with EBV negative cell lines. PD-L1 expression could be increased by exogenous and endogenous induction of LMP1 induced PD-L1. In agreement, expression of PD-L1 was suppressed by knocking down LMP1 in EBV positive cell lines. We further demonstrated that LMP1 up-regulated PD-L1 through STAT3, AP-1, and NF-κB pathways. Besides, IFN-γ was independent of but synergetic with LMP1 in up-regulating PD-L1 in NPC. Furthermore, we showed that PD-L1 was associated with worse disease-free survival in NPC patients. These results imply that blocking both the LMP1 oncogenic pathway and PD-1/PD-L1 checkpoints may be a promising therapeutic approach for EBV positive NPC patients.

Significant differences in the microbial biomass and diversity in soil have been observed following long-term fertilization, but little research has explored the co-occurrence patterns among microbial taxa or functions. Soil samples from four long-term experiments in China were collected, and an Illumina amplicon sequencing of 16S rDNA amplicon was performed to decipher the differences in interactions and network organization arising from fertilization with organic amendment (OA) or chemical fertilizer (CF) across the sampling sites. We thus aimed to extend the analysis beyond the basic inventory descriptions of the composition and diversity of the microbiome and toward an assessment of relationships and effects. Nonmetric multidimensional scaling analysis revealed a clear difference between the soil microbiomes associated with the OA and CF treatments. Fertilization can interact with the soil chemical properties and sampling site, thereby significantly impacting the associated soil microbiome. Distinct network structures indicated that the OA network was characterized by more functionally interrelated operational taxonomic units (OTUs) than the CF network. The topological roles of individual OTUs and key microbial populations were distinctly different, although the generalists mainly belonging to Actinobacteria and Proteobacteria were observed in both networks. The different fertilization treatments had distinct effects on the soil pH, which represented a key variable to impact the associated microbial community modularity. Most functional groups involving carbon-, nitrogen- and phosphorus-related metabolism and recycling and antibiotic biosynthesis were present at significantly high abundance in the organic-amended soils. All of the functions with statistical significance could be traced to corresponding responsible major modules in the CF network, but it was not the case in OA soil. These results suggested that the soil functions were decentralized and assigned to small groups in OA soil. Overall, we demonstrated that long-term organic amendment supports stronger functional potentials and more interactions within soil community relative to long-term chemical-only fertilization, which may be related to OA benefits to soil stability and buffering capacity. These results explore the correlations, inter-taxa and between major groups and functional traits, to gain a more integrated understanding of microbiome and ecological rules guiding soil community fostered by fertilization regimes.

Worldwide, banana production is severely hindered by Fusarium wilt, a devastating disease caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc). With no widely adopted efficient method of control or prevention, the emergence of a new Foc variant, tropical race 4 (Foc TR4), has led to the widespread destruction of plantations in Cavendish-producing areas. Recently, banana Fusarium wilt has been controlled by the consecutive application of biofertilizer (BIO) in newly reclaimed fields. In this study we examine the temporal effects of BIO versus compost application in newly converted banana fields on the composition and abundance of the rhizosphere bacterial and fungal communities and the survival of the biocontrol inoculant, Bacillus amyloliquefaciens NJN-6. Our findings show that BIO-amended rhizosphere soils increased the abundance of bacteria while decreasing fungal abundance. This corresponded to higher bacterial richness and diversity in the BIO amendment, while no trends were observed with the fungal community. Rhizosphere soil bacterial and fungal community composition were significantly different between BIO and compost amendment and treatment, not time, exhibited the largest impact. Other potential taxa involved in disease suppression were also identified, such as increased abundances of Sphingobium, Dyadobacter, and Cryptococcus and lower abundances of Fusarium, Ralstonia, and Burkholderia. Overall, decreased abundances of F. oxysporum and a lack of variability in the abundance of the biocontrol agent NJN-6 over three years contributed to disease suppression, in combination with alterations in fungal and bacterial composition and abundance, pointing to the sustainability of BIO as an amendment for disease suppression.

Extracellular matrix (ECM) plays a prominent role in establishing and maintaining an ideal microenvironment for tissue regeneration, and ECM scaffolds are used as a feasible alternative to cellular and molecular therapy in the fields of tissue engineering. Because of their advantages over tissue-derived ECM scaffolds, cultured cell-derived ECM scaffolds are beginning to attract attention, but they have been scarcely studied for peripheral nerve repair. Here we aimed to develop a tissue engineered nerve scaffold by reconstituting nerve cell-derived ECM with natural biomaterials. A protocol was adopted to prepare and characterize the cultured Schwann cell (SC)-derived ECM. A chitosan conduit and silk fibroin (SF) fibers were prepared, cultured with SCs for ECM deposition, and subjected to decellularization, followed by assembly into a chitosan/SF-based, SC-derived ECM-modified scaffold, which was used to bridge a 10 mm rat sciatic nerve gap. The results from morphological analysis as well as electrophysiological examination indicated that regenerative outcomes achieved by our developed scaffold were similar to those by an acellular nerve graft (namely a nerve tissue-derived ECM scaffold), but superior to those by a plain chitosan/SF scaffold. Moreover, blood and histopathological parameters confirmed the safety of scaffold modification by SC-derived ECM. Therefore, a hybrid scaffold based on joint use of acellular and classical biomaterials represents a promising approach to nerve tissue engineering.

To investigate the role of PD-L1 expression in tumor recurrence and metastasis in Chinese patients with breast cancer.Suitable tissue samples were available from 870 patients with breast cancer. Paraffin-embedded tumor sections were stained with PD-L1 antibody. The correlations between PD-L1 expression and clinical characteristics, ER/PR/HER2 status and survival parameters were analyzed. Kaplan-Meier and univariate Cox proportional hazards model analyses were used to compare the survival of patients with high PD-L1 expression and patients with no PD-L1 expression.The median follow-up time was 98 months(range, 17-265 months).The positive rate of PD-L1 expression in breast cancer was 21.7% (189/870). PD-L1 high expression was inversely associated with larger tumor size, higher tumor grade, more positive lymph node number, as well as negative ER and PR status. PD-L1 expression was particularly higher in TNBC compared with non-TNBC, although no statistical significance was observed. Nomogram logistic regression results based on clinical and pathological features showed that the following factors were more likely associated with high PD-L1 expression: patient age younger than 35 years, larger tumor size, lymphovascular invasion and advanced stage. Our data indicated that patients with high PD-L1 expression had poor DFS, DMFS and overall survival compared with those with no PD-L1 expression. Univariate Cox proportional hazards model analysis showed that PD-L1 was an independent prognostic factor for tumor prognosis.PD-L1 expression is an important indicator of unfavorable prognosis in breast cancer patients.

<h3>Importance</h3> Among all subtypes of breast cancer, triple-negative breast cancer has a relatively high relapse rate and poor outcome after standard treatment. Effective strategies to reduce the risk of relapse and death are needed. <h3>Objective</h3> To evaluate the efficacy and adverse effects of low-dose capecitabine maintenance after standard adjuvant chemotherapy in early-stage triple-negative breast cancer. <h3>Design, Setting, and Participants</h3> Randomized clinical trial conducted at 13 academic centers and clinical sites in China from April 2010 to December 2016 and final date of follow-up was April 30, 2020. Patients (n = 443) had early-stage triple-negative breast cancer and had completed standard adjuvant chemotherapy. <h3>Interventions</h3> Eligible patients were randomized 1:1 to receive capecitabine (n = 222) at a dose of 650 mg/m²twice a day by mouth for 1 year without interruption or to observation (n = 221) after completion of standard adjuvant chemotherapy. <h3>Main Outcomes and Measures</h3> The primary end point was disease-free survival. Secondary end points included distant disease-free survival, overall survival, locoregional recurrence-free survival, and adverse events. <h3>Results</h3> Among 443 women who were randomized, 434 were included in the full analysis set (mean [SD] age, 46 [9.9] years; T1/T2 stage, 93.1%; node-negative, 61.8%) (98.0% completed the trial). After a median follow-up of 61 months (interquartile range, 44-82), 94 events were observed, including 38 events (37 recurrences and 32 deaths) in the capecitabine group and 56 events (56 recurrences and 40 deaths) in the observation group. The estimated 5-year disease-free survival was 82.8% in the capecitabine group and 73.0% in the observation group (hazard ratio [HR] for risk of recurrence or death, 0.64 [95% CI, 0.42-0.95];<i>P</i> = .03). In the capecitabine group vs the observation group, the estimated 5-year distant disease-free survival was 85.8% vs 75.8% (HR for risk of distant metastasis or death, 0.60 [95% CI, 0.38-0.92];<i>P</i> = .02), the estimated 5-year overall survival was 85.5% vs 81.3% (HR for risk of death, 0.75 [95% CI, 0.47-1.19];<i>P</i> = .22), and the estimated 5-year locoregional recurrence-free survival was 85.0% vs 80.8% (HR for risk of locoregional recurrence or death, 0.72 [95% CI, 0.46-1.13];<i>P</i> = .15). The most common capecitabine-related adverse event was hand-foot syndrome (45.2%), with 7.7% of patients experiencing a grade 3 event. <h3>Conclusions and Relevance</h3> Among women with early-stage triple-negative breast cancer who received standard adjuvant treatment, low-dose capecitabine maintenance therapy for 1 year, compared with observation, resulted in significantly improved 5-year disease-free survival. <h3>Trial Registration</h3> ClinicalTrials.gov Identifier:NCT01112826

Maintaining stability of ecosystem functions in the face of global change calls for a better understanding regulatory factors of functionally specialized microbial groups and their population response to disturbance. In this study, we explored this issue by collecting soils from 54 managed ecosystems in China and conducting a microcosm experiment to link disturbance, elemental stoichiometry, and genetic resistance. Soil carbon:nitrogen:phosphorus (C:N:P) stoichiometry imparted a greater effect on the abundance of microbial groups associated with main C, N, and P biogeochemical processes in comparison with mean annual temperature and precipitation. Nitrogen cycling genes, including bacterial amoA-b, nirS, narG, and norB, exhibited the highest genetic resistance to N deposition. The amoA-a and nosZ genes exhibited the highest resistance to warming and drying-wetting cycles, respectively. Soil total C, N, and P contents and their ratios had a strong direct effect on the genetic resistance of microbial groups, which was dependent on mean annual temperature and precipitation. Specifically, soil C/P ratio was the main predictor of N cycling genetic resistance to N deposition. Soil total C and N contents and their ratios were the main predictors of P cycling genetic resistance to N deposition, warming, and drying-wetting. Overall, our work highlights the importance of soil stoichiometric balance for maintaining the ability of ecosystem functions to withstand global change.IMPORTANCE To be effective in predicting future stability of soil functions in the context of various external disturbances, it is necessary to follow the effects of global change on functionally specialized microbes related to C and nutrient cycling. Our study represents an exploratory effort to couple the stoichiometric drivers to microbial populations related with main C, N, and P cycling and their resistances to global change. The abundance of microbial groups involved in cellulose, starch, and xylan degradation, nitrification, N fixation, denitrification, organic P mineralization, and inorganic P dissolution showed a high stoichiometry dependency. Resistance of these microbial populations to global change could be predicted by soil C:N:P stoichiometry. Our work highlights that stoichiometric balance in soil C and nutrients is instrumental in maintaining the stability and adaptability of ecosystem functions under global change.

Abstract The network analysis has attracted increasing attention and interest from ecological academics, thus it is of great necessity to develop more convenient and powerful tools. For that reason, we have developed an R package, named “ggClusterNet,” to complete and display the network analysis in an easier manner. In that package, ten network layout algorithms are designed to better display the modules of microbiome network (randomClusterG, PolygonClusterG, PolygonRrClusterG, ArtifCluster, randSNEClusterG, PolygonModsquareG, PolyRdmNotdCirG, model_Gephi.2, model_igraph, and model_maptree). For the convenience of the users, many functions related to microbial network analysis, such as corMicor(), net_properties(), node_properties(), ZiPiPlot(), random_Net_compate(), are integrated to complete the network mining. Furthermore, the pipeline function named network.2() and corBionetwork() are also added for the quick achievement of the network or bipartite network analysis as well as their in‐depth mining. The ggClusterNet is publicly available via GitHub ( https://github.com/taowenmicro/ggClusterNet/ ) or Gitee ( https://gitee.com/wentaomicro/ggClusterNet ) for users' access. A complete description of the usages can be found on the manuscript's GitHub page ( https://github.com/taowenmicro/ggClusterNet/wiki ).

Triple-negative breast cancer (TNBC) is associated with poor prognosis and high probability of distant metastases. Tumor microenvironments play a pivotal role in tumor metastasis. Tumor-associated macrophages (TAMs) are one of the main cell components, and they are correlated with increasing metastatic risk. The aim of this study is to analyze the prognostic significance of the infiltration of TAMs in patients with TNBC.Immunohistochemical staining for cluster of differentiation (CD)68 (a marker for macrophages) was performed on tissue microarrays of operable breast cancer among 287 patients with TNBC, and the number of infiltrating TAMs was correlated with clinicopathological parameters.We found that TNBC with a large number of infiltrating TAMs had a significantly higher risk of distant metastasis, as well as lower rates of disease-free survival and overall survival than those with a smaller number of infiltrating TAMs. Multivariate analysis indicated that the number of infiltrating TAMs was a significant independent prognostic factor of disease-free survival (P=0.001) in all patients.Our results suggested that high infiltrating TAMs are a significantly unfavorable prognostic factor for patients with TNBC, and they could become a potentially useful prognostic marker for TNBC.

Abstract Panama disease caused by Fusarium oxysporum f. sp. cubense infection on banana is devastating banana plantations worldwide. Biological control has been proposed to suppress Panama disease, though the stability and survival of bio-control microorganisms in field setting is largely unknown. In order to develop a bio-control strategy for this disease, 16S rRNA gene sequencing was used to assess the microbial community of a disease-suppressive soil. Bacillus was identified as the dominant bacterial group in the suppressive soil. For this reason, B. amyloliquefaciens NJN-6 isolated from the suppressive soil was selected as a potential bio-control agent. A bioorganic fertilizer (BIO), formulated by combining this isolate with compost, was applied in nursery pots to assess the bio-control of Panama disease. Results showed that BIO significantly decreased disease incidence by 68.5%, resulting in a doubled yield. Moreover, bacterial community structure was significantly correlated to disease incidence and yield and Bacillus colonization was negatively correlated with pathogen abundance and disease incidence, but positively correlated to yield. In total, the application of BIO altered the rhizo-bacterial community by establishing beneficial strains that dominated the microbial community and decreased pathogen colonization in the banana rhizosphere, which plays an important role in the management of Panama disease.

Different fertilization managements of red soil, a kind of Ferralic Cambisol, strongly affected the soil properties and associated microbial communities. The association of the soil microbial community and functionality with long-term fertilization management in the unique low-productivity red soil ecosystem is important for both soil microbial ecology and agricultural production. Here, 454 pyrosequencing analysis of 16S recombinant ribonucleic acid genes and GeoChip4-NimbleGen-based functional gene analysis were used to study the soil bacterial community composition and functional genes involved in soil organic carbon degradation. Long-term nitrogen-containing chemical fertilization-induced soil acidification and fertility decline and significantly altered the soil bacterial community, whereas long-term organic fertilization and fallow management improved the soil quality and maintained the bacterial diversity. Short-term quicklime remediation of the acidified soils did not change the bacterial communities. Organic fertilization and fallow management supported eutrophic ecosystems, in which copiotrophic taxa increased in relative abundance and have a higher intensity of labile-C-degrading genes. However, long-term nitrogen-containing chemical fertilization treatments supported oligotrophic ecosystems, in which oligotrophic taxa increased in relative abundance and have a higher intensity of recalcitrant-C-degrading genes but a lower intensity of labile-C-degrading genes. Quicklime application increased the relative abundance of copiotrophic taxa and crop production, although these effects were utterly inadequate. This study provides insights into the interaction of soil bacterial communities, soil functionality and long-term fertilization management in the red soil ecosystem; these insights are important for improving the fertility of unique low-productivity red soil.

Previous studies in our laboratory have shown that when the N-terminus of interferon-α2b (IFN-α2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-α2b) was heterogeneous (migrated as doublets on non-reducing SDS–PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-α2b was ascribed to the structural disturbance between HSA and IFN-α2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-α2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-α2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS–PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-α2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-α2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 °C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-α2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively.

In the present study, 3 replanted black pepper orchards with continuously cropping histories for 10, 21, and 55 years in tropical China, were selected for investigating the effect of monoculture on soil physiochemical properties, enzyme activities, bacterial abundance, and bacterial community structures. Results showed long-term continuous cropping led to a significant decline in soil pH, organic matter contents, enzymatic activities, and resulted in a decrease in soil bacterial abundance. 454 pyrosequencing analysis of 16S rRNA genes revealed that the Acidobacteria and Proteobacteria were the main phyla in the replanted black pepper orchard soils, comprising up to 73.82% of the total sequences; the relative abundances of Bacteroidetes and Firmicutes phyla decreased with long-term continuous cropping; and at genus level, the Pseudomonas abundance significantly depleted after 21 years continuous cropping. In addition, bacterial diversity significantly decreased after 55 years black pepper continuous cropping; obvious variations for community structures across the 3 time-scale replanted black pepper orchards were observed, suggesting monoculture duration was the major determinant for bacterial community structure. Overall, continuous cropping during black pepper cultivation led to a significant decline in soil pH, organic matter contents, enzymatic activities, resulted a decrease in soil bacterial abundance, and altered soil microbial community membership and structure, which in turn resulted in black pepper poor growth in the continuous cropping system.

Several EGFR-tyrosine kinase inhibitors (EGFR-TKIs) including erlotinib, gefitinib, afatinib and icotinib are currently available as treatment for patients with advanced non-small-cell lung cancer (NSCLC) who harbor EGFR mutations. However, no head to head trials between these TKIs in mutated populations have been reported, which provides room for indirect and integrated comparisons.We searched electronic databases for eligible literatures. Pooled data on objective response rate (ORR), progression free survival (PFS), overall survival (OS) were calculated. Appropriate networks for different outcomes were established to incorporate all evidences. Multiple-treatments comparisons (MTCs) based on Bayesian network integrated the efficacy and specific toxicities of all included treatments.Twelve phase III RCTs that investigated EGFR-TKIs involving 1821 participants with EGFR mutation were included. For mutant patients, the weighted pooled ORR and 1-year PFS of EGFR-TKIs were significant superior to that of standard chemotherapy (ORR: 66.6% vs. 30.9%, OR 5.46, 95%CI 3.59 to 8.30, P<0.00001; 1-year PFS: 42.9% vs. 9.7%, OR 7.83, 95%CI 4.50 to 13.61; P<0.00001) through direct meta-analysis. In the network meta-analyses, no statistically significant differences in efficacy were found between these four TKIs with respect to all outcome measures. Trend analyses of rank probabilities revealed that the cumulative probabilities of being the most efficacious treatments were (ORR, 1-year PFS, 1-year OS, 2-year OS): erlotinib (51%, 38%, 14%, 19%), gefitinib (1%, 6%, 5%, 16%), afatinib (29%, 27%, 30%, 27%) and icotinib (19%, 29%, NA, NA), respectively. However, afatinib and erlotinib showed significant severer rash and diarrhea compared with gefitinib and icotinib.The current study indicated that erlotinib, gefitinib, afatinib and icotinib shared equivalent efficacy but presented different efficacy-toxicity pattern for EGFR-mutated patients. Erlotinib and afatinib revealed potentially better efficacy but significant higher toxicities compared with gefitinib and icotinib.

The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV-SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species.

Despite great progress in the fields of tissue engineering and stem cell therapy, the translational and preclinical studies are required to accelerate the clinical application of tissue engineered nerve grafts, as an alternative to autologous nerve grafts, for peripheral nerve repair. Rhesus monkeys (non-human primates) are more clinically relevant and more suitable for scaling up to humans as compared to other mammalians. Based on this premise, and considering a striking similarity in the anatomy and function between human and monkey hands, here we used chitosan/PLGA-based, autologous marrow mesenchymal stem cells (MSCs)-containing tissue engineered nerve grafts (TENGs) for bridging a 50-mm long median nerve defect in rhesus monkeys. At 12 months after grafting, locomotive activity observation, electrophysiological assessments, and FG retrograde tracing tests indicated that the recovery of nerve function by TENGs was more efficient than that by chitosan/PLGA scaffolds alone; histological and morphometric analyses of regenerated nerves further confirmed that the morphological reconstruction by TENGs was close to that by autografts and superior to that by chitosan/PLGA scaffolds alone. In addition, blood test and histopathological examination demonstrated that TENGs featured by addition of autologous MSCs could be safely used in the primate body. These findings suggest the efficacy of our developed TENGs for peripheral nerve regeneration and their promising perspective for clinical applications.

Introduction Treatment choice for NSCLC in China has not previously been reported. This paper explores the clinical practice and adherence to treatment guidelines for NSCLC. Methods A specifically designed questionnaire was used. It consisted of personal information of the responders and treatment details (patient identification data was excluded). Questionnaires were delivered to doctors in 12 major cities in China. Doctors were asked to answer the questionnaires based on real cases in their daily practice. Results 987 cases of NSCLC were included. In first-line chemotherapy, regimens were mostly platinum-based among which gemcitabine plus platinum was predominately used (27.4%), followed by docetaxel plus platinum (16.2%) and paclitaxel plus platinum (13.5%). In second-line therapy some were treated with single agents, such as docetaxel (12.9%), gefitinib (11.1%), pemetrexed (9.3%), and erlotinib (3.5%). 44.5% were with doublet therapy. Detection rate of epidermal growth factor receptor (EGFR) mutation was only 9.6% because of the limited prevalence of testing technology. EGFR mutation rate was 46.8%. EGFR-tyrosine kinase inhibitors (TKIs) were used more frequently as salvage (14.8%) rather than upfront therapy (5.3%). Conclusions This survey reveals the daily clinical treatment for NSCLC in China. Overall data showed modest adherence to the national guideline (NCCN guideline Chinese version) for first-line chemotherapy. We believe this survey is valuable to provide a reference for further clinical trial design and policy making.

Spinal cord injury (SCI) is a serious problem that primarily affects younger and middle-aged adults at its onset. To date, no effective regenerative treatment has been developed. Over the last decade, researchers have made significant advances in stem cell technology, biomaterials, nanotechnology, and immune engineering, which may be applied as regenerative therapies for the spinal cord. Although the results of clinical trials using specific cell-based therapies have proven safe, their efficacy has not yet been demonstrated. The pathophysiology of SCI is multifaceted, complex and yet to be fully understood. Thus, combinatorial therapies that simultaneously leverage multiple approaches will likely be required to achieve satisfactory outcomes. Although combinations of biomaterials with pharmacologic agents or cells have been explored, few studies have combined these modalities in a systematic way. For most strategies, clinical translation will be facilitated by the use of minimally invasive therapies, which are the focus of this review. In addition, this review discusses previously explored therapies designed to promote neuroregeneration and neuroprotection after SCI, while highlighting present challenges and future directions. To date there are no effective treatments that can regenerate the spinal cord after injury. Although there have been significant preclinical advances in bioengineering and regenerative medicine over the last decade, these have not translated into effective clinical therapies for spinal cord injury. This review focuses on minimally invasive therapies, providing extensive background as well as updates on recent technological developments and current clinical trials. This review is a comprehensive resource for researchers working towards regenerative therapies for spinal cord injury that will help guide future innovation.

Summary In the processes controlling ecosystem fertility, fungi are increasingly acknowledged as key drivers. However, our understanding of the rules behind fungal community assembly regarding the effect of soil fertility level remains limited. Using soil samples from typical tea plantations spanning c. 2167 km north‐east to south‐west across China, we investigated the assemblage complexity and assembly processes of 140 fungal communities along a soil fertility gradient. The community dissimilarities of total fungi and fungal functional guilds increased with increasing soil fertility index dissimilarity. The symbiotrophs were more sensitive to variations in soil fertility compared with pathotrophs and saprotrophs. Fungal networks were larger and showed higher connectivity as well as greater potential for inter‐module connection in more fertile soils. Environmental factors had a slightly greater influence on fungal community composition than spatial factors. Species abundance fitted the Zipf–Mandelbrot distribution (niche‐based mechanisms), which provided evidence for deterministic‐based processes. Overall, the soil fungal communities in tea plantations responded in a deterministic manner to soil fertility, with high fertility correlated with complex fungal community assemblages. This study provides new insights that might contribute to predictions of fungal community complexity.

Recent studies have shown the prognostic nutritional index (PNI) had prognostic value in some solid tumors. However, no studies have examined its prognostic role in small-cell lung cancer (SCLC) patients. In this retrospective study, 724 consecutive SCLC patients were included between 2006 and 2013. Demographic, clinical, and laboratory data were collected. The PNI was calculated as 10 × serum albumin value (g/dl) + .005 × peripheral lymphocyte count (per mm(3)). Univariate and multivariate analyses were performed to assess the prognostic value of relevant factors. The optimal cut-off value of PNI for OS stratification was determined to be 52.48. A total of 464 and 260 patients were assigned to low and high PNI groups, respectively. Compared with low PNI, high PNI was associated with older age, advanced stage, and elevated lactate dehydrogenase (LDH). Median overall survival (OS) was worse in the low PNI group (low vs high, 15.90 vs 25.27 months; HR, 0.62; p < 0.001). In multivariate analysis, stage, performance status, LDH, and PNI were independent prognostic factors for OS. Subgroup analysis showed PNI was generally a significant prognostic factor in different clinical situations. The assessment of PNI could assist the identification of patients with poor prognosis and be a hierarchical factor in the future SCLC clinical trials.

Our previous work demonstrated that application of a bio-organic fertilizer (BIO) to a banana mono-culture orchard with serious Fusarium wilt disease effectively decreased the number of soil Fusarium sp. and controlled the soil-borne disease. Because bacteria are an abundant and diverse group of soil organisms that responds to soil health, deep 16 S rRNA pyrosequencing was employed to characterize the composition of the bacterial community to investigate how it responded to BIO or the application of other common composts and to explore the potential correlation between bacterial community, BIO application and Fusarium wilt disease suppression. After basal quality control, 137,646 sequences and 9,388 operational taxonomic units (OTUs) were obtained from the 15 soil samples. Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria were the most frequent phyla and comprised up to 75.3% of the total sequences. Compared to the other soil samples, BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria, while Bacteroidetes were found in lower abundance. Meanwhile, on genus level, higher abundances compared to other treatments were observed for Gemmatimonas and Gp4. Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application, and they were all positively correlated with disease suppression. Cumulatively, the reduced Fusarium wilt disease incidence that was seen after BIO was applied for 1-year might be attributed to the general suppression based on a shift within the bacteria soil community, including specific enrichment of Gemmatimonas and Sphingomonas.

Abstract Transdermal delivery of water‐insoluble drugs via hydrogel‐based microneedle (MN) arrays is crucial for improving their therapeutic efficacies. However, direct loading of water‐insoluble drug into hydrophilic matrices remains challenging. Here, a biodegradable MN array patch that is fabricated from naturally derived polymer conjugates of gelatin methacryloyl and β ‐cyclodextrin (GelMA‐ β ‐CD) is reported. When curcumin, an unstable and water‐insoluble anticancer drug, is loaded as a model drug, its stability and solubility are improved due to the formation of an inclusion complex. The polymer‐drug complex GelMA‐ β ‐CD/CUR can be formulated into MN arrays with sufficient mechanical strength for skin penetration and tunable drug release profile. Anticancer efficacy of released curcumin is observed in three‐dimensional B16F10 melanoma models. The GelMA‐ β ‐CD/CUR MN exhibits relatively higher therapeutic efficacy through more localized and deeper penetrated manner compared with a control nontransdermal patch. In vivo studies also verify biocompatibility and degradability of the GelMA‐ β ‐CD MN arrays patch.

BackgroundPrevious results from our trial showed that adding oxaliplatin to radiotherapy (RT) increased survival in patients with locoregionally advanced nasopharyngeal carcinoma (NPC) at 2 years. Here, we present the data of long-term efficacy and late toxic effects.Patients and methodsBetween January 2001 and January 2003, 115 Patients with nonkeratinizing/undifferentiated locoregionally advanced NPC were randomly to receive either RT alone (n = 56) or plus concurrent oxaliplatin 70 mg/m2 weekly for six cycles (n = 59).ResultsAfter a median follow-up of 114 months (range 18–139 months), the 5-year overall survival (OS) and metastasis-free survival (MFS) rates in the concurrent chemoradiotherapy (CCRT) group were significantly higher than those observed in the RT-alone group (OS, 73.2% versus 60.2%, P = 0.028; MFS, 74.7% versus 63.0%, P = 0.027). However, CCRT did not improve locoregional failure-free survival significantly. Subgroup analyses showed that the superiorities of CCRT mainly existed in the T3-4N0-1 stage subgroup (OS: HR = 0.394, P = 0.034). The grade 3/4 late toxic effects were similar in the two groups.Conclusion(s)The long-term follow-up data confirms the role of CCRT as a treatment of locoregionally advanced NPC. Oxaliplatin can be considered as an alternative optional therapeutic regimen for these patients due to its high efficiency and low toxic effect.

Background: CD44 is widely used as a putative cancer stem cells (CSCs) marker for colorectal cancer (CRC). However, the prognostic role of CD44 in CRC remains controversial. Methods: We performed a systematic review and meta-analysis to evaluate the association of various CD44 isoforms and overall survival (OS) and clinicopathological features of CRC patients. Results: A total of 48 studies were included in the meta-analysis. Total CD44 isoforms overexpression was significantly correlated with worse OS of patients with CRC (HR = 1.32, 95% CI = 1.08-1.61, P = 0.007). In a stratified analysis, a higher level of either CD44v6 or CD44v2 had an unfavorable impact on OS (HRCD44v6 = 1.50, 95% CI = 1.10-2.14, P = 0.010; HRCD44v2 = 2.93, 95% CI = 1.49-5.77, P = 0.002). Additionally, CD44 was shown to be associated with some clinicopathological features, such as lymph node metastasis (ORCD44 = 1.56, 95% CI = 1.01-2.41, P = 0.044; ORCD44v6 = 1.97, 95% CI = 1.19-3.26, P = 0.008; ORTotal CD44 isoforms = 1.57, 95% CI = 1.15-2.14, P = 0.004), distant metastasis (ORCD44 = 2.90, 95% CI = 1.08-7.83, P = 0.035; ORTotal CD44 isoforms = 1.89, 95% CI = 1.02-3.53, P = 0.044). Moreover, a high level of CD44 showed a possible correlation with poor differentiation (ORTotal CD44 isoforms = 1.44, 95% CI = 1.00-2.08, P = 0.051), elevated level of CD44v6 tend to be correlated with tumor size (OR = 1.71, 95% CI = 0.99-2.96, P = 0.056). Conclusions: This meta-analysis demonstrated that CD44 overexpression might be an unfavorable prognostic factor for CRC patients and could be used to predict poor differentiation, lymph node metastasis and distant metastasis.

<h3>Background</h3> Immune-checkpoint inhibitors plus chemotherapy are emerging as effective first-line treatment in advanced non-small-cell lung carcinoma (NSCLC), but little is known about the magnitude of benefits and potential clinical predictors. <h3>Methods</h3> We performed a meta-analysis of randomized trials that compared PD-1/PD-L1 inhibitor plus chemotherapy with chemotherapy in first line of treatment for advanced NSCLC. The outcomes included progression-free survival (PFS), overall survival (OS), objective response rate (ORR) and treatment-related adverse events (AEs). A fixed-effect or random-effects model was adopted depending on between-study heterogeneity. <h3>Results</h3> Six trials involving 3144 patients were included. PD-1/PD-L1 inhibitor plus chemotherapy was significantly associated with improvement of PFS (hazards ratio [HR], 0.62; 95% CI 0.57–0.67; <i>P</i> &lt; .001), OS (HR, 0.68; 95% CI 0.53–0.87; <i>P</i> = .002) and ORR (relative ratio [RR], 1.56; 95% CI 1.29–1.89; <i>P</i> &lt; .001), irrespective of PD-L1 expression level. The significant predictor(s) for treatment benefit with combination therapy versus chemotherapy alone were PD-L1 expression level for PFS (<i>P</i> &lt; .001); types of checkpoint inhibitor for ORR (<i>P</i> &lt; .001); histology (<i>P</i> = .025), age (<i>P</i> = .038), gender (<i>P</i> &lt; .001), and types of checkpoint inhibitor (<i>P</i> &lt; .001) for OS. In safety analyses, PD-1/PD-L1 inhibitor plus chemotherapy had significantly higher incidence of adverse events (AEs) of grade 3 or higher (RR, 1.14; <i>P</i> = .007), AEs leading to treatment discontinuation (RR, 1.29; <i>P</i> = .022), serious AEs (RR 1.70; <i>P</i> = .006), immune mediated AEs of any grade (RR, 2.37; <i>P</i> &lt; .001), and immune mediated AEs of grade 3 or higher (RR, 3.71; <i>P</i> &lt; .001). <h3>Conclusions</h3> PD-1/PD-L1 inhibitor plus chemotherapy, compared with chemotherapy, is associated with significantly improved PFS, ORR, and OS in first-line therapy in NSCLC, at the expense of increased treatment-related AEs.

Maintaining soil fertility and the microbial communities that determine fertility is critical to sustainable agricultural strategies, and the use of different organic fertilizer regimes represents an important practice in attempts to preserve soil quality. However, little is known about the dynamic response of bacterial communities to fertilization regimes across crop growth stages. In this study, we examined microbial community structure and diversity across eight representative growth stages of wheat-rice rotation under four different fertilization treatments: no nitrogen fertilizer (NNF), chemical fertilizer (CF), organic-inorganic mixed fertilizer (OIMF) and organic fertilizer (OF). Quantitative PCR (QPCR) and high-throughput sequencing of bacterial 16S rRNA gene fragments revealed that growth stage as the best predictor of bacterial community abundance and structure. Additionally, bacterial community compositions differed between wheat and rice rotations. Relative to soils under wheat rotation, soils under rice rotation contained higher relative abundances (RA) of anaerobic and mesophilic microbes and lower RA of aerophilic microbes. With respect to fertilization regime, NNF plots had a higher abundance of nitrogen–fixing Cyanobacteria. OIMF had a lower abundance of ammonia-oxidizing Thaumarchaeota compared with CF. Application of chemical fertilizers (CF and OIMF treatments) significantly increased the abundance of some generally oligotrophic bacteria such those belonging to the Acidobacteria, while more copiotrophic of the phylum Proteobacteria increased with organic fertilizer application. A high correlation coefficient was found when comparing RA of Acidobacteria based upon QPCR versus sequence analysis, yet poor correlations were found for the Alpha- and Beta- Proteobacteria, highlighting the caution required when interpreting these molecular data. In total, crop, fertilization scheme and plant developmental stage all influenced soil microbial community structure, but not total levels of alpha diversity.

Fusarium wilt disease of banana, caused by the fungus Fusarium oxysporum f. sp. cubense race 4, is a serious soil-borne fungal disease that currently threatens worldwide banana production. No single agricultural practice has yet been developed to effectively manage this disease. In the present study, greenhouse experiments were conducted to evaluate the effect of an integrated biofertilizer application after ammonia fumigation to enhance control of Fusarium wilt disease in severely infected banana mono-cropped soils. Quantitative PCR and high-throughput sequencing were used to characterise soil microbial community structure and the results from both two-season experimental studies showed that biofertilizer application after ammonia fumigation significantly reduced the incidence of banana Panama disease by approximately 55% and promoted the plant biomass, compared to the control application of cow manure to non-fumigated soil. Ammonia fumigation significantly reduced total fungal and F. oxysporum abundances and bacterial and fungal diversities. Biofertilizer application after fumigation further depleted the abundance of the pathogen. Biofertilizer application and fumigation altered the soil microbial community composition with the bacterial community responding first to fumigation, while the fungal community responded first to fertilization. Although Bacillus, including our inoculated strain, was not enriched after biofertilization, putative beneficial microbes such as Paenibacillus, Virgibacillus, Nitrosomonas, and Nitrobacter, were significantly enriched by ammonia fumigation and biofertilizer application, and were significantly correlated with disease suppression or increased plant biomass. Furthermore, fumigation and biofertilization significantly increased the soil pH and nutrient contents, with concomitant effects on the microbial community. Overall, the observed disease suppression and increased plant biomass resulting from both soil fumigation and biofertilization after banana cropping can be attributed to the reduced abundance of F. oxysporum and general suppression from altered soil properties that may enable the establishment of a beneficial soil microbiome.

Background: Novel coronavirus disease (COVID-19) is spreading globally. Little is known about the risk factors for the clinical outcomes of COVID-19 in children. Methods: A retrospective case-control study was taken in children with severe acute respiratory syndrome coronary virus-2 infection in Wuhan Children’s Hospital. Risk factors associated with the development of COVID-19 and progression were collected and analyzed. Results: Eight of 260 children diagnosed with severe COVID-19 pneumonia were included in the study. Thirty-five children with COVID-19 infection matched for age, sex and date of admission, and who classified as non-severe type, were randomly selected from the hospital admissions. For cases with severe pneumonia caused by COVID-19, the most common symptoms were dyspnea (87.5%), fever (62.5%) and cough (62.5%). In laboratory, white blood cells count was significantly higher in severe children than non-severe children. Levels of inflammation bio-makers such as hsCRP, IL-6, IL-10 and D-dimer elevated in severe children compared with non-severe children on admission. The level of total bilirubin and uric acid clearly elevated in severe children compared with non-severe children on admission. All of severe children displayed the lesions on chest CT, more lung segments were involved in severe children than in non-severe children, which was only risk factor associated with severe COVID-19 pneumonia in multivariable analysis. Conclusions: More than 3 lung segments involved were associated with greater risk of development of severe COVID-19 in children. Moreover, the possible risk of the elevation of IL-6, high total bilirubin and D-dimer with univariable analysis could identify patients to be severe earlier.

A favorable microenvironment plays an important role in nerve regeneration. Extracellular matrix (ECM) derived from cultured cells or natural tissues can facilitate nerve regeneration in the presence of various microenvironmental cues, including biochemical, spatial, and biomechanical factors. This study, through proteomics and three-dimensional image analysis, determines that the components and spatial organization of the ECM secreted by bone marrow mesenchymal cells (BMSCs) are more similar to acellular nerves than those of the ECMs derived from Schwann cells (SCs), skin-derived precursor Schwann cells (SKP-SCs), or fibroblasts (FBs). ECM-modified nerve grafts (ECM-NGs) are engineered by co-cultivating BMSCs, SCs, FBs, SKP-SCs with well-designed nerve grafts used to bridge nerve defects. BMSC-ECM-NGs exhibit the most promising nerve repair properties based on the histology, neurophysiology, and behavioral analyses. The regeneration microenvironment formed by the ECM-NGs is also characterized by proteomics, and the advantages of BMSC-ECM-NGs are evidenced by the enhanced expression of factors related to neural regeneration and reduced immune response. Together, these findings indicate that BMSC-derived ECMs create a more superior microenvironment for nerve regeneration than that by the other ECMs and may, therefore, represent a potential alternative for the clinical repair of peripheral nerve defects.

Phase II trials showed the efficacy of anti-HER2 RC48-ADC (disitamab vedotin) for HER2-positive metastatic urothelial carcinoma (UC). This study evaluated RC48 alone verses in combination with immunotherapy for locally advanced or metastatic UC using real-world data.This retrospective, multicenter, real-world study included patients with locally advanced or metastatic UC who received RC48 in five hospitals in China between July 2021 and April 2022. The outcomes were progression-free survival (PFS), overall survival (OS), objective response rate (ORR), disease control rate (DCR), and adverse events.Thirty-six patients were included. The patients were 47-87 years, and 26 (72.2%) were male. Eighteen patients received RC48 alone, and 18 received RC48 combined with a programmed death-1 antibody. The median PFS was 5.4 months. The median OS was not reached. The 6-month and 1-year PFS rates were 38.8% and 15.5%, respectively. The 1-year OS rate was 79.6%. Fourteen (38.9%) patients achieved a partial response, and the ORR was 38.9%. Eleven patients had stable disease, and the DCR was 69.4%. The median PFS for patients who received RC48 combined with immunotherapy and those who received RC48 alone was 8.5 and 5.4 months, respectively. The main treatment-related adverse events included anemia, hypoesthesia, fatigue, and elevated transaminase. No treatment-related death occurred.RC48 alone or combined with immunotherapy might benefit patients with locally advanced or metastatic UC, regardless of impaired renal function.

Tissue engineered nerve grafts are considered as a promising alternative to autologous nerve grafts used for peripheral nerve repair. The differences between these two types of nerve grafts are mainly in the regenerative microenvironment established by them. To construct ideal tissue engineered nerve grafts, it is therefore required to develop a better way to introduce biochemical cues into a neural scaffold, as compared to single or combined use of support cells and growth factors. Here, we used a co-culture system of dorsal root ganglia and Schwann cells to create an in vitro formed nerve equivalent, which was introduced into a silk fibroin-based scaffold to furnish a tissue engineered nerve graft (TENG). At 4- and 12- weeks after the TENG was implanted to bridge a 10-mm-long sciatic nerve defect in rats, histological and functional assessments as well as Western blot analysis were performed to evaluate the influences of the TENG on peripheral nerve regeneration. We found that at an early stage of nerve regeneration, the TENG significantly accelerated axonal growth, and up-regulated expressions of N-cadherin and PMP22. Twelve weeks after nerve grafting, the TENG produced a further improved outcome of nerve regeneration and functional recovery, which was more close to that of the autologous nerve graft than that of the silk fibroin-based scaffold. The introduction of an in vitro cultured nerve equivalent into a scaffold might contribute to establishing a native-like microenvironment for nerve regeneration.

Thymic stromal lymphopoietin (TSLP), highly expressed in keratinocytes in atopic dermatitis patients and bronchial epithelial cells in asthma patients, plays a key role in allergic diseases. Two forms of TSLP mRNA (long and short) have been reported.We compared the expression of the long-form and total TSLP transcripts in primary human keratinocytes.Primary human keratinocytes were stimulated with Toll-like receptor (TLR) ligands, cytokines, and vitamin D receptor agonists. Gene expression was analyzed by quantitative real-time PCR. The amount of TSLP released was measured by ELISA.PolyI:C (TLR3 ligand), FSL-1 (TLR2-TLR6 ligand) and flagellin (TLR5 ligand) upregulated long-form TSLP expression, which predominantly contributed to upregulation of total TSLP expression. A glucocorticoid or an endosomal acidification inhibitor inhibited the polyI:C-dependent upregulation of total TSLP and the decrease of the total TSLP was due to the decrease of the long-form. An atopic cytokine milieu (TNF-α+IL-4+IL-13) or TNF-α alone also upregulated the long-form. These stimuli also induced the release of TSLP. In contrast, a high concentration of calcitriol (1,25-dihydroxyvitamin D(3), the active form of vitamin D(3)) or its analog MC903 upregulated total TSLP significantly but not the long-form, and did not induce the release of TSLP.TLR ligands or cytokines predominantly upregulate the gene expression of the long TSLP form, which contributes to the TSLP protein production, in primary human keratinocytes. Specific measurement of the long-form rather than total TSLP should be useful for accurate detection of functional human TSLP gene expression.

Tissue-engineered nerve grafts (TENGs) constitute a promising alternative to nerve autografts that are recognized as the gold standard for surgical repair of peripheral nerve gaps.To investigate the feasibility of using TENGs for bridging extra large peripheral nerve gaps in large animals.TENGs were constructed by incorporating autologous bone marrow mesenchymal stem cells (MSCs) into a neural scaffold that consisted of a chitosan conduit inserted with poly(lactic-co-glycolic acid) (PLGA) fibers. A 60-mm-long sciatic nerve gap in dogs was bridged by TENGs, chitosan/PLGA scaffolds, or nerve autografts. At 12 months postsurgery, behavioral analysis, electrophysiology, retrograde fluorogold tracing, and histological examination were performed.The outcomes of TENGs were similar to those of autografts and better than those of scaffolds alone.Introduction of autologous MSCs to a chitosan/PLGA scaffold improved the repair and rehabilitation of a large gap after peripheral nerve injury in dogs. Autologous MSCs may be a source of support cells for neural tissue engineering.

We aimed to investigate the efficacy and tolerability of sorafenib combined with cisplatin and 5-fluorouracil (5-FU) in patients with recurrent or metastatic nasopharyngeal carcinoma (NPC).It was a Simon two-stage designed trial. Chemotherapy-naive patients with recurrent or metastatic disease were enrolled. The regimen was sorafenib 400 mg orally b.i.d., cisplatin 80 mg/m(2) i.v. day 1, and 5-FU 1000 mg/m(2)/day CIV for 4 days, repeated every 21 days. After a maximum of six cycles of chemotherapy, patients received maintenance of sorafenib.In total, 54 patients were enrolled. The objective response rate reached 77.8%, including 1 complete response and 41 partial responses. The median progression-free survival was 7.2 months (95% CI 6.8-8.4 months), and the median overall survival was 11.8 months (95% CI 10.6-18.7 months). Major toxic effects included hand-foot skin reaction, myelosuppression, and gastrointestinal (GI) reaction. The incidence of hemorrhage was 22.2%, and one patient with liver metastases died of GI bleeding. Contrast-enhanced ultrasonography was carried out in a subset of patients with liver metastases.Combination of sorafenib, cisplatin (80 mg/m(2)) and 5-FU (3000 mg/m(2)) was tolerable and feasible in recurrent or metastatic NPC. Further randomized trials to compare sorafenib plus cisplatin and 5-FU with standard dose of cisplatin plus 5-FU in NPC are warranted.

Following peripheral nerve injury, transcriptional responses are orchestrated to regulate the expression of numerous genes in the lesioned nerve, thus activating the intrinsic regeneration program. To better understand the molecular regulation of peripheral nerve regeneration, we aimed at investigating the transcriptional landscape of dorsal root ganglia (DRGs) after sciatic nerve transection in rats. The cDNA microarray analysis was used to identify thousands of genes that were differentially expressed at different time points post nerve injury (PNI). The results from Euclidean distance matrix, principal component analysis, and hierarchical clustering indicated that 2 nodal transitions in temporal gene expressions could segregate 3 distinct transcriptional phases within the period of 14 d PNI. The 3 phases were designated as "a stress response phase", "a pre-regeneration phase", and "a regeneration phase", respectively, by referring to morphological observation of post-nerve-injury changes. The gene ontology (GO) analysis revealed the distinct features of biological process, cellular component, and molecular function at each transcriptional phase. Moreover, Ingenuity Pathway Analysis suggested that differentially expressed genes, mainly transcription factors and genes associated with neurite/axon growth, might be integrated into regulatory networks to mediate the regulation of peripheral nerve regeneration in a highly cooperative manner.

Programmed cell death-ligand 1 (PD-L1) and driver mutations are commonly seen in non-small-cell lung cancer (NSCLC). However, the prevelance of PD-L1 over-expression and its prognostic value in Epstein-Barr virus (EBV) associated pulmonary lymphoepithelioma-like carcinoma (LELC) remains poorly understood.A total of 214 NSCLC patients and 113 surgically treated pulmonary LELC patients were included. Paraffin-embedded tumor sections were stained with PD-L1 antibody. Correlations between PD-L1 expression and clinicopathological features as well as survival outcomes were analyzed.The frequency of PD-L1 over-expression in NSCLC was 51.4%. No significant association was observed between common driver mutations and PD-L1 over-expression. Remakably, the positive rate of PD-L1 in pulmonary LELC was 74.3%. High PD-L1 expression was associated with impaired disease-free survival (DFS) compared with low PD-L1 expression (p = 0.008). Multivariate analysis shows that PD-L1 expression level, N stage and M stage were independent prognostic factors for DFS. N stage and M stage but not PD-L1 expression level were significantly associated with overall survival (OS).PD-L1 over-expression was not related to common driver mutations in NSCLC. Pulmonary LELC have remarkably high incidence of PD-L1 expression. PD-L1 was a negative prognostic factor for DFS in surgically resected pulmonary LELC. These findings may provide a rationale for immunotarget therapy in this virus-associated lung cancer.

Silk fibroin (SF)-derived silkworms represent a type of highly biocompatible biomaterial for tissue engineering. We have previously investigated biocompatibility of SF with neural cells isolated from the central nervous system or peripheral nerve system in vitro, and also developed a SF-based nerve graft conduit or tissue-engineered nerve grafts by introducing bone marrow mesenchymal stem cells, as support cells, into SF-based scaffold and evaluated the outcomes of peripheral nerve repair in a rat model. As an extension of the previous study, the electrospun technique was performed here to fabricate SF-based neural scaffold inserted with silk fibres for bridging a 30-mm-long sciatic nerve gap in dogs. Assessments including functional, histological and morphometrical analyses were applied 12 months after surgery. All the results indicated that the SF-based neural scaffold group achieved satisfactory regenerative outcomes, which were close to those achieved by autologous nerve grafts as the golden-standard for peripheral nerve repair. Overall, our results raise a potential possibility for the translation of SF-based electrospun neural scaffolds as an alternative to nerve autografts into the clinic.

Lipoxygenases (LOXs) are important dioxygenases in cellular organisms. LOXs contribute to plant developmental processes and environmental responses. However, a systematic and comprehensive analysis has not been focused on the LOX gene family in poplar. Therefore, in the present study, we performed a comprehensive analysis of the LOX gene family in poplar.Using bioinformatics methods, we identified a total of 20 LOX genes. These LOX genes were clustered into two subfamilies. The gene structure and motif composition of each subfamily were relatively conserved. These genes are distributed unevenly across nine chromosomes. The PtLOX gene family appears to have expanded due to high tandem and low segmental duplication events. Microarray analysis showed that a number of PtLOX genes have different expression pattern across disparate tissues and under various stress treatments. Quantitative real-time PCR (qRT-PCR) analysis was further performed to confirm the responses to MeJA treatment of the 20 poplar LOX genes. The results show that the PtLOX genes are regulated by MeJA (Methyl jasmonate) treatment.This study provides a systematic analysis of LOX genes in poplar. The gene family analysis reported here will be useful for conducting future functional genomics studies to uncover the roles of LOX genes in poplar growth and development.

Biofuel-cropping systems, projected for large land areas, can potentially change their soil microbiome and the ecosystem services they catalyze. We determined the bacterial community composition and relevant soil properties for samples collected after 6 crop years at 0–10 cm, 10–25 cm, 25–50 cm, and 50–100 cm under corn, switchgrass, Miscanthus, and restored prairie, as well as 0–10 cm under six additional candidate biofuel crops in replicate side-by side plots. Deep sequencing of the 16S rRNA-V4 region established that soil bacterial communities were significantly differentiated by depth as determined by proportional OTU abundance and composition, UniFrac distance, and taxonomic and indicator analyses. The cropping system significantly impacted bacterial community composition within the top three layers, with corn and switchgrass communities the most different within the 0–25 cm and 25–50 cm depths, respectively. The effects of crop type and depth co-mingled, likely attributed to differences in rooting depth and biomass among crops. Individual phyla demonstrated varying patterns with depth, with significant proportional decreases of Proteobacteria, Actinobacteria, Planctomycetes, and Bacteroidetes but proportional increases of Firmicutes from shallow to deep soils. The Acidobacteria, Verrucomicrobia, and Chloroflexi peaked in abundance in the middle layers, whereas Thaumarchaeota decreased in abundance. Importantly, some classes within the Acidobacteria, Verrucomicrobia, and Firmicutes followed contrasting patterns with depth suggesting that they have different ecological specializations. Poplar, followed by soils with perennial crops contained the most C in the surface soils, with data indicating that these differences will become more pronounced with time.

Peripheral nervous system owns the ability of self-regeneration, mainly in its regenerative microenvironment including vascular network reconstruction. More recently, more attentions have been given to the close relationship between tissue regeneration and angiogenesis. To explore the overlap of molecular mechanisms and key regulation molecules between peripheral nerve regeneration and angiogenesis post peripheral nerve injury, integrative and bioinformatic analysis was carried out for microarray data of proximal stumps after sciatic nerve transection in SD rats. Nerve regeneration and angiogenesis were activated at 1 day immediately after sciatic nerve transection simultaneously. The more obvious changes of transcription regulators and canonical pathways suggested a phase transition between 1 and 4 days of both nerve regeneration and angiogenesis after sciatic nerve transection. Furthermore, 16 differentially expressed genes participated in significant biological processes of both nerve regeneration and angiogenesis, a few of which were validated by qPCR and immunofluorescent staining. It was demonstrated that STAT3, EPHB3, and Cdc42 co-expressed in Schwann cells and vascular endothelial cells to play a key role in regulation of nerve regeneration and angiogenesis simultaneously response to sciatic nerve transection. We provide a framework for understanding biological processes and precise molecular correlations between peripheral nerve regeneration and angiogenesis after peripheral nerve transection. Our work serves as an experimental basis and a valuable resource to further understand molecular mechanisms that define nerve injury-induced micro-environmental variation for achieving desired peripheral nerve regeneration.

Perennial grasses can assimilate nitrogen (N) fixed by non-nodulating bacteria living in the rhizosphere and the plant's own tissues, but many details of associative N fixation (ANF) remain unknown, including ANF's contribution to grass N nutrition, the exact location of fixation, and composition of the associated microbial community. We examined ANF in switchgrass (Panicum virgatum L.), a North American perennial grass, using 15N-enriched N2 isotopic tracer additions in a combination of in vitro, greenhouse, and field experiments to estimate how much N is assimilated, where fixation takes place, and the likely N-fixing taxa present. Using in vitro incubations, we documented fixation in root-free rhizosphere soil and on root surfaces, with average rates of 3.8 μg N g root−1 d−1 on roots and 0.81 μg N g soil−1 d−1 in soil. In greenhouse transplants, N fixation occurred only in the early growing season, but in the field, fixation was irregularly detectable throughout the 3-month growing season. Soil, leaves, stems, and roots all contained diazotrophs and incorporated fixed N2. Metagenomic analysis suggested that microbial communities were distinct among tissue types and influenced by N fertilizer application. A diverse array of microbes inhabiting the rhizosphere, and possibly aboveground tissues, appear to be episodically contributing fixed N to switchgrass.

Long-term vanilla monocropping often results in the occurrence of vanilla Fusarium wilt disease, seriously affecting its production all over the world. In the present study, vanilla exhibited significantly less Fusarium wilt disease in the soil of a long-term continuously cropped black pepper orchard. The entire fungal communities of bulk and rhizosphere soils between the black pepper-vanilla system (i.e., vanilla cropped in the soil of a continuously cropped black pepper orchard) and vanilla monoculture system were compared through the deep pyrosequencing. The results showed that the black pepper-vanilla system revealed a significantly higher fungal diversity than the vanilla monoculture system in both bulk and rhizosphere soils. The UniFrac-weighted PCoA analysis revealed significant differences in bulk soil fungal community structures between the two cropping systems, and fungal community structures were seriously affected by the vanilla root system. In summary, the black pepper-vanilla system harboured a lower abundance of F. oxysporum in the vanilla rhizosphere soil and increased the putatively plant-beneficial fungal groups such as Trichoderma and Penicillium genus, which could explain the healthy growth of vanilla in the soil of the long-term continuously cropped black pepper field. Thus, cropping vanilla in the soil of continuously cropped black pepper fields for maintaining the vanilla industry is executable and meaningful as an agro-ecological system.

Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice (Oryza sativa L. japonica) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr-facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome and might mark other cis-regulatory DNA elements that are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants. Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice (Oryza sativa L. japonica) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr-facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome and might mark other cis-regulatory DNA elements that are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants. Precursor proteins are typically inactive and could be converted into mature functional proteins through a series of posttranslational modifications (PTMs), which modulate diverse protein properties and functions (1Deribe Y.L. Pawson T. Dikic I. Post-translational modifications in signal integration.Nat. Struct. Mol. Biol. 2010; 17: 666-672Crossref PubMed Scopus (533) Google Scholar). PTMs have been associated with almost all known metabolic processes and cellular pathways in various ways (2Zhao Y. Jensen O.N. Modification-specific proteomics: Strategies for characterization of post-translational modifications using enrichment techniques.Proteomics. 2009; 9: 4632-4641Crossref PubMed Scopus (277) Google Scholar, 3Olsen J.V. Mann M. Status of large-scale analysis of post-translational modifications by mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 3444-3452Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). The major form of PTMs is covalent addition of functional chemical groups to one or more amino acids. PTMs can greatly increase the complexity of the proteome based on the presence of multiple modification sites within a protein, each with different types. Due to specific chemical reactivity, lysine (K) is one of the most common residues that is subject to PTMs (4Azevedo C. Saiardi A. Why always lysine? The ongoing tale of one of the most modified amino acids.Advances Biolog. Reg. 2016; 60: 144-150Crossref PubMed Scopus (52) Google Scholar), such as acetylation (Kac) (5Zhao S. Xu W. Jiang W. Yu W. Lin Y. Zhang T. Yao J. Zhou L. Zeng Y. Li H. Li Y. Shi J. An W. Hancock S.M. He F. Qin L. Chin J. Yang P. Chen X. Lei Q. Xiong Y. Guan K.L. Regulation of cellular metabolism by protein lysine acetylation.Science. 2010; 327: 1000-1004Crossref PubMed Scopus (1458) Google Scholar, 6Tropberger P. Pott S. Keller C. Kamieniarz-Gdula K. Caron M. Richter F. Li G.H. Mittler G. Liu E.T. Bühler M. Margueron R. Schneider R. Regulation of transcription through acetylation of H3K122 on the lateral surface of the histone octamer.Cell. 2013; 152: 859-872Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), methylation (Kme) (7Cao R. Wang L. Wang H. Xia L. Erdjument-Bromage H. Tempst P. Jones R.S. Zhang Y. Role of histone H3 lysine 27 methylation in polycomb-group silencing.Science. 2002; 298: 1039-1043Crossref PubMed Scopus (2831) Google Scholar, 8Peach S.E. Rudomin E.L. Udeshi N.D. Carr S.A. Jaffe J.D. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.Mol. Cell. Proteomics. 2012; 11: 128-137Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), malonylation (Kma) (9Peng C. Lu Z.K. Xie Z.Y. Cheng Z.Y. Chen Y. Tan M.J. Luo H. Zhang Y. He W. Yang K. Zwaans B.M.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J.B. Verdin E. Ye Y. Zhao Y.M. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10M111.012658Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 10Xie Z. Dai J. Dai L. Tan M. Cheng Z. Wu Y. Boeke J.D. Zhao Y. Lysine succinylation and lysine malonylation in histones.Mol. Cell Proteomics. 2012; 11: 100-107Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), propionylation (Kpro) (11Cheng Z. Tang Y. Chen Y. Kim S. Liu H. Li S. Gu W. Zhao Y. Molecular characterization of propionyllysines in non-histone proteins.Mol. Cell. Proteomics. 2009; 8: 45-52Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 12Zhang K. Chen Y. Zhang Z. Zhao Y. Identification and verification of lysine propionylation and butyrylation in yeast core histones using PTMap software.J. Proteome Res. 2009; 8: 900-906Crossref PubMed Scopus (113) Google Scholar), butyrylation (Kbu) (12Zhang K. Chen Y. Zhang Z. Zhao Y. Identification and verification of lysine propionylation and butyrylation in yeast core histones using PTMap software.J. Proteome Res. 2009; 8: 900-906Crossref PubMed Scopus (113) Google Scholar, 13Chen Y. Sprung R. Tang Y. Ball H. Sangras B. Kim S.C. Falck J.R. Peng J. Gu W. Zhao Y. Lysine propionylation and butyrylation are novel post-translational modifications in histones.Mol. Cell. Proteomics. 2007; 6: 812-819Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar), and succinylation (Ksucc) (10Xie Z. Dai J. Dai L. Tan M. Cheng Z. Wu Y. Boeke J.D. Zhao Y. Lysine succinylation and lysine malonylation in histones.Mol. Cell Proteomics. 2012; 11: 100-107Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar). With the development of high-specific antibodies and high-resolution MS techniques, increasing numbers of lysine modifications of both histone and non-histone proteins have been identified in the proteome. These modifications include Kac (15Kim S.C. Sprung R. Chen Y. Xu Y. Ball H. Pei J. Cheng T. Kho Y. Xiao H. Xiao L. Grishin N.V. White M. Yang X.J. Zhao Y. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey.Mol. Cell. 2006; 23: 607-618Abstract Full Text Full Text PDF PubMed Scopus (1217) Google Scholar, 16Jeffers V. Sullivan Jr., W.J. Lysine acetylation is widespread on proteins of diverse function and localization in the protozoan parasite Toxoplasma gondii.Eukaryot. Cell. 2012; 11: 735-742Crossref PubMed Scopus (88) Google Scholar, 17Nallamilli B.R. Edelmann M.J. Zhong X. Tan F. Mujahid H. Zhang J. Nanduri B. Peng Z. Global analysis of lysine acetylation suggests the involvement of protein acetylation in diverse biological processes in rice (Oryza sativa).PLOS One. 2014; 9: e89283Crossref PubMed Scopus (86) Google Scholar, 18Fang X. Chen W. Zhao Y. Ruan S. Zhang H. Yan C. Jin L. Cao L. Zhu J. Ma H. Cheng Z. Global analysis of lysine acetylation in strawberry leaves.Front. Plant Sci. 2015; 6: 739Crossref PubMed Scopus (54) Google Scholar), Ksucc (14Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (569) Google Scholar, 19Li X. Hu X. Wan Y. Xie G. Li X. Chen D. Cheng Z. Yi X. Liang S. Tan F. Systematic identification of the lysine succinylation in the protozoan parasite Toxoplasma gondii.J. Proteome Res. 2014; 13: 6087-6095Crossref PubMed Scopus (82) Google Scholar), Kme (20Cao X.J. Garcia B.A. Global proteomics analysis of protein lysine methylation.Curr. Protoc. Protein Sci. 2016; 86: 24.8.1-24.8.19Crossref Scopus (19) Google Scholar), and Kma (9Peng C. Lu Z.K. Xie Z.Y. Cheng Z.Y. Chen Y. Tan M.J. Luo H. Zhang Y. He W. Yang K. Zwaans B.M.M. Tishkoff D. Ho L. Lombard D. He T.C. Dai J.B. Verdin E. Ye Y. Zhao Y.M. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell. Proteomics. 2011; 10M111.012658Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Among the lysine modifications of the proteome, histone lysine modifications are essential for the control of gene expression by complex interactions of transcription factors binding to regulatory DNA elements, including promoters, enhancers, insulators, and silencers (21Perillo B. Ombra M.N. Bertoni A. Cuozzo C. Sacchetti S. Sasso A. Chiariotti L. Malorni A. Abbondanza C. Avvedimento E.V. DNA oxidation as triggered by H3K9me2 demethylation drives estrogen-induced gene expression.Science. 2008; 319: 202-206Crossref PubMed Scopus (414) Google Scholar, 22Bannister A.J. Kouzarides T. Regulation of chromatin by histone modifications.Cell Res. 2011; 21: 381-395Crossref PubMed Scopus (3415) Google Scholar). Specific histone modifications of the chromatin activate or repress regulatory DNA elements, thus playing an important role in transcriptional regulation (23Sabari B.R. Tang Z. Huang H. Yong-Gonzalez V. Molina H. Kong H.E. Dai L. Shimada M. Cross J.R. Zhao Y. Roeder R.G. Allis C.D. Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation.Mol. Cell. 2015; 58: 203-215Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). For example, H3K9ac, H3K4me3, and H3K4me2 mark active promoters, whereas repressed genes are marked by H3K27me3 or H3K9me2, and enhancers are commonly marked by H3K27ac and H3K4me1/2 (24Heintzman N.D. Stuart R.K. Hon G. Fu Y. Ching C.W. Hawkins R.D. Barrera L.O. Van Calcar S. Qu C. Ching K.A. Wang W. Weng Z. Green R.D. Crawford G.E. Ren B. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome.Nat. Genet. 2007; 39: 311-318Crossref PubMed Scopus (2409) Google Scholar, 25Zhou V.W. Goren A. Bernstein B.E. Charting histone modifications and the functional organization of mammalian genomes.Nat. Rev. Genet. 2011; 12: 7-18Crossref PubMed Scopus (840) Google Scholar). Lysine crotonylation (Kcr) 1The abbreviations used are:Kcrlysine crotonylationPTMposttranslational modificationnano-LC-MS/MSnanoscale liquid chromatography coupled to tandem mass spectrometryChIPchromatin immunoprecipitationDHSsDNase hypersensitive sitesIPA1ideal plant architecture1Kaclysine acetylationKmelysine methylationKmalysine malonylationKprolysine propionylationKbulysine butyrylationKsucclysine succinylationWBWestern blottingGOgene ontologyMACSmodel-based analysis of ChIP-SeqKEGGKyoto Encyclopedia of Genes and GenomesPPIsprotein-protein interactionqPCRquantitative polymerase chain reaction. 1The abbreviations used are:Kcrlysine crotonylationPTMposttranslational modificationnano-LC-MS/MSnanoscale liquid chromatography coupled to tandem mass spectrometryChIPchromatin immunoprecipitationDHSsDNase hypersensitive sitesIPA1ideal plant architecture1Kaclysine acetylationKmelysine methylationKmalysine malonylationKprolysine propionylationKbulysine butyrylationKsucclysine succinylationWBWestern blottingGOgene ontologyMACSmodel-based analysis of ChIP-SeqKEGGKyoto Encyclopedia of Genes and GenomesPPIsprotein-protein interactionqPCRquantitative polymerase chain reaction. is a newly discovered PTM that exists in mammals (26Tan M. Luo H. Lee S. Jin F. Yang J.S. Montellier E. Buchou T. Cheng Z. Rousseaux S. Rajagopal N. Lu Z. Ye Z. Khochbin S. Ren B. Zhao Y. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.Cell. 2011; 146: 1016-1028Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar, 27Bao X.C. Wang Y. Li X. Li X.M. Liu Z. Yang T.P. Wong C.F. Zhang J.W. Hao Q. Li X.D. Identification of 'erasers' for lysine crotonylated histone marks using a chemical proteomics approach.Elife. 2014; 3: e02999Crossref Scopus (197) Google Scholar). This histone modification has been identified in evolutionary distant eukaryotic organisms, such as yeast (Saccharomyces. cerevisiae) and invertebrate species including Caenorhabditis. elegans and Drosophila, as well as in mice and humans, suggesting that this modification is widely conserved (26Tan M. Luo H. Lee S. Jin F. Yang J.S. Montellier E. Buchou T. Cheng Z. Rousseaux S. Rajagopal N. Lu Z. Ye Z. Khochbin S. Ren B. Zhao Y. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.Cell. 2011; 146: 1016-1028Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). Crotonylation is a histone modification, involving a four-carbon length in the planar orientation (28Sun H.J. Liu X.W. Li F.F. Li W. Zhang J. Xiao Z.X. Shen L.L. Li Y. Wang F.L. Yang J.G. First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum.Sci. Rep. 2017; 7Google Scholar). This modification neutralizes the positive charge of the ε-amino group of lysine, leading to the possibility of charge-based cis-effects on the chromatin fiber, where the increased bulk and rigidity of the crotonyl group may result in an enhanced effect (23Sabari B.R. Tang Z. Huang H. Yong-Gonzalez V. Molina H. Kong H.E. Dai L. Shimada M. Cross J.R. Zhao Y. Roeder R.G. Allis C.D. Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation.Mol. Cell. 2015; 58: 203-215Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Therefore, histone Kcr is generally enriched in the regions of active promoters and potential enhancers in mammalian cells (26Tan M. Luo H. Lee S. Jin F. Yang J.S. Montellier E. Buchou T. Cheng Z. Rousseaux S. Rajagopal N. Lu Z. Ye Z. Khochbin S. Ren B. Zhao Y. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.Cell. 2011; 146: 1016-1028Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). In addition, Kcr has been shown to clearly mark autosomal testis-specific genes, which are activated in postmeiotic round spermatids (29Montellier E. Rousseaux S. Zhao Y. Khochbin S. Histone crotonylation specifically marks the haploid male germ cell gene expression program.Bioessays. 2012; 34: 187-193Crossref PubMed Scopus (84) Google Scholar). Recently, the global profiling of crotonylation on non-histone proteins has been reported in mammals and tobacco (28Sun H.J. Liu X.W. Li F.F. Li W. Zhang J. Xiao Z.X. Shen L.L. Li Y. Wang F.L. Yang J.G. First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum.Sci. Rep. 2017; 7Google Scholar, 30Wei W. Mao A. Tang B. Zeng Q. Gao S. Liu X. Lu L. Li W. Du J.X. Li J. Wong J. Liao L. Large-scale identification of protein crotonylation reveals its role in multiple cellular functions.J. Proteome Res. 2017; 16: 1743-1752Crossref PubMed Scopus (77) Google Scholar, 31Xu W. Wan J. Zhan J. Li X. He H. Shi Z. Zhang H. Global profiling of crotonylation on non-histone proteins.Cell Res. 2017; 27: 946-949Crossref PubMed Scopus (78) Google Scholar). Crotonylation of non-histone proteins is involved in different signaling pathways and cellular functions. To our knowledge, crotonylation of non-histone and histone proteins has rarely been reported in monocots. lysine crotonylation posttranslational modification nanoscale liquid chromatography coupled to tandem mass spectrometry chromatin immunoprecipitation DNase hypersensitive sites ideal plant architecture1 lysine acetylation lysine methylation lysine malonylation lysine propionylation lysine butyrylation lysine succinylation Western blotting gene ontology model-based analysis of ChIP-Seq Kyoto Encyclopedia of Genes and Genomes protein-protein interaction quantitative polymerase chain reaction. lysine crotonylation posttranslational modification nanoscale liquid chromatography coupled to tandem mass spectrometry chromatin immunoprecipitation DNase hypersensitive sites ideal plant architecture1 lysine acetylation lysine methylation lysine malonylation lysine propionylation lysine butyrylation lysine succinylation Western blotting gene ontology model-based analysis of ChIP-Seq Kyoto Encyclopedia of Genes and Genomes protein-protein interaction quantitative polymerase chain reaction. Rice (Oryza sativa) is one of the most important cereal crops in the world and represents a valuable model plant for the investigation of monocots in functional genome research (32Shi J. Dong A. Shen W.H. Epigenetic regulation of rice flowering and reproduction.Front. Plant Sci. 2014; 5: 803PubMed Google Scholar). Since crotonylated proteins have not yet been identified in rice, we initiated a systematic study to identify and investigate functional roles of the crotonylated proteins in Nipponbare, which is the first rice variety with a complete genomic sequence published (33Goff S.A. Ricke D. Lan T.H. Presting G. Wang R. Dunn M. Glazebrook J. Sessions A. Oeller P. Varma H. Hadley D. Hutchinson D. Martin C. Katagiri F. Lange B.M. Moughamer T. Xia Y. Budworth P. Zhong J. Miguel T. Paszkowski U. Zhang S. Colbert M. Sun W.L. Chen L. Cooper B. Park S. Wood T.C. Mao L. Quail P. Wing R. Dean R. Yu Y. Zharkikh A. Shen R. Sahasrabudhe S. Thomas A. Cannings R. Gutin A. Pruss D. Reid J. Tavtigian S. Mitchell J. Eldredge G. Scholl T. Miller R.M. Bhatnagar S. Adey N. Rubano T. Tusneem N. Robinson R. Feldhaus J. Macalma T. Oliphant A. Briggs S. A draft sequence of the rice genome (Oryza sativa L. ssp japonica).Science. 2002; 296: 92-100Crossref PubMed Scopus (2663) Google Scholar). In this study, we obtained the crotonylome of Nipponbare using high accuracy LC-MS/MS in combination with the enrichment of crotonylated peptides from digested cell lysates and subsequent peptide identification. In total, 1,265 crotonylated sites were identified in 690 proteins in Nipponbare. In addition, we conducted a genome-wide study of histone Kcr by ChIP-seq analysis with the pan anti-Kcr and H3K14cr antibodies. This information will broaden our understanding of the biological functions influenced by histone Kcr. In short, our findings provide significant insights into the range of functions regulated by lysine crotonylation in rice. O. sativa variety “Nipponbare” seeds were germinated at ambient temperature for 72 h. The germinated seeds were then sown and grown in water under greenhouse conditions (12 h light at 28 °C/12 h dark at 25 °C) with 70% humidity. Leaves of the two-week-old rice seedling were sampled for protein and ChIP-DNA isolation. Green seedlings and albino seedlings derived from another culture of Nipponbare were grown in Murashige and Skoog plates containing 0.5% naphthylacetic acid, 3% sucrose, and 0.5% agar. Protein extraction, trypsin digestion and HPLC fractionation were conducted using a procedure described by Xue et al. (34Xue C. Liu S. Chen C. Zhu J. Yang X.B. Zhou Y. Guo R. Liu X.Y. Gong Z.Y. Global proteome analysis links lysine acetylation to diverse functions in Oryza sativa.Proteomics. 2018; 18: 1700036Crossref Scopus (29) Google Scholar). Leaf samples were ground under liquid nitrogen and sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (8 m urea, 1% Triton-100, 65 mm DTT, and 0.1% Protease Inhibitor Mixture). The remaining debris was removed by centrifugation (20,000 g, 4 °C, 10 min). Finally, the protein was precipitated with cold 15% TCA for 2 h at −20 °C. After centrifugation (12,000 g, 4 °C, 10 min), the supernatant was discarded. The remaining precipitate was washed three times with cold acetone. The protein was redissolved in buffer (8 m urea, 100 mm NH4CO3, pH 8.0). For digestion, the protein solution was reduced with 10 mm DTT for 1 h at 37 °C and alkylated with 20 mm iodoacetamide for 45 min at room temperature in darkness. For trypsin digestion, the protein samples were diluted by adding 100 mm NH4CO3 to reduce the urea concentration to below 2 m. The protein concentration was determined with BCA kit (P0011-1, Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instructions. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for an overnight digestion, followed by an addition of 1:100 trypsin-to-protein mass ratio for another 4-h digestion. The samples were then separated into 80 fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6 mm inner diameter, 250 mm length). Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mm ammonium bicarbonate with a pH of 10 over 80 min. The peptides were then combined into eight fractions and dried by vacuum centrifugation. To enrich Kcr peptides, tryptic peptides dissolved in NETN buffer (100 mm NaCl, 1 mm EDTA, 50 mm Tris-HCl, 0.5% NP-40, pH 8.0) were incubated with prewashed antibody beads (PTM BioLabs, HangZhou, China PTM-501) at 4 °C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with ddH2O. The bound peptides were eluted from the beads with 0.1% TFA. The eluted fractions were combined and vacuum-dried. The resulting peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturer's instructions prior to LC-MS/MS analysis. Enrichment of lysine peptides were analyzed using LC-MS/MS as described by Xue et al. (34Xue C. Liu S. Chen C. Zhu J. Yang X.B. Zhou Y. Guo R. Liu X.Y. Gong Z.Y. Global proteome analysis links lysine acetylation to diverse functions in Oryza sativa.Proteomics. 2018; 18: 1700036Crossref Scopus (29) Google Scholar). Briefly, peptides were dissolved in 0.1% formic acid and directly loaded onto a reversed-phase precolumn (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient was as follows: 6% to 22% solvent B (0.1% formic acid in 98% acetonitrile) for 24 min, 22% to 35% for 8 min then increased to 80% over 5 min and held at 80% for 3 min. The gradient was generated at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by Q ExactiveTM hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). The peptides were subjected to a nano electrospray ionization source followed by tandem mass spectrometry (MS/MS) using Q ExactiveTM (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using a normalized collision energy setting of 28; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 2e4 in the MS survey scan with 10.0-s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control was used to prevent overfilling of the ion trap; 5e4 ions were accumulated for generation of MS/MS spectra. The MS scans were performed over the range of 350 to 1,800 m/z. The resulting tandem MS data were processed using the MaxQuant with integrated Andromeda search engine (v.1.4.1.2) described by Zhou et al. (35Zhou S. Yang Q. Yin C. Liu L. Liang W. Systematic analysis of the lysine acetylome in Fusarium graminearum.BMC Genomics. 2016; 17: 1019Crossref PubMed Scopus (35) Google Scholar) according to a method described by He et al. (36He D. Wang Q. Li M. Damaris R.N. Yi X. Cheng Z. Yang P. Global proteome analyses of lysine acetylation and succinylation reveal the widespread involvement of both modification in metabolism in the embryo of germinating rice seed.J. Proteome Res. 2016; 15: 879-890Crossref PubMed Scopus (99) Google Scholar). Tandem mass spectra were searched against the UniProt_Oryza sativa database (UniProt Oryza sativa subsp. japonica) concatenated with the reverse decoy database. The search database consisted of the UniProt Oryza sativa subsp. japonica proteome set (including 63,195 protein sequences) downloaded from UniProtKB (http://www.uniprot.org) in July 2014. Trypsin/P was specified as the cleavage enzyme with allowances set for up to four missing cleavages, five modifications per peptide and five charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation of Cys was specified as a fixed modification. Oxidation of Met, crotonylation of lysine, and acetylation on protein N-terminal were specified as variable modifications. False discovery rate thresholds for proteins, peptides, and modification sites were set at 1%. Minimum peptide length was set at 7. All the other MaxQuant parameters were set to default values and the site localization probability was set at >0.75. The phylogenetic tree was constructed using the neighbor-joining method with MEGA Version 6.0 (37Tamura K. Stecher G. Peterson D. Filipski A. Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0.Mol. Biol. Evol. 2013; 30: 2725-2729Crossref PubMed Scopus (33504) Google Scholar) (gaps/missing data treatment: pairwise deletion, bootstrap: 1,000). Human and mouse p300 genes were used as an outgroup. All p300 protein sequences were aligned using T-coffee (38Keller O. Kollmar M. Stanke M. Waack S. A novel hybrid gene prediction method employing protein multiple sequence alignments.Bioinformatics. 2011; 27: 757-763Crossref PubMed Scopus (297) Google Scholar) with default options. The analysis of sequences around the Kcr site was performed based on Shen et al. (39Shen Z. Wang B. Luo J. Jiang K. Zhang H. Mustonen H. Puolakkainen P. Zhu J. Ye Y. Wang S. Global-scale profiling of differential expressed lysine acetylated proteins in colorectal cancer tumors and paired liver metastases.J. Proteomics. 2016; 142: 24-32Crossref PubMed Scopus (21) Google Scholar). For all proteins, Motif-X was used to analyze the model of sequences constituted by amino acids in specific positions of modifier 21-mers (10 amino acids upstream and downstream of the site). And all the database protein sequences were used as background database parameters, while other parameters were set to the default. All the crotonylation substrate categories obtained after enrichment were collated along with their p values and then filtered for those categories that were enriched in at least one of the clusters with p value <0.05. This filtered p value matrix was transformed by the x = −log10 (p value). The results were visualized in a heat map generated using “heatmap.2.” Gene ontology (GO) annotation of the proteome was achieved with reference to the UniProt-gene ontology annotation (GOA) database (www.http://www.ebi.ac.uk/GOA/) using a procedure described by Xue et al. (34Xue C. Liu S. Chen C. Zhu J. Yang X.B. Zhou Y. Guo R. Liu X.Y. Gong Z.Y. Global proteome analysis links lysine acetylation to diverse functions in Oryza sativa.Proteomics. 2018; 18: 1700036Crossref Scopus (29) Google Scholar). Proteins were classified by GO annotation based on three categories: biological process, cellular component, and molecular function. The Encyclopedia of Genes and Genomes (KEGG) database was used to identify enriched pathways using the Functional Annotation Tool of database for annotation, visualization and integrated discovery (DAVID) against the background of Nipponbare, following a detailed procedure described by Xue et al. (34Xue C. Liu S. Chen C. Zhu J. Yang X.B. Zhou Y. Guo R. Liu X.Y. Gong Z.Y. Global proteome analysis links lysine acetylation to diverse functions in Oryza sativa.Proteomics. 2018; 18: 1700036Crossref Scopus (29) Google Scholar). GO annotation and enriched pathways with a corrected p value <0.05 w

Summary Chemoautotrophic ammonia‐oxidizers and nitrite‐oxidizers are responsible for a significant amount of soil nitrate production. The identity and composition of these active nitrifiers in soils under different long‐term fertilization regimes remain largely under‐investigated. Based on that soil nitrification potential significantly decreased in soils with chemical fertilization (CF) and increased in soils with organic fertilization (OF), a microcosm experiment with DNA stable isotope probing was further conducted to clarify the active nitrifiers. Both ammonia‐oxidizing archaea (AOA) and bacteria (AOB) were found to actively respond to urea addition in soils with OF and no fertilizer (CK), whereas only AOB were detected in soils with CF. Around 98% of active AOB were Nitrosospira cluster 3a.1 in all tested soils, and more than 90% of active AOA were Nitrososphaera subcluster 1.1 in unfertilized and organically fertilized soils. Nitrite oxidation was performed only by Nitrospira ‐like bacteria in all soils. The relative abundances of Nitrospira lineage I and VI were 32% and 61%, respectively, in unfertilized soils, and that of Nitrospira lineage II was 97% in fertilized soils, indicating long‐term fertilization shifted the composition of active Nitrospira ‐like bacteria in response to urea. This finding indicates that different fertilizer regimes impact the composition of active nitrifiers, thus, impacting soil nitrification potential.

Tissue-engineered nerve grafts (TENGs) are the most promising way for repairing long-distance peripheral nerve defects. Chitosan and poly (lactic-co-glycolic acid) (PLGA) scaffolds are considered as the promising materials in the pharmaceutical and biomedical fields especially in the field of tissue engineering. To further clarify the effects of a chitosan conduit inserted with various quantity of poly (lactic-co-glycolic acid) (PLGA) scaffolds, and their degrades on the peripheral nerve regeneration, the chitosan nerve conduit inserted with different amounts of PLGA scaffolds were used to repair rat sciatic nerve defects. The peripheral nerve regeneration at the different time points was dynamically and comprehensively evaluated. Moreover, the influence of different amounts of PLGA scaffolds on the regeneration microenvironment including inflammatory response and cell state were also revealed. The modest abundance of PLGA is more instrumental to the success of nerve regeneration, which is demonstrated in terms of the structure of the regenerated nerve, reinnervation of the target muscle, nerve impulse conduction, and overall function. The PLGA scaffolds aid the migration and maturation of Schwann cells. Furthermore, the PLGA and chitosan degradation products in a correct ratio neutralize, reducing the inflammatory response and enhancing the regeneration microenvironment. The balanced microenvironment regulated by the degradants of appropriate PLGA scaffolds and chitosan conduit promotes peripheral nerve regeneration. The findings represent a further step towards programming TENGs construction, applying polyester materials in regenerative medicine, and understanding the neural regeneration microenvironment.

As a widely used fungicide, the environmental fate of carbendazim and its residues in agricultural products have caused great concern. However, its effects on soil microbial communities are largely unknown. Herein, we used high-throughput sequencing to reveal the effects of high and low dose of carbendazim and its degrading strain, Rhodococcus qingshengii strain djl-6, on the composition, diversity, and interrelationship of soil bacterial and fungal communities in short- and medium-term under laboratory conditions. The results showed that carbendazim exhibited an increased negative impact on bacterial communities and reduced the proportion of dominant fungal phylum Ascomycota during a 14-day incubation period. Only the impacts of low-dose carbendazim (2 mg·kg−1 dry soil) on fungal community were weakened. Network analysis showed that carbendazim increased the connectivity and modularity of microbial co-occurrence networks. Strain djl-6 exhibited good potential for bioremediation of carbendazim-contaminated soils. Moreover, it driven the assembly of potential carbendazim-degrading consortia from indigenous microbial communities; and members of the genera Arthrobacter, Bacillus, Brevundimonas, Lysinibacillus, Massilia, Mycobacterium, Paenibacillus, and Pseudarthrobacter might be participated in the degradation of carbendazim. Taken together, our study provides a relatively comprehensive understanding of the effects of carbendazim and its degrading strain djl-6 on soil microbial communities.

Tumor-associated macrophages/microglia (TAMs) are prominent microenvironment components in human glioblastoma (GBM) that are potential targets for anti-tumor therapy. However, TAM depletion by CSF1R inhibition showed mixed results in clinical trials. We hypothesized that GBM subtype-specific tumor microenvironment (TME) conveys distinct sensitivities to TAM targeting.We generated syngeneic PDGFB- and RAS-driven GBM models that resemble proneural-like and mesenchymal-like gliomas, and determined the effect of TAM targeting by CSF1R inhibitor PLX3397 on glioma growth. We also investigated the co-targeting of TAMs and angiogenesis on PLX3397-resistant RAS-driven GBM. Using single-cell transcriptomic profiling, we further explored differences in TME cellular compositions and functions in PDGFB- and RAS-driven gliomas.We found that growth of PDGFB-driven tumors was markedly inhibited by PLX3397. In contrast, depletion of TAMs at the early phase accelerated RAS-driven tumor growth and had no effects on other proneural and mesenchymal GBM models. In addition, PLX3397-resistant RAS-driven tumors did not respond to PI3K signaling inhibition. Single-cell transcriptomic profiling revealed that PDGFB-driven gliomas induced expansion and activation of pro-tumor microglia, whereas TAMs in mesenchymal RAS-driven GBM were enriched in pro-inflammatory and angiogenic signaling. Co-targeting of TAMs and angiogenesis decreased cell proliferation and changed the morphology of RAS-driven gliomas.Our work identifies functionally distinct TAM subpopulations in the growth of different glioma subtypes. Notably, we uncover a potential responsiveness of resistant mesenchymal-like gliomas to combined anti-angiogenic therapy and CSF1R inhibition. These data highlight the importance of characterization of the microenvironment landscape in order to optimally stratify patients for TAM-targeted therapy.

Abstract Purpose: There is no research evidence demonstrate which is the better partner strategy, endocrine therapy or chemotherapy, to combine with anti-HER2 therapy as the first-line management of hormone receptor (HR)-positive (HR+) and HER2-positive (HER2+) metastatic breast cancer (MBC). We wished to ascertain if trastuzumab plus endocrine therapy is noninferior to trastuzumab plus chemotherapy. Patients and Methods: We conducted an open-label, noninferiority, phase III, randomized, controlled trial (NCT01950182) at nine hospitals in China. Participants, stratified by previous adjuvant endocrine therapy and disease status (recurrent disease vs. de novo metastasis), were assigned randomly (1:1) to receive trastuzumab plus endocrine therapy (per investigator's choice of oestrogen-receptor modulators or aromatase inhibitor, with/without concurrent ovarian suppression) or chemotherapy (per investigator's choice of taxanes, capecitabine, or vinorelbine). The primary endpoint was progression-free survival (PFS) with a noninferiority upper margin of 1.35 for the HR. The intention-to-treat population was used in primary and safety analyses. Results: A total of 392 patients were enrolled and assigned randomly to receive trastuzumab plus endocrine therapy (ET group, n = 196) or trastuzumab plus chemotherapy (CT group, n = 196). After a median follow-up of 30.2 months [interquartile range (IQR) 15.0–44.7], the median PFS was 19.2 months [95% confidence interval (CI), 16.7–21.7)] in the ET group and 14.8 months (12.8–16.8) in the CT group (hazard ratio, 0.88; 95% CI, 0.71–1.09; Pnoninferiority &amp;lt; 0.0001). A significantly higher prevalence of toxicity was observed in the CT group compared with the ET group. Conclusions: Trastuzumab plus endocrine therapy was noninferior to trastuzumab plus chemotherapy in patients with HR+HER2+ MBC.

Although the enrichment of antibiotic resistance genes (ARGs) in diverse organic soils have been explored, understanding of the ecological processes governing the composition of ARGs in long-term organically fertilized soils still remains limited across typical agricultural regions. Thus, the distribution and assembly of ARG profile in three typical agricultural soils (black soil, fluvo-aquic soil, and red soil) under long-term contrasting fertilization regimes (chemical-only vs organic-only) were investigated using high-throughput qPCR (HT-qPCR). The application of organic manure significantly increased the abundance and number of ARGs across soils, as compared to those with chemical fertilizer. Organic manure application enriched the abundance of mobile genetic elements (MGEs), which were positively associated with ARGs. In addition, it is long-term organic fertilizer that enriched the number and abundance of opportunist and specialist ARGs in the fluvo-aquic and red soils, but not black soils. The number and abundance of most generalist ARGs did not change significantly among different fertilization or soil types. The assembly process of the ARG profiles tends to be more deterministic in organically fertilized soils than in chemically fertilized soils. These results suggest that long-term organic fertilizer application may contribute to the persistence and health risk of the soil antibiotic resistomes (especially specialist ARGs).

In this paper, the composite of Ti3C2Tx and poly (3,4-propylenedioxythiophene) (PProDOT) is prepared via a simple in-situ chemical oxidative polymerization. The optimum ratio of Ti3C2Tx@PProDOT composite with an outstanding capacitance of 462 C g−1 (462 F g−1) at 1 A g−1 is obtained by adjusting the mass ratio of Ti3C2Tx and ProDOT. The asymmetric flexible supercapacitor with Ti3C2Tx@PProDOT (1:10)//AC exhibits the gravimetric capacity of 238 C g−1 at 1 A g−1, and the retention of initial capacity remains at 79.6% after 10,000 cycles. Meanwhile, the asymmetrical supercapacitor displays an energy density of 48.6 Wh kg−1 at a power density of 729.6 W kg−1. The improvement of these properties stems from that the PProDOT effectively inhibits the stacking of Ti3C2Tx sheets, which is advantageous to shortening the transport path of ions and promoting electron transport as well as the rapid redox reactions. Therefore, these characteristics enhance the electrochemical performance of composites.

Abstract Background Cotton Verticillium wilt, causing by Verticillium dahliae , has seriously affected the yield and quality of cotton. The incidence of Verticillium wilt in cotton fields has been on the rise for many years, especially after straw has been returned to the fields. Intercropping can reduce the incidence of soil borne diseases and is often used to control crop diseases, but the relationship between the effects of intercropping on microbial communities and the occurrence of plant diseases is unclear. This research explored the relationship between soil microbial community structure and Cotton Verticillium wilt in interplanting of cotton-onion, cotton-garlic, cotton-wheat and cotton monocultures. Amplicon sequencing applied to the profile of bacterial and fungal communities. Results The results showed that the disease index of Cotton Verticillium wilt was significantly reduced after intercropping with cotton-garlic and cotton-onion. Chao1 and Sobs indices were not significantly different in the rhizosphere soil and pre-plant soils of the four planting patterns, but the pre-plant fungal shannon index was significantly lower in the cotton-onion intercropping plot than in the other three plots. PCoA analysis showed that the soil microbial communities changed to a certain extent after intercropping, with large differences in the microbial communities under different cropping patterns. The abundance of Chaetomium was highest in the cotton-garlic intercropping before planting; the abundance of Penicillium was significantly higher in the cotton-wheat intercropping than in the other three systems. Conclusion Cotton-garlic and cotton-onion interplanting can control Cotton Verticillium wilt by affecting the soil microbial community. Fungi of the genera Chaetomium and Penicillium may be associated with plant disease resistance.

Due to its multi-principal component characteristics, high-entropy alloys exhibit excellent comprehensive mechanical properties and become a new generation of engineering structure alternative materials. At present, a single constitutive model is used to describe the change of flow stress in the study of hot deformation behavior of high-entropy alloys, and there are some problems such as inaccurate prediction accuracy or inapplicability of the model in some hot processing intervals. In this paper, three different constitutive models are combined to fully consider the applicability of the model in different temperature ranges, and the change of flow stress behavior is accurately described, which is of great significance for subsequent deformation mechanism research, hot processing process optimization, numerical simulation and so on. Thermal compression experiments on cast Ni28.5Co18.6Fe18.2Al16.3Cr10.5Mo4 Ti2.3Nb0.6W0.9C0.2 high-entropy alloy were carried out using a Gleeble-3800 thermal simulation tester with deformation temperatures in the range from 900 ℃ to 1150 ℃ and strain rates from 0.001 s−1 to 1 s−1. The constitutive relationships between true stress and strain, temperature and strain rate were established, while the heat deformation process of this alloy under different deformation conditions by Arrhennius, Modified Johnson-Cook and Modified Fields-Backofen models were characterized. The results indicate that Arrenius model describe the rheological behavior of the alloy better in a wider temperature range and at a wider strain rate range. Comparatively, the model shows the highest prediction accuracy in the high-temperature region and slightly lower accuracy in the mesothermal and low-temperature regions. The modified J-C and F-B models have better prediction accuracy in the low-temperature and medium-temperature regions, respectively. Due to the different deformation conditions, dislocation configurations such as dislocation tangles, dislocation networks, and subcrystalline junctions can be observed in the plastic deformation region. The dislocation density of this alloy increases with increasing strain rate and decreasing deformation temperature after deformation.

Abstract Growing evidence supports the analgesic efficacy of electroacupuncture (EA) in managing chronic neuropathic pain (NP) in both patients and NP models induced by peripheral nerve injury. However, the underlying mechanisms remain incompletely understood. Ferroptosis, a novel form of programmed cell death, has been found to be activated during NP development, while EA has shown potential in promoting neurological recovery following acute cerebral injury by targeting ferroptosis. In this study, to investigate the detailed mechanism underlying EA intervention on NP, male Sprague‐Dawley rats with chronic constriction injury (CCI)‐induced NP model received EA treatment at acupoints ST36 and GV20 for 14 days. Results demonstrated that EA effectively attenuated CCI‐induced pain hypersensitivity and mitigated neuron damage and loss in the spinal cord of NP rats. Moreover, EA reversed the oxidative stress‐mediated spinal ferroptosis phenotype by upregulating reduced expression of xCT, glutathione peroxidase 4 (GPX4), ferritin heavy chain (FTH1) and superoxide dismutase (SOD) levels, and downregulating increased expression of acyl‐CoA synthetase long‐chain family member 4 (ACSL4), malondialdehyde levels and iron overload. Furthermore, EA increased the immunofluorescence co‐staining of GPX4 in neurons cells of the spinal cord of CCI rats. Mechanistic analysis unveiled that the inhibition of antioxidant pathway of Nrf2 signalling via its specific inhibitor, ML385, significantly countered EA's protective effect against neuronal ferroptosis in NP rats while marginally diminishing its analgesic effect. These findings suggest that EA treatment at acupoints ST36 and GV20 may protect against NP by inhibiting neuronal ferroptosis in the spinal cord, partially through the activation of Nrf2 signalling.

Nitric oxide (NO), an important cell messenger molecule, is formed endogenously in the lung airway. Three individual genes of NO synthase (NOS), which represent brain NOS (bNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), have been reported in the cultured lung epithelium. Although studies in vivo showed that bNOS and iNOS were expressed and localized in the cytoplasm of bronchial epithelium, the expression and localization of eNOS remains to be determined. Therefore, we employed an eNOS monoclonal antibody whose immunospecificity was tested by both Western blot and preadsorption immunohistochemistry to immunostain rat lungs from fetus to adult. The results showed that eNOS immunoreactivity began to appear in the lung epithelium within 2 hr after birth. Six hours later (8 hr after birth), the NOS immunoreaction was concentrated near the surface of the ciliated epithelial cells. This staining pattern appeared in lungs at Day 1, Week 1, Week 2, and in adult rats. By electron microscopy, eNOS immunoreactivity was confirmed within ciliated epithelium and was shown to be associated with the basal microtubule membrane of the cilia. Nonciliated cells were not stained. Type II epithelial cells also contain eNOS immunoreactivity, which is primarily associated with rough endoplasmic reticulum, and free ribosomes. However, macrophages in the lungs lacked eNOS immunoreactivity. This study demonstrated that eNOS was postnatally expressed in rat bronchial ciliated epithelium. The localization of eNOS at the basal membrane of ciliary microtubules suggests that eNOS may be involved in the function of epithelial cilia, consistent with previous physiological studies.

In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssa1p, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssa1p, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJ1p/PDI, YDJ1p/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.

We discuss theoretical and experimental approaches to three distinct developmental systems that illustrate how theory can influence experimental work and vice-versa. The chosen systems - Drosophila melanogaster, bacterial pattern formation, and pigmentation patterns - illustrate the fundamental physical processes of signaling, growth and cell division, and cell movement involved in pattern formation and development. These systems exemplify the current state of theoretical and experimental understanding of how these processes produce the observed patterns, and illustrate how theoretical and experimental approaches can interact to lead to a better understanding of development. As John Bonner said long ago'We have arrived at the stage where models are useful to suggest experiments, and the facts of the experiments in turn lead to new and improved models that suggest new experiments. By this rocking back and forth between the reality of experimental facts and the dream world of hypotheses, we can move slowly toward a satisfactory solution of the major problems of developmental biology.'

To evaluate the efficacy, predictability, stability, safety, and complications of secondary anterior iris fixation of the Artisan iris-fixated intraocular lens (IOL) to correct aphakia in eyes without sufficient capsule support.Department of Ophthalmology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China.Cohort study.Eyes having implantation of aphakic iris-fixated IOLs for aphakia correction were followed for 3 years.The study evaluated 72 eyes (72 patients). After 3 years, the uncorrected distance visual acuity improved in all eyes (P<.05); 53 eyes (73.6%) reached 20/40 or better. Two eyes had a postoperative corrected distance visual acuity (CDVA) worse than the preoperative CDVA due to postoperative ischemic optic neuropathy and retinal detachment, respectively. The mean spherical equivalent (SE) decreased from 11.65 diopters (D) ± 1.21 (SD) to -0.58 ± 0.56 D (P<.05); the SE at the last follow-up was within ±1.00 D of the target refraction in 63 eyes (87.5%). The mean endothelial cell loss 3 years postoperatively was 9.78%. There was no significant postoperative intraocular pressure increase throughout the follow-up. Twelve patients (16.7%) reported glare and halos during night driving. Iris pigment precipitates on the IOLs occurred in 4 eyes (5.6%) 3 years postoperatively. No other serious complications occurred.Three-year results indicate that secondary implantation of aphakic IOLs is effective, predictable, and safe for the correction of aphakia in eyes without capsule support. However, longer follow-up with a larger cohort is necessary to confirm these conclusions.

Breast cancer research and treatment by different subtypes is an inevitable trend. We investigated the clinicopathologic features and outcomes of different breast cancer subtypes in Southern China. A total of 5809 patients with invasive ductal carcinomas were identified. Immunohistochemical (IHC) markers for estrogen receptor ( ER ), progesterone receptor ( PR ), Her2/neu, and Ki‐67 proliferation index were used to classify cases into five molecular subtypes. Clinicopathologic characteristics and survival rates were analyzed retrospectively. Of all patients, 31.1% were luminal A subtype, 30.4% luminal B (high Ki‐67), 13.1% luminal B (Her2/neu+), 9.0% Her2/neu and 16.5% triple negative subtype. Luminal B (high Ki‐67) presented primarily in premenopausal patients with the lowest average age (43.0 years). Her2/neu positive tumors were more closely associated with aggressive features including increased tumor size, positive lymph node status and lymphvascular invasion ( LVI ). Triple negative subtype was characterized by poorer histologic grade. Her2/neu positive cases had presented the worst 5‐year disease‐free survival ( DFS ) and overall survival ( OS ). Multivariate analyses of OS and DFS suggested that there were different negative prognostic factors for the five subtypes. The benefit of the cyclophosphamide, methotrexate, and 5‐fluorouracil (5FU) (CMF) regimen was equal to that of anthracycline‐based and Taxane‐based regimens for patients with luminal A subtype and triple negative subtype, but inferior to anthracycline‐based and Taxane‐based regimens for those with two luminal B subtypes and Her2/neu subtype. The prognostic significance of traditional markers may differ among subtypes. This study revealed the distinct clinicopathologic characteristics, systemic therapy benefits, prognostic factors and survival rate among different breast cancer subtypes.

China's land use has undergone many changes over the past 300 years due to the significant transformations caused by natural and human factors and their impact on regional climate and the environment. This comprehensive review of recent state-of-the-art studies of China's land-use changes during that period concentrates on cropland, forest, grassland and urban areas. While most small-scale studies have reconstructed information from historical archive data and focused on a specific time period, large-scale studies have tended to rely on inverse modeling techniques to interpret land-use change dynamics based on remote-sensing data for example, the global land-use products of the History Database of the Global Environment (HYDE) and Center for Sustainability and the Global Environment (SAGE) datasets. All studies have shown that the cropland areas in China increased between 1700 and 1950, although they indicate different magnitudes and rates. A decrease in forest coverage was also reported in all studies. Little information was available on urban and grassland areas over the same period. Rapid urbanization in China has been particularly evident in the past 50 years. Meanwhile, spatially explicit reconstructions of historical land-use change in China since 1700 remain highly uncertain due to the lack of reliable data. Extensive work on primary data collection is required, including land-use records and drivers for future change.

IntroductionThe use of bevacizumab combined with chemotherapy represents a recent advance in clinical oncology for significantly improving the survival of patients who have non-small cell lung cancer (NSCLC). There is an unmet need for biomarkers that can predict response to such treatment and identify patients sensitive to it. Our study was designed to investigate the predictive value of intratumoral microvascular density (MVD) in patients with NSCLC treated with bevacizumab.MethodsSixteen patients with NSCLC who underwent chemotherapy combined with bevacizumab were included into this study. Paraffin-embedded tumor samples were sectioned and stained immunohistochemically for the blood vessel markers CD34 and CD31 to characterize the intratumoral vasculature. A computerized image analysis program was used to quantitatively calculate the intratumoral MVD. Treatment response was evaluated by computed tomography scanning.ResultsTwo types of blood vessels, undifferentiated (CD31+/CD34−) and differentiated (CD34+), were identified. A positive correlation was found between the largest percentage of tumor shrinkage and the MVD of undifferentiated (CD31+/CD34−) vessels, with Spearman correlation coefficient being 0.576 (p = 0.019). No correlation between tumor shrinkage and differentiated vessel MVD (CD34+) was found. Moreover, seven of the eight patients with more undifferentiated vessels showed a partial response, versus only one of the seven patients with fewer undifferentiated vessels (p = 0.009).ConclusionsThere are two major types of microvessel in lung cancer vasculature. The MVD of undifferentiated vessels is a favorable predictor for patients with NSCLC treated with a chemotherapy regimen plus bevacizumab, with a higher MVD value correlating with better treatment response. Further studies are needed to verify the predictive role of MVD in treatment of NSCLC with bevacizumab.

Retinoblastoma is the most common intraocular malignancy that occurs during childhood; however, the mechanism underlying retinoblastoma proliferation and progression remains unclear. MicroRNAs (miRNAs) play an important role in the regulation of a myriad of biological processes in various types of cancer. In this study, we performed microarray analysis followed by qRT-PCR using four classes of retinoblastoma tissues with increasing cTNM classification stages to identify crucial miRNAs whose expression was correlated with retinoblastoma progression. miR-125a-5p was downregulated, and its expression levels were inversely correlated with cell proliferation in retinoblastoma compared with adjacent non-tumor retinal tissues. The overexpression of miR-125a-5p significantly suppressed cell proliferation and tumor formation in retinoblastoma. We further identified the transcriptional co-activator with PDZ binding motif (TAZ) as a direct target of miR-125a-5p. Importantly, TAZ levels were inversely correlated with miRNA-125a-5p expression, and TAZ promoted retinoblastoma cell proliferation. Moreover, the overexpression of miR-125a-5p led to a decrease in TAZ expression and downstream EGFR signaling pathway activation both in vitro and vivo. Finally, TAZ overexpression in retinoblastoma cells overexpressing miR-125a-5p restored retinoblastoma cell proliferation and EGFR pathway activation. Taken together, our data demonstrated that miR-125a-5p functions as an important tumor suppressor that suppresses the EGFR pathway by targeting TAZ to inhibit tumor progression in retinoblastoma. Thus, the miR-125a-5p/TAZ/EGFR axis may be a potential therapeutic target for retinoblastoma.

Latest study showed that a novel translocation between programmed cell death ligand 1 (PD-L1) (cluster of differentiation 274) and TP63 (tumor protein 63) can be found in diffuse large B-cell lymphoma (DLBCL), resulting in their conjunct overexpression in tumor cells at RNA level. However, the expressed pattern of these 2 genes at protein level in DLBCL remains largely unknown, and the clinical relevance of PD-L1 and TP63 expression in DLBCL are also unclear. Tumor tissues from 76 Chinese DLBCL patients were immunostained for programmed cell death 1 (PD-1), PD-L1, and TP63 using the EnVision system. Clinical relevance of PD-1, PD-L1, and TP63 in 74 DLBCL were analyzed by chi-square test, the Kaplan–Meier curves with log rank test, and Cox's proportional hazards regression model. PD-1 was mainly expressed in tumor-infiltrating lymphocytes (TILs) of 39.5% patients. PD-L1 was expressed in tumor cells of 26.3% patients, and TP63 was immunostained in nucleoli of tumor cells of 31.6% cases. PD-1 expression was significantly associated with the patients’ gender and B symptoms (P = 0.032, P = 0.026). DLBCL with PD-L1 or TP63 expression in tumor cells showed low International Prognostic Index (IPI) score (P = 0.007, P = 0.009). PD-1+ TILs was related to prolonged overall survival rate (OS) of DLBCL patients (P = 0.02), whereas PD-L1 expression was associated with worse clinical outcome of patients (P = 0.049). Immunoreactivity of TP63 was not correlated with patients’ survival time. Besides, PD-1 expression, patients’ age, Ann Arbor stage, and IPI score were significant prognostic markers for OS, but PD-L1 and TP63 had no prognostic significance. PD-1, PD-L1, and TP63 are frequently expressed in DLBCL. PD-1/PD-L1/TP63 blockade may be a potential therapeutic strategy for some patients.

In this study, a facile process for efficient and scalable fabrication of biomaterials conduits with seamless sidewall and longitudinally aligned structure on luminal inwall by combining use of lyophilization and micromolding method was firstly developed and reported. The micropatterned chitosan conduits possessed better mechanical matching with the normal sciatic nerve. The highly aligned microstructure could accelerate the orientation growth of newborn tissue, whereas the porous sidewall is anticipated to be beneficial for loading biofactors and reducing nutrition leakage or neurite exogenesis. The as prepared chitosan conduits with micropatterned inwall are proven to significantly promote the regeneration of 10 mm-gapped sciatic nerve of rat. Taken together, the study provides an important experiment and application basis for design and development of new generation of artificial implants for peripheral nerve regeneration, which may also substantially extend current knowledge concerning the engineered biomaterials conduits for tissue engineering and regenerative medicine.

The echinococcosis is prevalent in 10 provinces /autonomous region in western and northern China. Epidemiological survey of echinococcosis in China in 2012 showed the average prevalence of four counties in Tibet Autonomous Region (TAR) is 4.23%, much higher than the average prevalence in China (0.24%). It is important to understand the transmission risks and the prevalence of echinococcosis in human and animals in TAR.A stratified and proportionate sampling method was used to select samples in TAR. The selected residents were examined by B-ultrasonography diagnostic, and the faeces of dogs were tested for the canine coproantigen against Echinococcus spp. using enzyme-linked immunosorbent assay. The internal organs of slaughtered domestic animals were examined by visual examination and palpation. The awareness of the prevention and control of echinococcosis among of residents and students was investigated using questionnaire. All data were inputted using double entry in the Epi Info database, with error correction by double-entry comparison, the statistical analysis of all data was processed using SPSS 21.0, and the map was mapped using ArcGIS 10.1, the data was tested by Chi-square test and Cochran-Armitage trend test.A total of 80 384 people, 7564 faeces of dogs, and 2103 internal organs of slaughtered domestic animals were examined. The prevalence of echinococcosis in humans in TAR was 1.66%, the positive rate in females (1.92%) was significantly higher than that in males (1.41%), (χ2 = 30.31, P < 0.01), the positive rate of echinococcosis was positively associated with age (χ2trend = 423.95, P < 0.01), and the occupational populations with high positive rates of echinococcosis were herdsmen (3.66%) and monks (3.48%). The average positive rate of Echinococcus coproantigen in TAR was 7.30%. The positive rate of echinococcosis in livestock for the whole region was 11.84%. The average awareness rate of echinococcosis across the region was 33.39%.A high prevalence of echinococcosis is found across the TAR, representing a very serious concern to human health. Efforts should be made to develop an action plan for echinococcosis prevention and control as soon as possible, so as to control the endemic of echinococcosis and reduce the medical burden on the population.

Extracellular/acellular matrix has been attracted much research interests for its unique biological characteristics, and ACM modified neural scaffolds shows the remarkable role of promoting peripheral nerve regeneration. In this study, skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) were used as parent cells to generate acellular(ACM) for constructing a ACM-modified neural scaffold. SKP-SCs were co-cultured with chitosan nerve guidance conduits (NGC) and silk fibroin filamentous fillers, followed by decellularization to stimulate ACM deposition. This NGC-based, SKP-SC-derived ACM-modified neural scaffold was used for bridging a 10 mm long rat sciatic nerve gap. Histological and functional evaluation after grafting demonstrated that regenerative outcomes achieved by this engineered neural scaffold were better than those achieved by a plain chitosan-silk fibroin scaffold, and suggested the benefits of SKP-SC-derived ACM for peripheral nerve repair.

This study aimed to evaluate whether ginsenosides Rb1 (20-S-protopanaxadiol aglycon) and Rg1 (20-S-protopanaxatriol aglycon) have mitochondrial protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in primary mouse astrocytes and to explore the mechanisms involved. The OGD/R model was used to mimic the pathological process of cerebral ischemia-reperfusion in vitro. Astrocytes were treated with normal conditions, OGD/R, OGD/R plus Rb1, or OGD/R plus Rg1. Cell viability was measured to evaluate the cytotoxicity of Rb1 and Rg1. Intracellular reactive oxygen species (ROS) and catalase (CAT) were detected to evaluate oxidative stress. The mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP) were measured to evaluate mitochondrial function. The activities of the mitochondrial respiratory chain (MRC) complexes I–V and the level of cellular adenosine triphosphate (ATP) were measured to evaluate oxidative phosphorylation (OXPHOS) levels. Cell viability was significantly decreased in the OGD/R group compared to the control group. Rb1 or Rg1 administration significantly increased cell viability. Moreover, OGD/R caused a significant increase in ROS formation and, subsequently, it decreased the activity of CAT and the mtDNA copy number. At the same time, treatment with OGD/R depolarized the MMP in the astrocytes. Rb1 or Rg1 administration reduced ROS production, increased CAT activity, elevated the mtDNA content, and attenuated the MMP depolarization. In addition, Rb1 or Rg1 administration increased the activities of complexes I, II, III, and V and elevated the level of ATP, compared to those in the OGD/R groups. Rb1 and Rg1 have different chemical structures, but exert similar protective effects against astrocyte damage induced by OGD/R. The mechanism may be related to improved efficiency of mitochondrial oxidative phosphorylation and the reduction in ROS production in cultured astrocytes.

Thrombosis is a life-threatening pathological condition in which blood clots form in blood vessels, obstructing or interfering with blood flow. Thrombolytic agents (TAs) are enzymes that can catalyze the conversion of plasminogen to plasmin to dissolve blood clots. The plasmin formed by TAs breaks down fibrin clots into soluble fibrin that finally dissolves thrombi. Several TAs have been developed to treat various thromboembolic diseases, such as pulmonary embolisms, acute myocardial infarction, deep vein thrombosis, and extensive coronary emboli. However, systemic TA administration can trigger non-specific activation that can increase the incidence of bleeding. Moreover, protein-based TAs are rapidly inactivated upon injection resulting in the need for large doses. To overcome these limitations, various types of nanocarriers have been introduced that enhance the pharmacokinetic effects by protecting the TA from the biological environment and targeting the release into coagulation. The nanocarriers show increasing half-life, reducing side effects, and improving overall TA efficacy. In this work, the recent advances in various types of TAs and nanocarriers are thoroughly reviewed. Various types of nanocarriers, including lipid-based, polymer-based, and metal-based nanoparticles are described, for the targeted delivery of TAs. This work also provides insights into issues related to the future of TA development and successful clinical translation.

Ethanol-induced gastric mucosal injury is a common gastrointestinal disorder. Polysaccharides separated from herbs have been shown to be effective for ethanol-induced gastric mucosal injury, but whether the polysaccharides from Dendrobium officinale Kimura & Migo leaves (LDOP-1) protected mucosa from ethanol-induced injury remains unknown. Thus, the present study carried out gastric mucosal protection and the mechanism of LDOP-1 in vivo and vitro. The chemical composition of LDOP-1 was a heteropolysaccharide comprising mannose, galacturonic acid, glucose, galactose, and arabinose at a molar ratio of 2.0:1.1:0.7:0.5:0.4. Pharmacological results showed that LDOP-1 significantly reduced gastric mucosal injury score and pathological injury, improved antioxidant capacity, reduced the level of reactive oxygen species, and reversed the apoptosis of GES-1 in vivo and vitro. Research showed that LDOP-1 pretreatment upregulated the expression level of p-AMPK, LC3β, HO-1, and Beclin-1; downregulated the expression level of p-mTOR and p62; and reversed the expression level of caspase3, Bax, and Bcl-2. This study was the first to demonstrate that LDOP-1 could protect against ethanol-induced gastric mucosal injury via the AMPK/mTOR signaling pathway in vitro and vivo.

Abstract Maleimides and succinimides are all vital scaffolds in biological fields and various natural products. Maleimide derivatives have been extensively used as coupling partners for various organic transformations, affording a broad array of important molecular architectures including succinimides. In the past decade, a variety of efficient chelation‐assisted strategies have been employed for the selective addition of C−H bonds to maleimides giving succinimides, which are also highly important building blocks in organic synthesis. This Review provides an overview of research progress relating to maleimides participated transition‐metal‐catalyzed group‐directed C−H alkylation from 2012 to 2021. Significant advances in this field were highlighted, diverse transition metal catalysts, organic substrates contained different directing groups, reaction mechanisms as well as synthetic applications are systematically discussed. In addition, the limitations and intractable issues need to be solved in the future are also pointed out.

Denitrification is an important global N cycle process. The gene encoding NosZ that converts nitrous oxide (N2O) to N2 has been widely used as a biomarker to study denitrifying communities. However, conventional PCR primers target a limited range of the genetically diverse Clade I nosZ, and the amplicons are too long for sequencing on current NGS platforms. To address these issues, we developed a new PCR primer set that amplifies a 355-bp region of Clade I nosZ and captures broader taxonomic coverage than conventional primers in in silico tests. When compared with the widely used nosZF_nosZR_Rich_2003 set using the same soil samples and the same sequencing depth, the new set retrieved genes from four times more unique species, with consistently higher general diversity-based metrics. The new primer set performed well with different sequencing platforms (Ion Torrent and Illumina), and among a wide variety of soils from polar to tropical, desert to agricultural, and surface to a very low biomass subsoil, with significant differences in denitrifying community diversity and composition. This new primer set for Clade I together with the primers recently reported for Clade II by Chee-Sanford et al. (J Microbiol Meth 172:105908, 2020) provides a more comprehensive assessment of denitrifier gene hosts, their ecological patterns, and the degree of novelty in retrieved gene sequences.

Salmonella enterica remains one of the leading causes of foodborne bacterial disease. Retail meat is a major source of human salmonellosis. However, comparative genomic analyses of S. enterica isolates from retail meat from different sources in China are lacking. A total of 341 S. enterica strains were isolated from retail meat in sixteen districts of Beijing, China, at three different time points (January 1st, May 1st, and October 1st) in 2017. Comparative genomics was performed to investigate the genetic diversity, virulence and antimicrobial resistance gene (ARG) profiles of these isolates. The most common serotype was S. Enteritidis (203/341, 59.5%), which dominated among isolates from three different time points during the year. Laboratory retesting confirmed the accuracy of the serotyping results predicted by the Salmonella In Silico Typing Resource (SISTR) (96.5%). The pangenome of the 341 S. enterica isolates contained 13,931 genes, and the core genome contained 3,635 genes. Higher Salmonella phage 118970 sal3 (219/341, 64.2%) and Gifsy-2 (206/341, 60.4%) prevalence contributed to the diversity of the accessory genes, especially those with unknown functions. IncFII(S), IncX1, and IncFIB(S) plasmid replicons were more common in these isolates and were major sources of horizontally acquired foreign genes. The virulence gene profile showed fewer virulence genes associated with type III secretion systems in certain isolates from chicken. A total of 88 different ARGs were found in the 341 isolates. Three beta-lactamases, namely, bla CTX – M – 55 ( n = 15), bla CTX – M – 14 ( n = 11), and bla CTX – M – 65 ( n = 11), were more prevalent in retail meats. The emergence of qnrE1 and bla CTX – M – 123 indicated a potential increase in the prevalence of retail meats. After the prohibition of colistin in China, three and four isolates were positive for the colistin resistance genes mcr-1.1 and mcr-9 , respectively. Thus, we explored the evolution and genomic features of S. enterica isolates from retail meats in Beijing, China. The diverse ARGs of these isolates compromise food security and are a clinical threat.

Injectable shear-thinning biomaterials (STBs) have attracted significant attention because of their efficient and localized delivery of cells as well as various molecules ranging from growth factors to drugs. Recently, electrostatic interaction-based STBs, including gelatin/LAPONITE® nanocomposites, have been developed through a simple assembly process and show outstanding shear-thinning properties and injectability. However, the ability of different compositions of gelatin and LAPONITE® to modulate doxorubicin (DOX) delivery at different pH values to enhance the effectiveness of topical skin cancer treatment is still unclear. Here, we fabricated injectable STBs using gelatin and LAPONITE® to investigate the influence of LAPONITE®/gelatin ratio on mechanical characteristics, capacity for DOX release in response to different pH values, and cytotoxicity toward malignant melanoma. The release profile analysis of various compositions of DOX-loaded STBs under different pH conditions revealed that lower amounts of LAPONITE® (6NC25) led to higher pH-responsiveness capable of achieving a localized, controlled, and sustained release of DOX in an acidic tumor microenvironment. Moreover, we showed that 6NC25 had a lower storage modulus and required lower injection forces compared to those with higher LAPONITE® ratios. Furthermore, DOX delivery analysis in vitro and in vivo demonstrated that DOX-loaded 6NC25 could efficiently target subcutaneous malignant tumors via DOX-induced cell death and growth restriction.

Soil plays a crucial role as a significant reservoir for antibiotic resistance genes (ARGs). Despite the extensive research conducted in this area, there remains a need for further investigation, particularly concerning diverse types of agricultural soil. This comprehensive study examined Fusarium wilt diseased and healthy soil samples to investigate the spectrum of ARGs and identify bacteria potentially harboring these genes. The level of antibiotic resistance (AR) in the two soil types was detected by using culture plate counts (PC). We quantified and identified antibiotic resistant bacteria (ARB) against nine antibiotics. From the PC results, the relative number of ARB (number of ARB/total number culturable bacteria) resistant to erythromycin, lincomycin, sulfonamides, streptomycin sulfate, kanamycin, and enrofloxacin in the diseased soil was significantly higher than that in the healthy soil (One-way ANOVA, p < 0.01). The GeoChip (version 5.0) was employed to identify ARGs and antibiotic biosynthetic genes (ABGs). Interestingly, despite the presence of similar ARGs in both soil types, the Fusarium wilt diseased soil demonstrated a significantly elevated abundance of the tetracycline (tetX) resistance gene compared to the healthy soil. A robust correlation (r ≥ 0.8) between multidrug efflux pumps and ARGs indicates that natural antibiotics as selective pressures for transportation mechanisms. Our analysis identified diverse ARB phylotypes and multiple ARGs in both soil types. Incorporating these soil types into ARGs risk assessments and relevant monitoring is essential for a comprehensive understanding of AR dynamics in various environments.

Osteoarthritis (OA) is a common chronic inflammatory disorder. Effective remodeling of inflammatory microenvironment in the joint is a promising strategy to prevent OA. However, current drugs remain unsatisfactory due to a lack of targeted and effective ways for relieving inflammatory conditions in OA joints. Bortezomib (BTZ), a proteasome inhibitor, could effectively inhibit proinflammatory cytokines but with poor accumulation in the inflammatory tissues. To overcome the shortcomings of BTZ delivery and to improve the efficacy of OA therapy, herein, we designed a novel nanomedicine (denoted as BTZ@PTK) by the co-assembly of BTZ and an amphiphilic copolymer (denoted as PTK) with ROS-cleaved thioketal (TK) linkages. The TK units in BTZ@PTK are first cleaved by the excessive ROS at OA sites, and then triggered the controlled release of BTZ, resulting in the accurate delivery and the inflammatory microenvironment remodeling. Accordingly, BTZ@PTK suppressed ROS generation and proinflammatory cytokines while promoting M1 macrophage apoptosis in lipopolysaccharide (LPS)-activated RAW264.7 macrophages or LPS/IFN-γ-treated primary macrophages, which leads to a better effect than BTZ. In OA mice, BTZ@PTK passively accumulates into inflamed joints to attenuate pain sensitivity and gait abnormality. Importantly, BTZ@PTK treatment successfully ameliorates synovitis with the reduction of synovial hyperplasia and synovitis scores by suppressing M1 macrophage polarization and promoting M1 macrophage apoptosis in the synovium, thereby delaying cartilage damage. Collectively, BTZ@PTK can effectively modulate inflammatory microenvironment for OA recession by activating M1 macrophage apoptosis and inhibiting M1macrophage-mediated inflammatory response.

Download This Paper Open PDF in Browser Add Paper to My Library Share: Permalink Using these links will ensure access to this page indefinitely Copy URL Copy DOI

Protein kinase B (AKT) signaling frequently is deregulated in human cancers and plays an important role in nasopharyngeal carcinoma (NPC). This preclinical study investigated the effect of MK-2206, a potent allosteric AKT inhibitor, on human NPC cells in vitro and in vivo.The effect of MK-2206 on the growth and proliferation of CNE-1, CNE-2, HONE-1, and SUNE-1 cells was assessed by Cell Counting Kit 8 and colony formation assay. Flow cytometry was performed to analyze cell cycle and apoptosis. The effects of MK-2206 on the AKT pathway were analyzed by Western blotting. Autophagy induction was evaluated via electron microscopy and Western blot. To test the effects of MK-2206 in vivo, CNE-2 cells were subcutaneously implanted into nude mice. Tumor-bearing mice were treated orally with MK-2206 or placebo. Tumors were harvested for immunohistochemical analysis.In vitro, MK-2206 inhibited the four NPC cell line growths and reduced the sizes of the colonies in a dose-dependent manner. At 72 and 96 hours, the half maximal inhibitory concentration (IC50) values of MK-2206 in CNE-1, CNE-2, and HONE-1 cell lines were 3-5 μM, whereas in SUNE-1, IC50 was less than 1 μM, and MK-2206 induced cell cycle arrest at the G1 phase. However, our study found no evidence of apoptosis. MK-2206 induced autophagy in NPC cells, as evidenced by electron microscopy and Western blot, and inhibited the growth of tumors that were subcutaneously implanted in mice. Inhibition of downstream phosphorylation through the PRAS40 and S6 pathways seems to be the main mechanism for the MK-2206-induced growth inhibition.Our preclinical study suggests that MK-2206's antiproliferative effect may be useful for NPC treatment; however, strategies for reinforcing this effect are needed to maximize clinical benefit.

Cucumber plants subjected to consecutive monoculture for 9 years were found to suffer from severe Fusarium wilt disease, caused by the soil-borne fungus Fusarium oxysporum f. sp. Cucumerinum J. H. Owen. In the present study, greenhouse experiments were performed to evaluate the influence of ammonia gas fumigation on Fusarium wilt suppression, fungal abundance and fungal community composition. Results showed that ammonia gas fumigation remarkably reduced disease incidence from 80% to 27%, resulting in a four-fold increase in yield, compared to the control. Total fungal abundance declined dramatically after fumigation and reached the lowest level at day 32, at 243 times lower than the control. Moreover, fumigation significantly increased soil fungal diversity, though it also decreased considerably coinciding with cucumber growth. Fumigation also significantly altered soil fungal community composition, relative to the control. Fusarium was strongly inhibited by fumigation in both relative abundance (3.8 times lower) and targeted quantification (a decrease of 167 fold). Collectively, the application of ammonia gas fumigation to control Fusarium wilt of cucumber resulted in a re-assembly of the fungal community to resemble that of a non-disease conducive consortium. Additional strategies, such as bioorganic fertilizer application, may still be required to develop sustainable disease suppression following fumigation.

Extracellular/acellular matrix-containing neural scaffolds represent a promising design of a tissue engineered nerve graft (TENG) for peripheral nerve repair. In this study, we engineered a composite neural scaffold by culturing dog bone marrow mesenchymal stem cells (BMSCs) onto the surface of a chitosan/silk fibroin-based scaffold and then exposing the cell culture to decellularization to deposit acellular matrix (ACM) coatings on the scaffold. This natural biomaterial-based, cell-derived ACM-coated neural scaffold, as a novel nerve graft, was used to bridge a 60 mm long nerve gap in a dog sciatic nerve. At 12 months after grafting, behavioral, functional, and histological evaluation indicated that our developed neural scaffold achieved satisfactory regenerative outcomes, which were very close to those achieved by autologous nerve grafts, the accepted golden standard for peripheral nerve repair. Moreover, additional therapeutic benefits produced by the modification of a neural scaffold with BMSC-derived ACM may be associated with the unique neural activity of the ACM, as evidenced by in vitro experimental findings that the ACM significantly enhanced axonal regrowth and Schwann cell proliferation. Our results will provide a further experimental basis for the translation of ACM-containing neural scaffolds into the clinic.

Abstract Lysine acetylation (Kac) is an important protein post‐translational modification in both eukaryotes and prokaryotes. Herein, we report the results of a global proteome analysis of Kac and its diverse functions in rice ( Oryza sativa ). We identified 1353 Kac sites in 866 proteins in rice seedlings. A total of 11 Kac motifs are conserved, and 45% of the identified proteins are localized to the chloroplast. Among all acetylated proteins, 38 Kac sites are combined in core histones. Bioinformatics analysis revealed that Kac occurs on a diverse range of proteins involved in a wide variety of biological processes, especially photosynthesis. Protein–protein interaction networks of the identified proteins provided further evidence that Kac contributes to a wide range of regulatory functions. Furthermore, we demonstrated that the acetylation level of histone H3 (lysine 27 and 36) is increased in response to cold stress. In summary, our approach comprehensively profiles the regulatory roles of Kac in the growth and development of rice.

Abstract The microenvironment of peripheral nerve regeneration consists of multiple neurotrophic factors, adhesion molecules, and extracellular matrix molecules, secreted by unique glial cells in the peripheral nerve system (PNS)-Schwann cell (SCs). Following peripheral nerve injury (PNI), local IGF-1 production is upregulated in SCs and denervated muscle during axonal sprouting and regeneration. Regulation of IGF-1/IGF-1R signaling is considered as a potentially targeted therapy of PNI. We previously identified a group of novel miRNAs in proximal nerve following rat sciatic nerve transection. The present work focused on the role of miR-129 in regulation of IGF-1 signaling after sciatic nerve injury. The temporal change profile of the miR-129 expression was negatively correlated with the IGF-1 expression in proximal nerve stump and dorsal root ganglion (DRG) following sciatic nerve transection. An increased expression of miR-129 inhibited proliferation and migration of SCs, and axonal outgrowth of DRG neurons, which was inversely promoted by silencing of the miR-129 expression. The IGF-1 was identified as one of the multiple target genes of miR-129, which exerted negative regulation of IGF-1 by translational suppression. Moreover, knockdown of IGF-1 attenuated the promoting effects of miR-129 inhibitor on proliferation and migration of SCs, and neurite outgrowth of DRG neurons. Overall, our data indicated that miR-129 own the potential to regulate the proliferation and migration of SCs by targeting IGF-1, providing further insight into the regulatory role of miRNAs in peripheral nerve regeneration. The present work not only provides new insight into miR-129 regulation of peripheral nerve regeneration by robust phenotypic modulation of neural cells, but also opens a novel therapeutic window for PNI by mediating IGF-1 production. Our results may provide further experimental basis for translation of the molecular therapy into the clinic.

To answer which epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is the best choice for advanced non-small cell lung cancer (NSCLC) EGFR mutants.16 phase III randomized trials involving 2962 advanced NSCLC EGFR mutants were enrolled. Multiple treatment comparisons showed different EGFR-TKIs shared equivalent curative effect in terms of all outcome measures among the overall, chemo-naïve and previously treated patients. Rank probabilities showed that erlotinib and afatinib had potentially better efficacy compared with gefitinib in both of the overall and chemo-naïve patients. Potentially survival benefit of erlotinib was also observed in previously treated patients compared with gefitinib. Additionally, EGFR-TKI showed numerically greater survival benefit in 19 Del compared with chemotherapy, while it was opposite in 21 L858R. Furthermore, afatinib, erlotinib and gefitinib had high, moderate and low risk of rash & diarrhea, respectively, while the occurrence of elevated liver transaminase was more common in gefitinib.Data of objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and adverse events were extracted from included studies. Efficacy and toxicity of all included treatments were integrated by network meta-analyses.Our study indicated a high efficacy-high toxicity pattern of afatinib, a high efficacy-moderate toxicity pattern of erlotinib and a medium efficacy-moderate toxicity pattern of gefitinib. Recommended EGFR-TKI should be suggested according to patients' tolerability and therapeutic efficacy in clinical practice. Moreover, the treatment for advanced EGFR-positive NSCLC might be different between 19 Del and 21 L858R.

Abstract Archaeal (AOA) and bacterial (AOB) ammonia-oxidizer responses to long-term field fertilization in a Mollisol soil were assessed through pyrosequencing of amoA genes. Long-term fertilization treatments including chemical fertilizer (NPK), NPK plus manure (NPKM) and no fertilization over 23 years altered soil properties resulting in significant shifts in AOA and AOB community composition and abundance. NPK exhibited a strong influence on AOA and AOB composition while the addition of manure neutralized the community change induced by NPK. NPK also led to significant soil acidification and enrichment of Nitrosotalea . Nitrosospira cluster 9 and 3c were the most abundant AOB populations with opposing responses to fertilization treatments. NPKM had the largest abundance of ammonia-oxidizers and highest potential nitrification activity (PNA), suggesting high N loss potential due to a doubling of nutrient input compared to NPK. PNA was strongly correlated to AOA and AOB community composition indicating that both were important in ammonium oxidization in this Mollisol soil. Total N and organic C were the most important factors driving shifts in AOA and AOB community composition. The AOA community was strongly correlated to the activities of all sugar hydrolysis associated soil enzymes and was more responsive to C and N input than AOB.

It has been shown that material surface topography greatly affects cell attachment, growth, proliferation, and differentiation. However, the underlying molecular mechanisms for cell-material interactions are still not understood well. Here, two kinds of butterfly wings with different surface architectures were employed for addressing such an issue. Papilio ulysses telegonus (P.u.t.) butterfly wing surface is composed of micro/nanoconcaves, whereas Morpho menelaus (M.m.) butterfly wings are decorated with grooves. RSC96 cells grown on M.m. wings showed a regular sorting pattern along with the grooves. On the contrary, the cells seeded on P.u.t. wings exhibited random arrangement. Transcriptome sequencing and bioinformatics analysis revealed that huntingtin (Htt)-regulated lysosome activity is a potential key factor for determining cell growth behavior on M.m. butterfly wings. Gene silence further confirmed this notion. In vivo experiments showed that the silicone tubes fabricated with M.m. wings markedly facilitate rat sciatic nerve regeneration after injury. Lysosome activity and Htt expression were greatly increased in the M.m. wing-fabricated graft-bridged nerves. Collectively, our data provide a theoretical basis for employing butterfly wings to construct biomimetic nerve grafts and establish Htt lysosome as a crucial regulator for cell-material interactions.

High-salinity acetonitrile (ACN)-containing wastewater is a typical chemical wastewater with high salinity, high volatility, high toxicity and nitrogen-rich organic matter. To efficiently treat high-salinity ACN wastewater without generating secondary pollution, a novel zero-discharge treatment system was developed based on the combination of pervaporation (PV) with a membrane-aerated bioreactor (MAB). Integrated (IMABS) and separate (SMABS) MAB systems were constructed and compared. In these two systems, the ACN was separated from the high-salinity ACN wastewater by PV. The separated ACN entered the MAB for biodegradation, and the high-salinity water remaining after separating ACN was reused for industrial production. The effects of the hydraulic retention time (HRT), salt content of the feed liquid, initial ACN concentration of the feed liquid, and aeration pressure on the process performance were investigated. The ACN separation and removal by SMABS were superior to those by IMABS. With an initial feed liquid ACN concentration of 2000 mg/L and salt content (as NaCl) of 5 wt%, ACN was not detected in the feed liquid of SMABS over an 8-day operation, and the high-salinity water remaining after ACN separation satisfied the requirements for reuse. The corresponding ACN permeation flux and ACN removal rate at 8 days were 0.131 g/(m2·h) and 98.75%, respectively. This research shows that the zero-discharge treatment of high-salinity ACN wastewater can be realized by the combination of PV with an MAB.

Abstract Injectable shear‐thinning biomaterials (iSTBs) have great potential for in situ tissue regeneration through minimally invasive therapeutics. Previously, an iSTB was developed by combining gelatin with synthetic silicate nanoplatelets (SNPs) for potential application to hemostasis and endovascular embolization. Hence, iSTBs are synthesized by varying compositions of gelatin and SNPs to navigate their material, mechanical, rheological, and bioactive properties. All compositions (each component percentage; 1.5–4.5%/total solid ranges; 3–9%) tested are injectable through both 5 Fr general catheter and 2.4 Fr microcatheter by manual pressure. In the results, an increase in gelatin contents causes decrease in swellability, increase in freeze‐dried hydrogel scaffold porosity, increase in degradability and injection force during iSTB fabrication. Meanwhile, the amount of SNPs in composite hydrogels is mainly required to decrease degradability and increase shear thinning properties of iSTB. Finally, in vitro and in vivo biocompatibility tests show that the 1.5–4.5% range gelatin–SNP iSTBs are not toxic to the cells and animals. All results demonstrate that the iSTB can be modulated with specific properties for unmet clinical needs. Understanding of mechanical and biological consequences of the changing gelatin–SNP ratios through this study will shed light on the biomedical applications of iSTB on specific diseases.

Abstract Fusarium wilt is a devastating disease which impacts watermelon production. Soil fumigation using dazomet followed by biological organic fertilizer was applied to suppress the Fusarium wilt disease. We propose that fumigation suppresses the soil indigenous community, especially the soil-borne pathogens, while the utilization of bio-organic fertilizer facilitates the recovery of the soil microbiome to a beneficial, suppressive state through the introduction of plant growth-promoting microorganisms. Greenhouse experiment showed that applied biological organic fertilizer after dazomet fumigation effective restrain the disease incidence with a 93.6% disease control. Fumigation strongly decreased soil microbial diversity and altered relative taxa abundances, suggesting the possibility of niche release by the resident soil microbial community. Fumigation followed by bio-fertilizer transformed the soil microbial community composition and resulted in higher relative abundances of beneficial microbial groups such as Bacillus (8.5%) and Trichoderma (13.5%), coupled with lower Fusarium abundance compared to other treatments. Network analysis illustrated that soil fumigation decreased interactions within the soil microbial community with less nodes and links while bio-fertilizer addition promoted node interactions. In addition, bio-fertilizer addition after fumigation resulted in the beneficial species becoming the key network connectors. Collectively, fumigation appears to release the resident soil niche resulting in lower diversity while the beneficial microbes introduced by bio-fertilizer addition colonize these niches, leading to a more complex community with fewer pathogens that suppresses Fusarium wilt disease incidence.

Stimulator of interferon genes (STING) has emerged as a key regulator of innate immunity, recognizing intracellular exogenous and endogenous DNA. Recent findings reveal that STING has multiple cell-specific immune functions in various pathological settings, including pathogenic infections, cancer, and autoimmune diseases. Here, we hypothesize that this unique location of STING in the mitochondria-associated membrane (MAM) might lead to the specificity of the cellular functions of STING mediated by mitochondria-ER communication. This new insight suggests that STING on the MAM might act as a hub that translates multiple cues on MAM into distinct cellular responses. This innovative view of STING biology might impart insights for future putative treatments in cancer and immune diseases that have been attributed to STING dysfunction.

We report a unique graphene architecture combining the advantages of highly vertical alignments and covalently bonded interfaces to improve the heat transfer ability of PVDF, achieving a thermal conductivity enhancement per 1 wt% filler loading of up to 1659.

ABSTRACT High salt is a major environmental factor that threatens plant growth and development. Increasing evidence indicates that histone acetylation is involved in plant responses to various abiotic stress; however, the underlying epigenetic regulatory mechanisms remain poorly understood. In this study, we revealed that the histone deacetylase OsHDA706 epigenetically regulates the expression of salt stress response genes in rice ( Oryza sativa L.). OsHDA706 localizes to the nucleus and cytoplasm and OsHDA706 expression is significantly induced under salt stress. Moreover, oshda706 mutants showed a higher sensitivity to salt stress than the wild‐type. In vivo and in vitro enzymatic activity assays demonstrated that OsHDA706 specifically regulates the deacetylation of lysines 5 and 8 on histone H4 (H4K5 and H4K8). By combining chromatin immunoprecipitation and mRNA sequencing, we identified the clade A protein phosphatase 2 C gene, OsPP2C49 , which is involved in the salt response as a direct target of H4K5 and H4K8 acetylation. We found that the expression of OsPP2C49 is induced in the oshda706 mutant under salt stress. Furthermore, the knockout of OsPP2C49 enhances plant tolerance to salt stress, while its overexpression has the opposite effect. Taken together, our results indicate that OsHDA706, a histone H4 deacetylase, participates in the salt stress response by regulating the expression of OsPP2C49 via H4K5 and H4K8 deacetylation.