Yanyan Zheng

Transcription factors (TFs) are proteins that regulate the expression of target genes by binding to specific cis-elements in promoter regions. Transcriptional regulators (TRs) also regulate the expression of target genes; however, they operate indirectly via interaction with the basal transcription apparatus (e.g., TFs), or by altering the accessibility of DNA to TFs via chromatin remodeling. Another type of regulatory proteins, protein kinases (PKs), function in signal transduction pathways and alter the activity of target proteins by phosphorylating them. These three important classes of regulatory proteins have been associated with numerous aspects of plant growth and development (Gapper et al., 2014Gapper N.E. Giovannoni J.J. Watkins C.B. Understanding development and ripening of fruit crops in an ‘omics’ era.Hortic. Res. 2014; 1: 14034Crossref PubMed Scopus (52) Google Scholar, Xu and Zhang, 2015Xu J. Zhang S. Mitogen-activated protein kinase cascades in signaling plant growth and development.Trends Plant Sci. 2015; 20: 56-64Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar), and response to biotic and abiotic stimuli (Zhang et al., 2013Zhang Y. Lubberstedt T. Xu M. The genetic and molecular basis of plant resistance to pathogens.J. Genet. Genomics. 2013; 40: 23-35Crossref PubMed Scopus (70) Google Scholar, Mickelbart et al., 2015Mickelbart M.V. Hasegawa P.M. Bailey-Serres J. Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability.Nat. Rev. Genet. 2015; 16: 237-251Crossref PubMed Scopus (576) Google Scholar). Effective and accurate identification and classification of these genes is important for understanding their evolution, biological functions, and regulatory networks. Currently, more than 100 plant genomes have been sequenced and regulatory proteins have been systematically identified from several of these plant genomes. Databases presenting these regulatory proteins, especially TFs, have been developed, such as PlnTFDB (Pérez-Rodríguez et al., 2010Pérez-Rodríguez P. Riaño-Pachón D.M. Corrêa L.G.G. Rensing S.A. Kersten B. Mueller-Roeber B. PlnTFDB: updated content and new features of the plant transcription factor database.Nucleic Acids Res. 2010; 38: D822-D827Crossref PubMed Scopus (542) Google Scholar) and PlantTFDB (Jin et al., 2013Jin J. Zhang H. Kong L. Gao G. Luo J. PlantTFDB 3.0: a portal for the functional and evolutionary study of plant transcription factors.Nucleic Acids Res. 2013; 42: D1182-D1187Crossref PubMed Scopus (669) Google Scholar). However, annotations of TF/TR families and the associated classification rules have been inconsistent among different studies. For example, the PlantTFDB does not include TRs that are presented in PlnTFDB. As another example, the forbidden domain (a domain that the specific TF families should not contain) of the C2H2 family is annotated as an RNase_T domain in PlantTFDB but as a PHD domain in PlnTFDB. Presently, while the collection of genome sequences is rapidly expanding, cataloged and annotated TFs/TRs vary across different databases due to inconsistent identification and characterization criteria with serious consequences for genome-scale and targeted analyses. Furthermore, in contrast to many studies focusing on specific families of plant regulators, computational tools for identification and classification of these regulatory proteins on a genome scale are very limited. In this study, we systemically compared TF/TR classification rules used in different databases, and derived a set of consensus rules based on the available literature for accurate plant TF/TR identification and classification. For plant PKs, we directly used the HMM profiles developed by Lehti-Shiu and Shiu, 2012Lehti-Shiu M.D. Shiu S.-H. Diversity, classification and function of the plant protein kinase superfamily.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2012; 367: 2619-2639Crossref PubMed Scopus (206) Google Scholar to provide a comprehensive classification system. These consensus rules for TF/TR classification and HMM profiles for PK classification were implemented in iTAK (http://bioinfo.bti.cornell.edu/tool/itak), a computational program that provides consistency and uniformity on the identification and classification of plant TFs, TRs, and PKs. To construct consensus rules for TF/TR prediction and classification, we compared Pfam domain assignment rules between PlnTFDB and PlantTFDB. The families in PlantTFcat (Dai et al., 2013Dai X. Sinharoy S. Udvardi M. Zhao P.X. PlantTFcat: an online plant transcription factor and transcriptional regulator categorization and analysis tool.BMC Bioinformatics. 2013; 14: 321Crossref PubMed Scopus (98) Google Scholar) and AtTFDB (Yilmaz et al., 2011Yilmaz A. Mejia-Guerra M.K. Kurz K. Liang X. Welch L. Grotewold E. AGRIS: Arabidopsis gene regulatory information server, an update.Nucleic Acids Res. 2011; 39: D1118-D1122Crossref PubMed Scopus (249) Google Scholar) were used as supporting evidence, as they use different methods for domain identification, therefore cannot be directly compared with PlnTFDB and PlantTFDB. A family annotation was considered more reliable if it had been assigned in both PlnTFDB and PlantTFDB, while a family unique to a single database was considered to be less reliable and required more evidence to support its identity. Under this criterion, 57 TF families/subfamilies were considered reliable, while 25 were considered less reliable (Supplemental Table 1). Comparison of domain assignment rules for the reliable families between PlnTFDB and PlantTFDB indicated that most were consistent, but rules of several subfamilies were missing in PlnTFDB. For example, PlantTFDB defines the AP2/ERF family to comprise three subfamilies, AP2, ERF, and RAV, while PlnTFDB only defines an AP2-EREBP family. In this example, the domain assignment rules used in PlantTFDB provide more details about the relationship between the superfamily and subfamily of AP2/ERF. Therefore, we adopted the rules for both the AP2/ERF superfamily and the three subfamilies (Figure 1A ). Similarly, the rules for the NF-Y (CCAAT) and MADS families were also adopted from PlantTFDB as they provide more detailed TF classification. In addition to handling missing rules in either PlnTFDB or PlantTFDB, we updated the domain assignment rules for several families including Homeobox (HB), BSD, and LIM, based on literature review (Figure 1A). In PlantTFDB, the HB superfamily is divided into five subfamilies: HD-ZIP, TALE, WOX, HB-PHD, and HB-other. In our consensus rule set, HB-TALE was further divided into two subfamilies, HB-BELL and HB-KNOX. We made this assignment because members of HB-BELL and HB-KNOX have different domains: HB-BELL contains POX and HB-KN domains, while HB-KNOX has a KNOX1 and a KNOX2 domain. In our study, the HD-ZIP_I/II domain that typifies the HB-HD-ZIP subfamily was replaced by the HALZ domain, which was specifically built from homeodomain-leucine zipper proteins. In addition, we updated the classification rule for the BSD family to require both a BSD and a PH_TFIIH domain, instead of requiring only the BSD domain as done by PlantTFDB and PlnTFDB. Finally, the LIM subfamily was updated to require two LIM domains (Weiskirchen and Günther, 2003Weiskirchen R. Günther K. The CRP/MLP/TLP family of LIM domain proteins: acting by connecting.Bioessays. 2003; 25: 152-162Crossref PubMed Scopus (85) Google Scholar) (Figure 1A; Supplemental Table 1). In reviewing the literature for the 25 TF families that were supported by only one of PlantTFDB and PlnTFDB, we found that six (WD40-like, TIG, FHA, Sigma70-like, TAZ, and mTERF) were inaccurately categorized as TFs (Supplemental Table 1). We excluded WD40-like since WD-repeat proteins perform diverse functions and using the existing rule would result in the identification of many non-TF WD40-like proteins. Based on multiple sequence alignments of TIG proteins, it was difficult to distinguish TFs from the TIG superfamily of proteins, which include not only TFs but also kinases and membrane proteins. Similarly, the FHA domain is present in a functionally diverse range of proteins that include not only TFs but also kinases, phosphatases and kinesins, and plant TFs containing an FHA domain have not been found. Sigma70-like proteins were also excluded from the TF category since they do not themselves bind to promoters; rather they function as components of an RNA polymerase holoenzyme involved in binding a core RNA polymerase to specific promoters. Finally, mTERF and TAZ were categorized as TRs instead of TFs because TAZ proteins function as coactivators alongside other regulatory proteins, and mTERF proteins exert a broad range of regulatory activities while not binding directly to promoters. To achieve low and balanced false positive and false negative rates, we excluded these six families from the rule set that defines TFs. Furthermore, the DDT family, which was categorized as TR in PlnTFDB, was categorized as TF. Based on a literature review, the remaining 16 TF families were included, resulting in a set of consensus rules that included 72 families/subfamilies for plant TF classification (Supplemental Table 1). The TR family classification rules were adopted from those used in PlnTFDB with support from PlantTFcat. Excluding the aforementioned DDT family, a total of 23 TR families including TAZ and mTERF that were incorrectly classified into TFs were derived from PlnTFDB. All these TR families were reviewed and accepted based on literature support (Supplemental Table 2). We developed the iTAK program base on the consensus rules we derived for the identification and classification of plant TFs/TRs/PKs (Supplemental Figure 1). To evaluate the performance of iTAK, the predicted Arabidopsis TFs with iTAK were systemically compared with those identified in PlantTFDB and PlnTFDB. The three datasets shared a total of 1602 TFs, accounting for approximately 90% of the TFs in PlnTFDB, PlantTFDB, and iTAK (Figure 1B). Although the majority of them were commonly identified as TFs and classified into the same families, we did observe some inconsistencies. The inconsistencies were mainly between PlnTFDB and PlantTFDB, while the iTAK classifications were consistent with one of the two other databases, with the exception of two genes, AT1G50680 and AT1G51120, which were assigned to the B3 family using iTAK, rather than to the AP2/ERF-RAV family by PlnTFDB and PlantTFDB, because they only contained B3 domains (Supplemental Table 3). This minor difference may reflect the recent update of the AP2 HMM profile in the Pfam database. Overall, the high consistency between iTAK and other studies indicates the high accuracy of TF identification and classification by iTAK. A total of 112, 28, and 14 TFs were identified only in PlnTFDB, PlantTFDB, or iTAK, respectively (Figure 1C; Supplemental Table 4). Five of the 14 iTAK-specific TFs were from the DDT family, which were inaccurately categorized as TRs in the other databases. Of the PlnTFDB-specific genes, 64 belonged to the mTERF, FHA, sigma70-like, and TAZ families, which should not be categorized as TFs. After eliminating these discrepant families, 48, 28, and 9 genes were predicted only by PlnTFDB, PlantTFDB, or iTAK, respectively. Furthermore, 87, 64, and 23 TFs were not identified by PlantTFDB, PlnTFDB, and iTAK, respectively, but were predicted and assigned to the same families by the other two (Figure 1D–1F). The smaller number of unique and missing identifications by iTAK indicates it achieved a better balance between false positives and false negatives. The reason that iTAK did not identify the 23 TFs was mainly due to the significance cutoff of the required domains (Supplemental Information; Supplemental Table 5). The identified TFs/TRs were also compared with other datasets, further supporting the high accuracy of iTAK (Supplemental Information). In summary, we have derived a set of consensus domain assignment rules for accurate identification and classification of plant TFs and TRs. We have developed a novel bioinformatics tool, iTAK, to facilitate genome-wide identification and classification of plant TFs, TRs, and PKs, and a comprehensive database for these regulatory proteins from sequenced plant species (Supplemental Information). These provide valuable tools and resources for the research community to study transcriptional regulations and signaling networks. This work was supported in part by a seed grant from the Association of Independent Plant Institutes (AIPI) to S.Y.R., P.Z., and Z.F. and grants from the National Science Foundation (IOS-0923312, IOS-1025642, and IOS-1339287 to Z.F., DBI-0960897 and DBI-1458597 to P.X.Z. and IOS-1026003 to S.Y.R.) and Department of Energy (DE-SC0008769) to S.Y.R.

Corn (Zea mays) responds to salt stress via changes in gene expression, metabolism and physiology. This adaptation is achieved through the regulation of gene expression at the transcriptional and post-transcriptional levels. MicroRNAs (miRNAs) have been found to act as key regulating factors of post-transcriptional gene expression. However, little is known about the role of miRNAs in plants' responses to abiotic stresses.A custom microparaflo microfluidic array containing release version 10.1 plant miRNA probes (http://microrna.sanger.ac.uk/) was used to discover salt stress-responsive miRNAs using the differences in miRNA expression between the salt-tolerant maize inbred line 'NC286' and the salt-sensitive maize line 'Huangzao4'. Key Results miRNA microarray hybridization revealed that a total of 98 miRNAs, from 27 plant miRNA families, had significantly altered expression after salt treatment. These miRNAs displayed different activities in the salt response, and miRNAs belonging to the same miRNA family showed the same behaviour. Interestingly, 18 miRNAs were found which were only expressed in the salt-tolerant maize line, and 25 miRNAs that showed a delayed regulation pattern in the salt-sensitive line. A gene model was proposed that showed how miRNAs could regulate the abiotic stress-associated process and the gene networks coping with the stress.Salt-responsive miRNAs are involved in the regulation of metabolic, morphological and physiological adaptations of maize seedlings at the post-transcriptional level. The miRNA genotype-specific expression model might explain the distinct salt sensitivities between maize lines.

High oil and protein content make tetraploid peanut a leading oil and food legume. Here we report a high-quality peanut genome sequence, comprising 2.54 Gb with 20 pseudomolecules and 83,709 protein-coding gene models. We characterize gene functional groups implicated in seed size evolution, seed oil content, disease resistance and symbiotic nitrogen fixation. The peanut B subgenome has more genes and general expression dominance, temporally associated with long-terminal-repeat expansion in the A subgenome that also raises questions about the A-genome progenitor. The polyploid genome provided insights into the evolution of Arachis hypogaea and other legume chromosomes. Resequencing of 52 accessions suggests that independent domestications formed peanut ecotypes. Whereas 0.42-0.47 million years ago (Ma) polyploidy constrained genetic variation, the peanut genome sequence aids mapping and candidate-gene discovery for traits such as seed size and color, foliar disease resistance and others, also providing a cornerstone for functional genomics and peanut improvement.

In development, timing is of the utmost importance, and the timing of developmental processes often changes as organisms evolve. In human evolution, developmental retardation, or neoteny, has been proposed as a possible mechanism that contributed to the rise of many human-specific features, including an increase in brain size and the emergence of human-specific cognitive traits. We analyzed mRNA expression in the prefrontal cortex of humans, chimpanzees, and rhesus macaques to determine whether human-specific neotenic changes are present at the gene expression level. We show that the brain transcriptome is dramatically remodeled during postnatal development and that developmental changes in the human brain are indeed delayed relative to other primates. This delay is not uniform across the human transcriptome but affects a specific subset of genes that play a potential role in neural development.

Changes in gene expression levels determine differentiation of tissues involved in development and are associated with functional decline in aging. Although development is tightly regulated, the transition between development and aging, as well as regulation of post-developmental changes, are not well understood. Here, we measured messenger RNA (mRNA), microRNA (miRNA), and protein expression in the prefrontal cortex of humans and rhesus macaques over the species' life spans. We find that few gene expression changes are unique to aging. Instead, the vast majority of miRNA and gene expression changes that occur in aging represent reversals or extensions of developmental patterns. Surprisingly, many gene expression changes previously attributed to aging, such as down-regulation of neural genes, initiate in early childhood. Our results indicate that miRNA and transcription factors regulate not only developmental but also post-developmental expression changes, with a number of regulatory processes continuing throughout the entire life span. Differential evolutionary conservation of the corresponding genomic regions implies that these regulatory processes, although beneficial in development, might be detrimental in aging. These results suggest a direct link between developmental regulation and expression changes taking place in aging.

Over the course of ontogenesis, the human brain and human cognitive abilities develop in parallel, resulting in a phenotype strikingly distinct from that of other primates. Here, we used microarrays and RNA-sequencing to examine human-specific gene expression changes taking place during postnatal brain development in the prefrontal cortex and cerebellum of humans, chimpanzees, and rhesus macaques. We show that the most prominent human-specific expression change affects genes associated with synaptic functions and represents an extreme shift in the timing of synaptic development in the prefrontal cortex, but not the cerebellum. Consequently, peak expression of synaptic genes in the prefrontal cortex is shifted from <1 yr in chimpanzees and macaques to 5 yr in humans. This result was supported by protein expression profiles of synaptic density markers and by direct observation of synaptic density by electron microscopy. Mechanistically, the human-specific change in timing of synaptic development involves the MEF2A-mediated activity-dependent regulatory pathway. Evolutionarily, this change may have taken place after the split of the human and the Neanderthal lineages.

While multiple studies have reported the accelerated evolution of brain gene expression in the human lineage, the mechanisms underlying such changes are unknown. Here, we address this issue from a developmental perspective, by analyzing mRNA and microRNA (miRNA) expression in two brain regions within macaques, chimpanzees, and humans throughout their lifespan. We find that constitutive gene expression divergence (species differences independent of age) is comparable between humans and chimpanzees. However, humans display a 3-5 times faster evolutionary rate in divergence of developmental patterns, compared to chimpanzees. Such accelerated evolution of human brain developmental patterns (i) cannot be explained by life-history changes among species, (ii) is twice as pronounced in the prefrontal cortex than the cerebellum, (iii) preferentially affects neuron-related genes, and (iv) unlike constitutive divergence does not depend on cis-regulatory changes, but might be driven by human-specific changes in expression of trans-acting regulators. We show that developmental profiles of miRNAs, as well as their target genes, show the fastest rates of human-specific evolutionary change, and using a combination of computational and experimental methods, we identify miR-92a, miR-454, and miR-320b as possible regulators of human-specific neural development. Our results suggest that different mechanisms underlie adaptive and neutral transcriptome divergence, and that changes in the expression of a few key regulators may have been a major driving force behind rapid evolution of the human brain.

Epigenetic modifications are thought to be important for gene expression changes during development and aging. However, besides the Sir2 histone deacetylase in somatic tissues and H3K4 trimethylation in germlines, there is scant evidence implicating epigenetic regulations in aging. The insulin/IGF-1 signaling (IIS) pathway is a major life span regulatory pathway. Here, we show that progressive increases in gene expression and loss of H3K27me3 on IIS components are due, at least in part, to increased activity of the H3K27 demethylase UTX-1 during aging. RNAi of the utx-1 gene extended the mean life span of C. elegans by ~30%, dependent on DAF-16 activity and not additive in daf-2 mutants. The loss of utx-1 increased H3K27me3 on the Igf1r/daf-2 gene and decreased IIS activity, leading to a more "naive" epigenetic state. Like stem cell reprogramming, our results suggest that reestablishment of epigenetic marks lost during aging might help "reset" the developmental age of animal cells.

While splicing differences between tissues, sexes and species are well documented, little is known about the extent and the nature of splicing changes that take place during human or mammalian development and aging. Here, using high-throughput transcriptome sequencing, we have characterized splicing changes that take place during whole human lifespan in two brain regions: prefrontal cortex and cerebellum. Identified changes were confirmed using independent human and rhesus macaque RNA-seq data sets, exon arrays and PCR, and were detected at the protein level using mass spectrometry. Splicing changes across lifespan were abundant in both of the brain regions studied, affecting more than a third of the genes expressed in the human brain. Approximately 15% of these changes differed between the two brain regions. Across lifespan, splicing changes followed discrete patterns that could be linked to neural functions, and associated with the expression profiles of the corresponding splicing factors. More than 60% of all splicing changes represented a single splicing pattern reflecting preferential inclusion of gene segments potentially targeting transcripts for nonsense-mediated decay in infants and elderly.

Article31 July 2018free access Source DataTransparent process Zika virus elicits inflammation to evade antiviral response by cleaving cGAS via NS1-caspase-1 axis Yanyan Zheng MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Qingxiang Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yaoxing Wu orcid.org/0000-0002-3639-0202 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ling Ma Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhenzhen Zhang Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Tao Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Shouheng Jin MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yuanchu She MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yi-Ping Li Corresponding Author [email protected] orcid.org/0000-0001-6011-3101 Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China Search for more papers by this author Jun Cui Corresponding Author [email protected] orcid.org/0000-0002-8000-3708 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yanyan Zheng MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Qingxiang Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yaoxing Wu orcid.org/0000-0002-3639-0202 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Ling Ma Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Zhenzhen Zhang Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Search for more papers by this author Tao Liu MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Shouheng Jin MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yuanchu She MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Yi-Ping Li Corresponding Author [email protected] orcid.org/0000-0001-6011-3101 Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China Search for more papers by this author Jun Cui Corresponding Author [email protected] orcid.org/0000-0002-8000-3708 MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China Search for more papers by this author Author Information Yanyan Zheng1,‡, Qingxiang Liu1,‡, Yaoxing Wu1,‡, Ling Ma2, Zhenzhen Zhang2, Tao Liu1, Shouheng Jin1, Yuanchu She1, Yi-Ping Li *,2,3 and Jun Cui *,1 1MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China 2Institute of Human Virology, Key Laboratory of Tropical Diseases Control Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China 3Department of Infectious Disease, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 20 87335085; E-mail: [email protected] *Corresponding author. Tel: +86 20 39943429; E-mail: [email protected] EMBO J (2018)37:e99347https://doi.org/10.15252/embj.201899347 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Viral infection triggers host innate immune responses, which primarily include the activation of type I interferon (IFN) signaling and inflammasomes. Here, we report that Zika virus (ZIKV) infection triggers NLRP3 inflammasome activation, which is further enhanced by viral non-structural protein NS1 to benefit its replication. NS1 recruits the host deubiquitinase USP8 to cleave K11-linked poly-ubiquitin chains from caspase-1 at Lys134, thus inhibiting the proteasomal degradation of caspase-1. The enhanced stabilization of caspase-1 by NS1 promotes the cleavage of cGAS, which recognizes mitochondrial DNA release and initiates type I IFN signaling during ZIKV infection. NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKVin vitro and in vivo. Taken together, our work unravels a novel antagonistic mechanism employed by ZIKV to suppress host immune response by manipulating the interplay between inflammasome and type I IFN signaling, which might guide the rational design of therapeutics in the future. Synopsis Zika virus promotes NLRP3 inflammasome activation by stabilizing caspase-1 to suppress cGAS-mediated type I IFN signaling. The non-structural protein NS1 enhances ZIKV-induced NLRP3 inflammasome activation. NS1 stabilizes caspase-1 by blocking its proteasomal degradation. NS1 recruits USP8 to cleave K11-linked poly-ubiquitin chains from caspase-1 at Lys134. ZIKV enhances inflammasome activation to benefit its infection by inhibiting type I IFN signaling. NS1-mediated stabilization of caspase-1 promotes the cleavage of cGAS. Introduction Zika virus (ZIKV) is an arthropod-borne flavivirus in the Flaviviridae family, which was initially discovered from Rhesus macaque in Uganda in 1947 (Dick et al, 1952). ZIKV contains a positive-sense single-stranded RNA genome and is closely related to several other important viruses that cause disease globally, including Dengue (DENV), hepatitis C, yellow fever, West Nile, and Japanese encephalitis viruses (Pierson & Diamond, 2013). ZIKV genome encodes a single polyprotein which can be processed to produce three structural (C, prM, and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins (Pierson & Diamond, 2013). ZIKV infection was originally thought as a mild and self-limiting viral illness and caught little attention (Rossi et al, 2016; Miner & Diamond, 2017). However, it became a global health emergency since accumulating evidence has suggested that ZIKV infection is associated with the increasing incidence of microcephaly in newborns and Guillain–Barré syndrome during the outbreak of ZIKV in Brazil from 2015 (Ioos et al, 2014; Petersen et al, 2016; Rubin et al, 2016). Type I interferon (IFN) response serves as the first line of defense to combat viral infection (Schneider et al, 2014). Recently, we and other groups have demonstrated that ZIKV evolved several strategies to counter human IFN antiviral response (Fernandez-Garcia et al, 2009; Grant et al, 2016; Kumar et al, 2016; Wu et al, 2017; Xia et al, 2018). The IFN-antagonistic strategies can be mainly divided into two types (Fernandez-Garcia et al, 2009): The first strategy employed by ZIKV is to reduce and delay the activation of IFN production. For example, we recently reported that NS1 and NS4B of ZIKV blocked virus-mediated IFN signaling by targeting TBK1 (Wu et al, 2017). Xia et al showed the similar result of NS1 and found that residue 188 is critical for the inhibition of IFN (Xia et al, 2018). ZIKV could also utilize the other strategy to evade innate immunity by antagonizing IFN-mediated downstream signaling transduction. It has been reported that NS5 of ZIKV promoted the degradation of STAT2 (Grant et al, 2016; Kumar et al, 2016), while our study also revealed that NS2B3 can degrade JAK1 (Wu et al, 2017), thus inhibiting JAK-STAT signaling and coincidentally impairing downstream ISG expressions. Taken together, different non-structural proteins of ZIKV attenuate innate antiviral response at different levels of IFN signaling pathway and cooperatively assist ZIKV to evade host immune response (Bowen et al, 2018). To infect the fetus and affect the neural development of fetus, ZIKV should first cross the placental barrier and reach the fetus (Li et al, 2016; Miner et al, 2016). However, there is no commonly accepted mechanism employed by ZIKV to achieve mother-to-fetus transmission so far. One of the possible mechanisms utilized by ZIKV is to use monocytes as the carrier (Parekh et al, 2010; Khaiboullina et al, 2017). Monocytes are able to detect several kinds of pathogens and respond with the activation of inflammasome, a large signaling protein complex whose assembly usually requires the protein apoptosis-associated speck-like protein containing a CARD (ASC), caspases, and scaffold proteins (such as NLRP3 or AIM2) (Martinon et al, 2009). Once activated, the inflammasome triggers the activation of the cysteine protease caspase-1 to prompt the maturation and secretion of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 (Park et al, 2007; Liu et al, 2016). IL-1β serves the central role in inflammatory response and initiates a series of innate immune responses (Dinarello, 2009). Recently, accumulating evidence has revealed that ZIKV can infect monocytes and result in the activation of inflammasome pathway (Khaiboullina et al, 2017; Tricarico et al, 2017; Wang et al, 2018). However, the correlation between inflammasome activation and ZIKV infection remains incompletely understood. Here, we report that ZIKV activates host inflammasome responses by increasing the stabilization of caspase-1. Interestingly, ZIKV NS1 targets caspase-1 and removes its K11-linked ubiquitin chains at lysine (Lys) 134 by recruiting deubiquitinase (DUB) USP8. Consequently, caspase-1 targets to cGAS for cleavage, which results in reduced type I IFN signaling and enhanced ZIKV replication. Furthermore, NLRP3 deficiency increases type I IFN production and strengthens host resistance to ZIKV in vivo. Taken together, we identify a novel function of ZIKV NS1 in regulating the stability of caspase-1 and therefore reveal a mechanism by which ZIKV evades host antiviral response via initiating inflammasome activation. Our findings will facilitate the development of antiviral inhibitors and vaccine design toward novel strategies against ZIKV infection. Results ZIKV infection induces NLRP3 inflammasome activation The inflammasome plays a key role in host innate immune responses by promoting pro-caspase-1 cleavage to generate the active subunits p20 and p10, leading to the maturation and secretion of IL-1β. In order to determine whether ZIKV infection activates the inflammasomes, we measured IL-1β secretion from unprimed or lipopolysaccharide (LPS)-primed THP-1 cells (a human monocyte cell line) infected with ZIKV (Asian lineage strain GZ01 (GenBank No. KU820898) if not specified). Immunoblot and ELISA analyses demonstrated that ZIKV infection induced the release of IL-1β from both unprimed and LPS-primed THP-1 cells (Fig 1A and B). NLRP3 and AIM2 are known to play important roles in inflammasome activation by virus infection (Martinon et al, 2009). We next generated NLRP3 and AIM2 knockout (KO) THP-1 cells by CRISPR/Cas9 technology (Fig EV1A), and KO efficiency was functionally validated by LPS plus ATP or poly (dA:dT) treatment as positive or negative controls. We found that ZIKV-induced IL-1β and IL-18 secretion as well as caspase-1 cleavage was abrogated in NLRP3 KO THP-1 cells but not in AIM2 KO THP-1 cells (Figs 1C and EV1B–D). To further confirm the induction of NLRP3 inflammasome by ZIKV, we examined IL-1β secretion in two different clones of NLRP3 or AIM2 KO cells at various time points after ZIKV infection and got consistent results (Figs 1D and EV1E). Moreover, we found that knockdown of NLRP3 by two different siRNAs suppressed IL-1β secretion after ZIKV infection in human peripheral blood mononuclear cells (PBMCs) from two healthy donors (Figs 1E and EV1F). We also examined another ZIKV recombinant (MR766)—a historical African lineage strain (Dick et al, 1952; Schwarz et al, 2016), which differs from GZ01 by 11% at nucleotide level. We found that both African strain MR766 and Asian strain GZ01 induced NLRP3 inflammasome activation (Fig 1F). In addition, we compared ZIKV with Dengue virus serotype 2 (DENV-2) strain 16681 (Pu et al, 2011), a flavivirus closely related to ZIKV, and found that compared with ZIKV, DENV-2 only induced weak inflammasome activation (Fig 1F). Taken together, these results demonstrate that ZIKV infection specifically activates the NLRP3 inflammasome. Figure 1. The non-structural protein NS1 of ZIKV enhances ZIKV-induced NLRP3 inflammasome activation ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 h) THP-1 cells infected with ZIKV (MOI = 1) for 36 h. Uninfected cells serve as mock control. Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated, or primed by LPS (500 ng/ml, 3 h), after ZIKV infection (MOI = 1) for 36 h. ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1, or AIM2 KO#1 THP-1 cells left untreated (UT), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) stimulation, or stimulated with poly (dA:dT) (1 mg/ml, 6 h), or infected with ZIKV (MOI = 1, 36 h). ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI = 1) at the indicated time points. ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI = 1) for 36 h. ELISA of IL-1β in the supernatant of WT, NLRP3 KO, or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI = 1) or DENV-2 (MOI = 1) infection for 36 h. ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 h or mock control. The cells were then treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM) treatment for 6 h. Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI = 1) or mock control for 24 h. The cells were then treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM) stimulation for 6 h. Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1, and pro-IL-1β together with NS1. Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 h. ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 h) or poly (I:C) (2 mg/ml, 6 h), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) or poly (I:C) (2 mg/ml, 6 h) stimulation. Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) or poly (I:C) (2 mg/ml, 6 h) stimulation. Data information: In (B, H–J, L), data are representative of three independent experiments. In (A, C, D–G, K), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embj201899347-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The non-structural protein NS1 of ZIKV enhances ZIKV-induced NLRP3 inflammasome activation, related to Fig 1 A. Immunoblot analysis of extracts of wild-type (WT) and two different clones of NLRP3 or AIM2 knockout (KO) THP-1 cells by the indicated antibodies. B. ELISA of IL-18 in the supernatants of WT, NLRP3 KO#1, or AIM2 KO#1 THP-1 cells left untreated (UT), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) stimulation, or stimulated with poly (dA:dT) (1 mg/ml, 6 h), or infected with ZIKV (MOI = 1, 36 h). C, D. Immunoblot analysis of the cleavage of caspase-1 and IL-1β in WT, NLRP3 KO#1 (C) or AIM2 KO#1 (D) THP-1 cells left untreated (UT), or pre-treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM, 6 h) stimulation, or stimulated with poly (dA:dT) (1 mg/ml, 6 h), or infected with ZIKV (MOI = 1, 36 h). E. ELISA of IL-1β in the supernatants of two clones of AIM2 KO THP-1 cells infected with ZIKV (MOI = 1) for the indicated time points. F. Immunoblot analysis of extracts of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI = 1) for 36 h by the indicated antibodies. G. THP-1 cells were pre-infected with ZIKV (MOI = 1) for 12 h. The cells were then treated with LPS (500 ng/ml, 3 h) followed by ATP (5 mM) stimulation for the indicated time points. Cell death was measured by LDH (lactate dehydrogenase) release. H. Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids encoding NLRP3, ASC, pro-caspase-1, and pro-IL-1β together with NS2B3 (left) or NS4B (right). I. Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of Flag-NS1-inducible unprimed THP-1 cells stimulated by LPS (500 ng/ml, 3 h), ATP (5 mM, 6 h), or poly (I:C) (2 mg/ml, 6 h), respectively. Data information: In (A, C, D, F, H, I), data are representative of three independent experiments. In (B, E, G), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; ***P < 0.001. Download figure Download PowerPoint A number of viruses have evolved mechanisms to prolong their intracellular survival by inhibiting the inflammasome activation (Komune et al, 2011; Cheong et al, 2015). However, we observed that ZIKV infection promotes the activation of inflammasomes, since more cleaved IL-1β was detected in THP-1 cells infected with ZIKV followed by LPS and ATP treatment to activate NLRP3 inflammasome (Fig 1G and H). Meanwhile, ZIKV infection did not affect the inflammasome activation-induced cell death (Fig EV1G). Together, these results suggest that ZIKV infection triggers NLRP3 inflammasome activation. The non-structural protein NS1 of ZIKV enhances inflammasome activation Next, we set out to unveil the underlying mechanisms through which ZIKV promotes inflammasome activation. The non-structural protein 1 (NS1) is the major host-interaction molecule that functions in flavivirus replication, pathogenesis, and immune evasion (Hilgenfeld, 2016). To evaluate whether NS1 of ZIKV plays a role in inflammasome activation, we utilized human embryonic kidney (HEK) 293T cells in which the NLRP3 inflammasome is deficient and can be reconstituted. Overexpression of NS1 but not NS2B3 or NS4B of ZIKV led to a profound inflammasome activation, as characterized by the increased secretion of mature caspase-1 and IL-1β into the culture medium (Figs 1I and EV1H). To further confirm these results, we generated a doxycycline (Dox)-inducible NS1 THP-1 cell line (Fig 1J). After LPS priming and ATP or poly (I:C) stimulation, more mature IL-1β and cleaved caspase-1 could be detected when NS1 was induced to express (Figs 1K and L, and EV1I). Taken together, these results demonstrate that NS1 further facilitates NLRP3 inflammasome activation during ZIKV infection. The enhanced inflammasome activation by NS1 benefits ZIKV infection In light of the critical role of the inflammasome in the host antiviral responses, we turned our attention to its impact on ZIKV replication. Surprisingly, we observed a significantly higher accumulation of ZIKV viral RNA in LPS and ATP pre-treated THP-1 cells (Fig 2A). Consistently, knockout (KO) of NLRP3 in THP-1 cells resulted in lower viral replication (Fig 2B). To further confirm this finding, we examined ZIKV replication in bone marrow-derived macrophages (BMDMs) from NLRP3-deficient (Nlrp3−/−) mice (Fig 2C) by qRT–PCR as well as plaque titration assay. The results showed that the level of ZIKV RNA (Fig 2D) and virus titer (Fig 2E) was significantly lower in Nlrp3−/− BMDMs, compared to that of wild-type (WT) BMDMs. We next generated caspase-1 KO THP-1 cells (Fig 2F) and found that caspase-1 deficiency also decreased ZIKV replication (Fig 2G). Moreover, the use of Ac-YVAD-cmk (YVAD for short), a specific inhibitor of caspase-1, attenuated ZIKV replication (Fig EV2A), indicating that the reinforcing effect of NLRP3 inflammasome on ZIKV replication relied on the enzyme activity of caspase-1. Consistently, NS1-induced up-regulation of ZIKV replication could be blocked by YVAD (Fig 2H), indicating that NS1 also functions through caspase-1 activity. To validate these findings in human primary cells, we knocked down NLRP3 by siRNA in PBMCs from two different healthy donors (Fig 2I). We observed lower viral replication after knockdown of NLRP3 (Fig 2J). Furthermore, YVAD treatment in PBMCs also suppressed ZIKV replication (Fig 2K). We next examined whether MR766 strain shared the same property as GZ01 strain and found that NLRP3 deficiency inhibited the viral replication of both strains (Fig EV2B). Since NLRP3 inflammasome activation is sufficient to drive pro-IL-1β processing and secretion, we therefore assessed the contribution of IL-1β to this course. However, we found that the replication of ZIKV was not affected by IL-1β treatment (Fig EV2C). Taken together, these data suggest that the enhanced NLRP3 inflammasome activation by NS1 benefits to ZIKV replication, which is dependent on the activity of caspase-1 but not IL-1β. Figure 2. NS1 enhances inflammasome activation to benefit ZIKV infection Relative qRT–PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 h) and ATP (5 mM, 6 h), followed by ZIKV infection (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI = 1) for the indicated time points. Immunoblot analysis of extracts of WT or Nlrp3−/− BMDMs by the indicated antibodies. Relative qRT–PCR analysis of ZIKV RNA in WT or Nlrp3−/− BMDMs infected with ZIKV (MOI = 1) for the indicated time points. Plaque titration of ZIKV in supernatants of WT or Nlrp3−/− BMDMs infected with ZIKV (MOI = 1) for the indicated time points. Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies. Relative qRT–PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μM) for 3 h followed by ZIKV infection (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors. Relative qRT–PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI = 1) for 36 h. qRT–PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20 μM) for 3 h followed by ZIKV infection (MOI = 1) for 36 h. Data information: In (C, F), data are representative of three independent experiments. In (A, B, D, E, G–K), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. NS1 enhances inflammasome activation by inhibiting the proteasomal degradation of caspase-1 to benefit ZIKV infection, related to Figs 2 and 3 Relative qRT–PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with Ac-YVAD-cmk (20 μM) for 3 h, followed by ZIKV infection (MOI = 1) for the indicated time points. Relative qRT–PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (GZ01) or ZIKV (MR766) (MOI = 1) for 36 h. Relative qRT–PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with IL-1β (2.5 ng/ml) for 3 h, followed by ZIKV infection (MOI = 1) for the indicated time points. Immunoblot analysis of THP-1 cells infected with ZIKV (GZ01), ZIKV (MR766), or DENV-2 (MOI = 1) for 36 h by the indicated antibodies. Intensity analysis of the bands in Fig 3F from the three independent experiments. Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), 3-methyladenine (3MA) (10 mM), chloroquine (CQ) (50 mM), or NH4Cl (20 mM) for 6 h. Confocal microscopy analysis of the co-localization of proteasome subunit PSMD14 and caspase-1 in HeLa cells transfected with or without NS1 followed by CHX (100 μg/ml) and MG132 (10 mM) treatment for 3 h. Scale bar is 10 μm. Intensity analysis of the bands in Fig 3I from three independent experiments. Data information: In (D, F), data are representative of three independent experiments. In (A–C, E, H), data are mean values ± SEM (n = 3). NS (non-significant), P > 0.05; ***P < 0.001. Download figure Download PowerPoint NS1 inhibits the proteasomal degradation of caspase-1 To dissect the molecular mechanisms by which NS1 enhances inflammasome and caspase-1 activation, we sought to determine whether NS1 directly interacts with inflammasome components. Co-immunoprecipitation experiments showed that NS1 had a strong interaction with caspase-1 in 293T (Fig 3A) and Flag-NS1-inducible THP-1 cells (Fig 3B). Meanwhile, we repeatedly observed an increased protein level of caspase-1 after increasing the effect of NS1 on caspase-1 abundance, and we repeated this work both in THP-1 and in 293T cells. After NS1 overexpression, we observed a considerable accumulation of caspase-1 but not of any other proteins such as IL-1β and NLRP3 (Fig 3C and D). In addition, RT–PCR showed that caspase-1 mRNA abundance was not altered with NS1 overexpression (Fig 3D). We did not observe the accumulation effect of caspase-1 by DENV-2 NS1 protein, although DENV is a flavivirus closely related to ZIKV (Fig 3E). In line with this result, we observed increased protein abundance of caspase-1 after the infection of both strains of ZIKV but not DENV-2 (Fig EV2D). We next performed cycloheximide (CHX) chase assay to determine whether NS1 affects the stability of caspase-1 (Figs 3F and EV2E) and found that NS1 stabilized caspase-1 protein by delaying its degradation. To reveal which degradation system is responsible for the degradation of caspase-1, we assessed the caspase-1 stability in the presence of different inhibitors and found that NS1-mediated stabilization of caspase-1 was blocked by the proteasome inhibitors MG132, lactacystin, or carfilzomib, but not by the autophagy inhibitor (3MA) or lysosome inhibitor chloroquine (CQ) and NH4Cl (Figs 3G and EV2F). We also performed confocal microscopy analysis to visualize the co-localization of caspase-1 with the proteasome and observed that caspase-1 co-localized with the proteasome subunit PSMD14. However, the co-localizations were diminished in the presence of NS1, suggesting that the degradation of caspase-1 via proteasome pathway can be inhibited by NS1 (Fig EV2G). Collectively, these results demonstrate that NS1 protects caspase-1 from proteasomal degradation. Figure 3. NS1 inhibits the proteasomal degradation of caspase-1 Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1, and Flag-IL-1β. WCL, whole-cell lysates. Co-immunopre

Abstract Perfluoroalkyl substances (PFAS) are widely used in various manufacturing processes. Accumulation of these chemicals has adverse effects on human health, including inflammation in multiple organs, yet how PFAS are sensed by host cells, and how tissue inflammation eventually incurs, is still unclear. Here, we show that the double-stranded DNA receptor AIM2 is able to recognize perfluorooctane sulfonate (PFOS), a common form of PFAS, to trigger IL-1β secretion and pyroptosis. Mechanistically, PFOS activates the AIM2 inflammasome in a process involving mitochondrial DNA release through the Ca 2+ -PKC-NF-κB/JNK-BAX/BAK axis. Accordingly, Aim2 −/ − mice have reduced PFOS-induced inflammation, as well as tissue damage in the lungs, livers, and kidneys in both their basic condition and in an asthmatic exacerbation model. Our results thus suggest a function of AIM2 in PFOS-mediated tissue inflammation, and identify AIM2 as a major pattern recognition receptor in response to the environmental organic pollutants.

Tomato is one of the most important vegetable crops in the world and is a model plant used to study the ripening of climacteric fleshy fruit. During the ripening process of tomato fruit, flavor and aroma metabolites, color, texture and plant hormones undergo significant changes. However, low temperatures delayed the ripening process of tomato fruit, inhibiting flavor compounds and ethylene production. Metabolomics and transcriptomics analyses of tomato fruit stored under low temperature (LT, 5 °C) and room temperature (RT, 25 °C) were carried out to investigate the effects of storage temperature on the physiological changes in tomato fruit after harvest. The results of transcriptomics changes revealed that the differentially expressed genes (DEGs) involved in tomato fruit ripening, including several kinds of transcription factors (TFs) (TCP, WRKY, MYB and bZIP), enzymes involved in cell wall metabolism [beta-galactosidase (β-GAL), pectinesterase (PE) and pectate lyase (PL), cellulose and cellulose synthase (CESA)], enzymes associated with fruit flavor and aroma [acetyltransferase (AT), malic enzyme (ME), lipoxygenase(LOX), aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH) and hexokinase (HK)], genes associated with heat stress protein 70 and genes involved in the production of plant hormones such as Ethylene responsive factor 1 (ERF1), Auxin/indoleacetic acids protein (AUX/IAA), gibberellin regulated protein. Based on the above results, we constructed a regulatory network model of the effects of different temperatures during the fruit ripening process. According to the analysis of the metabolomics results, it was found that the contents of many metabolites in tomato fruit were greatly affected by storage temperature, including, organic acids (L-tartaric acid, a-hydroxyisobutyric acid and 4-acetamidobutyric acid), sugars (melezitose, beta-D-lactose, D-sedoheptulose 7-phosphate, 2-deoxyribose 1-phosphate and raffinose) and phenols (coniferin, curcumin and feruloylputrescine). This study revealed the effects of storage temperature on postharvest tomato fruit and provided a basis for further understanding of the molecular biology and biochemistry of fruit ripening.

Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the HBG1 and HBG2 (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny.We performed a tiling CRISPR-Cas9 screen of the HBG1 and HBG2 promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923.In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period.CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).

Piwi-interacting RNA (piRNA) are small RNA abundant in the germline across animal species. In fruit flies and mice, piRNA have been implicated in maintenance of genomic integrity by transposable elements silencing. Outside of the germline, piRNA have only been found in fruit fly ovarian follicle cells. Previous studies have further reported presence of multiple piRNA-like small RNA (pilRNA) in fly heads and a small number of pilRNA have been reported in mouse tissues and in human NK cells. Here, we analyze high-throughput small RNA sequencing data in more than 130 fruit fly, mouse and rhesus macaque samples. The results show widespread presence of pilRNA, displaying all known characteristics of piRNA in multiple somatic tissues of these three species. In mouse pancreas and macaque epididymis, pilRNA abundance was compatible with piRNA abundance in the germline. Using in situ hybridizations, we further demonstrate pilRNA co-localization with mRNA expression of Piwi-family genes in all macaque tissues. Further, using western blot, we have shown the expression of Miwi protein in mouse pancreas. These findings indicate that piRNA-like molecules might play important roles outside of the germline.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes severe liver diseases, such as cirrhosis and hepatocellular carcinoma. HCV uses an RNA-dependent RNA polymerase to replicate its genome and an internal ribosomal entry site to translate its proteins. HCV infection is characterized by an increase in the concentrations of reactive oxygen species (ROS), the effect of which on HCV replication has yet to be determined. In this report, we investigated the effect of ROS on HCV replication, using a bicistronic subgenomic RNA replicon and a genomic RNA that can replicate in human hepatoma cells. The treatment with peroxide at concentrations that did not deplete intracellular glutathione or induce cell death resulted in significant decreases in the HCV RNA level in the cells. This response could be partially reversed by the antioxidant N-acetylcysteine. Further studies indicated that such a suppressive response to ROS was not due to the suppression of HCV protein synthesis or the destabilization of HCV RNA. Rather, it occurred rapidly at the level of RNA replication. ROS appeared to disrupt active HCV replication complexes, as they reduced the amount of NS3 and NS5A in the subcellular fraction where active HCV RNA replication complexes were found. In conclusion, our results show that ROS can rapidly inhibit HCV RNA replication in human hepatoma cells. The increased ROS levels in hepatitis C patients may therefore play an important role in the suppression of HCV replication.

Background and AimsAnaerobic or low oxygen conditions occur when maize plants are submerged or subjected to flooding of the soil. Maize survival under low oxygen conditions is largely dependent on metabolic, physiological and morphological adaptation strategies; the regulation mechanisms of which remain unknown. MicroRNAs (miRNAs) play critical roles in the response to adverse biotic or abiotic stresses at the post-transcriptional level. The aim of this study was to understand submergence-responsive miRNAs and their potential roles in submerged maize roots.

During microRNA (miRNA) maturation in humans and flies, Drosha and Dicer cut the precursor transcript, thereby producing a short RNA duplex. One strand of this duplex becomes a functional component of the RNA-Induced Silencing Complex (RISC), while the other is eliminated. While thermodynamic asymmetry of the duplex ends appears to play a decisive role in the strand selection process, the details of the selection mechanism are not yet understood.Here, we assess miRNA strand selection bias in humans and fruit flies by analyzing the sequence composition and relative expression levels of the two strands of the precursor duplex in these species. We find that the sequence elements associated with preferential miRNA strand selection and/or rejection differ between the two species. Further, we identify another feature that distinguishes human and fly miRNA processing machinery: the relative accuracy of the Drosha and Dicer enzymes.Our result provides clues to the mechanistic aspects of miRNA strand selection in humans and other mammals. Further, it indicates that human and fly miRNA processing pathways are more distinct than currently recognized. Finally, the observed strand selection determinants are instrumental in the rational design of efficient miRNA-based expression regulators.

The male sterility of thermosensitive genic male sterile (TGMS) lines of wheat (Triticum aestivum) is strictly controlled by temperature. The early phase of anther development is especially susceptible to cold stress. MicroRNAs (miRNAs) play an important role in plant development and in responses to environmental stress. In this study, deep sequencing of small RNA (smRNA) libraries obtained from spike tissues of the TGMS line under cold and control conditions identified a total of 78 unique miRNA sequences from 30 families and trans-acting small interfering RNAs (tasiRNAs) derived from two TAS3 genes. To identify smRNA targets in the wheat TGMS line, we applied the degradome sequencing method, which globally and directly identifies the remnants of smRNA-directed target cleavage. We identified 26 targets of 16 miRNA families and three targets of tasiRNAs. Comparing smRNA sequencing data sets and TaqMan quantitative polymerase chain reaction results, we identified six miRNAs and one tasiRNA (tasiRNA-ARF [for Auxin-Responsive Factor]) as cold stress-responsive smRNAs in spike tissues of the TGMS line. We also determined the expression profiles of target genes that encode transcription factors in response to cold stress. Interestingly, the expression of cold stress-responsive smRNAs integrated in the auxin-signaling pathway and their target genes was largely noncorrelated. We investigated the tissue-specific expression of smRNAs using a tissue microarray approach. Our data indicated that miR167 and tasiRNA-ARF play roles in regulating the auxin-signaling pathway and possibly in the developmental response to cold stress. These data provide evidence that smRNA regulatory pathways are linked with male sterility in the TGMS line during cold stress.

Kernel row number (KRN) is an important component of yield during the domestication and improvement of maize and controlled by quantitative trait loci (QTL). Here, we fine-mapped a major KRN QTL, KRN4, which can enhance grain productivity by increasing KRN per ear. We found that a ~3-Kb intergenic region about 60 Kb downstream from the SBP-box gene Unbranched3 (UB3) was responsible for quantitative variation in KRN by regulating the level of UB3 expression. Within the 3-Kb region, the 1.2-Kb Presence-Absence variant was found to be strongly associated with quantitative variation in KRN in diverse maize inbred lines, and our results suggest that this 1.2-Kb transposon-containing insertion is likely responsible for increased KRN. A previously identified A/G SNP (S35, also known as Ser220Asn) in UB3 was also found to be significantly associated with KRN in our association-mapping panel. Although no visible genetic effect of S35 alone could be detected in our linkage mapping population, it was found to genetically interact with the 1.2-Kb PAV to modulate KRN. The KRN4 was under strong selection during maize domestication and the favorable allele for the 1.2-Kb PAV and S35 has been significantly enriched in modern maize improvement process. The favorable haplotype (Hap1) of 1.2-Kb-PAV-S35 was selected during temperate maize improvement, but is still rare in tropical and subtropical maize germplasm. The dissection of the KRN4 locus improves our understanding of the genetic basis of quantitative variation in complex traits in maize.

Sustained tumor progression has been attributed to a distinct population of tumor-propagating cells (TPCs). To identify TPCs relevant to lung cancer pathogenesis, we investigated functional heterogeneity in tumor cells isolated from Kras-driven mouse models of non-small-cell lung cancer (NSCLC). CD24(+)ITGB4(+)Notch(hi) cells are capable of propagating tumor growth in both a clonogenic and an orthotopic serial transplantation assay. While all four Notch receptors mark TPCs, Notch3 plays a nonredundant role in tumor cell propagation in two mouse models and in human NSCLC. The TPC population is enriched after chemotherapy, and the gene signature of mouse TPCs correlates with poor prognosis in human NSCLC. The role of Notch3 in tumor propagation may provide a therapeutic target for NSCLC.

Head smut, caused by the fungus Sphacelotheca reiliana (Kühn) Clint, is a devastating global disease in maize, leading to severe quality and yield loss each year. The present study is the first to conduct a genome-wide association study (GWAS) of head smut resistance using the Illumina MaizeSNP50 array. Out of 45,868 single nucleotide polymorphisms in a panel of 144 inbred lines, 18 novel candidate genes were associated with head smut resistance in maize. These candidate genes were classified into three groups, namely, resistance genes, disease response genes, and other genes with possible plant disease resistance functions. The data suggested a complicated molecular mechanism of maize resistance against S. reiliana. This study also suggested that GWAS is a useful approach for identifying causal genetic factors for head smut resistance in maize.

Abstract Lung cancer remains the most common cause of cancer-related death worldwide and it continues to lack effective treatment. The increasingly large and diverse public databases of lung cancer gene expression constitute a rich source of candidate oncogenic drivers and therapeutic targets. To define novel targets for lung adenocarcinoma, we conducted a large-scale meta-analysis of genes specifically overexpressed in adenocarcinoma. We identified an 11-gene signature that was overexpressed consistently in adenocarcinoma specimens relative to normal lung tissue. Six genes in this signature were specifically overexpressed in adenocarcinoma relative to other subtypes of non–small cell lung cancer (NSCLC). Among these genes was the little studied protein tyrosine kinase PTK7. Immunohistochemical analysis confirmed that PTK7 is highly expressed in primary adenocarcinoma patient samples. RNA interference–mediated attenuation of PTK7 decreased cell viability and increased apoptosis in a subset of adenocarcinoma cell lines. Further, loss of PTK7 activated the MKK7–JNK stress response pathway and impaired tumor growth in xenotransplantation assays. Our work defines PTK7 as a highly and specifically expressed gene in adenocarcinoma and a potential therapeutic target in this subset of NSCLC. Cancer Res; 74(10); 2892–902. ©2014 AACR.

Lipids are prominent components of the nervous system. Here we performed a large-scale mass spectrometry-based analysis of the lipid composition of three brain regions as well as kidney and skeletal muscle of humans, chimpanzees, rhesus macaques, and mice. The human brain shows the most distinct lipid composition: 76% of 5,713 lipid compounds examined in our study are either enriched or depleted in the human brain. Concentration levels of lipids enriched in the brain evolve approximately four times faster among primates compared with lipids characteristic of non-neural tissues and show further acceleration of change in human neocortical regions but not in the cerebellum. Human-specific concentration changes are supported by human-specific expression changes for corresponding enzymes. These results provide the first insights into the role of lipids in human brain evolution.

Summary Maize plant height is closely associated with biomass, lodging resistance and grain yield. Determining the genetic basis of plant height by characterizing and cloning plant height genes will guide the genetic improvement of crops. In this study, a quantitative trait locus ( QTL ) for plant height, qPH3.1 , was identified on chromosome 3 using populations derived from a cross between Z ong3 and its chromosome segment substitution line, SL 15. The plant height of the two lines was obviously different, and application of exogenous gibberellin A 3 removed this difference. QTL mapping placed qPH3.1 within a 4.0 cM interval, explaining 32.3% of the phenotypic variance. Furthermore, eight homozygous segmental isolines ( SIL s) developed from two larger F 2 populations further narrowed down qPH3.1 to within a 12.6 kb interval. ZmGA3ox2 , an ortholog of OsGA3ox2 , which encodes a GA3 β ‐ hydroxylase, was positionally cloned. Association mapping identified two polymorphisms in ZmGA3ox2 that were significantly associated with plant height across two experiments. Quantitative RT ‐ PCR showed that SL 15 had higher ZmGA3ox2 expression relative to Z ong3. The resultant higher GA 1 accumulation led to longer internodes in SL 15 because of increased cell lengths. Moreover, a large deletion in the coding region of ZmGA3ox2 is responsible for the dwarf mutant d1‐6016 . The successfully isolated qPH3.1 enriches our knowledge on the genetic basis of plant height in maize, and provides an opportunity for improvement of plant architecture in maize breeding.

Low temperature is a major limiting factor in rice growth and development. Mapping of quantitative trait loci (QTLs) controlling cold tolerance is important for rice breeding. Recent studies have suggested that bulked segregant analysis (BSA) combined with next-generation sequencing (NGS) can be an efficient and cost-effective way for QTL mapping. In this study, we employed NGS-assisted BSA to map QTLs conferring cold tolerance at the seedling stage in rice. By deep sequencing of a pair of large DNA pools acquired from a very large F3 population (10,800 individuals), we obtained ∼450,000 single nucleotide polymorphisms (SNPs) after strict screening. We employed two statistical methods for QTL analysis based on these SNPs, which yielded consistent results. Six QTLs were mapped on chromosomes 1, 2, 5, 8 and 10. The three most significant QTLs on chromosomes 1, 2 and 8 were validated by comparison with previous studies. Two QTLs on chromosomes 2 and 5 were also identified previously, but at the booting stage rather than the seedling stage, suggesting that some QTLs may function at different developmental stages, which would be useful for cold tolerance breeding in rice. Compared with previously reported QTL mapping studies for cold tolerance in rice based on the traditional approaches, the results of this study demonstrated the advantages of NGS-assisted BSA in both efficiency and statistical power.

Among other factors, changes in gene expression on the human evolutionary lineage have been suggested to play an important role in the establishment of human-specific phenotypes. However, the molecular mechanisms underlying these expression changes are largely unknown. Here, we have explored the role of microRNA (miRNA) in the regulation of gene expression divergence among adult humans, chimpanzees, and rhesus macaques, in two brain regions: prefrontal cortex and cerebellum. Using a combination of high-throughput sequencing, miRNA microarrays, and Q-PCR, we have shown that up to 11% of the 325 expressed miRNA diverged significantly between humans and chimpanzees and up to 31% between humans and macaques. Measuring mRNA and protein expression in human and chimpanzee brains, we found a significant inverse relationship between the miRNA and the target genes expression divergence, explaining 2%-4% of mRNA and 4%-6% of protein expression differences. Notably, miRNA showing human-specific expression localize in neurons and target genes that are involved in neural functions. Enrichment in neural functions, as well as miRNA-driven regulation on the human evolutionary lineage, was further confirmed by experimental validation of predicted miRNA targets in two neuroblastoma cell lines. Finally, we identified a signature of positive selection in the upstream region of one of the five miRNA with human-specific expression, miR-34c-5p. This suggests that miR-34c-5p expression change took place after the split of the human and the Neanderthal lineages and had adaptive significance. Taken together these results indicate that changes in miRNA expression might have contributed to evolution of human cognitive functions.

MicroRNA-mediated gene regulation is important in many physiological processes. Here we explore the roles of a microRNA, miR-941, in human evolution. We find that miR-941 emerged de novo in the human lineage, between six and one million years ago, from an evolutionarily volatile tandem repeat sequence. Its copy-number remains polymorphic in humans and shows a trend for decreasing copy-number with migration out of Africa. Emergence of miR-941 was accompanied by accelerated loss of miR-941-binding sites, presumably to escape regulation. We further show that miR-941 is highly expressed in pluripotent cells, repressed upon differentiation and preferentially targets genes in hedgehog- and insulin-signalling pathways, thus suggesting roles in cellular differentiation. Human-specific effects of miR-941 regulation are detectable in the brain and affect genes involved in neurotransmitter signalling. Taken together, these results implicate miR-941 in human evolution, and provide an example of rapid regulatory evolution in the human linage.

Cancer-associated fibroblasts (CAF) have been reported to support tumor progression by a variety of mechanisms. However, their role in the progression of non-small cell lung cancer (NSCLC) remains poorly defined. In addition, the extent to which specific proteins secreted by CAFs contribute directly to tumor growth is unclear. To study the role of CAFs in NSCLCs, a cross-species functional characterization of mouse and human lung CAFs was conducted. CAFs supported the growth of lung cancer cells in vivo by secretion of soluble factors that directly stimulate the growth of tumor cells. Gene expression analysis comparing normal mouse lung fibroblasts and mouse lung CAFs identified multiple genes that correlate with the CAF phenotype. A gene signature of secreted genes upregulated in CAFs was an independent marker of poor survival in patients with NSCLC. This secreted gene signature was upregulated in normal lung fibroblasts after long-term exposure to tumor cells, showing that lung fibroblasts are "educated" by tumor cells to acquire a CAF-like phenotype. Functional studies identified important roles for CLCF1-CNTFR and interleukin (IL)-6-IL-6R signaling in promoting growth of NSCLCs. This study identifies novel soluble factors contributing to the CAF protumorigenic phenotype in NSCLCs and suggests new avenues for the development of therapeutic strategies.

Bacterial wilt caused by Ralstonia solanacearum is a ruinous soilborne disease affecting more than 450 plant species. Efficient control methods for this disease remain unavailable to date. This study characterized a novel nucleotide-binding site-leucine-rich repeat resistance gene AhRRS5 from peanut, which was up-regulated in both resistant and susceptible peanut cultivars in response to R. solanacearum. The product of AhRRS5 was localized in the nucleus. Furthermore, treatment with phytohormones such as salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (ET) increased the transcript level of AhRRS5 with diverse responses between resistant and susceptible peanuts. Abiotic stresses such as drought and cold conditions also changed AhRRS5 expression. Moreover, transient overexpression induced hypersensitive response in Nicotiana benthamiana. Overexpression of AhRRS5 significantly enhanced the resistance of heterogeneous tobacco to R. solanacearum, with diverse resistance levels in different transgenic lines. Several defence-responsive marker genes in hypersensitive response, including SA, JA and ET signals, were considerably up-regulated in the transgenic lines as compared with the wild type inoculated with R. solanacearum. Nonexpressor of pathogenesis-related gene 1 (NPR1) and non-race-specific disease resistance 1 were also up-regulated in response to the pathogen. These results indicate that AhRRS5 participates in the defence response to R. solanacearum through the crosstalk of multiple signalling pathways and the involvement of NPR1 and R gene signals for its resistance. This study may guide the resistance enhancement of peanut and other economic crops to bacterial wilt disease.

Polysaccharides named as PP1, PP2, PP3, PP4 and PP5 were extracted and isolated from the rhizomes of Panax japonicus C.A. Meyer, a well-known Chinese traditional medicine, by controlling the final concentration of solution to precipitate the polysaccharides. The molecular weight of polysaccharides was determined by HGPLC chromatography system. The monosaccharide composition was analyzed by Gas chromatography on an Agilent 6890A instrument using a DB-35MS column and flame-ionization detector. All of the polysaccharides were heteropolysaccharides and consisted of arabinose, glucose and galactose. The content of arabinose increased with the increasing of ethanol concentration and PP5 had the most arabinose content in these samples. IR spectra indicated obvious characteristic peaks of polysaccharide, the presence of uronic acids. Their antioxidant activities were evaluated by various established in vitro systems, including scavenging activity of superoxide anion, hydroxyl radical, ABTS and DPPH radicals. Both samples showed inhibitory effects on superoxide, hydroxyl, ABTS and DPPH radical. And PP5 shows more clearly and relatively stronger capacity than other polysaccharides on the protective effect of DNA damage.

The Cucurbitaceae is one of the most genetically diverse plant families in the world. Many of them are important vegetables or medicinal plants and are widely distributed worldwide. The rapid development of sequencing technologies and bioinformatic algorithms has enabled the generation of genome sequences of numerous important Cucurbitaceae species. This has greatly facilitated research on gene identification, genome evolution, genetic variation and molecular breeding of cucurbit crops. So far, genome sequences of 18 different cucurbit species belonging to tribes Benincaseae, Cucurbiteae, Sicyoeae, Momordiceae and Siraitieae have been deciphered. This review summarizes the genome sequence information, evolutionary relationship, and functional genes associated with important agronomic traits (e.g., fruit quality). The progress of molecular breeding in cucurbit crops and prospects for future applications of Cucurbitaceae genome information are also discussed.

Bell pepper is prone to chilling injury during cold storage. In this study, the plant hormone, methyl jasmonate (MeJA), which can stimulate the expression of plant defense genes, was used to investigate its effects on the transcriptome, metabolome and proteome after 6 days at 4 ℃. Differentially expressed (DE) mRNAs that were identified with plant hormone signaling included the transcription factor MYC2, and those associated with antioxidant properties, membrane lipids and cell wall modification, fruit quality and an important transcription factors pathway -- calmodulin-binding transcription activator - c-repeat-binding factor - transcription factor ICE1 (CMAT-CaCBF1A-ICE1). Effects on the proteome analysis were mainly associated with glutathione metabolism, biosynthesis of unsaturated fatty acids and fatty acid metabolism. Differentially expressed metabolites were identified as acids, phenolics, and capsaicin. Our study suggests that MeJA treatment mitigates chilling injury of bell peppers though inhibiting the MYC2-JA signaling pathway, enhancing the ASA-GSH cycle, reducing membrane lipid damage, suppressing cell wall disassembly, and activating the CMAT-CBF-ICE pathway. These results provide a theoretical basis for further study on chilling injury of bell pepper fruit.

Agricultural production has improved with the development of water deficit (WD) irrigation technology, but the impact of the application of WD irrigation on product quality is still understudied. The effects of WD irrigation on the transcriptome and metabolome of fruit have been investigated by treating tomato plants with control or WD irrigation during the growing period. WD irrigation promoted the accumulation of reducing sugars, total acids, vitamin C (VC) and soluble solid content (SSC) of the fruit, which increased the sweetness and enriched their taste. Transcripts of genes involved in fruit quality including color, texture and flavor, as well as plant hormones were affected by WD irrigation. Also, a combined analysis of multiple omics revealed that the genes and metabolites involved in galactose metabolism, starch and sucrose metabolism were affected by WD. This study provides insights into the effects of WD irrigation techniques on tomato fruit quality, which will provide new directions for the improvement of other fruit quality in the future.

Soil waterlogging is a major environmental stress that suppresses maize (Zea mays) growth and yield. To identify quantitative trait loci (QTL) associated with waterlogging tolerance at the maize seedling stage, a F2 population consisting of 288 F(2:3) lines was created from a cross between two maize genotypes, 'HZ32' (waterlogging-tolerant) and 'K12' (waterlogging-sensitive).The F2 population was genotyped and a base-map of 1710.5 cM length was constructed with an average marker space of 11.5 cM based on 177 SSR (simple sequence repeat) markers. QTL associated with root length, root dry weight, plant height, shoot dry weight, total dry weight and waterlogging tolerance coefficient were identified via composite interval mapping (CIM) under waterlogging and control conditions in 2004 (EXP.1) and 2005 (EXP.2), respectively.Twenty-five and thirty-four QTL were detected in EXP.1 and EXP.2, respectively. The effects of each QTL were moderate, ranging from 3.9 to 37.3 %. Several major QTL determining shoot dry weight, root dry weight, total dry weight, plant height and their waterlogging tolerance coefficient each mapped on chromosomes 4 and 9. These QTL were detected consistently in both experiments. Secondary QTL influencing tolerance were also identified and located on chromosomes 1, 2, 3, 6, 7 and 10. These QTL were specific to particular traits or environments. Although the detected regions need to be mapped more precisely, the findings and QTL found in this study may provide useful information for marker-assisted selection (MAS) and further genetic studies on maize waterlogging tolerance.

Waterlogging of plants leads to low oxygen levels (hypoxia) in the roots and causes a metabolic switch from aerobic respiration to anaerobic fermentation that results in rapid changes in gene transcription and protein synthesis. Our research seeks to characterize the microRNA-mediated gene regulatory networks associated with short-term waterlogging. MicroRNAs (miRNAs) are small non-coding RNAs that regulate many genes involved in growth, development and various biotic and abiotic stress responses. To characterize the involvement of miRNAs and their targets in response to short-term hypoxia conditions, a quantitative real time PCR (qRT-PCR) assay was used to quantify the expression of the 24 candidate mature miRNA signatures (22 known and 2 novel mature miRNAs, representing 66 miRNA loci) and their 92 predicted targets in three inbred Zea mays lines (waterlogging tolerant Hz32, mid-tolerant B73, and sensitive Mo17). Based on our studies, miR159, miR164, miR167, miR393, miR408 and miR528, which are mainly involved in root development and stress responses, were found to be key regulators in the post-transcriptional regulatory mechanisms under short-term waterlogging conditions in three inbred lines. Further, computational approaches were used to predict the stress and development related cis-regulatory elements on the promoters of these miRNAs; and a probable miRNA-mediated gene regulatory network in response to short-term waterlogging stress was constructed. The differential expression patterns of miRNAs and their targets in these three inbred lines suggest that the miRNAs are active participants in the signal transduction at the early stage of hypoxia conditions via a gene regulatory network; and crosstalk occurs between different biochemical pathways.

Human evolution is characterized by the rapid expansion of brain size and drastic increase in cognitive capabilities. It has long been suggested that these changes were accompanied by modifications of brain metabolism. Indeed, human-specific changes on gene expression or amino acid sequence were reported for a number of metabolic genes, but actual metabolite measurements in humans and apes have remained scarce. Here, we investigate concentrations of more than 100 metabolites in the prefrontal and cerebellar cortex in 49 humans, 11 chimpanzees, and 45 rhesus macaques of different ages using gas chromatography-mass spectrometry (GC-MS). We show that the brain metabolome undergoes substantial changes, both ontogenetically and evolutionarily: 88% of detected metabolites show significant concentration changes with age, whereas 77% of these metabolic changes differ significantly among species. Although overall metabolic divergence reflects phylogenetic relationships among species, we found a fourfold acceleration of metabolic changes in prefrontal cortex compared with cerebellum in the human lineage. These human-specific metabolic changes are paralleled by changes in expression patterns of the corresponding enzymes, and affect pathways involved in synaptic transmission, memory, and learning.

Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys.

Although Neanderthals are extinct, fragments of their genomes persist in contemporary humans. Here we show that while the genome-wide frequency of Neanderthal-like sites is approximately constant across all contemporary out-of-Africa populations, genes involved in lipid catabolism contain more than threefold excess of such sites in contemporary humans of European descent. Evolutionally, these genes show significant association with signatures of recent positive selection in the contemporary European, but not Asian or African populations. Functionally, the excess of Neanderthal-like sites in lipid catabolism genes can be linked with a greater divergence of lipid concentrations and enzyme expression levels within this pathway, seen in contemporary Europeans, but not in the other populations. We conclude that sequence variants that evolved in Neanderthals may have given a selective advantage to anatomically modern humans that settled in the same geographical areas.

To better understand the physiological and biochemical mechanisms of waterlogging tolerance, waterlogging effects on lipid peroxidation and the activity of antioxidative enzymes were investigated in leaves and roots of two maize genotypes, HZ32 (waterlogging-tolerant) and K12 (waterlogging-sensitive). Potted maize plants were waterlogged at the second leaf stage under glasshouse conditions. Leaves and roots were harvested 1 d before and 2, 4, 6, 8 and 10 d after the start of waterlogging treatment. Through comparing the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT) and guaiacol peroxidase (POD) between waterlogging-tolerant and waterlogging-sensitive genotype, we deduced that CAT was the most important H2O2 scavenging enzyme in leaves, while APX seemed to play a key role in roots. POD, APX, GR and CAT activities in conjunction with SOD seem to play an essential protective role in the O2T and H2O2 scavenging process. Lipid peroxidation was enhanced significantly only in K12 (P < 0.001) and there was no difference (P > 0.05) in HZ32 up to 6 d after waterlogging stress. These results indicated that oxidative stress may play an important role in waterlogging-stressed maize plants and that the greater protection of HZ32 leaves and roots from waterlogging-induced oxidative damage results, at least in part, through the maintenance of increased antioxidant enzyme activity.

Poria cocos F.A.Wolf is a Chinese traditional medicine used to treat chronic gastritis, edema, nephrosis, gastric atony, and acute gastroenteric catarrh. Polysaccharides are the main active component of P. cocos. We obtained polysaccharides PCP-1, PCP-2, and PCP-3 from the degradation of P. cocos polysaccharides (PCP) with different concentrations of H2O2 solution. Molecular weights were determined by high performance size exclusion chromatography. HPLC analysis of monosaccharide composition confirmed that PCP-1, PCP-2, and PCP-3 are heteropolysaccharides composed of glucose and arabinose. IR spectra indicated obvious characteristic peaks of polysaccharides. The antioxidant activities of these polysaccharides were evaluated by established in vitro systems, including scavenging activity of hydroxyl radicals, ABTS radicals, and ferrous ions. The degradation polysaccharides exhibited obvious and concentration-dependent antioxidant properties. In addition, DNA binding analysis showed that PCP-1 had a stronger capacity than other polysaccharides to interact with DNA. However, each polysaccharide had a certain capacity for DNA damage protection.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that repress the expression of their target genes post-transcriptionally. MiRNAs participate in the regulation of a variety of biological processes, including development and diseases. However, the functional role and molecular mechanism by which miRNAs regulate skeletal muscle development and differentiation are not fully understood. In this report, we identified miR-23a as a key regulator of skeletal muscle differentiation. Using bioinformatics analyses, miR-23a is predicted to target multiple adult fast myosin heavy chain (Myh) genes, including Myh 1, 2 and 4. Luciferase reporter assays show that miR-23a directly targets the 3' untranslated regions (UTRs) of these mRNAs. Interestingly, the expression level of mature miR-23a is inversely correlated with myogenic progression in mouse skeletal muscle. Both gain- and loss-of-function studies using C2C12 myoblasts demonstrate that miR-23a inhibits myogenic differentiation. These findings therefore reveal a novel role of miR-23a in regulating myogenic differentiation via inhibiting the expression of fast myosin heavy chain isoforms.

MicroRNAs (miRNAs) are critical post‐transcriptional modulators of gene expression involving in plant responses to abiotic stress. However, the regulation of miRNA in the morphological response to waterlogging is poorly understood in maize. In this study, we detected miRNAs and their targets that expressed in waterlogged crown roots of maize seedlings in two inbred lines (Hz32 and Mo17) by RNA sequencing. A total of 61 mature miRNAs were found including 36 known maize (zma) miRNAs and 25 potential novel miRNA candidates. Comparison of miRNA expression in both waterlogged and control crown roots revealed 32 waterlogging‐responsive miRNAs, most were consistently downregulated under waterlogging in the two inbred lines. We identified the miRNA targets through degradome sequencing. Many known miRNA targets involving in transcription regulation and reactive oxygen species elimination were found in the degradome libraries, and 17 targets of 10 newly detected miRNAs were identified as well. Moreover, the miRNA‐mediated pathways that respond to waterlogging and regulate the induction of crown roots were discussed. This study is a comprehensive survey of responsive miRNAs in waterlogged maize crown roots. The results will help to understand the miRNA expression in response to waterlogging and miRNA‐mediated regulation of morphological adaptation to waterlogging in maize.

Soil waterlogging is one of the major abiotic stresses adversely affecting maize growth and yield. To identify dynamic expression of genes or quantitative trait loci (QTL), QTL associated with plant height, root length, root dry weight, shoot dry weight and total dry weight were identified via conditional analysis in a mixed linear model and inclusive composite interval mapping method at three respective periods under waterlogging and control conditions. A total of 13, 19 and 23 QTL were detected at stages 3D|0D (the period during 0–3 d of waterlogging), 6D|3D and 9D|6D, respectively. The effects of each QTL were moderate and distributed over nine chromosomes, singly explaining 4.14–18.88% of the phenotypic variation. Six QTL (ph6-1, rl1-2, sdw4-1, sdw7-1, tdw4-1 and tdw7-1) were identified at two consistent stages of seedling development, which could reflect a continuous expression of genes; the remaining QTL were detected at only one stage. Thus, expression of most QTL was influenced by the developmental status. In order to provide additional evidence regarding the role of corresponding genes in waterlogging tolerance, mapping of Expressed Sequence Tags markers and microRNAs were conducted. Seven candidate genes were observed to co-localize with the identified QTL on chromosomes 1, 4, 6, 7 and 9, and may be important candidate genes for waterlogging tolerance. These results are a good starting point for understanding the genetic basis for selectively expressing of QTL in different stress periods and the common genetic control mechanism of the co-localized traits.

Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non-small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1(HI) subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.

Abstract The agronomical relevant tomato- Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.

With the development of new high-throughput DNA sequencing technologies and decreasing costs, large gene expression datasets are being generated at an accelerating rate, but can be complex to visualize. New, more interactive and intuitive tools are needed to visualize the spatiotemporal context of expression data and help elucidate gene function. Using tomato fruit as a model, we have developed the Tomato Expression Atlas to facilitate effective data analysis, allowing the simultaneous visualization of groups of genes at a cell/tissue level of resolution within an organ, enhancing hypothesis development and testing in addition to candidate gene identification. This atlas can be adapted to different types of expression data from diverse multicellular species.The Tomato Expression Atlas is available at http://tea.solgenomics.net/ . Source code is available at https://github.com/solgenomics/Tea .jr286@cornell.edu or lam87@cornell.edu.Supplementary data are available at Bioinformatics online.

Most primary auxin response genes are classified into three families: AUX/IAA, GH3 and SAUR genes. Few studies have been conducted on Arabidopsis thaliana SAUR genes, possibly due to genetic redundancy among different subfamily members. Data mining on arabidopsis transcriptional profiles indicates that the SAUR41 subfamily members of SMALL AUXIN UP RNA genes are, strikingly, induced by an inhibitory phytohormone, abscisic acid (ABA). We aimed to reveal the physiological roles of arabidopsis SAUR41 subfamily genes containing SAUR40, SAUR41, SAUR71 and SAUR72.Transcriptional responses of arabidopsis SAUR41 genes to phytohormones were determined by quantitative real-time PCR. Knock out of SAUR41 genes was carried out with the CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing technique. The saur41/40/71/72 quadruple mutants, SAUR41 overexpression lines and the wild type were subjected to ultrastructural observation, transcriptome analysis and physiological characterization.Transcription of arabidopsis SAUR41 subfamily genes is activated by ABA but not by gibberellic acids and brassinosteroids. Quadruple mutations in saur41/40/71/72 led to reduced cell expansion/elongation in cotyledons and hypocotyls, opposite to the overexpression of SAUR41; however, an irregular arrangement of cell size and shape was observed in both cases. The quadruple mutants had increased transcription of calcium homeostasis/signalling genes in seedling shoots, and the SAUR41 overexpression lines had decreased transcription of iron homeostasis genes in roots and increased ABA biosynthesis in shoots. Notably, both the quadruple mutants and the SAUR41 overexpression lines were hypersensitive to salt stress during seedling establishment, whereas specific expression of SAUR41 under the ABA-responsive RD29A (Responsive to Desiccation 29A) promoter in the quadruple mutants rescued the inhibitory effect of salt stress.The SAUR41 subfamily genes of arabidopsis are ABA inducible to modulate cell expansion, ion homeostasis and salt tolerance. Our work may provide new candidate genes for improvement of plant abiotic stress tolerance.

Singlet oxygen (1O2), the major reactive oxygen species (ROS) produced in chloroplasts, has been demonstrated recently to be a highly versatile signal that induces various stress responses. In the fluorescent (flu) mutant, its release causes seedling lethality and inhibits mature plant growth. However, these drastic phenotypes are suppressed when EXECUTER1 (EX1) is absent in the flu ex1 double mutant. We identified SAFEGUARD1 (SAFE1) in a screen of ethyl methanesulfonate (EMS) mutagenized flu ex1 plants for suppressor mutants with a flu-like phenotype. In flu ex1 safe1, all 1O2-induced responses, including transcriptional rewiring of nuclear gene expression, return to levels, such as, or even higher than, those in flu Without SAFE1, grana margins (GMs) of chloroplast thylakoids (Thys) are specifically damaged upon 1O2 generation and associate with plastoglobules (PGs). SAFE1 is localized in the chloroplast stroma, and release of 1O2 induces SAFE1 degradation via chloroplast-originated vesicles. Our paper demonstrates that flu-produced 1O2 triggers an EX1-independent signaling pathway and proves that SAFE1 suppresses this signaling pathway by protecting GMs.

Prostaglandin E2 (PGE2), an arachidonic acid pathway metabolite produced by cyclooxygenase (COX)-1/2, has been shown to impair anti-tumor immunity through engagement with one or more E-type prostanoid receptors (EP1-4). Specific targeting of EP receptors, as opposed to COX-1/2 inhibition, has been proposed to achieve preferential antagonism of PGE2–mediated immune suppression. Here we describe the anti-tumor activity of MF-766, a potent and highly selective small-molecule inhibitor of the EP4 receptor. EP4 inhibition by MF-766 synergistically improved the efficacy of anti-programmed cell death protein 1 (PD-1) therapy in CT26 and EMT6 syngeneic tumor mouse models. Multiparameter flow cytometry analysis revealed that treatment with MF-766 promoted the infiltration of CD8+ T cells, natural killer (NK) cells and conventional dendritic cells (cDCs), induced M1-like macrophage reprogramming, and reduced granulocytic myeloid-derived suppressor cells (MDSC) in the tumor microenvironment (TME). In vitro experiments demonstrated that MF-766 restored PGE2-mediated inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in THP-1 cells and human blood, and PGE2-mediated inhibition of interleukin (IL)-2-induced interferon (IFN)-γ production in human NK cells. MF-766 reversed the inhibition of IFN-γ in CD8+ T-cells by PGE2 and impaired suppression of CD8+ T-cells induced by myeloid-derived suppressor cells (MDSC)/PGE2. In translational studies using primary human tumors, MF-766 enhanced anti-CD3-stimulated IFN-γ, IL-2, and TNF-α production in primary histoculture and synergized with pembrolizumab in a PGE2 high TME. Our studies demonstrate that the combination of EP4 blockade with anti-PD-1 therapy enhances antitumor activity by differentially modulating myeloid cell, NK cell, cDC and T-cell infiltration profiles.

Myelofibrosis (MF) is a disease associated with high unmet medical needs because allogeneic stem cell transplantation is not an option for most patients, and JAK inhibitors are generally effective for only 2 to 3 years and do not delay disease progression. MF is characterized by dysplastic megakaryocytic hyperplasia and progression to fulminant disease, which is associated with progressively increasing marrow fibrosis. Despite evidence that the inflammatory milieu in MF contributes to disease progression, the specific factors that promote megakaryocyte growth are poorly understood. Here, we analyzed changes in the cytokine profiles of MF mouse models before and after the development of fibrosis, coupled with the analysis of bone marrow populations using single-cell RNA sequencing. We found high interleukin 13 (IL-13) levels in the bone marrow of MF mice. IL-13 promoted the growth of mutant megakaryocytes and induced surface expression of transforming growth factor β and collagen biosynthesis. Similarly, analysis of samples from patients with MF revealed elevated levels of IL-13 in the plasma and increased IL-13 receptor expression in marrow megakaryocytes. In vivo, IL-13 overexpression promoted disease progression, whereas reducing IL-13/IL-4 signaling reduced several features of the disease, including fibrosis. Finally, we observed an increase in the number of marrow T cells and mast cells, which are known sources of IL-13. Together, our data demonstrate that IL-13 is involved in disease progression in MF and that inhibition of the IL-13/IL-4 signaling pathway might serve as a novel therapeutic target to treat MF.

Momordica charantia L. var. abbreviata Ser. (Mca), known as bitter gourd or bitter melon, is a Momordica variety with medicinal value and belongs to the Cucurbitaceae family. In view of the lack of genomic information on bitter gourd and other Momordica species and to promote Mca genomic research, we assembled a 295.6-Mb telomere-to-telomere (T2T) high-quality Mca genome with six gap-free chromosomes after Hi-C correction. This genome is anchored to 11 chromosomes, which is consistent with the karyotype information, and comprises 98 contigs (N50 of 25.4 Mb) and 95 scaffolds (N50 of 25.4 Mb). The Mca genome harbors 19 895 protein-coding genes, of which 45.59% constitute predicted repeat sequences. Synteny analysis revealed variations involved in fruit quality during the divergence of bitter gourd. In addition, assay for transposase-accessible chromatin by high-throughput sequencing and metabolic analysis showed that momordicosides and other substances are characteristic of Mca fruit pulp. A combined transcriptomic and metabolomic analysis revealed the mechanisms of pigment accumulation and cucurbitacin biosynthesis in Mca fruit peels, providing fundamental molecular information for further research on Mca fruit ripening. This report provides a new genetic resource for Momordica genomic studies and contributes additional insights into Cucurbitaceae phylogeny.

Abstract Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), can significantly affect wheat production. Cloning resistance genes is critical for efficient and effective breeding of stripe rust resistant wheat cultivars. One resistance gene ( Yr10 CG ) underlying the Pst resistance locus Yr10 has been cloned. However, following haplotype and linkage analyses indicate the presence of additional Pst resistance gene(s) underlying/near Yr10 locus. Here, we report the cloning of the Pst resistance gene YrNAM in this region using the method of sequencing trait-associated mutations (STAM). YrNAM encodes a non-canonical resistance protein with a NAM domain and a ZnF-BED domain. We show that both domains are required for resistance. Transgenic wheat harboring YrNAM gene driven by its endogenous promoter confers resistance to stripe rust races CYR32 and CYR33. YrNAM is an ancient gene and present in wild wheat species Aegilops longissima and Ae. sharonensis ; however, it is absent in most wheat cultivars, which indicates its breeding value.

Hepatitis B virus (HBV) core promoter contains a binding site for nuclear receptors. A natural double mutation in this binding site, which changes nucleotide (nt) 1765 from A to T and nt 1767 from G to A, selectively abolishes the binding of several nuclear receptors without affecting that of HNF4. This double mutation also creates a binding site for the transcription factor HNF1 and changes two amino acids in the overlapping X protein sequence. In this study, we have examined the roles of HNF1, HNF4, and the X protein in the regulation of the core promoter activities in Huh7 hepatoma cells. Our results indicate that HNF4 could stimulate the expression of the precore RNA and the core RNA from the core promoter of both the wild-type (WT) HBV and the double mutant, although its effect on the former was more prominent. In contrast, HNF1, which did not affect the WT core promoter, suppressed the precore RNA expression of the double mutant. Further analysis using HBV genomic constructs, with and without the ability to express the X protein, indicates that the X protein did not affect the HNF4 activity on the core promoter and affected the HNF1 activity on the core promoter of only the double mutant. Thus, our results indicate that the phenotypic differences of HBV WT and double-mutant core promoters are at least partially due to the differential activities of HNF1, HNF4, and the X protein on these two promoters.

HBV is a major risk factor for hepatocellular carcinoma (HCC). However, whether HBV can directly cause HCC or only indirectly via the induction of chronic liver inflammation has been controversial. By using transgenic mice carrying the entire HBV genome as a model, we now demonstrate that HBV by itself is an inefficient carcinogen. However, it can efficiently promote hepatocarcinogenesis initiated by the carcinogen diethylnitrosamine (DEN). This effect of HBV does not involve chronic liver inflammation, is apparently due to enhanced hepatocellular apoptosis and compensatory regeneration following DEN treatment, and does not require the HBV X protein. Conclusion: Our results demonstrate a direct role of HBV in a hepatocarcinogenesis pathway that involves the interaction between this virus and a dietary carcinogen. (HEPATOLOGY 2007;45:16–21.)

Plants respond to low oxygen stress, particularly that caused by waterlogging, by altering transcription and translation. Previous studies have mostly focused on revealing the mechanism of the response at the early stage, and there is limited information about the transcriptional profile of genes in maize roots at the late stage of waterlogging. The genetic basis of waterlogging tolerance is largely unknown. In this study, the transcriptome at the late stage of waterlogging was assayed in root cells of the tolerant inbred line HZ32, using suppression subtractive hybridization (SSH). A forward SSH library using RNA populations from four time points (12 h, 16 h, 20 h and 24 h) after waterlogging treatment was constructed to reveal up-regulated genes, and transcriptional and linkage data was integrated to identify candidate genes for waterlogging tolerance.Reverse Northern analysis of a set of 768 cDNA clones from the SSH library revealed a large number of genes were up-regulated by waterlogging. A total of 465 ESTs were assembled into 296 unigenes. Bioinformatic analysis revealed that the genes were involved in complex pathways, such as signal transduction, protein degradation, ion transport, carbon and amino acid metabolism, and transcriptional and translational regulation, and might play important roles at the late stage of the response to waterlogging. A significant number of unigenes were of unknown function. Approximately 67% of the unigenes could be aligned on the maize genome and 63 of them were co-located within reported QTLs.The late response to waterlogging in maize roots involves a broad spectrum of genes, which are mainly associated with two response processes: defense at the early stage and adaption at the late stage. Signal transduction plays a key role in activating genes related to the tolerance mechanism for survival during prolonged waterlogging. The crosstalk between carbon and amino acid metabolism reveals that amino acid metabolism performs two main roles at the late stage: the regulation of cytoplasmic pH and energy supply through breakdown of the carbon skeleton.

Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize.

Maize kernel row number might be dominated by a set of large additive or partially dominant loci and several small dominant loci and can be accurately predicted by fewer than 300 top KRN-associated SNPs. Kernel row number (KRN) is an important yield component in maize and directly affects grain yield. In this study, we combined linkage and association mapping to uncover the genetic architecture of maize KRN and to evaluate the phenotypic predictability using these detected loci. A genome-wide association study revealed 31 associated single nucleotide polymorphisms (SNPs) representing 17 genomic loci with an effect in at least one of five individual environments and the best linear unbiased prediction (BLUP) over all environments. Linkage mapping in three F2:3 populations identified 33 KRN quantitative trait loci (QTLs) representing 21 QTLs common to several population/environments. The majority of these common QTLs that displayed a large effect were additive or partially dominant. We found 70% KRN-associated genomic loci were mapped in KRN QTLs identified in this study, KRN-associated SNP hotspots detected in NAM population and/or previous identified KRN QTL hotspots. Furthermore, the KRN of inbred lines and hybrids could be predicted by the additive effect of the SNPs, which was estimated using inbred lines as a training set. The prediction accuracy using the top KRN-associated tag SNPs was obviously higher than that of the randomly selected SNPs, and approximately 300 top KRN-associated tag SNPs were sufficient for predicting the KRN of the inbred lines and hybrids. The results suggest that the KRN-associated loci and QTLs that were detected in this study show great potential for improving the KRN with genomic selection in maize breeding.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Long non-coding RNAs (lncRNAs) have been confirmed to play a critical regulatory role in various biological processes including carcinogenesis, which indicates that lncRNAs are valuable biomarkers and therapeutic targets. The novel lncRNA prostate cancer non-coding RNA 1 (PRNCR1) is located in the susceptible genomic area of CRC, however the functional role of PRNCR1 remains unknown. Thus, we aimed to investigate the clinical significance and biological function of PRNCR1 in CRC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expression profile of PRNCR1 in CRC tissues and cell lines. An antisense oligonucleotide (ASO) was designed to knock down PRNCR1. In a cohort of 63 patients, PRNCR1 was significantly overexpressed in CRC tissues compared with the expression in adjacent tissues, with an average fold increase of 10.55 (P=0.006). Additionally, a high level of PRNCR1 was associated with large tumor volume (P<0.05). Based on receiver operating characteristic curve (ROC), we found that the area under the curve (AUC) of PRNCR1 was 0.799 while the AUC of conventional biomarker CEA-CA199 was 0.651, indicating that PRNCR1 could be a sensitive diagnostic biomarker of CRC. Compared with the normal human colorectal epithelial cell line (FHC), PRNCR1 was upregulated in most CRC cell lines (HCT116, SW480, LoVo and HT-29). After knockdown of PRNCR1 by ASO, CRC cell proliferation ability was significantly inhibited. We further found that PRNCR1 knockdown induced cell cycle arrest in the G0/G1 phase and a significant decrease in the proportion of cells in the S phases. In contrast, PRNCR1 knockdown did not affect cell apoptosis or invasive ability. Hence, these data indicate that PRNCR1 promotes the proliferation of CRC cells and is a potential oncogene of CRC.

Whiteflies threaten agricultural crop production worldwide, are polyphagous in nature, and transmit hundreds of plant viruses. Little is known how whitefly gene expression is altered due to feeding on plants infected with a semipersistently transmitted virus. Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae) is transmitted by the whitefly (Bemisia tabaci) in a semipersistent manner and infects several globally important agricultural and ornamental crops, including tomato.To determine changes in global gene regulation in whiteflies after feeding on tomato plants infected with a crinivirus (ToCV), comparative transcriptomic analysis was performed using RNA-Seq on whitefly (Bemisia tabaci MEAM1) populations after 24, 48, and 72 h acquisition access periods on either ToCV-infected or uninfected tomatoes. Significant differences in gene expression were detected between whiteflies fed on ToCV-infected tomato and those fed on uninfected tomato among the three feeding time periods: 447 up-regulated and 542 down-regulated at 24 h, 4 up-regulated and 7 down-regulated at 48 h, and 50 up-regulated and 160 down-regulated at 72 h. Analysis revealed differential regulation of genes associated with metabolic pathways, signal transduction, transport and catabolism, receptors, glucose transporters, α-glucosidases, and the uric acid pathway in whiteflies fed on ToCV-infected tomatoes, as well as an abundance of differentially regulated novel orphan genes. Results demonstrate for the first time, a specific and temporally regulated response by the whitefly to feeding on a host plant infected with a semipersistently transmitted virus, and advance the understanding of the whitefly vector-virus interactions that facilitate virus transmission.Whitefly transmission of semipersistent viruses is believed to require specific interactions between the virus and its vector that allow binding of virus particles to factors within whitefly mouthparts. Results provide a broader understanding of the potential mechanism of crinivirus transmission by whitefly, aid in discerning genes or loci in whitefly that influence virus interactions or transmission, and subsequently facilitate development of novel, genetics-based control methods against whitefly and whitefly-transmitted viruses.

BackgroundAllergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness.ObjectiveWe sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness.MethodsWe assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model.ResultsInflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl− currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor–TMEM16A–voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity.ConclusionsA G protein-coupled receptor–TMEM16A–voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli. Allergic inflammation has long been implicated in asthmatic hyperresponsiveness of airway smooth muscle (ASM), but its underlying mechanism remains incompletely understood. Serving as G protein-coupled receptor agonists, several inflammatory mediators can induce membrane depolarization, contract ASM, and augment cholinergic contractile response. We hypothesized that the signal cascade integrating on membrane depolarization by the mediators might involve asthmatic hyperresponsiveness. We sought to investigate the signaling transduction of inflammatory mediators in ASM contraction and assess its contribution in the genesis of hyperresponsiveness. We assessed the capacity of inflammatory mediators to induce depolarization currents by electrophysiological analysis. We analyzed the phenotypes of transmembrane protein 16A (TMEM16A) knockout mice, applied pharmacological reagents, and measured the Ca2+ signal during ASM contraction. To study the role of the depolarization signaling in asthmatic hyperresponsiveness, we measured the synergistic contraction by methacholine and inflammatory mediators both ex vivo and in an ovalbumin-induced mouse model. Inflammatory mediators, such as 5-hydroxytryptamin, histamine, U46619, and leukotriene D4, are capable of inducing Ca2+-activated Cl− currents in ASM cells, and these currents are mediated by TMEM16A. A combination of multiple analysis revealed that a G protein-coupled receptor–TMEM16A–voltage-dependent Ca2+ channel signaling axis was required for ASM contraction induced by inflammatory mediators. Block of TMEM16A activity may significantly inhibit the synergistic contraction of acetylcholine and the mediators and hence reduces hypersensitivity. A G protein-coupled receptor–TMEM16A–voltage-dependent Ca2+ channel axis contributes to inflammatory mediator-induced ASM contraction and synergistically activated TMEM16A by allergic inflammatory mediators with cholinergic stimuli.

RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.

Dipeptidyl peptidase IV (DPP-IV) has an important role in blood glucose metabolism. Silver carp swim bladder protein was hydrolyzed using papain, bromelain, Alcalase 2.4 L, Neutrase, and Flavourzyme. The swim bladder hydrolysate obtained using 5 h hydrolysis of Neutrase showed the highest DPP-IV inhibition (81 ± 2%) at 5 mg/mL. This hydrolysate was purified sequentially using ultrafiltration, gel filtration chromatography, and RP-HPLC. Peptide sequences from the RP-HPLC fractions were identified using liquid chromatography tandem mass spectrometry. The most potent DPP-IV inhibitory peptide WGDEHIPGSPYH (IC50: 0.35 ± 0.01 mM) showed an uncompetitive inhibition mode. Docking analysis showed that the binding site of WGDEHIPGSPYH was located inside the cavity of the DPP-IV monomer, which was close to the catalytic site. WGDEHIPGSPYH and its hydrolysate IPGSPY were transported intact across a Caco-2 cell monolayer. These two peptides were tested for their effects on Caco-2 and INS-1 cells and both showed good inhibition for soluble DPP-IV and promoted insulin secretion.

Significance Guanylate-binding proteins (GBPs) belong to interferon-inducible GTPases and mediate a broad spectrum of innate immune responses against various pathogens. Their protective functions require oligomerization induced by nucleotide binding and/or catalysis, but the actual molecular mechanisms are still elusive. Here, we report the crystal structures of human GBP5 (hGBP5) in both its nucleotide-free state and nucleotide-bound state, as well as nucleotide-free human GBP2 (hGBP2). We show that hGBP5 forms a closed face-to-face dimer upon GTP loading. This closed conformation is crucial to its anti–HIV-1 activity. Furthermore, with hGBP2 structure and SAXS validation, we propose a plausible working model for GTP-induced assembly of GBPs. Our findings lay the foundation to better understand the molecular mechanisms of GBPs and their immune functions.

Green pepper is regarded as one of the globally essential commercial crops with bright color and rich nutrition. However, green pepper is susceptible to mechanical injuries as a result of vibration, compression and impact events during postharvest handling and transportation. These injuries can induce physiological and biochemical abnormalities in fruit, thus accelerating fruit senescence and leading to economic losses. Calcium chloride (CaCl2) treatment is a safe, effective and economical method to maintain fruit quality during postharvest storage. Here, the impact of calcium treatment on the fruit quality of mechanically injured green pepper was investigated. The results indicated that calcium treatment maintained the sensory quality and hardness of green pepper fruit, inhibited redness development and elevation in the level of malondialdehyde (MDA). The combination of transcriptomics, metabolomics and assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) revealed treatment effects of the expression of key genes related to fruit color change, fruit softening, active oxygen metabolism and hormone response, as well as changes of metabolites concentrations, and chromatin accessibility changes. A construction of regulatory network model of calcium treatment on post-harvest mechanical injury of green pepper fruit was developed based on these effects. Overall, the study provides valuable insights into the impact of mechanical injury on fruit after harvest and its amelioration by calcium.

Several physiological changes occur during fruit storage, which include the regulation of genes, metabolisms and transcription factors. In this study, we compared 'JF308' (a normal tomato cultivar) and 'YS006' (a storable tomato cultivar) to determine the difference in accumulated metabolites, gene expression, and accessible chromatin regions through metabolome, transcriptome, and ATAC-seq analysis. A total of 1006 metabolites were identified in two cultivars. During storage time, sugars, alcohols and flavonoids were found to be more abundant in 'YS006' compared to 'JF308' on day 7, 14, and 21, respectively. Differentially expressed genes, which involved in starch and sucrose biosynthesis were observed higher in 'YS006'. 'YS006' had lower expression levels of CesA (cellulose synthase), PL (pectate lyase), EXPA (expansin) and XTH (xyglucan endoglutransglucosylase/hydrolase) than 'JF308'. The results showed that phenylpropanoid pathway, carbohydrate metabolism and cell wall metabolism play important roles in prolonging the shelf life of tomato (Solanum lycopersicum) fruit. The ATAC-seq analysis revealed that the most significantly up-regulated transcription factors during storage were TCP 2,3,4,5, and 24 in 'YS006' compared to 'JF308' on day 21. This information on the molecular regulatory mechanisms and metabolic pathways of post-harvest quality changes in tomato fruit provides a theoretical foundation for slowing post-harvest decay and loss, and has theoretical importance and application value in breeding for longer shelf life cultivars.

Abstract Panicle architecture is a key determinant of rice grain yield and is mainly determined at the 1-2 mm young panicle stage. Here, we investigated the transcriptome of the 1-2 mm young panicles from 275 rice varieties and identified thousands of genes whose expression levels were associated with panicle traits. Multimodel association studies suggested that many small-effect genetic loci determine spikelet per panicle (SPP) by regulating the expression of genes associated with panicle traits. We found that alleles at cis -expression quantitative trait loci of SPP-associated genes underwent positive selection, with a strong preference for alleles increasing SPP. We further developed a method that integrates the associations of cis - and trans -expression components of genes with traits to identify causal genes at even small-effect loci and construct regulatory networks. We identified 36 putative causal genes of SPP, including SDT (MIR156j) and OsMADS17 , and inferred that OsMADS17 regulates SDT expression, which was experimentally validated. Our study reveals the impact of regulatory variants on rice panicle architecture and provides new insights into the gene regulatory networks of panicle traits.

The effects of exogenous NO generated from sodium nitroprusside (SNP) on hyacinth bean have been investigated with metabolomic, transcriptomic, and Assay for Transposase-Accessible Chromatin with high-throughput Sequencing (ATAC-seq) analyses. Comparative analysis revealed 421 and 253 differentially accumulated metabolites (DAMs) in control 0 d vs control 8 d, and control 0 d vs SNP 8 d, respectively. Flavonoid biosynthesis accounted for 50% and 66% of the DAMs in the two comparative groups, respectively. The key flavonoid synthesis genes (PAL, CHS, CHI) were downregulated in NO treated beans. NO also downregulated genes related to cell wall degradation, lignin synthesis, plant pathogen interaction, and ethylene signal transduction. ATAC-seq analysis identified 14 co-enriched transcription factors from the ERF /DREB family, suggesting their role in regulating bean senescence. Overall, NO regulated flavonoid metabolism, cell wall degradation, lignin synthesis, plant hormones, flavonoid biosynthesis, plant pathogen interaction genes, and the ERF /DREB transcription factor family. These findings provide information about postharvest quality regulation of bean senescence.

The development of cationic polymers that simulate antimicrobial peptides to treat bacterial infections has received much research interest. In order to obtain polymers that can not only eradicate bacteria but also inhibit biofilm formation, without inducing bacterial drug resistance, a series of cationic polymers have been developed. Despite recent progress, the chemical structures of these polymers are stable, making them recalcitrant to biodegradation and metabolism within organisms, potentially inducing long-term toxicity. To overcome this limitation, herein, a novel strategy of designing biodegradable polyurethanes with tertiary amines and quaternary ammonium salts via condensation polymerization and post-functionalizing them is reported. These polymers were found to exhibit potent antibacterial activity against Staphylococcus aureus and Escherichia coli, effectively prevent the formation of Staphylococcus aureus biofilms, act quickly and effectively against bacteria and display no resistance after repeated use. In addition, the potent in vivo antibacterial effects of these antimicrobial polyurethanes in a mouse model with methicillin-resistant Staphylococcus aureus skin infection are demonstrated.

Due to its unique structure, articular cartilage has limited abilities to undergo self-repair after injury. Additionally, the repair of articular cartilage after injury has always been a difficult problem in the field of sports medicine. Previous studies have shown that the therapeutic use of mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) has great potential for promoting cartilage repair. Recent studies have demonstrated that most transplanted stem cells undergo apoptosis in vivo, and the apoptotic EVs (ApoEVs) that are subsequently generated play crucial roles in tissue repair. Additionally, MSCs are known to exist under low-oxygen conditions in the physiological environment, and these hypoxic conditions can alter the functional and secretory properties of MSCs as well as their secretomes. This study aimed to investigate whether ApoEVs that are isolated from adipose-derived MSCs cultured under hypoxic conditions (hypoxic apoptotic EVs [H-ApoEVs]) exert greater effects on cartilage repair than those that are isolated from cells cultured under normoxic conditions. Through in vitro cell proliferation and migration experiments, we demonstrated that H-ApoEVs exerted enhanced effects on stem cell proliferation, stem cell migration, and bone marrow derived macrophages (BMDMs) M2 polarization compared to ApoEVs. Furthermore, we utilized a modified gelatine matrix/3D-printed extracellular matrix (ECM) scaffold complex as a carrier to deliver H-ApoEVs into the joint cavity, thus establishing a cartilage regeneration system. The 3D-printed ECM scaffold provided mechanical support and created a microenvironment that was conducive to cartilage regeneration, and the H-ApoEVs further enhanced the regenerative capacity of endogenous stem cells and the immunomodulatory microenvironment of the joint cavity; thus, this approach significantly promoted cartilage repair. In conclusion, this study confirmed that a ApoEVs delivery system based on a modified gelatine matrix/3D-printed ECM scaffold together with hypoxic preconditioning enhances the functionality of stem cell-derived ApoEVs and represents a promising approach for promoting cartilage regeneration.

Two strategies of metabolic engineering have been used, individually or combined, to alter the metabolic flux to improve the production of S-adenosyl-l-methionine (SAM) in Pichia pastoris. One is over expressing SAM synthase by knock-in technique, the other is the disruption of cystathionine-β synthase (CBS) by knock-out technique. Strain Gsam with ectopic SAM synthase gene produced 20 times of SAM comparing to the starter strain GS115. Disruption of CBS in GS115 only doubled its SAM production. However, disruption of CBS in Gsam results in a robust increase of SAM production, more than 56 times of the strain GS115. Thus, we report for the first time a synergistic effect on the production of SAM in yeast by the combination of knock-in and knock-out techniques. Furthermore, we optimize the cultural conditions for the genetically modified strain Gsam-cbs to produce SAM. The maximum yield of SAM reaches 3.6 g/L in shake flask and 13.5 g/L in a 5 L fermentor, indicating that it could be used for industrial fermentation to produce large scale of SAM.

ABSTRACT The male sterility of a wheat thermosensitive genic male sterile (TGMS) line is strictly controlled by temperature. When the TGMS line BS366 was exposed to 10 °C from the pollen mother cell stage to the meiosis stage, a few pollen grains were formed and devoid of starch. We report here a large‐scale transcriptomic study using the Affymetrix wheat GeneChip to follow gene expression in BS366 line anthers in response to cold stress. Notably, many cytoskeletal signaling components were gradually induced in response to cold stress in BS366 line anthers. However, the cytoskeleton‐associated genes that play key roles in the dynamic organization of the cytoskeleton were dramatically repressed. Histological studies revealed that the separation of dyads occurred abnormally during male meiosis I, indicating defective male meiotic cytokinesis. Fluorescence labelling and subcellular histological observations revealed that the phragmoplast was defectively formed and the cell plate was abnormally assembled during meiosis I under cold stress. Based on the transcriptomic analysis and observations of characterized histological changes, our results suggest that cold stress repressed transcription of cytoskeleton dynamic factors and subsequently caused the defective cytokinesis during meiosis I. The results may explain the male sterility caused by low temperature in wheat TGMS lines.

Transcription is the first step connecting genetic information with an organism's phenotype. While expression of annotated genes in the human brain has been characterized extensively, our knowledge about the scope and the conservation of transcripts located outside of the known genes' boundaries is limited. Here, we use high-throughput transcriptome sequencing (RNA-Seq) to characterize the total non-ribosomal transcriptome of human, chimpanzee, and rhesus macaque brain. In all species, only 20–28% of non-ribosomal transcripts correspond to annotated exons and 20–23% to introns. By contrast, transcripts originating within intronic and intergenic repetitive sequences constitute 40–48% of the total brain transcriptome. Notably, some repeat families show elevated transcription. In non-repetitive intergenic regions, we identify and characterize 1,093 distinct regions highly expressed in the human brain. These regions are conserved at the RNA expression level across primates studied and at the DNA sequence level across mammals. A large proportion of these transcripts (20%) represents 3′UTR extensions of known genes and may play roles in alternative microRNA-directed regulation. Finally, we show that while transcriptome divergence between species increases with evolutionary time, intergenic transcripts show more expression differences among species and exons show less. Our results show that many yet uncharacterized evolutionary conserved transcripts exist in the human brain. Some of these transcripts may play roles in transcriptional regulation and contribute to evolution of human-specific phenotypic traits.

Waterlogging strongly affects agronomic performance of maize (Zea mays L.). In order to investigate the suitable selection criteria of waterflooding tolerant genotypes, and identify the most susceptible stage and the best continuous treatment time to waterlogging, 20 common maize inbred lines were subjected to successive artificial waterflooding at seedling stage, and waterlogging tolerance coefficient (WTC) was used to screen waterflooding tolerant genotypes. In addition, peroxidase (POD) activities and malondialdehyde (MDA) contents were measured for 6 of 20 lines. The results showed that the second leaf stage (V2) was the most susceptible stage, and 6 d after waterflooding was the best continuous treatment time. Dry weight (DW) of both shoots and roots of all lines were significantly reduced at 6 d time-point of waterlogging, compared to control. POD activities and MDA contents were negatively and significantly correlated, and the correlation coefficient was −0.9686 (P < 0.0001). According to the results, WTC of shoot DW can be used for practical screening as a suitable index, which is significantly different from control and waterlogged plants happened 6 d earlier. Furthermore, leaf chlorosis, MDA content and POD activities could also be used as reference index for material screening. The implications of the results for waterlogging-tolerant material screening and waterlogging-tolerant breeding have been discussed in maize.

Epigenetic modifications are critical determinants of cellular and developmental states. Epigenetic changes, such as decreased H3K27me3 histone methylation on insulin/IGF1 genes, have been previously shown to modulate lifespan through gene expression regulation. However, global epigenetic changes during aging and their biological functions, if any, remain elusive. Here, we examined the histone modification H3K4 dimethylation (H3K4me2) in the prefrontal cortex of individual rhesus macaques at different ages by chromatin immunoprecipitation, followed by deep sequencing (ChIP-seq) at the whole genome level. Through integrative analysis of the ChIP-seq profiles with gene expression data, we found that H3K4me2 increased at promoters and enhancers globally during postnatal development and aging, and those that correspond to gene expression changes in cis are enriched for stress responses, such as the DNA damage response. This suggests that metabolic and environmental stresses experienced by an organism are associated with the progressive opening of chromatin. In support of this, we also observed increased expression of two H3K4 methyltransferases, SETD7 and DPY30, in aged macaque brain.

Mice in which lung epithelial cells can be induced to express an oncogenic KrasG12D develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of KrasG12D tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ∼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured KrasG12D tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.

Nitric oxide (NO) and mitogen-activated protein kinases (MAPKs) are signal molecules involved in the disease resistance of plants. To investigate the role of tomato MAPKs in the NO-mediated defense response, mature green tomatoes (Lycopersicon esculentum Mill. cv. Qian-xi) were treated with a MAPKs inhibitor (1,4-diamino-2,3-dicyano-1,4-bis(o-amino-phenylmercapto) butadiene (U0126)), NO donor sodium nitroprusside (SNP), and SNP plus U0126. Treatment with U0126 increased the incidence of disease and size of lesion areas in the tomato fruits after being inoculated with Botrytis cinerea. NO enhanced the resistance of the tomato fruits against Botrytis cinerea invasion and the activities of nitric oxide synthase, Chitinase, β-1,3-glucanase, polyphenol oxidase, and phenylalanine ammonia-lyase. However, the effects of NO on disease resistance were weakened by the MAPKs inhibitor. Meanwhile, the relative expression of LeMAPK1, LeMAPK2, and LeMAPK3 in the (SNP + U0126)-treated fruits was lower than that in the SNP-treated fruits. The results suggest that LeMAPK1/2/3 are involved in NO-induced disease resistance of tomato fruits against Botrytis cinerea.

Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at p<0.05 & 0.01, but hemicelluloses did not show any significant impact on hexoses yields. Comparative analysis of five standard pairs of corn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H2SO4 at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%-23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at p<0.05, leading to a high biomass digestibility. Hence, this study could suggest an optimal approach for genetic modification of plant cell walls in bioenergy corn.

Significance The polar overdominance inheritance of callipyge sheep is an unusual mode of non-Mendelian inheritance. We established a mouse line with deletion of the microRNA (miR) 379/miR-544 cluster and assessed the role of this cluster in the inheritance. Our results showed that the maternally expressed miR-379/miR-544 cluster might regulate skeletal muscle growth through the imprinted Delta-like 1 homolog ( Dlk1 ) gene. This report revealed a molecular mechanism of the polar overdominance inheritance.

The mechanism of maternal in vivo haploid induction is not fully understood. In this study, the young embryos were identified by morphology, cytology and simple sequence repeat (SSR) markers at different developmental stages in the cross HZ514 (sweet corn) × HZI1 (inducer). The results indicated that the low seed setting rate was determined by the inducer pollen during the process of fertilization. The mosaic endosperm kernels and the different percentages of aneuploidy, mixploidy, lagged chromosome, micronuclei, chromosomal bridge and ring chromosome were found in the cross; 7.37% of the haploid embryos carried chromosome segments from HZI1. About 1% twin seedlings resulted from the cross and were analyzed by cytology and SSR markers. Four pairs of twin seedlings had different chromosome numbers (2n = 20 and 2n = 10–20) and there were some chromosome fragments from HZI1. Aneuploidy, mixploidy and the abnormal chromosomes occurred in the in vivo haploid induction by HZI1, which is the cytological basis for haploid induction and indicates that the inducer's chromosomes are prone to be lost during mitotic and meiotic divisions. Morphological, cellular and molecular evidences reveal that complete or partial chromosome elimination from inducer HZI1 controls the maize in vivo haploid induction. The link refers to the raw data from: Morphological, cellular and molecular evidences of chromosome random elimination in vivo upon haploid induction in maize. Current Plant Biology. Raw data for phenotype, maker sequence and cytology could be directly downloaded by the link: http://dx.doi.org/10.5061/dryad.bt963

Response surface method (RSM) was employed to optimize the extraction conditions of polysaccharides from Rhizoma Panacis Majoris (the rhizomes of Panax japonicus C. A. Mey. var. major (Burk.) C. Y. Wu et K. M. Feng) (RPMP), a well-known Chinese traditional medicine. In order to obtain the optimal processing parameters, a three-variable Box–Behnken designs (BBD) were applied for experimental designs. RSM analysis indicated the good correspondence between experimental and predicted values, the optimal conditions for the yield of polysaccharides were as follows: the ultrasound time is 31.15 min, extraction temperature is 92.50 °C, and the ratio of water to raw material is 40 mL/g. The maximum value (13.87 ± 0.16%) of the yield of polysaccharides was obtained under these optimal conditions. The molecular weight (MW) was determined to be 1.48 × 105(± 0.39%) Da by HPSEC-MALLS-RID chromatography system. FT-IR spectra demonstrated obvious characteristic peaks of polysaccharides. The antioxidant activities of RPMP were investigated including scavenging activity of hydrogen radicals, ABTS radicals, and free radicals of superoxide anion in vitro, and the results exhibited that RPMP had a good potential for antioxidant.

Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom compost infected by red bread mold, exhibited antagonistic activities against Neurospora sitophila. Antifungal activity against phytopathogenic fungi was also observed. The maximum antifungal activity was reached during the stationary phase. This antifungal activity was stable over a wide pH and temperature range and was not affected by proteases. Assay of antifungal activity in vitro indicated that a purified antifungal substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In addition, treatment with strain B154 in A. bisporus mushroom compost infected with N. sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-associated laser desorption ionization time-of-flight mass spectrometry, and electrospray ionization tandem mass spectrometry analyses revealed a molecular weight consistent with 1498.7633 Da. The antifungal compound might belong to a new type of lipopeptide fengycin.

The molecular mechanism of down-regulated microRNA-145 (miR-145) expression in lung adenocarcinoma (LAC) remains largely unknown. We hypothesized that aberrant hyper-methylation of the CpG sites silenced the expression of miR-145 in LAC. In consideration of its pivotal role in LAC development and progression, we also evaluated the clinical utility of miR-145 as a prognostic marker. We assessed the DNA methylation status of the miR-145 promoter region in 20 pairs of LAC and the matched non-tumor specimens. We subsequently applied our own LAC tissue microarray containing 92 pairs of tumor and non-tumor tissues with long time follow-up records to evaluate whether miR-145 is a potential prognostic marker in LAC. The Sequenom EpiTYPER MassArray analysis showed that miR-145 was down-regulated in human LAC tissues accompanied by increased DNA methylation of its upstream region, which was further validated by the data from TCGA database. Significance was observed between miR-145 expression and clinic-pathologic parameters. Univariate and multivariate analysis revealed that miR-145 expression level was an independent risk factor for both OS and DFS in LAC patients. Taken together, DNA hyper-methylation in the miR-145 promoter region reduced its expression in LAC and miR-145 expression level might serve as a novel prognostic biomarker.

Mitogen-activated protein kinases (MAPKs) are major components of defense signaling pathways that transduce extracellular stimuli into intracellular responses in plants. Our previous study indicated that SlMPK1/2/3 were associated with nitric oxide-induced defense response in tomato fruit. In this study, we determine whether SlMPK1/2/3 influence the tomato fruit's innate immunity and whether plant hormones and reactive oxygen species (ROS) are involved in SlMPK1/2/3 defense signaling pathways. Treatment with 10 μM U0126 significantly inhibited the relative expression of SlMPK1, SlMPK2, and SlMPK3 (P < 0.05). U0126-treated fruit showed higher concentrations of auxin indole acetic acid (IAA), abscisic acid (ABA), and gibberellic acid (GA), but a lower concentration of methyl jasmonate (MeJA). The activities of defense enzymes, including β-1,3-glucanases (GLU), chitinase (CHI), phenylalanine ammonia lyase (PAL), and polyphenol oxidase (PPO), decreased after U0126 treatment. Meanwhile, H2O2 content increased, and catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) activities decreased after U0126 treatment. U0126 treatment enhanced the susceptibility of tomato fruit to Botrytis cinerea and resulted in more severe gray mold rot. These results demonstrate that inhibition of SlMPK1/2/3 disrupts tomato fruit defense signaling pathways and enhances the susceptibility to B. cinerea and also that plant hormones and ROS are associated with SlMPK1/2/3 defense signaling pathways.

WRKY transcription factors play an important role in cold defense of plants. However, little information is available about the cold-responsive WRKYs in tomato (Solanum lycopersicum). In the present study, a complete characterization of this gene family was described. Eighty WRKY genes in the tomato genome were identified. Almost all WRKY genes contain putative stress-responsive cis-elements in their promoter regions. Segmental duplications contributed significantly to the expansion of the SlWRKY gene family. Transcriptional analysis revealed notable differential expression in tomato tissues and expression patterns under cold stress, which indicated wide functional divergence in this family. Ten WRKYs in tomato were strongly induced more than 2-fold during cold stress. These genes represented candidate genes for future functional analysis of WRKYs involved in the cold-related signal pathways. Our data provide valuable information about tomato WRKY proteins and form a foundation for future studies of these proteins, especially for those that play an important role in response to cold stress.

The bacterial wilt of peanut (Arachis hypogaea L.) caused by Ralstonia solanacearum is a devastating soil-borne disease that seriously restricted the world peanut production. However, the molecular mechanism of R. solanacearum-peanut interaction remains largely unknown. We found that R. solanacearum HA4-1 and PeaFJ1 isolated from peanut plants showed different pathogenicity by inoculating more than 110 cultivated peanuts. Phylogenetic tree analysis demonstrated that HA4-1 and PeaFJ1 both belonged to phylotype I and sequevar 14M, which indicates a high degree of genomic homology between them. Genomic sequencing and comparative genomic analysis of PeaFJ1 revealed 153 strain-specific genes compared with HA4-1. The PeaFJ1 strain-specific genes consisted of diverse virulence-related genes including LysR-type transcriptional regulators, two-component system-related genes, and genes contributing to motility and adhesion. In addition, the repertoire of the type III effectors of PeaFJ1 was bioinformatically compared with that of HA4-1 to find the candidate effectors responsible for their different virulences. There are 79 effectors in the PeaFJ1 genome, only 4 of which are different effectors compared with HA4-1, including RipS4, RipBB, RipBS, and RS_T3E_Hyp6. Based on the virulence profiles of the two strains against peanuts, we speculated that RipS4 and RipBB are candidate virulence effectors in PeaFJ1 while RipBS and RS_T3E_Hyp6 are avirulence effectors in HA4-1. In general, our research greatly reduced the scope of virulence-related genes and made it easier to find out the candidates that caused the difference in pathogenicity between the two strains. These results will help to reveal the molecular mechanism of peanut-R. solanacearum interaction and develop targeted control strategies in the future.

Preeclampsia (PE) is a multi-organ syndrome that onsets in the second half of pregnancy. It is the second leading cause of maternal death globally. The homeostasis of zinc (Zn) levels is important for feto-maternal health.We aimed to collect all studies available to synthesize the evidence regarding the association between maternal Zn levels and the risk of preeclampsia.A systematic review and meta-analysis was conducted via searching seven electronic databases [PubMed, Web of Science, Embase, African Journals Online (AJOL), ClinicalTrial.gov, and two Chinese databases: Wanfang and Chinese National Knowledge Infrastructure, CNKI]. Studies reporting maternal serum Zn levels in pregnant women with or without preeclampsia were included. Eligible studies were assessed through Newcastle-Ottawa Scale (NOS) and the meta-analysis was performed via RevMan and Stata. The random-effects method (REM) was used for the meta-analysis with 95% confidence interval (CI). The pooled result was assessed using standard mean difference (SMD). The heterogeneity test was carried out using I2 statistics, and the publication bias was evaluated using Begg's and Egger's test. Meta-regression and sensitivity analysis was performed via Stata software.A total of 51 studies were included in the final analysis. 6,947 participants from 23 countries were involved in our study. All studies went through the quality assessment. The pooled results showed that maternal serum Zn levels were lower in preeclamptic women than in healthy pregnant women (SMD: -1.00, 95% CI: -1.29, -0.70). Sub-group analysis revealed that geographical, economic context, and disease severity may further influence serum Zn levels and preeclampsia.There are significant between-study heterogeneity and publication bias among included studies.A lower level of maternal Zn was associated with increased risks of preeclampsia. The associations were not entirely consistent across countries and regions worldwide.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=337069, Identifier: CRD42022337069.

The replication of hepatitis B virus (HBV) can be regulated by a variety of factors, including hormones, growth factors, and cytokines. However, the molecular mechanisms of these regulations are largely unknown. Ras is a small GTPase that responds to many of these external stimuli. In this study, we investigated the possible effect of Ras on the replication of HBV. Our results indicated that activated Ras could suppress the replication of HBV in both Huh7 and HepG2 cells. This suppression was independent of the X protein and most likely occurred at the transcriptional level. Deletion-mapping analysis of the HBV core promoter and its upstream ENI and ENII enhancers revealed multiple elements responsive to activated Ras. This suppression of HBV replication by activated Ras was apparently mediated by the mitogen-activated protein (MAP) kinase pathway, as it was accompanied by activation of ERK1/2 and abolished by the MEK1/2 inhibitor U0126. Our results thus indicate that external stimuli may suppress HBV replication through the Ras-MAP kinase pathway.

The yield of maize grain is a highly complex quantitative trait that is controlled by multiple quantitative trait loci (QTLs) with small effects, and is frequently influenced by multiple genetic and environmental factors. Thus, it is challenging to clone a QTL for grain yield in the maize genome. Previously, we identified a major QTL, qKNPR6, for kernel number per row (KNPR) across multiple environments, and developed two nearly isogenic lines, SL57-6 and Ye478, which differ only in the allelic constitution at the short segment harboring the QTL. Recently, qKNPR6 was re-evaluated in segregating populations derived from SL57-6×Ye478, and was narrowed down to a 2.8 cM interval, which explained 56.3% of the phenotypic variance of KNPR in 201 F(2∶3) families. The QTL simultaneously affected ear length, kernel weight and grain yield. Furthermore, a large F(2) population with more than 12,800 plants, 191 recombinant chromosomes and 10 overlapping recombinant lines placed qKNPR6 into a 0.91 cM interval corresponding to 198Kb of the B73 reference genome. In this region, six genes with expressed sequence tag (EST) evidence were annotated. The expression pattern and DNA diversity of the six genes were assayed in Ye478 and SL57-6. The possible candidate gene and the pathway involved in inflorescence development were discussed.

Hydrogen peroxide (H2O2) acts as a signaling molecule in response to cold stress. Mitogen-activated protein kinases (MAPKs) and C-repeat/dehydration-responsive factor (CBF) play important roles in cold response regulation. To investigate the roles of MAPKs and CBF in H2O2-induced chilling tolerance, tomato (Solanum lycopersicum cv. Ailsa Craig) plants were treated with 1 mM H2O2 before chilling treatment. The results showed that H2O2 treatment protected subcellular structure, increased concentrations of abscisic acid (ABA), zeatin riboside (ZR), and methyl jasmonate (MeJA), but decreased the concentration of gibberellic acid (GA3). Furthermore, 1 mM H2O2 treatment enhanced the activities of antioxidant enzymes. Meanwhile, relative expressions of SlMAPK1/2/3 and SlCBF1 in H2O2-treated plants were higher than those in the control. Our findings suggest that H2O2 treatment might enhance the chilling tolerance of tomato plants by activating SlMAPK1/2/3 and SlCBF1 gene expression and by regulating phytohormone concentrations and antioxidant enzyme activities.

Sickle cell disease (SCD) is a monogenic disorder characterized by recurrent episodes of severe bone pain, multi-organ failure, and early mortality. Although medical progress over the past several decades has improved clinical outcomes and offered cures for many affected individuals living in high-income countries, most SCD patients still experience substantial morbidity and premature death. Emerging technologies to manipulate somatic cell genomes and insights into the mechanisms of developmental globin gene regulation are generating potentially transformative approaches to cure SCD by autologous hematopoietic stem cell (HSC) transplantation. Key components of current approaches include ethical informed consent, isolation of patient HSCs, in vitro genetic modification of HSCs to correct the SCD mutation or circumvent its damaging effects, and reinfusion of the modified HSCs following myelotoxic bone marrow conditioning. Successful integration of these components into effective therapies requires interdisciplinary collaborations between laboratory researchers, clinical caregivers, and patients. Here we summarize current knowledge and research challenges for each key component, emphasizing that the best approaches have yet to be developed.

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease that limits peanut production and quality, but the molecular mechanisms of the peanut response to R. solanacearum remain unclear. In this study, we reported the first work analyzing the transcriptomic changes of the resistant and susceptible peanut leaves infected with R. solanacearum HA4-1 and its type III secretion system mutant strains by the cutting leaf method at different timepoints (0, 24, 36, and 72 h post inoculation). A total of 125,978 differentially expressed genes (DEGs) were identified and subsequently classified into six groups to analyze, including resistance-response genes, susceptibility-response genes, PAMPs induced resistance-response genes, PAMPs induced susceptibility-response genes, T3Es induced resistance-response genes, and T3Es induced susceptibility-response genes. KEGG enrichment analyses of these DEGs showed that plant-pathogen interaction, plant hormone signal transduction, and MAPK signaling pathway were the outstanding pathways. Further analysis revealed that CMLs/CDPKs-WRKY module, MEKK1-MKK2-MPK3 cascade, and auxin signaling played important roles in the peanut response to R. solanacearum. Upon R. solanacearum infection (RSI), three early molecular events were possibly induced in peanuts, including Ca2+ activating CMLs/CDPKs-WRKY module to regulate the expression of resistance/susceptibility-related genes, auxin signaling was induced by AUX/IAA-ARF module to activate auxin-responsive genes that contribute to susceptibility, and MEKK1-MKK2-MPK3-WRKYs was activated by phosphorylation to induce the expression of resistance/susceptibility-related genes. Our research provides new ideas and abundant data resources to elucidate the molecular mechanism of the peanut response to R. solanacearum and to further improve the bacterial wilt resistance of peanuts.