Biology subjects

Li, L.

Publications and source records attributed to Li, L..

At least 19 recordsLinked to original sources

bioRxiv2018

SmbHLH37 functions antagonistically with SmMYC2 in regulating jasmonate-mediated biosynthesis of phenolic acids in Salvia miltiorrhiza

Jasmonates (JAs) are integral to various defense responses and induce biosynthesis of many secondary metabolites. MYC2, a basic helix-loop-helix (bHLH) transcription factor (TF), acts as a transcriptional activator of JA signaling. MYC2 is repressed by the JASMONATE ZIM-domain (JAZ) proteins in the absence of JA, but de-repressed by the protein complex SCFCOI1 on perception of JA. We previously reported that overexpression of SmMYC2 promotes the production of salvianolic acid B (Sal B) in Salvia miltiorrhiza. However, the responsible molecular mechanism is unclear. Here, we showed that SmMYC2 binds to and activates the promoters of its target genes SmTAT1, SmPAL1, and SmCYP98A14 to activate Sal B accumulations. SmbHLH37, a novel bHLH gene significantly up-regulated by constitutive expression of SmMYC2, was isolated from S. miltiorrhiza for detailed functional characterization. SmbHLH37 forms a homodimer and interacts with SmJAZ3/8. Overexpression of SmbHLH37 substantially decreased yields of Sal B. SmbHLH37 binds to the promoters of its target genes SmTAT1 and SmPAL1 and blocks their expression to suppress the pathway for Sal B biosynthesis. These results indicate that SmbHLH37 negatively regulates JA signaling and functions antagonistically with SmMYC2 in regulating Sal B biosynthesis in S. miltiorrhiza.

plant biology↗

bioRxiv2018

The seven transmembrane domain protein MoRgs7 functions in surface perception and undergoes coronin MoCrn1-dependent endocytosis in complex with Gα subunit MoMagA to promote cAMP signaling and appressorium formation in Magnaporthe oryzae

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of G subunits, thereby regulating G-protein mediated signaling. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, together with G MoMagA but not MoRgs8, undergoes endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobic cues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.\n\nAuthor summaryThe 7-TM domain is considered the hallmark of GPCR proteins, which activate G proteins upon ligand binding and undergo endocytosis for regeneration or recycling. Among eight RGS and RGS-like proteins of M. oryzae, MoRgs7 and MoRgs8 contain the 7-TM domain in addition to the RGS domain. We found that MoRgs7 can form hydrophobic interactions with the hydrophobic surface. This interaction is important in sensing hydrophobic cues by the fungus. We also found that, in response to surface hydrophobicity, MoRgs7 couples with G subunit MoMagA to undergo endocytosis leading to the activation of cAMP signaling. Moreover, we found that such an endocytic event requires functions of the actin-binding protein MoCrn1. Our results revealed MoRgs7 functions as a GPCR-like receptor protein to sense surface cues and activate signaling required for pathogenesis, providing new insights into G-protein regulatory mechanisms in this and other pathogenic fungi.

microbiology↗

bioRxiv2018

GWAS using 2b-RAD sequencing identified three mastitis important SNPs via two-stage association analysis in Chinese Holstein cows.

BackgroundBovine mastitis is a key disease restricting developing global dairy industry. Genomic wide association studies (GWAS) provided a convenient way to understand the biological basis of mastitis and better prevent or treat the disease. 2b-RADseq is a reduced-representation sequencing that offered a powerful method for genome-wide genetic marker development and genotyping. This study, GWAS using two-stage association analysis identified mastitis important genes single nucleotide polymorphisms (SNP) in Chinese Holstein cows.\n\nResultsIn the selected Chinese Holstein cows population, we identified 10,058 SNPs and predicted their allele frequencies. In stage I, 42 significant SNPs screened out in Chinese Holstein cows via Bayesian (P<0.001), while logistic regression model identified 51 SNPs (P<0.01). Twenty-seven significant SNPs appeared simultaneously in both analytical models, which of them only three significant SNPs (rs75762330, C>T, PIC=0.2999; rs88640083, A>G, PIC=0.1676; rs20438858, G>A, PIC=0.3366) located in non-coding region (introns and intergenic) screened out associated with inflammation or immune response. GO enrichment analysis showed that they annotated to three genes (PTK2B, SYK and TNFRSF21), respectively. Stage II? case-control study used to verify three important SNPs associated with dairy cows mastitis traits in independent population. Data suggested that the correlation between these three SNPs (rs75762330, P<0.025; rs88640083, P<0.005; rs20438858, P<0.001) and mastitis traits in dairy cows were consistent with stage I.\n\nConclusionTwo-stage association analysis approved that three significant SNPs associated with mastitis traits in Chinese Holstein cows. Gene function analysis indicated that three genes (PTK2B, SYK and TNFRSF21) involved in inflammation and immune response of dairy cows. Suggesting that they as new candidate genes have an impact on mastitis susceptibility (PTK2B and SYK, OR>1) or resistance (TNFRSF21, OR<1) in Chinese Holstein cows.

genomics↗

bioRxiv2018

Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

developmental biology↗

bioRxiv2018

Process-specific somatic mutation distributions vary with three-dimensional genome structure

Somatic mutations arise during the life history of a cell. Mutations occurring in cancer driver genes may ultimately lead to the development of clinically detectable disease. Nascent cancer lineages continue to acquire somatic mutations throughout the neoplastic process and during cancer evolution (Martincorena and Campbell, 2015). Extrinsic and endogenous mutagenic factors contribute to the accumulation of these somatic mutations (Zhang and Pellman, 2015). Understanding the underlying factors generating somatic mutations is crucial for developing potential preventive, therapeutic and clinical decisions. Earlier studies have revealed that DNA replication timing (Stamatoyannopoulos et al., 2009) and chromatin modifications (Schuster-Bockler and Lehner, 2012) are associated with variations in mutational density. What is unclear from these early studies, however, is whether all extrinsic and exogenous factors that drive somatic mutational processes share a similar relationship with chromatin state and structure. In order to understand the interplay between spatial genome organization and specific individual mutational processes, we report here a study of 3000 tumor-normal pair whole genome datasets from more than 40 different human cancer types. Our analyses revealed that different mutational processes lead to distinct somatic mutation distributions between chromatin folding domains. APOBEC- or MSI-related mutations are enriched in transcriptionally-active domains while mutations occurring due to tobacco-smoke, ultraviolet (UV) light exposure or a signature of unknown aetiology (signature 17) enrich predominantly in transcriptionally-inactive domains. Active mutational processes dictate the mutation distributions in cancer genomes, and we show that mutational distributions shift during cancer evolution upon mutational processes switch. Moreover, a dramatic instance of extreme chromatin structure in humans, that of the unique folding pattern of the inactive X-chromosome leads to distinct somatic mutation distribution on X chromosome in females compared to males in various cancer types. Overall, the interplay between three-dimensional genome organization and active mutational processes has a substantial influence on the large-scale mutation rate variations observed in human cancer.

genomics↗

bioRxiv2018

Vegfa expression is activated through positive and negative transcriptional regulatory networks controlled by the ETS factor Etv6 in vivo

VEGFA signaling is crucial for physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VEGFA have been uncovered, vegfa transcriptional regulation in vivo remains unclear. Here we show that the ETS transcription factor, Etv6, positively regulates vegfa expression during Xenopus blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits the expression of the vegfa repressor, foxo3. Surprisingly, it also directly activates the expression of the vegfa activator, klf4. Finally, it indirectly binds to the vegfa promoter where it co-localizes with Klf4. Klf4 deficiency downregulates vegfa expression and significantly decreases Etv6 binding to the vegfa promoter, indicating that Klf4 recruits Etv6 to the vegfa promoter. Thus, our work uncovers a dual function for Etv6, as both a transcriptional repressor and activator, in controlling a major signaling pathway involved in blood and endothelial development in vivo. Given the established relationships between development and cancer, this elaborate gene regulatory network may inform new strategies for the treatment of VEGFA-dependent tumorigenesis.

developmental biology↗

bioRxiv2018

A Sparse Unreliable Distributed Code Underlies the Limits of Behavioral Discrimination

The cortical code that underlies perception must enable subjects to perceive the world at timescales relevant for behavior. We find that mice can integrate visual stimuli very quickly (<100 ms) to reach plateau performance in an orientation discrimination task. To define features of cortical activity that underlie performance at these timescales, we measured single unit responses in the mouse visual cortex at timescales relevant to this task. In contrast to high contrast stimuli of longer duration, which elicit reliable activity in individual neurons, stimuli at the threshold of perception elicit extremely sparse and unreliable responses in V1 such that the activity of individual neurons do not reliably report orientation. Integrating information across neurons, however, quickly improves performance. Using a linear decoding model, we estimate that integrating information over 50-100 neurons is sufficient to account for behavioral performance. Thus, at the limits of perception the visual system is able to integrate information across a relatively small number of highly unreliable single units to generate reliable behavior.

neuroscience↗

bioRxiv2018

An Extreme Value Theory Model of Cross-Modal Sensory Information Integration in Modulation of Vertebrate Visual System Functions

We propose a computational model of vision that describes the integration of cross-modal sensory information between the olfactory and visual systems in zebrafish based on the principles of the statistical extreme value theory. The integration of olfacto-retinal information is mediated by the centrifugal pathway that originates from the olfactory bulb and terminates in the neural retina. Motivation for using extreme value theory stems from physiological evidence suggesting that extremes and not the mean of the cell responses direct cellular activity in the vertebrate brain. We argue that the visual system, as measured by retinal ganglion cell responses in spikes/sec, follows an extreme value process for sensory integration and the increase in visual sensitivity from the olfactory input can be better modeled using extreme value distributions. As zebrafish maintains high evolutionary proximity to mammals, our model can be extended to other vertebrates as well.

neuroscience↗

bioRxiv2018

Molecular characterization of the viral structural gene of the first dengue virus type 1 outbreak in Xishuangbanna, a border area of China, Burma and Laos

In the context of recent arbovirus epidemics, dengue fever is becoming a greater concern around the world. In August 2017, Xishuangbanna, which is a border city of China, Burma and Laos, had its first major dengue outbreak. A total of 156 serum samples from febrile patients were collected; 97 DENV positive serum samples were screened out, and viral RNAs were successfully and directly extracted, including 77 cases from China and 20 cases from Myanmar. Phylogenetic analysis revealed that all of the strains were classified as DENV-1. There are eight epidemic dengue strains from Myanmar and 74 from Jinghong, Xishuangbanna, that were classified as cluster 1, which are the most similar to the strain of China Guangzhou 2011. There are three epidemic strains from Xishuangbanna Mengla that were classified as cluster 2, which have the closest relationship to the strain of China Hubei 2014. However, there are 12 epidemic strains from Myanmar that were classified as cluster 3, which have the closest relationship to the strain of Laos from 2008, which shows that there is a recycling epidemic trend of DENV in China. There were 236 mutations in the base, which caused 31 nonsynonymous mutations in the DENV structural protein C/prM/E genes when the strain of Xishuangbanna and Myanmar were compared with the DENV-1SS. There is no clear homologous recombination signal among these stains. Homology modeling possibly predicted a three-dimensional structure of the structural protein of these strains and revealed that they had the same three-dimensional structure and all had five predicted protein binding sites, but there are differences in binding site 434 (DENV-1SS: Thr434, DV-Jinghong: Ser434, DV-Myanmar: Ser434, DV-Mengla: Ser434). The results of the molecular clock phylogenetic and demographic reconstruction analysis show that DENV-1 became highly diversified in 1972 followed by a slightly decreased period until 2017. In conclusion, our study lays the foundation for studying the global evolution and prevalence of DENV.\n\nAuthor SummaryDengue fever (DF) is a mosquito-borne illness caused by a flavivirus. Human infections with Dengue virus (DENV) could cause fever, cutaneous rash and malaise. Xishuangbanna, which is located in the southwestern Yunnan Province and is a border city with China, Burma and Laos, was reported to have outbreak of DENV in 2013 and 2015 with different types. However, there was a large outburst of dengue in May 2017. To understand the genetic characterization, potential source and evolution of the virus, 156 serum samples were analyzed. We focused on: (i) Phylogenetic analysis of the structural protein genes sequences; (ii) Mutation, recombination analysis and predicted protein binding sites of the structural protein genes; (iii) Molecular clock and demographic reconstruction of global dengue virus serotype 1(DENV-1). Our results indicated that this is the first outbreak of DENV-1 in Xishuangbanna, dengue epidemic strains on the Burma border of China show diversification, we found a virulence site changed from I to T(amino acid position: 440), which may lead to weakened virulence of the epidemic strains. We found that the evolution of DENV-1 is dominated by regional evolution. Whats more, DENV-1 became highly diversified in 1972 followed by a slightly decreased period until 2017.

epidemiology↗

bioRxiv2018

COMPASS Family Histone Methyltransferase ASH2L Mediates Corticogenesis via Transcriptional Regulation of Wnt Signalling

Cell fate specification in neural progenitor cells (NPCs) is orchestrated via extrinsic and intrinsic molecular programs, and histone methylation in these decisions has been ascribed to a crucial function regulating gene expression. Here, we show that the COMPASS family histone methyltransferase co-factor ASH2L is required in NPCs proliferation and upper layer cortical projection neurons production and position. Deletion of Ash2l impairs trimethylation of H3K4 and transcriptional machinery specifically for subsets of Wnt-{beta}-catenin signalling, disrupting their transcription and consequently inhibiting the proliferation ability of NPCs in late stages of neurogenesis. Consistently, Ash2l conditional mutants exhibit thinning neocortex with reduced upper layer neurons and altered neuronal position. Moreover, overexpressing {beta}-catenin after Ash2l elimination or knockdown can rescue the proliferation deficiency of NPCs both in vivo and in vitro. These results demonstrate an essential and highly specific role for Ash2l in controlling NPCs proliferation and late-born neurons lamination in corticogenesis via transcriptionally regulating Wnt-{beta}-catenin signalling, and provide clues to how the COMPASS family epigenetic factors coordinate cell fate determination during cortex development.

neuroscience↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2018

Defining the core essential genome of Pseudomonas aeruginosa

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. It is becoming clear that a major limitation was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a novel statistical model, FiTnEss, to classify genes as essential versus non-essential across all strain-media combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of 4 strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

microbiology↗

bioRxiv2018

Systems therapeutics analyses identify genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer

The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop genomic signatures for sensitivity or resistance to specific drugs. We analyzed TCGA data for lung adenocarcinoma (LUAD) patients and identified a subset where xanthine dehydrogenase expression correlated with decreased survival. We tested allopurinol, a FDA approved drug that inhibits xanthine dehydrogenase on a library of human Non Small Cell Lung Cancer (NSCLC) cell lines from CCLE and identified sensitive and resistant cell lines. We utilized the gene expression profiles of these cell lines to identify six-gene signatures for allopurinol sensitive and resistant cell lines. Network building and analyses identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 were verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinolsensitive NSCLC lines, and four allopurinol-resistant lines susceptible to combination therapy. We found that drug treatment of all cell lines yielded responses as predicted by the genomic signatures. We searched the library of patient derived NSCLC tumors from Jackson Laboratory to identify tumors that would be predicted to be sensitive or resistant to allopurinol treatment. Both patient derived tumors predicted to be allopurinol sensitive showed the predicted sensitivity, and the predicted resistant tumors were sensitive to combination therapy. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

systems biology↗

bioRxiv2018

CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1{beta}. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1{beta} in murine neutrophils through its ITIM receptor. We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1-/- mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G-) express IL-6 in the early response (<2 hours) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and {beta}-actin in response to LPS to produce IL-6. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and {beta}-actin from RP105. This novel pathway and regulation of IL-6 producing by CEACAM1 defines a novel role for monocytes in the fever of mice to LPS.\n\nAUTHOR SUMMARYFever is one of the most common signs of the immune response to pathogens. The fever response to LPS or endotoxin of gram-negative bacteria is mediated by the combined action of two cytokines, IL-1{beta} and IL-6. Regulation of their production in response to LPS is an important area of investigation. While we previously showed that the regulation of IL-1{beta} production in neutrophils is through the lymphocyte receptor CEACAM1, we were interested if a similar mechanism operated for IL-6. Using a mouse model in which the CEACAM1 gene was knocked out, we show that IL-6 is over-produced compared to normal mice, and that monocytes, rather than neutrophils were the principal IL-6 producing cells. Surprisingly, murine monocytes do not express TLR4, the most commonly studied receptor for LPS, but instead express the low affinity LPS receptor, RP105, a receptor common expressed on B-cells. Furthermore, we show that bone marrow monocytes are rapidly released into the blood and home to tissues throughout the body in response to LPS. These findings explain much of the confusion in the literature concerning the immediate source of IL-6 and the distinct differences between murine and human monocytes in their in responses to LPS.

immunology↗

bioRxiv2018

Evolutionarily developed connections compromised in schizophrenia

The genetic basis and uniquely human character of schizophrenia has led to the notion of human brain evolution to have resulted in vulnerability to the disorder. We examined schizophrenia-related changes in brain connectivity in the context of evolutionary changes in human brain wiring by comparing in-vivo neuroimaging data from humans, chimpanzees and macaque monkeys. We find that evolutionary changes in human connectome organization overlap with the pattern of schizophrenia-related changes in brain connectivity, with connections evolutionary enhanced in the human brain showing significantly more involvement in schizophrenia pathology than connections shared between humans and non-human primates (effects shown in three independent patient-control datasets). Our findings suggest that the evolution of brain wiring in support of complex brain function in humans may have come at the cost of an increased vulnerability to brain dysfunction in disease.

neuroscience↗

bioRxiv2018

Heterodimerization of UNC-13/RIM regulates synaptic vesicle release probability but not priming

UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13Ls C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.

neuroscience↗

bioRxiv2018

A novel maize gene, glossy6 involved in epicuticular wax deposition and drought tolerance

Epicuticular waxes, long-chain hydrocarbon compounds, form the outermost layer of plant surfaces in most terrestrial plants. The presence of epicuticular waxes protects plants from water loss and other environmental stresses. Cloning and characterization of genes involved in the regulation, biosynthesis, and extracellular transport of epicuticular waxes on to the surface of epidermal cells have revealed the molecular basis of epicuticular wax accumulation. However, intracellular trafficking of synthesized waxes to the plasma membrane for cellular secretion is poorly understood. Here, we characterized a maize glossy (gl6) mutant that exhibited decreased epicuticular wax load, increased cuticle permeability, and reduced seedling drought tolerance relative to wild type. We combined an RNA-sequencing based mapping approach (BSR-Seq) and chromosome walking to identify the gl6 candidate gene, which was confirmed via the analysis of multiple independent mutant alleles. The gl6 gene represents a novel maize glossy gene containing a conserved, but uncharacterized domain. Functional characterization suggests that the GL6 protein may be involved in the intracellular trafficking of epicuticular waxes, opening a door to elucidating the poorly understood process by which epicuticular wax is transported from its site of biosynthesis to the plasma membrane.\n\nSIGNIFICANCE STATEMENTPlant surface waxes provide an essential protective barrier for terrestrial plants. Understanding the composition and physiological functions of surface waxes, as well as the molecular basis underlying wax accumulation on plant surfaces provides opportunities for the genetic optimization of this protective layer. Genetic studies have identified genes involved in wax biosynthesis, extracellular transport, as well as spatial and temporal regulation of wax accumulation. In this study, a maize mutant, gl6 was characterized that exhibited reduced wax load on plant surfaces, increased water losses, and reduced seedling drought tolerance compared to wild type controls. The gl6 gene is a novel gene harboring a conserved domain with an unknown function. Quantification and microscopic observation of wax accumulation as well as subcellular localization of the GL6 protein provided evidence that gl6 may be involved in the intracellular trafficking of waxes, opening a door for studying this necessary yet poorly understood process for wax loading on plant surfaces.

plant biology↗

bioRxiv2018

Structural insight into the mechanism of neuraminidase inhibitor-resistant mutations in human-infecting H10N8 Influenza A virus

The emergence of drug resistance in avian influenza virus (AIV) is a serious concern for public health. Neuraminidase (NA) isolated from a fatal case of avian-origin H10N8 influenza virus infection was found to carry a drug-resistant mutation, NA-Arg292Lys (291 in N8 numbering). In order to understand the full potential of H10N8 drug resistance, the virus was first passaged in the presence of the most commonly used neuraminidase inhibitors (NAIs), oseltamivir and zanamivir. As expected, the Arg292Lys substitution was detected after oseltamivir treatment, however a novel Val116Asp substitution (114 in N8 numbering) was selected by zanamivir treatment. Next generation sequencing (NGS) confirmed that the mutations arose early (after passages 1-3) and became dominant in the presence of the NAI inhibitors. Extensive crystallographic studies revealed that N8-Arg292Lys resistance results mainly from loss of interactions with the inhibitor carboxylate, while rotation of Glu276 was not impaired as observed in the N9-Arg292Lys, a group 2 NA structure. In the case of Val116Asp, the binding mode between oseltamivir and zanamivir is different. Asp151 forms stabilized hydrogen bond to guanidine group of zanamivir, which may compensate the resistance caused by Val116Asp. By contrast, the amino group of oseltamivir is too short to maintain this hydrogen bond, which result in resistant. Moreover, the oseltamivir-zanamivir hybrid inhibitor MS-257 displays higher effectiveness to Val116Asp than oseltamivir, which support this notion.\n\nAuthor SummaryAside from vaccination, NAIs are currently the only alternative for the clinical treatment and prophylaxis of influenza. Understanding the mechanisms of resistance is critical to guide in drug development. In this study, two drug-resistant NA substitutions, Val116Asp and Arg292Lys, were discovered from oseltamivir and zanamivir treatment of H10N8 virus. Crystal structural analyses revealed two distinct mechanisms of these two resistant mutations and provide the explanation for the difference in susceptibility of different NAIs. Zanamivir and laninamivir were more effective against the resistant variants than oseltamivir, and Arg292Lys results in more serious oseltamivir resistance in N9 than N8 subtype. This study is well-correlated to influenza pandemic/epidemic pre-warning, as the discovery of inhibitor resistant viruses will help for new drug preparedness.

microbiology↗