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Hu, Q.

Publications and source records attributed to Hu, Q..

At least 127 records · Page 7Linked to original sources
bioRxiv2020

A GTP-state specific cyclic peptide inhibitor of the GTPase Gαs

The G protein-coupled receptor (GPCR) cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable receptors and enzymes with the exception of the G subunit, Gs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 1012 cyclic peptides in order to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gs, respectively. Both macrocyclic peptides fine-tune Gs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/3 pocket and block effector interactions. The Gs inhibitors showed strong activity in cellular contexts through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gs provides a path for further development of state-dependent GTPase inhibitors.

biochemistry↗
bioRxiv2020

The SEM-4 transcription factor is required for regulation of the oxidative stress response in Caenorhabditis elegans

Oxidative stress causes damage to cells by creating reactive oxygen species (ROS) and the overproduction of ROS have been linked to the onset of premature ageing. We previously found that a brap-2 (BRCA1 associated protein 2) mutant significantly increases the expression of phase II detoxification enzymes in C. elegans. An RNAi suppression screen to identify transcription factors involved in the production of gst-4 mRNA in brap-2 worms identified SEM-4 as a potential candidate. Here, we show that knockdown of sem-4 suppresses the activation of gst-4 caused by the mutation in brap-2. We also demonstrate that sem-4 is required for survival upon exposure to oxidative stress and that SEM-4 is required for expression of the transcription factor SKN-1C. These findings identify a novel role for SEM-4 in ROS detoxification by regulating expression of SKN-1C and the phase II detoxification genes. Article SummaryReactive oxygen species have been implicated as a harmful agent in many age-related diseases as well as an important signaling molecule. The transcription factor SKN-1 in C. elegans is an important regulator of reactive oxygen species levels. Here we show that the transcription factor SEM-4 is required to activate the expression of skn-1c and promote phase II detoxification gene expression. These findings identify a novel role for SEM-4 in regulating reactive oxygen species levels.

genetics↗
bioRxiv2020

Epigenetic transgenerational effects on RNAi in Arabidopsis

Epigenetic states and even certain environmental responses in nematodes, mammals and seed plants can be inherited over successive generations. RNAi in plants is an epigenetic form of RNA degradation that is normally reset early in development. In contrast, competence, i.e., the susceptibility of a transgene for RNAi, is believed to be genetically determined and faithfully transmitted over many generations. Unexpectedly, we found that the same yellow fluorescent protein (YFP) transgene in a monogenic, homozygous line of Arabidopsis exhibited two epigenetic states of RNAi: type I, which is initiated in rosette leaves and is stable in later developmental stages, and, type II, which is initiated after floral induction and is unstable. The incidence of type I plants descended from type I parents decreased over three successive generations of self-fertilized plants. By the third generation, a substantial fraction of plants from type I parents exhibited either the type II phenotype or remained high-YFP expressing throughout sporophytic development, indicating that silencing had been completely lost. Similarly, plants descended from type II parents showed a progressive shift from the type II phenotype and a complete loss of silencing. These results show that the silencing types (or states) of parental plants have significant influence on both silencing determination and silencing types (or states) of their offspring, however, this influence fades through several generations of self; as regardless of the types of the parental lines, competence for RNA silencing tends to loss after generations. Thus, we propose that RNA silencing in plants can be the target of transgenerational effects. While the underlying mechanisms are still unknown, our findings in the Arabidopsis RNA silencing system complement the current understanding of heritable RNAi in C. elegans, that this phenomenon is likely a common mechanism in Eukaryote systems.

plant biology↗
bioRxiv2020

The histone variant H2A.Z is required to establish normal patterns of H3K27 methylation in Neurospora crassa

Neurospora crassa contains a minimal Polycomb repression system, which provides rich opportunities to explore Polycomb-mediated repression across eukaryotes and enables genetic studies that can be difficult in plant and animal systems. Polycomb Repressive Complex 2 is a multi-subunit complex that deposits mono-, di-, and tri-methyl groups on lysine 27 of histone H3, and tri-methyl H3K27 is a molecular marker of transcriptionally repressed facultative heterochromatin. In mouse embryonic stem cells and multiple plant species, H2A.Z has been found to be co-localized with H3K27 methylation. H2A.Z is required for normal H3K27 methylation in these experimental systems, though the regulatory mechanisms are not well understood. We report here that Neurospora crassa mutants lacking H2A.Z or SWR-1, the ATP-dependent histone variant exchanger, exhibit a striking reduction in levels of H3K27 methylation. RNA-sequencing revealed downregulation of eed, encoding a subunit of PRC2, in an hH2Az mutant compared to wild type and overexpression of EED in a {Delta}hH2Az;{Delta}eed background restored most H3K27 methylation. Reduced eed expression leads to region-specific losses of H3K27 methylation suggesting that EED-dependent mechanisms are critical for normal H3K27 methylation at certain regions in the genome. AUTHOR SUMMARYEukaryotic DNA is packaged with histone proteins to form a DNA-protein complex called chromatin. Inside the nucleus, chromatin can be assembled into a variety of higher-order structures that profoundly impact gene expression. Polycomb Group proteins are important chromatin regulators that control assembly of a highly condensed form of chromatin. The functions of Polycomb Group proteins are critical for maintaining stable gene repression during development of multicellular organisms, and defects in Polycomb proteins are linked to disease. There is significant interest in elucidating the molecular mechanisms that regulate the activities of Polycomb Group proteins and the assembly of transcriptionally repressed chromatin domains. In this study, we used a model fungus to investigate the regulatory relationship between a histone variant, H2A.Z, and a conserved histone modifying enzyme complex, Polycomb Repressive Complex 2 (PRC2). We found that H2A.Z is required for normal expression of a PRC2 component. Mutants that lack H2A.Z have defects in chromatin structure at some parts of the genome, but not others. Identification of PRC2-target domains that are differentially dependent on EED provides insights into the diverse mechanisms that regulate assembly and maintenance of facultative heterochromatin in a simple model system. Data Reference NumbersGSE146611

genetics↗
bioRxiv2020

Evolution of cellular diversity in primary motor cortex of human, marmoset monkey, and mouse

The primary motor cortex (M1) is essential for voluntary fine motor control and is functionally conserved across mammals. Using high-throughput transcriptomic and epigenomic profiling of over 450,000 single nuclei in human, marmoset monkey, and mouse, we demonstrate a broadly conserved cellular makeup of this region, whose similarity mirrors evolutionary distance and is consistent between the transcriptome and epigenome. The core conserved molecular identity of neuronal and non-neuronal types allowed the generation of a cross-species consensus cell type classification and inference of conserved cell type properties across species. Despite overall conservation, many species specializations were apparent, including differences in cell type proportions, gene expression, DNA methylation, and chromatin state. Few cell type marker genes were conserved across species, providing a short list of candidate genes and regulatory mechanisms responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allowed the Patch-seq identification of layer 5 (L5) corticospinal Betz cells in non-human primate and human and characterization of their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell type diversity in M1 across mammals and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

neuroscience↗
bioRxiv2020

An integrated transcriptomic and epigenomic atlas of mouse primary motor cortex cell types

Single cell transcriptomics has transformed the characterization of brain cell identity by providing quantitative molecular signatures for large, unbiased samples of brain cell populations. With the proliferation of taxonomies based on individual datasets, a major challenge is to integrate and validate results toward defining biologically meaningful cell types. We used a battery of single-cell transcriptome and epigenome measurements generated by the BRAIN Initiative Cell Census Network (BICCN) to comprehensively assess the molecular signatures of cell types in the mouse primary motor cortex (MOp). We further developed computational and statistical methods to integrate these multimodal data and quantitatively validate the reproducibility of the cell types. The reference atlas, based on more than 600,000 high quality single-cell or -nucleus samples assayed by six molecular modalities, is a comprehensive molecular account of the diverse neuronal and non-neuronal cell types in MOp. Collectively, our study indicates that the mouse primary motor cortex contains over 55 neuronal cell types that are highly replicable across analysis methods, sequencing technologies, and modalities. We find many concordant multimodal markers for each cell type, as well as thousands of genes and gene regulatory elements with discrepant transcriptomic and epigenomic signatures. These data highlight the complex molecular regulation of brain cell types and will directly enable design of reagents to target specific MOp cell types for functional analysis.

neuroscience↗
bioRxiv2020

Genetic structure, function and evolution of capsule biosynthesis loci in Vibrio parahaemolyticus

Capsule-forming extracellular polysaccharides are crucial to bacterial host colonization, invasion, immune evasion and ultimately pathogenicity. Due to warming ocean waters and human encroachment of coastal ecosystems, Vibrio parahaemolyticus has emerged as a globally important food-borne enteropathogen implicated in acute gastroenteritis, wound infections, and septic shock. Conventionally, the antigenic properties of lipopolysaccharide (LPS, O antigen) and capsular polysaccharide (CPS, K antigen) have provided a basis for serotyping V. parahaemolyticus, while disclosure of genetic elements encoding 13 O-serogroups have allowed molecular serotyping methods to be developed. However, the genetic structure of CPS loci for 71 K-serogroups has remained unidentified, limiting progress in understanding its roles in V. parahaemolyticus pathophysiology. In this study, we identified and characterized the genetic structure and their evolutionary relationship of CPS loci of 40 K-serogroups through whole genome sequencing of 443 V. parahaemolyticus strains. We found a distinct pattern of CPS gene cluster across different K-serogroups, and expanded its new right-border by identifying glpX as a key gene conserved across all serotypes. A total of 217 genes involved in CPS biosynthesis were annotated. Functional contents and genetic structure of the 40 K-serogroups were analyzed. Based on inferences from species trees and gene trees, we proposed an evolution model of the CPS gene clusters of 40 K-serogroups. Horizontal gene transfer by recombination from other Vibrio species, gene duplication and nonsense mutations are likely to play instrumental roles in the evolution of CPS in V. parahaemolyticus. It is the first time, to the best of our knowledge, that a large-scale of CPS gene clusters of different K-serogroups in V. parahaemolyticus have been identified and characterized in evolutionary contexts. This work should help advance understanding on the variation of CPS in V. parahaemolyticus, and provide a framework for developing diagnostically relevant serotyping methods. Author summaryDue to warming ocean waters and human encroachment of coastal ecosystems, Vibrio parahaemolyticus has emerged as a globally important food-borne enteropathogen. However, the genetic structure of CPS loci for 71 K-serogroups V. parahaemolyticus have remained unidentified, limiting progress in understanding its roles in V. parahaemolyticus pathophysiology. In this study, we identified and characterized the genetic structure of CPS loci of 40 K-serogroups through whole genome sequencing of 443 V. parahaemolyticus strains. We expanded and identified its new right-border by identifying glpX as a key gene conserved across all serotypes. We proposed an evolution model of the CPS gene clusters of 40 K-serogroups. We also found horizontal gene transfer by recombination from other Vibrio species, gene duplication and nonsense mutations are likely to play instrumental roles in the evolution of CPS in V. parahaemolyticus. It is the first time, to the best of our knowledge, that a large-scale of CPS loci of different K-serogroups in V. parahaemolyticus have been identified and characterized in evolutionary contexts. This work should help advance understanding on the variation of CPS in V. parahaemolyticus, and provide a framework for developing diagnostically relevant serotyping methods.

microbiology↗
bioRxiv2020

Common postzygotic mutational signature in multiple healthy adult tissues related to embryonic hypoxia

Postzygotic mutations are acquired in all of the normal tissues throughout an individuals lifetime and hold clues for identifying mutagenesis causing factors. The process and underlying mechanism of postzygotic mutations in normal tissues is still poorly understood. In this study, we investigated postzygotic mutation spectra in healthy individuals by optimized ultra-deep exome sequencing of time series samples from the same volunteer and samples from different individuals. In cells of blood, sperm, and muscle, we resolved three common types of mutational signature. Two of them are known to represent clock-like mutational processes, and their proportions in mutation profiles associated with polymorphisms of epigenetic regulation genes, suggesting the contribution of personal genetic backgrounds to underlying biological process. Notably, the third signature, characterized by C>T transitions at GpCpN sites, tends to be a feature of diverse normal tissues. Mutations of this type were likely to occur early in embryo development even before the tissue differentiation, as indicated by their relatively high allele frequencies, sharing variants between multiple tissues, and lacking of age-related accumulation. Almost all tumors shown in public datasets did not have this signature detected except for 19.6% of clear cell renal cell carcinoma samples, which featured by activation of the hypoxia-induced signaling pathway. Moreover, in vitro activation of HIF signaling pathway successfully introduced the corresponding mutation profile of this signature in a culture-expanded human embryonic stem cell line. Therefore, embryonic hypoxia may explain this novel signature across multiple normal tissues. Our study suggest hypoxic conditions in the early stage of embryo development may be a crucial factor for the C>T transitions at GpCpN sites and individual genetic background also related to shaping human postzygotic mutation profiles.

genetics↗
bioRxiv2020

Giant African snail genomes provide insights into molluscan whole-genome duplication and aquatic-terrestrial transition

Whole-genome duplication (WGD) has been observed across a wide variety of eukaryotic groups, contributing to evolutionary diversity and environmental adaptability. Mollusks are the second largest group of animals, and are among the organisms that have successfully adapted to the nonmarine realm through aquatic-terrestrial (A-T) transition, and no comprehensive research on WGD has been reported in this group. To explore WGD and the A-T transition in Mollusca, we assembled a chromosome-level reference genome for the giant African snail Achatina immaculata, a global invasive species, and compared the genomes of two giant African snails (A. immaculata and Achatina fulica) to the other available mollusk genomes. The chromosome-level macrosynteny, colinearity blocks, Ks peak and Hox gene clusters collectively suggested the occurrence of a WGD event shared by A. immaculata and A. fulica. The estimated timing of this WGD event ([~]70 MYA) was close to the speciation age of the Sigmurethra-Orthurethra (within Stylommatophora) lineage and the Cretaceous-Tertiary (K-T) mass extinction, indicating that the WGD reported herein may have been a common event shared by all Sigmurethra-Orthurethra species and could have conferred ecological adaptability and genomic plasticity allowing the survival of the K-T extinction. Based on macrosynteny, we deduced an ancestral karyotype containing 8 conserved clusters for the Gastropoda-Bivalvia lineage. To reveal the mechanism of WGD in shaping adaptability to terrestrial ecosystems, we investigated gene families related to the respiration, aestivation and immune defense of giant African snails. Several mucus-related gene families expanded early in the Stylommatophora lineage, functioning in water retention, immune defense and wound healing. The hemocyanins, PCK and FBP families were doubled and retained after WGD, enhancing the capacity for gas exchange and glucose homeostasis in aestivation. After the WGD, zinc metalloproteinase genes were highly tandemly duplicated to protect tissue against ROS damage. This evidence collectively suggests that although the WGD may not have been the direct driver of the A-T transition, it provided an important legacy for the terrestrial adaptation of the giant African snail.

genomics↗
bioRxiv2020

Ultra-deep sequencing differentiates patterns of skin clonogenic mutations associated with sun-exposure status

Non-melanoma skin cancer is the most common human malignancy and is primarily caused by exposure to ultraviolet (UV) radiation. The earliest detectable precursor of UV-mediated skin cancer is the growth of cell groups harboring clonal mutation (CM) in clinically normal appearing skin. Systematic evaluation of CMs is crucial to understand early photo-carcinogenesis. Previous studies confirmed the presence of CMs in sun-exposed skin. However, the relationship between UV-exposure and the accumulation of CMs, and the correlation of CMs with skin cancer risk remain poorly understood. To elucidate the exact molecular and clinical effects of long-term UV-exposure on skin, we performed targeted ultra-deep sequencing in 450 individual-matched sun-exposed (SE) and non-sun-exposed (NE) epidermal punch biopsies obtained from clinically normal skin from 13 donors. A total of 638 CMs were identified, including 298 UV-signature mutations (USMs). The numbers of USMs per sample were three times higher in the SE samples and were associated with significantly higher variant allele frequencies (VAFs), compared with the NE samples. We identified genomic regions in TP53, NOTCH1 and GRM3 where mutation burden was significantly associated with UV-exposure. Six mutations were almost exclusively present in SE epidermis and accounted for 42% of the overall difference between SE and NE mutation burden. We defined Cumulative Relative Clonal Area (CRCA), a single metric of UV-damage calculated by the overall relative percentage of the sampled skin area affected by CMs. The CRCA was dramatically elevated by a median of 11.2 fold in SE compared to NE samples. In an extended cohort of SE normal skin samples from patients with a high- or low-burden of cutaneous squamous cell carcinoma (cSCC), the SE samples in high-cSCC patients contained significantly more USMs than SE samples in low-cSCC patients, with the difference mostly conferred by mutations from low-frequency clones (defined by VAF≤1%) but not expanded clones (VAF>1%). Our studies of differential mutational features in normal skin between paired SE/NE body sites and high/low-cSCC patients provide novel insights into the carcinogenic effect of UV exposure, and suggest CMs might be used to develop novel biomarkers for predicting cancer risk.Significance statement In UV radiation exposed skin, mutations fuel clonal cell growth. We established a sequencing-based method to objectively assess the mutational differences between sun-exposed (SE) and non-sun-exposed (NE) areas of normal human skin. Striking differences, in both the numbers of mutations and variant allele frequencies, were found between SE and NE areas. Furthermore, we identified specific genomic regions where mutation burden is significantly associated with UV-exposure status. These findings revealed previously unknown mutational patterns associated with UV-exposure, providing important insights into UV radiation’s early carcinogenic effects. Additionally, in an extended cohort, we identified preliminary association between normal skin mutation burden and cancer risk. These findings pave the road for future development of quantitative measurement of subclinical UV damage and skin cancer risk.Competing Interest StatementThe authors have declared no competing interest.Common AbbreviationsUVUltravioletCMClonogenic mutationNMSCNonmelanoma skin cancerSESun-exposedNENon-sun-exposedUSMUV-signature mutationNUSMNon-UV-signature mutationCRCACumulative Relative Clonal AreacSCCCutaneous squamous cell carcinomaAKActinic keratosisSNVSingle nucleotide variant Indels – Insertions/deletionsDNVDinucleotide variantCSNVCluster of single nucleotide variantMACMulti-Nucleotide Variant Annotation CorrectorVAFVariant allele frequencyView Full Text

cancer biology↗
bioRxiv2020

CTDSP1 inhibitor rabeprazole regulates DNA-PKcs dependent topoisomerase I degradation and irinotecan drug resistance in colorectal cancer

Irinotecan specifically targets topoisomerase I (topoI), and is used to treat various solid tumors, but only 13-32% of patients respond to the therapy. Now, it is understood that the rapid rate of topoI degradation in response to irinotecan causes irinotecan resistance. We have published that the deregulated DNA-PKcs kinase cascade ensures rapid degradation of topoI and is at the core of the drug resistance mechanism of topoI inhibitors, including irinotecan. We also identified CTD small phosphatase 1 (CTDSP1) (a nuclear phosphatase) as a primary upstream regulator of DNA-PKcs in response to topoI inhibitors. Previous reports showed that rabeprazole, a proton pump inhibitor (PPI) inhibits CTDSP1 activity. The purpose of this study was to confirm the effects of rabeprazole on CTDSP1 activity and its impact on colon cancer. Using HCT116 and HT29, with high and low CTDSP1 expression respectively and a retrospective analysis of patients receiving irinotecan with or without rabeprazole have indicated the effect of CTDSP1 in irinotecan response. These results indicate that CTDSP1 promotes sensitivity to irinotecan and rabeprazole prevents this effect, resulting in drug resistance. To ensure the best chance at effective treatment, rabeprazole may not be a suitable PPI for cancer patients treated with irinotecan.

cancer biology↗
bioRxiv2019

An epigenetic LTR-retrotransposon insertion in the upstream region of BnSHP1.A9 controls quantitative pod shattering resistance in Brassica napus

Seed loss resulting from pod shattering is a major problem in oilseed rape (Brassica napus L.) production worldwide. However, the molecular mechanisms underlying pod shatter resistance are not well understood. Here we show that the pod shatter resistance at quantitative trait locus, qSRI.A9.1 is controlled by a SHATTERPROOF1 (SHP1) paralog in B. napus (BnSHP1.A9). Expression analysis by quantitative RT-PCR showed that BnSHP1.A9 was specifically expressed in flower buds, flowers and developing siliques in the oilseed rape line (R1) carrying the qSRI.A9.1 allele with negative effect, but not expressed in any tissue of the line (R2) carrying the positive effect qSRI.A9.1 allele. Transgenic plants constitutively expressing BnSHP1.A9 alleles from pod resistant and pod shattering parental lines showed that both alleles are responsible for pod shattering via promoting lignification of enb layer, which indicated allelic difference of BnSHP1.A9 gene per se is not the causal factor of the QTL. The upstream sequence of BnSHP1.A9 in the promotor region harboring highly methylated long terminal repeat retrotransposon insertion (LTR, 4803bp) in R2 repressed the expression of BnSHP.A9, and thus contributed to the positive effect on pod shatter resistance. Genetic and association analysis revealed that the copia LTR retrotransposon based marker BnSHP1.A9-R2 can be used for breeding for pod shatter resistant varieties and reducing the loss of seed yield in oilseed rape.

genetics↗
bioRxiv2019

Pathway engineering in yeast for synthesizing the complex polyketide bikaverin

Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recoded, constructed and characterized the biosynthetic pathway of the formation of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We used a green fluorescent protein (GFP) tagging strategy to identify the low expression of Bik1 (polyketide synthase) as the bottleneck step in the pathway, and a promoter exchange strategy to increase expression of Bik1 and bikaverin yield. To further increase product yield, we used an enzyme-fusion strategy to couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to a dramatic improvement of Bikaverin yield of nearly 60-fold. This study demonstrates that the biosynthesis of complex polyketides biosynthesis can be established and efficiently engineered in S. cerevisiae, highlighting the great potential for natural product synthesis and large-scale fermentation in yeast.

synthetic biology↗
bioRxiv2019

E-cadherin binds glycosylated sodium-taurocholate cotransporting polypeptide to facilitate hepatitis B virus entry

Hepatitis B virus (HBV) continues to pose a serious public health risk and is one of the major causes of chronic liver disease and hepatocellular carcinoma. Current antiviral therapy does not effectively eradicate HBV and, thus, further investigation into the mechanisms employed by HBV to allow for invasion of host cells, is critical for the development of novel therapeutic agents. Sodium-taurocholate cotransporting polypeptide (NTCP) has been identified as a functional receptor for HBV. However, the specific mechanism by which HBV and NTCP interact remains unclear. Herein we show that the expression of E-cadherin was upregulated in cells expressing HBV, while knockdown of E-cadherin in HepG2-NTCP cells, HepaRG cells and primary human hepatocytes served to significantly inhibit infection by HBV and HBV pseudotyped particles. Alternatively, exogenous E-cadherin expression was found to significantly enhance HBV uptake by HepaRG cells. Further, mechanistic studies identified glycosylated NTCP localized to the cell membrane via E-cadherin binding, which subsequently allowed for more efficient binding between NTCP and the preS1 of the large HBV surface proteins. E-cadherin was also found to play a key role in establishing and maintaining hepatocyte polarity, which is essential for efficient HBV infection. These observations suggest that E-cadherin facilitates HBV entry through regulation of NTCP distribution and hepatocyte polarity.\n\nAuthor SummaryHepatitis B Virus (HBV) still seriously endangers public health. It is very important to understand the mechanism of HBV invading host cells for developing new therapy target. Sodium-taurocholate cotransporting polypeptide (NTCP) is the key receptor mediating HBV invasion, while other molecules also exhibit important roles in ensuring efficient and productive HBV infection. This study reports that E-cadherin facilitates HBV entry by directly interacting with glycosylated NTCP to mediate its distribution on the hepatocyte membrane and also affects the efficacy of HBV invasion by influncing hepatocyte polarity.

microbiology↗
bioRxiv2019

Genome analysis and Hi-C assisted assembly of Elaeagnus angustifolia L., a deciduous tree belonging to Elaeagnaceae

Elaeagnus angustifolia L. is a deciduous tree of the Elaeagnaceae family. It is widely used in the study of abiotic stress tolerance in plants and for the improvement of desertification-affected land due to its characteristics of drought resistance, salt tolerance, cold resistance, wind resistance, and other environmental adaptation. Here, we report the complete genome sequencing using the Pacific Biosciences (PacBio) platform and Hi-C assisted assembly of E. angustifolia. A total of 44.27 Gb raw PacBio sequel reads were obtained after filtering out low-quality data, with an average length of 8.64 Kb. And 39.56 Gb clean reads was obtained, with a sequencing coverage of 75x, and Q30 ratio > 95.46%. The 510.71 Mb genomic sequence was mapped to the chromosome, accounting for 96.94% of the total length of the sequence, and the corresponding number of sequences was 269, accounting for 45.83% of the total number of sequences. The genome sequence study of E. angustifolia can be a valuable source for the comparative genome analysis of the Elaeagnaceae family members, and can help to understand the evolutionary response mechanisms of the Elaeagnaceae to drought, salt, cold and wind resistance, and thereby provide effective theoretical support for the improvement of desertification-affected land.

genetics↗
bioRxiv2019

Drosophila Trpm mediates calcium influx during egg activation

Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germline specific RNAi knockdown, and germline specific CRISPR/Cas9 knockout to identify the Transient receptor potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed reduced egg hatchability in trpm germline knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since the mouse Trpm ortholog was recently reported also to be involved in calcium influx during egg activation and in further embryonic development our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.\n\nSignificanceA rise in intracellular free calcium is a conserved feature of the egg-to-embryo transition in almost all animals. In Drosophila, as in vertebrates, the rise starts at one end of the egg, and then travels across the egg in a wave. The Drosophila calcium rise is mediated by an influx of calcium, due to the action of mechanically-gated ion channels. Here we identify the ion channel that is critical for the calcium entry as TRPM. TRPM is the ortholog of the channel recently shown to mediate the post-fertilization calcium influxes needed to sustain calcium oscillations in fertilized mouse eggs, suggesting a deep homology - despite species differences in the trigger for egg activation.

developmental biology↗
bioRxiv2019

Dysregulated Alternative Splicing Landscape Identifies Intron Retention as a Hallmark and Spliceosome as a Therapeutic Vulnerability in Aggressive Prostate Cancer

Dysregulation of mRNA alternative splicing (AS) has been implicated in development and progression of hematological malignancies. Here we describe the first comprehensive AS landscape in the spectrum of human prostate cancer (PCa) development, progression and therapy resistance. We find that the severity of splicing dysregulation correlates with disease progression and establish intron retention (IR) as a hallmark of PCa stemness and aggressiveness. Systematic interrogation of 274 splicing-regulatory genes (SRGs) uncovers prevalent SRG mutations associated with, mainly, copy number variations leading to mis-expression of ~68% of SRGs during PCa evolution. Consequently, we identify many SRGs as prognostic markers associated with splicing disruption and patient outcome. Interestingly, androgen receptor (AR) controls a splicing program distinct from its transcriptional regulation. The spliceosome modulator, E7107, reverses cancer aggressiveness and abolishes the growth of castration-resistant PCa (CRPC) models. Altogether, we establish aberrant AS landscape caused by dysregulated SRGs as a novel therapeutic vulnerability for CRPC.\n\nStatement of significanceWe present the first comprehensive AS landscape during PCa evolution and link genomic and transcriptional alterations in SRGs to global splicing dysregulation. AR regulates splicing in pri-PCa and CRPC distinct from its transcriptional regulation. Intron retention is a hallmark for and spliceosome represents a therapeutic vulnerability in aggressive PCa.

cancer biology↗
bioRxiv2019

TMC1 confers a leak conductance to modulate excitability of auditory hair cells in mammals

Hearing sensation relies on the mechano-electrical transducer (MET) channel of cochlear hair cells, in which Transmembrane channel-like 1 (TMC1) and TMC2 have been proposed to be the pore-forming subunits. Meanwhile it has been reported that TMCs regulate other biological processes in a variety of lower organisms ranging from sensations to motor functions. However, it is still an open question whether TMCs play roles other than their function in MET in mammals. In this study, we report that in mouse hair cells TMC1, but not TMC2, provides a background leak conductance, with properties distinct from those of the MET channels. By cysteine substitution, 4 amino acids of TMC1 are characterized critical for the leak conductance. The leak conductance is essential for action potential firing and tonotopic along the cochlear coil. Taken together, our results suggest that TMC1 confers a background leak conductance that modulates membrane excitability in cochlear hair cells.

cell biology↗