Biology subjects

Yang, F.

Publications and source records attributed to Yang, F..

At least 19 recordsLinked to original sources

bioRxiv2018

Differential gene expression, including Sjfs800, in Schistosoma japonicum females before, during, and after male-female pairing

Schistosomiasis is a prevalent but neglected tropical disease caused by parasitic trematodes of the genus Schistosoma, with the primary disease-causing species being S. haematobium, S. mansoni, and S. japonicum. Male-female pairing of schistosomes is necessary for sexual maturity and the production of a large number of eggs, which are primarily responsible for schistosomiasis dissemination and pathology. Here, we used microarray hybridization, bioinformatics, quantitative PCR, in situ hybridization, and gene silencing assays to identify genes that play critical roles in S. japonicum reproduction biology, particularly in vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. Microarray hybridization analyses generated a comprehensive set of genes differentially transcribed before and after male-female pairing. Although the transcript profiles of females were similar 16 and 18 days after host infection, marked gene expression changes were observed at 24 days. The 30 most abundantly transcribed genes on day 24 included those associated with vitellarium development. Among these, genes for female-specific 800 (fs800), eggshell precursor protein, and superoxide dismutase (cu-zn-SOD) were substantially upregulated. Our in situ hybridization results in female S. japonicum indicated that cu-zn-SOD mRNA was highest in the ovary and vitellarium, eggshell precursor protein mRNA was expressed in the ovary, ootype, and vitellarium, and Sjfs800 mRNA was observed only in the vitellarium, localized in mature vitelline cells. Knocking down the Sjfs800 gene in female S. japonicum by approximately 60% reduced the number of mature vitelline cells, decreased rates of pairing and oviposition, and decreased the number of eggs produced in each male-female pairing by about 50%. These results indicate that Sjfs800 is essential for vitellarium development and egg production in S. japonicum and suggest that Sjfs800 regulation may provide a novel approach for the prevention or treatment of schistosomiasis.\n\nAuthor SummarySchistosomiasis is a common but largely unstudied tropical disease caused by parasitic trematodes of the genus Schistosoma. The eggs of schistosomes are responsible for schistosomiasis transmission and pathology, and the production of these eggs is dependent on the pairing of females and males. In this study, we determined which genes in Schistosoma japonicum females were differentially expressed before and after pairing with males, identifying the 30 most abundantly expressed of these genes. Among these 30 genes, we further characterized those in female S. japonicum that were upregulated after pairing and that were related to reproduction and vitellarium development, a process that affects male-female pairing, sexual maturation, and subsequent egg production. We identified three such genes, S. japonicum female-specific 800 (Sjfs800), eggshell precursor protein, and superoxide dismutase, and confirmed that the mRNAs for these genes were primarily localized in reproductive structures. By using gene silencing techniques to reduce the amount of Sjfs800 mRNA in females by about 60%, we determined that Sjfs800 plays a key role in development of the vitellarium and egg production. This finding suggests that regulation of Sjfs800 may provide a novel approach to reduce egg counts and thus aid in the prevention or treatment of schistosomiasis.

genomics↗

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

Proteomic Profile of TGF-β1 treated Lung Fibroblasts identifies Novel Markers of Activated Fibroblasts in the Silica Exposed Rat Lung

We performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) on control and TGF-{beta}1-exposed rat lung fibroblasts to identify proteins differentially expressed between cell populations. A total of 1648 proteins were found to be differentially expressed in response to TGF-{beta}1 treatment and 196 proteins were expressed at [≥] 1.2 fold relative to control. Guided by these results, we next determined whether similar changes in protein expression were detectable in the rat lung after chronic exposure to silica dust. Of the five proteins selected for further analysis, we found that levels of all proteins were markedly increased in the silica-exposed rat lung, including the proteins for the very low density lipoprotein receptor (VLDLR) and the transmembrane (type I) heparin sulfate proteoglycan called syndecan 2 (SDC2). Because VLDLR and SDC2 have not, to our knowledge, been previously linked to the pathobiology of silicosis, we next examined whether knockdown of either gene altered responses to TGF-{beta}1 in MRC-5 lung fibroblasts. Interestingly, we found knockdown of either VLDLR or SDC2 dramatically reduced collagen production to TGF-{beta}1, suggesting that both proteins might play a novel role in myofibroblast biology and pathogenesis of silica-induced pulmonary fibrosis. In summary, our findings suggest that performing LC-MS/MS on TGF-{beta}1 stimulated lung fibroblasts can uncover novel molecular targets of activated myofibroblasts in silica-exposed lung.\n\nHighlightsWe identified 196 proteins differentially expressed between control and TGF-{beta}1 treated fibroblastsby LC-MS/MS.\n\nSeveral proteins identified by LC-MS/MS were also found to be differentially expressed in whole lung tissues and isolated fibroblasts after chronic exposure to silica dust, including the very low density lipoprotein receptor (VLDLR) and the transmembrane type I heparan sulfate proteoglycan called syndecan 2\n\nKnockdown of SDC2 or VLDLR markedly inhibited collagen production in MRC-5 fibroblasts, suggesting a novel pathogenic role for these proteins in myofibroblast biology.

pharmacology and toxicology↗

bioRxiv2018

Microbial Cells Harboring a Mitochondrial Gene Are Capable of CO2 Capture

Global warming is escalating with increased temperatures reported worldwide. Given the enormous land mass on the planet, biological capture of CO2 remains a viable approach to mitigate the crisis as it is economical and easy to implement. In this study, a gene capable of CO2 capture was identified via selection in minimal media. This mitochondrial gene named as OG1 encodes the OK/SW-CL.16 protein and shares homology with cytochrome oxidase subunit III of various species and PII uridylyl-transferase from Loktanella vestfoldensis SKA53. CO2 capture experiments indicate that {delta}13C was substantially higher in the cells harboring the gene OG1 than the control in the nutrition-poor media. This study suggests that CO2 capture using engineered microorganisms in barren land can be exploited to address the soaring CO2 level in the atmosphere, opening up vast land resources to cope with global warming.\n\nIMPORTANCEGlobal warming crisis is deteriorating with increased CO2 levels in the atmosphere each year. Action must be taken before catastrophic consequences occur in the not-so-distant future. Biological capture of CO2 is a feasible approach to alleviate the current crisis. We have identified a mitochondrial gene which demonstrated CO2 utilization capability. Data presented in this study suggest that CO2 capture using engineered microorganisms can be harnessed to address the ever-rising CO2 level in the atmosphere.

bioengineering↗

bioRxiv2018

New genetic variants associated with major adverse cardiovascular events in patients with acute coronary syndromes and treated with clopidogrel and aspirin

ImportanceAlthough a few studies have reported the effects of several polymorphisms on major adverse cardiovascular events (MACE) in patients with acute coronary syndromes (ACS) and those undergoing percutaneous coronary intervention (PCI), these genotypes account for only a small fraction of the variation and evidence is insufficient. This study aims to identify new genetic variants associated with MACE by large-scale sequencing data.\n\nObjectiveTo identify the genetic variants that caused MACE.\n\nDesignAll patients in this study were allocated to dual antiplatelet therapy for up to 12 months and have the follow-up duration of 18 months.\n\nSettingA two-stage association study was performed.\n\nParticipantsWe evaluated the associations of genetic variants and MACE in 1961 patients with ACS undergoing PCI (2009-2012), including high-depth whole exome sequencing of 168 patients in the discovery cohort and high-depth targeted sequencing of 1793 patients in the replication cohort.\n\nMain Outcomes and MeasureThe primary clinical efficacy endpoint was the major adverse cardiovascular events (MACE) composite endpoint, including cardiovascular death, myocardial infarction (MI), stroke (CT or MR scan confirmed) and repeated revascularization (RR).\n\nResultsWe discovered and confirmed six new genotypes associated with MACE in patients with ACS. Of which, rs17064642 at MYOM2 increased the risk of MACE (hazard ratio [HR] 2.76; P = 2.95 x 10-9) and reached genome-wide significance. The other five suggestive variants were KRTAP10-4 (rs201441480), WDR24 (rs11640115), ECHS1 (rs140410716), AGAP3 (rs75750968) and NECAB1 (rs74569896). Notably, the expressions of MYOM2 and ECHS1 are down-regulated in both animal models and patients with phenotypes related to MACE. Importantly, we developed the first superior classifier for predicting MACE and achieved high predictive accuracy (0.809).\n\nConclusions and RelevanceWe identified six new genotypes associated with MACE and developed a superior classifier for predicting MACE. Our findings shed light on the pathogenesis of cardiovascular outcomes and may help clinician to make decision on the therapeutic intervention for ACS patients.\n\nTrial RegistrationThis study has been registered in the Chinese Clinical Trial Registry (http://www.chictr.org.cn, Registration number: ChiCTR-OCH-11001198).

genetics↗

bioRxiv2018

PIRD: Pan immune repertoire database

MotivationT and B cell receptors (TCRs and BCRs) play a pivotal role in the adaptive immune system by recognizing an enormous variety of external and internal antigens. Understanding these receptors is critical for exploring the process of immunoreaction and exploiting potential applications in immunotherapy and antibody drug design. Although a large number of samples have had their TCR and BCR repertoires sequenced using high-throughput sequencing in recent years, very few databases have been constructed to store these kinds of data. To resolve this issue, we developed a database.\n\nResultsWe developed a database, the Pan Immune Repertoire Database (PIRD), located in China National GeneBank (CNGBdb), to collect and store annotated TCR and BCR sequencing data, including from Homo sapiens and other species. In addition to data storage, PIRD also provides functions of data visualisation and interactive online analysis. Additionally, a manually curated database of TCRs and BCRs targeting known antigens (TBAdb) was also deposited in PIRD.\n\nAvailability and ImplementationPIRD can be freely accessed at https://db.cngb.org/pird.

immunology↗

bioRxiv2018

High Genetic Potential for Proteolytic Decomposition in Northern Peatland Ecosystems

AbstractNitrogen (N) is a scarce nutrient commonly limiting primary productivity. Microbial decomposition of complex carbon (C) into small organic molecules (e.g., free amino acids) has been suggested to supplement biologically-fixed N in high latitude peatlands. We evaluated the microbial (fungal, bacterial, and archaeal) genetic potential for organic N depolymerization in peatlands at Marcell Experimental Forest (MEF) in northern Minnesota. We used guided gene assembly to examine the abundance and diversity of protease genes; and further compared to those of N-fixing (nifH) genes in shotgun metagenomic data collected across depth at two distinct peatland environments (bogs and fens). Microbial proteases greatly outnumbered nifH genes with the most abundant gene families (archaeal M1 and bacterial Trypsin) each containing more sequences than all sequences attributed to nifH. Bacterial protease gene assemblies were diverse and abundant across depth profiles, indicating a role for bacteria in releasing free amino acids from peptides through depolymerization of older organic material and contrasting the paradigm of fungal dominance in depolymerization in forest soils. Although protease gene assemblies for fungi were much less abundant overall than for bacteria, fungi were prevalent in surface samples and therefore may be vital in degrading large soil polymers from fresh plant inputs during early stage of depolymerization. In total, we demonstrate that depolymerization enzymes from a diverse suite of microorganisms, including understudied bacterial and archaeal lineages, are likely to play a substantial role in C and N cycling within northern peatlands.\n\nImportanceNitrogen (N) is a common limitation on primary productivity, and its source remains unresolved in northern peatlands that are vulnerable to environmental change. Decompositionof complex organic matter into free amino acids has been proposed as an important N source, but the genetic potential of microorganisms mediating this process has not been examined. Such information can elucidate possible responses of northern peatlands to environmental change. We show high genetic potential for microbial production of free amino acids across a range of microbial guilds. In particular, the abundance and diversity of bacterial genes encoding proteolytic activity suggests a predominant role for bacteria in regulating productivity and contrasts a paradigm of fungal dominance of organic N decomposition. Our results expand our current understanding of coupled carbon and nitrogen cycles in north peatlands and indicate that understudied bacterial and archaeal lineages may be central in this ecosystems response to environmental change.

ecology↗

bioRxiv2018

The malaria-protective human glycophorin structural variant DUP4 shows somatic mosaicism and association with hemoglobin levels

Glycophorin A and glycophorin B are red blood cell surface proteins that are both receptors for the parasite Plasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A - glycophorin B fusion gene, and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and reveal somatic variation in copy number of the glycophorin A-glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation, and show that it is associated with a malarial-related phenotype in a non-hospitalized population.\n\nSignificance statementPrevious work has identified a human complex genomic structural variant called DUP4, which includes two novel glycophorin A-glycophorin B fusion genes, is associated with a profound protection against severe malaria. In this study, we present data showing the molecular basis of this complex variant. We also show evidence of somatic variation in the copy number of the fusion genes. We develop a simple robust assay for this variant and demonstrate that DUP4 is at an appreciable population frequency in Tanzania and that it is associated with higher hemoglobin levels in a malaria-endemic village. We suggest that DUP4 is therefore protective against malarial anemia.

genetics↗

bioRxiv2018

Aerobic removal of microcystin-LR by a novel native effective bacterial community designated as YFMCD4 isolated from Lake Taihu

Microcystins (MCs) are a group of monocyclic heptapeptide hepatotoxins produced by species of cyanobacteria. MC-LR is the most toxic and frequently detected MCs variant in water, which poses a great threat to the natural ecosystem and public health. Its important to seek environment-friendly and cost-efficient methods to remove MC-LR. To investigate the MC-degrading capacities of a novel indigenous bacterial community designated as YFMCD4 and the influence of environmental factors including various temperatures, MC concentrations and pH on the MC-degrading activities, the concentration of MC-LR was measured by high performance liquid chromatography. In addition, the MC-degrading mechanism containing the degradation pathway and products of YFMCD4 was studied using HPLC coupled with an ultra-high resolution LTQ Orbitrap Velos Pro ETD mass spectrometry equipped with electrospray ionization interface. The data showed MC-LR can be removed at the maximum rate of 0.5 {micro}g/(ml{middle dot}h) by YFMCD4 containing Alcaligenes faecalis and Stenotrophomonas acidaminiohila. The MC-degrading rates of YFMCD4 were significantly affected by different temperatures, pH and MC-LR concentrations. Two intermediates of a tetrapeptide and Adda appeared in the degradation process. These results illustrate that the novel bacterial community YFMCD4 can remove MC-LR effectively and completely, which indicates YFMCD4 possesses a significant potential to be used in bioremediation of water bodies contaminated by MC-LR.

ecology↗

bioRxiv2018

Corilagin controls post-parasiticide schistosome egg-induced liver fibrosis by inhibiting Stat6 signalling pathway

This study aims to explore the effect of Corilagin (Cor) on post-parasiticide schistosome egg-induced hepatic fibrosis through the Stat6 signalling pathway in vitro and in vivo. Cellular and animal models were established and treated by Corilagin. The inhibitory effect of Corilagin was also confirmed in RAW264.7 cells in which Stat6 was overexpressed based on the GV367-Stat6-EGFP lentiviral vector system and in which Stat6 was knock-downed by gene specific siRNAs. As a result, Corilagin prevented increases in the protein level of Phospho-Stat6 (P-Stat6). Both the mRNA and protein levels of the downstream mediators SOCS1, KLF4, and PPAR{gamma}/{delta} were markedly suppressed after Corilagin treatment. Expression of ARG1 and FIZZ1/Retnla, Ym1, TGF-{beta} and PDGF in serum were also inhibited by Corilagin. The pathological changes, area of granulomas of liver sections, and degree of hepatic fibrosis were significantly alleviated in the Corilagin group. The areas of CD68- and CD206-positive cells stained by immunofluorescence were significantly decreased by Corilagin. In conclusion, Corilagin can suppress post-parasiticide schistosome egg-induced hepatic fibrosis by inhibiting the Stat6 signalling pathway and provide a new therapeutic strategy for schistosomiasis liver fibrosis.

pharmacology and toxicology↗

bioRxiv2018

Local Polyploidy Phenomenon in Escherichia coli and its significance in genetic engineering

Classic Helmstetter & Cooper model asserted that the multifork phenomenon in the process of replication. However, the impacts of the multifork on the evolution and genetic engineering are still vague. Here, we employed CRISPR/Cas9 technology to knock-out eighteen Escherichia coli chromosomal fragments (over 50 kb) that represent all areas of the chromosome. We demonstrated that a single cell could have wild-type, color-reporter, and antibiotic-resistant genes simultaneously in the same locus of the different branches of the duplication forks after multiple rounds of deletions and replacements. This phenomenon that a single cell had different genotypes in its local polyploid chromosomes, which was similar to eukaryotic heterozygote, was named as local polyploidy. Under a defined selective pressure condition, offspring cells containing at least a copy of conditionally beneficial mutation could be enriched, and other alleles could be kept silently and peacefully in the duplication fork(s) of the same cell. The significance of this phenomenon in the genetic engineering was discussed.

microbiology↗

bioRxiv2018

Tandem duplications lead to loss of fitness effects in CRISPR-Cas9 data

CRISPR-Cas9 gene-editing is widely used to study gene function and is being advanced for therapeutic applications. Structural rearrangements are a ubiquitous feature of cancers and their impact on CRISPR-Cas9 gene-editing has not yet been systematically assessed. Utilising CRISPR-Cas9 knockout screens for 163 cancer cell lines, we demonstrate that targeting tandem amplified regions is highly detrimental to cellular fitness, in contrast to amplifications caused by chromosomal duplications which have little to no effect. Genomically clustered Cas9 double-strand DNA breaks are associated with a strong gene-independent decrease in cell fitness. We systematically identified collateral vulnerabilities in 25% of cancer cells, introduced by tandem amplifications of tissue non-expressed genes. Our analysis demonstrates the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage, with implications for the use of CRISPR-Cas9 gene-editing technology, and how resulting collateral vulnerabilities are a generalisable strategy to target cancer cells.

genomics↗

bioRxiv2018

ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks

Mutations in the ATM tumor suppressor confer hypersensitivity to DNA-damaging agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase poison topotecan. Thus, we establish that loss of terminal components of the non-homologous end-joining (NHEJ) machinery or the BRCA1-A complex specifically confers topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase inhibitor olaparib is due to delayed homologous recombination repair at DNA-replication-fork-associated double-strand breaks (DSBs), resulting in toxic NHEJ-mediated chromosome fusions. Accordingly, restoring legitimate repair in ATM-deficient cells, either by preventing NHEJ DNA ligation or by enhancing DSB-resection by BRCA1-A complex inactivation, markedly suppresses this toxicity. Our work suggests opportunities for patient stratification in ATM-deficient cancers and when using ATM inhibitors in the clinic, and identifies additional therapeutic vulnerabilities that might be exploited when such cancers evolve drug resistance.\n\nOne Sentence SummaryATM counteracts toxic NHEJ at broken replication forks

cell biology↗

bioRxiv2018

Effects of Xiantao sewage treatment on microbial community structure of shallow groundwater in wetland

In order to solve the problem of urban domestic sewage treated by semi natural wetland method with high connectivity between surface water and underground water in the flood diversion channel, Whether there is a blank study on the pollution of shallow groundwater. The community structure and abundances of CODCr, total nitrogen, total phosphorus and microorganism were added to the surface water. Environmental factors such as CODCr, TN, TP, NH4+-N and microbial community and abundance indices were monitored through surface water and groundwater. In this study, physical and chemical indicators, microbial diversity and community structure of 12 water samples from Xiantao artificial wetland were studied by using the Illumina Miseq sequencing technique and the resulting microbial data were statistically analyzed in combination with environmental variables. The results showed that CODCr concentration had a very significant positive correlation with total microbial communities (r=0.530, P<0.01), NH4+-N and TN were significantly positively correlated (r=0.337 and 0.325, P<0.05). In addition, CODCr concentration was highly positively correlated with abundant groups (r=0.520, P<0.05), NH4+-N and TN were significantly positively correlated (r=0.325 and 0.304, P<0.05). For rare taxa, they might be more sensitive to the environment than their abundant groups. The relative abundance of the rare group is 0 at the depth of 10m, so we should carefully evaluate microbial reaction (rare group) environmental conditions in the future.

microbiology↗

bioRxiv2018

Molecular mechanisms controlling the multistage post-translational processing of endogenous Nrf1α/TCF11 proteins to yield distinct proteoforms within the coupled positive and negative feedback circuits

In an attempt to terminate the chaotic state of the literature on Nrf1/TCF11 with various confused molecular masses, we herein establish a generally acceptable criterion required for identification of its endogenous full-length proteins and derivative isoforms expressed differentially in distinct experimental cell lines. Further work has been focused on the molecular mechanisms that dictate the successive multistate post-translational modifications (i.e. glycosylation by OST, deglycosylation by NGLY, and ubiquitination by Hrd1) of this CNC-bZIP protein and its proteolytic processing to yield multiple isoforms. Several lines of experimental evidence have demonstrated that the nascent Nrf1/TCF11 polypeptide (non-glycosylated) is transiently translocated into the endoplasmic reticulum (ER), in which it becomes an inactive glycoprotein-A, and also folded in a proper topology within and around membranes. Thereafter, dynamic repositioning of the ER-resident domains in Nrf1 glycoprotein is driven by p97-fueled retrotranslocation into extra-ER compartments. Therein, glycoprotein of Nrf1 is allowed for digestion into a deglycoprotein-B and then its progressive proteolytic processing by cytosolic DDI-1/2 and proteasomes to yield distinct proteoforms (i.e. protein-C/D). The processing is accompanied by removal of a major N-terminal ~12.5-kDa polypeptide from Nrf1. Interestingly, our present study has further unraveled that coupled positive and negative feedback circuits exist between Nrf1 and its cognate target genes, including those encoding its regulators p97, Hrd1, DDI-1 and proteasomes. These key players are differentially or even oppositely involved in diverse cellular signalling responses to distinct extents of ER-derived proteotoxic and oxidative stresses induced by different concentrations of proteasomal inhibitors.

biochemistry↗

bioRxiv2018

PP-2, a src-kinase inhibitor, is a potential corrector for F508del-CFTR in cystic fibrosis

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common mutation in CF, an in-frame deletion of phenylalanine 508, leads to a trafficking defect and endoplasmic reticulum retention of the protein where it becomes targeted for degradation. Successful clinical deployments of ivacaftor and ivacaftor/lumacaftor combination have been an exciting translational development in treating CF. However, their therapeutic effects are variable between subjects and remain insufficient. We used the Library of Integrated Network-based Cellular Signatures (LINCS) database as our chemical pool to screen for candidates. For in silico screening, we integrated connectivity mapping and CF systems biology to identify candidate therapeutic compounds for CF. Following in silico screening, we validated our candidate compounds with (i) an enteroid-based compound screening assay using CF ({Delta}F508/{Delta}F508-CFTR) patient-derived enteroids, (ii) short-circuit current analysis using polarized CF primary human airway epithelial cells and (iii) Western blots to measure F508-del-CFTR protein maturation. We identified 184 candidate compounds with in silico screening and tested 24 of them with enteroid-based forskolin-induced swelling (FIS) assay. The top hit compound was PP2, a known src-kinase inhibitor that induced swelling in enteroid comparable to known CF corrector (lumacaftor). Further validation with Western blot and short-circuit current analysis showed that PP-2 could correct mutant CFTR mis-folding and restore CFTR-mediated transmembrane current. We have identified PP2, a known src-kinase inhibitor, as a novel corrector of {Delta}F508-CFTR. Based on our studies and previous reports, src kinase inhibition may represent a novel paradigm of multi-action therapeutics - corrector, anti-inflammatory, and anti-infective - in CF.

systems biology↗

bioRxiv2018

Single-molecule optical mapping enables accurate molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral Muscular Dystrophy (FSHD) is a common adult muscular dystrophy in which the muscles of the face, shoulder blades and upper arms are among the most affected. FSHD is the only disease in which \"junk\" DNA is reactivated to cause disease, and the only known repeat array-related disease where fewer repeats cause disease. More than 95% of FSHD cases are associated with copy number loss of a 3.3kb tandem repeat (D4Z4 repeat) at the subtelomeric chromosomal region 4q35, of which the pathogenic allele contains less than 10 repeats and has a specific genomic configuration called 4qA. Currently, genetic diagnosis of FSHD requires pulsed-field gel electrophoresis followed by Southern blot, which is labor-intensive, semi-quantitative and requires long turnaround time. Here, we developed a novel approach for genetic diagnosis of FSHD, by leveraging Bionano Saphyr single-molecule optical mapping platform. Using a bioinformatics pipeline developed for this assay, we found that the method gives direct quantitative measurement of repeat numbers, can differentiate 4q35 and the highly paralogous 10q26 regions, can determine the 4qA/4qB allelic configuration, and can quantitate levels of post-zygotic mosaicism. We evaluated this approach on 5 patients (including two with post-zygotic mosaicism) and 2 patients (including one with post-zygotic mosaicism) from two separate cohorts, and had complete concordance with Southern blots, but with improved quantification of repeat numbers resolved between haplotypes. We concluded that single-molecule optical mapping is a viable approach for molecular diagnosis of FSHD and may be applied in clinical diagnostic settings once more validations are performed.

genomics↗

bioRxiv2018

SlARF10, an auxin response factor, is required for chlorophyll and sugar accumulation during tomato fruit development

Tomato green fruits photosynthesis contributes to fruit growth and carbon economy. Tomato auxin response factor 10 (SlARF10) is one of the members of ARF family. Our results showed that SlARF10 locates in the nucleus and has no transcriptional activity. SlARF10 was expressed in various tomato tissues, but highly expressed in green fruit. Up-regulation of SlARF10 produced dark green phenotype of fruits, whereas down-regulation of SlARF10 had light green phenotype. Autofluorescence and chlorophyll content analysis confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation in tomato fruits. Up-regulation of SlARF10 increased the photochemical potential in tomato leaves and fruits. Furthermore, the SlARF10 up-regulating lines displayed improved accumulation of starch in fruits, whereas SlARF10 suppressed lines had inhibited starch accumulation. Up-regulation of SlARF10 increased the expression of AGPases, the starch biosynthesis genes. SlARF10 up-regulating lines had increased accumulation of SlGLK1 and SlGLK2 transcripts in fruits. The promoter sequence of SlGLK1 gene had two conserved ARF binding sites. SlARF10 may regulate the expression of SlGLK1, thus controlling chlorophyll accumulation, photosynthesis rates and sugars synthesis in fruits. Our study provided more insight on the link between auxin signaling, chloroplastic activity and sugar metabolism during the development of tomato fruits.\n\nAbbreviations\n\nHighlightSlARF10 played an important role in the chlorophyll accumulation and photosynthesis in tomato fruits. SlARF10 was involved in starch accumulation by controlling the expression of starch synthesis related enzyme genes. SlARF10 may regulate the expression of SlGLK1, thus controlling chlorophyll accumulation, photosynthesis rates and sugars synthesis in tomato fruits.

plant biology↗