Biology subjects

Yang, K.

Publications and source records attributed to Yang, K..

143 records · Page 8Linked to original sources

bioRxiv2020

Room-temperature-storable PCR Mixes for SARS-CoV-2 Detection

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] in Wuhan, China, which then rapidly spread globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. We found that freeze-dried PCR mixes with [~]1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18[~]25{degrees}C) for 28 days, and for 14 and 10 days when stored at 37{degrees}C and 56{degrees}C, respectively. The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories. This method can also be applied to the generation of freeze-dried PCR mixes for the detection of other pathogens.

microbiology↗

bioRxiv2020

Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

Perturbations in the early life gut microbiome are associated with increased risk to complex immune disorder like inflammatory bowel diseases. We previously showed maternal antibiotic-induced gut dysbiosis vertically passed to offspring increases experimental colitis risk in IL-10 gene deficient (IL-10-/-) mice. While this could arise from emergence of pathobionts or loss/lack of essential microbes needed for appropriate immunological education, our findings suggest the latter. A dominant Bacteroides strain absent following antibiotic-induced perturbation was cultivated from murine fecal samples. Addition of this strain into mice with antibiotic-induced dysbiosis significantly promoted immune tolerance and reduced incidence of colitis in IL-10-/- mice, but only if engrafted early in life, and not during adulthood. Thus, key members of the gut microbiome are essential for development of immune tolerance to commensal microbes in early life and their addition in presence of gut dysbiosis during this period can reduce colitis risk in genetically prone hosts. HighlightsO_LISpecific gut microbes promote early life immune tolerance to key commensal microbes C_LIO_LILoss of early life keystone microbes increases colitis risk in genetically prone hosts C_LIO_LIEmergence of absent commensal microbes late in life worsened colitis outcome C_LIO_LIEarly life exposure to a missing keystone Bacteroides strain reduced colitis risk C_LI

microbiology↗

bioRxiv2020

Structure-based drug design, virtual screening and high-throughput screening rapidly identify antiviral leads targeting COVID-19

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan1-4. Currently there is no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (Mpro). Mpro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus5,6. Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus Mpro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of Mpro. Six of these inhibit Mpro with IC50 values ranging from 0.67 to 21.4 M. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases where no specific drugs or vaccines are available.

biochemistry↗

bioRxiv2020

A variant in SMOC2, inhibiting BMP signaling by competitively binding to BMPR1B, causes multiple epiphyseal dysplasia

Previously study showed that SMOC, a matricellular protein, inhibits BMP signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In our study, exome sequencing revealed a missense mutation (c.1076T>G, p.Leu359Arg) in EC domain of SMOC2 in a Chinese family with multiple epiphyseal disease (MED). The pathogenicity of this SMOC2 variant was verified by Smoc2L359R/L359R knock-in mice. Of note, decreasing phosphorylation of SMAD1/5/9 was detected in growth plates and primary chondrocytes from Smoc2L359R/L359R mice. Furthermore, binding affinity of mutant SMOC2 with collagen IX and HSPG in the extracellular matrix of cartilage were reduced while binding affinity with BMPRIB was intact. In addition, in contrast to previously results, that SMOC2 cannot antagonize BMP activity in the presence of a constitutively activated BMP receptor. These results support that SMOC2 with p.Leu359Arg variant act as an antagonist of canonical BMP pathway by competitively binding with BMP receptors.

genomics↗

bioRxiv2020

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

cell biology↗

bioRxiv2019

Prediction and Characterization of RXLR Effectors in Pythium Species

Being widely existed in oomycetes, the RXLR effector features conserved RXLR-dEER motifs in its N terminal. Every known Phytophthora or Hyaloperonospora pathogen harbors hundreds of RXLRs. In Pythium species, however, none of the RXLR effectors has been characterized yet. Here, we developed a stringent method for de novo identification of RXLRs and characterized 359 putative RXLR effectors from nine tested Pythium species. Phylogenetic analysis revealed a single superfamily formed by all oomycetous RXLRs, suggesting they descent from a common ancestor. RXLR effectors from Pythium and Phytophthora species exhibited similar sequence features, protein structures and genome locations. In particular, the mosquito biological agent P. guiyangense contains a significantly larger RXLR repertoire than the other eight Pythium species examined, which may result from gene duplication and genome rearrangement events as indicated by synteny analysis. Expression pattern analysis of RXLR-encoding genes in the plant pathogen P. ultimum detected transcripts from the vast majority of predicted RXLRs with some of them being induced at infection stages. One such RXLRs showed necrosis-inducing activity. Furthermore, all predicted RXLRs were cloned from two biocontrol agents P. oligandrum and P. periplocum. Three of them were found to encode effectors inducing defense response in Nicotiana benthamiana. Taken together, our findings represent the first complete synopsis of Pythium RXLR effectors, which provides critical clues on their evolutionary patterns as well as the mechanisms of their interactions with diverse hosts. Author summaryPathogens from the Pythium genus are widespread across multiple ecological niches. Most of them are soilborne plant pathogens whereas others cause infectious diseases in mammals. Some Pythium species can be used as biocontrol agents for plant diseases or mosquito management. Despite that phylogenetically close oomycete pathogens secrete RXLR effectors to enable infection, no RXLR protein was previously characterized in any Pythium species. Here we developed a stringent method to predict Pythium RXLR effectors and compared them with known RXLRs from other species. All oomycetous RXLRs form a huge superfamily, which indicates they may share a common ancestor. Our sequence analysis results suggest that the expansion of RXLR repertoire results from gene duplication and genome recombination events. We further demonstrated that most predicted Pythium RXLRs can be transcribed and some of them encode effectors exhibiting pathogenic or defense-inducing activities. This work expands our understanding of RXLR evolution in oomycetes in general, and provides novel insights into the molecular interactions between Pythium pathogens and their diverse hosts.

microbiology↗

bioRxiv2019

CRISPR screening of porcine sgRNA library identified host factors essential for Japanese encephalitis virus replication

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, key host genes involved in the JEV life cycle and cell death are poorly understood. Here, we designed 85,674 single guide RNAs (sgRNAs) targeting 17,743 protein-coding genes, 11,053 long ncRNAs (lncRNAs), and 551 microRNAs (miRNAs) in the porcine genome, and subsequently developed a porcine sgRNA library and genome-scale CRISPR/Cas9 knockout (PigGeCKO) system. These sgRNAs were delivered into porcine kidney-15 (PK-15) cells that constitutively express Cas9, positive selection screening of the resulting PigGeCKO cell collection for resistance to JEV-induced cell death led to the identification of several previously unreported genes required for JEV infection. We conducted follow-up studies to verify the dependency of JEV on these genes, and identified functional contributions for six of the many candidate JEV-related host genes, including an endoplasmic reticulum membrane protein complex subunit 3 (EMC3) and calreticulin (CALR). Additionally, we identified that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPG sulfurylation, facilitated JEV entry into PK-15 cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this work deepens our basic understanding of flavivirus infection and identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to prevent and treat diseases caused by Japanese encephalitis virus.

genetics↗

bioRxiv2019

The methodological quality of animal studies: A cross-sectional study based on the SYRCLEs risk of bias tool

ObjectiveTo assess the methodological quality of animal studies published in China and abroad using the SYRCLEs risk of bias tool, and to provide references to improve the methodological quality of animal studies to encourage high quality preclinical studies.\n\nMethodsAn electronic search was performed in the Chinese Scientific Citation Database (CSCD) and Web of Science from 2014 to October 2018. Document screening and data extraction were performed independently by four researchers. The methodological quality of the included studies was assessed using the SYRCLEs risk of bias tool. Statistical analysis was performed using SPSS23.\n\nResultsA total of 2764 animal studies were included. Of the studies, 984 were published in English and 1780 were in Chinese. The citation frequency of more than 90% of the included studies was less than 5. The results of methodological quality assessment showed that 36.36% (8/22) of the sub-items were rated as \"low risk\" in more than 50% of the included studies, of which 75% (6/8) were rated as \"low risk\" in more than 80% of the included studies. A total of 59.09% (13/22) of the sub-items were rated as \"low risk\" in less than 30% of the included studies, of which 92.31% (12/13) were rated as \"low risk\" in less than 10% of the included studies. The incidence of \"low risk\" Chinese studies regarding performance bias, detection bias and reporting bias were lower than English studies. For foreign studies, more attention should be paid to selection bias, attrition bias, and reporting bias.\n\nConclusionWe identified limitations in the methodological quality of animal experiment studies published in China and abroad. We therefore suggest that it is necessary to take targeted measures to popularize the SYRCLEs risk of bias tool to effectively improve the design and implementation of animal experiments, and guide study development.

scientific communication and education↗

bioRxiv2019

Does Sex Modify an Association of Electrophysiological Substrate with Sudden Cardiac Death? The Atherosclerosis Risk in Communities (ARIC) Study

BackgroundSex is a well-recognized risk factor for sudden cardiac death (SCD). Sex differences in electrophysiological (EP) substrate of SCD are known. However, it remains unknown whether sex can modify an association of EP substrate with SCD.\n\nMethodsParticipants from the Atherosclerosis Risk in Communities study with analyzable ECGs (n=14,725; age, 54.2{+/-}5.8 yrs; 55% female, 74% white) were included. EP substrate was characterized by traditional 12-lead ECG (heart rate, QRS, QTc, Cornell voltage), spatial ventricular gradient (SVG) and sum absolute QRST integral (SAI QRST) metrics. Two competing outcomes were adjudicated SCD and nonSCD. Interaction of ECG metrics with sex was studied in Cox proportional hazards and Fine-Gray competing risk models. Relative hazard ratio (RHR) and relative sub-hazard ratio (RSHR) with a 95% confidence interval for SCD and nonSCD risk for women relative to men were calculated. Model 1 was adjusted for prevalent cardiovascular disease (CVD) and risk factors. Time-updated model 2 was additionally adjusted for incident non-fatal CVD.\n\nResultsOver a median follow-up of 24.4 years, there were 530 SCDs (incidence 1.72 (1.58-1.88)/1000 person-years) and 2,178 nonSCDs (incidence 7.09; (6.80-7.39)/ 1000 person-years). Women experienced a greater than men risk of SCD associated with Cornell voltage (RHR 1.18(1.06-1.32); P=0.003), SAI QRST (RHR 1.16(1.04-1.30); P=0.007), area SVG magnitude (RHR 1.24(1.05-1.45); P=0.009), and peak SVG magnitude (RHR 1.22(1.04-1.44); P=0.018), independently from incident CVD. Greater risk of SCD for women than men associated with QRS duration (RHR 1.24(1.07-1.44); P=0.004) and QTc (RSHR 1.15(1.02-1.30); P=0.025) was explained by incident CVD. Furthermore, women had greater odds of SCD associated with heart rate (RSHR 1.19(1.01-1.40); P=0.036), independently of incident CVD.\n\nConclusionsSex modifies an association of EP substrate with SCD. In women, global EP substrate is associated with up to 27% greater risk of SCD than in men. Development of sex-specific risk scores of SCD is necessary. Further studies of mechanisms behind sex differences in EP substrate of SCD are warranted.

epidemiology↗

bioRxiv2019

A universal scaling method for biodiversity-ecosystem functioning relationships

Global biodiversity is declining at rates faster than at any other point in human history. Experimental manipulations of biodiversity at small spatial scales have demonstrated that communities with fewer species consistently produce less biomass than higher diversity communities. However, understanding how the global extinction crisis is likely to impact global ecosystem functioning will require applying these local and largely experimental findings to natural systems at substantially larger spatial and temporal scales. Here we propose that we can use two simple macroecological patterns - the species area curve and the biomass-area curve - to upscale the species richness-biomass relationship. We demonstrate that at local spatial scales, each additional species will contribute more to biomass production with increasing area sampled because the species-area curve saturates and the biomass-area curve increases monotonically. We use species-area and biomass-area curves from a Minnesota grassland and a Panamanian tropical dry forest to examine the species richness - biomass relationship at three and ten sampling extents, respectively. In both datasets, the observed relationship between biodiversity and biomass production at every sampling extent was predicted from simple species-area and biomass-area relationships. These findings suggest that macroecological patterns like the species-area curve underpin the scaling of biodiversity-ecosystem functioning research and can be used to predict these relationships at the global scales where they are relevant for species loss.

ecology↗

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

Synthetic chromosome fusion: effects on genome structure and function

As part of the Synthetic Yeast 2.0 (Sc2.0) project, we designed and synthesized synthetic chromosome I. The total length of synI is [~]21.4% shorter than wild-type chromosome I, the smallest chromosome in Saccharomyces cerevisiae. SynI was designed for attachment to another synthetic chromosome due to concerns of potential instability and karyotype imbalance. We used a variation of a previously developed, robust CRISPR-Cas9 method to fuse chromosome I to other chromosome arms of varying length: chrIXR (84kb), chrIIIR (202kb) and chrIVR (1Mb). All fusion chromosome strains grew like wild-type so we decided to attach synI to synIII. Through the investigation of three-dimensional structures of fusion chromosome strains, unexpected loops and twisted structures were formed in chrIII-I and chrIX-III-I fusion chromosomes, which depend on silencing protein Sir3. These results suggest a previously unappreciated 3D interaction between HMR and the adjacent telomere. We used these fusion chromosomes to show that axial element Red1 binding in meiosis is not strictly chromosome size dependent even though Red1 binding is enriched on the three smallest chromosomes in wild-type yeast, and we discovered an unexpected role for centromeres in Red1 binding patterns.

synthetic biology↗

bioRxiv2018

Neurofibromin is essential to maintain metabolic function and sustain life in the adult mouse

The consequences of pathogenic variants in the NF1 gene can manifest in numerous tissues as a result of loss of neurofibromin protein function(s). A known function of NF1 is negative regulation of p21ras signaling via a GTPase activating (Ras-GAP) domain. Besides modulation of Ras signaling as a tumor suppressor, other functions of this multi-domain protein are less clear. Biallelic inactivation of NF1 leads to an embryonic lethal phenotype, while neurofibromin is expressed at varying levels in most tissues beyond developmental stages. Taking advantage of the mouse genetics toolkit, we established novel tamoxifen-inducible systemic knockout Nf1 mouse models (C57BL/6) to gain a better understanding of the role of Nf1 in the adult (3-4 months) mouse. Following inactivation of floxed Nf1 alleles, adult CAGGCre-ERTM;Nf14F/4F mice lose function of Nf1 systemically. Both male and female animals do not survive beyond 11 days post-tamoxifen induction and exhibit histological changes in multiple tissues. During this acute crisis, CAGGCre-ERTM;Nf14F/4F mice are not able to maintain body temperature or body mass, and expend all adipose tissue; however, they continue to consume food and absorb calories comparable to littermate-paired controls. Targeted metabolite analyses and indirect calorimetry studies revealed altered fat metabolism, amino acid metabolism and energy expenditure, with animals undergoing metabolic crisis and torpor-like states. Thermoneutral conditions accelerated the acute, lethal phenotype coincident with lower food intake. This study reveals that systemic loss of neurofibromin in the adult mouse induces metabolic dysfunction and lethality, thus highlighting potential functions of this multi-domain protein in addition to tumor suppression.

genetics↗

bioRxiv2018

Loss of CREST leads to neuroinflammatory responses and ALS-like motor defects in mice

Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease with fast progression. Mutations of the CREST gene (also known as SS18L1) are identified in sporadic ALS patients. Whether CREST mutations may lead to ALS remained largely unclear. In this study, we showed that the ALS-related CREST-Q388X mutation exhibited loss-of-function effects. Importantly, we found that microglial activation were prevalent in CREST haploinsufficieny mice and the Q394X mice mimicking the human CREST Q388X mutation. Furthermore, we showed that both CREST haploinsufficieny and the Q394X mice displayed deficits in motor coordination. Finally, we identified the critical role of CREST-BRG1 complex in repressing the expression of immune-related cytokines including Ccl2 and Cxcl10 in neurons, via histone deacetylation, providing the molecular mechanisms underlying inflammatory responses lack of CREST. These findings indicate that elevated inflammatory responses in a subset of ALS may be caused by neuron-derived factors, suggesting potential therapeutic methods through inflammation pathways.\n\nIn BriefCheng et al. discovered that neuronal loss of CREST reduces the protein level of FUS, de-represses the transcriptional inhibition of chemokine genes which in turn causes microglial activation and proinflammation, and ultimately leads to axonal degeneration of motor neurons and impairment of locomotion.

neuroscience↗

bioRxiv2017

Spindle asymmetry drives non-Mendelian chromosome segregation

Genetic elements compete for transmission through meiosis, when haploid gametes are created from a diploid parent. Selfish elements can enhance their transmission through meiotic drive, in violation of Mendels Law of Segregation. In female meiosis, selfish elements drive by preferentially attaching to the egg side of the spindle, which implies some asymmetry between the two sides of the spindle, but molecular mechanisms underlying spindle asymmetry are unknown. Here we show that CDC42 signaling from the cell cortex regulates microtubule tyrosination to induce spindle asymmetry, and non-Mendelian segregation depends on this asymmetry. These signals depend on cortical polarization directed by chromosomes, which are positioned near the cortex to allow the asymmetric cell division. Thus, selfish meiotic drivers exploit the asymmetry inherent in female meiosis to bias their transmission.

cell biology↗

bioRxiv2017

A toolbox of immunoprecipitation-grade monoclonal antibodies against human transcription factors.

A key component to overcoming the reproducibility crisis in biomedical research is the development of readily available, rigorously validated and renewable protein affinity reagents. As part of the NIH Protein Capture Reagents Program (PCRP), we have generated a collection of 1406 highly validated, immunoprecipitation (IP) and/or immunoblotting (IB) grade, mouse monoclonal antibodies (mAbs) to 736 human transcription factors. We used HuProt human protein microarrays to identify mAbs that recognize their cognate targets with exceptional specificity. Using an integrated production and validation pipeline, we validated these mAbs in multiple experimental applications, and have distributed them to the Developmental Studies Hybridoma Bank (DSHB) and several commercial suppliers. This study allowed us to perform a meta-analysis that identified critical variables that contribute to the generation of high quality mAbs. We find that using full-length antigens for immunization, in combination with HuProt analysis, provides the highest overall success rates. The efficiencies built into this pipeline ensure substantial cost savings compared to current standard practices.

biochemistry↗

bioRxiv2017

Assessment of the World Largest Afforestation Program: Success, Failure, and Future Directions

The Three-North Afforestation Program (TNAP), initiated in 1978 and scheduled to be completed in 2050, is the worlds largest afforestation project and covers 4.07 x 106 km2 (42.4%) of China. We systematically assessed goals and outcomes of the first 30 years of the TNAP using high-resolution remote sensing and ground survey data. With almost 23 billion dollars invested between 1978 and 2008, the forested area within the TNAP region increased by 1.20 x 107 ha, but the proportion of high quality forests declined by 15.8%. The establishment of shelterbelts improved crop yield by 1.7%, much lower than the 5.9% expected once all crop fields are fully protected by shelterbelts. The area subjected to soil erosion by water decreased by 36.0% from 6.72 x 107 to 4.27 x 107 ha; the largest reductions occurred in areas where soil erosion had been most severe and forests contributed more than half of this improvement. Desertification area increased from 1978 to 2000 but decreased from 2000 to 2008; the 30-year net reduction was 13.0% (4.05x106 ha), with 8.0% being accounted for by afforestation in areas with only slight, prior desertification. In addition to its direct impacts, the TNAP has enhanced peoples awareness of environmental protection and attracted consistent attention and long-term commitment from the Chinese government to the restoration and protection of fragile ecosystems in the vast Three-North region. The significant decline in forest quality, limited success in reducing desertification, and low coverage of shelterbelts are aspects of the TNAP in need of re-assessment, and additional ca. 34 billion dollars will be needed to ensure the completion of the TNAP.

ecology↗