Biology subjects

Liao, Y.

Publications and source records attributed to Liao, Y..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Coronavirus endoribonuclease nsp15 induces host cellular protein synthesis shutoff

The endoribonuclease (EndoU) nsp15 of coronaviruses plays an important role in evasion of host innate immune responses by reducing the abundance of viral double-stranded RNA, whereas less is known about potential host cellular targets of nsp15. In this study, we show that cellular protein synthesis is inhibited upon over-expression of nsp15 from four genera of coronaviruses and this is accompanied by nuclear retention of the poly(A) binding protein cytoplasmic 1 (PABPC1). We also show that the EndoU activity of nsp15 is indispensable for both, inhibition of protein synthesis and PABPC1 nuclear relocation. FISH analysis using oligo-dT probes, revealed an overlap between the localization of cellular mRNA and that of overexpressed nsp15 in some cells, suggesting that, when expressed alone, nsp15 may target host mRNA. When investigating the association of nsp15 on protein shut off in the context of a viral infection, we observed that the{gamma} -coronavirus infectious bronchitis virus (IBV), induced host translation shutoff in an p-eIF2-independent manner and mainly retained PABPC1 in the cytoplasm, whereas the nsp15 EndoU-deficient IBV accumulated viral dsRNA and caused p-PKR-p-eIF2-dependent host protein translation shutoff, accompanied with PABPC1 nuclear relocation or stress granule (SG) localization. This phenomenon suggests that during infection with wild type IBV, although the cellular translation is inhibited, initiation of viral mRNA translation leads to PABPC1 binding to viral mRNA, thereby preventing its nuclear entry; during infection with nsp15 EndoU-deficient IBV however, the eIF2-dependent host protein translation shutoff prevents both host and viral mRNA translation initiation, releasing PABPC1 from binding to cytosolic and viral mRNA, thereby relocating it to the nucleus or to SG. Altogether, this study reveals unique yet conserved mechanisms of host protein shutoff that add to our understanding of how coronaviruses regulate host protein expression through a mechanism that involves catalytically active nsp15 EndoU, and describes how nsp15 may target both, viral and host mRNA. Author summaryIt has been reported that coronavirus infection suppresses host protein translation, - and {beta}- coronavirus nsp1 is responsible for inhibition of host gene expression. However, for{gamma} - and{delta} -coronavirus, there is no nsp1 and the underlying mechanisms by which virus regulates host translation are not well characterized. Here, we show that coronavirus endoribonuclease nsp15 is responsible for the inhibition of host translation by targeting to host factors, meanwhile it helps virus bypass the PKR-eIF2 mediated host translation shutoff, which is harmful for virus gene expression, by reducing the accumulation of viral dsRNA. This novel finding gives insight how does nsp15 targets to both host factors and viral RNA, to facilitate virus replication. Moreover, the novel function of nsp15 is found to be conserved among coronaviruses, revealing the essential role of this endoribonuclease in hijacking host translation machinery for virus replication.

microbiology↗

bioRxiv2023

Extracellular vesicles of Euryarchaeida: precursor to eukaryotic membrane trafficking

Since their discovery, extracellular vesicles (EVs) have changed our view on how organisms interact with their extracellular world. EVs are able to traffic a diverse array of molecules across different species and even domains, facilitating numerous functions. In this study, we investigate EV production in Haloferax volcanii, as representative for Euryarchaeida. We uncover that EVs enclose RNA, with specific transcripts preferentially enriched, including those with regulatory potential, and conclude that EVs can act as an RNA communication system between haloarchaea. We demonstrate the key role of an EV-associated Ras superfamily GTPase for EV formation in H. volcanii that is also present across other diverse evolutionary branches of Archaea. Ras superfamily GTPases are key players in eukaryotic intracellular vesicle formation and trafficking mechanisms that have been crucial for the emergence of Eukaryotes. Therefore, we propose that archaeal EV formation could reveal insights into the origin of the compartmentalized eukaryotic cell.

microbiology↗

bioRxiv2023

The intracellular lifestyle of an archaeal symbiont

DPANN Archaea are a diverse group of organisms typically characterised by small cells and reduced genomes. To date, all cultivated DPANN Archaea are ectosymbionts that require direct cell contact with an archaeal host species for proliferation. However, the dynamics of DPANN - host interactions and the impacts of these interactions on the host species are poorly understood. Here, we show that one DPANN archaeon (Candidatus Nanohaloarchaeum antarcticus) engages in parasitic interactions with its host (Halorubrum lacusprofundi) that result in host cell lysis. Our data also suggest that these interactions involve invasion of the host cell by the nanohaloarchaeon. This is the first reported instance of such a predatory-like lifestyle amongst Archaea and indicates that some DPANN Archaea may interact with host populations in a manner similar to viruses.

microbiology↗

bioRxiv2022

Evaluation of antibody kinetics and durability in health subjects vaccinated with inactivated COVID-19 vaccine (CoronaVac): A cross-sectional and cohort study in Zhejiang, China

BackgroundAlthough inactivated COVID-19 vaccines are proven to be safe and effective in the general population, the dynamic response and duration of antibodies after vaccination in the real world should be further assessed. MethodsWe enrolled 1067 volunteers who had been vaccinated with one or two doses of CoronaVac in Zhejiang Province, China. Another 90 healthy adults without previous vaccinations were recruited and vaccinated with three doses of CoronaVac, 28 days and 6 months apart. Serum samples were collected from multiple timepoints and analyzed for specific IgM/IgG and neutralizing antibodies (NAbs) for immunogenicity evaluation. Antibody responses to the Delta and Omicron variants were measured by pseudovirus-based neutralization tests. ResultsOur results revealed that binding antibody IgM peaked 14-28 days after one dose of CoronaVac, while IgG and NAbs peaked approximately 1 month after the second dose then declined slightly over time. Antibody responses had waned by month 6 after vaccination and became undetectable in the majority of individuals at 12 months. Levels of NAbs to live SARS-CoV-2 were correlated with anti-SARS-CoV-2 IgG and NAbs to pseudovirus, but not IgM. Homologous booster around 6 months after primary vaccination activated anamnestic immunity and raised NAbs 25.5-fold. The NAb inhibition rate subsequently rose to 36.0% for Delta (p=0.03) and 4.3% for Omicron (p=0.004), and the response rate for Omicron rose from 7.9% (7/89) to 17.8% (16/90). ConclusionsTwo doses of CoronaVac vaccine resulted in limited protection over a short duration. The homologous booster slightly increased antibody responses to the Delta and Omicron variants; therefore, the optimization of booster procedures is vital. FundingKey Research and Development Program of Zhejiang Province; Key Program of Health Commission of Zhejiang Province/ Science Foundation of National Health Commission; Major Program of Zhejiang Municipal Natural Science Foundation.

immunology↗

bioRxiv2022

UBAP2L-dependent coupling of PLK1 localization and stability during mitosis

PLK1 is an important regulator of mitosis whose protein levels and activity fluctuate during the cell cycle. PLK1 dynamically localizes to various mitotic structures to regulate chromosome segregation. However, the signaling pathways linking localized PLK1 activity to its protein stability remain elusive. Here, we identify the Ubiquitin-Binding Protein 2-Like (UBAP2L) that controls both, the localization and the protein stability of PLK1.We demonstrate that UBAP2L is a spindle-associated protein whose depletion leads to severe mitotic defects. UBAP2L depleted cells are characterized by increased PLK1 protein levels and abnormal PLK1 accumulation in several mitotic structures such as kinetochores, centrosomes and mitotic spindle. UBAP2L deficient cells exit mitosis and enter the next interphase in the presence of aberrant PLK1 kinase activity. The C-terminal domain of UBAP2L mediates its function on PLK1 independently of its role in stress response signaling. Importantly, the mitotic defects of UBAP2L depleted cells are largely rescued upon chemical inhibition of PLK1. Overall, our data suggest that UBAP2L is required to finetune the ubiquitin-mediated PLK1 turnover during mitosis as a means to maintain genome fidelity.

cell biology↗

bioRxiv2022

Development of an affinity-enhanced clinical candidate TCR targeting NY-ESO-1 with optimal potency and high specificity

The clinical success of T-cell receptor (TCR)-based immunotherapy depends on the efficacy and specificity of TCRs. Naturally occurring TCRs have limited anti-tumor potency due to their low affinity for tumor antigens. Affinity enhancement is a promising strategy to generate highly potent TCRs. However, it is concerned that affinity-enhanced TCRs are prone to lose specificity. We isolated low affinity TCRs specific for NY-ESO-1157-165/HLA-A*02:01 from peripheral blood mononuclear cells of healthy donors. An affinity-enhanced TCR candidate with optimal affinity and specificity was generated using phage display and an extensive set of in vitro and in vivo assays. Alanine scanning mutagenesis showed that the TCR candidate retained specificity by making extensive contacts to the side chains of NY-ESO-1157-165 peptide. Adoptive transfer of T cells engineered with this candidate (termed TAEST16001) significantly inhibited tumor growth in subcutaneous, metastatic, and patient-derived xenograft (PDX) mouse tumor models. This study demonstrates that sophisticated engineering and screening techniques can be utilized to generate a clinical candidate TCR with potent anti-tumor activity without losing specificity. TAEST16001 was approved by the Center for Drug Evaluation (CDE) as the first TCR-based immunotherapy clinical trial in China (ClinicalTrials.gov Identifier: NCT03159585).

immunology↗

bioRxiv2022

MAP4Ks inhibition promotes retinal neuron regeneration from Muumlller glia in adult mice

Mammalian Muller glia (MG) possess limited regenerative capacities. However, the intrinsic capacity of mammalian MG to transdifferentiate to generate mature neurons without transgenic manipulations remains speculative. Here we show that MAP4K4, MAP4K6 and MAP4K7, which are conserved Misshapen subfamily of ste20 kinases homologs, repress YAP activity in mammalian MG and therefore restrict their ability to be reprogrammed. However, by treating with a small molecule inhibitor of MAP4K4/6/7, mouse MG regain their ability to proliferate and enter into a retinal progenitor cell (RPC)-like state after NMDA-induced retinal damage; such plasticity was lost in YAP knockout MG. Moreover, spontaneous trans-differentiation of MG into retinal neurons expressing both amacrine and retinal ganglion cell (RGC) markers occurs after inhibitor withdrawal. Taken together, these findings suggest that MAP4Ks block the reprogramming capacity of MG in a YAP-dependent manner in adult mammals, which provides a novel avenue for the pharmaceutical induction of retinal regeneration in vivo.

neuroscience↗

bioRxiv2022

Long-term passaging of replication competent pseudo-typed SARS-CoV-2 reveals the antiviral breadth of monoclonal and bispecific antibody cocktails

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses challenges to the effectiveness of neutralizing antibodies. Rational design of antibody cocktails is a realizable approach addressing viral immune evasion. However, evaluating the breadth of antibody cocktails is essential for understanding the development potential. Here, based on a replication competent vesicular stomatitis virus model that incorporates the spike of SARS-CoV-2 (VSV-SARS-CoV-2), we evaluated the breadth of a number of antibody cocktails consisting of monoclonal antibodies and bispecific antibodies by long-term passaging the virus in the presence of the cocktails. Results from over two-month passaging of the virus showed that 9E12+10D4+2G1 and 7B9-9D11+2G1 from these cocktails were highly resistant to random mutation, and there was no breakthrough after 30 rounds of passaging. As a control, antibody REGN10933 was broken through in the third passage. Next generation sequencing was performed and several critical mutations related to viral evasion were identified. These mutations caused a decrease in neutralization efficiency, but the reduced replication rate and ACE2 susceptibility of the mutant virus suggested that they might not have the potential to become epidemic strains. The 9E12+10D4+2G1 and 7B9-9D11+2G1 cocktails that picked from the VSV-SARS-CoV-2 system efficiently neutralized all current variants of concern and variants of interest including the most recent variants Delta and Omicron, as well as SARS-CoV-1. Our results highlight the feasibility of using the VSV-SARS-CoV-2 system to develop SARS-CoV-2 antibody cocktails and provide a reference for the clinical selection of therapeutic strategies to address the mutational escape of SARS-CoV-2.

cell biology↗

bioRxiv2022

PinR as a Stress Inducible Inhibitory Transcription Factor in Stopping Stress-induced Mutagenesis Bacterial Evolution of Antibiotic Resistance

A recent work reports that the single treatment of {beta}-lactams can cause a SOS-independent superfast evolution of multi-drug resistance in the DNA repair deficiency Escherichia coli (E. coli), but the mechanism is not yet clear. Here, we find that the induction of PinR, a lambdoid prophage Rac, is involved in this process and facilitates the evolution of antibiotic resistance in DNA repair deficiency bacteria through the repression on the transcription of antioxidative genes and the thereafter ROS burst in cells. It is highlighted that the bacteriophage PinR can orchestrate the mutagenesis induced by the overaccumulation of ROS in cells. More importantly, we for the first time demonstrate that the deletion of pinR can avoid the rapid evolution of antibiotic resistance induced by either the single or long-term exposure to antibiotics, while strategies to target RecA, e.g., the inactivation on RecA, can be safely implemented to disarm the bacterial resistance to other antibiotics. Therefore, from a drug development perspective, our work suggests future studies on the "evolutionary potentiators" towards a safe and more effective strategy to be developed for infectious disease treatment.

microbiology↗

bioRxiv2022

Efficient Neutralization of SARS-CoV-2 Omicron and Other VOCs by a Broad Spectrum Antibody 8G3

Numerous mutations in the spike protein of SARS-CoV-2 B.1.1.529 Omicron variant pose a crisis for antibody-based immunotherapies. The efficacy of emergency use authorized (EUA) antibodies that developed in early SARS-CoV-2 pandemic seems to be in flounder. We tested the Omicron neutralization efficacy of an early B cell antibody repertoire as well as several EUA antibodies in pseudovirus and authentic virus systems. More than half of the antibodies in the repertoire that showed good activity against WA1/2020 previously had completely lost neutralizing activity against Omicron, while antibody 8G3 displayed non-regressive activity. EUA antibodies Etesevimab, Casirivimab, Imdevimab and Bamlanivimab were entirely desensitized by Omicron. Only Sotrovimab targeting the non-ACE2 overlap epitope showed a dramatic decrease activity. Antibody 8G3 efficiently neutralized Omicron in pseudovirus and authentic virus systems. The in vivo results showed that Omicron virus was less virulent than the WA1/2020 strain, but still caused deterioration of health and even death in mice. Treatment with 8G3 quickly cleared virus load of mice. Antibody 8G3 also showed excellent activity against other variants of concern (VOCs), especially more efficient against authentic Delta plus virus. Collectively, our results suggest that neutralizing antibodies with breadth remains broad neutralizing activity in tackling SARS-CoV-2 infection despite the universal evasion from EUA antibodies by Omicron variant.

cell biology↗

bioRxiv2022

Deciphering the developmental patterns of white matter tracts in a diffusion MRI based fetal brain atlas

White matter of the fetal brain undergoes rapid development to form the early structural connections. Diffusion MRI has shown to be a useful tool to depict the fetal brain white matter in utero, and many studies have observed increasing fractional anisotropy and decreasing mean diffusivity in the fetal brain during the second-to-third trimester, whereas others reported non-monotonic changes. Unbiased diffusion MRI atlases of the fetal brain are important for characterizing developmental trajectories of white matter and providing normative references for in-utero diagnosis of prenatal abnormalities. To date, the sole fetal brain diffusion MRI atlas was collected from a Caucasian/mixed population, and was constructed based on the diffusion tensor model with limited spatial resolution. In this work, we proposed a fiber orientation distribution (FOD) based pipeline for the generation of fetal brain diffusion MRI atlases, which showed better registration accuracy than diffusion tensor-based pipeline. Based on the FOD pipeline, we constructed the first Chinese fetal brain diffusion MRI atlas using 89 normal fetal diffusion MRI scans at GA between 24 and 38 weeks. Complex non-monotonic trends of tensor- and FOD-derived microstructural parameters in eight white matter tracts were observed, which jointly pointed to different phases of microstructural development. Specifically, we speculated that the turning point of the diffusivity trajectory may correspond to the starting point of pre-myelination, based on which, the developmental order of white matter tracts can be mapped and the order was in agreement with order of myelination from histological studies. The normative atlas also provided a reference for detection of abnormal white matter development, such as that in congenital heart disease. Therefore, the high-order fetal brain diffusion MRI atlas established in this study depicted the spatiotemporal pattern of early white matter development, and findings may help decipher the distinct microstructural events in utero.

neuroscience↗

bioRxiv2022

Genome-wide in vivo CRISPR screens identify GATOR1 as a TP53 induced tumour suppressor

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC (which is abnormally highly expressed in [~]70% of human cancers) we have used a genome-wide CRISPR knockout screen in E{micro}-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discovered that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerated c-MYC-driven lymphoma development in mice. Low expression of the GATOR1 complex genes correlated with poor survival outcomes for human patients with high MYC- expressing cancers. Murine lymphomas lacking GATOR1 were highly sensitive to mTOR inhibitors as a single agent therapy, both in vitro and in vivo. These findings identify inhibition of mTORC1 as a potent tumor suppressive mechanism in c-MYC-driven lymphomagenesis and suggest a new avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition. Key PointsO_LIIn vivo CRISPR/Cas9 whole genome knockout screens identified the GATOR1 complex as a potent suppressor of c-MYC-driven lymphomagenesis. C_LIO_LIGATOR1 deficiency in MYC-driven lymphomas confers sensitivity to mTOR inhibition, suggesting a novel therapeutic approach. C_LI

cancer biology↗

bioRxiv2022

Comprehensive Evaluation of ACE2-Fc Combination with Neutralization Antibody on Broad Protection against SARS-CoV-2 and Its Variants

Emerging SARS-CoV-2 variants are threatening the efficacy of antibody therapies. Combination treatments including ACE2-Fc have been developed to overcome the evasion of neutralizing antibodies (NAbs) in individual cases. Here we conducted a comprehensive evaluation of this strategy by combining ACE2-Fc with NAbs of diverse epitopes on the RBD. NAb+ACE2-Fc combinations efficiently neutralized HIV-based pseudovirus carrying the spike protein of the Delta or Omicron variants, achieving a balance between efficacy and breadth. In an antibody escape assay using replication-competent VSV-SARS-CoV-2-S, all the combinations had no escape after fifteen passages. By comparison, all the NAbs without combo with ACE2-Fc had escaped within six passages. Further, the VSV-S variants escaped from NAbs were neutralized by ACE2-Fc, revealing the mechanism of NAb+ACE2-Fc combinations survived after fifteen passages. We finally examined ACE2-Fc neutralization against pseudovirus variants that were resistant to the therapeutic antibodies currently in clinic. Our results suggest ACE2-Fc is a universal combination partner to combat SARS-CoV-2 variants including Delta and Omicron.

cell biology↗

bioRxiv2021

The 3D architecture of the pepper (Capsicum annum) genome and its relationship to function and evolution

The architecture of topologically associating domains (TADs) varies across plant genomes. Understanding the functional consequences of this diversity requires insights into the pattern, structure, and function of TADs. Here, we present a comprehensive investigation of the 3D genome organization of pepper (Capsicum annuum) and its association with gene expression and genomic variants. We report the first chromosome-scale long-read genome assembly of pepper and generate Hi-C contact maps for four tissues. The contact maps indicate that 3D structure varies somewhat across tissues, but generally the genome was segregated into subcompartments that were correlated with transcriptional state. In addition, chromosomes were almost continuously spanned by TADs, with the most prominent found in large genomic regions that were rich in retrotransposons. A substantial fraction of TAD boundaries were demarcated by chromatin loops, suggesting loop extrusion is a major mechanism for TAD formation; many of these loops were bordered by genes, especially in highly repetitive regions, resulting in gene clustering in three dimensional space. Integrated analysis of Hi-C profiles and transcriptomes showed that change in 3D chromatin structures (e.g. subcompartments, TADs, and loops) was not the primary mechanism contributing to differential gene expression between tissues, but chromatin structure does play a role in transcription stability. TAD boundaries were significantly enriched for breaks of synteny and depletion of sequence variation, suggesting that TADs constrain patterns of genome structural evolution in plants. Together, our work provides insights into principles of 3D genome folding in large plant genomes and its association with function and evolution.

genomics↗

bioRxiv2021

Broad ultra-potent neutralization of SARS-CoV-2 variants by monoclonal antibodies specific to the tip of RBD

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with ACE2 interface, which gives 2G1 ability to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar IC50 in vitro. In SARS-CoV-2 and Beta- and Delta-variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 could be potentially capable of dealing with emerging SARS-CoV-2 variants in future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

cell biology↗

bioRxiv2021

Interleukin-6 Signaling Mediates Cartilage Degradation and Pain in Post-Traumatic Osteoarthritis

Osteoarthritis (OA) and post-traumatic OA (PTOA) are prevalent joint disorders and leading causes of chronic pain. The disease pathology of OA/PTOA is caused by imbalanced catabolic and anabolic responses and pro-inflammatory changes; however, their connection to pain is not well studied. Since IL-6 is involved in cartilage degradation and conditions of inflammatory pain, we set out to identify whether IL-6 and IL-6 signaling mechanisms contribute to both PTOA-associated cartilage degradation and pain. We performed a modified destabilization of the medial meniscus (DMM) surgery, a model of PTOA, on conventional IL-6 KO and control mice and assessed both cartilage degradation and pain-associated phenotypes. Genetic removal of Il6 in males attenuates PTOA-associated cartilage catabolism, decreases innervation of soft tissues associated with the knee joint, and reduces nociceptive pain signaling, without improving subchondral bone sclerosis or chondrocyte apoptosis. We further demonstrate that specific downstream mediators of IL-6 signaling, the Janus kinases (JAKs), are critical in regulating both cartilage catabolism and pain signaling. We identified STAT3 as a key regulator of cartilage catabolism downstream of JAK; however, inhibition of STAT3 decreases cartilage anabolism while enhancing pain signals. ERK was found to be important for neurite outgrowth and pain signaling; however, inhibition of ERK was less effective in reducing cartilage catabolism. Therefore, our data demonstrate that IL-6 mediates both PTOA-associated cartilage degradation and pain, and provides critical details regarding the downstream mediators of IL-6 signaling as therapeutic targets for disease-modifying osteoarthritis drugs. Single Sentence Summary IL-6 mediates PTOA-associated cartilage degradation and pain via specific downstream signaling mechanisms in a gender specific manner.

pathology↗

bioRxiv2021

A SUBTILISIN-LIKE SERINE PROTEASE1 (OsSUBSrP1), plays an important role in the wax and cutin pathway, is essential for panicle development in rice

Panicle degeneration is a severe physiological defect and causes reduction in grain yield. In this study, we characterized and presented the functional analysis of our previously reported mutant apa1331 (apical panicle abortion1331) that showed apical spikelet degeneration. The anthers from the apical spikelets of apa1331 were degenerated, pollen-less and showed lack of cuticle formation. Transverse sections showed normal meiosis till stage 5-6, however, defects in post-meiotic microspore development were found at stage 8-9 in apa1331. Measurement of wax and cutin analysis showed a significant reduction in anthers of apa1331 compared to Wildtype (WT). Quantification of H2O2 and MDA has indicated the excessive ROS (reactive oxygen species) in apa1331. Trypan blue staining, and TUNEL assay revealed cell death and excessive DNA fragmentation in apa1331. Map-based cloning and Mutmap analysis identified a candidate gene (LOC_Os04g40720) that is a SUBTILISIN-LIKE SERINE PROTEASE (OsSUBSrP1) which harbored an SNP (A>G) in apa1331. CRISPR-mediated knock-out lines of OsSUBSrP1 displayed spikelet degeneration comparable to apa1331. Global gene expression analysis revealed a significant downregulation of wax and cutin biosynthesis genes e.g., OsWDA1, OsMS2 and OsCER4 in apa1331. Our study reports the novel role of SUBSrP1 in ROS-mediated cell death in panicle development. Highlights1. OsSUBSrP1 plays an important role in maintaining ROS-mediated programmed cell death. 2. OsSUBSrP1 is essential for apical spikelet development. 3. OsSUBSrP1 regulates the expression of wax and cutin biosynthesis pathway genes.

molecular biology↗

bioRxiv2021

Development and Application of Cas13a-based Diagnostic Assay for Neisseria Gonorrhoeae Detection and Identification of Azithromycin Resistance

Gonorrhea caused by Neisseria gonorrhoeae has spread world-wide. Antimicrobial-resistant strains have emerged to an alarming level to most antibiotics including to the ceftriaxone-azithromycin combination, currently recommended as first-line dual therapy. Rapid testing for antimicrobial resistance will contribute to clinical decision-making for rational drug use and will slow this trend. Herein, we developed a Cas13a-based assay for N. gonorrhoeae detection (porA target) and azithromycin resistance identification (A2059G and C2611T point mutations). We evaluated the sensitivity and specificity of this method, and 10 copies per reaction can be achieved in porA detection and C2611T identification, with no cross-reactions. Comparison of the Cas13a-based assay (porA target) with Roche Cobas 4800 assay (n=23 urine samples) revealed 100% concordance. Isolated N. gonorrhoeae strains were used to validate the identification of A2059G and C2611T resistance mutations. All tested strains (8 A2059G strains, 8 C2611T strains, and 8 wild-type strains) were successfully distinguished by our assay and verified by testing MIC for azithromycin and sequencing the 23S rRNA gene. We adopted lateral flow for the SHERLOCK assay readout, which showed a visible difference between test group and NC group results. To further evaluate the capability of our assay, we tested 27 urethral swabs from patients with urethritis for N. gonorrhoeae detection and azithromycin-resistance identification. Of these, 62.96% (17/27) strains were detected with no mutant strains and confirmed by sequencing. In conclusion, the novel Cas13a-based assay for rapid and accurate N. gonorrhoeae detection combined with azithromycin drug resistance testing is a promising assay for application in clinical practice.

microbiology↗