Biology subjects

Dong, Y.

Publications and source records attributed to Dong, Y..

At least 181 records · Page 10Linked to original sources

bioRxiv2023

Convergence of resistance and evolutionary responses in Escherichia coli and Salmonella enterica co-inhabiting chicken farms in China

Sharing among different pathogens and commensals inhabiting same hosts and environments has significant implications for antimicrobial resistance (AMR), especially in settings with high antimicrobial exposure. We analysed 661 E. coli and S. enterica isolates collected within and across hosts and environments, in 10 Chinese chicken farms over 2.5 years using novel data-mining methods. Most isolates within same hosts possessed same clinically relevant AMR-carrying mobile genetic elements (plasmids: 70.6%, transposons: 78%), which also showed recent common evolution. Machine learning revealed known and novel AMR-associated mutations and genes underlying resistance to 28 antimicrobials and primarily associated with resistance in Escherichia coli and susceptibility in Salmonella enterica. Many of these genes were essential and affected same metabolic processes in both species, albeit with varying degrees of phylogenetic penetration. Multi-modal strategies are crucial to investigate the interplay of mobilome, resistance and metabolism in cohabiting bacteria, especially in ecological settings where community-driven resistance selection occurs.

genomics↗

bioRxiv2023

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Engineering improved B-family DNA polymerases to incorporate 3'-O-modified nucleotide reversible terminators is limited by an insufficient understanding of the structural determinants that define polymerization efficiency. To explore the key mechanism for unnatural nucleotide incorporation, we engineered a B-family DNA polymerase from Thermococcus Kodakaraenis (KOD pol) by using semi-rational design strategies. We first scanned the active pocket of KOD pol through site-directed saturation mutagenesis and combinatorial mutations and identified a variant Mut_C2 containing five mutation sites (D141A, E143A, L408I, Y409A, A485E) using a high-throughput microwell-based screening method. Mut_C2 demonstrated high catalytic efficiency in incorporating 3-O-azidomethyl-dATP labeled with a Cy3 dye, whereas the wild-type KOD pol failed to incorporate it. Computational simulations were then conducted towards the DNA binding region of KOD pol to predict additional mutations with enhanced catalytic activity, which were subsequently experimentally verified. By a stepwise combinatorial mutagenesis approach, we obtained an eleven-mutation variant, named Mut_E10 by introducing additional mutations to the Mut_C2 variant. Mut_E10, which carried six specific mutations (S383T, Y384F, V389I, V589H, T676K, and V680M) within the DNA-binding region, demonstrated over 20-fold improvement in kinetic efficiency as compared to Mut_C2. In addition, Mut_E10 demonstrated satisfactory performance in two different sequencing platforms (BGISEQ-500 and MGISEQ-2000), indicating its potential for commercialization. Our study demonstrates that an effective enhancement in its catalytic efficiency towards modified nucleotides can be achieved efficiently through combinatorial mutagenesis of residues in the active site and DNA binding region of DNA polymerase. These findings contribute to a comprehensive understanding of the mechanisms that underlie the incorporation of modified nucleotides by DNA polymerase. The beneficial mutation sites, as well as the nucleotide incorporation mechanism identified in this study, can provide valuable guidance for the engineering of other B-family DNA polymerases.

bioengineering↗

bioRxiv2023

Molecular understanding of anthocyanin biosynthesis activated by PAP1 in engineered red Artemisia annua cells and regulation of 2, 4-dichlorophenoxyacetic acid

Artemisia annua is an effective antimalarial medicinal crop. We have established anthocyanin-producing red cell cultures from this plant with the overexpression of Production of Anthocyanin Pigment 1 (PAP1) encoding a R2R3MYB transcription factor. To understand the molecular mechanism by which PAP1 activated the entire anthocyanin pathway, we mined the genomic sequences of A. annua and obtained eight promoters of the anthocyanin pathway genes. Sequence analysis identified four types of AC cis-elements from six promoters, the MYB response elements (MRE) bound by PAP1. In addition, six promoters were determined to have at least one G-Box cis-element. Eight promoters were cloned for activity analysis. Duel luciferase assays showed that PAP1 significantly enhanced the promoting activity of seven promoters, indicating that PAP1 turned on the biosynthesis of anthocyanins via the activation of these pathway gene expression. To understand how 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin, regulates the PAP1-activated anthocyanin biosynthesis, five different concentrations (0, 0.05, 0.5, 2.5, and 5 M) were tested to characterize anthocyanin production and profiles. The resulting data showed that the concentrations tested decreased the fresh weight of callus growth, anthocyanin levels, and the production of anthocyanins per petri dish. HPLC-qTOF-MS/MS based profiling showed that these concentrations did not alter anthocyanin profiles. Real time RT-PCR was completed to characterize the expression PAP1 and four representative pathway genes. The results showed that the five concentrations reduced the expression levels of the constitutive PAP1 transgene and three pathway genes significantly and eliminated chalcone synthase gene in expression either significantly or slightly. These data indicate that the constitutive PAP1 expression depends on gradients added in the medium. Based on these findings, the regulation of 2,4-D is discussed for anthocyanin engineering in red cells of A. annua. ConclusionPromoters of eight anthocyanin pathway genes were cloned. Four types of AC cis-elements were identified from six promoters and G-Box elements were also determined from six promoters. PAP1 enhanced the activity of eight promoters. 2,4-D downregulated the expression of the constitutive PAP1 transgene leading to the decrease the biosynthesis of anthocyanins.

plant biology↗

bioRxiv2023

Phosphorylation of pyruvate dehydrogenase marks the inhibition of in vivo neuronal activity

For decades, the expression of immediate early genes (IEGs) such as c-fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

neuroscience↗

bioRxiv2023

VirRep: accurate identification of viral genomes from human gut metagenomic data via a hybrid language representation learning framework

Accurate identification of viral genomes from metagenomic data provides a broad avenue for studying viruses in the human gut. Here, we introduce VirRep, a novel virus identification method based on a hybrid language representation learning framework. VirRep employs a context-aware encoder and a composition-focused encoder to incorporate the learned knowledge and known biological insights to better describe the source of a DNA sequence. We benchmarked VirRep on multiple human gut virome datasets under different conditions and demonstrated significant superiority than state-of-the-art methods and even combinations of them. A comprehensive validation has also been conducted on real human gut metagenomes to show the great utility of VirRep in identifying high-quality viral genomes that are missed by other methods.

bioinformatics↗

bioRxiv2023

ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

Gasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family. One-Sentence SummaryGSDMD palmitoylation is induced by ROS and required for pore formation

immunology↗

bioRxiv2023

IMPACT: a web server for exploring immunotherapeutic predictive and cancer prognostic biomarkers

Immune checkpoint inhibitors (ICIs) are a breakthrough in oncology treatment, and studies of screening predictive biomarkers of ICIs are emerging. We developed a web server named IMPACT (http://impact.brbiotech.com/) to thoroughly explore immunotherapeutic predictive or prognostic biomarkers. IMPACT contains a large dataset of 6,276 patients treated with ICIs and integrates 11 well-designed function modules, enabling an in-depth solution for biomarkers exploration. Compared with the existing tools, IMPACT was implemented with one exclusive module for interaction analysis and several optimized conventional functions for discovering novel biomarkers. Specifically, the interaction analysis of biomarker-treatment effect is essential to determine whether a biomarker is predictive and/or prognostic for ICIs. Moreover, several optimized functions allow complicated biomarker exploration, including customized selections of variant types in more detail, automatically screening meaningful co-mutations among multiple genes, and selecting cut-off values for gene expression biomarkers. In summary, IMPACT is a comprehensive analysis resource to facilitate biomarker research of ICIs.

bioinformatics↗

bioRxiv2023

Metabolic engineering of anthocyanin pathway in Artemisia annua cells does not exclude the expression of artemisinin pathway

Artemisia annua is the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs),; however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated from A. annua. In this study, our goals were to engineer anthocyanins in A. annua cells and to understand the artemisinin biosynthesis in anthocyanin-producing cells. Arabidopsis PAP1 (AtPAP1) was used to engineer four types of transgenic anthocyanin-producing A. annua (TAPA1 to 4) cells. Three wild type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from DHAA in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells of A. annua.

plant biology↗

bioRxiv2022

Molecular insights into the gating mechanisms of voltage-gated calcium channel CaV2.3

High-voltage-activated R-type CaV2.3 channel plays pivotal roles in many physiological activities and is implicated in epilepsy, convulsions, and other neurodevelopmental impairments. Here, we determine the high-resolution cryo-electron microscopy (cryo-EM) structure of human CaV2.3 in complex with the 2{delta}1 and {beta}1 subunits. The VSDII is stabilized in the resting state. Electrophysiological experiments elucidate that the conformational change of VSDII in response to variation in membrane potential is not required for channel activation, whereas the other VSDs are essential for channel opening. The intracellular gate is blocked by the W-helix. A pre-W-helix adjacent to the W-helix can significantly regulate closed-state inactivation (CSI) by modulating the association and dissociation of the W-helix with the gate. Electrostatic interactions formed between the negatively charged domain on S6II, which is exclusively conserved in the CaV2 family, and nearby regions at the alpha-interacting domain (AID) and S4-S5II helix are identified. Further functional analyses indicate that these interactions are critical for the open-state inactivation (OSI) of CaV2 channels.

biophysics↗

bioRxiv2022

Signaling Node at TSC2 S1365 Potently Regulates T-Cell Differentiation and Improves Adoptive Cellular Cancer Therapy

MTORC1 integrates signaling from the immune microenvironment to regulate T-cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex potently regulates mTORC1 activation. CD8+ T-cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate potent effector T cells; however sustained mTORC1 activation prevents generation of long-lived memory CD8+ T-cells. Here we show manipulating TSC2 at Ser1365 potently regulates activated but not basal mTORC1 signaling in T cells. Unlike non-stimulated TSC2 knockout cells, CD8+ T-cells expressing mutant TSC2-S1365A (SA) have normal basal mTORC1 activity. PKC and T-cell Receptor (TCR) stimulation induces TSC2 S1365 phosphorylation and preventing this with the SA mutation markedly increases mTORC1 activation and T-cell effector function. Consequently, CD8+ SA T-cells display greater effector responses while retaining their capacity to become long-lived memory T-cells. CD8+ SA T-cells also display enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, CD8+ SA T-cells used as adoptive cell therapy have greater anti-tumor immunity than WT CD8+ T-cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T-cells. The TSC2-SA mutation enhances both T-cell effector function and long-term persistence/memory formation, supporting a novel approach to engineer better CAR-T cells to treat cancer.

immunology↗

bioRxiv2022

How do Vampires Suck Blood?

Lampreys are blood-sucking vampires in the marine. From a survival perspective, it is expected that lamprey buccal gland exhibits a repository of pharmacologically active components to modulate the hosts homeostasis, inflammatory and immune responses. Several proteins have been found to function as anticoagulants, ion channel blockers, and immune suppressors in lampreys, while small metabolites have never been explored in detail. In this study, by analyzing the metabolic profiles of 14 different lamprey tissues, we have identified two groups of blood-sucking-associated metabolites, i.e., kynurenine pathway metabolites and prostaglandins, in the buccal gland and they can be injected into the host fish to ensure a steady and sustained blood flow to the feeding site. These findings demonstrate the complex nature of lamprey buccal gland and highlight the diversity in the mechanisms utilized for blood-sucking in lampreys. In addition, a lamprey spatial metabolomics database (https://www.lampreydb.com) was constructed to assist studies using lampreys as model animal. The database contains detailed qualitative, quantitative, and spatial distribution information of each detected metabolite, and users can easily query and check their metabolites of interest, and/or identify unknown peaks using the database. Significance StatementLampreys are one of the two surviving jawless vertebrate groups that hold the key to our understanding of the early vertebrate evolution, adaptive immune origin, and developmental neurobiology. Here, we applied a spatial metabolomics approach to study the lamprey-host interaction. Two groups of metabolites, i.e., kynurenine pathway metabolites and prostaglandins, were found in the lamprey buccal gland, which modulate the hosts homeostasis, inflammatory and immune responses. The establishment of the first tissue-wide spatial lamprey metabolomics database in this study facilitate future studies in biochemistry, clinical chemistry, natural product discovery, medicine, and metabolomics using lampreys as a model animal.

biochemistry↗

bioRxiv2022

MalariaSED: a deep learning framework to decipher the regulatory contributions of noncoding variants in malaria parasites

Malaria remains one of the deadliest infectious diseases. Transcriptional regulation effects of noncoding variants in this unusual genome of malaria parasites remain elusive. We developed a sequence-based, ab initio deep learning framework, MalariaSED, for predicting chromatin profiles in malaria parasites. The MalariaSED performance was validated by published ChIP-qPCR and TF motifs results. Applying MalariaSED to [~]1.3 million variants shows that geographically differentiated noncoding variants are associated with parasite invasion and drug resistance. Further analysis reveals chromatin accessibility changes at Plasmodium falciparum rings are partly associated with artemisinin resistance. MalariaSED illuminates the potential functional roles of noncoding variants in malaria parasites.

bioinformatics↗

bioRxiv2022

Conserved allosteric inhibition mechanism in SLC1 transporters

Excitatory Amino Acid Transporter 1 (EAAT1) is a plasma-membrane glutamate transporter belonging to the SLC1 family of solute carriers. It plays a key role in neurotransmitter transport and contributes to the regulation of the extracellular glutamate concentration in the mammalian brain. The structure of EAAT1 was determined using cryo-EM, in complex with UCPH-101, a highly potent and non-competitive inhibitor of EAAT1. Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid transporter, which regulates pools of amino acids such as glutamine, serine and alanine between intracellular and extracellular compartments in a Na+ dependent manner. ASCT2 also belongs to the SLC1 family and shares 58% sequence similarity with EAAT1. However, allosteric modulation of ASCT2 via non-competitive inhibitors is unknown. Here we explore the UCPH-101 inhibitory mechanisms of EAAT1 and ASCT2 by using rapid kinetic experiments. Our results show that UCPH-101 slows substrate translocation rather than substrate or Na+ binding, confirming a non-competitive inhibitory mechanism, but only partially inhibits wild-type ASCT2 with relatively low affinity. Guided by computational modeling using ligand docking and molecular dynamics (MD) simulations, we selected two residues involved in UCPH-101/EAAT1 interaction, which were mutated in ASCT2 (F136Y, I237M, F136Y/I237M) in the corresponding positions. We show that in the F136Y/I237M double mutant transporter, 100% of the inhibitory effect of UCPH-101 on anion current could be restored, and the apparent affinity was increased (Ki = 9.3 M), much closer to the EAAT1 value of 0.6 M. Finally, we identify a novel non-competitive ASCT2 inhibitor, identified through virtual screening and experimental testing against the allosteric site, further supporting its localization. Together, these data indicate that the mechanism of allosteric modulation is conserved between EAAT1 and ASCT2. Due to the difference in binding site residues between ASCT2 and EAAT1, these results raise the possibility that more potent, and potentially selective inhibitors can be designed that target the ASCT2 allosteric binding site.

biophysics↗

bioRxiv2022

High-throughput TCRB enrichment sequencing of human cord blood exhibited a distinct fetal T cell repertoire in the third trimester of pregnancy

Study questionWhat are the molecular characteristics during the maturation process of the human fetal immune system in the third trimester of pregnancy? Summary answerBoth the diversity and length of complementarity determining region 3 (CDR3s) in the fetal TCRB repertoire were less than those of adult CDR3s, and the fetal CDR3 length increased with gestation weeks in late pregnancy. What is known alreadyThe adaptive immune system recognizes various pathogens based on a large repertoire of T-cell receptors (TCR repertoire), but the maturation dynamics of the fetal TCR repertoire in the third trimester are largely unknown. The CDR3is the most diversified segment in the T-cell receptor {beta} chain (TCRB) that binds and recognizes the antigen. Study design, size, and durationThis was a basic research to assess the composing characteristics of TCRBs in core blood and the dynamic pattern with fetal development in the third trimester of pregnancy. Participants/materials, setting methodsHigh-throughput TCRB-enrichment sequencing was utilized to characterize the TCRB repertoire of cord blood at 24~38 weeks of gestational age (WGA) with nonpreterm fetuses and to investigate their difference compared with that of adult peripheral blood. Main results and the role of chanceCompared to the adult control, the fetal TCRB repertoire had a 4.8-fold lower number of unique CDR3s, a comparable Shannon diversity index (p=0.7387), a lower mean top clone rate (p < 0.001) and a constrictive top 1000 unique clone rates. Although all kinds of TCRBV and TCRBJ genes present in adult CDR3s were identified in fetuses, nearly half of these fragments showed a significant difference in usage. Moreover, the fetal TCRB repertoire held a shorter CDR3 length, and the CDR3 length showed a progressive increase with fetal development. Jensen-Shannon (JS) divergences of TCRBV and TCRBJ gene usage in dizygotic twins were much lower than those in unrelated pairs. In the parental-fetal pair, JS divergence of TCRBV gene usage was not obviously different, while that of TCRBJ gene usage was only slightly lower. Limitations, reasons for cautionThe sample size is limited due to the limited accessibility to cord blood in late pregnancy with healthy nonpreterm fetuses. Wider implications of the findingsOur findings reveal the unique properties of fetal TCRB repertoires in the third trimester, fill the gap in our understanding of the maturation process of prenatal fatal immunity, and deepen our understanding of the immunologically relevant problems in neonates. Study funding/competing interest(s)This work was supported by the National Natural Science Foundation of China (82171661) and Tianjin Municipal Science and Technology Special Funds for Enterprise Development (NO. 14ZXLJSY00320). The authors declare that they have no competing interests.

genetics↗

bioRxiv2022

CRISPR Mediated Transactivation in the Human Disease Vector Aedes aegypti

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ( dead) Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2). This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may lead to the development of innovative vector control tools.

bioengineering↗

bioRxiv2022

Single-cell transcriptomic profiling in inherited retinal degen-eration reveals distinct metabolic pathways in rod and cone photoreceptors

The cellular mechanisms underlying hereditary photoreceptor degeneration are still poorly understood. The aim of this study was to systematically map the transcriptional changes that occur in the degenerating mouse retina at the single cell level. To this end, we employed single cell RNA-sequencing (scRNA-seq) and retinal degeneration-1 (rd1) mice to profile the impact of the disease mutation on the diverse retinal cell types during early post-natal development. The transcriptome data allowed to annotate 43,979 individual cells grouped into 20 distinct retinal cell types. We further characterized cluster-specific metabolic and biological changes in individual cell types. Our results highlight Ca2+-signaling as relevant to hereditary photoreceptor degeneration. Though metabolic reprogramming in retina, known as Warburg effect, has been documented, further metabolic changes were noticed in rd1 mice. Such metabolic changes in rd1 mutation was likely regulated through mitogen-activated protein kinase (MAPK) pathway. By combining single-cell transcriptomes and immunofluorescence staining, our study revealed cell type-specific changes in gene expression, as well as interplay between Ca2+ induced cell death and metabolic pathways.

genomics↗

bioRxiv2022

Detection of Frog virus 3 via the system integrating RPA-CRISPR/Cas12a-SPM with deep learning

Frog virus 3 (FV3, genera Ranavirus, family Iridoviridae), a double-stranded DNA virus, results in irreparable damage to biodiversity and significant economic losses to aquaculture. Although the existing FV3 detection methods are of high sensitivity and specificity, the complex procedure and requirement of expensive instruments limit their practical implantation. Herein, we develop a fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for FV3. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, we achieve a limit of detection (LoD) of 100 aM (60.2 copies/L) by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, we build a smartphone microscopy (SPM) and achieve an LoD of 10 aM within 40 minutes. Four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32 are detectable by the proposed system. In addition, we deploy deep learning models for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100% and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, RPA-CRISPR/Cas12a combined with a smartphone readout and artificial intelligence (AI) assisted classification shows great potential for FV3 detection. This integrated system holds great promise for POC detection of aquatic DNA pathogens. HighlightsO_LIAn integrated DNA detection system is developed by combining RPA, CRISPR/Cas12a, smartphone microscopy, and deep learning. C_LIO_LIThe LoD of frog virus 3 is 10 aM within 40 min. C_LIO_LIThe detection system shows good performance on animal-derived samples. C_LI

molecular biology↗

bioRxiv2022

Structural principles of B-cell antigen receptor assembly

The B-cell antigen receptor (BCR) is composed of a membrane-bound immunoglobulin (mIg) of class M, D, G, A or E for antigen recognition and a disulfide-linked heterodimer between Ig and Ig{beta} (Ig/{beta}, also known as CD79A and CD79B) that functions as the signalling entity. The organizing principle of BCR assembly remains elusive. Here we report the cryo-electron microscopy structures of the intact IgM class BCR at 8.2 [A] resolution and its Fab-deleted form (IgM BCR{Delta}Fab) at 3.6 [A] resolution. At the ectodomain (ECD), Ig and Ig{beta} position their respective Ig folds roughly in parallel with an approximate 2-fold symmetry, which is distinct from structures of Ig{beta}/{beta} homodimers. Unlike previous predictions, the BCR structure displays an asymmetric arrangement, in which the Ig/{beta} ECD heterodimer mainly uses Ig to associate with C{micro}3-C{micro}4 domains of one heavy chain ({micro}HC) while leaving the other heavy chain ({micro}HC) empty. The transmembrane domain (TMD) helices of the two {micro}HCs also deviate from the 2-fold symmetry of the C{micro}3-C{micro}4 domain dimer and form together with the TMD helices of the Ig/{beta} heterodimer a tight 4-helix bundle. The asymmetry at the TMD helices prevents the recruitment of two Ig/{beta} heterodimers. Surprisingly, the connecting peptides (CPs) between the ECD and TMD are braided together through striking charge complementarity, resulting in intervening of the CP of {micro}HC in between those of Ig and Ig{beta} and crossover of the TMD relative to ECD for the Ig/{beta} heterodimer, to guide the TMD assembly. Interfacial analyses suggest that the IgM BCR structure we present here may represent a general organizational architecture of all BCR classes. Our studies thus provide a structural platform for understanding B-cell signalling and for designing rational therapies against BCR-mediated diseases.

immunology↗