Biology subjects

Tang, Q.

Publications and source records attributed to Tang, Q..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Detyrosination enrichment on microtubule subsets is established by the interplay between a stochastically-acting enzyme and microtubule stability

Microtubules in cells consist of functionally diverse subpopulations carrying distinct post-translational modifications (PTMs). Akin to the histone code, the tubulin code regulates a myriad of microtubule functions ranging from intracellular transport to chromosome segregation. Yet, how individual PTMs only occur on subsets of microtubules to contribute to microtubule specialization is not well understood. In particular, microtubule detyrosination, which is the removal of the C-terminal tyrosine on -tubulin subunits, marks the stable population of microtubules and modifies how microtubules interact with other microtubule-associated proteins to regulate a wide range of cellular processes. Previously, we found that, in certain cell types, only a small subpopulation of microtubules is highly enriched with the detyrosination mark ([~]30%) and that detyrosination spans most of the length of a microtubule, often adjacent to a completely tyrosinated microtubule. How the activity of a cytosolic detyrosinase, Vasohibin (VASH) leads to only a small subpopulation of highly detyrosinated microtubules is unclear. Here, using quantitative super-resolution microscopy, we visualized nascent microtubule detyrosination events in cells consisting of 1-3 detyrosinated -tubulin subunits after Nocodazole washout. Microtubule detyrosination accumulates slowly and in a disperse pattern across the microtubule length. By visualizing single molecules of VASH in live cells, we found that VASH engages with microtubules stochastically on a short time scale suggesting limited removal of tyrosine per interaction, consistent with the super-resolution results. Combining these quantitative imaging results with simulations incorporating parameters from our experiments, we propose a stochastic model for cells to establish a subset of detyrosinated microtubules via a detyrosination-stabilization feedback mechanism.

cell biology↗

bioRxiv2022

Structures of LIG1 uncover a lack of sugar discrimination against a ribonucleotide at the 3'-end of nick DNA

Human DNA ligase I (LIG1) is the main replicative ligase that seals Okazaki fragments and finalizes DNA repair pathways by joining canonical 3-OH and 5-P ends of the nick DNA in a three-step ligation reaction. Ribonucleotides can be misincorporated by DNA polymerases resulting in a nick with 3-ribonucleotide while RNase H2 mediated cleavage leaves a nick harboring 5-ribonucleotide during ribonucleotide excision repair. However, how LIG1 surveils DNA ends with a "wrong" sugar at atomic resolution is unknown. Here, we determine X-ray structures of LIG1/nick DNA complexes with 3- or 5-single ribonucleotide during different stages of the ligation reaction. Our LIG1/5-rG:C structure reveals a global conformational change, which discriminates against 5-RNA/DNA junctions at the initial step when the ligase-AMP intermediate is formed. Furthermore, we capture LIG1/3-RNA-DNA heteroduplexes that are tolerated at the active site where AMP is transferred to nick DNA (step 2) and final phosphodiester bond formation occurs (step 3). Finally, we demonstrate the mutagenic and defective ligation of the nick DNA with 3- and 5-ribonucleotide, respectively, in vitro. Together, these results uncover how LIG1 encounters ribonucleotides embedded into genome during nuclear replication and the last step of DNA repair pathways to maintain genome integrity.

biochemistry↗

bioRxiv2022

Response of the organellar and nuclear (post)transcriptomes of Arabidopsis to drought stress

Plants have evolved sophisticated mechanisms to cope with drought, which involve massive changes in nuclear gene expression. However, little is known about the roles of post-transcriptional processing of nuclear or organellar transcripts and how meaningful these changes are. To address these issues, we used long non-coding RNA-sequencing to monitor (post)transcriptional changes during different times of drought exposure in Arabidopsis Col-0 and a mutant (protein phosphatase 7-like, pp7l), from which we demonstrated that it can survive long periods of drought stress. The changes detected in the pp7l mutant were marginal, while in the wild type chloroplast transcript levels were globally reduced, editing efficiency dropped, but splicing was not affected. Mitochondrial transcripts were slightly elevated, while editing and splicing were unchanged. Also, transcriptional activation of transposable elements played only a minor role. Conversely, alternative splicing (AS) affected nearly 2,000 genes (11% of expressed nuclear genes). Of these, 25% underwent isoform switching, and 15% were regulated solely at the level of AS, representing transcripts that would have gone unnoticed in a microarray-based approach. Our data show that AS enhances proteome diversity to counteract drought stress and represent a valuable resource that will facilitate the development of new strategies to improve plant performance under drought. Moreover, altering the relative contributions of spliced isoforms might enhance drought resistance. For instance, our data imply that accumulation of a non-functional FLM (FLOWERING LOCUS M) isoform - and not the ratio of functional isoforms as suggested for temperature responses - accounts for the early-flowering phenotype under drought conditions.

plant biology↗

bioRxiv2022

stAPAminer: Mining Spatial Patterns of Alternative Polyadenylation for Spatially Resolved Transcriptomic Studies

Alternative polyadenylation (APA) contributes to transcriptome complexity and gene expression regulation, which has been implicated in various cellular processes and diseases. Single-cell RNA-seq (scRNA-seq) has led to the profile of APA at the single-cell level, however, the spatial information of cells is not preserved in scRNA-seq. Alternatively, spatial transcriptomics (ST) technologies provide opportunities to decipher the spatial context of the transcriptomic landscape within single cells and/or across tissue sections. Pioneering studies on ST have unveiled potential spatially variable genes and/or splice isoforms, however, the pattern of APA usages in spatial contexts remains unappreciated. Here, we developed a toolkit called stAPAminer for mining spatial patterns of APA from spatial barcoded ST data. APA sites were identified and quantified from the ST data. Particularly, an imputation model based on K-nearest neighbors algorithm was designed for recovering APA signals. Then APA genes with spatial patterns of APA usage variation were identified. By analyzing the well-established ST data of mouse olfactory bulb (MOB), we present a detailed view of spatial APA usage across morphological layers of MOB with stAPAminer. We complied a comprehensive list of genes with spatial APA dynamics and obtained several major spatial expression patterns representing spatial APA dynamics in different morphological layers. Extending this analysis to two additional replicates of the MOB ST data, we found that spatial APA patterns of many genes are reproducible among replicates. stAPAminer employs the power of ST for exploring transcriptional atlas of spatial APA patterns with spatial resolution, which is available at https://github.com/BMILAB/stAPAminer.

bioinformatics↗

bioRxiv2022

Hi-TrAC reveals fractal nesting of super-enhancers

Eukaryotic genome spatial folding plays a key role in genome function. Decoding the principles and dynamics of 3D genome organization depends on improving technologies to achieve higher resolution. Chromatin domains have been suggested as regulatory micro-environments, whose identification is crucial to understand the genome architecture. We report here that our recently developed method, Hi-TrAC, which specializes in detecting chromatin loops among genomic accessible regulatory regions, allows us to examine active domains with limited sequencing depths at a high resolution. Hi-TrAC can detect active sub-TADs with a median size of 100kb, most of which harbor one or two cell specifically expressed genes and regulatory elements such as super-enhancers organized into nested interaction domains. These active sub-TADs are characterized by highly enriched signals of histone mark H3K4me1 and chromatin-binding proteins, including Cohesin complex. We show that knocking down core subunit of the Cohesin complex using shRNAs in human cells or decreasing the H3K4me1 modification by deleting the H3K4 methyltransferase Mll4 gene in mouse Th17 cells disrupted the sub-TADs structure. In summary, Hi-TrAC serves as a compatible and highly responsive approach to studying dynamic changes of active sub-TADs, allowing us more explicit insights into delicate genome structures and functions. Highlights- Hi-TrAC detects active sub-TADs with a median size of 100 kb. - Hi-TrAC reveals a block-to-block interaction pattern between super-enhancers, and fractal structures within super-enhancers. - Active sub-TADs are disrupted by the knockdown of RAD21. - Active sub-TADs interaction densities are decreased by the knockout of Mll4.

genomics↗

bioRxiv2022

Sniff Olfactometer (SO) Protocols

Most olfactometers used to study human olfaction have stimulus durations of more than 1 second and often lasting minutes(Dravnieks 1975; Leland et al. 2001; Schmidt and Cain 2010). During long stimulations, olfactory receptor responses and their resulting behaviors are modulated by adaptation and habituation to the stimulus(Pellegrino et al. 2017; Rankin 2009; Wilson and Linster 2008). For example, EOG results from the first deorphanized olfactory receptor tissue reached a maximum in [~]1 s, dropping to 1/2 maximum in the next second, and showing little signal reduction until the stimulation stopped after 6 seconds(Zhao et al. 1998). Longer stimulations can result in complete habituation; receptors still respond even though the behavior shows complete habituation (Barwich 2014). To minimize the effects of adaption and habituation on stimulus responses, the sniff olfactometer (SO) combined the precision of a blast olfactometer with the gentleness of a stream olfactometer by blasting a brief odorant puff (70ms duration) into a subjects self-imposed inhalation air stream(Rochelle 2017; Rochelle et al. 2017b; Wyckoff and Acree 2017). Here we describe SO protocols for threshold determinations of odorants in aqueous headspaces using odorant recognition probabilities associated with Log(odorant-concentrations(Rochelle et al. 2017a)). During a single trial a subject, preconditioned to associate a veridical name with a given odor (e.g., a pyrazine with "nuts" when the odor was detected and "not nuts" if it wasnt) was cued to "inhale" and 750ms later, a 15ml-70ms puff of odorant headspace was delivered into their inhalation airstream. A session consisted of 12 randomized double-blind trials of 3 different odorant concentrations. Additional sessions with different concentrations were conducted until the response probability to the samples ranged from below 0.2 to above 0.8. The robustness of the fitted function and the size of their confidence intervals depended on the difference between the concentrations of the odorants during a single session: small differences in sample concentration resulted in the data failing to fit a logistic function; larger concentration differences resulted in a better fit to the model. However, if one of the stimuli had no odorant at all i.e., a blank, the response to the blank was random.

animal behavior and cognition↗

bioRxiv2022

Mice lacking dopamine production in neurotensin receptor 1 neurons voluntarily undergo time-restricted feeding of high fat diet and resist obesity

The introduction of processed foods high in fat and sugars has caused a dramatic increase in obesity in humans. Diet-induced obesity (DIO) can be modeled in laboratory mice by increasing the fat content of their diet. Previously, it was determined that mice lacking dopamine receptor 1 (Drd1) are completely resistant to DIO and do not eat as much food during the day as control mice. Surprisingly, when Drd1 is restored to the suprachiasmatic nucleus (SCN), which is the central regulator of circadian rhythms, these mice increase day-eating and become obese. The source of dopamine in the SCN is the ventral tegmental area (VTA), but the genetic identity of the dopamine neurons is unknown. Here we create conditional deletion mutants for tyrosine hydroxylase (TH) using neurotensin receptor 1 (Ntsr1) Cre and other Cre drivers and measure feeding and body weight homeostasis on standard and high fat diets. Control mice were susceptible to DIO and overate during the day whereas Ntsr1-Cre conditional knockouts for TH mice did not increase day-eating, nor did they gain much weight on HFD. We used an adeno-associated virus to selectively restore TH to the VTA Ntsr1 neurons and observed an increase in body weight and increased day-eating of HFD. These results implicate VTA Ntsr1 dopamine neurons as promoting out-of-phase feeding behavior on a high fat diet that could be an important contributor to diet-induced obesity.

neuroscience↗

bioRxiv2022

Hi-TrAC reveals fine-scale chromatin structures organized by transcription factors

The three-dimensional genomic structure plays a critical role in gene expression, cellular differentiation, and pathological conditions. It is pivotal to elucidate fine-scale chromatin architectures, especially interactions of regulatory elements, to understand the temporospatial regulation of gene expression. In this study, we report Hi-TrAC as a proximity ligation free, robust, and sensitive technique to profile genome-wide chromatin interactions at high-resolution among accessible regulatory elements. Hi-TrAC detects chromatin looping among accessible chromatin regions at single nucleosome resolution. With almost half-million identified loops, we constructed a comprehensive interaction network of regulatory elements across the genome. After integrating chromatin binding profiles of transcription factors, we discovered that cohesin complex and CTCF are responsible for organizing long-range chromatin loops, related to domain formation; whereas ZNF143 and HCFC1 are involved in structuring short-range chromatin loops between regulatory elements, which directly regulate gene expression. Thus, we developed a new methodology to identify a delicate and comprehensive network of cis-regulatory elements, revealing the complexity and a division of labor of TFs in chromatin looping for genome organization and gene expression.

genomics↗

bioRxiv2022

Increased α-ketoglutarate as a missing link from the C3-C4 intermediate state to C4 photosynthesis in the genus Flaveria

As a complex trait, C4 photosynthesis has multiple independent origins in evolution. Phylogenetic evidence and theoretical analysis suggest that C2 photosynthesis, which is driven by glycine decarboxylation in the bundle sheath cell, may function as a bridge from C3 towards C4 photosynthesis. However, the exact molecular mechanism underlying the transition between C2 photosynthesis towards C4 photosynthesis remains elusive. Here, we provide multiple evidence suggesting a role of higher -ketoglutarate (AKG) concentration during this transition. Metabolomic data of 12 Flaveria species, including multiple photosynthetic types, show that AKG concentration initially increases in the C3-C4 intermediate with a further increase in C4 species. Petiole feeding of AKG increased the concentrations of C4 related metabolites in C3-C4 and C4 species but not the activity of C4 related enzymes. Sequence analysis shows that glutamate synthase (Fd-GOGAT), which catalyzes the generation of glutamate using AKG, was under strong positive selection during the evolution of C4 photosynthesis. Simulations with a constraint-based model for C3-C4 intermediate further show that decreasing the activity of Fd-GOGAT facilitates the transition from a C2-dominant to a C4-dominant CO2 concentrating mechanisms. All these provide an insight into the mechanistic switch from C3-C4 intermediate to C4 photosynthesis.

plant biology↗

bioRxiv2022

Metformin decreases Cyp26a1 to prevent hepatocarcinogenesis through down- regulating CD8+ T cells

Hepatocellular carcinoma (HCC) is a highly heterogeneous cancer, which limits the selectivity of prevention and treatment. Preclinical and clinical studies suggested that in patients with diabetes, prolonged use of metformin, the AMPK activator, was associated with a reduction of HCC incidence. This association promotes us to investigate the possible functions and mechanisms of metformin in HCC without diabetes backgrounds. Here, we found that several unique pathways that changed during chronic liver injury of Fah-/- mice, including glucose metabolic process and retinol metabolism. Further, metformin suppressed the tumor formation in chronic liver injury of Fah-/- mice. RNA sequencing, in vivo and in vitro experiments showed that metformin suppressed Cyp26a1 gene expression of hepatocyte. Moreover, the down-regulation of Cyp26a1 leads to the increased level of all-trans-retinoic acid (atRA), which could suppress the tumor formation in our model. On the other hand, flow multicolor analysis showed that the cell number and proportion of cancer promoting (pro-tumor) CD8+ T cells increased significantly during chronic liver injury in Fah-/- mice, and both metformin and atRA treatment could reduce the number and proportion of pro-tumor CD8+ T cells. We also found metformin decreased the Cyp26a1 expression through the AMPK/JNK/c-Jun pathway. In short, the association between the metformin and atRA may explain the commonness of their anti-tumor activities. Our findings highlight the importance of targeting the precancerous microenvironment for the prognosis, prevention and treatment of HCC.

cancer biology↗

bioRxiv2022

SARS-CoV-2 triggers Golgi fragmentation via downregulation of GRASP55 to facilitate viral trafficking

The COVID-19 pandemic is caused by SARS-CoV-2, an enveloped RNA virus. Despite extensive investigation, the molecular mechanisms for its assembly and secretion remain largely elusive. Here, we show that SARS-CoV-2 infection induces global alterations of the host endomembrane system, including dramatic Golgi fragmentation. SARS-CoV-2 virions are enriched in the fragmented Golgi. Disrupting Golgi function with small molecules strongly inhibits viral infection. Significantly, SARS-CoV-2 infection down-regulates GRASP55 but up-regulates TGN46 protein levels. Surprisingly, GRASP55 expression reduces both viral secretion and spike number on each virion, while GRASP55 depletion displays opposite effects. In contrast, TGN46 depletion only inhibits viral secretion without affecting spike incorporation into virions. TGN46 depletion and GRASP55 expression additively inhibit viral secretion, indicating that they act at different stages. Taken together, we show that SARS-CoV-2 alters Golgi structure and function to control viral assembly and secretion, highlighting the Golgi as a potential therapeutic target for blocking SARS-CoV-2 infection.

cell biology↗

bioRxiv2022

Crippled Coronavirus: 5'-PolyU targeted Oligo prevents development of infectious Virions

Current RNA viral pandemic of COVID-19 has been worsened by rapidly spreading viral variants. To inhibit mutation-based development of new escape variants, elements that are indispensable for the virus may be targeted. The 5-polyU tract of the antigenome offers one such target. Host cells do not harbor 5-polyU tracts on any of their transcripts, making the tract an attractive virus-specific target. We hypothesize that inhibiting the 5-polyU by complementary oligonucleotide can limit the use of the tract as template for virus to generate 3 polyA tails of RNA viruses. Here, we used a frameshift-inducing DNA oligonucleotide with 3 polyAs to target the 5-polyU tract of mouse coronavirus (MHV-A59). Results from assays for double stranded RNA (dsRNA) synthesis, infectivity of released virions, and syncytium formation indicate that the oligonucleotide treatment prevented generation of infectious virions. Our results show a unique mode of action of the designed 3-polyA oligonucleotide against mouse coronavirus which leaves host cells unaffected. This strategy can be adopted for the development of novel classes of oligonucleotide-based drugs that inhibit the production of infectious RNA viruses, including the coronaviruses. Since the 5-polyU tract is conserved and is essential for variants of coronaviruses, this strategy can potentially address coronavirus variant emergence as well.

microbiology↗

bioRxiv2022

Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) {beta} gap filling and an incorrect nucleotide insertion by pol{beta} creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3'-mismatches at atomic resolution remains undefined. Here, we determined X-ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the LIG1-adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3'-strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3'-preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP-Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at the final BER steps where a multi-protein complex including pol{beta}, LIG1, and APE1 can maintain accurate repair.

biochemistry↗

bioRxiv2021

Genome-wide characterization of SARS-CoV-2 cytopathogenic proteins in the search of antiviral targets

Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify antiviral targets through genome-wide characterization of SARS-CoV-2 proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins including eight non-structural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis, and caused activation of pro-inflammatory response with production of the cytokines TNF-, IL-6, and IFN-{beta}1, possibly through the activation of NF-{kappa}B. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, {Delta}G188. Compared to wild type ORF3a, the {Delta}G188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19. SignificanceThe ongoing SARS-CoV-2 pandemic has claimed over 5 million lives with more than 250 million people infected world-wide. While vaccines are effective, the emergence of new viral variants could jeopardize vaccine protection. Antiviral drugs provide an alternative to battle against COVID-19. Our goal was to identify viral therapeutic targets that can be used in antiviral drug discovery. Utilizing a genome-wide functional analysis in a fission yeast cell-based system, we identified twelve viral candidates, including ORF3a, which cause cellular oxidative stress, inflammation and apoptosis and necrosis that contribute to COVID-19. Our findings indicate that antiviral agents targeting ORF3a could greatly impact COVID-19.

molecular biology↗

bioRxiv2021

LungCancer3D: A Comprehensive Database for Integrating Lung Cancer Chromatin Architecture with Other Multi-omics

With the breakthrough of chromatin conformation capture technologies in recent years, the importance of three-dimensional (3D) genome structure in gene expression, cell function regulation, disease occurrence, and development has been gradually recognized. To provide a comprehensive visualization of chromatin architecture and other multi-omics data for lung cancer research, we have constructed a comprehensive database, LungCancer3D (http://www.lungcancer3d.net). This web-based tool focuses on displaying human lung cancer-related HiC data along with a variety of other publicly available data, such as RNA-seq, scRNA-seq, ATAC-seq, ChIP-seq, DNA methylation, DNA mutation, and copy number variations. Researchers can visualize these diverse multi-omics data directly through the genome browser and discover how the genes expression is regulated at diverse levels. For example, we have demonstrated that the high expression level of C-MYC in lung cancer may be caused by the distant enhancer introduced by the de novo chromatin loops in lung cancer cells to bind the C-MYC promoter. The integrated multi-omics analyses through the LungCancer3D website can reveal the mechanisms underlying lung cancer development and provide potential targets for lung cancer therapy.

bioinformatics↗

bioRxiv2021

Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-{zeta} signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

immunology↗

bioRxiv2021

TNFa and IL-6 promote ex-vivo proliferation of lineage-committed human regulatory T cells

Treg therapy is being tested in clinical trials in transplantation and autoimmune diseases, however, the impact of inflammation on Tregs is unclear. In this study, we challenged human Tregs ex-vivo with pro-inflammatory cytokines, TNF and IL-6. These cytokines enhanced Treg proliferation induced by anti-CD3 and anti-CD28 or CD28 superagonist (CD28SA) while maintaining high expression of FOXP3 and HELIOS, demethylated FOXP3 enhancer, and low expression of cytokines IFNg, IL-4 and IL-17. Blocking TNF receptor signaling using etanercept or deletion of TNF receptor 2 using CRISPR/Cas9 blunted Treg proliferation and attenuated FOXP3 and HELIOS expression, revealing the importance of TNFR2 signaling in Treg proliferation and lineage stability. The robust proliferation induced by CD28SA with IL-6 and TNF may be adopted for the expansion of therapeutic Tregs. Metabolomics analysis showed that Tregs expanded with CD28SA plus cytokines had more active glycolysis and oxidative phosphorylation, increased energy production, and higher antioxidant potential. Finally, CD28SA plus cytokine-expanded Tregs had comparable suppressive activity in vitro and in vivo in a humanized mouse model of graft-versus-host-disease when compared to Tregs expanded using the conventional protocol. These results demonstrate that human Tregs positively respond to proinflammatory cytokines with enhanced proliferation without compromising their lineage identity or function.

immunology↗

bioRxiv2021

The Achilles' heel of coronaviruses: targeting the 5' Polyuridines tract of the antigenome to inhibit Mouse coronavirus virus-induced cell death

The current coronavirus pandemic situation is worsened by the rapidly-spreading SARS-CoV-2 virus variants. Identification of viral targets that are indispensable for the virus can be targeted to inhibit mutation-based new escape variant development. The 5-polyU tract of the antigenome offers such a target. Host cells do not harbor 5-polyU tracts on any of their transcripts, making the tract an attractive, virus-specific target. Inhibiting the 5-polyU can limit the use of the tract as template to generate 3 polyA tails of +RNAs of coronaviruses. Here, a modified DNA oligo with 3 polyAs is used to target the 5-polyU tract in mouse coronavirus (MHV-A59). The oligo treatment in mouse 17CL-1 cells infected with MHV-A59 significantly prevented virus-induced cell deaths. This proof-of-concept result shows a unique mode of action against mouse coronavirus without affecting host cells, and can be used for the development of novel classes of drugs that inhibit coronavirus infection in host cells, specifically by the COVID-19-causing virus SARS-CoV-2. In addition, as the 5-polyU tract is immediately generated upon infection, the tag can also be targeted for reliable early detection of viral infection.

microbiology↗