Biology subjects

Lai, J.

Publications and source records attributed to Lai, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Bone marrow haematopoietic stem cells influence liver homeostatic networks and cancer development after dietary intervention

A holistic understanding of anti-tumor immunity requires examination of systemic immunity beyond the tumor microenvironment. This link between systemic and tumor immune activity is underexplored. We demonstrate that a stronger Type I interferon response in human liver tumors predicts better survival and correlates with the immune response in the adjacent non-tumor liver. Further, in patients with liver cancer, clonal hematopoiesis (CH) (a marker of systemic immune activation) is associated with a trend towards improved survival. In a mouse model of liver cancer, basal liver immune responses correlate with the degree of bone marrow hematopoietic stem and progenitor cell responses. A higher systemic and liver immune response, marked by innate myeloid cell infiltration reduces tumor burden, while sub-optimal systemic and liver immunity corresponded with a higher tumor burden. Our findings indicate that the state of bone marrow hematopoiesis impacts liver tumor outcomes, offering both therapeutic and prognostic opportunities.

systems biology↗

bioRxiv2022

Brainstem speech encoding is dynamically shaped online by fluctuations in cortical α state

Experimental evidence in animals demonstrates cortical neurons innervate subcortex bilaterally to tune brainstem auditory coding. Yet, the role of the descending (corticofugal) auditory system in modulating earlier sound processing in humans during speech perception remains unclear. Here, we measured EEG activity as listeners performed speech identification tasks in different noise backgrounds designed to tax perceptual and attentional processing. Brainstem speech coding might be tied to attention and arousal states (indexed by cortical power) that actively modulate the interplay of brainstem-cortical signal processing. When speech-evoked brainstem frequency-following responses (FFRs) were categorized according to cortical states, low FFRs in noise were found to be weaker, correlated positively with behavioral response times, more "decodable" via classifiers, and associated indirectly with other signal-in-noise perceptual performance. Our data provide evidence for online corticofugal interplay in humans and establish that brainstem sensory representations are continuously yoked to the ebb and flow of cortical states to dynamically update perceptual processing.

neuroscience↗

bioRxiv2021

Outsourced hearing in spiders by using their webs as auditory sensors

Hearing is a fundamental sense of many animals, including all mammals, birds, some reptiles, amphibians, fish, and arthropods. The auditory organs of these animals are extremely diverse in anatomy after hundreds of millions of years of evolution, yet all are made up of cellular tissues and are morphologically part of bodies of animals. Here we show hearing in the orb-weaving spider, Larinioides sclopetarius is not constrained by the organisms body but is extended through outsourcing hearing to its extended phenotype, the proteinaceous, self-manufactured orb-web. We find the wispy, wheel-shaped orb-web acts as a hyperacute acoustic "antenna" to capture the sound-induced air particle movements that approach the maximum physical efficiency, better than the acoustic responsivity of all previously known eardrums. By sensing the motion of web threads, the spider remotely detects and localizes the source of an incoming airborne acoustic wave such as those emitted by approaching prey or predators. By outsourcing its acoustic sensors to its web, the spider is released from body size constraints and permits the araneid spider to increase its sound-sensitive surface area enormously, up to 10,000 times greater than the spider itself. The spider also enables the flexibility to functionally adjust and regularly regenerate its "external eardrum" according to its needs. The "outsourcing" and "supersizing" of auditory function in spiders provides unique features for studying extended and regenerative sensing, and designing novel acoustic flow detectors for precise fluid dynamic measurement and manipulation.

animal behavior and cognition↗

bioRxiv2021

Computational design of highly signaling active membrane receptors through de novo solvent-mediated allosteric networks

Protein catalysis and allostery require the atomic-level orchestration and motion of residues, ligand, solvent and protein effector molecules, but the ability to design protein activity through precise protein-solvent cooperative interactions has not been demonstrated. Here, we report the design of a dozen novel membrane receptors catalyzing G-protein nucleotide exchange through diverse de novo engineered allosteric pathways mediated by cooperative networks of intra-protein, protein-ligand and solvent molecule interactions. Consistent with the predictions, designed protein activities strongly correlated with the level of solvent-mediated interaction network plasticity at flexible transmembrane helical interfaces. Several designs displayed considerably enhanced thermostability and activity compared to related natural receptors. The most stable and active variant crystallized in an unforeseen signaling active conformation, in excellent agreement with the design models. The allosteric network topologies of the best designs bear limited similarity to those of natural receptors and reveal a space of allosteric interactions larger than previously inferred from natural proteins. The approach should prove useful for engineering proteins with novel complex protein catalytic and signaling activities.

biophysics↗

bioRxiv2021

Tuft cell-produced cysteinyl leukotrienes and IL-25 synergistically drivelung type 2 inflammation

Aeroallergen sensing by airway epithelial cells can trigger pathogenic immune responses leading to chronic type 2 inflammation, the hallmark of airway diseases such as asthma. Airway tuft cells are specialized chemosensory epithelial cells and the dominant source of the epithelial cytokine IL-25 in the trachea and of cysteinyl leukotrienes (CysLTs) in the naive murine nasal mucosa. The interaction of IL-25 and CysLTs and the contribution of tuft cell-derived CysLTs to the development of allergen-triggered inflammation in the airways has not been clarified. Here we show that inhalation of LTC4 in combination with a subthreshold dose of IL-25 leads to dramatic synergistic induction of type 2 inflammation throughout the lungs, causing rapid eosinophilia, dendritic cell (DC) and inflammatory type 2 innate lymphoid cell (ILC2) expansion, and goblet cell metaplasia. While lung eosinophilia is dominantly mediated through the classical CysLT receptor CysLT1R, type 2 cytokines and activation of innate immune cells require signaling through both CysLT1R and CysLT2R. Tuft cell-specific deletion of the terminal enzyme requisite for CysLT production, Ltc4s, was sufficient to reduce both the innate immune response in the lung - eosinophilia, KLRG1+ ILC2 activation and DC recruitment - and the systemic immune response in the draining lymph nodes after inhalation of the mold aeroallergen Alternaria. Our findings identify surprisingly potent synergy of CysLTs and IL-25 downstream of aeroallergen-trigged activation of airway tuft cells leading to a highly polarized type 2 immune response and further implicate airway tuft cells as powerful modulators of type 2 immunity in the lungs. One Sentence SummaryTuft cells produce two highly synergistic mediators: LTC4 and IL-25 to cooperatively induce allergen-driven airway inflammation.

immunology↗

bioRxiv2021

Single-nucleus transcriptomic analyses reveal microglial activation underlying cerebellar degeneration in Ataxia Telangiectasia

While ATM loss-of-function has long been identified as the genetic cause of Ataxia Telangiectasia (A-T), how this genetic mutation leads to selective and progressive degeneration of cerebellar Purkinje and granule neurons remains unclear. ATM expression is enriched in microglia, the resident immune cell of the central nervous system, throughout cerebellar development and adulthood. Microglial activation has been strongly implicated in neurodegenerative disease and observed in rodent and cellular models of ATM deficiency. Here, we find evidence of prominent inflammation of microglia in cerebellum from A-T patients using single-nucleus RNA-sequencing. A-T microglia have transcriptomic signatures of aging and neurodegenerative disease associated microglia. Pseudotime analysis revealed that activation of A-T microglia preceded upregulation of apoptosis related genes in granule and Purkinje neurons, and microglia exhibited increased neurotoxic cytokine signaling to granule and Purkinje neurons in A-T. To confirm these findings experimentally, we studied microglia and neurons that we generated from A-T patient vs. control induced pluripotent stem cells (iPSCs). Transcriptomic profiling of A-T iPSC-derived microglia revealed cell-intrinsic microglial activation of cytokine production and innate immune response pathways compared to controls. Furthermore, adding A-T microglia to co-cultures with either control or A-T iPSC-derived neurons was sufficient to induce cytotoxicity. Taken together, these studies reveal that cell-intrinsic microglial activation may play a critical role in the development and progression of neurodegeneration in Ataxia Telangiectasia.

neuroscience↗

bioRxiv2021

Genome of the giant panda roundworm illuminates its host shift and parasitic adaptation

Baylisascaris schroederi, a bamboo-feeding giant panda (Ailuropoda melanoleuca)-specific roundworm (ascaridoid) parasite, is the causative agent of baylisascariasis, which represents a leading reason for the mortality of wild giant panda populations and therefore poses a significant threat to giant panda conservation. Here we present a 293-Mb chromosome-level genome assembly of B. schroederi to inform its biology, including host adaptations. Comparative genomics revealed an evolutionary trajectory accompanied by host-shift events in ascaridoid parasite lineages after host separations, suggesting their potential transmissions and fast adaptations to hosts. Genomic and anatomical lines of evidence, including expansion and positive selection of genes related to cuticle and basal metabolisms, indicates that B. schroederi undergoes specific adaptations to survive in the sharp-edged bamboo enriched gut of giant panda by structurally increasing its cuticle thickness and efficiently utilizing host nutrients during gut parasitism. Also, we characterized the secretome and predicted potential drug and vaccine targets for new interventions. Overall, this genome resource provides new insights into the host adaptation of B. schroederi to giant panda as well as the host-shift events in ascaridoid parasite lineages. These findings also add to our knowledge on the unique biology of the giant panda roundworm and aid the development of much-needed novel strategies for the control of baylisascariasis and thus the protection of giant panda populations.

genomics↗

bioRxiv2021

LYRUS: A Machine Learning Model for Predicting the Pathogenicity of Missense Variants

Single amino acid variations (SAVs) are a primary contributor to variations in the human genome. Identifying pathogenic SAVs can aid in the diagnosis and understanding of the genetic architecture of complex diseases, such as cancer. Most approaches for predicting the functional effects or pathogenicity of SAVs rely on either sequence or structural information. Nevertheless, previous analyses have shown that methods that depend on only sequence or structural information may have limited accuracy. Recently, researchers have attempted to increase the accuracy of their predictions by incorporating protein dynamics into pathogenicity predictions. This study presents < Lai Yang Rubenstein Uzun Sarkar > (LYRUS), a machine learning method that uses an XGBoost classifier selected by TPOT to predict the pathogenicity of SAVs. LYRUS incorporates five sequence-based features, six structure-based features, and four dynamics-based features. Uniquely, LYRUS includes a newly-proposed sequence co-evolution feature called variation number. LYRUSs performance was evaluated using a dataset that contains 4,363 protein structures corresponding to 20,307 SAVs based on human genetic variant data from the ClinVar database. Based on our dataset, the LYRUS classifier has a higher accuracy, specificity, F-measure, and Matthews correlation coefficient (MCC) than alternative methods including PolyPhen2, PROVEAN, SIFT, Rhapsody, EVMutation, MutationAssessor, SuSPect, FATHMM, and MVP. Variation numbers used within LYRUS differ greatly between pathogenic and neutral SAVs, and have a high feature weight in the XGBoost classifier employed by this method. Applications of the method to PTEN and TP53 further corroborate LYRUSs strong performance. LYRUS is freely available and the source code can be found at https://github.com/jiaying2508/LYRUS.

bioinformatics↗

bioRxiv2021

Maxent modeling for predicting the potential distribution of global talaromycosis

Talaromycosis, an invasive mycosis caused by Talaromyces marneffei (Tm), has rapidly increased in recent years, becoming an emerging pathogenic fungal disease. However, The driving factors and potential distribution of global talaromycosis is still unclear. Here, we developed maxent ecology model using environmental variables, Rhizomys distribution and HIV/AIDS epidemic to forecast ecological niche of talaromycosis worldwhile, as well as Identify the drivering factors. The constructed model had excellent performance with the area under the curve (AUC) of the receiver operating curve (ROC) of 0.997 in training data and 0.991 in testing data. Our model revealed that Rhizomys distribution, mean temperature of warmest quarter, precipitation of wettest month, HIV/AIDS epidemic and mean temperature of driest quarter were the top 5 important variables affecting talaromycosis distribution. In addition to traditional talaromycosis epidemic areas (South of the Yangtze River in China, Southeast Asian and North and Northeast India), our model also identified other potential epidemic regions, inculding parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Our findings has redefined global talaromycosis, discovered hidden high-risk areas and prorvided insights about driving factors of talaromycosis distribution, which will help inform surveillance strategies and improve the effectiveness of public health interventions against Tm infections. Author SummaryOur study aims to explore the spatial ecology of talaromycosis worldwhile. The diseases burden of Talaromycosis, a neglected zoonotic disease, is continuously rising in recent years because of the sheer size of susceptible population in the setting of increased globalization, rising HIV prevalence, and emerging iatrogenic immunodeficiency conditions. Here, we used historic reported talaromycosis cases from 1964 to 2017, combined with environmental factors, Rhizomys distribution and HIV/AIDS epidemic to build an maxent ecology model to define the ecological niche of talaromycosis, then predicting the potential distribution of the disease. The ecological niche of talaromycosis is characterized by a concentrated distribution, which can be cognitively divided into two regions: traditional talaromycosis epidemic areas (South of the Yangtze River in China, Southeast Asian and North and Northeast India), while other potential epidemic regions were predicted in parts of the North of the Yangtze River, Central America, West Coast of Africa, East Coast of South America, the Korean Peninsula and Japan. Our model also identified 5 driving factors affecting talaromycosis distribution. These findings will help demonstrate the global distribution of talaromycosis, discover hidden high-risk areas, and improve the effectiveness of public health interventions against Tm infections.

ecology↗

bioRxiv2021

rdacca.hp: an R package for generalizing hierarchical and variation partitioning in multiple regression and canonical analysis

O_LICanonical analysis, a generalization of multiple regression to multiple response variables, is widely used in ecology. Because these models often involve large amounts of parameters (one slope per response per predictor), they pose challenges to model interpretation. Currently, multi-response canonical analysis is constrained by two major challenges. Firstly, we lack quantitative frameworks for estimating the overall importance of single predictors. Secondly, although the commonly used variation partitioning framework to estimate the importance of groups of multiple predictors can be used to estimate the importance of single predictors, it is currently computationally constrained to a maximum of four predictor matrices. C_LIO_LIWe established that commonality analysis and hierarchical partitioning, widely used for both estimating predictor importance and improving the interpretation of single-response regression models, are related and complementary frameworks that can be expanded for the analysis of multiple-response models. C_LIO_LIIn this application, we aim at: a) demonstrating the mathematical links between commonality analysis, variation and hierarchical partitioning; b) generalizing these frameworks to allow the analysis of any number of responses, predictor variables or groups of predictor variables in the case of variation partitioning; and c) introducing and demonstrating the usage of the R package rdacca.hp that implements these generalized frameworks. C_LI

ecology↗

bioRxiv2021

The immunophilin protein FKBPL and its peptide derivatives are novel regulators of vascular integrity and inflammation via NF-kappaB signaling

A breakdown in vascular integrity and excessive inflammation are hallmarks of serious pathological conditions including sepsis, acute respiratory distress syndrome (ARDs) and most recently, severe COVID-19. FK506 - binding protein like (FKBPL) is a member of the immunophilin protein superfamily with potent anti-tumor activity through inhibition of angiogenesis and cancer stemness. An FKBPL-based 23mer peptide, ALM201, displayed a good safety and pharmacokinetic profile in a Phase 1a oncology clinical trial and was subsequently designated orphan drug status by the FDA in ovarian cancer. Here we describe a novel role for FKBPL and its peptides in regulating vascular integrity and cytokine production though modulating NF-{kappa}B signaling. FKBPL knockdown promoted endothelial cell barrier permeability, which was further exacerbated upon stimulation with lipopolysaccharide (LPS) and accompanied by increased expression of TNF mRNA and phosphorylation of p65(RelA). Whilst treatment with the FKBPL based pre-clinical peptide, AD-01, increased VE-cadherin endothelial tight junctions following LPS stimulation. Bone marrow derived macrophages (BMDM) from FKBPL haploinsufficient mice (Fkbpl+/-) also demonstrated increased phosphorylation of p65(RelA) in response to LPS stimulation compared to wild-type mice. Furthermore, treatment with AD-01 inhibited p65(RelA) phosphorylation following LPS stimulation resulting in reduced NF-{kappa}B target gene expression and proinflammatory cytokine production. In an in vivo LPS survival model, Fkbpl+/- mice have reduced survival compared to wild-type mice. Moreover, treatment of wild-type mice with the clinical FKBPL-based peptide, ALM201, following LPS injection resulted in a 100% survival rate in mice at experimental endpoint, as well as an abrogation of production of pro-inflammatory cytokines, TNF and IL-6, in peritoneal lavage washings. Analysis of human genetic biobanks found an association between common genetic variants associated with FKBPL and traits associated with inflammatory disorders such as psoriasis, rheumatoid arthritis and high lymphocyte count. In summary, for the first time, we describe a novel role for FKBPL as a regulator of inflammation and vascular integrity through modulating NF-{kappa}B signaling and FKBPL based therapies demonstrate potent anti-inflammatory activity.

immunology↗

bioRxiv2021

Talaromyces marneffei promotes M2 polarization of human macrophages by downregulating SOCS3 expression and activating TLR9 pathway

Little is known about how Talaromyces marneffei, a thermally dimorphic fungus that causes substantial morbidity and mortality in Southeast Asia, evades the human immune system. Polarization of macrophages into fungal-inhibiting M1 and fungal-promoting M2 types has been shown to play an important role in the innate immune response against fungal pathogens. This mechanism has not been defined for T. marneffei. Here, we demonstrated that T. marneffei promotes its survival in human macrophages by inducing them towards M2 polarization. Our investigations of the mechanism revealed that T. marneffei infection led to SOCS3 protein degradation by inducing tyrosine phosphorylation, thereby relieving the inhibitory effect of SOCS3 on p-STAT6, a key factor for M2 polarization. Our SOCS3-overexpression experiments showed that SOCS3 is a positive regulator of M1 polarization and plays an important role in limiting M2 polarization. Furthermore, we found that inhibition of TLR9 pathway partially blocked T. marneffei-induced M2 polarization and significantly enhanced the killing activity of macrophages against T. marneffei. Collectively, these results reveal a novel mechanism by which T. marneffei evades the immune response of human macrophages.

microbiology↗

bioRxiv2020

Antigen-driven clonal selection shapes the persistence of HIV-1 infected CD4+ T cells in vivo

Clonal expansion of infected CD4+ T cells is a major mechanism of HIV-1 persistence and a barrier to cure. Potential causes are homeostatic proliferation, effects of HIV-1 integration, and interaction with antigens. Here we show that it is possible to link antigen responsiveness, full proviral sequence, integration site, and T cell receptor {beta}-chain (TCR{beta}) sequence to examine the role of recurrent antigenic exposure in maintaining the HIV-1 reservoir. We isolated Cytomegalovirus (CMV)- and Gag-responding CD4+ T cells from 10 treated individuals. Proviral populations in CMV-responding cells were dominated by large clones, including clones harboring replication-competent proviruses. TCR{beta} repertoires showed high clonality driven by converging adaptive responses. Although some proviruses were in genes linked to HIV-1 persistence (BACH2, STAT5B, MKL1), proliferation of infected cells under antigenic stimulation occurred regardless of the site of integration. Paired TCR{beta}-integration site analysis showed that infection could occur early or late in the course of a clones response to antigen and could generate infected cell populations too large to be explained solely by homeostatic proliferation. Together these findings implicate antigen-driven clonal selection as a major factor in HIV-1 persistence, a finding that will be a difficult challenge to eradication efforts.

microbiology↗

bioRxiv2020

Transcriptome Profiling of different types of human respiratory tract cells infected by SARS-CoV-2 Highlight an unique Role for Inflammatory and Interferon Response

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) at the end of 2019 has caused a large global outbreak and now become a major public health issue. Lack of data underlying how the human host interacts with SARS-CoV-2 virus. In the current study, We performed Venn-analysis, Gene ontology (GO), KEGG pathway analysis and Protein-protein interaction analysis of whole transcriptome studies with the aim of clarifying the genes and pathways potentially altered during human respiratory tract cells infected with SARS-CoV-2. We selected four studies through a systematic search of the Gene Expression Omnibus (GEO) database or published article about SARS-CoV-2 infection in different types of respiratory tract cells. We found 36 overlapping upregulated genes among different types of cells after viral infection. Further functional enrichment analysis revealed these DEGs are most likely involved in biological processes related to inflammatory response and response to cytokine, cell component related to extracellular space and I-kappaB/NF-kappaB complex, molecular function related to protein binding and cytokine activity. KEGG pathways analysis highlighted altered conical and casual pathways related to TNF, NF-kappa B, Cytokine-cytokine receptor interaction and IL17 signaling pathways during SARS-CoV-2 infection with CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, IL32, CX3CL1, CCL20, IRF1, NFKB2 and NFKB1A up-regulated which may explain the inflammatory cytokine storms associated with severe cases of COVID-19.

immunology↗

bioRxiv2020

Characterization and establishment of a novel EBV strain simultaneously associated with nasopharyngeal carcinoma and B-cell lymphoma

Epstein-Barr virus (EBV) - the prototypical human tumor virus - is responsible for 1-2% of the global cancer burden, but divergent strains seem to exist in different geographical regions with distinct predilections for causing lymphoid or epithelial malignancies. Here we report the establishment and characterization of Yu103, an Asia Pacific EBV strain with a highly remarkable provenance of being derived from nasopharyngeal carcinoma biopsy but subsequently propagated in human B-lymphoma cells and xenograft models. Unlike previously characterized EBV strains which are either predominantly B-lymphotropic or epitheliotropic, Yu103 evinces an uncanny capacity to infect and transform both B-lymphocytes and nasopharyngeal epithelial cells. Genomic and phylogenetic analyses indicated that Yu103 EBV lies midway along the spectrum of EBV strains known to drive lymphomagenesis or carcinogenesis, and harbors molecular features which likely account for its unusual properties. To our knowledge, Yu103 EBV is currently the only EBV isolate shown to drive human nasopharyngeal carcinoma and B-lymphoma, and should therefore provide a powerful novel platform for research on EBV-driven hematological and epithelial malignancies.

cancer biology↗

bioRxiv2019

Short-term Effects of Vagus Nerve Stimulationon Learning and Evoked Activity in AuditoryCortex

Chronic vagus nerve stimulation (VNS) can facilitate learning of sensory and motor behaviors. VNS is believed to trigger release of neuromodulators, including norepinephrine and acetylcholine, which can mediate cortical plasticity associated with learning. Most previous work has studied effects of VNS over many days, and less is known about how acute VNS influences neural coding and behavior over the shorter term. To explore this question, we measured effects of VNS on learning of an auditory discrimination over 1-2 days. Ferrets implanted with cuff electrodes on the vagus nerve were trained by classical conditioning on a tone frequency-reward association. One tone was associated with reward while another tone, was not. The frequencies and reward associations of the tones were changed every two days, requiring learning of a new relationship. When the tones (both rewarded and non-rewarded) were paired with VNS, rates of learning increased on the first day following a change in reward association. To examine VNS effects on auditory coding, we recorded single- and multi-unit neural activity in primary auditory cortex (A1) of passively listening animals following brief periods of VNS (20 trials/session) paired with tones. Because afferent VNS induces changes in pupil size associated with fluctuations in neuromodulation, we also measured pupil during recordings. After pairing VNS with a neurons best-frequency (BF) tone, responses in a subpopulation of neurons were reduced. Pairing with an off-BF tone or performing VNS during the inter-trial interval had no effect on responses. We separated the change in A1 activity into two components, one that could be predicted by fluctuations in pupil and one that persisted after VNS and was not accounted for by pupil. The BF-specific reduction in neural responses remained, even after regressing out changes that could be explained by pupil. In addition, the size of VNS-mediated changes in pupil predicted the magnitude of persistent changes in the neural response. This interaction suggests that changes in neuromodulation associated with arousal gate the long-term effects of VNS on neural activity. Taken together, these results support a role for VNS in auditory learning and help establish VNS as a tool to facilitate neural plasticity.

neuroscience↗

bioRxiv2019

Macrocyclic colibactin induces DNA double-strand breaks via copper-mediated oxidative cleavage

Colibactin is an as-yet-uncharacterized human gut bacterial genotoxin, whose biosynthesis is linked to clb genomic island that distributes widespread in pathogenic and commensal human enterobacteria. Colibactin-producing gut microbes promote colon tumor formation and enhance progression of colorectal cancer (CRC) via DNA double-strand breaks (DSBs)-induced cellular senescence and death; however, the chemical basis contributing to the pathogenesis at the molecular level remains elusive. Here we report the discovery and the mechanism of action of colibactin-645 as the highly sought final colibactin metabolite with a novel molecular scaffold. Colibactin-645 recapitulates its previously assumed genotoxicity and cytotoxicity, exhibiting a strong DNA DSBs activity in vitro and in human cell cultures via a unique copper-mediated oxidative mechanism. We also present a complete model for colibactin biosynthesis, revealing an unprecedented dual function of the aminomalonate-utilizing polyketide synthases. This work thus provides the first molecular basis for colibactins genotoxic activity and facilitates further mechanistic study of colibactin-related CRC incidence and prevention.

microbiology↗

bioRxiv2018

Assessing intra-lab precision and inter-lab repeatability of outgrowth assays of HIV-1 latent reservoir size

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a \"gold standard\" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates - as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir - rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.\n\nAuthor summaryThe latent reservoir of resting CD4+ T cells is a major, if not the primary, obstacle to curing HIV. Quantitative viral outgrowth assays (QVOAs) are used to measure the latent reservoir in ART-suppressed HIV-infected people. Using QVOA is difficult, however, as the fraction of cells constituting the latent reservoir is typically about one in one million, far lower than other infectious disease biomarkers. To study reliability of these assays, we distributed 75 PBMC samples from five ART-suppressed HIV-infected participants among four labs, each conducting QVOA and following prespecified sample batching procedures. Using a Bayesian statistical method, we analyzed detailed assay output to understand how results varied within batches, between batches, and between labs. We found that, if batch variation can be controlled (i.e., a lab assays all samples in one batch), typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. We also found that freezing, storing, and thawing samples for later analysis caused no more than a 2-fold change in results. These outcomes, and the statistical methods developed to obtain them, should lead towards more precise and powerful assessments of HIV cure strategies.

microbiology↗