Biology subjects

Thomas, A.

Publications and source records attributed to Thomas, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

The 5:2 diet does not increase adult hippocampal neurogenesis or enhance spatial memory in mice

New neurones are generated throughout life in the mammalian brain in a process known as adult hippocampal neurogenesis (AHN). Since this phenomenon grants a high degree of neuroplasticity influencing learning and memory and mood related behaviour, identifying factors that regulate AHN may be important for ameliorating age-related cognitive decline and neurodegeneration. Calorie restriction (CR), in the absence of malnutrition, has been shown to enhance AHN and improve hippocampal-dependent memory, mediated by the stomach hormone, ghrelin. Intermittent fasting (IF), a dietary strategy offering more flexibility than conventional CR, also promotes aspects of AHN. The 5:2 diet is a popular form of IF linked to a range of health benefits, however its effects on AHN and spatial memory are not well characterised. We hypothesised that the 5:2 diet would enhance AHN in a ghrelin-dependent manner. To assess this, we used immunohistochemistry to quantify new adult-born neurones and new neural stem cells (NSCs) in the hippocampal DG of adolescent and adult wild-type and mice lacking the ghrelin receptor following six weeks on a 5:2 diet. We report an age-related decline in neurogenic processes and identify a novel role for ghrelin-receptor in regulating the formation of new adult born NSCs in an age-dependent manner. However, the 5:2 diet did not affect new neurone or NSC formation in the DG. Consistent with this finding the 5:2 diet did not alter performance on a spatial learning and memory task. These data suggest that the 5:2 diet used in this study does not increase AHN or improve associated spatial memory function. HighlightsO_LI5:2 diet does not increase adult hippocampal neurogenesis C_LIO_LI5:2 diet does not enhance spatial memory performance C_LIO_LIThere is an age-related decline in adult hippocampal neurogenesis C_LIO_LIGhrelin-receptor regulates new neural stem cell and new neurone number in an age-related manner C_LIO_LIGhrelin-receptor is required for intact spatial memory C_LI

neuroscience↗

bioRxiv2022

Origin and dynamics of Mycobacterium tuberculosis subpopulations that predictably generate drug tolerance and resistance

Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis (Mtb) population. We developed a multi-transwell robotic system to perform numerous parallel cultures of genetically barcoded Mtb exposed to steady-state concentrations of rifampicin to uncover these difficult to eliminate minority populations. We found that tolerance repeatedly emerged from at least two subpopulations of barcoded cells, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed glpK phase variation mutants were associated with up to 16 % of the resistant cultures. Drug tolerance and resistance were eliminated in{Delta} ruvA mutant consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug tolerant subpopulations whose elimination may be critical to developing rapid and resistance free cures. ImportanceTuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter into a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult to eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.

microbiology↗

bioRxiv2022

CasDinG is an ATP-dependent 5'-3' DNA helicase with accessory domains essential for type IV CRISPR immunity

CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. However, the enzymatic activities of CasDinG are unknown. Here we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP- and metal-dependent 5-3 DNA helicase. The crystal structure of CasDinG reveals a helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these CasDinG domains, we first identified the preferred PAM sequence (5-GNAWN-3 on the 5-side of the target) with a plasmid library containing all combinations of the five nucleotides upstream of the target sequence. Plasmid clearance assays (using a 5-GGAAA-3 PAM) with CasDinG domain mutants demonstrated the vFeS and arch accessory domains are both essential for type IV immunity. These results provide a needed structural and biochemical framework for understanding the type IV-A CRISPR system.

biochemistry↗

bioRxiv2022

Rationally designed Gla-domainless FXa as TFPI bait in hemophilia

Gla-domainless factor Xa (GD-FXa) was proposed as a trap to the endogenous anticoagulant Tissue Factor Pathway Inhibitor (TFPI) to restore thrombin generation in hemophilia. Using computational chemistry and experimental approaches, we previously showed that S195A GD-FXa also binds TFPI and restores ex vivo coagulation in hemophilia plasmas. To design a GD-FXa variant with improved anti-TFPI activity and identify suitable sites for mutagenesis, we performed molecular dynamics simulations. The calculations identified residues R150FXa and K96FXa as cold-spots of interaction between GD-FXa and the K2 domain of TFPI. In the three-dimensional model, both residues are facing TFPI hydrophobic residues and are thus potential candidates for mutagenesis into hydrophobic residues to favor an improved protein-protein interaction. Catalytically inactive GD-FXa variants containing the S195A mutation and additional mutations as K96Y, R150I, R150G and R150F were produced to experimentally confirm these computational hypotheses. Among these mutants, the R150FFXA showed increased affinity for TFPI as theoretically predicted, and was also more effective than S195A GD-FXa in restoring coagulation in FVIII deficient plasmas. Moreover, the R150 mutants lost interaction with antithrombin, which is favorable to extend their half-life.

bioengineering↗

bioRxiv2022

Subtyping of Small Cell Lung Cancer using plasma cell-free nucleosomes

Emerging data on small cell lung cancer (SCLC), an aggressive malignancy with exceptionally poor prognosis, support subtypes driven by distinct transcription regulators, which engender unique therapeutic vulnerabilities. However, the translational potential of these observations is limited by access to tumor biopsies. Here, we leverage chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) on 442 plasma samples from individuals with advanced SCLC, neuroendocrine carcinomas (NEC), non-SCLC cancers, and healthy adults. Beyond providing reliable estimates of SCLC circulating free DNA tumor fraction, cfChIP-seq captures the unique epigenetic states of SCLC tissue- and cell-of-origin. Comparison of cfChIP-seq signals to matched tumor transcriptomes reveals genome-wide concordance, establishing a direct link between gene expression in the tumor and plasma cell-free nucleosomes. Exploiting this link, we develop a classifier that discriminates between SCLC lineage-defining transcription factor subtypes based on cfChIP-seq data. This work sets the stage to non-invasively profile SCLC transcriptomes using plasma cfDNA histone modifications.

cancer biology↗

bioRxiv2022

Extended practice improves the accuracy and efficiency of goal-directed reaching guided by supplemental kinesthetic vibrotactile feedback

Prior studies have shown that providing task-specific vibrotactile feedback (VTF) during reaching and stabilizing with the arm can immediately improve the accuracy and efficiency. However, such studies typically evaluate performance after less than 1 hour of practice using VTF. Here we tested the effects of extended practice using supplemental kinesthetic VTF on goal-directed reaching with the arm. Healthy young adults performed a primary reaching task and a secondary choice reaction task individually and as a dual-task. The reaching task was performed under three feedback conditions: visual feedback, proprioceptive feedback, and with supplemental kinesthetic VTF applied to the non-moving arm. We compared performances before, during, and after approximately 10 hours of practice on the VTF-guided reaching task, distributed across 20 practice sessions. Upon initial exposure to VTF-guided reaching, participants were immediately able to use the VTF to improve reaching accuracy. Performance improvements were retained from one practice session to the next. After 10 hours of practice, the accuracy and temporal efficiency of VTF-guided reaching were equivalent to or better than reaching performed without vision or VTF. However, hand paths during VTF-guided reaching exhibited a persistent strategy whereby movements were decomposed into discrete sub-movements along the cardinal axes of the VTF interface. Dual-tasking capability also improved, such that the primary and secondary tasks we performed more concurrently after extended practice. Our results demonstrate that extended practice on VTF-guided reaching can yield performance improvements that accrue in a manner increasingly resistant to dual-task interference.

neuroscience↗

bioRxiv2022

EZH2 interacts with MEG3 to directly regulate integrin signaling and endothelial cell function

Enhancer of Zeste Homologue 2 (EZH2) modulates gene transcription during endothelial cell (EC) dysfunction, via interaction with non-coding RNAs (ncRNAs). Thus, EZH2 can act as a rheostat in deposition of histone H3K27 trimethylation (H3K27me3) to repress many genes. We profiled EZH2-RNA interactions using formaldehyde/UV assisted cross-linking ligation and sequencing of hybrids (FLASH-seq) in primary human ECs. Transcriptome-wide EZH2-associated ncRNAs and RNA-RNA interactome were obtained. This approach revealed EZH2 directly binding maternally expressed gene (MEG3) and MEG3:MEG3 hybrid structures. By chromatin immunoprecipitation with sequencing (ChIP-seq) following depletion of MEG3, we discovered that MEG3 targets and controls recruitment of EZH2/H3K27me3 onto a regulatory region of integrin subunit alpha 4 (ITGA4). MEG3 knockdown or pharmacological inhibition of EZH2 de-repressed ITGA4, whilst improving endothelial cell function in vitro, and increasing ITGA4 expression in vivo. Our study demonstrates new role for MEG3, as instrumental in epigenetic regulation of EC function by EZH2, through targeting of integrin-dependent signalling.

molecular biology↗

bioRxiv2022

Hypoxic memory of tumor intrinsic type I interferon suppression promotes breast cancer metastasis

Hypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis and has been associated with tumor progression and metastasis. Most of the well-known hypoxia effects are mediated through hypoxia-inducible factors (HIFs), but the long-lasting effect of hypoxia beyond the immediate HIF regulation remains less understood. Here we show that hypoxia exerts a prolonged effect to promote metastasis. Using breast cancer patient-derived circulating tumor cell (CTC) lines and common breast cancer cell lines, we found that hypoxia downregulates tumor intrinsic type I interferon (IFN) signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells, via both HIF-dependent and HIF-independent mechanisms. Hypoxia induced IFN/AP suppression can last longer than the hypoxic exposure, presenting a "hypoxic memory" phenotype. Hypoxic memory of IFN/AP downregulation is established by specific hypoxic priming, and cells with hypoxic memory have an enhanced ability for tumorigenesis and metastasis. The histone deacetylase inhibitor (HDACi) Entinostat can erase the hypoxic memory and improve the immune clearance of tumor cells when combined with checkpoint immunotherapies in a syngeneic breast cancer mouse model. These results point to a novel mechanism for hypoxia facilitated tumor progression, through a long-lasting memory that provides advantages for CTCs during the metastatic cascade. Significance: We revealed a novel hypoxic memory of prolonged suppression of tumor intrinsic type I IFN and AP signals that promote tumorigenesis and metastasis, suggesting novel mechanistic understanding of the immune evasive properties of CTCs.

cancer biology↗

bioRxiv2022

BET inhibition sensitizes immunologically-cold Rb-deficient prostate cancer to immune checkpoint blockade

PurposeNon-T cell-inflamed immunologically "cold" tumor microenvironments (TME) are associated with poor responsiveness to immune checkpoint blockade (ICB), and can be sculpted by tumor cell genomics. Here we evaluated how Retinoblastoma (Rb) tumor suppressor loss of function (LOF), one of the most frequent alterations in human cancer and associated with lineage plasticity, poor prognosis and therapeutic outcomes, alters the TME, and whether therapeutic strategies targeting the molecular consequences of Rb loss enhance ICB efficacy. Experimental DesignWe performed bioinformatics analysis to elucidate the impact of endogenous Rb LOF on the immune TME in human primary and metastatic tumors. Next, we utilized isogenic murine models of Rb-deficient prostate cancer (PC) for in vitro and in vivo mechanistic studies to examine how Rb loss and bromodomain and extraterminal (BET) domain inhibition (BETi) reprograms the immune landscape, and evaluated in vivo therapeutic efficacy of BETi, singly and in combination with ICB and androgen deprivation therapy. ResultsRb loss was enriched in non-T cell-inflamed tumors, and Rb-deficient murine tumors demonstrated decreased immune infiltration in vivo. The BETi JQ1 increased immune infiltration into the TME through enhanced tumor cell STING/NF-{kappa}B activation and type I interferon (IFN) signaling within tumor cells, resulting in differential macrophage and T cell-mediated tumor growth inhibition and sensitization of Rb-deficient PC to ICB. ConclusionsBETi can reprogram the immunologically cold Rb-deficient TME via STING/NF-{kappa}B/IFN signaling to sensitize Rb-deficient PC to ICB. These data provide the mechanistic rationale to test combinations of BETi and ICB in clinical trials of Rb-deficient PC. STATEMENT OF TRANSLATIONAL RELEVANCERb LOF is one of the most common genomic alterations in human cancer, occurring in approximately 1/3 of advanced malignancies, Furthermore, loss of Rb correlates with enhanced aggressiveness and poor therapeutic outcomes. In this study, we demonstrate that loss of Rb is also associated with an immunosuppressive tumor microenvironment and lack of responsiveness to immune checkpoint blockade (ICB). As a strategy to overcome Rb LOF induced immunosuppression, we have demonstrated that BETi treatment drives STING/NF-{kappa}B signaling and type I interferon production within tumor cells, resulting in immune-mediated tumor control in Rb-deficient PC, which is accentuated by the combination with ICB and ADT. These findings provide a roadmap for maximizing the clinical translation of BET inhibitors into the clinic to treat aggressive-variant Rb-deficient PC.

cancer biology↗

bioRxiv2022

DNA methylation signatures of Alzheimer's disease neuropathology in the cortex are primarily driven by variation in non-neuronal cell-types

Alzheimers disease (AD) is a chronic neurodegenerative disease characterized by the progressive accumulation of amyloid-beta and neurofibrillary tangles of tau in the neocortex. Utilizing extensive neuropathology data from the Brains for Dementia Research (BDR) cohort we performed the most systematic epigenome-wide association study (EWAS) of multiple measures of AD neuropathology yet undertaken, profiling DNA methylation in two cortical regions from 631 donors. We meta-analyzed our results with those from previous studies of DNA methylation in AD cortex (total n = 2,013 donors), identifying 334 cortical differentially methylated positions (DMPs) associated with AD pathology including methylomic variation at novel loci not previously implicated in dementia. We subsequently characterized DNA methylation in purified nuclei populations - enriched for neurons, oligodendrocytes and microglia - exploring the extent to which cortex AD-associated DMPs reflect differences manifest in specific cell populations. We find that the majority of DMPs identified in bulk cortex tissue actually reflect DNA methylation differences occurring in non-neuronal cells, with dramatically increased effect sizes observed in microglia-enriched nuclei populations. Our study highlights the power of utilizing multiple measures of neuropathology to identify epigenetic signatures of AD and the importance of characterizing disease-associated variation in purified neural cell-types.

genetics↗

bioRxiv2022

Sex differences in insular gyri responses to the cold pressor challenge

IntroductionSex differences in autonomic control may contribute to physiological sex differences in cardiovascular disease, thermoregulation, and the experience of pain. We previously showed sex differences in functional mapping of autonomic responses across the insula, a central autonomic control region. Anterior insula response patterns to sympathetic activation (Valsalva) and parasympathetic withdrawal (handgrip) differed between men and women. We here assessed sex differences in autonomic insula gyrus responses to a cold pressor challenge, which involves temperature and pain regulation. MethodsFunctional MRI (fMRI) involved a 1-minute right-foot cold pressor challenge in 22 women (age; mean{+/-}std: 50{+/-}4 yrs), and 39 men (45{+/-}3 yrs). Regions of interest (ROI) comprised left and right anterior short gyrus (ASG), middle short gyrus (MSG), posterior short gyrus (PSG), anterior long gyrus (ALG), and posterior long gyrus (PLG). Two-second time intervals of fMRI signal responses per ROI and concurrently recorded heart rate and blood oxygen saturation (SaO2) were tested for within- and between-group effects over time (repeated measures ANOVA P[≤]0.05). We tested sex differences in 1) each ROI; 2) lateralization effects; and 3) posterior-to-anterior gradients in responses. ResultsNo sex differences emerged in heart rate or SaO2. Women showed larger signal changes in several gyri than men after cold pressor onset and offset. Most consistently, women showed a greater right-over-left hemisphere dominance in cold pressor response compared to men in the MSG, PSG and ALG. Greater right-over-left hemisphere anterior-to-posterior dominance was more pronounced in men. ConclusionsThe findings confirm anterior and right-hemispheric dominance of insular responses during sympathetic activation that includes pain and cold. Although distinct transient male-female differences in fMRI responses appeared during the cold pressor, unlike the Valsalva and handgrip, no sex differences in ASG lateralization emerged, suggesting that the previously found sex differences may not solely relate to regulating blood pressure responses.

neuroscience↗

bioRxiv2022

Vertical HIV-1 transmission in the setting of maternal broad and potent antibody responses

Despite the worldwide availability of antiretroviral therapy (ART), approximately 150,000 pediatric HIV infections continue to occur annually. ART can dramatically reduce HIV mother-to-child transmission (MTCT), but inconsistent drug access and adherence, as well as primary maternal HIV infection during pregnancy and lactation are major barriers to eliminating vertical HIV transmission. Thus, immunologic strategies to prevent MTCT, such as an HIV vaccine, will be required to attain an HIV-free generation. A primary goal of HIV vaccine research has been to elicit broadly neutralizing antibodies (bnAbs) given the ability of passive bnAb immunization to protect against sensitive strains, yet we previously observed that HIV-transmitting mothers have more plasma neutralization breadth than non-transmitting mothers. Additionally, we have identified infant transmitted/founder (T/F) viruses that escape maternal bnAb responses. In this study, we examine a cohort of postpartum HIV-transmitting women with neutralization breadth to determine if certain maternal bnAb specificities drive the selection of infant T/F viruses. Using HIV pseudoviruses that are resistant to neutralizing antibodies targeting common bnAb epitopes, we mapped the plasma bnAb specificities of this cohort. Significantly more transmitting women with plasma bnAb activity had a mappable plasma bnAb specificity (six of seven, or 85.7%), compared to that of non-transmitting women with plasma bnAb activity (seven of twenty-one, or 33.3%, p=0.029 by 2-sided Fisher exact test). Our study suggests that having multispecific broad activity and/or uncommon epitope-specific bnAbs in plasma may be associated with protection against the vertical HIV transmission in the setting of maternal bnAb responses. ImportanceAs mother to child transmission (MTCT) of HIV plays a major part in the persistence of the HIV/AIDS epidemic and bnAb-based passive and active vaccines are a primary strategy for HIV prevention, research in this field is of great importance. While previous MTCT research has investigated the neutralizing antibody activity of HIV-infected women, this is to our knowledge the largest study identifying differences in bnAb specificity of maternal plasma between transmitting and non-transmitting women. Here, we show that among HIV-infected women with broad and potent neutralization activity, more postpartum-transmitting women had a mappable plasma broadly neutralizing antibody (bnAb) specificity, compared to that of non-transmitting women, suggesting that the non-transmitting women more often have multi-specific bnAb responses or bnAb responses that target uncommon epitopes. Such responses may be required for protection against vertical HIV transmission in the setting of maternal bnAb responses.

immunology↗

bioRxiv2021

Computational analysis reveals similarities and differences between SCLC subtypes

Small cell lung cancer (SCLC) is a neuroendocrine malignancy with dismal survival rates. Previous studies have revealed inter and intra tumoral heterogeneity of SCLC driven by neuroendocrine differentiation and multiple gene expression signatures have been proposed to classify the distinct SCLC molecular subtypes However, few questions remain unanswered: a) how many SCLC subtypes exist? b) how similar or different are these subtypes?, c) which gene list(s) can be used to identify those specific subtypes? Here, we show that irrespective of the three gene sets (33 genes, 50 genes, 105 genes) proposed in different studies to classify SCLC into different subtypes, the markers of phenotypic heterogeneity in SCLC form a "teams" like pattern of co-expressed modules. Moreover, the 105 geneset could classify SCLC cell lines into five clusters, three of which can be distinctly mapped to the SCLC-A, SCLC-N and SCLC-Y subtypes. Intriguingly, we noticed a high degree of similarity in the transcriptional landscape of two non-neuroendocrine subtypes: SCLC-Y and SCLC-I*, as well as in their enrichment of EMT. Thus, our analysis elucidates the landscape of phenotypic heterogeneity enabling diverse SCLC subtypes.

cancer biology↗

bioRxiv2021

The Chloroflexi supergroup is metabolically diverse and representatives have novel genes for non-photosynthesis based CO2 fixation

The Chloroflexi superphylum have been investigated primarily from the perspective of reductive dehalogenation of toxic compounds, anaerobic photosynthesis and wastewater treatment, but remain relatively little studied compared to their close relatives within the larger Terrabacteria group, including Cyanobacteria, Actinobacteria, and Firmicutes. Here, we conducted a detailed phylogenetic analysis of the phylum Chloroflexota, the phylogenetically proximal candidate phylum Dormibacteraeota, and a newly defined sibling phylum proposed in the current study, Eulabeiota. These groups routinely root together in phylogenomic analyses, and constitute the Chloroflexi supergroup. Chemoautotrophy is widespread in Chloroflexi. Two Form I Rubisco ancestral subtypes that both lack the small subunit are prevalent in ca. Eulabeiota and Chloroflexota, suggesting that the predominant modern pathway for CO2 fixation evolved in these groups. The single subunit Form I Rubiscos are inferred to have evolved prior to oxygenation of the Earths atmosphere and now predominantly occur in anaerobes. Prevalent in both Chloroflexota and ca. Eulabeiota are capacities related to aerobic oxidation of gases, especially CO and H2. In fact, aerobic and anaerobic CO dehydrogenases are widespread throughout every class-level lineage, whereas traits such as denitrification and reductive dehalogenation are heterogeneously distributed across the supergroup. Interestingly, some Chloroflexota have a novel clade of group 3 NiFe hydrogenases that is phylogenetically distinct from previously reported groups. Overall, the analyses underline the very high level of metabolic diversity in the Chloroflexi supergroup, suggesting the ancestral metabolic platform for this group enabled highly varied adaptation to ecosystems that appeared in the aerobic world.

microbiology↗

bioRxiv2021

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies have the potential to induce broadly reactive nAbs. Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets of neutralizing antibodies (NAbs), the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-neutralizing antibodies, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the V2 loops C-strand. Notably, a 167N mutation improved V2-sensitivity. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing other glycans in some cases had local and global "ripple" effects on glycan maturation and sequon occupation in the gp120 outer domain and gp41. V2 mAb CH01 selectively bound trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a N49 glycan perturbs gp41 glycans via a distal glycan network effect, increasing FP NAb sensitivity - and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) [~]25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express [~]30-40 spikes. Overall, we identified 7 diverse trimers with a range of sensitivities to two targets that should enable rigorous testing of immunofocusing vaccine concepts. Author SummaryDespite almost 40 years of innovation, an HIV vaccine to induce antibodies that block virus infection remains elusive. Challenges include the unparalleled sequence diversity of HIVs surface spikes and its dense sugar coat that limits antibody access. However, a growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints. To date, these kinds of antibodies have been difficult to induce by vaccination. However, two antibody targets, one at the spike apex and another at the side of the spikes are more forgiving in their demands for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites for later use as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that the spikes, when expressed on particles, are infectious, allowing us to appraise our vaccine designs in an ideal manner; ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine.

immunology↗

bioRxiv2021

Critical Assessment of Metagenome Interpretation - the second round of challenges

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the community-driven initiative for the Critical Assessment of Metagenome Interpretation (CAMI). In its second challenge, CAMI engaged the community to assess their methods on realistic and complex metagenomic datasets with long and short reads, created from [~]1,700 novel and known microbial genomes, as well as [~]600 novel plasmids and viruses. Altogether 5,002 results by 76 program versions were analyzed, representing a 22x increase in results. Substantial improvements were seen in metagenome assembly, some due to using long-read data. The presence of related strains still was challenging for assembly and genome binning, as was assembly quality for the latter. Taxon profilers demonstrated a marked maturation, with taxon profilers and binners excelling at higher bacterial taxonomic ranks, but underperforming for viruses and archaea. Assessment of clinical pathogen detection techniques revealed a need to improve reproducibility. Analysis of program runtimes and memory usage identified highly efficient programs, including some top performers with other metrics. The CAMI II results identify current challenges, but also guide researchers in selecting methods for specific analyses.

bioinformatics↗

bioRxiv2021

Incorporation of unnatural amino acid for the tagging of cannabinoid receptors 1 and 2 reveals receptor roles in regulating cAMP levels

The role of CB1/CB2 co-expression in cell signaling remains elusive. We established a simplified mammalian cell model system in which expression of CB1 or CB2 can be easily monitored under a confocal microscope. For this, we applied amber codon suppression in live cells to incorporate a single trans-cyclooctene (TCO) bearing amino acid in one of the extracellular loops of CB1 or CB2, followed by fluorescent labeling via click chemistry. We employed genetically encoded biosensors to measure the roles of CB1 and/or CB2 in regulating intracellular calcium ([Ca2+]i) and cAMP ([cAMP]i) levels. We show that the agonist-mediated activation of tagged-CB1 or -CB2 can transiently elevate [Ca2+]i levels. However, when the two receptors were co-expressed in the same cell, CB2 no longer signaled through calcium although CB1-mediated transient elevation of [Ca2+]i levels was unaffected. Because of the existence of crosstalk between calcium and cAMP signaling, we measured the effects of CB1 and/or CB2 in regulating adenylate cyclase activity. We found that the expression of CB1 increased forskolin-induced [cAMP]i levels compared to non-transfected cells. Conversely, CB2 expression decreased stimulated [cAMP]i levels under the same conditions. Finally, co-expressed CB1 and CB2 receptors showed additive yet opposing effects on stimulated [cAMP]i levels. These observations suggest that co-expressed CB1/CB2 act locally as a pair in regulating cell excitability by modulating stimulated [cAMP]i levels.

cell biology↗

bioRxiv2021

Female fruitflies use gustatory cues to exhibit reproductive plasticity in response to the social environment

Animals can exhibit remarkable reproductive plasticity in response to their social surroundings, with profound fitness consequences. The study of such plasticity in females, particularly in same-sex interactions, has been severely neglected. Here we measured the impact of variation in the pre-mating social environment on reproductive success in females and tested the underlying mechanisms involved. We used the Drosophila melanogaster model system to test the effect of varying female group size prior to mating and deployed physical and genetic methods to manipulate the perception of different social cues and sensory pathways. We found that socially isolated females were significantly more likely to retain unfertilised eggs before mating, but to show the opposite pattern and lay significantly more fertilised eggs in the 24h after mating, in comparison to grouped females. More than 48h of exposure to other females was necessary for this socially-induced plasticity to be expressed. Neither olfactory nor visual cues were involved in mediating these responses. Instead, we found that females detected other females through direct contact with the deposits they leave behind, even in the absence of eggs. The results demonstrate that females show striking reproductive plasticity in response to their social surroundings and that the nature of their plastic reproductive responses, and the cues they use, differ markedly from those of males. The results emphasise the stark contrasts in how each sex realises reproductive success.

evolutionary biology↗