Biology subjects

Moore, J.

Publications and source records attributed to Moore, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

State-Threatened Gopher Tortoise (Gopherus polyphemus) Gut Microbiome Analysis Reveals Health Insights into Southeastern Florida Population

The gopher tortoise (Gopherus polyphemus), endemic to the southeastern part of the United States, is a keystone species that provides ecological support to over 350 species. Deforestation, urbanization, road mortality, and disease, over the past 100 years, have caused population decline by over 80%, causing Florida to declare the gopher tortoise a threatened species. Little is known about the gopher tortoise gut microbiome, which could play a major role in tortoise health. This study aimed to better understand the health and biology of this threatened keystone species through the characterization and analysis of their gut microbiomes in the Abacoa Greenway of Southeastern Florida using next-generation sequencing to survey the gut microbiome. Major findings include: high levels of alpha diversity; lack of significant difference of male and female alpha diversity and taxonomic composition profiles and, counterintuitively, male beta diversity was less disparate than female beta diversity; phyla associated with fiber fermentation short-chain fatty acid metabolism, Firmicutes and Bacteroidetes, predominated all samples. Notable probiotic taxa present included: Lachnospiraceae and Clostridium butyricum. Potentially pathogenic taxa included: Helicobacter sp. and Mollicutes sp. Pathogenetic taxa undetected: gopher tortoise Helicobacter, and Mycoplasma spp. These results add to the understudied reptiles and tortoise gut microbiome.

microbiology↗

bioRxiv2024

Mining Yeast Diversity Unveils Novel Targets for Improved Heterologous Laccase Production in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a widely utilized host cell for recombinant protein production due to its well studied and annotated genome, its ability to secrete large and post-translationally modified proteins, fast growth and cost-effective culturing. However, recombinant protein yields from S. cerevisiae often fall behind that of other host systems. To address this, we developed a high throughput screen of wild, industrial and laboratory S. cerevisiae isolates to identify strains with a natural propensity for greater recombinant protein production, specifically focussing on laccase multicopper oxidases from the fungi Trametes trogii and Myceliophthora thermophila. Using this method, we identified 20 non-laboratory strains with higher capacity to produce active laccase. Interestingly, lower levels of laccase mRNA were measured in most cases, indicating that the drivers of elevated protein production capacity lie beyond the regulation of recombinant gene expression. We characterized the identified strains using complementary genomic and proteomic approaches to reveal several potential pathways driving the improved expression phenotype. Gene ontology analysis suggests broad changes in cellular metabolism, specifically in genes/proteins involved in carbohydrate catabolism, thiamine biosynthesis, transmembrane transport and vacuolar degradation. Targeted deletions of the hexose transporter HXT11 and the Coat protein complex II interacting paralogs PRM8 and 9, involved in ER to Golgi transport, resulted in significantly improved laccase production from the S288C laboratory strain. Whereas the deletion of the Hsp110 SSE1 gene, guided by our proteomic analysis, also led to higher laccase activity, we did not observe major changes of the protein homeostasis network within the strains with higher laccase activity. This study opens new avenues to leverage the vast diversity of Saccharomyces cerevisiae for recombinant protein production, as well as offers new strategies and insights to enhance recombinant protein yields of current strains.

molecular biology↗

bioRxiv2024

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1s known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.

molecular biology↗

bioRxiv2024

Harnessing genome prediction in Brassica napus through a nested association mapping population

Genome prediction (GP) significantly enhances genetic gain by improving selection efficiency and shortening crop breeding cycles. Using a nested association mapping (NAM) population a set of diverse scenarios were assessed to evaluate GP for vital agronomic traits in B. napus. GP accuracy was examined by employing different models, marker sets, population sizes, marker densities, and incorporating genome-wide association (GWAS) markers. Eight models, including linear and semi-parametric approaches, were tested. The choice of model minimally impacted GP accuracy across traits. Notably, two models, rrBLUP and RKHS, consistently yielded the highest prediction accuracies. Employing a training population of 1500 lines or more resulted in increased prediction accuracies. Inclusion of single nucleotide absence polymorphism (SNaP) markers significantly improved prediction accuracy, with gains of up to 15%. Utilizing the Brassica 60K Illumina SNP array, our study effectively revealed the genetic potential of the B. napus NAM panel. It provided estimates of genomic predictions for crucial agronomic traits through varied prediction scenarios, shedding light on achievable genetic gains. These insights, coupled with marker application, can advance the breeding cycle acceleration in B. napus. Core ideasO_LIGenome prediction (GP) enhances genetic gains by improving selection efficiency and shortening breeding cycles. C_LIO_LIFactors influencing GP accuracy include model choice, marker types, and population size. C_LIO_LIInclusion of SNaP markers and highly significant GWAS markers improves prediction accuracy, shedding light on achievable genetic gains. C_LI Plain SummaryGenome prediction (GP) is a powerful tool that helps us improve crops more efficiently. In this study, we assessed how well GP works for predicting important traits in Brassica napus plants. We tested different models and marker sets to see which ones were most accurate. We found that two models, rrBLUP and RKHS, were consistently the best. Also, including certain types of genetic markers, like SNaP markers and highly significant GWAS markers, improved the predictions. Overall, our study shows that GP can help us understand the genetic potential of B. napus plants and improve breeding strategies, which can be exploited to develop better varieties more quickly, which is good news for farmers and the food supply.

genomics↗

bioRxiv2024

Beta3 accelerates microtubule plus end maturation through a divergent lateral interface

{beta}-tubulin isotypes exhibit similar sequences but different activities, suggesting that limited sequence divergence is functionally important. We investigated this hypothesis for TUBB3/{beta}3, a {beta}-tubulin linked to aggressive cancers and chemoresistance in humans. We created mutant yeast strains with {beta}-tubulin alleles that mimic variant residues in {beta}3 and find that residues at the lateral interface are sufficient to alter microtubule dynamics and response to microtubule targeting agents. In HeLa cells, {beta}3 overexpression decreases the lifetime of microtubule growth, and this requires residues at the lateral interface. These microtubules exhibit a shorter region of EB binding at the plus end, suggesting faster lattice maturation, and resist stabilization by paclitaxel. Resistance requires the H1-S2 and H2-S3 regions at the lateral interface of {beta}3. Our results identify the mechanistic origins of the unique activity of {beta}3 tubulin and suggest that tubulin isotype expression may tune the rate of lattice maturation at growing microtubule plus ends in cells.

cell biology↗

bioRxiv2024

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes

Background & AimsAging is associated with a significant decline in mitochondrial function in the liver, leading to an increased risk of liver disease. This study examines age-related changes in the mitochondrial structure of human and murine livers using a combination of Serial Block-Face Scanning Electron Microscopy (SBF-SEM) and mass spectrometry approaches. MethodsThis study integrates mitochondrial structure analysis in a murine model with an analysis of liver architecture, lipogenesis, and genetically regulated gene expression in human cohorts. We explored the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex using SBF-SEM, three-dimensional reconstruction with Amira software, and mass spectrometry techniques. ResultsAging leads to a reduction in mitochondrial size and complexity, resulting in changes in the metabolomic and lipidomic profiles of murine liver cells that are comparable to those observed in aged human samples. We find that genetically modeled expression of MICOS complex genes OPA1 and CHCHD3 is associated with chronic liver disease phenotypes within a large biobank population. Furthermore, we observed dysregulated mitochondrial calcium handling and increased oxidative stress due to the disruption of the MICOS complex. ConclusionOur study highlights the age-associated decline in mitochondrial complexity and metabolic regulation within the aging murine liver and the human population. We have identified that these changes are partially attributable to the age-related loss of the MICOS complex. Impact and implicationsThis study offers new insights into the changes to mitochondrial ultrastructure that occur during aging. Using SBF-SEM, the quantification of young and aged murine mitochondrial structure was performed, which had previously been an underexplored avenue for measuring mitochondrial changes. The discovery of mitochondrial ultrastructural changes, in conjunction with measurements of age-associated metabolic alterations and gene association data, provides a model for how changes in MICOS expression may modulate age-related impairment of hepatic mitochondria. These results provide a new model by which changes in MICOS protein expression may both cause and be a potential therapeutic target for age-related impairment in hepatic function. HighlightsDecreased modeled expression of CHCHD3 in individuals of European genetic ancestry is linked to liver transplant and cirrhosis, while decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic liver disease and cirrhosis. Aging alters liver lipid accumulation, MICOS mRNA levels, and disease markers. Aging reduces the volume and complexity of murine liver ultrastructure. Aging and diet significantly alter the MICOS complex in mice. Knockdown of Mic60 and Chchd6 lowers Ca2+ uptake, retention, and induces oxidative stress in HepG2 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/599846v3_ufig1.gif" ALT="Figure 1000"> View larger version (47K): org.highwire.dtl.DTLVardef@1cdd61corg.highwire.dtl.DTLVardef@a3fb74org.highwire.dtl.DTLVardef@1d1ad36org.highwire.dtl.DTLVardef@c2e55f_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver aging causes metabolic, lipidomic, and mitochondrial structural alterations, reflecting age-dependent losses in the MICOS complex. Key components of the MICOS complex (MIC60, CHCHD3 and CHCHD6) are illustrated.

physiology↗

bioRxiv2024

The ER Thioredoxin-Related Transmembrane Protein TMX2 Controls Redox-Mediated Tethering of ER-Mitochondria Contacts (ERMCS)

Thioredoxin-related transmembrane proteins (TMX) of the endoplasmic reticulum (ER) have emerged as key regulators of ER membrane properties. Within the ER lumen, TMX proteins and other ER redox enzymes determine oxidative conditions, which control the formation of ER-mitochondria membrane contacts (ERMCS) and determine their function. ERMCS exhibit cytoplasmic redox nanodomains, derived from ER and mitochondrial reactive oxygen species (ROS), whose mechanistic regulation is uncharacterized. Our research has identified the ER protein TMX2, which uses its unique cytosolic thioredoxin domain to prevent cytosolic sulfenylation of mitochondrial outer membrane proteins such as TOM70 through a functional interaction with peroxiredoxin-1 (PRDX1). By doing so, TMX2 interferes with the TOM70 ERMCS tethering function and reduces mitochondrial Ca2+ flux and metabolism. Recently, TMX2 mutations have been identified to cause a neurodevelopmental disorder with microcephaly, cortical malformations, and spasticity (NEDMCMS). Using TMX2-mutated NEDMCMS patient cells, we demonstrate that compromising TMX2 through mutation reproduces mitochondrial defects. In a fly in vivo model, TMX2 knockdown manifests predominantly in glial cells. Our results therefore provide important mechanistic insight into NEDMCMS and mechanistically link TMX2-mediated control of ERMCS to brain development and function. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/589228v2_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@809587org.highwire.dtl.DTLVardef@1d8ef0corg.highwire.dtl.DTLVardef@135fdc6org.highwire.dtl.DTLVardef@1a7f7b6_HPS_FORMAT_FIGEXP M_FIG C_FIG The transmembrane thioredoxin-related TMX2 prevents TOM70 sulfenylation at ERMCS, thus maintaining normal mitochondria metabolism in wild-type cells. TMX2 knockout leads to TOM70 sulfenylation and tight ERMCS formation. This then increases ROS production, unbalances mitochondrial lipids, and relatively shifts OXPHOS electron supply to complex II.

cell biology↗

bioRxiv2024

Broad-Spectrum Coronavirus Inhibitors Discovered by Modeling Viral Fusion Dynamics

Broad-spectrum therapeutics capable of inhibiting SARS-CoV-2, its variants, and related coronaviruses hold promise in curbing the spread of COVID-19 and averting future pandemics. Here, we employed a multidisciplinary approach that included molecular dynamics simulation (MDS) and artificial intelligence (AI)-based docking predictions to identify potent inhibitors that target a conserved region within the SARS-CoV-2 spike protein that mediates membrane fusion by undergoing large-scale mechanical rearrangements. In silico binding screens honed in on this region, leading to the discovery of FDA-approved drugs and novel molecules predicted to disrupt spike protein conformational changes. These compounds significantly inhibited SARS-CoV-2 infection and blocked the entry of spike protein-bearing pseudotyped , {beta}, {gamma}, {delta} variants as well as SARS-CoV and MERS-CoV in cultured human ACE2-expressing cells. The optimized lead compound significantly inhibited SARS-CoV2 infection in mice when administered orally.

microbiology↗

bioRxiv2024

Single-cell genomics and regulatory networks for 388 human brains

Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet, little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multi-omics datasets into a resource comprising >2.8M nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550K cell-type-specific regulatory elements and >1.4M single-cell expression-quantitative-trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized [~]250 disease-risk genes and drug targets with associated cell types. Summary Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/585576v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@70b24org.highwire.dtl.DTLVardef@6c9eddorg.highwire.dtl.DTLVardef@131fae2org.highwire.dtl.DTLVardef@4e0f1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Lack of TGFβ signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade

BackgroundThe efficacy of immune checkpoint blockade (ICB) depends on restoring immune recognition of cancer cells that have evaded immune surveillance. At the time of diagnosis, patients with lymphocyte-infiltrated cancers are the most responsive to ICB, yet a considerable fraction of patients have immune-poor tumors. MethodsWe analyzed transcriptomic data from IMvigor210, TCGA, and TISMO datasets to evaluate the predictive value of {beta}Alt, a score representing the negative correlation of signatures consisting of transforming growth factor beta (TGF{beta}) targets and genes involved in error-prone DNA repair. The immune context of {beta}Alt was assessed by evaluating tumor-educated immune signatures. An ICB-resistant, high {beta}Alt preclinical tumor model was treated with a TGF{beta} inhibitor, radiation, and/or ICB and assessed for immune composition and tumor control. ResultsHere, we show that high {beta}Alt is associated with an immune-poor context yet is predictive of ICB response in both humans and mice. A high {beta}Alt cancer in which TGF{beta} signaling is compromised generates a TGF{beta} rich, immunosuppressive tumor microenvironment. Accordingly, preclinical modeling showed that TGF{beta} inhibition followed by radiotherapy could convert an immune-poor, ICB-resistant tumor to an immune-rich, ICB-responsive tumor. Mechanistically, TGF{beta} blockade in irradiated tumors activated natural killer cells that were required to recruit lymphocytes to respond to ICB. In support of this, natural killer cell activation signatures were also increased in immune-poor mouse and human tumors that responded to ICB. ConclusionsThese studies suggest that loss of TGF{beta} competency identifies a subset of cold tumors that are candidates for ICB. Our mechanistic studies show that inhibiting TGF{beta} activity converts high {beta}Alt, cold tumors into ICB-responsive tumors via NK cells. Thus, a biomarker consisting of combined TGF{beta}, DNA repair, and immune context signatures provides a means to prospectively identify patients whose cancers may be converted from cold to hot, which could be exploited for therapeutic treatment. O_LIWhat is already known on this topic - For some cancer patients, response to ICB provides durable tumor control. Current biomarkers are insufficient to reliably predict the immunotherapy response for most patients, particularly those whose tumors lack lymphocytic infiltration. C_LIO_LIWhat this study adds - The {beta}Alt score, which reports a DNA damage deficiency caused by lack of TGF{beta} signaling, predicts response to ICB in clinical trial data from IMvigor210 metastatic bladder cancer patients and for melanoma patients. Notably, transcriptomic assessment of the immune context shows that these are immune-poor, so-called "cold" tumors. Preclinical modeling indicates that alleviating TGF{beta} inhibition of NK cells is critical to relieving immunosuppression. C_LIO_LIHow this study might affect research, practice or policy - Our work identifies a novel tumor phenotype consisting of cancer cells that have lost TGF{beta} signaling and gained error-prone DNA repair embedded in a TGF{beta} rich, immune-poor microenvironment, which is conserved across cancer types in humans and among preclinical tumor models. Patients whose immune-poor tumors have high {beta}Alt scores are strong candidates for ICB and radiotherapy combinations that may be further augmented by TGF{beta} inhibition. Hence, the {beta}Alt score can be used to stratify immune-poor cancer patients for optimal therapeutic strategies. C_LI

cancer biology↗

bioRxiv2024

The Neuron as a Direct Data-Driven Controller

In the quest to model neuronal function amidst gaps in physiological data, a promising strategy is to develop a normative theory that interprets neuronal physiology as optimizing a computational objective. This study extends the current normative models, which primarily optimize prediction, by conceptualizing neurons as optimal feedback controllers. We posit that neurons, especially those beyond early sensory areas, act as controllers, steering their environment towards a specific desired state through their output. This environment comprises both synaptically interlinked neurons and external motor sensory feedback loops, enabling neurons to evaluate the effectiveness of their control via synaptic feedback. Utilizing the novel Direct Data-Driven Control (DD-DC) framework, we model neurons as biologically feasible controllers which implicitly identify loop dynamics, infer latent states and optimize control. Our DD-DC neuron model explains various neurophysiological phenomena: the shift from potentiation to depression in Spike-Timing-Dependent Plasticity (STDP) with its asymmetry, the duration and adaptive nature of feedforward and feedback neuronal filters, the imprecision in spike generation under constant stimulation, and the characteristic operational variability and noise in the brain. Our model presents a significant departure from the traditional, feedforward, instant-response McCulloch-Pitts-Rosenblatt neuron, offering a novel and biologically-informed fundamental unit for constructing neural networks.

neuroscience↗

bioRxiv2024

Indirect effects of habitat amount mediated by habitat configuration determine bat diversity at the landscape-scale in Peninsular Malaysia

The impacts of fragmentation are often overlooked in landscape studies investigating how habitat loss impacts biodiversity, despite the casual relationship linking both processes. As habitat loss is the primary cause of fragmentation, understanding the inter-related effects of these twin processes on biodiversity is key to minimise biodiversity loss. Here we assess how habitat amount and configuration influence insectivorous bat assemblages, considering both the direct effects of these processes, as well as the indirect effects of habitat amount mediated through configuration. Bats were acoustically surveyed along independent gradients of habitat amount (forest cover) and configuration (number of patches and edge density) across 28 insular landscapes embedded within a Malaysian hydroelectric reservoir. Using Structural Equation Modelling, the direct and indirect effects of habitat amount were examined on bat sonotype richness, total, and guild-specific activity (forest, edge and open-space foragers). Forest cover had a direct and positive effect on sonotype richness and forest forager activity. The quadratic relationship linking edge density and forest cover was strong and overall positive, but while below 30% of forest cover, increasingly forested landscapes had increasingly high edge densities, the opposite pattern was observed in more forested landscapes. On the other hand, increasingly forested landscapes consistently harboured less patches. Owing to the overall low habitat amount in our landscapes and negative edge effects, the indirect effects of forest cover (mediated through edge density) were therefore negative on sonotype richness, outweighing any positive direct effect. The number of patches had little effect on the bat assemblage, except on total activity which was higher in landscapes harbouring more forest patches. As a result, negative indirect effects of forest cover mediated through number of patches were only observed on total activity. Our results highlight that, in natural settings, habitat amount can hardly be altered without influencing habitat configuration, thereby preventing any independent management of these threats. Minimising habitat loss is therefore essential to balance the associated prevailing negative effects of fragmentation on insectivorous bats across tropical forests.

ecology↗

bioRxiv2023

A human prenatal skin cell atlas reveals immune cell regulation of skin morphogenesis

Human prenatal skin is populated by innate immune cells including macrophages, and whether they act solely in immunity or have additional functions in morphogenesis is unclear. We assembled the first comprehensive multi-omic reference atlas of prenatal human skin (7-16 post-conception weeks), combining single cell and spatial transcriptomic data, to characterise the skins microenvironmental cellular organisation. This revealed that crosstalk between non-immune and immune cells underpins formation of hair follicles, has implications for scarless wound healing, and is critical for skin angiogenesis. We benchmarked a skin organoid model, derived from human embryonic stem (ES) and induced pluripotent stem (iPS) cells, against prenatal and adult skin, demonstrating close recapitulation of the epidermal and dermal skin components during hair follicle development. Notably, the skin organoid lacked immune cells and had markedly diminished endothelial cell heterogeneity and quantity. From our in vivo skin cell atlas data, we found that macrophages and macrophage-derived growth factors play a key role in driving endothelial development prenatally. Indeed, vascular network formation was enhanced following transfer of autologous iPS-derived macrophages into both endothelial cell angiogenesis assays and skin organoid cultures. In summary, innate immune cells moonlight as key players in skin morphogenesis beyond their conventional immune roles, a function they achieve via extensive crosstalk with non-immune cells. Finally, we leveraged our human prenatal skin cell atlas to further our understanding of the pathogenesis of genetic hair and skin disorders.

developmental biology↗

bioRxiv2023

Glycan heterogeneity as a cause of the persistent fraction in HIV-1 neutralization

Neutralizing antibodies (NAbs) to multiple epitopes on the HIV-1 envelope glycoprotein (Env) have been isolated from infected persons. The potency of NAbs is more often measured than the size of the persistent fraction of infectivity at maximum neutralization, which may also influence preventive efficacy by active or passive immunization and the therapeutic outcome of the latter. HIV-1 CZA97.012, a clone of a Clade C isolate, is neutralized to [~]100% by many NAbs. But here NAb PGT151, directed to a fusion-peptide epitope, was shown to leave a persistent fraction of 15%. NAb PGT145, ligating the Env-trimer apex, was less potent but more effective. We sought explanations of the different persistent fractions by depleting pseudoviral populations of the most PGT151- and PGT145-reactive virions. Thereby, neutralization by the non-depleting NAb increased; it decreased by the depleting NAb. Furthermore, depletion by PGT151 increased sensitivity to autologous neutralization by sera from rabbits immunized with soluble native-like CZA97.012 trimer: substantial persistent fractions were reduced. NAbs in these sera target epitopes comprising residue D411 at the V4-{beta}19 transition in a defect of the glycan shield on CZA97.012 Env. Affinity-fractionated soluble native-like CZA97.012 trimer showed commensurate antigenic differences in analyses by ELISA and surface plasmon resonance. We then demonstrated glycan differences between PGT151- and PGT145-purified trimer fractions by mass spectrometry, providing one explanation for the differential antigenicity. These differences were interpreted in relation to a new structure at 3.4-[A] resolution of the soluble CZA97.012 trimer determined by cryo-electron microscopy. PGT151-purified trimer showed a closed conformation, refuting apex opening as the cause of reduced PGT145 binding. The evidence suggests that differences in binding and neutralization after trimer purification or PV depletion with PGT145 or PGT151 are caused by variation in glycosylation, and that some glycan variants confer antigenic heterogeneity through direct effects on antibody contacts, whereas others act allosterically. Author SummaryNeutralizing antibodies block the entry of HIV-1 into cells and protect against HIV-1 infection in animal models. Therefore, a goal of vaccination is to elicit antibodies that potently neutralize most HIV-1 variants. Such antibodies suppress virus levels when given to HIV-1-infected patients. Their potency is often measured as the concentration that gives 50% or 80% neutralization. But higher degrees of neutralization are needed to protect an organism from infection. And for some antibodies a ceiling is reached, so that even with increased concentrations a constant fraction of infectious virus persists. We studied the carbohydrate moieties on the envelope glycoprotein, which is the sole target for neutralizing antibodies, of one HIV-1 isolate of the most widespread subtype, Clade C, prevalent in Africa and Asia. We show how differences in carbohydrates can contribute to persistent infectivity, because distinct carbohydrates fit different antibodies. With a new three-dimensional structure of the entry-mediating protein from the Clade-C isolate, we illustrate that some carbohydrate differences occur exactly where the antibodies bind, whereas others are located elsewhere and can act indirectly. When we combined two neutralizing antibodies the persistent infectivity shrank. Our results reinforce the need for multiple specificities of neutralizing antibodies in prevention and therapy.

immunology↗

bioRxiv2023

TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the /{beta}-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

cell biology↗

bioRxiv2023

Reprogramming method does not impact the neuronal differentiation potential of 16p11.2 deletion patient iPSCs

A major impediment to the actualization of the induced pluripotent stem cell (iPSC)-based personalized medicine revolution is the lack of widely accepted standard operating procedures (SOPs) across different groups and institutions. The various methods employed can include choice of starting materials, reprogramming agents, and culture conditions, with each of these factors hypothesized to influence the reprogramming efficiency and transcriptional identity of iPSCs. As such, we systematically compared iPSC reprogramming procedures using cells derived from the somatic cells of three patients with 16p11.2 deletion syndrome (16p11.2del) and found remarkable similarity among the different methods. FACS analysis revealed that regardless of somatic cell type (fibroblast, lymphocyte, erythroblast), route of reprogramming factor introduction (mRNA, Sendai virus, episome), donor sex, or facility (Rutgers, NYSCF), 16p11.2del patient iPSCs were viable as high purity cultures expressing pluripotency marker proteins. This observation was supported at the transcript level by qPCR analysis, which demonstrated the ability for the iPSCs to differentiate into all three embryonic germ cell lineages after 12 days in culture as embryoid bodies. NGN2-mediated differentiation of these iPSCs produced functional neurons that formed active synaptic networks as revealed by multi-electrode array (MEA) recordings. Importantly, no group-wise comparisons among the reprogramming methods yielded consistent statistically significant differences, indicating that these procedures are equally capable of producing pluripotent stem cells that can efficiently differentiate into mature, functional neurons. This work highlights the utility of these reprogramming methods and supports the use of differentially reprogrammed iPSCs for direct comparative studies of human neurodevelopment.

developmental biology↗

bioRxiv2023

Alpha-tubulin regulation by 5 introns in S. cerevisiae

Across eukaryotic genomes, multiple - and {beta}-tubulin genes require regulation to ensure sufficient production of tubulin heterodimers. Features within these gene families that regulate expression remain underexplored. Here we investigate the role of the 5 intron in regulating -tubulin expression in S. cerevisiae. We find that the intron in the -tubulin, TUB1, promotes -tubulin expression and cell fitness during microtubule stress. The role of the TUB1 intron depends on proximity to the TUB1 promoter and sequence features that are distinct from the intron in the alternative -tubulin isotype, TUB3. These results lead us to perform a screen to identify genes that act with the TUB1 intron. We identified several genes involved in chromatin remodeling, /{beta}-tubulin heterodimer assembly, and the spindle assembly checkpoint. We propose a model where the TUB1 intron promotes expression from the chromosomal locus, and that this may represent a conserved mechanism for tubulin regulation under conditions that require high levels of tubulin production. Article Summary and {beta}-tubulin proteins are encoded by families of genes that must be coordinately regulated to supply the {beta} heterodimers that form microtubules. This study by Wethekam and Moore identifies a role for the early intron in the budding yeast -tubulin, TUB1, in promoting gene function. A genetic screen reveals new tubulin regulators that act through the TUB1 intron. The results establish new layers of -tubulin regulation that may be conserved across eukaryotes.

genetics↗

bioRxiv2023

WebAtlas pipeline for integrated single cell and spatial transcriptomic data

Single cell and spatial transcriptomics illuminate complementary features of tissues. However, online dissemination and exploration of integrated datasets is challenging due to the heterogeneity and scale of data. We introduce the WebAtlas pipeline for user-friendly sharing and interactive navigation of integrated datasets. WebAtlas unifies commonly used atlassing technologies into the cloud-optimised Zarr format and builds on Vitessce to enable remote data navigation. We showcase WebAtlas on the developing human lower limb to cross-query cell types and genes across single cell, sequencing- and imaging-based spatial transcriptomic data.

bioinformatics↗