Biology subjects

Howe, A.

Publications and source records attributed to Howe, A..

16 recordsLinked to original sources

bioRxiv2026

Highly conserved talin isoforms encode distinct mechanical responses and ligand-binding capabilities

Talin is a central mechanosensitive adaptor that links integrins to the actin cytoskeleton and converts mechanical force into biochemical signals. Vertebrates express two highly homologous talin isoforms, talin-1 and talin-2, yet the molecular basis for their nonredundant functions remains poorly understood. Here, we investigate how isoform-specific sequence differences alter the mechanical response and ligand-binding capabilities of the talin rod. Using single-molecule force spectroscopy and biochemical binding assays, we show that talin-2 preserves the overall mechanical architecture of talin-1 but exhibits distinct mechanical responses in key rod regions. Talin-2 R7-R9 exhibits a previously unobserved three-domain cooperative unfolding pathway, while an alternative sequential pathway reveals early unfolding of R9 at substantially lower force than in talin-1. Strikingly, talin-2 R4-R6 has acquired two additional mechanically regulated interactions: a previously unrecognized vinculin-binding site in R5 and an A-kinase anchoring protein (AKAP) region within R5-R6 that recruits protein kinase A (PKA). Together with the conserved R9 AKAP site, this establishes two spatially separated, mechanically regulated PKA-anchoring regions within the talin-2 scaffold. These findings establish that isoform-specific sequence divergence not only reshapes the mechanical response of the talin scaffold but also expands its repertoire of force-regulated interactions, providing a mechanism for the assembly of different structural and signaling complexes in an isoform-dependent manner.

biophysics↗

bioRxiv2026

The ecological context of enzymatic variation

Among the challenges in understanding microbial ecosystems is the presence of physics at vastly different scales. Reactions, catalyzed by enzymes but regulated at the level of the cell, propel the flux of carbon and nitrogen through our atmosphere. Compounding this is the presence of pervasive enzyme sequence variation; this variation has been shown to contain coevolving modes of amino acids which encode the enzyme's evolutionary history. In this work, we take steps towards bridging the gap between the enzymatic variation present in an ecosystem and its subsequent activity. We employ the reduction of nitrate by NarG as a model system, which acts as an essential step in the nitrogen cycle by mediating both the return of di-nitrogen to the atmosphere and the assimilation of nitrate into biomass. Considering both metagenomic reconstructions as well as functional data from soil nitrate reducers, we find that sequence variants of enzymes obey predictable responses to environmental fluctuations. That is, while prior community-level metagenomic studies have characterized the response of bacterial strains to the environment, our study provides an enzyme variant level sequence-to-response map. Further, we demonstrate that a simple statistical model can predict the organismal phenotype from variant sequence; in soil samples not originally seen by that model, the prediction of a variant's reduction rate correlates with how much cells with that variant grow in abundance.

biophysics↗

bioRxiv2026

Could microbes be the architects of improved soil structure under Miscanthus x giganteus?

The perennial grass Miscanthus x giganteus (miscanthus) offers a sustainable alternative to traditional biomass feedstocks while improving key soil health parameters, including aggregation. Aggregate stability results from dynamic soil-plant-microbe interactions, yet the relative importance of each factor remains an active research question. Building on previous observations that miscanthus alters soil structure to improve water-holding capacity and aggregate stability, we characterized the communities of soil bacteria and arbuscular mycorrhizal fungi (AMF) across three sites in Iowa, USA, comparing miscanthus to annual maize (Zea mays L.) and non-cropped perennial turfgrass (Poa spp.). We examined whether microbiomes co-varied with soil aggregation and, if so, whether plant cover identity or life history categorization better explained the observed patterns. Bacterial and AMF communities varied across sites and plant types, with signals that life history and plant cover identity both mattered. Aggregate stability aligned with a perennial-annual divergence in microbial beta diversity, while finer-scale differences in community composition and network structure were plant-specific. Soils under perennial plants were enriched in microbial groups positively correlated with aggregate stability; we identified 61 bacterial and 8 AMF "architect" taxa for future study. Within- and cross-kingdom co-occurrence network analysis revealed greater complexity under perennial plants: 1.9-fold more network links in miscanthus bacteria-bacteria networks than in maize, and 1.7-fold more in turfgrass AMF-AMF networks. Miscanthus fundamentally shapes microbial interactions, particularly among bacteria, relating to improved soil physical structure. Understanding these soil-plant-microbe feedbacks advances the development of biomass feedstocks with a portfolio of soil health benefits for next-generation biofuels and bioproducts. IMPORTANCEPerennial bioenergy crops can provide the raw material for biofuels and bioproducts while simultaneously improving soil health. Miscanthus x giganteus (miscanthus) efficiently stabilizes soil aggregates, potentially leading to higher water retention and erosion resistance. Understanding the microbial contributions to these outcomes is key to building resilient, sustainable bioenergy systems. This study highlights the connections between communities of soil microbes--bacteria and arbuscular mycorrhizal fungi--across three sites and three plant covers, including miscanthus, maize, and turfgrass. We identify a guild of potential "microbial architects" linked to soil aggregation and show more interconnected microbial networks under the perennial plant covers compared to annual maize. These insights shed light on the interactions between soil biological communities and soil physical and chemical properties. More broadly, the results may inform efforts to harness plant-associated microbiomes for sustainable biomass production.

microbiology↗

bioRxiv2026

Metabolically engineered oilcane reshapes rhizosphere microbial guilds while preserving broad functional capacity

Metabolic engineering of crops can redirect host carbon flux, but its consequences for microbiomes remain unclear. Here, we show that engineering oilcane for triacylglycerol (TAG) accumulation reshapes rhizosphere microbial guilds across greenhouse and field environments while preserving functional capacity. Using 36 rhizosphere metagenomes from wild-type sugarcane and engineered oilcane accessions, we reconstructed metagenome-assembled genomes and linked community turnover with shifts in functional potential. Oilcane rhizospheres exhibited taxonomic restructuring relative to wild-type plants, driven primarily by turnover rather than nestedness and marked by genotype-dependent replacement of microbial guilds. These patterns were strongest in accession 1566 and amplified under field conditions. Despite these compositional shifts, broad patterns of functional potential remained similarly distributed, whereas pathway-level differences were evident in energy production and conversion, lipid transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, and signal transduction. These findings extend evaluation of engineered crops beyond host traits alone to include microbiome-scale responses.

microbiology↗

bioRxiv2025

Dairy manure analysis reveals significant risk of Antibiotic resistance from Extracellular DNA in Manure storage Pit

AbstractDairy manure pit storage systems are significant reservoirs for antimicrobial resistance genes (ARGs). These genes occur in both intracellular DNA (iDNA) and extracellular DNA (exDNA), but their distribution across these categories in fresh (loafing pen surface) and pit-stored dairy manure has not been previously characterized. To address this gap, we quantified the abundance of six ARGs (tetG, tetM, tetX- tetracyline, sul1-sulfonamide, and ermB-macrolide) and three mobile genetic elements (MGEs) (intI1, intI2, and intI3) in iDNA and exDNA extracted from fresh and pit-stored manure collected at a dairy farm in Iowa. While total DNA yields were lower in pit-stored relative to fresh manure samples, exDNA-to-iDNA ratios were significantly elevated across all genes (p<0.001), indicating relative enrichment of exDNA during storage. Notably, tetM exhibited a higher free (unattached) to bound (surface-attached) exDNA ratio in pit samples, which suggested an increased potential for gene transfer in the pit. Correlation network analysis revealed similar numbers of strong ARG-MGE associations in pit and fresh exDNA, but lower interconnectivity in pit exDNA. Merging fresh and pit datasets showed wider ARG-MGE associations: intI1 and intI3 strongly co-occurred with tetracyclines and macrolide resistance in iDNA, while sul1 correlated with MGEs only in the exDNA network. Microbial community profiling showed similar taxa in exDNA across manure types, while iDNA communities diverged significantly. This result could support that exDNA is relatively stable over time and in varying environments, and that iDNA is relatively more reflective of selective pressures. Overall, our results highlight exDNA as a critical but overlooked reservoir of resistance determinants, warranting further investigation and targeted management strategies in dairy systems. ImportanceTo date, extracellular DNA (exDNA) has been shown to contribute to the spread of antibiotic resistance genes (ARGs) in the environment; however, few studies have evaluated its enrichment in dairy pit-stored manure systems. This study demonstrates that dairy manure pits concentrate exDNA during dairy manure storage and serve as a reservoir for ARGs, along with mobile genetic elements that can facilitate subsequent gene transfer. The results of this study are a strong rationale for further investigation and targeted management strategies of exDNA in manure pits.

microbiology↗

bioRxiv2025

Substrate recognition by human separase

The protein complex cohesin encircles the sister chromatids in early mitosis1. At anaphase onset, sister separation is triggered by cleavage of the cohesin subunit SCC1/RAD21 by the cysteine protease separase2-5. SCC1 contains two cleavage sites, where cleavage is stimulated by SCC1 phosphorylation5,6. The molecular mechanisms of substrate recognition and cleavage are only partly understood7. Here, we determined a series of cryoEM structures of human separase in apo-or substrate-bound forms that, together with biochemical analysis, provide novel insights into the regulation of separase cleavage activity. We verify the first SCC1 cleavage site and reassign the second site. We show that multiple substrates, including separase autocleavage sites8,9 and the two SCC1 cleavage sites, interact with several docking sites in separase, including four phosphate-binding sites. We also describe the structural basis of the interaction between the cohesin subunit SA1/A2 and separase, which promotes cleavage at the second site in SCC1. Finally, using cross-linking mass spectrometry and cryoEM, we propose a model of how cohesin is targeted by human separase. Our work provides an extensive functional and structural framework that explains one of the most fundamental events in cell division.

biochemistry↗

bioRxiv2023

Molecular architecture and conservation of an immature human endogenous retrovirus

A significant part of the human genome consists of endogenous retroviruses sequences. Human endogenous retrovirus K (HERV-K) is the most recently acquired endogenous retrovirus, is activated and expressed in many cancers and amyotrophic lateral sclerosis and possibly contributes to the aging process. To understand the molecular architecture of endogenous retroviruses, we determined the structure of immature HERV-K from native virus-like particles (VLPs) using cryo-electron tomography and subtomogram averaging (cryoET STA). The HERV-K VLPs show a greater distance between the viral membrane and immature capsid lattice, correlating with the presence of additional peptides, SP1 and p15, between the capsid (CA) and matrix (MA) proteins compared to the other retroviruses. The resulting cryoET STA map of the immature HERV-K capsid at 3.2 [A] resolution shows a hexamer unit oligomerized through a 6-helix bundle which is further stabilized by a small molecule in the same way as the IP6 in immature HIV-1 capsid. The HERV-K immature CA hexamer assembles into the immature lattice via highly conserved dimmer and trimer interfaces, whose interactions were further detailed through all-atom molecular dynamics simulations and supported by mutational studies. A large conformational change mediated by the flexible linker between the N-terminal and the C-terminal domains of CA occurs between the immature and the mature HERV-K capsid protein, analogous to HIV-1. Comparison between HERV-K and other retroviral immature capsid structures reveals a highly conserved mechanism for the assembly and maturation of retroviruses across genera and evolutionary time.

microbiology↗

bioRxiv2023

ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface

Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

microbiology↗

bioRxiv2022

Ultrastructural characterization of a viral RNA and G-protein containing, membranous organelle formed in respiratory syncytial virus infected cells

Withdrawal statementThe authors have withdrawn their manuscript because since deposition of this preprint, they have become aware of the possibility that the findings reported may be an artefact. Carter et al. (J Struct Biol 2018 Jan;201(1):15-25. doi: 10.1016/j.jsb.2017.10.009) have reported that multilamellar bodies exhibit autofluorescence under cryogenic conditions. Thus, there is a possibility that stress induced by virus infection may stimulate formation of the features reported in this article, and autofluorescence, rather than specific labelling led them to identify the puncta as compartments containing RSV proteins or genomes. They are working to address these issues. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

microbiology↗

bioRxiv2021

Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples

Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed a method entitled the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a system implementing high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant genes representing a full-spectrum of antibiotic resistance classes commonly seen in environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within environmental samples originated from manure, soil, and animal feces, in addition to a mock-community used as a control to test performance. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate the obstacles that many researchers in this area currently face.

microbiology↗

bioRxiv2021

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

microbiology↗

bioRxiv2020

Land-use and forest floor explain prokaryotic metacommunity structuring and spatial turnover in Amazonian forest-to-pasture conversion areas

Advancing extensive cattle production shifts the forest landscape and is considered one of the main drivers against biodiversity conservation in the Brazilian Amazonia. Considering soil as an ecosystem it becomes vital to identify the effects of land-use changes on soil microbial communities, structure, as well as its ecological functions and services. Herein, we explored relationships between land-use, soil types and forest floor (i.e., association between litter, root layer and bulk soil) on the prokaryotic metacommunity structuring in the Western Amazonia. Sites under high anthropogenic pressure were evaluated along a gradient of {+/-} 800 km. Prokaryotic metacommunity are synergistically affected by soil types and land-use systems. Especially, the gradient of soil fertility and land-use shapes the structuring of the metacommunity and determines its composition. Forest-to-pasture conversion increases alpha, beta, and gamma diversities when considering only the prokaryotes from the bulk soil. Beta diversity was significantly higher in all forests when the litter and root layer were taken into account with the bulk soil. Our argumentation is that the forest floor harbors a prokaryotic metacommunity that adds at the regional scale of diversity a spatial turnover hitherto underestimated. Our findings highlight the risks of biodiversity loss and, consequently, the soil microbial diversity maintenance in tropical forests.

microbiology↗

bioRxiv2020

SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

microbiology↗

bioRxiv2020

Identification and evolution of Cas9 tracrRNAs

Cas9 trans-activating CRISPR RNAs (tracrRNAs) form distinct structures essential for target recognition and cleavage and dictate exchangeability between orthologous proteins. As non-coding RNAs that are often apart from the CRISPR array, their identification can be arduous. In this paper, a new bioinformatic method for the detection of Cas9 tracrRNAs is presented. The approach utilizes a co-variance model (CM) based on both sequence homology and predicted secondary structure to locate tracrRNAs. This method predicts a tracrRNA for 98% of CRISPR-Cas9 systems identified by us. The identified tracrRNAs exhibit wide variation in sequence identity, however, CM analyses allow 94.7% to be categorized into just 10 related groups. Finally, association between Cas9 amino acid sequence-based phylogeny and tracrRNA secondary structure is evaluated, revealing strong evidence that secondary structure is evolutionarily conserved among Cas9 lineages. Altogether, our findings provide insight into Cas9 tracrRNA evolution and efforts to characterize the tracrRNA of new Cas9 systems.

evolutionary biology↗

bioRxiv2020

Statistical model integrating interactions into genotype-phenotype association mapping: an application to reveal 3D-genetic basis underlying Autism

Biological interactions are prevalent in the functioning organisms. Correspondingly, statistical geneticists developed various models to identify genetic interactions through genotype-phenotype association mapping. The current standard protocols in practice test single variants or single regions (that contain multiple local variants) sequentially along the genome, followed by functional annotations that involve various aspects including interactions. The testing of genetic interactions upfront is rare in practice due to the burden of testing a huge number of combinations, which lead to the multiple-test problem and the risk of overfitting. In this work, we developed interaction-integrated linear mixed model (ILMM), a novel model that integrates a priori knowledge into linear mixed models. ILMM enables statistical integration of genetic interactions upfront and overcomes the problems associated with combination searching. Three dimensional (3D) genomic interactions assessed by Hi-C experiments have led to unprecedented biological discoveries. However, the contribution of 3D genomic interactions to the genetic basis of complex diseases has yet to be quantified. Using 3D interacting regions as a priori information, we conducted both simulations and real data analysis to test ILMM. By applying ILMM to whole genome sequencing data for Autism Spectrum Disorders, or ASD (MSSNG) and transcriptome sequencing data (GTEx), we revealed the 3D-genetic basis of ASD and 3D-eQTLs for a substantial proportion of gene expression in brain tissues. Moreover, we have revealed a potential mechanism involving distal regulation between FOXP2 and DNMT3A conferring the risk of ASD. Software is freely available in our GitHub: https://github.com/theLongLab/Jawamix5

genetics↗

bioRxiv2020

MetaFunPrimer: primer design for targeting genes observed in metagenomes

High throughput primer design is needed to simultaneously design primers for multiple genes of interest, such as a group of functional genes. We have developed MetaFunPrimer, a bioinformatic pipeline to design primer targets for genes of interests, with a prioritization based on ranking the presence of gene targets in references, such as metagenomes. MetaFunPrimer takes inputs of protein and nucleotide sequences for gene targets of interest accompanied by a set of reference metagenomes or genomes for determining genes of interest. Its output is a set of primers that may be used to amplify genes of interest. To demonstrate the usage and benefits of MetaFunPrimer, a total of 78 HT-qPCR primer pairs were designed to target observed ammonia monooxygenase subunit A (amoA) genes of ammonia-oxidizing bacteria (AOB) in 1,550 soil metagenomes. We demonstrate that these primers can significantly improve targeting of amoA-AOB genes in soil metagenomes compared to previously published primers. IMPORTANCEAmplification-based gene characterization allows for sensitive and specific quantification of functional genes. Often, there is a large diversity of genes represented for a function of interest, and multiple primers may be necessary to target associated genes. Current primer design tools are limited to designing primers for only a few genes of interest. MetaFunPrimer allows for high throughput primer design for functional genes of interest and also allows for ranking gene targets by their presence and abundance in environmental datasets. This tool enables high throughput qPCR approaches for characterizing functional genes.

bioinformatics↗