Biology subjects

Walker, D.

Publications and source records attributed to Walker, D..

At least 19 recordsLinked to original sources

bioRxiv2026

SOX9-mediated G1 elongation confers reserve stem cell-associated injury resistance in human intestinal stem cells

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

cell biology↗

bioRxiv2026

Expression and Engineering of Conductive Cytochrome Nanowires

Electrically conductive protein nanowires produced by metal-reducing bacteria have attracted interest as sustainable electronic materials, but their study and engineering have been limited by difficulties in expression, purification, and genetic manipulation. Here we establish Shewanella oneidensis as a heterologous host for production of Geobacter sulfurreducens OmcZ nanowires and progress a complementary in vitro assembly strategy that yields highly pure nanowire preparations. This platform enabled systematic engineering of OmcZ, revealing extensive tolerance to sequence variation and faciliating the design of enhanced-conductivity variants. Guided by comparative analysis of environmental OmcZ homologs, we generated a chimeric nanowire, OmcZ+, that exhibited a [~]3.5-fold increase in bulk conductivity while retaining the overall structure of the parent nanowire. Cryo-electron microscopy revealed unexpected architectural plasticity in OmcZ+ wires, including non-linear dendritic and pentameric assemblies mediated by the solvent-exposed heme VII, suggesting previously unrecognized modes of cytochrome nanowire organization. Incorporation of engineered OmcZ variants into water evaporation-induced electricity generators produced power densities up to [~]25.3 W cm-{superscript 2} and enabled high-performance operation in saline environments, including seawater and human sweat. Together, these results establish a versatile platform for the production, structural analysis, and engineering of cytochrome nanowires, providing a foundation for the development of programmable biological electronic materials.

bioengineering↗

bioRxiv2026

Exploring the diversity and community structure of the Testudines fecal mycobiome

Most gut microbiome studies have focused on bacteria, leaving a knowledge gap regarding gut associated fungi. We assessed fungal diversity in the gastrointestinal tract of the reptilian order Testudines (turtles and tortoises) using samples from 6 families, 19 genera, and 27 species. A highly diverse community affiliated with 17 phyla and 157 orders was encountered, with four phyla (Neocallimastigomycota, Chytridiomycota, Ascomycota, and Basidiomycota) representing 89.13% of the community. Neocallimastigomycota was identified in host families Testudinidae (land tortoises), Chelidae, Chelydridae, Emydidae, Geoemydidae, and Kinosternidae, with higher relative abundances in Testudinidae (40.18{+/-}37.97%) compared to all other families combined (2.71{+/-}4.04%). Neocallimastigomycota sequences were mostly affiliated with orders Testudinimycetales in the host family Testudinidae and Neocallimastigales in other host families. Chytridiomycota was identified in all host families, but was more ubiquitous and abundant in Kinosternidiae (45.17{+/-}34.12%), and exhibited a high level of variability across samples. Dikarya communities were highly diverse, with 108 orders identified, and, similar to Chytridiomoycota, exhibited a highly stochastic distribution pattern. Representatives of multiple yet-uncultured phyla (Candidatus "Algovoracomycota", "Sedimentomastigomycota", "Tartumycota" and "Cantoromastigomycota") were identified, as well as eight novel orders in Chytridiomycota and Rozellomycota. Deterministic selection shaped community assembly in the host family Testudinidae, while the process was more stochastic in other host families. Distinct community structure was driven by differences in abundance and identity of the Neocallimastigomycota when comparing Testudinidae to. Our results describe a diverse and dynamic fungal community, shaped by the co-occurrence of autochthonous (resident) and transient (allochthonous) members of the gut microbiome. ImportanceFungi are known to inhabit the gastrointestinal tract (GIT) of humans and mammals. However, information on the fungal community in the GIT of reptiles is relatively sparse. We investigated the diversity and community structure of fungi in the reptilian order Testudines. We conducted a culture-independent diversity survey on fecal samples obtained from 27 different host species. We identify representatives of 17 fungal phyla. As well, we demonstrate that the anaerobic gut fungi (phylum Neocallimastigomycota) are not restricted to the family Testudinidae (land tortoises) as previously suggested, but could successfully colonize and inhabit all other testudines families, including those exhibiting a predominantly omnivorous or carnivorous lifestyles. In addition, we expand on the known fungal diversity by identifying additional representatives of multiple recently described yet-uncultured phyla, and describe multiple novel orders and classes within existing phyla. Collectively, this effort adds to the growing body of knowledge of mycobiomes in underexplored animal hosts.

microbiology↗

bioRxiv2026

Energy-precision trade-off in mitotic oscillators revealed by ATP modulation in artificial cells

The temporal precision of biochemical oscillators is fundamentally constrained by the energy dissipated to suppress molecular fluctuations, a widely predicted trade-off governing information processing across biology and physics, from molecular motors to kinetic proofreading to computing. Yet, experimental validation in complex biological oscillators remains elusive due to challenges of systematically modulating energy while quantifying stochastic dynamics across large ensembles. Here, we establish a high-throughput droplet-microfluidics platform to reconstitute mitotic oscillations from Xenopus laevis egg extracts within thousands of sub-nanoliter compartments. By precisely tuning ATP across a broad free-energy landscape and developing an analytical framework that decouples intrinsic phase diffusion from quenched period heterogeneity, we uncover a hidden trade-off linking metabolic budget, oscillation speed, and precision. While speed peaks non-monotonically near physiological ATP levels and declines toward both high and low bifurcation limits, precision increases monotonically with energy. These findings provide direct experimental evidence that mitotic timing is actively shaped by energy budgets. Intriguingly, embryonic cell cycles are not optimized for maximum fidelity, but for a metabolic compromise maintaining just enough coherence for synchronous yet rapid divisions, placing the endogenous ATP budget near an energetic optimum balancing speed and accuracy. Our integrated artificial-cell and analytical strategy provides a generalizable framework for mapping thermodynamic limits in non-equilibrium biological dynamics.

biophysics↗

bioRxiv2025

A Novel Prognostic Metric Resolves the MYCN enigma in Silico and Points to a Biosynthetic Regulatory Shift Driven by MYCN Amplification in Neuroblastoma

Neuroblastoma (NB) is the most common extra-cranial solid tumour in children. Although MYCN amplification is typically indicative of a poor prognosis, the expression level of MYCN has a non-monotonic relationship with the clinical outcome. This paper proposes an explanation for this phenomenon, which is called the MYCN enigma in the literature. The p53/MYCN metric, which is a measure of the p53 protein level relative to the MYCN protein level in a NB tumour, is the key concept. Our hypothesis is that the metric has a positive relationship with the outcome of a patient. It is presented as a binary classification system, where the prognosis is favourable when p53/MYCN>3.5. The mathematical model presented in this paper describes the dynamics between MYCN, p53, ARF, and MDM2; their genes; and their mRNA transcripts. Simulations were carried out by solving the model numerically in a series of initial value problems. The results are aligned with a list of clinical and experimental observations. We extracted association rules with the Apriori algorithm and explored the parametric space stochastically. Assuming that MYCN enables biogenesis in NB tumours, our results support the prediction that MYCN amplification shifts the balance towards producing MYCN. When a wild-type tumour is deficient in MYCN, stress responses may restore biogenesis in general and preferentially produce MYCN in a negative feedback loop. Another prediction is that MYCN - amplified tumours, without treatment, require hard-to-attain biosynthetic rates and stress responses to achieve good outcomes.

systems biology↗

bioRxiv2025

Receptor-substrate competition for the TonB homologue FusB suggests a model for ferredoxin import in Pectobacterium spp.

TonB-dependent uptake systems of Gram-negative bacterial pathogens constitute prominent virulence factors, allowing nutrient acquisition, primarily siderophore-bound iron, to cross the highly impermeable outer membrane (OM). Remarkably, the ferredoxin uptake system (Fus) of Pectobacteriaceae, a group of soft rot-inducing plant pathogens, imports an entire folded host protein into the periplasm and extracts its bound iron for growth. The inner membrane protein FusB, a TonB homologue, plays two roles in facilitating ferredoxin import. First, like other TonBs, it remodels the globular plug domain obstructing the lumen of the OM receptor FusA to allow ferredoxin passage. Unusually for a TonB protein, FusB then interacts directly with the FusA-bound ferredoxin substrate to facilitate its transport into the periplasm. In this work, we describe structures of FusB-ferredoxin and homodimeric FusB complexes and determine the key features of the binding interfaces formed by FusB with FusA and ferredoxin. We postulate that under resting conditions FusB exists a homodimer, stabilised by an intermolecular R241-D322 salt bridge. The homodimer dissociates when the "FusB-box" of FusA outcompetes one protomer, and FusA D53 displaces FusB D322. Upon ferredoxin binding, FusB undergoes a structural rearrangement, expanding its {beta}-sheet from three to four strands. In agreement with the proposed sequence of events, ferredoxin binding displaces the receptor (FusA) from FusB with Arg241 forming an intramolecular salt bridge with Asp322 to stabilise the newly formed {beta}-hairpin of FusB. We propose a mechanistic model for ferredoxin import in which FusB Arg241 acts as a molecular switch, and two distinct regions function as interaction hotspots.

biophysics↗

bioRxiv2025

FOXO1 inhibition and FADD knockdown have opposing effects on anticancer drug-induced cytotoxicity and p21 expression in osteosarcoma

Forkhead box class O1 (FOXO1) and fas-associated death domain (FADD) regulate cell death pathways and homeostatic processes such as cell cycle progression and apoptosis. FADD phosphorylation promotes nuclear localization of FOXO1 and FOXO1 regulates FADD expression. Therefore, it is plausible that FOXO1 and FADD have synergistic or antagonistic effects on cell cycle regulation and the response to anticancer drug treatment in cancer cells. In the present study we report that AS1842856-mediated inhibition of FOXO1 reverses anticancer drug-induced cytotoxicity while FADD knockdown increases anticancer drug-induced cytotoxicity in osteosarcoma (OS). Reversed anticancer drug-induced cytotoxicity was accompanied with G2/M cell cycle arrest and increased expression of p21. The anti-cancer function of FOXO1 was further supported by the observation that OS cells that express higher basal levels of FOXO1 had increased sensitivity to camptothecin-induced cytotoxicity. FADD knockdown reversed FOXO1 inhibition-induced increase in p21 expression. The results presented in this study indicate that FOXO1 has a tumor suppressor function while FADD has a tumor promoting function in OS following anticancer drug treatment. The experimental approach used in this investigation also indicates that FADD antagonists the effect of FOXO1 on p21 expression in OS.

cancer biology↗

bioRxiv2025

Identification of a BACH1 lung cancer signature: A novel tool for understanding BACH1 biology and identifying new inhibitors.

The transcription factor BACH1 is a transcriptional repressor with a central role in regulating oxidative stress and anti-inflammatory pathways, emerging as a promising therapeutic target for multiple conditions, including neoplastic malignancies, neurodegenerative disorders, ischemia-reperfusion injuries and sickle cell disease. In the field of cancer BACH1 has gained significant attention, with BACH1 overexpression correlating with poor prognosis and metastasis across various cancer types; however, despite this increasing relevance of BACH1, no universal pro-metastatic mechanism or transcriptional signature for BACH1 has been identified which is a major limitation for this growing field. To address this, we performed RNA-Seq coupled with ChIP-Seq in BACH1-proficient and BACH1-deficient lung cancer cells, identifying a set of common BACH1 directly regulated genes, which we thoroughly validated in a large panel of cancer cells. This novel lung cancer BACH1 transcriptional signature is highly sensitive and specific to BACH1 perturbations (both genetic and pharmacological) and does not respond to NRF2 modulation, underscoring its specificity. This signature not only represents a robust surrogate for BACH1 activity, but we also provide evidence of its potential value as a tool to i) identify novel BACH1 inhibitors, and ii) provide insights into BACH1s pro-metastatic role.

cancer biology↗

bioRxiv2024

Absolute abundance unveils Basidiobolus as a cross-domain bridge indirectly bolstering gut microbiome homeostasis

The host microbiome is integral to metabolism, immune function, and resilience against pathogens. However, reliance on relative abundance (RA) to estimate host-associated microbiomes introduces compositional biases, while limited tools for absolute abundance (AA) quantification hinder broader applications. To address these challenges, we developed DspikeIn (https://github.com/mghotbi/DspikeIn), an R package paired with a versatile wet-lab methodology for AA quantification. Using RA and AA to compare core microbiome distributions across herpetofauna orders and their natural histories revealed starkly distinct results, driven by aggregate effects, including inherited compositional biases in RA and additional multifactorial influences. Focusing on two closely related Desmognathus species demonstrated that AA quantification enhanced resolution in differential abundance analyses and minimized false discovery rates (FDR) when identifying enriched taxa in their gut microbiomes. Keystone taxa identified through network associations also differed between RA and AA data. For example, Lactococcus and Cetobacterium were core members in Anura and Caudata, while Basidiobolus and Mortierella were core to Chelonia and Squamata, facilitating host adaptation to diverse environments, insights undetectable with RA data. AA-based network analysis further revealed that removing the Basidiobolus subnetwork increased negative interactions, highlighting its role in promoting gut homeostasis through cross-domain connectivity. Despite low redundancy, the Basidiobolus node exhibited high betweenness, efficiency, and degree, serving as a critical bridge linking disconnected nodes or modules and indirectly supporting microbiome stability, consistent with Burts structural hole theory. DspikeIn represents a transformative tool for microbiome research, enabling the transition from RA to AA quantification and delivering more accurate, consistent, and comparable results across studies. Graphical abstract DspikeIn cheatsheet O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/630554v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@150900forg.highwire.dtl.DTLVardef@21cd90org.highwire.dtl.DTLVardef@13ead1borg.highwire.dtl.DTLVardef@1d6fa84_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

LC8 enhances 53BP1 foci through heterogeneous bridging of 53BP1 oligomers

53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.

biophysics↗

bioRxiv2024

Substrate Rigidity Modulates Segmentation Clock Dynamics in Isolated Presomitic Mesoderm Cells

The segmentation clock, a genetic oscillator in the presomitic mesoderm (PSM), is known to be influenced by biochemical signals, yet its potential regulation by mechanical cues remains unclear. The complex PSM microenvironment has made it challenging to isolate the effects of mechanical signals on clock behavior. Here we investigated how mechanical stimuli affect clock oscillations by culturing zebrafish PSM cells on bioengineered elastic substrates (PDMS micropost arrays) with tunable rigidities ranging from 0.6 to 1,200 kPa. We observed an inverse sigmoidal relationship between substrate rigidity and the percentage of oscillating PSM cells, with a switching rigidity threshold between 3-6 kPa. The oscillation periods of oscillating PSM cells showed a consistently broad distribution across the substrate rigidity conditions tested. Moreover, these oscillatory PSM cells exhibited distinct biophysical properties, including reduced motility, contractility, and sustained circularity, compared to non-oscillating ones. These findings highlight a role of cell-substrate interactions in regulating segmentation clock behavior, providing insights into the mechanobiology of somitogenesis. HighlightsO_LIOscillatory behaviors of single PSM cells respond to substrate rigidity in a switch-like manner, transitioning from an oscillatory state to a quiescent state at a critical rigidity threshold between 2.9 kPa and 6 kPa. C_LIO_LIIncreased substrate rigidity significantly suppresses the percentage of oscillating PSM cells, while oscillation period and cycle number show no consistent rigidity-dependent trend. C_LIO_LINon-oscillating PSM cells exhibit distinct biophysical properties compared to oscillating ones, including reduced circularity, a polarized and elongated morphology, greater motility, and increased contractility. C_LIO_LIAggregates of PSM cells exhibit similar trends in response to substrate rigidity changes, except for increased oscillation percentages across different rigidity conditions, suggesting a potential interplay between cell-cell and cell-matrix communications in influencing clock oscillation behavior. C_LI

developmental biology↗

bioRxiv2024

Efficient and reproducible generation of human iPSC-derived cardiomyocytes using a stirred bioreactor

In the last decade human iPSC-derived cardiomyocytes (hiPSC-CMs) proved to be valuable for cardiac disease modeling and cardiac regeneration, yet challenges with scale, quality, inter-batch consistency, and cryopreservation remain, reducing experimental reproducibility and limiting clinical translation. Here, we report a robust cardiac differentiation protocol that uses Wnt modulation and a stirred suspension bioreactor to produce on average 124 million hiPSC-CMs with >90% purity using a variety of hiPSC lines (19 differentiations; 10 iPSC lines). After controlled freeze and thaw, bioreactor-derived CMs (bCMs) showed high viability (>90%), interbatch reproducibility in cellular morphology, function, drug response and ventricular identity, which was further supported by single cell transcriptomes. bCMs on microcontact printed substrates revealed a higher degree of sarcomere maturation and viability during long-term culture compared to monolayer-derived CMs (mCMs). Moreover, functional investigation of bCMs in 3D engineered heart tissues showed earlier and stronger force production during long-term culture, and robust pacing capture up to 4 Hz when compared to mCMs. bCMs derived from this differentiation protocol will expand the applications of hiPSC-CMs by providing a reproducible, scalable, and resource efficient method to generate cardiac cells with well-characterized structural and functional properties superior to standard mCMs.

cell biology↗

bioRxiv2024

Single cell RNA-sequencing of Ewing sarcoma tumors demonstrates transcriptional heterogeneity and clonal evolution.

Ewing sarcoma is the second most common bone cancer in children, accounting for 2% of pediatric cancer diagnoses. Patients who present with metastatic disease at the time of diagnosis have a dismal prognosis, compared to the >70% 5-year survival of those with localized disease. Here, we utilized single cell RNA-sequencing to characterize the transcriptional landscape of primary Ewing sarcoma tumors and surrounding tumor microenvironment (TME). Copy-number analysis identified subclonal evolution within patients prior to treatment. Primary tumor samples demonstrate a heterogenous transcriptional landscape with several conserved gene expression programs, including those composed of genes related to proliferation and EWS targets. Single cell RNA-sequencing and immunofluorescence of circulating tumor cells at the time of diagnosis identified TSPAN8 as a novel therapeutic target.

cancer biology↗

bioRxiv2024

Stratifying macrophages based on their infectious burden identifies novel host targets for intervention during Crohns disease associated adherent-invasive Escherichia coli infection

Bacterial infection is a dynamic process resulting in a heterogenous population of infected and uninfected cells. These cells respond differently based on their bacterial load and duration of infection. In the case of infection of macrophages with Crohns disease (CD) associated adherent-invasive Escherichia coli (AIEC), understanding the drivers of pathogen success may allow targeting of cells where AIEC replicate to high levels. Here we show that stratifying immune cells based on their bacterial load identifies novel pathways and therapeutic targets not previously associated with AIEC when using a traditional homogeneous infected population approach. Using flow cytometry-based cell sorting we stratified cells into those with low or high intracellular pathogen loads, or those which were bystanders to infection. Immune cells transcriptomics revealed a diverse response to the varying levels of infection while pathway analysis identified novel intervention targets that were directly related to increasing intracellular AIEC numbers. Chemical inhibition of identified targets reduced AIEC intracellular replication or inhibited secretion of tumour necrosis factor alpha (TNF), a key cytokine associated with AIEC infection. Our results have identified new avenues of intervention in AIEC infection that may also be applicable to CD through the repurposing of already available inhibitors. Additionally, they highlight the applicability of immune cell stratification post-infection as an effective approach for the study of microbial pathogens.

microbiology↗

bioRxiv2023

p75NTR neurotrophin receptor function is redundant for development, growth and fertility in the rat.

The p75NTR neurotrophin receptor has positive and negative roles regulating cell survival in the nervous system. Unambiguous interpretation of p75NTR function in vivo has been complicated, however, by residual expression of alternate forms of p75NTR protein in initial p75NTR knock-out mouse models. As rats are the preferred rodent for studying brain and behaviour, and to simplify interpretation of the knock-out phenotype, we report here the generation of a mutant rat devoid of the p75NTR protein. TALEN-mediated recombination in embryonic stem cells (ESCs) was used to flank exon 2 of p75NTR with Lox P sites and produce transgenic rats carrying either un-recombined floxed p75NTREx2-fl, or recombined, exon-2 deleted p75NTREx2-{Delta} alleles. Crossing p75NTREx2-fl rats with a Cre-deleter strain efficiently removed exon 2 in vivo. Excision of exon 2 causes a frameshift after p75NTR Gly23 and eliminated p75NTR protein expression. Rats lacking p75NTR were healthy, fertile, and histological analysis did not reveal significant changes in cellular density or overall structure in their brains. Thus, p75NTR function appears largely dispensable for normal development, growth and basal homeostasis in the rat. The availability of constitutive and conditional p75NTREx2-{Delta} rats should, however, provide new opportunities to investigate specific roles of p75NTR upon injury and during regeneration.

genetics↗

bioRxiv2023

Herptile gut microbiomes: a natural system to study multi-kingdom interactions between filamentous fungi and bacteria

Reptiles and amphibians (herptiles) represent some of the more endangered and threatened species on the planet and numerous conservation strategies are being implemented with the goal of ensuring species recovery. Little is known, however, about the wild gut microbiome of herptiles and how it relates to the health of wild populations. Here we report results from both a broad survey of hosts and a more intensive sampling of hosts and geography of fungi and bacteria associated with herptile gut microbiomes. We demonstrate that bacterial communities sampled from frogs, lizards and salamanders are structured by the host higher level taxonomy and that the fungus Basidiobolus is a common and natural component of these wild gut microbiomes. Intensive sampling of multiple hosts across the ecoregions of Tennessee revealed that geography and host:geography interactions are strong predictors of distinct Basidiobolus OTUs present within a given host. Co-occurrence analyses of Basidiobolus and bacterial community diversity supports a correlation and interaction between Basidiobolus and bacteria, suggesting that Basidiobolus may play a role in structuring the bacterial community. We further the hypothesis that this interaction is advanced by unique specialized metabolism originating from horizontal gene transfer from bacteria to Basidiobolus, and demonstrate that Basidiobolus is capable of producing a diversity of specialized metabolites including small cyclic peptides. IMPORTANCEThis work significantly advances our understanding of interactions in herptile microbiomes; the role that fungi play as a structural and functional member of herptile gut microbiomes; and the chemical functions that structure host:microbiome phenotypes. We also provide an important observational system of how the gut microbiome represents a unique environment that selects for novel metabolic functions through horizontal gene transfer between fungi and bacteria. Such studies are needed to better understand the complexity of gut microbiomes in nature and will inform conservation strategies for threatened species of herpetofauna.

ecology↗

bioRxiv2023

A Refined Phylochronology of the Second Plague Pandemic in Western Eurasia

Although dozens of ancient Yersinia pestis genomes and a vast corpus of documentary data are available, the origin and spread of consecutive outbreaks of the Second Plague Pandemic in Europe (14th-18th c.) are still poorly understood. For the majority of ancient genomes, only radiocarbon dates spanning several decades are available, hampering an association with historically recorded plague outbreaks. Here, we present new genomic evidence of the Second Pandemic from 11 sites in England, Estonia, the Netherlands, Russia, and Switzerland yielding 11 Y. pestis genomes with >4-fold mean coverage dating to between 1349 and 1710. In addition, we present a novel approach for integrating the chronological information retrieved from phylogenetic analysis with their respective radiocarbon dates, based on a novel methodology offering more precise dating intervals. Together with a fine-grained analysis of documentarily recorded plague outbreaks, this allows us to tentatively associate all available Y. pestis genomes of the Second Pandemic with historically documented plague outbreaks. Through these combined multidisciplinary analytical efforts, our newly sequenced genomes can be attributed to the Black Death in Cambridge (England), the pestis tertia or pestis quarta in the late 14th century (Estonia), previously unknown branches emerging in the 15th century (Estonia, the Netherlands and England), and a widespread pandemic in Eastern Europe around 1500 (western Russia), which all seem to have originated from one or multiple reservoirs located in Central Europe. While the latter continued to harbour a major Y. pestis lineage at least until the 1630s, represented by new genomes of the Thirty Years War plague (Switzerland), another lineage consecutively spread into Europe between the 17th and 18th century from the Ottoman Empire, as evidenced by a genome associated with the Great Northern War plague (Estonia). By combining phylogenetic analysis with a systematic historical reconstruction based on textual sources and an innovative phylogenetically informed radiocarbon modelling (PhIRM), we offer a new groundbreaking interdisciplinary approach that solves several fundamental methodological challenges associated with phylogenetic and spatio-temporal reconstruction of historical pandemics.

genomics↗

bioRxiv2023

Dysregulation of N-terminal acetylation causes cardiac arrhythmia and cardiomyopathy

BACKGROUNDN-terminal-acetyltransferases catalyze N-terminal acetylation (Nt-acetylation), an evolutionarily conserved co-translational modification. Nt-acetylation regulates diverse signaling pathways, yet little is known about its effects in the heart. To gain insights, we studied NAA10-related syndrome, in which mutations in NAA10, which catalyzes Nt-acetylation, causes severe QT prolongation, hypotonia, and neurodevelopmental delay. METHODSWe identified a missense variant in NAA10 (c.10C>A; p.R4S) that segregated with severe QT prolongation, arrhythmia, cardiomyopathy, and sudden death in a large kindred. We developed patient-derived and genome-edited human induced pluripotent stem cell (iPSC) models and deeply phenotyped iPSC-derived cardiomyocytes (iPSC-CMs) to dissect the mechanisms underlying NAA10-mediated cardiomyocyte dysfunction. RESULTSThe NAA10-R4S mutation reduced enzymatic activity, decreased expression levels of NAA10/NAA15 proteins, and destabilized the NatA complex. In iPSC-CM models of NAA10 dysfunction, dysregulation of the late sodium and slow rectifying potassium currents caused severe repolarization abnormalities, consistent with clinical QT prolongation and increased risk for arrhythmogenesis. Engineered heart tissues generated from mutant NAA10 cell lines had significantly decreased contractile force and sarcomeric disorganization, consistent with the cardiomyopathic phenotype in the identified family members. Diastolic calcium levels were increased with corresponding alterations in calcium handling pathways. We identified small molecule and genetic therapies that reversed the effects of NAA10 dysregulation of iPSC-CMs. CONCLUSIONSOur study defines novel roles of Nt-acetylation in cardiac ion channel regulation and delineates mechanisms underlying QT prolongation, arrhythmia, and cardiomyopathy caused by NAA10 dysfunction.

cell biology↗