Biology subjects

Smith, C. J.

Publications and source records attributed to Smith, C. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Adolescent blockade of complement signaling in the lateral septum increases social novelty seeking behavior in male mice

Social behaviors are critical for survival and change dramatically over the lifespan. Adolescence is a critical period of development during which social novelty seeking peaks before declining into adulthood. Adolescence is also a time of pronounced neural circuit refinement as excess synapses are eliminated. One critical mechanism supporting this maturation of neural circuits is microglial pruning of synapses through the classical complement signaling cascade. However, it remains unclear how microglial pruning of the neural circuitry supporting social novelty preference shapes the trajectory of this behavior during adolescence. To address this, we blocked microglial complement-dependent pruning during adolescence by injecting neutrophil inhibitory factor (NIF; blocks the adhesion of ligands to CD11b/C3 receptor) in the lateral septum (LS), a key node in the social circuitry supporting social novelty preference, in male mice. We found that NIF administration into the LS during adolescence increased preference for the novel social chamber over the familiar as compared to control PBS administration. LS-NIF treatment had no impact on anxiety-like behavior in the light-dark box test and no effect on sociability. LS-NIF treatment also decreased the expression of immune-related genes in the LS as compared to PBS treatment. These data support the hypothesis that complement-dependent microglial synaptic elimination in the LS is critical for the developmental progression of social novelty preference.

neuroscience↗

bioRxiv2026

Activity-resolved microbial community profiling using rpoB gene and transcript sequencing

Determining which members of a microbial community are metabolically active remains a central challenge in microbial ecology. Although the 16S rRNA gene is the dominant marker for bacterial community profiling, it cannot reliably distinguish active cells from dormant or dead populations. As a result, complementary phylogenetic markers whose transcript abundance more closely reflects cellular activity are needed. Here, we systematically evaluated 80 Bacterial protein-coding marker genes and identified rpoB, encoding the beta subunit of bacterial RNA polymerase, as the optimal candidate. We designed a new primer pair (1528F 2041R) from a curated database of 305,274 unique rpoB sequences and validated it for quantitative PCR and amplicon sequencing of DNA and RNA templates. The rpoB qPCR assay achieved a limit of quantification two orders of magnitude lower than the benchmark 16S rRNA assay, for which a limit of detection could not be determined because of no-template-control amplification. In soil and sediment communities, rpoB recovered community composition comparable to 16S rRNA while providing a quantitative activity signal: rpoB cDNA:DNA ratios correlated significantly with taxon-level transcript abundance (R squared between 0.22 and 0.29, p < 0.001), whereas 16S rRNA ratios did not (p > 0.5). In a biological activated carbon biofilter experiment, rpoB transcript abundance tracked the decline in dissolved organic carbon removal rates across a 72 hour time series (correlation coefficients between 0.84 and 0.99), whereas 16S rRNA transcripts were uninformative (correlation coefficients between -0.4 and 0.98). These results establish rpoB as a quantitatively robust, activity-responsive complement to 16S rRNA for linking community composition to ecosystem processes.

microbiology↗

bioRxiv2026

Connectome quality converges predictably to reveal optimal stopping points during proofreading

Volumetric electron microscopy (EM) has become a critical approach to generating high-resolution reconstructions of brain tissue. As the size of EM volumes increase, use of automated image segmentation within the reconstruction pipeline has become essential, although it generates errors that need correction. The proofreading and correcting of these errors has since become the dominant cost driver in the pipeline, but precisely estimating the sufficient number of proofreading edits to enable meaningful scientific analyses of the reconstructed neuronal networks remains a challenge. We present a fast, computationally inexpensive way to estimate the progress of a connectomic proofreading effort without requiring a priori knowledge of ground truth. We show that simple global graph invariants converge predictably to asymptotic limits with increasing numbers of proofreading edits, informing a quantitative "pencils down" criterion for proofreading completeness. We illustrate our method on two datasets in different stages of proofreading progress, a zebrafish spinal cord and the hemibrain Drosophila melanogaster dataset. Our method reduces the uncertainty associated with the planning and prioritization of proofreading activities and enables data owners to accurately predict and budget the amount of proofreading necessary for their scientific questions.

neuroscience↗

bioRxiv2026

Physical contact reveals a hidden layer of cortical architecture

Neurons interact at synapses, but they also communicate through physical contact and proximity, including diffusion, glia-mediated interactions, and ephaptic coupling. Standard connectomes map synapses, but cannot capture the full set of cell-cell contacts that can support these pathways. Here we extract contactomes from two large mouse visual cortex volumes at nanoscale resolution and quantify every cell-cell contact, the shared surface area of each contact, and the relationship between contact and synaptic connectivity. We find that contactomes are 5 - 10x denser than synaptic graphs, revealing that neurons physically contact a much larger set of potential neighbors than they synaptically connect to. We further find that most nearby potential neighbors are already in physical contact, indicating that local structural change would add few new candidate synaptic partners. Finally, we find that astrocytes form a single large syncytium-like network that spans the tissue and directly contacts nearly all neurons, and that glial processes lie within a micron or two of almost every synapse, indicating that synapses reside within a pervasive glia-shaped microenvironment. Together, these results show that physical contact forms a distinct layer of brain architecture that extends far beyond the synaptic connectome.

neuroscience↗

bioRxiv2026

Blood-brain barrier dysfunction predicts cognitive trajectory after ischemic stroke

Ischemic stroke doubles the risk of dementia.1-4 Stroke severity and location affect cognition early,5,6 but late dementia risk is not related to infarct characteristics, nor is it reduced by preventing additional strokes,3,6,7 and its mechanism is unknown. We identified a plasma proteomic signature of chronic stroke that was consistent with blood-brain barrier (BBB) dysfunction, including a 58% decrease in plasma levels of platelet-derived growth factor B and downregulation of its pathway compared to healthy controls. During 2 years of follow-up, the stroke-specific proteome was accentuated in stroke survivors who subsequently declined in the processing speed/executive function cognitive domain. To test BBB function, we performed dynamic contrast-enhanced MRI 6-9 months after stroke in an additional cohort and found 1.7-fold higher whole brain BBB leakage compared to controls. Finally, we compared autopsy tissue from people with infarcts and dementia at death to those with infarcts and no dementia. Those who died with dementia had dramatic loss of vascular mural cell coverage compared to those without dementia (median 0.7% vs. 27%). Thus, our proteomic, functional, and structural data implicate chronic BBB dysfunction in cognitive decline late after stroke, revealing potential proteomic and imaging biomarkers and, importantly, a novel target for intervention.

neuroscience↗

bioRxiv2025

Microglial MyD88-dependent signaling influences extracellular matrix development and interneuron maturation in the hippocampus

Parvalbumin interneurons (PVIs) are disrupted across diverse neurodevelopmental disorders, highlighting their vulnerability to developmental perturbations. Inflammation can perturb PVI development and function, and inflammatory mechanisms are often propagated within the brain by microglia. Yet the microglial mechanisms linking inflammatory signals to interneuron development are unclear. To test the role of microglial innate immune signaling in PVI development, we used mice lacking toll-like receptor adaptor MyD88 specifically in microglia. MyD88-deficient microglia showed reduced inflammatory responses but increased early-life phagocytosis of inhibitory synaptic material. In adulthood, males without microglial MyD88 exhibited increased hippocampal PVI density, increased extracellular matrix (ECM) deposition, increased inhibitory signaling, and impaired discrimination behaviors. We determined the cytokine interleukin (IL)-33, which normally drives adult microglial remodeling of the ECM, is developmentally regulated in the hippocampus. MyD88-deficient microglia fail to respond to IL-33, leading to reduced remodeling of the ECM component aggrecan. These results reveal microglial immune signaling via MyD88 regulates hippocampal inhibitory circuit development in a sex-specific manner.

neuroscience↗

bioRxiv2025

Genetic dissection of microglia cannibalism reveals an IL10 signaling axis controls microglia lifespan

The development of complex organs, like the brain, demands a robust system for tissue remodeling and cellular debris clearance. In the brain, this function is performed by microglia, which must clear diverse debris substrates, including that caused by cell death. Although the subsequent fate of these phagocytic microglia is a critical regulatory point that impacts whether the brain resolves a debris environment, the genetic mechanisms that control microglia fate after debris clearance remain mostly unknown. To address this, we conducted a large-scale CRISPR screen in zebrafish using a custom-built robotic confocal microscope. We selected candidate genes from a single-cell RNA sequencing dataset of embryonic mouse microglia. This screen identified several modulators of microglial lifespan and cannibalism that are enriched in mouse and zebrafish microglia, including interleukin-10 receptor beta (il10rb), a receptor subunit for the cytokine IL10. Perturbation of il10, il10rb, and downstream signaling molecules JAK/STAT in zebrafish reduced microglial death. Expression analysis in mouse and zebrafish confirmed that microglia express both il10 and il10rb. Given the established role of IL10 in lysosomal remodeling, we hypothesized that it regulates microglial survival through lysosomal acidification. While il10rb perturbation did not alter lysosome number or size, it caused a significant reduction in LysoTracker-positive lysosomes, indicating decreased lysosomal acidification. Inhibiting v-ATPase also reduced microglial death, reinforcing the link between lysosomal pH and cell fate. Our findings reveal a cytokine-regulated mechanism where lysosomal dynamics determine the survival of phagocytic microglia. We propose that a necroptosis-cannibalism process functions as a quality control mechanism for microglial turnover, which is critical for refining neuroimmune cell function in the brain.

neuroscience↗

bioRxiv2025

Effects of combined prenatal exposure to air pollution and maternal stress on immune and dopaminergic gene expression in the gut-brain axis

Air pollution and maternal stress during pregnancy are both risk factors for neurodevelopmental disorders and often converge on the same communities. Epidemiological and animal studies suggest that maternal psychosocial stress may worsen the effects of air pollutants on neurodevelopmental outcomes. Previous work utilizing a mouse model of combined prenatal exposure to diesel exhaust particles (DEP) and maternal stress (MS) has found numerous sex-specific effects of DEP/MS exposure on neuroimmune outcomes, dopamine receptors, the gut-brain axis, and social behavior. However, it is unclear how broadly the immune landscape is shifted in the brain and intestinal epithelium following DEP/MS. Here, we analyzed immune gene expression in 5 brain regions important for social behavior and in 3 regions of the intestinal epithelium in both male and female offspring following either DEP/MS or control exposure. We found several interesting overall patterns. First, changes in expression of immune genes such as CD11b and Tlr4 were concentrated in the nucleus accumbens and hippocampus. Tlr4 and Il-17ra mRNA also increased in the jejunum and colon following DEP/MS, but only in females. Second, in the nucleus accumbens, catecholamine-O-methyltransferase (Comt) and dopamine transporter 1 (Slc6a3) gene expression were increased following DEP/MS indicating increased dopamine degradation at and reuptake from the synapse, respectively. Additionally, Drd2 mRNA was decreased following DEP/MS in males. Finally, we observed numerous sex differences in immune gene expression regardless of treatment in both the brain and gut. Together, these findings suggest the nucleus accumbens is a key site for neuroimmune and dopaminergic changes following DEP/MS exposure and indicate persistent female-specific changes in intestinal immunity following these prenatal exposures. HighlightsO_LICd11b and Tlr4 mRNA is altered in the NAc and Hipp following DEP/MS C_LIO_LISeveral genes indicate reduced dopamine signaling in the NAc following DEP/MS in both sexes C_LIO_LIIntestinal Tlr4 and IL-17ra mRNA is increased in females only following DEP/MS C_LI

neuroscience↗

bioRxiv2025

Effects of combined prenatal exposure to air pollution and maternal stress on social behavior and oxytocin and vasopressin systems in male and female mice

Prenatal exposures to air pollution and maternal psychosocial stress are each associated with increased risk of neurodevelopmental disorders, including autism spectrum disorder (ASD) and epidemiological work suggests that concurrent exposure to these risk factors may be particularly harmful. This is important given that the same populations often bear the brunt of both toxicant and psychosocial stress burdens. Social impairments are a defining symptom in ASD. Previous work modeling combined prenatal exposure to diesel exhaust particles (DEP) and maternal stress (MS) in rodents has found male-biased social deficits in offspring, as well as changes to neuroimmune processes and the gut microbiome. However, the precise neural circuits on which these exposures converge to impact social behavior is unclear. Oxytocin (OXT) and vasopressin (AVP) are neuropeptides critical to the regulation of social behavior across species, signaling primarily at the oxytocin receptor (Oxtr) and vasopressin V1a receptor (V1aR) in the brain. Here, we hypothesized that OXT and/or AVP expression would be reduced in the brain following DEP/MS exposure. Following prenatal exposure to DEP/MS or the vehicle/control condition (VEH/CON), we measured maternal and offspring outcomes during the perinatal period, social and anxiety-like behavior during adolescence, OXT and AVP cell/fiber density and Oxtr and Avpr1a mRNA expression in early adulthood in several brain regions in both males and females. We observed a decrease in interaction time in DEP/MS males as compared to VEH/CON in the sociability assay and a decrease in social novelty preference in DEP/MS females as compared to VEH/CON. No effects of sex or treatment were observed on OXT or AVP cell number or fiber density in the hypothalamic regions assessed. However, numerous sex differences were observed in Oxtr and Avpr1a mRNA. Moreover, Avpr1a mRNA was significantly increased following DEP/MS exposure in the nucleus accumbens in both sexes and tended to increase in the dorsal hippocampus. Conversely, Avpr1a mRNA tended to decrease in the amygdala in both sexes following DEP/MS exposure. Together, these findings suggest that DEP/MS exposure has a stronger impact on female social behavior than previously observed. Moreover, while DEP/MS exposure does not appear to impact OXT or AVP expression in the brain, V1aR expression is modulated by DEP/MS exposure in several brain regions. HighlightsO_LIPrenatal DEP/MS reduces social novelty preference in females C_LIO_LIPrenatal DEP/MS does not alter OXT or AVP cell number in the PVN C_LIO_LIPrenatal DEP/MS does not alter OXT or AVP fiber density in the LH, AH, or MPOA C_LIO_LIPrenatal DEP/MS increases Avpr1a mRNA in the NAc C_LIO_LIPrenatal DEP/MS tends to decrease Avrp1a mRNA in the AMY and increase in dHipp C_LI

neuroscience↗

bioRxiv2025

sox17 mutagenesis reveals microglia can be impacted by genetic maternal effects

Embryonic development is precisely shaped by maternal and zygotic factors. These maternal factors exert their influence through maternal effects, a phenomenon where an offsprings phenotype is determined, at least in part, by the mothers environment and genotype. While environmental maternal effects can cause phenotypes that present both early and later in life, genetic maternal effects generally induce phenotypes in the earliest embryonic stages. Here, we reveal a genetic maternal effect that influences the development of cells that arise after early embryogenesis, highlighting that specific cell types can be susceptible to late-onset genetic maternal effects. Using zebrafish to study microglia, the resident immune cells of the brain, we identified a mutation in sry-related HMG box gene-17 (sox17) that exhibits a maternal effect phenotype that presents as a reduction of microglia in the brain and precursors in the yolk sac. We demonstrate that sox17 is expressed in microglia and their yolk sac precursors and is maternally-loaded. We show that sox17 restoration via embryonic injection reverses the maternal effect on microglia and yolk sac cells in sox17 mutants. To identify additional genes interacting with sox17, we nominated genes from scRNA sequencing analysis of mouse embryonic microglia to perform a genetic screen using CRISPR mutagenesis and a custom-built robot that captures confocal images of the zebrafish brain in high-throughput. This screen identified f11r.1, gas6, and mpp1 as modifiers of microglia abundance in the embryonic brain, which we demonstrated are also expressed in zebrafish microglia. Transcriptional and mutant analyses with these new modifiers suggest that sox17 positively regulates mpp1 transcription. These results demonstrate that microglia are susceptible to genetic maternal effects, in addition to their known sensitivity to environmental maternal effects. Our findings reveal a late-onset phenotype associated with the maternal genotype, expanding the recognized impact of genetic maternal effects beyond initial embryo viability and into long-term vigor.

neuroscience↗

bioRxiv2025

Advanced Age in Mice Exacerbates Sepsis-Induced Inflammation, Vascular Permeability, and Multi-Organ Dysfunction

Sepsis is a life-threatening syndrome marked by a dysregulated immune response to an infection and significant endothelial vascular permeability, often leading to multi-organ failure. Elderly patients are particularly vulnerable to sepsis, with higher morbidity and mortality rates. We hypothesized that advanced age exacerbates sepsis-induced inflammation and endothelial vascular permeability, resulting in a delayed recovery, persistent inflammation, and sustained organ injury. Using a polymicrobial sepsis model in young (3-month-old) and aged (18-month-old) C57BL/6 mice, sepsis was induced via intraperitoneal cecal slurry (CS) injection. Outcomes were assessed during the acute (24-hour; 1.6mg/g CS) and recovery (8-day; 1.0 mg/g CS) phases. During the acute phase, aged mice exhibited worse physiologic dysfunction, higher systemic (plasma TNF-a: young septic 202.1 pg/mL [17.44, 398.9] vs. aged septic 482.6 pg/mL [279.8, 711.7]; p = 0.0352 Mann-Whitney) and organ-specific inflammation, increased endothelial injury and vascular permeability, as well as greater kidney and liver dysfunction compared to young mice. During recovery, aged mice had sustained physiologic dysfunction, prolonged systemic and organ-specific inflammation, and sustained organ injury (kidney tissue NGAL: young septic 291.5 RE [203.7, 373.2] vs. aged septic 821 RE [456, 1258] protein normalized to beta actin; p = 0.0008 Mann-Whitney) compared to young mice. These results support the hypothesis that advanced age worsens sepsis severity and outcomes and delays recovery, emphasizing the need for aged models and multi-organ evaluations to develop effective therapies for this vulnerable population.

physiology↗

bioRxiv2025

Prenatal exposure to environmental stressors alters gut macrophage development and gastrointestinal function of male offspring

Gastrointestinal (GI) dysfunction is a frequently reported comorbidity of neurodevelopmental disorders (NDDs). Early-life inflammatory challenges from the environment (e.g. infection, toxicants) can increase risk for NDDs but the impact of such stressors on the developing GI tract is not well understood. We investigated possible mechanisms by which GI comorbidities occur in response to environmental stressors using our well-characterized model of combined gestational exposure to air pollution (diesel exhaust particles, DEP) and maternal stress (MS), which induces social deficits in male but not female offspring. We show that DEP/MS disrupts normal GI development, leading to altered small intestine morphology in neonatal males, but not females. Recent evidence shows that resident macrophages of the gut prune enteric neurons during a precise postnatal window. We found decreased pruning of gut enteric neurons by the resident macrophages of the muscularis externa in DEP/MS exposed males at postnatal day 14. In line with this, we saw the expression of motor neuron-associated genes spike in males at the same postnatal time point following DEP/MS exposure. Finally, we assessed the motor function of the GI tract of these animals and observed dysmotility in DEP/MS males only. Taken together, these findings establish intestinal macrophages as a mediator of GI development that is sensitive to early-life perturbations from the environment, highlighting a potential mechanism connecting NDDs with comorbid GI dysfunction.

developmental biology↗

bioRxiv2025

An ultrastructural map of a spinal sensorimotor circuit reveals the potential of astroglia modulation

Information flow through circuits is dictated by the precise connectivity of neurons and glia. While a single astrocyte can contact many synapses, how glial-synaptic interactions are arranged within a single circuit to impact information flow remains understudied. Here, we use the local spinal sensorimotor circuit in zebrafish as a model to understand how neurons and astroglia are connected in a vertebrate circuit. With semi-automated cellular reconstructions and automated approaches to map all the synaptic connections, we identified the precise synaptic connections of the local sensorimotor circuit, from dorsal root ganglia neurons to spinal interneurons and finally to motor neurons. This revealed a complex network of interneurons that interact in the local sensorimotor circuit. We then mapped the glial processes within tripartite synapses in the circuit. We demonstrate that tripartite synapses are equally distributed across the circuit, supporting the idea that glia can modulate information flow through the circuit at different levels. We show that multiple astroglia, including bona fide astrocytes, contact synapses within a single sensory neurons circuit and that each of these astroglia can contact multiple parts of the circuit. This detailed map reveals an extensive network of connected neurons and astroglia that process sensory stimuli in a vertebrate. We then utilized this ultrastructural map to model how synaptic thresholding and glial modulation could alter information flow in circuits. We validated this circuit map with GCaMP6s imaging of dorsal root ganglia, spinal neurons and astroglia. This work provides a foundational resource detailing the ultrastructural organization of neurons and glia in a vertebrate circuit, offering insights in how glia could influence information flow in complex neural networks.

neuroscience↗

bioRxiv2024

Specificity, length, and luck: How genes are prioritized by rare and common variant association studies

Standard genome-wide association studies (GWAS) and rare variant burden tests are essential tools for identifying trait-relevant genes. Although these methods are conceptually similar, we show by analyzing association studies of 209 quantitative traits in the UK Biobank that they systematically prioritize different genes. This raises the question of how genes should ideally be prioritized. We propose two prioritization criteria: 1) trait importance -- how much a gene quantitatively affects a trait; and 2) trait specificity -- a genes importance for the trait under study relative to its importance across all traits. We find that GWAS prioritize genes near trait-specific variants, while burden tests prioritize trait-specific genes. Because non-coding variants can be context specific, GWAS can prioritize highly pleiotropic genes, while burden tests generally cannot. Both study designs are also affected by distinct trait-irrelevant factors, complicating their interpretation. Our results illustrate that burden tests and GWAS reveal different aspects of trait biology and suggest ways to improve their interpretation and usage.

genomics↗

bioRxiv2024

Single-cell multi-omic analysis of fibrolamellar carcinoma reveals rewired cell-to-cell communication patterns and unique vulnerabilities

Fibrolamellar carcinoma (FLC) is a rare malignancy disproportionately affecting adolescents and young adults with no standard of care. FLC is characterized by thick stroma, which has long suggested an important role of the tumor microenvironment. Over the past decade, several studies have revealed aberrant markers and pathways in FLC. However, a significant drawback of these efforts is that they were conducted on bulk tumor samples. Consequently, identities and roles of distinct cell types within the tumor milieu, and the patterns of intercellular communication, have yet to be explored. In this study we unveil cell-type specific gene signatures, transcription factor networks, and super-enhancers in FLC using a multi-omics strategy that leverages both single-nucleus ATAC-seq and single-nucleus RNA-seq. We also infer completely rewired cell-to-cell communication patterns in FLC including signaling mediated by SPP1-CD44, MIF-ACKR3, GDF15-TGFBR2, and FGF7-FGFR. Finally, we validate findings with loss-of-function studies in several models including patient tissue slices, identifying vulnerabilities that merit further investigation as candidate therapeutic targets in FLC.

cancer biology↗

bioRxiv2024

Design and testing of a PCR assay to quantify coliform bacteria in drinking water

Here we present an enhanced lacZ qPCR assay targeting {beta}-galactosidase, encoded by lacZ, to assess coliforms in drinking water. Coliform bacteria are a diverse group of over 80 species, commonly found in enteric environments, and act as indicators of water supply contamination. Due to the significant variation among coliforms, and the size of the group designing a single target was challenging, as previous studies have demonstrated. To address this, a coliform lacZ sequence database was created, and the phylogeny of the group was reviewed. The degree of phylogenetic differences both between and among different coliform genera is indeed large, raising questions about their current definition. Using the database, current and new primer sets were tested for specificity and coverage both in silico and in vitro. The de novo primer set, LZ1, was found to be the most effective for qPCR. When compared directly with traditional culture-based methods and flow cytometry total cell counts, the LZ1 lacZ qPCR assay quantified E. coli in drinking water down to a concentration of 1.82 x 103 cfu/100mL, equivalent to 360 cfu per qPCR reaction. ImportanceColiform bacteria and E. coli are key indicators of faecal contamination in drinking water. Compliance with U.K., U.S.A., or European regulations requires that drinking water be free of these organisms. However, the culture-based methods used to quantify these bacteria have remained unchanged for over 100 years, relying on the culturability of the organisms, sampling only a small volume at a single time point, and lacking direct correlation to pathogenic microbes. There is a pressing need for a rapid, high-throughput analysis capable of detecting coliforms more representatively, without relying solely on culturability. By analysing larger volumes or more replicates, this qPCR assay could potentially be used to assess the risk of drinking water contamination over longer periods of time, offering a more comprehensive evaluation compared to traditional, culture-dependent methods.

microbiology↗

bioRxiv2024

Cerebrovascular Health Mediates Processing Speed Change Through Anterior White Matter Alterations: A UK Biobank Study

Cerebrovascular disease is associated with an increased likelihood of developing dementia. While cardiovascular risk factors are modifiable and may reduce the risk of later-life cognitive dysfunction, the relationship between cerebrovascular risk factors, brain integrity and cognition remains poorly characterised. Using a large UK Biobank sample of predominantly middle-aged adults, without neurological disease, our structural equation mediation models showed that poor cerebrovascular health, indicated by the presence of cerebrovascular risk factors, was associated with slowed processing speed. This effect was best explained by anterior white matter microstructure changes, rather than posterior changes. Effects were also significantly reduced when considering other forms of cognition, demonstrating both regional- and cognitive-specificity of our effects. Critically, our findings also demonstrate that including measures of risk factor duration may be particularly important for improving estimations of cerebrovascular burden. In summary, our study demonstrates the specific impact of early cerebrovascular burden on brain structure and cognitive function, highlighting the necessary next steps for improving cerebrovascular burden quantification and improving clinical predictions.

neuroscience↗

bioRxiv2024

Novel roles for pirin proteins and a 2-ketoglutarate:ferredoxin oxidoreductase ortholog in Bacteroides fragilis central metabolism and comparison of metabolic mutants susceptibility to antimicrobials metronidazole and amixicile.

The regulation of the central metabolism and fermentation pathways and its effect on antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is not completely understood. In this study we show that B. fragilis encodes for two iron-dependent redox-sensitive regulatory pirin protein genes, pir1 and pir2, whose mRNA expression are upregulated following oxygen exposure and growth in iron-limiting conditions.{Delta} pir1 and{Delta} pir2 mutants showed altered short-chain fatty acids production compared to the parent strain. Overexpression of Pir1 and Pir2 increased susceptibility to metronidazole (MTZ), and to amixicile (AMIX), a novel inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobes. We hypothesized these observations were a result of a modulatory effect of Pir1 and Pir2 proteins. Consistent with this, we showed that Pir1 forms direct protein-protein interactions with PFOR. In addition, AlphaFold2-based structural analysis predicts that Pir1 and Pir2 form stable interactions with several enzymes of the central metabolism including the 2-ketoglutate:ferredoxin oxidoreductases (KFOR), Kor1AB and Kor2CDAEBG. A series of metabolic mutants and electron transport chain inhibitors were used to show a wide-ranging effect of bacterial metabolism on MTZ and AMIX susceptibility. There was no cross-resistance between MTZ resistant strains with AMIX susceptibility. Furthermore, we show that AMIX is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. This investigation led to the discovery that the kor2AEBG genes are essential for growth, and we present evidence that kor2AEBG genes have dual-functions including the reductive synthesis of 2-ketoglutarate via reverse TCA cycle. Support for a novel Kor2AEBG activity comes from the findings showing that addition of compounds containing oleic acid stimulated growth of the{Delta} kor2AEBG mutant. However, the metabolic activity that bypasses KorAEBG function remains to be defined. Collectively our investigation reveals new information on B. fragilis central metabolism and its modulatory control by pirin proteins. These data provide new genetic and metabolic knowledge which may be leveraged for the future development of new narrow-spectrum antimicrobials.

microbiology↗