Biology subjects

Smith, T. J.

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

At least 19 recordsLinked to original sources

bioRxiv2026

The Q1K Integrated EEG and Eye-Tracking Experimental Test Battery for Open Autism Science

The Quebec 1000 Families (Q1K) platform has been designed to recruit, phenotype, and collect biospecimens from a large cohort of families with at least one member with autism spectrum disorder or a related neurodevelopmental condition. As part of the Q1K protocol, an experimental test battery was developed and validated using simultaneous high-density electroencephalography (EEG) and eye tracking (ET). We report on the general approach and design principles, multimodal EEG/ET integration, the tasks, and their validation, providing a blueprint for implement such a project. It also describes the cohort and its methods as a reference for future studies using this dataset. By releasing openly this experimental test battery, we aim to support task standardization and multi-project data pooling in autism and related neurodevelopmental disorders.

neuroscience↗

bioRxiv2026

Synaptic activity controls local exposure of an 'eat-me' signal via ANO3-ITPR1 signaling

Neuronal synapses are eliminated during brain development and disease through pruning by glial cells. Individual synapses are marked for engulfment by eat-me signals, which include externalized phosphatidylserine. In apoptotic cells, phosphatidylserine externalization is driven by caspase-dependent activation of Xkr scramblases1 and inactivation of specific flippases2. Localized caspase activation at neuronal synapses can mediate spatially-restricted synaptic phosphatidylserine exposure leading to synapse pruning by glial cells during development and neurodegeneration3-6. It is unknown which caspase-regulated flippases and scramblases promote synaptic phosphatidylserine exposure and whether there are any caspase-independent mechanisms of phosphatidylserine exposure relevant for synapse elimination. To address this question, we here develop a scalable CRISPR screening approach, COMPASS-seq (compartment-anchored sgRNA screen sequencing), to uncover the genetic underpinnings of subcellular phenotypes. COMPASS-seq is compatible with in vitro and in vivo systems and a wide range of subcellular compartments; we here apply it to the neuronal synapse. We discover that inhibition of the caspase-independent, calcium-activated anoctamin ANO3 (TMEM16C) is sufficient to increase synapse numbers in vivo. ANO3 co-localizes with IP3 receptor 1 (ITPR1), a calcium channel in the endoplasmic reticulum, to form a postsynaptic signaling platform that drives spatially restricted phosphatidylserine exposure at synapses. Activation of the ITPR1 calcium channel activity is sufficient to drive synaptic phosphatidylserine exposure via an ANO3-dependent but caspase-independent mechanism. Our results suggest a mechanism for integrating synaptic activity information to control synaptic pruning. The role of ANO3 in regulating synapses could shed light on the mechanisms underlying its numerous associations with both dementia7 and other neurological diseases8,9.

neuroscience↗

bioRxiv2026

Non-canonical stringent response signaling mediates antimicrobial fatty acid sensitivity in Staphylococcus aureus

Staphylococcus aureus is a leading cause of skin and soft tissue infections, yet colonization of healthy skin is limited by multiple defenses, including antimicrobial fatty acids (AFAs) produced by host cells and the resident microbiota. The mechanisms by which S. aureus overcomes these lipid-based defenses remain incompletely understood. Here, we show that mutations truncating the essential stringent response regulator Rel confer broad tolerance to both host-and microbially-derived AFAs in diverse S. aureus strains. Unlike classical stringent response activation, C-terminal Rel truncations do not induce typical (p)ppGpp-dependent transcriptional changes but instead enhance activity of the alternative sigma factor SigB and the staphylococcal accessory regulator SarA. This SigB-SarA regulatory cascade promotes transcriptional remodeling, including upregulation of pyrimidine biosynthesis genes, and coincides with alterations to cell envelope structure. Moreover, in the absence of SigB or SarA, tolerance can be restored through mutations in the serine/threonine phosphatase Stp1, highlighting additional pathways that modulate cell envelope-mediated resistance to AFAs. These findings identify a previously unrecognized consequence of Rel mutation in S. aureus; whereby small truncations may promote survival in AFA-rich host environments facilitating skin colonization and infection. ImportanceStaphylococcus aureus is a major cause of skin and soft tissue infections, and persistent skin colonization is a significant risk factor for infection. Antimicrobial fatty acids (AFAs), produced by microbes and host cells on human skin, normally limit S. aureus colonization. The mechanisms that allow this pathogen to overcome these lipid defenses are incompletely understood. We show that mutations truncating the essential stringent response regulator Rel enable S. aureus to tolerate both host-and microbially derived AFAs. These Rel variants, which have been identified in clinical isolates, alter cell envelope properties through the transcriptional regulator SarA rather than activating a classical stringent response. Our findings reveal a previously unrecognized adaptation that may facilitate S. aureus survival on the skin and promote infection.

microbiology↗

bioRxiv2025

Murine norovirus allosteric escape mutants mimic gut activation.

Murine norovirus (MNV) undergoes large conformational changes in response to the environment. The T=3 icosahedral capsid is composed of 180 copies of [~]58 kDa VP1 that has N-terminal (N), shell (S) and C-terminal protruding (P) domains. In phosphate buffered saline, the P domains are loosely tethered to the shell and float [~]15[A] above the surface. At conditions found in the gut (i.e. low pH with high metal ion and bile salt concentrations) the P domain rotates and drops onto the shell with intra P domain changes that enhance receptor interactions while blocking antibody binding. Two of our monoclonal antibodies (2D3 and 4F9) have broad strain recognition and the only escape mutants, V339I and D348E, are located on the CD loop and [~]20 [A] from the epitope. Here we determined the cryo-EM structures of V339I and D348E at neutral pH +/- metal ions and bile salts. These allosteric escape mutants are constitutively in the activated state without the addition of metal ions or bile salts, thus explaining how they escape neutralization. Dynamic simulations of the P domain further suggest that movement of the CD loop may be the rate limiting step in the conformational change and that V339I increases the motion of the AB/EF loops compared to wt, making it easier for the virus to transition to the activated state. These findings have important implications for norovirus vaccine design since they uncover a form of the viral capsid that should lend superior immune protection against subsequent challenge by wild type virus. ImportanceImmune protection from norovirus infection is notoriously transient in both humans and mice. Our results strongly suggest that this is likely because the activated form of the virus found in gut conditions is not recognized by antibodies created in the circulation. By reversibly presenting one structure in the gut and a completely different antigenic structure in circulation, gut tissue can be infected in subsequent challenges, while extra intestinal organs are protected. We find here that allosteric escape mutants to the most broadly neutralizing antibodies thwart recognition by transitioning to the activated state without the need of gut conditions (i.e. bile, low pH, or metal ions). These findings are significant because it is now feasible to present the activated form of the virus to the immune system (for example as a vaccine) to better protect the gut tissue for longer periods of time.

microbiology↗

bioRxiv2024

Adaptation to skin mycobiota promotes antibiotic tolerance in Staphylococcus aureus

The microbiota can promote host health by inhibiting pathogen colonization, yet how host-resident fungi, or the mycobiota, contribute to this process remains unclear. The human skin mycobiota is uniquely stable compared to other body sites and dominated by yeasts of the genus Malassezia. We observe that colonization of human skin by Malassezia sympodialis significantly reduces subsequent colonization by the prominent bacterial pathogen Staphylococcus aureus. M. sympodialis secreted products possess potent bactericidal activity against S. aureus and are sufficient to impair S. aureus skin colonization. This bactericidal activity requires an acidic environment and is exacerbated by free fatty acids, demonstrating a unique synergy with host-derived epidermal defenses. Leveraging experimental evolution to pinpoint mechanisms of S. aureus adaptation in response to the skin mycobiota, we identified multiple mutations in the stringent response regulator Rel that promote survival against M. sympodialis. Similar Rel alleles have been reported in S. aureus clinical isolates, and natural Rel variants are sufficient for tolerance to M. sympodialis antagonism. Partial stringent response activation underlies tolerance to clinical antibiotics, with both laboratory-evolved and natural Rel variants conferring multidrug tolerance. These findings demonstrate the ability of the mycobiota to mediate pathogen colonization resistance, identify new mechanisms of bacterial adaptation in response to fungal antagonism, and reveal the potential for microbiota-driven evolution to shape pathogen antibiotic susceptibility. Highlights- M. sympodialis reduces colonization of human skin by S. aureus - Bactericidal activity of M. sympodialis is exacerbated by features of the skin niche - S. aureus Rel variants are sufficient for tolerance to Malassezia antagonism - Evolved tolerance to yeast antagonism coincides with S. aureus multidrug tolerance

microbiology↗

bioRxiv2023

Reconstitution of a Biofilm Adhesin System from a Sulfate-Reducing Bacterium in Pseudomonas fluorescens

Biofilms of the sulfate reducing bacterium (SRB) Desulfovibrio vulgaris Hildenborough (DvH) can facilitate metal corrosion in various industrial and environmental settings leading to substantial economic losses; however, the mechanisms of biofilm formation by DvH are not yet well-understood. Evidence suggests that a large adhesin, DvhA, may be contributing to biofilm formation in DvH. The dvhA gene and its neighbors encode proteins that resemble the Lap system, which regulates biofilm formation by Pseudomonas fluorescens, including a LapG-like protease DvhG and effector protein DvhD, which has key differences from the previously described LapD. By expressing the Lap-like adhesion components of DvH in P. fluorescens, our data support the model that the N-terminal fragment of the large adhesin DvhA serves as an adhesin "retention module" and is the target of the DvhG/DvhD regulatory module, thereby controlling cell-surface location of the adhesin. By heterologously expressing the DvhG/DvhD-like proteins in a P. fluorescens background lacking native regulation ({Delta}lapG{Delta}lapD) we also show that cell surface regulation of the adhesin is dependent upon the intracellular levels of c-di-GMP. This study provides insight into the key players responsible for biofilm formation by DvH, thereby expanding our understanding of Lap-like systems. SignificanceCorrosion leads to [~]2.5 trillion US dollars in economic losses, 20% of which are estimated to be microbially induced. Biofilms of sulfate reducing bacteria (SRB), especially the genus Desulfovibrio, are important members of the corrosion consortium and accelerate deterioration of metals. Understanding how biofilms are formed by SRB can provide important clues to mitigate this challenge. In this study, we used genetic and biochemical tools to investigate the mechanism of biofilm formation by Desulfovibrio vulgaris Hildenborough. Our study reveals critical genes responsible for regulating the secretion of a large adhesin known to be required for biofilm formation.

microbiology↗

bioRxiv2023

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in E. coli, Trm2 in S. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2{Delta} cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

biochemistry↗

bioRxiv2023

Behavioral Paradigm for the Evaluation of Stimulation-Evoked Somatosensory Perception Thresholds in Rats

Intracortical microstimulation (ICMS) of the somatosensory cortex via penetrating microelectrode arrays (MEAs) can evoke cutaneous and proprioceptive sensations for restoration of perception in individuals with spinal cord injuries. However, ICMS current amplitudes needed to evoke these sensory percepts tend to change over time following implantation. Animal models have been used to investigate the mechanisms by which these changes occur and aid in the development of new engineering strategies to mitigate such changes. Non-human primates are commonly the animal of choice for investigating ICMS, but ethical concerns exist regarding their use. Rodents are a preferred animal model due to their availability, affordability, and ease of handling, but there are limited choices of behavioral tasks for investigating ICMS. In this study, we investigated the application of an innovative behavioral go/no-go paradigm capable of estimating ICMS-evoked sensory perception thresholds in freely moving rats. We divided animals into two groups, one receiving ICMS and a control group receiving auditory tones. Then, we trained the animals to nose-poke - a well-established behavioral task for rats - following either a suprathreshold ICMS current-controlled pulse train or frequency-controlled auditory tone. Animals received a sugar pellet reward when nose-poking correctly. When nose-poking incorrectly, animals received a mild air puff. After animals became proficient in this task, as defined by accuracy, precision, and other performance metrics, they continued to the next phase for perception threshold detection, where we varied the ICMS amplitude using a modified staircase method. Finally, we used non-linear regression to estimate perception thresholds. Results indicated that our behavioral protocol could estimate ICMS perception thresholds based on [~]95% accuracy of rat nose-poke responses to the conditioned stimulus. This behavioral paradigm provides a robust methodology for evaluating stimulation-evoked somatosensory percepts in rats comparable to the evaluation of auditory percepts. In future studies, this validated methodology can be used to study the performance of novel MEA device technologies on ICMS-evoked perception threshold stability using freely moving rats or to investigate information processing principles in neural circuits related to sensory perception discrimination.

neuroscience↗

bioRxiv2023

Mitigating a TDP-43 proteinopathy using RNA-targeting CRISPR effector proteins

The TDP-43 proteinopathies, which include amyotrophic lateral sclerosis and frontotemporal dementia, are a devastating group of neurodegenerative disorders that are characterized by the mislocalization and aggregation of TDP-43. Here we demonstrate that RNA-targeting CRISPR effector proteins, a programmable class of gene silencing agents that includes the Cas13 family of enzymes and Cas7-11, can be used to mitigate TDP-43 pathology when programmed to target ataxin-2, a modifier of TDP-43-associated toxicity. In addition to inhibiting the aggregation and transit of TDP-43 to stress granules, we find that the in vivo delivery of an ataxin-2-targeting Cas13 system to a mouse model of TDP-43 proteinopathy improved functional deficits, extended survival, and reduced the severity of neuropathological hallmarks. Further, we benchmark RNA-targeting CRISPR platforms against ataxin-2 and find that high-fidelity forms of Cas13 possess improved transcriptome-wide specificity compared to Cas7-11 and a first-generation effector. Our results demonstrate the potential of CRISPR technology for TDP-43 proteinopathies.

bioengineering↗

bioRxiv2023

GATA1 knockout in human pluripotent stem cells generates enhanced neutrophils to investigate extracellular trap formation

Human pluripotent stem cell (hPSC)-derived tissues can be used to model diseases and validate targets in cell types that are challenging to harvest and study at-scale, such as neutrophils. Neutrophil dysregulation, specifically unbalanced neutrophil extracellular trap (NET) formation, plays a critical role in the prognosis and progression of multiple diseases, including COVID-19. hPSCs can provide a limitless supply of neutrophils (iNeutrophils) to study these processes and discover and validate targets in vitro. However, current iNeutrophil differentiation protocols are inefficient and generate heterogeneous cultures consisting of different granulocytes and precursors, which can confound the study of neutrophil biology. Here, we describe a method to dramatically improve iNeutrophils yield, purity, functionality, and maturity through the deletion of the transcription factor GATA1. GATA1 knockout (KO) iNeutrophils are nearly identical to primary neutrophils in cell surface marker expression, morphology, and host defense functions. Unlike wild type (WT) iNeutrophils, GATA1 KO iNeutrophils generate NETs in response to the physiologic stimulant lipopolysaccharide (LPS), suggesting they could be used as a more accurate model when performing small-molecule screens to find NET inhibitors. Furthermore, through CRSPR/Cas9 deletion of CYBB we demonstrate that GATA1 KO iNeutrophils are a powerful tool in quickly and definitively determining involvement of a given protein in NET formation.

immunology↗

bioRxiv2022

A mucin-regulated adhesin determines the intestinal biogeography and inflammatory character of a bacterial symbiont.

In a healthy gut, microbes are often aggregated with host mucus, yet the molecular basis for this organization and its impact on intestinal health are unclear. Mucus is a viscous physical barrier separating resident microbes from epithelia, but also provides glycan cues that regulate microbial behaviors. Using experimental evolution, we discovered a mucin-sensing pathway in an Aeromonas symbiont of zebrafish, Aer01. In response to the mucin-associated glycan N-acetylglucosamine, a sensor kinase regulates expression of a mucin-binding adhesin we named MbpA. When MbpA is disrupted, Aer01 colonizes to normal levels, but is largely planktonic and elicits increased intestinal inflammation, traits which are reversed by increasing cell surface MpbA. MbpA-like adhesins are common in human-associated bacteria and expression of an Akkermansia muciniphila MbpA-like adhesin in MbpA-deficient Aer01 restored lumenal aggregation and reversed its pro-inflammatory character. Our work demonstrates how resident bacteria use mucin glycans to modulate behaviors congruent with host health.

microbiology↗

bioRxiv2022

Disaggregation as an interaction mechanism among intestinal bacteria

The gut microbiome contains hundreds of interacting species that together influence host health and development. The mechanisms by which intestinal microbes can interact, however, remain poorly mapped and are often modeled as spatially unstructured competitions for chemical resources. Recent imaging studies examining the zebrafish gut have shown that patterns of aggregation are central to bacterial population dynamics. In this study, we focus on bacterial species of genera Aeromonas and Enterobacter. Two zebrafish gut derived isolates, Aeromonas ZOR0001 (AE) and Enterobacter ZOR0014 (EN), when mono-associated with the host, are highly aggregated and located primarily in the intestinal midgut. An Aeromonas isolate derived from the commensal strain, Aeromonas-MB4 (AE-MB4), differs from the parental strain in that it is composed mostly of planktonic cells localized to the anterior gut. When challenged by AE-MB4, clusters of EN rapidly fragment into non-motile, slow-growing, dispersed individual cells with overall abundance two orders of magnitude lower than the mono-association value. In the presence of a certain set of additional gut bacterial species, these effects on EN are dampened. In particular, if AE-MB4 invades an already established multi-species community, EN persists in the form of large aggregates. These observations reveal an unanticipated competition mechanism based on manipulation of bacterial spatial organization, namely dissolution of aggregates, and provide evidence that multi-species communities may facilitate stable intestinal co-existence. SignificanceThe diverse microbial communities inhabiting vertebrate intestines influence many aspects of their hosts health and development. The rules governing community assembly and even the types of interactions that are possible among gut microbes remain poorly understood, however, with the role of spatial aggregation in interspecies dynamics being especially unclear. To address this, we performed live imaging studies of the larval zebrafish gut, focusing on two spatially distinct bacterial species that exhibit strong competition, discovering that the latter can induce a striking, rapid disintegration of aggregates of the former. This competition is attenuated in the presence of a particular set of additional bacterial species. Our findings reveal an unanticipated interaction mechanism and highlight that diverse communities may stabilize the gut microbiome.

microbiology↗

bioRxiv2022

Secreted Aeromonas GlcNAc binding protein GbpA stimulates epithelial cell proliferation in the zebrafish intestine

In response to microbiota colonization, the intestinal epithelia of many animals exhibit increased rates of cell proliferation. We used gnotobiotic larval zebrafish to identify a secreted factor from the mutualist Aeromonas veronii that is sufficient to promote intestinal epithelial cell proliferation. This secreted A. veronii protein is a homologue of the Vibrio cholerae GlcNAc binding protein GbpA, which was identified as a chitin-binding colonization factor in mice. GbpA was subsequently shown to be a lytic polysaccharide monooxygenase (LPMO) that can degrade recalcitrant chitin. Our phenotypic characterization of gbpA deficient A. veronii found no alterations in the mutant cells biogeography in the zebrafish intestine and only a modest competitive disadvantage in chitin-binding and colonization fitness when competed against the wild type strain. These results argue against the model of GbpA being a secreted adhesin that binds simultaneously to bacterial cells and GlcNAc, and instead suggests that GbpA is part of a bacterial GlcNAc utilization program. We show that the host proliferative response to GbpA occurs in the absence of bacteria upon exposure of germ-free zebrafish to preparations of native GbpA secreted from either A. veronii or V. cholerae or recombinant A. veronii GbpA. Furthermore, domain 1 of A. veronii GbpA, containing the predicted LPMO activity, is sufficient to stimulate intestinal epithelial proliferation. We conjecture that intestinal epithelia upregulate their rates of renewal in response to secreted bacterial GbpA proteins as an adaptive strategy for coexisting with bacteria that can degrade glycan constituents of the protective intestinal lining.

microbiology↗

bioRxiv2022

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Chemical modifications to protein encoding messenger RNA (mRNA) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been identified by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. To overcome these challenges, we improved the mRNA purification and LC-MS/MS pipelines to identify new S. cerevisiae mRNA modifications and quantify 50 ribonucleosides in a single analysis. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and revealed four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells and used a reconstituted translation system to discern the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

biochemistry↗

bioRxiv2021

Murine norovirus capsid plasticity - Glycochenodeoxycholic acid stabilizes P domain dimers and triggers escape from antibody recognition

The murine norovirus (MNV) capsid protein is the target for various neutralizing antibodies binding to distal tips of its protruding (P)-domain. The bile acid glycochenodeoxycholic acid (GCDCA), an important co-factor for murine norovirus (MNV) infection, has recently been shown to induce conformational changes in surface-loops and a contraction of the virion. Here, we employ protein NMR experiments using stable isotope labeled MNV P-domains to shed light on underlying molecular mechanisms. We observe two separate sets of NMR resonance signals for P-domain monomers and dimers, permitting analysis of the corresponding exchange kinetics. Unlike human norovirus GII.4 P-dimers, which exhibit a half-life in the range of several days, MNV P-dimers are very short lived with a half-life of about 17 s. Addition of GCDCA shifts the equilibrium towards the dimeric form by tightly binding to the P-dimers. In MNV virions GCDCA-mediated stabilization of the dimeric arrangement of P-domains generates a more ordered state, which in turn may entropically assist capsid contraction. Numerous long-range chemical shift perturbations (CSPs) upon addition of GCDCA reflect allosteric conformational changes as a feature accompanying dimer stabilization. In particular, CSPs indicate rearrangement of the EF loop, a target for various neutralizing antibodies. Indeed, treating MNV virions with GCDCA prior to neutralizing antibody exposure abolishes neutralization. These findings advance our understanding of GCDCA-induced structural changes of MNV capsids and experimentally support an intriguing viral immune escape mechanism relying on GCDCA-triggered conformational changes of the P-dimer. Significance StatementThis study sheds light on the role of glycochenodeoxycholic acid (GCDCA) in promoting murine norovirus (MNV) infection and immune escape. Binding of GCDCA to the dimeric P-domain has been well characterized by crystallography and cryo EM studies, showing that upon GCDCA binding, a 90{degrees} rotation of the P-domain occurs, which results in its collapse onto the underlying shell of the virus. Our NMR experiments now reveal P-dimer stability as a new dimension of plasticity of MNV capsids and suggest that capsid contraction is entropically assisted. Conformational changes as a feature of P-dimer stabilization eliminate recognition by neutralizing antibodies, no longer being able to prevent infection. These findings highlight key differences between human and MNV capsid structures, promote our understanding of MNV infection on a molecular level, and reveal a novel immune escape mechanism.

biochemistry↗

bioRxiv2020

Evidence for nutrient-dependent regulation of the COPII coat by O-GlcNAcylation

O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is a dynamic form of intracellular glycosylation common in animals, plants and other organisms. O-GlcNAcylation is essential in mammalian cells and is dysregulated in myriad human diseases, such as cancer, neurodegeneration and metabolic syndrome. Despite this pathophysiological significance, key aspects of O-GlcNAc signaling remain incompletely understood, including its impact on fundamental cell biological processes. Here, we investigate the role of O-GlcNAcylation in the coat protein II complex (COPII), a system universally conserved in eukaryotes that mediates anterograde vesicle trafficking from the endoplasmic reticulum. We identify new O-GlcNAcylation sites on Sec24C, Sec24D and Sec31A, core components of the COPII system, and provide evidence for potential nutrient-sensitive pathway regulation through site-specific glycosylation. Our work suggests a new connection between metabolism and trafficking through the conduit of COPII protein O-GlcNAcylation.

cell biology↗

bioRxiv2020

MapA, a second large RTX adhesin, contributes to biofilm formation by Pseudomonas fluorescens

Mechanisms by which cells attach to a surface and form a biofilm are diverse and differ greatly between organisms. The Gram-negative, Gammaproteobacterium Pseudomonas fluorescens attaches to a surface through the localization of the large type 1-secreted RTX adhesin LapA to the outer surface of the cell. LapA localization to the cell surface is controlled by the activities of a periplasmic protease, LapG and an inner-membrane spanning cyclic di-GMP responsive effector protein, LapD. A previous study identified a second, LapA-like protein encoded in the P. fluorescens Pf0-1 genome: Pfl01_1463. However, deletion of this gene had no discernible phenotype under our standard laboratory growth conditions. Here, we identified specific growth conditions wherein, Pfl01_1463, hereafter called MapA (Medium Adhesion Protein A) is a functional adhesin contributing to biofilm formation. This adhesin, like LapA, appears to be secreted through a Lap-related type 1 secretion machinery. We show MapA involvement in biofilm formation is also controlled by LapD and LapG, and that the differing roles of LapA and MapA in biofilm formation are achieved, at least in part, through the differences in the sequences of the two adhesins and their differential, cyclic di-GMP-dependent transcriptional regulation. This differential regulation appears to lead to different distributions of the expression of lapA and mapA within a biofilm. Our data indicate that the mechanisms by which a cell forms a biofilm and the components of a biofilm matrix can differ depending on growth conditions in the biofilm. ImportanceAdhesins are critical for the formation and maturation of bacterial biofilms. We identify a second adhesin in P. fluorescens, called MapA, which appears to play a role in biofilm maturation and whose regulation is distinct from the previously reported LapA adhesin, which is critical for biofilm initiation. Analysis of bacterial adhesins show that LapA-like and MapA-like adhesins are found broadly in Pseudomonads and related organisms, indicating that the utilization of different suites of adhesins may be broadly important in the Gammaproteobacteria.

microbiology↗

bioRxiv2019

Integrated meditation and exercise therapy: A randomized controlled trial of a combined non-pharmacological intervention reduces disability and pain in patients with chronic low back pain

Integrative and complementary non-pharmacological treatments have proven efficacious in treating both the physiological and psychological symptoms of chronic pain conditions but the potential of many combined therapies is unknown. This study examined the effects of a combined intervention of mindfulness meditation followed by aerobic walking exercise in chronic low back pain (cLBP) patients. We hypothesized that meditation before exercise would reduce disability and pain by increasing mindfulness prior to physical activity. Thirty-eight adults completed either meditation and exercise treatment (MedExT) (n=18) or an audiobook control condition (n=20). Over a 4-week period, participants in the MedExT group performed 12-17 minutes of guided meditation followed by 30 minutes of moderate intensity walking exercise 5 days per week. Measures of disability, pain, mindfulness and anxiety were taken at baseline and post-intervention. Ratings of pain were also assessed by participant self-report, before and after each intervention session. Following MedExT, participants showed significant improvement in our primary outcome of disability compared to the control group (p<0.05). From pre to post-intervention, MedExT also increased mindfulness (p<0.05), but had no significant effect on quantitative sensory testing on the low back. Mean ratings of low back pain intensity and unpleasantness significantly improved with MedExT from before the study to during participation, respectively (intensity p<0.05; unpleasantness p<0.05). Overall, four weeks of MedExT produced substantive changes in disability, mindfulness and measures of pain intensity and unpleasantness.

clinical trials↗