Biology subjects

Miles, J.

Publications and source records attributed to Miles, J..

17 recordsLinked to original sources

bioRxiv2025

Virally encoded interleukin-6 (vIL-6) coordinates with human IL-6 to modulate cytokine expression during KSHV infection

Kaposi Sarcoma-Associated Herpesvirus (KSHV) is characterized by its ability to establish lifelong infections that can lead to disease in immunocompromised individuals. Understanding the complex interactions between KSHV, the host cellular machinery, and the immune response is essential for unraveling the mechanisms underlying viral persistence and pathogenesis. KSHV extensively remodels the host gene expression landscape by inducing widespread RNA decay via the viral endoribonuclease SOX. While the vast majority of cellular mRNAs are degraded by SOX, select transcripts including Interleukin 6 (IL-6) escape degradation. Interestingly, KSHV encodes a viral homolog of IL-6, known as viral interleukin-6 (vIL-6). Given the importance of IL-6 for KSHV biology and its ability to escape SOX-mediated decay, we explored how vIL-6 expression is regulated upon KSHV infection. Our data demonstrate that unlike IL-6, vIL-6 3UTR does not provide protection from SOX degradation, yet vIL-6 mRNA steady state levels remain high throughout KSHV lytic cycle, suggesting a distinct transcriptional regulation. To assess the contributions of vIL-6 and IL-6 in modulating host gene expression, inflammatory signaling and KSHV-driven pathogenesis, we used a vIL-6 null viral mutant combined with an IL-6 knock down to perform transcriptomic analyses. This analysis revealed that depletion of IL-6, vIL-6 or both results in distinct alterations of the host gene expression environment, with differential effects on chemokine signaling and related pathways as well as a synergistic effect when both IL-6 and vIL-6 are present. Furthermore, functional assays confirmed vIL-6 alone is sufficient to activate key signaling pathways and pro-inflammatory responses, including the JAK/STAT pathway. Collectively, our findings suggest a mechanistic synergy between vIL-6 and IL-6 that may enhance KSHV-mediated immune modulation and contribute to viral persistence and tumorigenesis.

microbiology↗

bioRxiv2025

IL-6 Evades KSHV-Mediated Hyperadenylation repression via CRM1-Dependent Nuclear Export.

RNA turnover is critical for regulating cellular homeostasis, with nuclear export representing a key step in mRNA fate. During infection by Kaposis Sarcoma-associated Herpesvirus (KSHV), widespread mRNA decay is mediated by the viral endonuclease SOX, which depletes cytoplasmic transcripts and induces secondary nuclear RNA processing defects. One such defect includes transcript hyperadenylation, which promotes nuclear retention and decay. However, a subset of mRNAs escapes both SOX degradation and nuclear retention, raising questions about their export mechanisms. Here, we investigate how KSHV infection impacts mRNA poly(A) tail length and nuclear export dynamics using poly(A)-sequencing in KSHV-positive cells. Our data confirm a global increase in poly(A) tail length during KSHV infection, yet we identified a group of hyperadenylated transcripts that remain localized in the cytoplasm, suggesting active evasion of nuclear retention. Notably, we focused on interleukin-6 (IL-6), an mRNA known to escape SOX-mediated decay. Using G/I tailing and sPAT assays, we show that IL-6 is hyperadenylated yet, exported. We demonstrate that its export is dependent upon the CRM1 nuclear export pathway, rather than through the canonical NXF1-NXT1 pathway. Inhibition of CRM1 impairs IL-6 nuclear export and reduces steady-state mRNA levels, implicating CRM1 export in the stabilization of this transcript. Our findings reveal a previously unrecognized mechanism by which select host mRNAs like IL-6, bypass KSHV-imposed nuclear export block, thereby preserving their cytoplasmic function during infection. This study highlights viral manipulation of RNA processing and export pathways as a critical determinant of transcript fate and identifies CRM1 as a key mediator of selective transcript preservation during KSHV infection. SIGNIFICANCE STATEMENTKaposis sarcoma-associated herpesvirus (KSHV) globally disrupts host gene expression by inducing host shutoff which triggers a global repression of the host transcriptome via widespread RNA decay, RNA, nascent transcript hyperadenylation and nuclear export blocks, yet a subset of transcripts escape this repression. This study reveals that despite acquiring long poly(A) tails, select host mRNAs such as IL-6 evade nuclear retention by using the alternative CRM1-dependent export pathway and remain stable in the cytoplasm. These findings challenge the prevailing model that hyperadenylation alone dictates nuclear decay and uncover a selective mechanism by which crucial host transcripts bypass KSHV-mediated gene repression. Understanding this selective escape provides new insights into host-virus interactions and highlights CRM1 as a potential therapeutic target in KSHV-associated diseases.

microbiology↗

bioRxiv2024

Nuclear RNA forms an interconnected network of transcription-dependent and tunable microgels

Living cells are organised through active, non-equilibrium processes, yet the physical principles governing their material states remain unclear. The cell nucleus has been described as either fluid-like or solid-like, but these behaviours have not been reconciled within a single framework. Here we show that nascent RNA and the multivalent RNA-binding protein SAF-A assemble into a nucleus-spanning microgel network maintained near the sol-gel transition. This active network exhibits scale- and time-dependent viscoelasticity and naturally reconciles solid-like and fluid-like responses. By integrating super-resolution imaging with mechanistic polymer modelling, we identify RNA production and degradation as control parameters that tune network connectivity. The model predicts enhanced susceptibility to modest perturbations near criticality, which we confirm experimentally by shifting the system between sol-like and gel-like regimes. Measurements of molecular transport, combined with simulations, reveal size-dependent diffusion and transient trapping within gel pores, establishing direct physical consequences of the near-critical state. Our results identify RNA metabolism as an energy-driven regulator of nuclear material properties and position the nucleus as a living example of an active microgel poised near criticality.

molecular biology↗

bioRxiv2023

Massively parallel mutant selection identifies genetic determinants of Pseudomonas aeruginosa colonization of Drosophila melanogaster

Pseudomonas aeruginosa is recognized for its ability to colonize diverse habitats and cause disease in a variety of hosts, including plants, invertebrates, and mammals. Understanding how this bacterium is able to occupy wide-ranging niches is important for deciphering its ecology. We used transposon sequencing (Tn-Seq, also known as INSeq) to identify genes in P. aeruginosa that contribute to fitness during colonization of Drosophila melanogaster. Our results reveal a suite of critical factors, including those that contribute to polysaccharide production, DNA repair, metabolism, and respiration. Comparison of candidate genes with fitness determinants discovered in previous studies of P. aeruginosa identified several genes required for colonization and virulence determinants that are conserved across hosts and tissues. This analysis provides evidence for both the conservation of function of several genes across systems, as well as host-specific functions. These findings, which represent the first use of transposon sequencing of a gut pathogen in Drosophila, demonstrate the power of Tn-Seq in the fly model system and advance existing knowledge of intestinal pathogenesis by D. melanogaster, revealing bacterial colonization determinants that contribute to a comprehensive portrait of P. aeruginosa lifestyles across habitats. ImportanceDrosophila melanogaster is a powerful model for understanding host-pathogen interactions. Research with this system has yielded notable insights into mechanisms of host immunity and defense, many of which emerged from analysis of bacterial mutants defective for well-characterized virulence factors. These foundational studies - and advances in high-throughput sequencing of transposon mutants - support unbiased screens of bacterial mutants in the fly. To investigate mechanisms of host-pathogen interplay and exploit the tractability of this model host, we used a high-throughput, genome-wide mutant analysis to find genes that enable a pathogen, P. aeruginosa, to colonize the fly. Our analysis reveals critical mediators of P. aeruginosa establishment in its host, some of which are required across fly and mouse systems. These findings demonstrate the utility of massively parallel mutant analysis and provide a platform for aligning the fly toolkit with comprehensive bacterial genomics.

microbiology↗

bioRxiv2022

Evaluating Stored Platelet Shape Change Using Imaging Flow Cytometry

Platelets are routinely stored at room temperature for 5-7 days before transfusion. Stored platelet quality is traditionally assessed by Kunickis morphology score. This method requires extensive training, experience, and is highly subjective. Moreover, the number of laboratories familiar with this technique is decreasing. Cold storage of platelets has recently regained interest because of potential advantages such as reduced bacterial growth and preserved function. However, platelets exposed to cold temperatures change uniformly from a discoid to a spherical shape, reducing the morphology score outcomes to a binary "spheres versus discs" during cooling. We developed a simpler, unbiased screening tool to measure temperature-induced platelet shape change using imaging flow cytometry. When reduced to two dimensions, spheres appear circular, while discs are detected on a spectrum from fusiform to circular. We defined circular events as having a transverse axis of > 0.8 of the longitudinal axis and fusiform events [≤] 0.8 of the longitudinal axis. We show that most EGTA-treated platelets and spherical beads are detected in the gate for circular events, while fresh platelets are found mainly in the gate for fusiform events. Using this assay, mouse and human platelets show a temperature and time-dependent, two-dimensional shape change from fusiform to circular, consistent with their three-dimensional change from discs to spheres. The method we describe here is a valuable tool for detecting shape change differences in response to agonists or temperature and will help screen for therapeutic measures to mitigate the cold-induced storage lesion. Plain language summaryO_ST_ABSWhat is the context?C_ST_ABSO_LIPlatelets for transfusion are currently stored for 5-7 days at room temperature, increasing the risk for bacterial growth C_LIO_LICold storage reduces the risk for bacterial growth but reduces circulation time C_LIO_LIStored platelet quality can be assessed by the light microscopy-based Morphology Score, first described in the 1970s C_LIO_LIDownsides of the Morphology Score include subjectivity, extensive training, and reduced availability in platelet laboratories. C_LI What is new?O_LIIn this study, we provide data showing that the Morphology score is reduced to a binary spheres versus discs response in cold-exposed platelets C_LIO_LIWe developed an imaging flow cytometry-based approach to quantify platelets response to cold based on the two-dimensional projection of the three-dimensional shapes, i.e., fusiform (discoid) versus circular (discoid and spherical) C_LIO_LIWe provide validation of this approach in mouse and human platelets C_LIO_LIWe provide evidence for increased temperature sensitivity in mouse platelets C_LI What is the impact?O_LIThis study provides an easy and unbiased tool for laboratories working on circumventing the cold-induced storage lesion or documenting spherical shape change in general C_LI

pathology↗

bioRxiv2022

Nonstructural protein 1 (nsp1) widespread RNA decay phenotype varies among Coronaviruses

AO_SCPLOWBSTRACTC_SCPLOWExtensive remodeling of the host gene expression environment by coronaviruses nsp1 proteins is a well-documented and conserved piece of the coronavirus-host takeover battle. However, whether and how the underlying mechanism of regulation or the transcriptional target landscape differ amongst coronaviruses remains mostly uncharacterized. In this study we use comparative transcriptomics to investigate the diversity of transcriptional targets between four different coronavirus nsp1 proteins (from MERS, SARS1, SARS2 and 229E). In parallel, we performed Affinity Purification followed by Mass-Spectrometry to identify common and divergent interactors between these different nsp1. For all four nsp1 tested, we detected widespread RNA destabilization, confirming that both - and {beta}-Coronavirus nsp1 broadly affect the host transcriptome. Surprisingly, we observed that even closely related nsp1 showed little similarities in the clustering of genes targeted. Additionally, we show that the RNA targeted by nsp1 from the -CoV 229E partially overlapped with MERS nsp1 targets. Given MERS nsp1 preferential targeting of nuclear transcripts, these results may indicate that these nsp1 proteins share a similar targeting mechanism. Finally, we show that the interactome of these nsp1 proteins differ widely. Intriguingly, our data indicate that the 229E nsp1, which is the smallest of the nsp1 proteins tested here, interacts with the most host proteins, while MERS nsp1 only engaged with a few host proteins. Collectively, our work highlights that while nsp1 is a rather well-conserved protein with conserved functions across different coronaviruses, its precise effects on the host cell is virus specific. SO_SCPLOWIGNIFICANCEC_SCPLOWCoronaviruses extensively co-opt their host gene expression machinery in order to quicky benefit from the host resources. The viral protein nsp1 plays a major role in this takeover as nsp1 is known to induce a widespread shutdown of the host gene expression, both at the RNA and the translational level. Previous work characterized the molecular basis for nsp1-mediated host shutdown. However, this was mostly conducted in the context of {beta}-coronaviruses and in particular SARS-CoV1, CoV2 and MERS due to the important public health burden that these viruses represent. Here instead, we explored the impact of nsp1 on the host using a comparative approach, defining the influence of 4 nsp1 protein from - and {beta}-coronaviruses. We delineated the impact of these 4 nsp1 on the host transcriptome and mapped their interactome. We revealed that host target range and interactomes vary widely among different nsp1, suggesting a viral-specific targeting. Understanding how these differences shape infection will be important to better inform antiviral drug development.

microbiology↗

bioRxiv2022

Transcellular chaperone signaling is an intercellular stress-response distinct from the HSF-1 mediated HSR

Organismal proteostasis is maintained by intercellular signaling processes including cell nonautonomous stress responses such as transcellular chaperone signaling (TCS). When TCS is activated upon tissue-specific knockdown of hsp-90 in the C. elegans intestine, heat-inducible hsp-70 is induced in muscle cells at the permissive temperature resulting in increased heat stress resistance and lifespan extension. However, our understanding of the molecular mechanism and signaling factors mediating transcellular activation of hsp-70 expression from one tissue to another is still in its infancy. Here we conducted a combinatorial approach using transcriptome RNA-Seq profiling and a forward genetic mutagenesis screen to elucidate how stress-signaling from the intestine to the muscle is regulated. We find that the TCS-mediated "gut-to-muscle" induction of hsp-70 expression is suppressed by HSF-1 and instead relies on transcellular-X-cross-tissue (txt) genes. We identify a key role for the PDZ-domain guanylate cyclase txt-1 and the homeobox transcription factor ceh-58 as signaling hubs in the stress receiving muscle cells to initiate hsp-70 expression and facilitate TCS-mediated heat stress resistance and lifespan extension. Our results provide a new view on cell-nonautonomous regulation of "inter-tissue" stress responses in an organism that highlight a key role for the gut. Our data suggest that the HSF-1-mediated HSR is switched off upon TCS activation, in favor of an intercellular stress-signaling-route to safeguard survival.

genetics↗

bioRxiv2021

Black Dots: Microcontact-Printed, Reference-Free Traction Force Microscopy

Measuring the traction forces produced by cells provides insight into their behavior and physiological function. Here, we developed a technique (dubbed black dots) that microcontact prints a fluorescent micropattern onto a flexible substrate to measure cellular traction forces without constraining cell shape or needing to detach the cells. To demonstrate our technique, we assessed human platelets, which can generate a large range of forces within a population. We find platelets that exert more force have more spread area, are more circular, and have more uniformly distributed F-actin filaments. As a result of the high yield of data obtainable by this technique, we were able to evaluate multivariate mixed effects models with interaction terms and conduct a clustering analysis to identify clusters within our data. These statistical techniques demonstrated a complex relationship between spread area, circularity, F-actin dispersion, and platelet force, including cooperative effects that significantly associate with platelet traction forces. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/454500v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1da5eeeorg.highwire.dtl.DTLVardef@18e939aorg.highwire.dtl.DTLVardef@17c7198org.highwire.dtl.DTLVardef@105c97c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2021

Enhanced lipogenesis through Pparγ helps cavefish adapt to food scarcity.

Nutrient availability varies seasonally and spatially in the wild. The resulting nutrient limitation or restricted access to nutrients pose a major challenge for every organism. While many animals, such as hibernating animals, evolved strategies to overcome periods of nutrient scarcity, the cellular mechanisms of these strategies are poorly understood. Cave environments represent an extreme example of nutrient deprived environments since the lack of sunlight and therefore primary energy production drastically diminishes the nutrient availability. Here, we used Astyanax mexicanus, which includes river-dwelling surface fish and cave adapted cavefish populations to study the genetic adaptation to nutrient limitations. We show that cavefish populations store large amounts of fat in different body regions when fed ad libitum in the lab. We found higher expression of lipogenesis genes in cavefish livers when fed the same amount of food as surface fish, suggesting an improved ability of cavefish to use lipogenesis to convert available energy into triglycerides for storage into adipose tissue. Moreover, the lipid metabolism regulator, Peroxisome proliferator-activated receptor {gamma} (Ppar{gamma}), is upregulated at both transcript and protein levels in cavefish livers. Chromatin Immunoprecipitation sequencing (ChIP seq) showed that Ppar{gamma} binds cavefish promoter regions of genes to a higher extent than surface fish. Finally, we identified two possible regulatory mechanisms of Ppar{gamma} in cavefish: higher amounts of ligands of the nuclear receptor, and nonsense mutations in per2, a known repressor of Ppar{gamma}. Taken together, our study reveals that upregulated Ppar{gamma} promotes higher levels of lipogenesis in the liver and contributes to higher body fat accumulation in cavefish populations, an important adaptation to nutrient limited environments.

evolutionary biology↗

bioRxiv2020

Immunogenomic profile of colorectal cancer response to immune checkpoint blockade

Colorectal cancers (CRCs) show variable response to immune checkpoint blockade, which can only partially be explained by the variability of tumour mutational burden. To dissect the cellular and molecular determinants of response we performed a multi-omic screen of 721 cancer regions from patients treated with Pembrolizumab (KEYNOTE 177 clinical trial) or Nivolumab. Multi-regional whole exome, RNA and T-cell receptor sequencing show that, within hypermutated CRCs, response to both anti-PD1 agents is not positively associated with tumour mutational burden but with high clonality of immunogenic mutations, expanded T cells, low activation of the WNT pathway and active immune escape mechanisms. Coupling high-dimensional imaging mass cytometry with multiplexed immunofluorescence and computational spatial analysis, we observe that responsive hypermutated CRCs are rich in cytotoxic and proliferating PD1-expressing CD8 cells interacting with high-density clusters of PDL1-expressing antigen presenting macrophages. We propose that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages thus promoting cytotoxic anti-tumour activity.

cancer biology↗

bioRxiv2020

The KSHV ORF20 Protein Interacts With The Viral Processivity Factor ORF59 And Promote Viral Reactivation.

AO_SCPLOWBSTRACTC_SCPLOWUpon KSHV lytic reactivation, rapid and widespread amplification of viral DNA (vDNA) triggers significant nuclear reorganization. As part of this striking shift in nuclear architecture, viral replication compartments are formed as sites of lytic vDNA production along with remarkable spatial remodeling and relocalization of cellular and viral proteins. These viral replication compartments house several lytic gene products that coordinate viral gene expression, vDNA replication, and nucleocapsid assembly. The viral proteins and mechanisms that regulate this overhaul of the nuclear landscape during KSHV replication remain largely unknown. KSHVs ORF20 is a widely conserved lytic gene among all herpesviruses suggesting it may have a fundamental contribution to the progression of herpesviral infection. Here, we utilized a promiscuous biotin ligase proximity labeling method to identify the proximal interactome of ORF20, which includes several replication-associated viral proteins, one of which is ORF59, the KSHV DNA processivity factor. Using co-immunoprecipitation and immunofluorescence assays, we confirmed the interaction between ORF20 and ORF59 and tracked the localization of both proteins to KSHV replication compartments. To further characterize the function of ORF20, we generated an ORF20-deficient KSHV and compared its replicative fitness relative to wild type virus. Virion production was significantly diminished in the ORF20-deficient virus as observed by supernatant transfer assays. Additionally, we tied this defect in viable virion formation to a reduction in viral late gene expression. Lastly, we observed an overall reduction in vDNA replication in the ORF20-deficient virus implying a key role for ORF20 in the regulation of lytic replication. Taken together, these results capture the essential role of KSHV ORF20 in progressing viral lytic infection by regulating vDNA replication alongside other crucial lytic proteins within KSHV replication compartments.

microbiology↗

bioRxiv2020

Expansion of Functional Regulatory T Cells Using Soluble RAGE Prevents Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease with no cure. Therapeutic translation has been hampered by preclinical reproducibility. Here, short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent centers. Treatment with sRAGE increased regulatory T cells (Tregs) within islets, pancreatic lymph nodes and spleen, increasing islet insulin expression and function. Diabetes protection was abrogated by Treg depletion and shown to be dependent on antagonizing RAGE using knockout mice. Human Tregs treated with a RAGE ligand downregulated genes for suppression, migration and Treg homeostasis (FOXP3, IL7R, TIGIT, JAK1, STAT3, STAT5b, CCR4). Loss of suppressive function was reversed by sRAGE, where Tregs increased proliferation and suppressed conventional T cell division, confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes, showing efficacy at multiple research centers and in human T cells.

immunology↗

bioRxiv2019

Computer-assisted EEG diagnostic review for idiopathic generalized epilepsy

Epilepsy diagnosis can be costly, time-consuming and not uncommonly inaccurate. The reference standard diagnostic monitoring is continuous video-EEG monitoring, ideally capturing all events or concordant interictal discharges. Automating EEG data review would save time and resources, thus enabling more people to receive reference standard monitoring and also potentially herald a more quantitative approach to therapeutic outcomes. There is substantial research into automated detection of seizures and epileptic activity from EEG. However, automated detection software is not widely used in the clinic; and, despite numerous published algorithms, few methods have regulatory approval for detecting epileptic activity from EEG.\n\nThis study reports on a deep learning algorithm for computer-assisted EEG review. Deep, convolutional neural networks were trained to detect epileptic discharges using a pre-existing dataset of over 6000 labelled events in a cohort of 103 patients with idiopathic generalized epilepsy (IGE). Patients underwent 24-hour ambulatory outpatient EEG, and all data was curated and confirmed independently by two epilepsy specialists (Seneviratne et al, 2016). The resulting automated detection algorithm was then used to review diagnostic scalp-EEG for seven patients (four with IGE and three with events mimicking seizures) to validate performance in a clinical setting.\n\nThe automated detection algorithm showed state-of-the-art performance for detecting epileptic activity from clinical EEG, with mean sensitivity of >95% and corresponding mean false positive rate of 1 detection per minute. Importantly, diagnostic case studies showed that the automated detection algorithm reduced human review time by 80%-99%, without compromising event detection or diagnostic accuracy. The presented results demonstrate that computer-assisted review can increase the speed and accuracy of EEG assessment and has the potential to greatly improve therapeutic outcomes.

neuroscience↗

bioRxiv2019

An intergenic non-coding RNA promoter required for histone modifications in the human β-globin chromatin domain

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human {beta}-globin genes. Mutational analyses in {beta}-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult {delta}- and {beta}-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring {varepsilon}- and {gamma}-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of {beta}-like globin gene transcription during development.

genetics↗

bioRxiv2018

Evaluation of InSeq to Identify Genes Essential for Pseudomonas aeruginosa PGPR2 Corn Root Colonization

The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the TnSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5-to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.

genomics↗

bioRxiv2018

Hookworm-derived small molecule extracts suppress pathology in a mouse model of colitis and inhibit secretion of key inflammatory cytokines in primary human leukocytes

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Here we use a metabolomics approach to characterize the non-protein small molecule complement of hookworms. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of somatic tissue extracts revealed the presence of 52 polar metabolites and 22 non-polar components including short chain fatty acids (SCFA). Several of these small metabolites, notably the SCFA, have been shown to have anti-inflammatory properties in various diseases, including IBD. Using a murine model of colitis and human peripheral blood mononuclear cells, we demonstrate that somatic tissue extracts of the hookworm Ancylostoma caninum contain small molecules with anti-inflammatory activities. Of the five extracts tested, two of them significantly protected mice against T cell-mediated immunopathology and weight loss in a chemically-induced colitis model. Moreover, one of the anti-colitic extracts suppressed ex vivo production of inflammatory cytokines from primary human leukocytes. While the origin of the SCFA (parasite or host microbiota-derived) present in the hookworm somatic tissue extracts cannot be ascertained from this study, it is possible that A. caninum may be actively promoting an anti-inflammatory host microbiome by facilitating immune crosstalk through SCFA production.

microbiology↗

bioRxiv2017

Aberrant Cortical Activity In Multiple GCaMP6-Expressing Transgenic Mouse Lines

Transgenic mouse lines are invaluable tools for neuroscience but as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically-encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, though rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

neuroscience↗