Biology subjects

Johnson, J.

Publications and source records attributed to Johnson, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Computational Screening of Filamin Mechanical Binding Proteins using AlphaFold2

Filamins are dimeric actin binding protein that play a critical role in mechanical signaling. They contain a mechanosensory region (MSR) that naturally folds into a globular closed conformation. Under mechanical stress, the MSR unfolds into an open conformation, exposing binding sites for numerous proteins. Filamins are involved in diverse cellular functions, and their mechanical binding targets are highly context dependent. In this study, we employed AlphaFold2 modeling for screening proteins that specifically recognize the open conformation of filamins. We focused on the Drosophila melanogaster filamin, Cheerio, and conducted a biased screen to identify mechanical binding proteins. We selected the top 132 hits from the initial screening for further characterization. All identified binding proteins specifically recognize the open conformation of the MSR and not the closed conformation. Interestingly, the binding regions of these proteins lack obvious sequence similarity. While some false positives were identified, they could be effectively filtered out based on the secondary structure formed at the binding interface. This study provides a framework for identifying specifically filamin interactions in mechanosignaling.

bioinformatics↗

bioRxiv2025

On modeling ovarian aging and menopause timing

Mathematical modeling of ovarian aging and menopause timing has a long history, dating back a half-century to the models of Nobel Prize winner Robert G. Edwards. More recently, such models have been used to investigate clinical interventions for women, which underscores the importance of scientific rigor in model development and analysis. In this paper, we analyze a recent model published in the biophysics literature. We first correct an error which invalidates claims about menopause age in different populations. We then use stochastic analysis to show how this model is a reparameterization of a prior model and put it in the framework of several prior models, which enables the application of extreme value theory. We prove some general extreme value theory results and use them to obtain detailed estimates of menopause age in this model. In particular, we derive a new expected menopause age formula which is orders of magnitude more accurate than the previous heuristic estimate. We further obtain rigorous analytical estimates of the full menopause age distribution and all its moments. We conclude by using these mathematical results to elucidate the physiological sources of menopause age variability.

physiology↗

bioRxiv2025

Automated Quality Control of Time-Course Imaging from 3D in vitro cultures

Longitudinal imaging of 3D cell cultures like tumor organoids and spheroids offers crucial insights into cancer progression and treatment. However, spatial displacement during time-course imaging, caused by matrix detachment or experimental artifacts, can confound analyses. Existing computational methods struggle to address this issue. We present a new algorithm to evaluate data integrity and rectify mislabeling in longitudinal imaging of 3D cell culture. Our algorithm integrates permutation-based optimization with Procrustes analysis. By using X and Y coordinates of images, it accurately reorders, matches, and aligns object positions across time points, correcting for rotation, translation, and small movements. Validation with simulated data confirmed its accuracy and robustness. Applied to longitudinal imaging of tumor spheroids, our algorithm revealed frequent displacement amongst the spheroids between time points and corrected many mislabeled images. This computationally efficient and adaptable method needs no experimental adjustments and presents a readily accessible solution for data quality control. MotivationThree-dimensional (3D) in vitro models, such as tumor organoids and spheroids embedded in an extracellular matrix, are increasingly vital for studying normal and disease biology, including drug responses.1-3 A key advantage of these models is that imaging platforms can perform continuous longitudinal imaging to track phenotypic changes. However, common issues in 3D techniques, such as matrix shifts during experimental setup or image capture, can introduce technical artifacts that affect downstream analyses. Currently, no automated analytical approaches exist for assessing or correcting technical artifacts. Here, we introduce a robust, automated algorithm for assessing the quality of time-course image data and, in some cases, correcting object mislabeling to enable accurate tracking of individual spheroids over time. This approach relies only on image metadata, requiring no experimental modifications. It offers a readily implementable solution for improving data integrity and reproducibility and enhancing the reliability of longitudinal 3D cell culture studies.

bioengineering↗

bioRxiv2025

A split ALFA tag-nanobody system for protein localization and proximity proteomics in mycobacteria

Tuberculosis remains a globally significant infection and new insights into the biology of Mycobacterium tuberculosis are badly needed. Discovery of protein localization and protein complex composition are powerful approaches to determine protein function, but have not been widely applied in mycobacteria, in part due to technical barriers. Here we develop a multi-functional system that utilizes the ALFA tag and functional protein fusions to an anti-ALFA nanobody (NBALFA) to target proteins in fast and slow growing mycobacteria. Insertion of the ALFA epitope tag on the target protein, coupled with conditional expression of the NBALFA fused to a fluorescent protein faithfully recapitulates cytosolic and membrane protein localization by fluorescent microscopy in living cells. Targeted NBALFA can relocalize an ALFA tagged protein to inclusion bodies or the cytoplasmic membrane, demonstrating enforced protein localization. Finally, conditional expression of the NBALFA fused to TurboID for proximity proteomics allowed identification of known partner proteins of the RNA polymerase complex and the PKS13 mycolic acid biosynthesis protein. We conclude that the split ALFA tag-nanobody system is a flexible platform for discovery protein biology in mycobacteria.

microbiology↗

bioRxiv2025

Efficient gastrointestinal colonization by Campylobacter jejuni requires components of the ChuABCD heme transport system

Previous research demonstrated that Campylobacter jejuni encodes a heme utilization system that facilitates heme-dependent growth under iron-limiting conditions and that transcription of this system is induced during human infection. Despite these observations, it remained unknown whether the heme transport system is required for colonization and disease in a susceptible host. To address this, we created individual non-polar deletion mutants of each component of the heme transport system, as well as a total deletion of the inner membrane transporter, ChuBCD, and examined their ability to promote heme-dependent growth and iron uptake. From this work, we found that only the heme receptor, ChuA, was required for heme-dependent growth and iron acquisition, which supports earlier work of another group. Further, we examined whether intestinal colonization, immune activation, and pathology were altered during infection with these mutants. After establishing that elevated heme and chuABCD expression occurs during C. jejuni infection of IL-10-/- mice, we found that heme transport mutants exhibited significantly reduced fecal shedding and colonization of the cecum and colon. In addition, we found that neutrophil and macrophage recruitment and intestinal pathology often remained intermediately elevated despite decreased bacterial loads. These results suggest that heme utilization promotes efficient colonization and full pathogenicity in C. jejuni, but that neither is completely abrogated in its absence.

microbiology↗

bioRxiv2025

Senolytics Restore Hematopoietic Stem Cells Function in Sickle Cell Disease

Sickle Cell Disease (SCD) is a blood disorder affecting millions worldwide. Emerging evidence reveals that SCD pathophysiology increases risk of myeloid malignancies and hematopoietic stem cell (HSC) dysfunction, possibly due to pathological stress on bone marrow. To investigate this further, we interrogated mice and individuals with SCD and observed extended cell cycle times, oxidative stress, DNA damage, senescence, and dysregulation of molecular programs associated with these processes in bone marrow hematopoietic stem and progenitor cells (HSPCs). Human SCD HSPCs displayed poor hematopoietic potential ex vivo. SCD mice displayed a dramatic loss of transplantable bone marrow HSPCs, which was reversed upon treatment of SCD mice with the senolytic agent, ABT-263 (navitoclax). Thus, senolytics restore bone marrow function during SCD in mice and represent a novel strategy to improve bone marrow health in individuals with SCD and improve the safety of potentially curative gene therapies that utilize autologous HSPCs from individuals with SCD.

cell biology↗

bioRxiv2024

Elovl1 inhibition reduced very long chain fatty acids in a mouse model of adrenoleukodystrophy

Adrenoleukodystrophy (ALD) is a rare neurometabolic disease caused by mutations in the ABCD1 gene, which encodes for the peroxisomal very long chain fatty acid (VLCFAs) transporter. It is a debilitating disorder, which has a spectrum of clinical presentations. The most severe form is a rapidly progressing demyelinating disease called cerebral ALD or CALD. Patients with cALD have a life expectancy of 2-4 years after onset and symptoms often manifest in childhood. The other forms are adrenomyeloneuropathy or AMN, which is a slower progressing degeneration of the spinal cord, and adrenal insufficiency (Addison disease). Since the accumulation of VLCFAs are a common factor in all ALD pathologies, we identified therapeutic approach that could correct this metabolic defect. We developed a substrate reduction therapy (SRT) for ALD in the form of an inhibitor of the lipid elongase principally responsible for the generation of VLCFAs, Elovl1. This small molecule was able to successfully reduce the accumulation of VLCFA in the brain and spinal cord of ABCD1-/y mice. We used single nuclei RNA seq to identify the pathways altered in the ABCD1-/y mouse and corrected with Elovl1 inhibition. Though many lipid metabolism genes and pathways were indeed corrected, treatment with the Elovl1 inhibitor unexpectedly led to profound transcriptional changes beyond correction of pathways altered by loss of ABCD1. These data suggest that Elovl1 inhibition may have broader consequences in ABCD1-/y mice than correction of lipid homeostasis.

pharmacology and toxicology↗

bioRxiv2024

SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo

In late 2019, SARS-CoV-2 spilled-over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions, and compare species, sex, age, virus and antibody detection assay, and types, frequency and length of clinical signs, demonstrating broad heterogeneity amongst infections. We then describe a SARS-CoV-2 outbreak in lions, tigers and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for four months. Lions had significantly more viral RNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health. ImportanceSurveillance and experimental testing have shown many animal species, including companion, wildlife, and conservatory, are susceptible to SARS-CoV-2. Early in the COVID-19 pandemic, big cats at zoological institutions were among the first documented cases of naturally infected animals; however, challenges in the ability to collect longitudinal samples in zoo animals have limited our understanding of SARS-CoV-2 kinetics and clearance in these settings. We measured SARS-CoV-2 infections over three months in lions, tigers and hyenas at Denver Zoo, and detected viral RNA, infectious virus, neutralizing antibodies, and recrudescence after initial clearance. We found lions had longer and higher levels of virus compared to the other species. All animals were infected by a rare viral lineage circulating in the human population, suggesting a single spillover followed by interspecies transmission. These data are important in better understanding natural SARS-CoV-2 spillover, spread and infection kinetics within multiple species of zoo animals.

microbiology↗

bioRxiv2024

Time-on-task-related decrements in performance in the rodent continuous performance test are not caused by physical disengagement from the task

Attention deficits, a hallmark of many neuropsychiatric disorders, significantly impair quality of life and functional outcome for patients. Continuous Performance Tests (CPTs) are widely used to assess attentional function in clinical settings and have been adapted for mice as the rodent Continuous Performance Test (rCPT). In this study, we combined traditional analyses of rCPT performance with markerless pose estimation using DeepLabCut and visual field analysis (VFA) to objectively measure the orientation of mice toward stimuli during rCPT sessions. Additionally, we extended session lengths to assess performance decrements over time. Our findings show that extending rCPT sessions from 45 to 90 minutes results in a significant decline in performance in male mice, which aligns with performance decrements observed in clinical research. Importantly, physical engagement with the task remained relatively stable throughout the session, even as performance deteriorated. This suggests that the performance decline specifically reflects a time-on-task (TOT)-dependent vigilance decrement rather than physical disengagement. We also investigated the effects of amphetamine, an FDA-approved treatment for attention-deficit/hyperactivity disorder (ADHD), on rCPT performance. Amphetamine significantly improved rCPT performance in male mice by reducing false alarms without modulating orientation or physical engagement with the task stimuli. Collectively, these findings validate a behavioral tracking platform for objectively measuring physical engagement in the rCPT and a task modification that accentuates TOT-dependent performance decrements, enhancing the translational value of the rCPT for studies related to human neuropsychiatric disorders.

neuroscience↗

bioRxiv2024

High-Throughput Assay for Predicting Diarrhea Risk Using a 2D Human Intestinal Stem Cell-Derived Model

Gastrointestinal toxicities (GITs) are the most prevalent adverse events (AE) reported in clinical trials, often resulting in dose-limitations that reduce drug efficacy and delay development and treatment optimization. Preclinical animal models do not accurately replicate human GI physiology, leaving few options for early detection of GI side effects prior to human studies. Development of an accurate model that predicts GIT earlier in drug discovery programs would better support successful clinical trial outcomes. Chemotherapeutics, which exhibit high rates of clinical GIT, frequently target mitotic cells. Therefore, we hypothesized that a model utilizing proliferative cell populations derived from human intestinal crypts would predict the occurrence of clinical GITs with high accuracy. Here, we describe the development of a multiparametric assay utilizing the RepliGut(R) Planar system, an intestinal stem cell-derived platform cultured in an accessible high throughput Transwell format. This assay addresses key physiological elements of GIT by assessing cell proliferation (EdU incorporation), cell abundance (DAPI quantification), and barrier function (TEER). Using this approach, we demonstrate that primary proliferative cell populations reproducibly respond to marketed chemotherapeutics at physiologic concentrations. To determine the ability of this model to predict clinical diarrhea risk, we evaluated a set of 30 drugs with known clinical diarrhea incidence in three human donors, comparing results to known plasma drug concentrations. This resulted in highly accurate predictions of diarrhea potential for each endpoint (balanced accuracy of 91% for DAPI, 90% for EdU, 88% for TEER) with minimal variation across human donors. In vitro toxicity screening using primary proliferative cells may enable improved safety evaluations, reducing the risk of AEs in clinical trials and ultimately lead to safer and more effective treatments for patients.

pharmacology and toxicology↗

bioRxiv2024

Phospho-proteomics identifies D-group MAP kinases as substrates of the Arabidopsis tyrosine phosphatase RLPH2

Despite being one of the few bona fide plant tyrosine phosphatases, RLPH2 has no known substrates. Utilizing phospho-proteomics, we identified the activation loop phospho-tyrosine of several D-group mitogen activated protein kinases (MPKs) as potential RLPH2 substrates. All Arabidopsis D-Group MPKs possess a TDY activation loop phosphorylation motif, whereas other MPKs (Groups A, B and C) contain a TEY motif. Our findings reveal that RLPH2 has a strong preference for aspartate (D) in the TXY motif, providing specificity for RLPH2 to exclusively target and dephosphorylate the D-Group MPKs. Additionally, D-Group MPKs contain a unique activation loop insertion that conforms to a protein phosphatase 1 (PP1) binding motif, with findings presented here confirming Arabidopsis PP1 phosphatases dock at this site. Intriguingly, only D-group MPKs among all identified Arabidopsis protein kinases possess this PP1 recruiting motif. Using multiple RLPH2 deficient plant lines, we demonstrate that RLPH2 represses seed dormancy release. Overall, this work highlights the power of phospho-proteomics in identifying substrates of this novel plant tyrosine phosphatase, while also revealing new complexities in the interactions between MPK activation loops and multiple phospho-mediated cell signaling events. One sentence summaryPhospho-proteomic analysis reveals Arabidopsis tyrosine phosphatase RLPH2 dephosphorylates the activation loop of D-Group mitogen activated protein kinases.

plant biology↗

bioRxiv2024

Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved post-translational modification that facilitates rapid and reversible modulation of enzyme activity, localization or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in non-tumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the epidermal growth factor receptor (EGFR) inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (PKM, PGAM1) and two novel (GSTP1, GLUD1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference (CRISPRi) system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous re-expression of wild-type, phosphomutant or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

biochemistry↗

bioRxiv2024

Characterization of West Nile virus Koutango lineage from Phlebotomine Sandflies in Kenya 2021

The West Nile virus (WNV), primarily transmitted by mosquitoes, is one of the most widespread flaviviruses globally, with past outbreaks occurring in the USA and Europe. Recent studies in parts of Africa, including Kenya, have identified the West Nile virus Koutango lineage (WN-KOUTV) among phlebotomine sandfly populations, however, our understanding of this virus remains limited. Hence, this study aimed to characterize WN-KOUTV from phlebotomine sandflies. Sandflies were sampled between 12-16th March 2021 from six villages in Baringo South, Kenya, using CDC light traps. Female sandflies were taxonomically identified and pooled based on genus. Virus isolation was performed in Vero cells. Viral genome was determined using next-generation sequencing. Phylogenetic and molecular clock analyses were done to decipher the viruss evolutionary relationships. Comparative analyses of amino acid sequences were performed to determine variations. Protein modeling in Pymol was conducted to elucidate variations in key protein regions. Evolutionary pressure analysis investigated the selection pressures on the virus. In vitro experiments were done to investigate the virus growth kinetics in mammalian (Vero-E6) and mosquito (C636) cells. We report the isolation of WN-KOUTV from Salabani Baringo South, Kenya. The isolated WN-KOUTV clustered with previously identified WN-KOUTV strains. Comparative analysis revealed unique amino acid at NS5 653. Diversifying pressure was acting NS3 267 of the WN-KOUTV lineage. WN-KOUTV replicates efficiently in Vero-E6 and C636 cells comparable to West Nile virus Lineage 1a, isolated from mosquitoes. The isolation of WN-KOUTV in sandflies points to them as potential vectors, however, vector competence studies would confirm this. The efficient replication in mammalian and mosquito cell lines elucidated its adaptability to host and vector. We speculate the close genetic relationship of WN-KOUTV strains is enabled by the bird migratory route between East and West Africa. If proven, this may point to a potential future pandemic pathway for this virus.

microbiology↗

bioRxiv2024

Resolved Genomes of Wastewater ESBL-Producing Escherichia coli and Metagenomic Analysis of Source Wastewater Samples

Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli pose a serious threat to human health because of their resistance to the most commonly prescribed antibiotics: penicillins and cephalosporins. In this study, we provide a genomic and metagenomic context for the determinant ESBL genes of E. coli isolated from various wastewater treatment utilities in Oregon, USA. Class A beta-lactamase genes on chromosomes (blaCTX-M, blaTEM) were clustered with antibiotic resistance genes associated with other classes of antibiotics (sulfonamides and aminoglycosides) along with insertional elements. ESBL genes such as blaCTX-M, blaTEM, and blaSHV were also detected on conjugable plasmids of IncF and IncI incompatibility types. One novel IncF plasmid (pSHV2A_ESBLF) was identified in which carried a multi-drug resistance genotype (blaSHV-2A, aadA22, aac(3), aph(6), tetA, and sul1) in addition to a mer (mercury resistance) operon, colicin, and aerobactin genes. Shotgun metagenomic analysis of the E. coli-originating wastewater samples showed the presence of class A beta-lactamases; however, the ESBL genes identified in the E. coli genomes were below the detection limits. Other ESBL-associated genes (i.e., blaOXA.11, blaFOX.7, and blaGES.17) were identified in the wastewater samples and their occurrences were correlated with the core microbial genera (e.g., Paraprevotella). In both the E. coli genomes and the wastewater samples, tetracycline, aminoglycoside, and beta-lactam resistance determinants frequently co-occurred. The unique combination of whole-genome and metagenomic analysis provides a holistic description of ESBL-producing organisms and genes in the Oregonian wastewater system.

microbiology↗

bioRxiv2024

The Chlamydia trachomatis Inc Tri1 interacts with TRAF7 to displace native TRAF7 interacting partners

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and of preventable blindness worldwide. This obligate intracellular pathogen replicates within a membrane-bound inclusion, but how it acquires nutrients from the host while avoiding detection by the innate immune system is incompletely understood. C. trachomatis accomplishes this in part through the translocation of a unique set of effectors into the inclusion membrane, the inclusion membrane proteins (Incs). Incs are ideally positioned at the host-pathogen interface to reprogram host signaling by redirecting proteins or organelles to the inclusion. Using a combination of co-affinity purification, immunofluorescence confocal imaging, and proteomics, we characterize the interaction between an early-expressed Inc of unknown function, Tri1, and tumor necrosis factor receptor associated factor 7 (TRAF7). TRAF7 is a multi-domain protein with a RING finger ubiquitin ligase domain and a C-terminal WD40 domain. TRAF7 regulates several innate immune signaling pathways associated with C. trachomatis infection and is mutated in a subset of tumors. We demonstrate that Tri1 and TRAF7 specifically interact during infection and that TRAF7 is recruited to the inclusion. We further show that the predicted coiled-coil domain of Tri1 is necessary to interact with the TRAF7 WD40 domain. Finally, we demonstrate that Tri1 displaces the native TRAF7 binding partners, mitogen activated protein kinase kinase kinase 2 (MEKK2) and MEKK3. Together, our results suggest that by displacing TRAF7 native binding partners, Tri1 has the capacity to alter TRAF7 signaling during C. trachomatis infection. ImportanceChlamydia trachomatis is the leading cause of bacterial sexually transmitted infections in the US and preventable blindness worldwide. Although easily treated with antibiotics, the vast majority of infections are asymptomatic and therefore go untreated, leading to infertility and blindness. This obligate intracellular pathogen evades the immune response, which contributes to these outcomes. Here, we characterize the interaction between a C. trachomatis secreted effector, Tri1, and a host protein involved in innate immune signaling, TRAF7. We identified host proteins that bind to TRAF7 and demonstrate that Tri1 can displace these proteins upon binding to TRAF7. Remarkably, the region of TRAF7 to which these host proteins bind is often mutated in a subset of human tumors. Our work suggests a mechanism by which Tri1 may alter TRAF7 signaling and has implications not only in the pathogenesis of C. trachomatis infections, but also in understanding the role of TRAF7 in cancer.

microbiology↗

bioRxiv2024

High phenotypic and genotypic plasticity among strains of the mushroom-forming fungus Schizophyllum commune

Schizophyllum commune is a mushroom-forming fungus notable for its distinctive fruiting bodies with split gills. It is used as a model organism to study mushroom development, lignocellulose degradation and mating type loci. It is a hypervariable species with considerable genetic and phenotypic diversity between the strains. In this study, we systematically phenotyped 16 dikaryotic strains for aspects of mushroom development and 18 monokaryotic strains for lignocellulose degradation. There was considerable heterogeneity among the strains regarding these phenotypes. The majority of the strains developed mushrooms with varying morphologies, although some strains only grew vegetatively under the tested conditions. Growth on various carbon sources showed strain-specific profiles. The genomes of seven monokaryotic strains were sequenced and analyzed together with six previously published genome sequences. Moreover, the related species Schizophyllum fasciatum was sequenced. Although there was considerable genetic variation between the genome assemblies, the genes related to mushroom formation and lignocellulose degradation were well conserved. These sequenced genomes will provide a solid basis for functional genomics analyses of the strains of S. commune.

microbiology↗

bioRxiv2024

Longitudinal profiling of the microbiome at four body sites reveals core stability and individualized dynamics during health and disease

To understand dynamic interplay between the human microbiome and host during health and disease, we analyzed the microbial composition, temporal dynamics, and associations with host multi-omics, immune and clinical markers of microbiomes from four body sites in 86 participants over six years. We found that microbiome stability and individuality are body-site-specific and heavily influenced by the host. The stool and oral microbiome were more stable than the skin and nasal microbiomes, possibly due to their interaction with the host and environment. Also, we identified individual-specific and commonly shared bacterial taxa, with individualized taxa showing greater stability. Interestingly, microbiome dynamics correlated across body sites, suggesting systemic coordination influenced by host-microbial-environment interactions. Notably, insulin-resistant individuals showed altered microbial stability and associations between microbiome, molecular markers, and clinical features, suggesting their disrupted interaction in metabolic disease. Our study offers comprehensive views of multi-site microbial dynamics and their relationship with host health and disease. Study HighlightsO_LIThe stability of the human microbiome varies among individuals and body sites. C_LIO_LIHighly individualized microbial genera are more stable over time. C_LIO_LIAt each of the four body sites, systematic interactions between the environment, the host and bacteria can be detected. C_LIO_LIIndividuals with insulin resistance have lower microbiome stability, a more diversified skin microbiome, and significantly altered host-microbiome interactions. C_LI

microbiology↗

bioRxiv2024

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome

While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. EF-Gs indispensability is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of E. coli EF-G induced by mutations that closely aligned with eEF2s Thr56 residue. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNAPhe-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which wasnt observed when the mutants interact solely with the sarcin/ricin loop. This study provides new insights into EF-Gs adaptability and sheds light on the modification mechanism of human eEF2.

biophysics↗