Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Metabolomic Profiles and Pathways in Osteoarthritic Human Cartilage: A Comparative Analysis with Healthy Cartilage.

Osteoarthritis (OA) is a chronic joint disease with heterogenous metabolic pathology. To gain insight into OA-related metabolism, metabolite extracts from healthy (n = 11) and end-stage osteoarthritic cartilage (n = 35) were analyzed using liquid chromatography-mass spectrometry metabolomic profiling. Specific metabolites and metabolic pathways, including lipid and amino acid pathways, were differentially regulated in osteoarthritis-derived and healthy cartilage. The detected alterations in amino acids and lipids highlighted key differences in bioenergetic resources, matrix homeostasis, and mitochondrial alterations in OA-derived cartilage compared to healthy cartilage. Moreover, the metabolomic profiles of osteoarthritic cartilage separated into four distinct endotypes, highlighting the heterogenous nature of OA metabolism and the diverse landscape within the joint in patients. The results of this study demonstrate that human cartilage has distinct metabolomic profiles in healthy and end-stage OA patients. By taking a comprehensive approach to assess metabolic differences between healthy and osteoarthritic cartilage and within osteoarthritic cartilage alone, several metabolic pathways with distinct regulation patterns were detected. Additional investigation may lead to the identification of metabolites that may serve as valuable indicators of disease status or potential therapeutic targets.

Metabolomic Profiles and Pathways in Osteoarthritic Human Cartilage: A Comparative Analysis with Healthy Cartilage.

Objective: Osteoarthritis (OA) is a chronic joint disease with heterogenous metabolic pathology. To gain insight into OA-related metabolism, healthy and end-stage osteoarthritic cartilage were compared metabolically to uncover disease-associated profiles, classify OA-specific metabolic endotypes, and identify targets for intervention for the diverse populations of individuals affected by OA. Design: Femoral head cartilage (n=35) from osteoarthritis patients were collected post-total joint arthroplasty. Healthy cartilage (n=11) was obtained from a tissue bank. Metabolites from all cartilage samples were extracted and analyzed using liquid chromatography-mass spectrometry metabolomic profiling. Additionally, cartilage extracts were pooled and underwent fragmentation analysis for biochemical identification of metabolites. Results: Specific metabolites and metabolic pathways, including lipid- and amino acid pathways, were differentially regulated between osteoarthritis-derived and healthy cartilage. The detected alterations of amino acids and lipids highlight key differences in bioenergetic resources, matrix homeostasis, and mitochondrial alterations in osteoarthritis-derived cartilage compared to healthy. Moreover, metabolomic profiles of osteoarthritic cartilage separated into four distinct endotypes highlighting the heterogenous nature of OA metabolism and diverse landscape within the joint between patients. Conclusions: The results of this study demonstrate that human cartilage has distinct metabolomic profiles between healthy and end-stage osteoarthritis patients. By taking a comprehensive approach to assess metabolic differences between healthy and osteoarthritic cartilage, and within osteoarthritic cartilage alone, several metabolic pathways with distinct regulation patterns were detected. Additional investigation may lead to the identification of metabolites that may serve as valuable indicators of disease status or potential therapeutic targets.

Metabolomic profiles of cartilage and bone reflect tissue type, radiography-confirmed osteoarthritis, and spatial location within the joint.

Osteoarthritis is the most common chronic joint disease, characterized by the abnormal remodeling of joint tissues including articular cartilage and subchondral bone. However, there are currently no therapeutic drug targets to slow the progression of disease because disease pathogenesis is largely unknown. Thus, the goals of this study were to identify metabolic differences between articular cartilage and subchondral bone, compare the metabolic shifts in osteoarthritic grade III and IV tissues, and spatially map metabolic shifts across regions of osteoarthritic hip joints. Articular cartilage and subchondral bone from 9 human femoral heads were obtained after total joint arthroplasty, homogenized and metabolites were extracted for liquid chromatography-mass spectrometry analysis. Metabolomic profiling revealed that distinct metabolic endotypes exist between osteoarthritic tissues, late-stage grades, and regions of the diseased joint. The pathways that contributed the most to these differences between tissues were associated with lipid and amino acid metabolism. Differences between grades were associated with nucleotide, lipid, and sugar metabolism. Specific metabolic pathways such as glycosaminoglycan degradation and amino acid metabolism, were spatially constrained to more superior regions of the femoral head. These results suggest that radiography-confirmed grades III and IV osteoarthritis are associated with distinct global metabolic and that metabolic shifts are not uniform across the joint. The results of this study enhance our understanding of osteoarthritis pathogenesis and may lead to potential drug targets to slow, halt, or reverse tissue damage in late stages of osteoarthritis.

Untargeted metabolomics profiling of oat (Avena sativa L.) and wheat (Triticum aestivum L.) infested with wheat stem sawfly (Cephus cinctus Norton) reveals differences associated with plant defense and insect nutrition.

Introduction: Wheat stem sawfly (WSS), Cephus cinctus Norton, is a major pest of common bread wheat (Triticum aestivum L.) and other cultivated cereals in North America. Planting of cultivars with solid stems has been the primary management strategy to prevent yield loss due to WSS infestation, however expression of this phenotype can vary depending on environmental conditions and solid stems hinder biological control of WSS via braconid parasitoids Bracon cephi (Gahan) and Bracon lissogaster Muesebeck. In the hollow stems of oat (Avena sativa L.), WSS larvae experience 100% mortality before they reach late instars, but the mechanisms for this observed resistance have not been characterized. Objective: The objective of this study was to explore additional sources of resistance outside of the historic solid stem phenotype. Methods: Here, we use an untargeted metabolomics approach to examine the response of the metabolome of two cultivars of oat and four cultivars of spring wheat to infestation by WSS. Using liquid chromatography-mass spectrometry (LC-MS), differentially expressed metabolites were identified between oat and wheat which were associated with the phenylpropanoid pathway, phospholipid biosynthesis and signaling, the salicylic acid signaling pathway, indole-3-acetic acid (IAA) degradation, and biosynthesis of 1,4-benzoxazin-3-ones (Bxs). Several phospho- and galacto- lipids were found in higher abundance in oat, and with the exception of early stem solidness cultivar Conan, both species experienced a decrease in abundance once infested. In all wheat cultivars except Conan, an increase in abundance was observed for Bxs HMDBOA-glc and DIBOA-ß-D-glucoside after infestation, indicating that this pathway is involved in wheat response to infestation in both solid and hollow stemmed cultivars. Differences between species in compounds involved in IAA biosynthesis, degradation and inactivation suggest that wheat may respond to infestation by inactivating IAA or altering the IAA pool in stem tissue. Conclusion: We propose that the species differences found here likely affect the survival of WSS larvae and may also be associated with differences in stem architecture at the molecular level. Our findings suggest pathways to focus on for future studies in elucidating plant response to WSS infestation.

The metabolome of individuals with knee osteoarthritis is influenced by 18-months of an exercise and weight loss intervention and sex: the IDEA trial.

Objective: The Intensive Diet and Exercise for Arthritis (IDEA) trial was conducted to evaluate the effects of diet and exercise on osteoarthritis (OA), the most prevalent form of arthritis. Various risk factors, such as obesity and sex, contribute to the debilitating nature of OA. While diet and exercise are known to improve OA symptoms, cellular and molecular mechanisms underlying these interventions, as well as effects of participant sex, remain elusive. Methods: Serum was obtained at three timepoints from IDEA participants assigned to groups of diet, exercise, or combined diet and exercise (n=10 per group). All serum metabolites were extracted and analyzed via liquid chromatography-mass spectrometry combined with metabolomic profiling. Extracted serum was pooled and fragmentation patterns were analyzed to identify metabolites that statistically differentially regulated between groups. Results: Changes in metabolism across male and female IDEA participants after 18-months of diet, exercise, and combined diet and excise intervention mapped to lipid, amino acid, carbohydrate, vitamin, and matrix metabolism. The diverse metabolic landscape detected across IDEA participants shows that intervention type impacts the serum metabolome of individuals with OA in distinct patterns. Moreover, differences in the serum metabolome corresponded with participant sex. Conclusions: These findings suggest that intensive weight loss among male and female subjects offers potential metabolic benefits for individuals with knee OA. This provides a deeper understanding of dysregulation occurring during OA development that may pave the way for improved interventions, treatments, and quality of life of those impacted by this disease.

A shift between mineral and nonmineral sources of iron and sulfur causes proteome-wide changes in Methanosarcina barkeri.

Iron (Fe) and sulfur (S) are required elements for life, and changes in their availability can limit the ecological distribution and function of microorganisms. In anoxic environments, soluble Fe typically exists as ferrous iron [Fe(II)] and S as sulfide (HS-). These species exhibit a strong affinity that ultimately drives the formation of sedimentary pyrite (FeS2). Recently, paradigm-shifting studies indicate that Fe and S in FeS2 can be made bioavailable by methanogens through a reductive dissolution process. However, the impact of the utilization of FeS2, as opposed to canonical Fe and S sources, on the phenotype of cells is not fully understood. Here, shotgun proteomics was utilized to measure changes in the phenotype of Methanosarcina barkeri MS grown with FeS2, Fe(II)/HS-, or Fe(II)/cysteine. Shotgun proteomics tracked 1,019 proteins overall, with 307 observed to change between growth conditions. Functional characterization and pathway analyses revealed these changes to be systemic and largely tangential to Fe/S metabolism. As a final step, the proteomics data were viewed with respect to previously collected transcriptomics data to deepen the analysis. Presented here is evidence that M. barkeri adopts distinct phenotypes to exploit specific sources of Fe and S in its environment. This is supported by observed protein abundance changes across broad categories of cellular biology. DNA adjacent metabolism, central carbon metabolism methanogenesis, metal trafficking, quorum sensing, and porphyrin biosynthesis pathways are all features in the phenotypic differentiation. Differences in trace metal availability attributed to complexation with HS-, either as a component of the growth medium [Fe(II)/HS-] or generated through reduction of FeS2, were likely a major factor underpinning these phenotypic differences.IMPORTANCEThe methanogenic archaeon Methanosarcina barkeri holds great potential for industrial bio-mining and energy generation technologies. Much of the biochemistry of this microbe is poorly understood, and its characterization will provide a glimpse into biological processes that evolved close to life's origin. The discovery of its ability to extract iron and sulfur from bulk, solid-phase minerals shifted a longstanding paradigm that these elements were inaccessible to biological systems. The full elucidation of this process has the potential to help scientists and engineers extract valuable metals from low-grade ore and mine waste generating energy in the form of methane while doing so.

Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor.

Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Aerobic methane synthesis and dynamics in a river water environment.

Reports of aerobic biogenic methane (CH4) have generated new views about CH4 sources in nature. We examine this phenomenon in the free-flowing Yellowstone river wherein CH4 concentrations were tracked as a function of environmental conditions, phototrophic microorganisms (using chlorophyll a, Chl a, as proxy), as well as targeted methylated amines known to be associated with this process. CH4 was positively correlated with temperature and Chl a, although diurnal measurements showed CH4 concentrations were greatest during the night and lowest during maximal solar irradiation. CH4 efflux from the river surface was greater in quiescent edge waters (71-94 µmol m-2 d) than from open flowing current (~ 57 µmol m-2 d). Attempts to increase flux by disturbing the benthic environment in the quiescent water directly below (~ 1.0 m deep) or at varying distances (0-5 m) upstream of the flux chamber failed to increase surface flux. Glycine betaine (GB), dimethylamine and methylamine (MMA) were observed throughout the summer-long study, increasing during a period coinciding with a marked decline in Chl a, suggesting a lytic event led to their release; however, this did not correspond to increased CH4 concentrations. Spiking river water with GB or MMA yielded significantly greater CH4 than nonspiked controls, illustrating the metabolic potential of the river microbiome. In summary, this study provides evidence that: (1) phototrophic microorganisms are involved in CH4 synthesis in a river environment; (2) the river microbiome possesses the metabolic potential to convert methylated amines to CH4; and (3) river CH4 concentrations are dynamic diurnally as well as during the summer active months.

Fe protein docking transduces conformational changes to MoFe nitrogenase active site in a nucleotide-dependent manner.

The reduction of dinitrogen to ammonia catalyzed by nitrogenase involves a complex series of events, including ATP hydrolysis, electron transfer, and activation of metal clusters for N2 reduction. Early evidence shows that an essential part of the mechanism involves transducing information between the nitrogenase component proteins through conformational dynamics. Here, millisecond time-resolved hydrogen-deuterium exchange mass spectrometry was used to unravel peptide-level protein motion on the time scale of catalysis of Mo-dependent nitrogenase from Azotobacter vinelandii. Normal mode analysis calculations complemented this data, providing insights into the specific signal transduction pathways that relay information across protein interfaces at distances spanning 100 Å. Together, these results show that conformational changes induced by protein docking are rapidly transduced to the active site, suggesting a specific mechanism for activating the metal cofactor in the enzyme active site.

Yeast Rad52 is a homodecamer and possesses BRCA2-like bipartite Rad51 binding modes.

Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.

Metabolic phenotypes reflect patient sex and injury status: A cross-sectional analysis of human synovial fluid.

OBJECTIVE: Osteoarthritis is a heterogeneous disease. The objective was to compare differences in underlying cellular mechanisms and endogenous repair pathways between synovial fluid (SF) from male and female participants with different injuries to improve the current understanding of the pathophysiology of downstream post-traumatic osteoarthritis (PTOA). DESIGN: SF from n = 33 knee arthroscopy patients between 18 and 70 years with no prior knee injuries was obtained pre-procedure and injury pathology assigned post-procedure. SF was extracted and analyzed via liquid chromatography-mass spectrometry metabolomic profiling to examine differences in metabolism between injury pathologies (ligament, meniscal, and combined ligament and meniscal) and patient sex. Samples were pooled and underwent secondary fragmentation to identify metabolites. RESULTS: Different knee injuries uniquely altered SF metabolites and downstream pathways including amino acid, lipid, and inflammatory-associated metabolic pathways. Notably, sexual dimorphic metabolic phenotypes were examined between males and females and within injury pathology. Cervonyl carnitine and other identified metabolites differed in concentrations between sexes. CONCLUSIONS: These results suggest that different injuries and patient sex are associated with distinct metabolic phenotypes. Considering these phenotypic associations, a greater understanding of metabolic mechanisms associated with specific injuries, sex, and PTOA development may yield data regarding how endogenous repair pathways differ between male and female injury types. Ongoing metabolomic analysis of SF in injured male and female patients can be performed to monitor PTOA development and progression.

Metabolic impact of polyphenol-rich aronia fruit juice mediated by inflammation status of gut microbiome donors in humanized mouse model.

Background: The Aronia melanocarpa fruit is emerging as a health food owing to its high polyphenolic content and associated antioxidant activity. Antioxidant-rich foods, such as Aronia fruit, may counter inflammatory stimuli and positively modulate the gut microbiome. However, a comprehensive study characterizing the impact of Aronia fruit supplementation has not been completed. Therefore, we completed analyses measuring the metabolic, microbial, and inflammatory effects of a diet supplemented with Aronia fruit juice. Method: Humanized mice were generated by colonizing gnotobiotic mice with microbiomes from human donors presenting disparate inflammation levels. Blood and fecal samples were collected throughout the course of an 8-week dietary intervention with either Aronia juice or a carbohydrate-matched beverage alone (2 weeks) or in combination with a high-fat diet to induce inflammation (6 weeks). Samples were analyzed using 16S rRNA gene sequencing (stool) and liquid chromatography-mass spectrometry (serum). Results: We demonstrated transfer of microbiome composition and diversity and metabolic characteristics from humans with low and high inflammation levels to second-generation humanized mice. Aronia supplementation provided robust protection against high-fat diet induced metabolic and microbiome changes that were dependent in part on microbiome donor. Aronia induced increases in bacteria of the Eggerthellaceae genus (7-fold) which aligns with its known ability to metabolize (poly)phenols and in phosphatidylcholine metabolites which are consistent with improved gut barrier function. The gut microbiome from a low inflammation phenotype donor provided protection against high-fat diet induced loss of microbiome ß-diversity and global metabolomic shifts compared to that from the high inflammation donor. Conclusion: These metabolic changes elucidate pathway-specific drivers of reduced inflammation stemming from both Aronia and the gut microbiota.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

An Aurora B-RPA signaling axis secures chromosome segregation fidelity.

Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.

Acute stress reduces population-level metabolic and proteomic variation.

BACKGROUND: Variation in omics data due to intrinsic biological stochasticity is often viewed as a challenging and undesirable feature of complex systems analyses. In fact, numerous statistical methods are utilized to minimize the variation among biological replicates. RESULTS: We demonstrate that the common statistics relative standard deviation (RSD) and coefficient of variation (CV), which are often used for quality control or part of a larger pipeline in omics analyses, can also be used as a metric of a physiological stress response. Using an approach we term Replicate Variation Analysis (RVA), we demonstrate that acute physiological stress leads to feature-wide canalization of CV profiles of metabolomes and proteomes across biological replicates. Canalization is the repression of variation between replicates, which increases phenotypic similarity. Multiple in-house mass spectrometry omics datasets in addition to publicly available data were analyzed to assess changes in CV profiles in plants, animals, and microorganisms. In addition, proteomics data sets were evaluated utilizing RVA to identify functionality of reduced CV proteins. CONCLUSIONS: RVA provides a foundation for understanding omics level shifts that occur in response to cellular stress. This approach to data analysis helps characterize stress response and recovery, and could be deployed to detect populations under stress, monitor health status, and conduct environmental monitoring.

Arsenic Exposure Causes Global Changes in the Metalloproteome of Escherichia coli.

Arsenic is a toxic metalloid with differential biological effects, depending on speciation and concentration. Trivalent arsenic (arsenite, AsIII) is more toxic at lower concentrations than the pentavalent form (arsenate, AsV). In E. coli, the proteins encoded by the arsRBC operon are the major arsenic detoxification mechanism. Our previous transcriptional analyses indicate broad changes in metal uptake and regulation upon arsenic exposure. Currently, it is not known how arsenic exposure impacts the cellular distribution of other metals. This study examines the metalloproteome of E. coli strains with and without the arsRBC operon in response to sublethal doses of AsIII and AsV. Size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) was used to investigate the distribution of five metals (56Fe, 24Mg, 66Zn, 75As, and 63Cu) in proteins and protein complexes under native conditions. Parallel analysis by SEC-UV-Vis spectroscopy monitored the presence of protein cofactors. Together, these data reveal global changes in the metalloproteome, proteome, protein cofactors, and soluble intracellular metal pools in response to arsenic stress in E. coli. This work brings to light one outcome of metal exposure and suggests that metal toxicity on the cellular level arises from direct and indirect effects.

Metabolomic Phenotypes Reflect Patient Sex and Injury Status: A Cross-Sectional Analysis of Human Synovial Fluid.

Background: Post-traumatic osteoarthritis (PTOA) is caused by knee injuries like anterior cruciate ligament (ACL) injuries. Often, ACL injuries are accompanied by damage to other tissues and structures within the knee including the meniscus. Both are known to cause PTOA but underlying cellular mechanisms driving disease remain unknown. Aside from injury, patient sex is a prevalent risk factor associated with PTOA. Hypothesis: Metabolic phenotypes of synovial fluid that differ by knee injury pathology and participant sex will be distinct from each other. Study Design: A cross-sectional study. Methods: Synovial fluid from n=33 knee arthroscopy patients between 18 and 70 years with no prior knee injuries was obtained pre-procedure and injury pathology assigned post-procedure. Synovial fluid was extracted and analyzed via liquid chromatography mass spectrometry metabolomic profiling to examine differences in metabolism between injury pathologies and participant sex. Additionally, samples were pooled and underwent fragmentation to identify metabolites. Results: Metabolite profiles revealed that injury pathology phenotypes were distinct from each other where differences in endogenous repair pathways that are triggered post-injury were detected. Specifically, acute differences in metabolism mapped to amino acid metabolism, lipid-related oxidative metabolism, and inflammatory-associated pathways. Lastly, sexual dimorphic metabolic phenotypes were examined between male and female participants, and within injury pathology. Specifically, Cervonyl Carnitine and other identified metabolites differed in concentration between sexes. Conclusions: The results of this study suggest that different injuries (e.g., ligament vs. meniscus), as well as sex are associated with distinct metabolic phenotypes. Considering these phenotypic associations, a greater understanding of metabolic mechanisms associated with specific injuries and PTOA development may yield data regarding how endogenous repair pathways differ between injury types. Furthermore, ongoing metabolomic analysis of synovial fluid in injured male and female patients can be performed to monitor PTOA development and progression. Clinical Relevance: Extension of this work may potentially lead to the identification of biomarkers as well as drug targets that slow, stop, or reverse PTOA progression based on injury type and patient sex.

Homodecameric Rad52 promotes single-position Rad51 nucleation in homologous recombination.

Homologous recombination (HR) is a pathway for the accurate repair of double-stranded DNA breaks. These breaks are resected to yield single-stranded DNA (ssDNA) that are coated by Replication Protein A (RPA). Saccharomyces cerevisiae Rad52 is a mediator protein that promotes HR by facilitating formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Canonically, Rad52 has been described to function by displacing RPA to promote Rad51 binding. However, in vitro, Rad51 readily forms a filament by displacing RPA in the absence of Rad52. Yet, in vivo, Rad52 is essential for HR. Here, we resolve how Rad52 functions as a mediator using single-particle cryo-electron microscopy and biophysical approaches. We show that Rad52 functions as a homodecamer and catalyzes single-position nucleation of Rad51. The N-terminal half of Rad52 is a well-ordered ring, while the C-terminal half is disordered. An intrinsic asymmetry within Rad52 is observed, where one or a few of the C-terminal halves interact with the ordered N-terminal ring. Within the C-terminal half, we identify two conserved charged patches that harbor the Rad51 and RPA interacting motifs. Interactions between these two charged patches regulate a ssDNA binding. These features drive Rad51 binding to a single position on the Rad52 decameric ring. We propose a Rad52 catalyzed single-position nucleation model for the formation of pre-synaptic Rad51 filaments in HR.

Gut microbiome dysbiosis drives metabolic dysfunction in Familial dysautonomia.

Familial dysautonomia (FD) is a rare genetic neurologic disorder caused by impaired neuronal development and progressive degeneration of both the peripheral and central nervous systems. FD is monogenic, with >99.4% of patients sharing an identical point mutation in the elongator acetyltransferase complex subunit 1 (ELP1) gene, providing a relatively simple genetic background in which to identify modifiable factors that influence pathology. Gastrointestinal symptoms and metabolic deficits are common among FD patients, which supports the hypothesis that the gut microbiome and metabolome are altered and dysfunctional compared to healthy individuals. Here we show significant differences in gut microbiome composition (16 S rRNA gene sequencing of stool samples) and NMR-based stool and serum metabolomes between a cohort of FD patients (~14% of patients worldwide) and their cohabitating, healthy relatives. We show that key observations in human subjects are recapitulated in a neuron-specific Elp1-deficient mouse model, and that cohousing mutant and littermate control mice ameliorates gut microbiome dysbiosis, improves deficits in gut transit, and reduces disease severity. Our results provide evidence that neurologic deficits in FD alter the structure and function of the gut microbiome, which shifts overall host metabolism to perpetuate further neurodegeneration.