Biology subjects

Evans, C.

Publications and source records attributed to Evans, C..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Mycobacterial lipoarabinomannan negatively interferes with macrophage responses to Aspergillus fumigatus in-vitro

IntroductionOver 1 million people have chronic pulmonary aspergillosis (CPA) secondary to pulmonary tuberculosis. Additionally, Aspergillus fumigatus (Af) has been reported as one of the most common pathogens associated with mycobacteria in patients with cystic fibrosis. Mycobacterial virulence factors, like lipoarabinomannan, have been shown to interfere with hosts intracellular pathways required for an effective immune response, however, the immunological basis for mycobacterial-fungal coinfection is still unknown. We therefore investigated the effect of lipoarabinomannan on macrophage responses against Af. MethodsBone marrow-derived macrophages (BMDMs) were stimulated with non-mannose-capped lipoarabinomannan (LAM) from Mycobacterium smegmatis or mannose-capped lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis for 2 hours and then infected with swollen Af conidia. Cell death was assessed by lactate dehydrogenase release. Cytokine release was measured in supernatant using Enzyme Linked Immuno-Sorbent Assay (ELISA). Colony forming units counting and time-lapse fluorescence microscopy was performed for studying conidia killing by macrophages. ResultsBMDMs stimulated with LAM showed increased cell death and inflammatory cytokine release in a dose-dependent manner, characterised by a significant increase of IL-1{beta} release. Time-lapse fluorescence microscopy and CFUs revealed that both LAM and ManLAM significantly decrease the capacity of macrophages to kill Af conidia within the first 6 hours of infection. ConclusionsThe mycobacterial virulence factor, lipoarabinomannan, disrupts macrophage capacity to efficiently clear Af at early stages of infection in-vitro.

immunology↗

bioRxiv2024

MICOS Complex Loss Governs Age-Associated Murine Mitochondrial Architecture and Metabolism in the Liver, While Sam50 Dictates Diet Changes

Background & AimsAging is associated with a significant decline in mitochondrial function in the liver, leading to an increased risk of liver disease. This study examines age-related changes in the mitochondrial structure of human and murine livers using a combination of Serial Block-Face Scanning Electron Microscopy (SBF-SEM) and mass spectrometry approaches. MethodsThis study integrates mitochondrial structure analysis in a murine model with an analysis of liver architecture, lipogenesis, and genetically regulated gene expression in human cohorts. We explored the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex using SBF-SEM, three-dimensional reconstruction with Amira software, and mass spectrometry techniques. ResultsAging leads to a reduction in mitochondrial size and complexity, resulting in changes in the metabolomic and lipidomic profiles of murine liver cells that are comparable to those observed in aged human samples. We find that genetically modeled expression of MICOS complex genes OPA1 and CHCHD3 is associated with chronic liver disease phenotypes within a large biobank population. Furthermore, we observed dysregulated mitochondrial calcium handling and increased oxidative stress due to the disruption of the MICOS complex. ConclusionOur study highlights the age-associated decline in mitochondrial complexity and metabolic regulation within the aging murine liver and the human population. We have identified that these changes are partially attributable to the age-related loss of the MICOS complex. Impact and implicationsThis study offers new insights into the changes to mitochondrial ultrastructure that occur during aging. Using SBF-SEM, the quantification of young and aged murine mitochondrial structure was performed, which had previously been an underexplored avenue for measuring mitochondrial changes. The discovery of mitochondrial ultrastructural changes, in conjunction with measurements of age-associated metabolic alterations and gene association data, provides a model for how changes in MICOS expression may modulate age-related impairment of hepatic mitochondria. These results provide a new model by which changes in MICOS protein expression may both cause and be a potential therapeutic target for age-related impairment in hepatic function. HighlightsDecreased modeled expression of CHCHD3 in individuals of European genetic ancestry is linked to liver transplant and cirrhosis, while decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic liver disease and cirrhosis. Aging alters liver lipid accumulation, MICOS mRNA levels, and disease markers. Aging reduces the volume and complexity of murine liver ultrastructure. Aging and diet significantly alter the MICOS complex in mice. Knockdown of Mic60 and Chchd6 lowers Ca2+ uptake, retention, and induces oxidative stress in HepG2 cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/599846v3_ufig1.gif" ALT="Figure 1000"> View larger version (47K): org.highwire.dtl.DTLVardef@1cdd61corg.highwire.dtl.DTLVardef@a3fb74org.highwire.dtl.DTLVardef@1d1ad36org.highwire.dtl.DTLVardef@c2e55f_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver aging causes metabolic, lipidomic, and mitochondrial structural alterations, reflecting age-dependent losses in the MICOS complex. Key components of the MICOS complex (MIC60, CHCHD3 and CHCHD6) are illustrated.

physiology↗

bioRxiv2024

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a868f0org.highwire.dtl.DTLVardef@1817bfborg.highwire.dtl.DTLVardef@1f2a1a3org.highwire.dtl.DTLVardef@520692_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Wheat NAC transcription factor NAC5-1 is a positive regulator of senescence

Wheat (Triticum aestivum L.) is an important source of both calories and protein in global diets, but there is a trade-off between grain yield and protein content. The timing of leaf senescence could mediate this trade-off as it is associated with both decline in photosynthesis and nitrogen remobilisation from leaves to grain. NAC transcription factors play key roles in regulating senescence timing. In rice, OsNAC5 expression is correlated with earlier senescence, but the role of the wheat ortholog in senescence had not been characterised. We verified that NAC5-1 is the ortholog of OsNAC5 and that it is expressed in senescing flag leaves in wheat. To characterise NAC5-1, we combined missense mutations in NAC5-A1 and NAC5-B1 from a TILLING mutant population and overexpressed NAC5-A1 in wheat. Mutation in NAC5-1 was associated with delayed onset of flag leaf senescence, while overexpression of NAC5-A1 was associated with slightly earlier onset of leaf senescence. DAP-seq was performed to locate transcription factor binding sites of NAC5-1. Analysis of DAP-seq and comparison with other studies identified putative downstream target genes of NAC5-1 which could be associated with senescence. This work showed that NAC5-1 is a positive transcriptional regulator of leaf senescence in wheat. Further research is needed to test the effect of NAC5-1 on yield and protein content in field trials, to assess the potential to exploit this senescence regulator to develop high-yielding wheat while maintaining grain protein content.

plant biology↗

bioRxiv2023

SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system

SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, is associated with a range of neurological manifestations including haemorrhage, thrombosis and ischaemic necrosis and encephalitits. However, the mechanism by which this occurs is unclear. Neurological disease associated with SARS-CoV-2 infection has been proposed to occur following direct infection of the central nervous system and/or indirect sequelae as a result of peripheral inflammation. We profiled ACE2 and TMPRSS2 in brain tissue from five healthy human donors, and observed expression of these proteins in astrocytes, neurons and choroid plexus epithelium within frontal cortex and medulla. Primary human astrocytes, neurons and choroid plexus epithelial cells supported productive SARS-CoV-2 infection in an ACE2- dependent manner. Infected cells supported the full viral lifecycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells, pericytes and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 is neurotropic, and this may in part explain the neurological sequelae of infection. ImportanceA subset of patients with COVID-19 develop neurological symptoms, but the underlying cause is poorly understood. We observed that cells within normal human brain express the SARS-CoV-2 entry factors ACE-2 and TMPRRS2, with expression mainly observed within astrocytes, neurons and choroid plexus epithelium. Primary human astrocytes, neurons and choroid plexus epithelial cells cultured in vitro supported the full SARS-CoV-2 life cycle with a range of SARS-CoV-2 variants. This study demonstrates that cells of the human central nervous system express SARS-CoV-2 entry factors in vivo and support viral infection in vitro, thus supporting a model where neurological symptoms seen in some COVID-19 patients may be as a result of direct viral infection of the central nervous system. Furthermore, these data highlight the importance of investigating the ability of therapeutics to clear virus from this potential reservoir of infection.

microbiology↗

bioRxiv2023

Transglobal spread of an ecologically significant sea urchin parasite

Mass mortality of the dominant coral reef herbivore Diadema antillarum in the Caribbean in the early 1980s led to a persistent phase shift from coral-to algal-dominated reefs. In 2022, a scuticociliate most closely related to Philaster apodigitiformis caused further mass mortality of D. antillarum across the Caribbean, leading to >95% mortality at affected sites. Mortality was also reported in the related species Diadema setosum in the Mediterranean in 2022, where urchins experienced gross signs compatible with scuticociliatosis. However, the causative agent of the Mediterranean outbreak has not yet been determined. In April 2023, mass mortality of D. setosum occurred along the Sultanate of Omans coastline. Urchins displayed signs compatible with scuticociliatosis including abnormal behavior, drooping and loss of spines, followed by tissue necrosis and death. Here we report the detection of an 18S rRNA gene sequence in abnormal urchins from Muscat, Oman that is identical to the Philaster strain responsible for D. antillarum mass mortality in the Caribbean. We also show that scuticociliatosis signs can be elicited in D. setosum by experimental challenge with the cultivated Philaster strain associated with Caribbean scuticociliatosis. These results demonstrate the Philaster sp. associated with D. antillarum mass mortality has rapidly spread to geographically distant coral reefs, compelling global-scale awareness and monitoring for this devastating condition through field surveys, microscopy, and molecular microbiological approaches, and prompting investigation of long-range transmission mechanisms.

ecology↗

bioRxiv2023

Local population structure in Cambridgeshire during the Roman occupation

The Roman period saw the empire expand across Europe and the Mediterranean, including much of what is today the United Kingdom. While there is written evidence of high mobility into and out of Britain for administrators, traders and the military, the impact of imperialism on local population structure is invisible in the textual record. The extent of genetic change that occurred in Britain before the Early Medieval Period and how closely linked by genetic kinship the local populations were, remains underexplored. Here, using genome-wide data from 52 ancient individuals from Cambridgeshire, we show low levels of genetic ancestry differentiation between Romano-British sites and lower levels of runs of homozygosity over 4 centimorgans (cM than in the Bronze Age and Neolithic. We find fourteen cases of genetic relatedness within and one between sites without evidence of patrilineal dominance and one case of temporary mobility within a family unit during the Late Romano-British period. We also show that the modern patterns of genetic ancestry composition in Modern Britain emerged after the Roman period.

genetics↗

bioRxiv2023

hnRNPUL1 ensures efficient Integrator-mediated cleavage of snRNAs and is mutated in amyotrophic lateral sclerosis

Integrator cleaves nascent RNA, triggering RNA polymerase II transcription termination, but how cleavage is regulated is poorly understood. Here we show hnRNPUL1 ensures efficient Integrator-mediated cleavage of nascent RNA downstream of snRNA genes and, in the case of U2 snRNA, binds a terminal stem-loop involved in this process. In the nucleoplasm, hnRNPUL1 binds U4 snRNA and SART3 and enables efficient reformation of the U4:U6 di-snRNP for further rounds of pre-mRNA splicing. Sustained hnRNPUL1 loss leads to reduced levels of snRNAs, defects in histone mRNA 3' end processing and loss of Cajal bodies. hnRNPUL1 binds RNA through multiple domains, including a globular central domain comprising tightly juxtaposed SPRY and dead polynucleotide kinase folds. This latter fold allows binding to 5'-monophosphorylated RNAs in a mutually exclusive manner with ATP binding and functions as an XRN2 antagonist when overexpressed. We identify a cohort of amyotrophic lateral sclerosis patients harbouring disruptive mutations in hnRNPUL1. SMN loss in spinal muscular atrophy and hnRNPUL1 loss both disrupt snRNP biogenesis, leading to motor neuron death, suggesting a common aetiology.

molecular biology↗

bioRxiv2023

Assessing generalizability of a dengue classifier across multiple datasets

Early diagnosis of dengue fever is important for individual treatment and monitoring disease prevalence in the population. To assist diagnosis, previous studies have proposed classification models to detect dengue from symptoms and clinical measurements. However, there has been little exploration of whether existing models can be used to make predictions for new populations. We trained logistic regression models on five publicly available dengue datasets from previous studies, using three explanatory variables identified as important in prior work: age, white blood cell count, and platelet count. These five datasets were collected at different times in different locations, with a variety of disease rates and patient ages. A model was trained on each dataset, and predictive performance and model calibration was evaluated on both the original (training) dataset, and the other (test) datasets from different studies. We further compared performance with larger models and other classification methods. In-sample area under the receiver operating characteristic curve (AUC) values for the logistic regression models ranged from 0.74 to 0.89, while out-of-sample AUCs ranged from 0.55 to 0.89. Matching age ranges in training/test datasets increased AUC values and balanced the sensitivity and specificity. Adjusting the predicted probabilities to account for differences in dengue prevalence improved calibration in 20/28 training-test pairs. Results were similar when other explanatory variables were included and when other classification methods (decision trees and support vector machines) were used. The in-sample performance of the logistic regression model was consistent with previous dengue classifiers, suggesting the chosen model is a good choice in a variety of settings and has decent overall performance. However, adjustments are required to make predictions on new datasets. Practitioners can use existing dengue classifiers in new settings but should be careful with different patient ages and disease rates.

pathology↗

bioRxiv2023

Tonic Meningeal Interleukin-10 Upregulates Delta Opioid Receptor to Prevent Relapse to Pain

Chronic pain often alternates between transient remission and relapse of severe pain. While most research on chronic pain has focused on mechanisms maintaining pain, there is a critical unmet need to understand what prevents pain from re-emerging in those who recover from acute pain. We found that interleukin (IL)-10, a pain resolving cytokine, is persistently produced by resident macrophages in the spinal meninges during remission from pain. IL-10 upregulated expression and analgesic activity of {delta}-opioid receptor ({delta}OR) in the dorsal root ganglion. Genetic or pharmacological inhibition of IL-10 signaling or {delta}OR triggered relapse to pain in both sexes. These data challenge the widespread assumption that remission of pain is simply a return to the naive state before pain was induced. Instead, our findings strongly suggest a novel concept that: remission is a state of lasting pain vulnerability that results from a long-lasting neuroimmune interactions in the nociceptive system.

neuroscience↗

bioRxiv2023

A network medicine approach to elucidate mechanisms underlying menopause-induced knee osteoarthritis

Post-menopausal women present with the highest incidence and morbidity of knee osteoarthritis (KOA), but no disease-modifying therapies are available. This treatment gap may be driven by the absence of menopause in preclinical studies, as rodents do not naturally maintain a menopausal phenotype. Here, we employed a chemically-induced menopause model to map the trajectory of KOA at the tissue and proteome levels and test therapeutics in silico. Middle-aged female mice were randomized to sesame oil (non-menopause) or 4-vinycyclohexene diepoxide (menopause) injections. Following comprehensive validation of our model, knees were collected across perimenopause and menopause for histology, and cartilage samples were micro-dissected for mass spectrometry proteomics. Menopause mice displayed aggravated cartilage degeneration and synovitis relative to non-menopause mice. An unbiased pathway analysis revealed progesterone as a predominant driver of pathological signaling cascades within the cartilage proteome. Network medicine-based analyses suggested that menopause induction amplifies chondrocyte senescence, actin cytoskeleton-based stress, and extracellular matrix disassembly. We then used in silico drug testing to evaluate how restoration of sex hormones impacted the cartilage network. The greatest restoration was observed with combined estradiol/progesterone treatment (i.e., hormone therapy), although in silico treatment with a senolytic drug also partially recovered the cartilage proteome. Taken together, our findings using a translatable female aging model demonstrate that menopausal aging induces progressive cartilage degeneration and amplifies age-related synovitis. These changes may be driven by a previously unappreciated role of progesterone loss and menopause-induced cellular senescence. Lastly, in silico treatment suggests an estradiol/progesterone cocktail or senolytics may attenuate menopause-induced cartilage pathology. One Sentence SummaryMenopause induces cartilage degradation, senescence, and extracellular matrix disassembly, while hormone therapy restores the cartilage proteome.

pathology↗

bioRxiv2021

Sexual and temporal variation in New Zealand bellbird song repertoires

How song repertoires vary within species and change over time is well studied in male songbirds. However, variation in female song repertoires remains largely unstudied despite female song being much more common and complex than once assumed. We investigated the song syllable repertoire of the New Zealand bellbird (Anthornis melanura), a species where both sexes have complex but sexually dimorphic song. We compared songs at individual and population levels to investigate sex and temporal variation of syllable repertoires. We detected 96 syllable types in the population over four years, of which 58% were unique to males, 32% unique to females and 9% were shared between the sexes. The population syllable repertoire of both sexes changed substantially across years with similar turnover rates (Jaccards similarity coefficients; female 52.9-69.0%; male 58.6-73.7%). Furthermore, many syllable types, unique to each sex, varied in prevalence within the population across years. The syllable repertoire sizes of individuals were higher for males than females (13-32, n = 7 and 6-16, n = 8, respectively). Although these sample sizes were low, the temporal variation in syllable prevalence and turnover for individuals were similar to patterns at the population level. Overall, male and female bellbirds exhibited similarities in temporal patterns of yearly repertoire composition, with rapid changes in syllable prevalence, but females had fewer syllable types than males. We suggest that these similarities and differences are consistent with male and female song repertoires being driven by similar but not identical selection pressures.

animal behavior and cognition↗

bioRxiv2021

Meta-analysis and multi-omics to elucidate pathogenic mechanisms of age-related knee osteoarthritis

Increased mechanistic insight into the pathogenesis of knee osteoarthritis (KOA) is needed to develop efficacious disease-modifying treatments. Though age-related pathogenic mechanisms are most relevant to the great majority of KOA seen clinically, the bulk of our mechanistic understanding of KOA has been derived using surgically induced post-traumatic OA (PTOA) models. Here, we took an integrated approach of meta-analysis and multi-omics to elucidate pathogenic mechanisms of age-related KOA in murine model. Protein-level data together with transcriptomic profiling revealed inflammation, autophagy, and cellular senescence as primary hallmarks of age-related KOA. Importantly, the molecular profiles of aged cartilage were unique from those in PTOA, with only 1% overlap between the two. At the nexus of aging hallmarks, Advanced Glycation End-Product (AGE)/Receptor for AGE emerged as intrinsically linked to age-related KOA. This pathway was further validated by mass spectrometry. Collectively, these findings implicate dysregulation of AGE-RAGE signaling as a key driver of age-related KOA.

molecular biology↗

bioRxiv2021

The impact of brain lesions on tDCS-induced electric field magnitude

BackgroundTranscranial direct current stimulation (tDCS) has been used to enhance motor and language rehabilitation following a stroke. However, improving the effectiveness of clinical tDCS protocols depends on understanding how lesions may influence tDCS-induced current flow through the brain. ObjectiveWe systematically investigated the effect of brain lesions on the magnitude of electric fields (e-mag) induced by tDCS, and how to overcome lesion-induced inter-individual variability in e-mag. MethodsWe simulated the effect of 630 different lesions - by varying lesion location, distance from the target region of interest (ROI), size and conductivity - on tDCS-induced e-mag in the brains of two participants. Current flow modelling was conducted for two tDCS montages commonly used in clinical applications, which target either primary motor cortex (M1) or Brocas area (BA44), respectively. We further explored how the inherent variability in e-mag that is introduced by inter-lesion differences can be overcome by individualising tDCS protocols. ResultsThe effect on absolute e-mag was highly dependent on lesion size, conductance and the distance from the target ROI. Larger lesions, with high conductivity, closer to the ROI caused e-mag changes of more than 30%. The sign of this change was determined by the location of the lesion. Specifically, lesions located in-line with the predominant direction of current flow increased e-mag in the ROI, whereas lesions located in the opposite direction caused a decrease. Lesions had a large impact on the optimal electrode configuration if attempting to maximise for the total e-mag in the ROI, but little impact if only the component of e-mag flowing radially inward to the cortex was maximised. Knowing the effect of a given lesion on e-mag also allows for individualising tDCS intensity to reduce variability. ConclusionsThese results demonstrate that tDCS-induced electric fields are profoundly influenced by lesion characteristics, and further exacerbate the known variability in e-mag across individuals. Additionally, the dependence of these results on the assigned conductance of the lesion underlines the need for improved estimates of lesion conductivity for current flow models. Our results highlight the need for individualised dose control of tDCS in the lesioned brain to overcome the substantial inter-individual variability in electric fields delivered to a cortical target region. Highlights- Lesions can alter tDCS-induced electric field magnitude (e-mag) in a target by 30% - Lesions can cause increases or decreases to e-mag - Direction of change depends on the position of the lesion relative to current flow - Lesion conductivity - the true value for which is unknown - also impacts change - E-mag variability can be reduced by individualising montage and stimulation intensity

neuroscience↗

bioRxiv2021

Age-related increase in matrix stiffness downregulates α-Klotho in chondrocytes and induces cartilage degeneration

Enhanced mechanistic insight into age-related knee osteoarthritis (KOA) is an essential step to promote successful translation of animal research to bedside interventions. To this end, the goal of these studies was to interrogate molecular mechanisms driving age-related KOA in a mouse model and correspond findings to human knee cartilage. Unbiased mass spectrometry proteomics of cartilage tissue revealed PI3K/Akt signaling was the predominant pathway disrupted over time in male, but not female, mice. This finding was consistent with a significantly accelerated KOA progression in males when compared to female counterparts. In probing for upstream regulators of these age-dependent alterations, we found that -Klotho, a suppressor of PI3K/Akt signaling and potent longevity protein, significantly decreased with aging in both mouse and human knee cartilage. Upstream of these alterations, we found that age-related increases in matrix stiffness initiated a cascade of altered nuclear morphology and downregulated -Klotho expression, ultimately impairing chondrocyte health. Conversely, reducing matrix stiffness increased -Klotho expression in chondrocytes, thus enhancing their chondrogencity and cartilage integrity. Collectively, our findings establish a novel mechanistic link between age-related alterations in ECM biophysical properties and regulation of cartilage health by -Klotho.

molecular biology↗

bioRxiv2021

Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections

SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3 of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. One Sentence SummaryThe recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.

genomics↗

bioRxiv2020

The molecular and metabolic program for adaptation of white adipocytesto cool physiologic temperatures

Although visceral adipocytes located within the bodys central core are maintained at ~37{degrees}C, adipocytes within bone marrow, subcutaneous, and dermal depots are found primarily within the peripheral shell, and generally exist at cooler temperatures. Responses of brown and beige/brite adipocytes to cold stress are well-studied; however, comparatively little is known about mechanisms by white adipocytes adapt to temperatures below 37{degrees}C. Here we report that adaptation of cultured adipocytes to 31{degrees}C, the temperature at which distal marrow adipose tissues and subcutaneous adipose tissues often reside, induces extensive changes in gene expression, increased anabolic and catabolic lipid metabolism, and elevated oxygen consumption with reduced reliance on glucose and preferential use of pyruvate, glutamine and fatty acids as energy sources. Cool temperatures up-regulate stearoyl-CoA desaturase-1 expression and monounsaturated lipid levels in cultured adipocytes and distal bone marrow adipose tissues, and stearoyl-CoA desaturase-1 activity is required for acquisition of maximal oxygen consumption at 31{degrees}C.

physiology↗

bioRxiv2020

periscope: sub-genomic RNA identification in SARS-CoV-2 ARTIC Network Nanopore Sequencing Data

We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "sub-genomic RNAs". sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5 UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5 end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.

bioinformatics↗