Biology subjects

Smith, R.

Publications and source records attributed to Smith, R..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

A compendium of Amplification-Related Gain Of Sensitivity (ARGOS) genes in human cancer

Chromosomal gains are among the most frequent somatic genetic alterations occurring in cancer. While the effect of sustained oncogene expression has been characterized, the impact of copy-number gains affecting collaterally-amplified "bystander" genes on cellular fitness remains less understood. To investigate this, we built a comprehensive map of dosage compensations across human cancers by integrating expression and copy number profiles from over 8,000 TCGA tumors and CCLE cell lines. Further, we analyzed the effect of gene overexpression across 17 human cancer ORF screens to provide an overview of genes that prove toxic to cancer cells when overexpressed. Combining these two independent approaches we propose a class of Amplification-Related Gain Of Sensitivity (ARGOS) genes. These genes are located in commonly amplified regions of the genome, have lower expression levels than expected by their copy-number status, and are toxic to cancer cells when overexpressed. We experimentally validated CDKN1A and RBM14 as high-confidence pan-cancer ARGOS genes in lung and breast cancer cell line models. We additionally suggest that RBM14s mechanism of toxicity involves altered DNA damage response and innate immune signaling processes following gene overexpression. Finally, we provide a comprehensive catalog of compensated, toxic, and ARGOS genes as a community resource.

cancer biology↗

bioRxiv2023

Retroelement-Age Clocks: Epigenetic Age Captured by Human Endogenous Retrovirus and LINE-1 DNA methylation states

Human endogenous retroviruses (HERVs), the remnants of ancient viral infections embedded within the human genome, and long interspersed nuclear elements 1 (LINE-1), a class of autonomous retrotransposons, are silenced by host epigenetic mechanisms including DNA methylation. The resurrection of particular retroelements has been linked to biological aging. Whether the DNA methylation states of locus specific HERVs and LINEs can be used as a biomarker of chronological age in humans remains unclear. We show that highly predictive epigenetic clocks of chronological age can be constructed from retroelement DNA methylation states in the immune system, across human tissues, and pan-mammalian species. We found retroelement epigenetic clocks were reversed during transient epigenetic reprogramming, accelerated in people living with HIV-1, responsive to antiretroviral therapy, and accurate in estimating long-term culture ages of human brain organoids. Our findings support the hypothesis of epigenetic dysregulation of retroelements as a potential contributor to the biological hallmarks of aging.

immunology↗

bioRxiv2023

Preclinical Characterization of the Omicron XBB.1.5-Adapted BNT162b2 COVID-19 Vaccine

As SARS-CoV-2 continues to evolve, increasing in its potential for greater transmissibility and immune escape, updated vaccines are needed to boost adaptive immunity to protect against COVID-19 caused by circulating strains. Here, we report features of the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine, which contains the same mRNA backbone as the original BNT162b2 vaccine, modified by the incorporation of XBB.1.5-specific sequence changes in the encoded prefusion-stabilized SARS-CoV-2 spike protein (S(P2)). Biophysical characterization of Omicron XBB.1.5 S(P2) demonstrated that it maintains a prefusion conformation that adopts a flexible and predominantly open one-RBD-up state, with high affinity binding to the human ACE-2 receptor. When administered as a 4th dose in BNT162b2-experienced mice, the monovalent Omicron XBB.1.5 vaccine elicited substantially higher serum neutralizing titers against pseudotyped viruses of Omicron XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and HV.1 sublineages and the phylogenetically distant BA.2.86 lineage than the bivalent Wild Type + Omicron BA.4/5 vaccine. Similar trends were observed against Omicron XBB sublineage pseudoviruses when the vaccine was administered as a 2-dose primary series in naive mice. Strong S-specific Th1 CD4+ and IFN{gamma}+ CD8+ T cell responses were also observed. These findings, together with prior experience with variant-adapted vaccine responses in preclinical and clinical studies, suggest that the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine is anticipated to confer protective immunity against dominant SARS-CoV-2 strains. ONE-SENTENCE SUMMARYThe monovalent Omicron XBB.1.5-adapted BNT162b2 mRNA vaccine encodes a prefusion-stabilized spike immunogen that elicits more potent neutralizing antibody responses against homologous XBB.1.5 and other circulating sublineage pseudoviruses compared to the bivalent Wild Type + Omicron BA.4/5 BNT162b2 vaccine, thus demonstrating the importance of annual strain changes to the COVID-19 vaccine.

immunology↗

bioRxiv2023

Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD-20 therapy

BackgroundPatients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as Rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. MethodsWe performed in depth antibody characterisation in sera collected four to six weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in Rituximab-treated primary vasculitis patients (n=14) using Luminex and pseudovirus neutralisation assays, whereas a novel microfluidic-based immunoassay was used to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. Comparative antibody profiling was performed at equivalent time points in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n=15) and in convalescent patients after WT SARS-CoV-2 infection (n=30). ResultsRituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in Rituximab-treated patients and in healthy individuals. In the Rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT (median [range] KD: 21.6 [9.7-38.8] nM vs 4.6 [2.3-44.8] nM, p=0.0004). By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in Rituximab-treated patients (median [range] KD: 1.05 [0.45-1.84] nM vs 20.25 [13.2-38.8] nM, p=0.0002), underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection which was not observed in Rituximab-treated patients, despite repeated vaccination. DiscussionOur results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in Rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.

immunology↗

bioRxiv2023

OMICmAge: An integrative multi-omics approach to quantify biological age with electronic medical records

Biological aging is a multifactorial process involving complex interactions of cellular and biochemical processes that is reflected in omic profiles. Using common clinical laboratory measures in ~30,000 individuals from the MGB-Biobank, we developed a robust, predictive biological aging phenotype, EMRAge, that balances clinical biomarkers with overall mortality risk and can be broadly recapitulated across EMRs. We then applied elastic-net regression to model EMRAge with DNA-methylation (DNAm) and multiple omics, generating DNAmEMRAge and OMICmAge, respectively. Both biomarkers demonstrated strong associations with chronic diseases and mortality that outperform current biomarkers across our discovery (MGB-ABC, n=3,451) and validation (TruDiagnostic, n=12,666) cohorts. Through the use of epigenetic biomarker proxies, OMICmAge has the unique advantage of expanding the predictive search space to include epigenomic, proteomic, metabolomic, and clinical data while distilling this in a measure with DNAm alone, providing opportunities to identify clinically-relevant interconnections central to the aging process.

bioinformatics↗

bioRxiv2023

Multifunction Fluorescence Open-Source In Vivo/In Vitro Imaging System (openIVIS)

The widespread availability and diversity of open-source microcontrollers paired with off-the-shelf electronics and 3D printed technology has led to the creation of a wide range of low-cost scientific instruments, including microscopes, spectrometers, sensors, data loggers, and other tools that can be used for research, education, and experimentation. These devices can be used to explore a wide range of scientific topics, from biology and chemistry to physics and engineering. In this study we designed and built a multifunction fluorescent open-source in-vivo/in-vitro imaging system (openIVIS) fluorescent imaging system that integrates a Raspberry Pi with commercial cameras and LEDs with 3D printed structures combined with an acrylic housing. Our openIVIS provides three excitation wavelengths of 460 nm, 520 nm, and 630 nm integrated with Python control software to enable fluorescent measurements across the full visible light spectrum. To demonstrate the various potential applications of our system, we tested its performance against a diverse set of experiments including laboratory type assays (measuring fluorescent dyes, using optical nanosensors, and DNA gel electrophoresis) to potentially fieldable applications (plant and mineral imaging). We also tested the potential use for a high school biology environment by imaging small animals and tracking their development over the course of a couple of weeks. Our system demonstrated its ability to measure a wide dynamic range fluorescent response from millimolar to picomolar concentrations in the same sample while measuring responses across visible wavelengths. These results demonstrate the power and flexibility of open-source hardware and software and how it can be integrated with customizable manufacturing to create low-cost scientific instruments with a wide range of applications. Our study provides a promising model for the development of low-cost instruments that can be used in both research and education.

bioengineering↗

bioRxiv2023

The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity

The clinical success of PARP1/2 inhibitors prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARP inhibitors. We show that the sensitivity of POLE4 knockouts is not due to a compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 deletion generates replication stress with the accumulation of single-stranded DNA gaps upon PARP inhibitor treatment. In POLE4 knockouts, replication stress leads to elevated DNA-PK signaling revealing a role of POLE4 in regulating DNA-PK activation. Moreover, POLE4 knockouts show synergistic sensitivity to the co-inhibition of ATR and PARP. Finally, POLE4 loss enhances the sensitivity of BRCA1-deficient cells to PARP inhibitors and counteracts acquired resistance consecutive to restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARP inhibitor driven therapies and hamper acquired PARP inhibitor resistance.

cell biology↗

bioRxiv2023

Robust collection and processing for label-free single voxel proteomics

With advanced mass spectrometry (MS)-based proteomics, genome-scale proteome coverage can be achieved from bulk tissues. However, such bulk measurement lacks spatial resolution and obscures important tissue heterogeneity, which make it impossible for proteome mapping of tissue microenvironment. Here we report an integrated wet collection of single tissue voxel and Surfactant-assisted One-Pot voxel processing method termed wcSOP for robust label-free single voxel proteomics. wcSOP capitalizes on buffer droplet-assisted wet collection of single tissue voxel dissected by LCM into the PCR tube cap and MS-compatible surfactant-assisted one-pot voxel processing in the collection cap. This convenient method allows reproducible label-free quantification of [~]900 and [~]4,600 proteins for single voxel from fresh frozen human spleen tissue at 20 m x 20 m x 10 m (close to single cells) and 200 m x 200 m x 10 m ([~]100 cells), respectively. 100s-1000s of protein signatures with differential expression levels were identified to be spatially resolved between spleen red and white pulp regions depending on the voxel size. Region-specific signaling pathways were enriched from single voxel proteomics data. Antibody-based CODEX imaging was used to validate label-free MS quantitation for single voxel analysis. The wcSOP-MS method paves the way for routine robust single voxel proteomics and spatial proteomics.

molecular biology↗

bioRxiv2023

Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response

Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9- mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing should be considered in the design of ex vivo autologous HSPC gene editing clinical trials. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/547089v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1dae519org.highwire.dtl.DTLVardef@1f6ddacorg.highwire.dtl.DTLVardef@535cbaorg.highwire.dtl.DTLVardef@ecbeaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. Here we show that PARP14 is dual function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a dramatic increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response. TeaserPARP14 is an antiviral PARP that combines ADP-ribosylation writer, reader and eraser functions in one polypeptide.

cell biology↗

bioRxiv2023

Neuronal alterations in AKT isotype expression in schizophrenia

Schizophrenia is characterized by substantial alterations in brain function, and previous studies suggest insulin signaling pathways, particularly involving AKT, are implicated in the pathophysiology of the disorder. This study demonstrates elevated mRNA expression of AKT1-3 in neurons from schizophrenia subjects, contrary to unchanged or diminished total AKT protein expression reported in previous postmortem studies, suggesting a potential decoupling of transcript and protein levels. Sex-specific differential AKT activity was observed, indicating divergent roles in males and females with schizophrenia. Alongside AKT, upregulation of PDK1, a critical component of the insulin signaling pathway, and several protein phosphatases known to regulate AKT were detected. Moreover, enhanced expression of the transcription factor FOXO1, a regulator of glucose metabolism, hints at possible compensatory mechanisms related to insulin signaling dysregulation. Findings were largely independent of antipsychotic medication use, suggesting inherent alterations in schizophrenia. These results highlight the significance of AKT and related signaling pathways in schizophrenia, proposing that these changes might represent a compensatory response to a primary defect of insulin signaling. This research underscores the need for a detailed understanding of these signaling pathways for the development of effective therapeutic strategies.

neuroscience↗

bioRxiv2023

Low-dose cytokine immunotherapy of solid cancers enabled by phagocytic-competent protein co-crystals

Protein therapeutics are often compromised by sub-optimal biodistribution contributing to poor efficacy and adverse events. Drug delivery mechanisms better able to target protein drugs to the disease site and provide localized, sustained release have the potential to transform therapeutic standards. PODS(R) crystals (PODS) are natural-mimetic, micron-scale protein co-crystals engineered to incorporate a protein cargo that can be sustainably released under the action of resident proteases. PODS are efficiently taken up by phagocytic cells with the cargo protein subsequently released in a bioactive form. Since blood-circulating phagocytic cells, including monocytes, are actively recruited into diseased and inflamed tissue, such as the tumour microenvironment, we postulated that monocyte/macrophage-mediated PODS delivery could be used as a molecular "Trojan horse" to efficiently deliver therapeutic proteins to target cells. This could improve the pharmacodynamics and pharmacokinetics of protein drug delivery to treat systemic and disseminated diseases. Interleukin-2 (IL-2) is notoriously toxic at the high doses required for therapeutic efficacy. Here, we demonstrate the therapeutic efficacy and tolerability of low doses of PODS containing IL-2 cargo (PODS-IL-2) administered intravenously in a mouse model of melanoma. We further demonstrate the therapeutic benefit of PODS delivering IL-2, interleukin-15 (IL-15) and interferon gamma (IFN-{gamma}) in a mouse model of renal cell carcinoma at two doses. Efficacy was seen in both doses with the higher dose generating rapid and complete rejection of the tumour in some of the mice treated with each cytokine. This study provides proof-of-concept for the utility of intravenously administered PODS to provide a generalised and widely applicable mechanism to effectively deliver protein drugs for the therapy of cancer and potentially other diseases.

cancer biology↗

bioRxiv2023

A meta-analysis of immune cell fractions at high resolution reveals novel associations with common phenotypes and health outcomes

AbstractO_ST_ABSBackgroundC_ST_ABSChanges in cell-type composition of complex tissues are associated with a wide range of diseases, environmental risk factors and may be causally implicated in disease development and progression. However, these shifts in cell-type fractions are often of a low magnitude, or involve similar cell-subtypes, making their reliable identification challenging. DNA methylation profiling in a tissue like blood is a promising approach to discover shifts in cell-type abundance, yet studies have only been performed at a relatively low cellular resolution and in isolation, limiting their power to detect these shifts in tissue composition. MethodsHere we derive a DNA methylation reference matrix for 12 immune cell-types in human blood and extensively validate it with flow-cytometric count data and in whole-genome bisulfite sequencing data of sorted cells. Using this reference matrix and Stouffers method, we perform a meta-analysis encompassing 25,629 blood samples from 22 different cohorts, to comprehensively map associations between the 12 immune-cell fractions and common phenotypes, including health outcomes. ResultsOur meta-analysis reveals many associations with age, sex, smoking and obesity, many of which we validate with single-cell RNA-sequencing. We discover that T-regulatory and naive T-cell subsets are higher in women compared to men, whilst the reverse is true for monocyte, natural killer, basophil and eosinophil fractions. In a large subset encompassing 5000 individuals we find associations with stress, exercise, sleep and health outcomes, revealing that naive T-cell and B-cell fractions are associated with a reduced risk of all-cause mortality independently of age, sex, race, smoking, obesity and alcohol consumption. We find that decreased natural killer cell counts are associated with smoking, obesity and stress levels, whilst an increased count correlates with exercise, sleep and a reduced risk of all-cause mortality. ConclusionsThis work derives and extensively validates a high resolution DNAm reference matrix for blood, and uses it to generate a comprehensive map of associations between immune cell fractions and common phenotypes, including health outcomes. AvailabilityThe 12 immune cell-type DNAm reference matrices for Illumina 850k and 450k beadarrays alongside tools for cell-type fraction estimation are freely available from our EpiDISH Bioconductor R-package http://www.bioconductor.org/packages/devel/bioc/html/EpiDISH.html

bioinformatics↗

bioRxiv2023

Interpretation of body condition index should be informed by natural history

Many studies assume that it is beneficial for individuals of a species to be heavier, or have a higher body condition index (BCI), without accounting for the physiological relevance of variation in the composition of different body tissues. We hypothesized that the relationship between BCI and masses of physiologically important tissues (fat and lean) would be conditional on annual patterns of energy acquisition and expenditure. We studied three species with contrasting ecologies in their respective natural ranges: an obligate hibernator (Columbian ground squirrel, Urocitellus columbianus), a facultative hibernator (black-tailed prairie dog, Cynomys ludovicianus), and a food-caching non-hibernator (North American red squirrel, Tamiasciurus hudsonicus). We measured fat and lean mass in adults of both sexes using quantitative magnetic resonance (QMR). We measured body mass and two measures of skeletal structure (zygomatic width and right hind foot length) to develop sex- and species-specific BCIs, and tested the utility of BCI to predict body composition in each species. Body condition indices were more consistently, and more strongly correlated, with lean mass than fat mass. The indices were most positively correlated with fat when fat was expected to be very high (pre-hibernation prairie dogs). In all cases, however, BCI was never better than body mass alone in predicting fat or lean mass. While the accuracy of BCI in estimating fat varied across the natural histories and annual energetic patterns of the species considered, measuring body mass alone was as effective, or superior in capturing sufficient variation in fat and lean in most cases.

physiology↗

bioRxiv2022

Exceptionally versatile respiratory metabolisms drive metabolite production by diverse gut bacteria

Respiratory reductases enable microbes to utilize molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae, Erysipelotrichaceae) that encode large arsenals of tens-to-hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta, and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate-profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

microbiology↗

bioRxiv2022

Identification of key residues of the DNA glycosylase OGG1 controlling efficient DNA scanning and recruitment to oxidized bases in living cells

The DNA-glycosylase OGG1 oversees the detection and clearance of the 7,8-dihydro-8-oxoguanine (8-oxoG), which is the most frequent form of oxidized base in the genome. This lesion is deeply buried within the double-helix and its detection requires careful inspection of the bases by OGG1 via a mechanism that remains only partially understood. By analyzing OGG1 dynamics in the nucleus of living human cells, we demonstrate that the glycosylase constantly scans the DNA by rapidly alternating between diffusion within the nucleoplasm and short transits on the DNA. This scanning process, that we find to be tightly regulated by the conserved residue G245, is crucial for the rapid recruitment of OGG1 at oxidative lesions induced by laser micro-irradiation. Furthermore, we show that residues Y203, N149 and N150, while being all involved in early stages of 8-oxoG probing by OGG1 based on previous structural data, differentially regulate the scanning of the DNA and recruitment to oxidative lesions.

cell biology↗

bioRxiv2022

Skeletal muscle DNA methylation and mRNA responses to a bout of higher versus lower load resistance exercise in previously trained men

We sought to determine the skeletal muscle genome-wide DNA methylation and mRNA responses to one bout of lower-load (LL) versus higher-load (HL) resistance exercise. Trained college-aged males (n=11, 23{+/-}4 years old, 4{+/-}3 years self-reported training) performed LL or HL bouts to failure separated by one week. The HL bout (i.e., 80 Fail) consisted of four sets of back squats and four sets of leg extensions to failure using 80% of participants estimated one-repetition maximum (i.e., est. 1-RM). The LL bout (i.e., 30 Fail) implemented the same paradigm with 30% of est. 1-RM. Vastus lateralis muscle biopsies were collected before, 3 hours, and 6 hours after each bout. Muscle DNA and RNA were batch-isolated and analyzed using the 850k Illumina MethylationEPIC array and Clariom S mRNA microarray, respectively. Performed repetitions were significantly greater during the 30 Fail versus 80 Fail (p<0.001), although total training volume (sets x reps x load) was not significantly different between bouts (p=0.571). Regardless of bout, more CpG site methylation changes were observed at 3-versus 6-hours post exercise (239,951 versus 12,419, respectively; p<0.01), and nuclear global ten-eleven translocation (TET) activity, but not global DNA methyltransferase activity, increased 3- and 6-hours following exercise regardless of bout. The percentage of genes significantly altered at the mRNA level that demonstrated opposite DNA methylation patterns was greater 3- versus 6-hours following exercise (~75% versus ~15%, respectively). Moreover, high percentages of genes that were up- or downregulated 6 hours following exercise also demonstrated significantly inversed DNA methylation patterns across one or more CpG sites 3 hours following exercise (65% and 82%, respectively). While 30 Fail decreased DNA methylation across various promoter regions versus 80 Fail, transcriptome-wide mRNA and bioinformatics indicated that gene expression signatures were largely similar between bouts. Bioinformatics overlay of DNA methylation and mRNA expression data indicated that genes related to "Focal adhesion", "MAPK signaling", and "PI3K-Akt signaling" were significantly affected at the 3- and 6-hour time points, and again this was regardless of bout. In conclusion, extensive molecular profiling suggests that post-exercise alterations in the skeletal muscle DNA methylome and mRNA transcriptome elicited by LL and HL training bouts to failure are largely similar, and this could be related to equal volumes performed between bouts.

molecular biology↗

bioRxiv2022

Is shape in the eye of the beholder? The reproducibility of geometric morphometric analyses on live animals

Geometric morphometrics is widely used to quantify morphological variation between biological specimens, but the fundamental influence of operator bias on data reproducibility is rarely considered, particularly in studies using photographs of live animals taken under field conditions. We examined this using four independent operators that applied an identical landmarking scheme to replicate photographs of 291 live Atlantic salmon (Salmo salar L.) from two rivers. Using repeated measures tests, we found significant inter-operator differences in mean body shape, suggesting that the operators introduced a systematic error despite following the same landmarking scheme. No significant differences were detected when the landmarking process was repeated by the same operator on a random subset of photographs. Importantly, in spite of significant operator bias, small but statistically significant morphological differences between fish from the two rivers were found consistently by all operators. Pairwise tests of angles of vectors of shape change showed that these between-river differences in body shape were analogous across operator datasets, suggesting a general reproducibility of findings obtained by geometric morphometric studies. In contrast, merging landmark data when fish from each river are digitised by different operators had a significant impact on downstream analyses, highlighting an intrinsic risk of bias. Overall, we show that, even when significant inter-operator error is introduced during digitisation, following an identical landmarking scheme can identify morphological differences between populations. This study indicates that operators digitising at least a sub-set of all data groups of interest may be an effective way of mitigating inter-operator error and potentially enabling data sharing.

zoology↗