Biology subjects

Reyes, L.

Publications and source records attributed to Reyes, L..

9 recordsLinked to original sources

bioRxiv2025

DNA Circle-sequencing lowers the single molecule sequencing error threshold and identifies ultrasonication as a source of DNA damage

DNA mutation is the ultimate source of all heritable genetic and phenotypic variation. On human timescales, DNA mutation results in the evolution of antibiotic resistance, viral resistance to vaccines, the emergence human cancers, and more. Precise measurement of DNA mutation is therefore desirable to rapidly detect and analyze low frequency mutations in a population of cells. Precise measurement of DNA mutation by high throughput sequencing has been hindered by sequencing error rates, which occur at rates 1x10-2 to 1x10-3 per base sequenced. Previous work on circle sequencing had pushed a putative sequencing error rate down to roughly 2.8x10-4 per base for genomic yeast DNA in the absence of DNA repair enzymes, where the expected number is 4.7x10-9 per base per generation. Through revision of the assay, we are now capable of taking 125ng of genomic DNA and obtaining mutation rate estimates with a sequencing resolution floor of 2x10-7 per base in the absence of repair enzymes, a resolution improvement of over 3 orders of magnitude. In practice, circle-seq recovers some of the mutation spectrum of mismatch-repair deficient E. coli, although some signature of C [->] T and G [->] A errors remain present. Curiously, it calls a mutation rate of 2x10-9 per base per generation, lower than expected, underscoring the difficulty of directly comparing mutation rates per base to MA mutation rates per site per generation. This protocol readily recognizes Covaris ultrasonication as mutagenic in library preps, with a mutation spectrum dominated by G:C [->] A:T, G:C [->] C:G, and G:C [->] T:A errors. The protocol is either detecting in vivo DNA damage that has yet to be repaired, is significantly biased in its ability to detect mutations, or is not yet sensitive enough to detect mutation rates of MMR-E. coli cells. The protocol may have some use in measuring mutation abundance from tissue samples, and in vitro sources of DNA damage. The protocol also highlights some biases inherent to single-stranded DNA (ssDNA) cyclization, including a 10-bp periodicity which echoes the constraints of dsDNA cyclization.

molecular biology↗

bioRxiv2025

Single cell proteomic analysis defines discrete neutrophil functional states in human glioblastoma

Neutrophils are vital innate immune cells shown to infiltrate glioblastomas, however we currently lack the molecular understanding of their functional states within the tumour niche. Given that neutrophils are known to display a prominent discordance between mRNA and protein abundance, we developed ultra-sensitive mini-bulk and single cell proteomic (SCP) workflows to study the heterogeneity of peripheral blood and tumour associated neutrophils (TAN) from patients with glioblastoma. Mini-bulk analysis enabled a deeper protein coverage of circulating immature, mature and TAN populations, defining signatures of maturity and demonstrating that TANs resemble mature circulating neutrophils. Analysis of the SCP data resulted in the detection of >1,100 proteins from a single TAN providing a detailed characterization of neutrophil subsets in glioblastoma. Our approach shows evidence of pathogenic and anti-tumorigenic clusters and discovers cell states invisible to scRNAseq, opening new opportunities to selectively target pro-tumoural neutrophil states.

immunology↗

bioRxiv2023

A Narrow Range of Transcript-error Rates Across the Tree of Life

The expression of genomically-encoded information is not error-free. Transcript-error rates are dramatically higher than DNA-level mutation rates, and despite their transient nature, the steady-state load of such errors must impose some burden on cellular performance. However, a broad perspective on the degree to which transcript-error rates are constrained by natural selection and diverge among lineages remains to be developed. Here, we present a genome-wide analysis of transcript-error rates across the Tree of Life using a modified rolling-circle sequencing method, revealing that the range in error rates is remarkably narrow across diverse species. Transcript errors tend to be randomly distributed, with little evidence supporting local control of error rates associated with gene-expression levels. A majority of transcript errors result in missense errors if translated, and as with a fraction of nonsense transcript errors, these are underrepresented relative to random expectations, suggesting the existence of mechanisms for purging some such errors. To quantitatively understand how natural selection and random genetic drift might shape transcript-error rates across species, we present a model based on cell biology and population genetics, incorporating information on cell volume, proteome size, average degree of exposure of individual errors, and effective population size. However, while this model provides a framework for understanding the evolution of this highly conserved trait, as currently structured it explains only 20% of the variation in the data, suggesting a need for further theoretical work in this area.

evolutionary biology↗

bioRxiv2023

Vertical transmission of African-lineage Zika virus through the fetal membranes in a rhesus Macaque (Macaca mulatta) model

Zika virus (ZIKV) can be transmitted vertically from mother to fetus during pregnancy, resulting in a range of outcomes, including severe birth defects and fetal/infant death. Potential pathways of vertical transmission in utero have been proposed but remain undefined. Identifying the timing and routes of vertical transmission of ZIKV may help us identify when interventions would be most effective. Furthermore, understanding what barriers ZIKV overcomes to effect vertical transmission may help improve models for evaluating infection by other pathogens during pregnancy. To determine the pathways of vertical transmission, we inoculated 12 pregnant rhesus macaques with an African-lineage ZIKV at gestational day 30 (term is 165 days). Eight pregnancies were surgically terminated at either seven or 14 days post-maternal infection. Maternal-fetal interface and fetal tissues and fluids were collected and evaluated with RT-qPCR, in situ hybridization for ZIKV RNA, immunohistochemistry, and plaque assays. Four additional pregnant macaques were inoculated and terminally perfused with 4% paraformaldehyde at three, six, nine, or ten days post-maternal inoculation. For these four cases, the entire fixed pregnant uterus was evaluated with in situ hybridization for ZIKV RNA. We determined that ZIKV can reach the MFI by six days post-infection and infect the fetus by ten days. Infection of the chorionic membrane and the extraembryonic coelomic fluid preceded infection of the fetus and the mesenchymal tissue of the placental villi. We did not find evidence to support a transplacental route of ZIKV vertical transmission via infection of syncytiotrophoblasts or villous cytotrophoblasts. The pattern of infection observed in the maternal-fetal interface provides evidence of vertical ZIKV transmission through the fetal membranes. Authors SummaryZika virus (ZIKV) can be vertically transmitted from mother to fetus during pregnancy resulting in adverse pregnancy outcomes. For vertical transmission to occur, ZIKV must overcome the barriers of the maternal-fetal interface, yet the exact pathway ZIKV takes remains undefined. The maternal-fetal interface consists of the maternal decidua, the placenta, and the fetal membranes. ZIKV could reach the fetus through the placenta if it can infect the layer of cells that are directly exposed to maternal blood. ZIKV could also reach the fetus by infecting the decidua and then the adjacent fetal membranes. To determine the pathways of ZIKV vertical transmission, we infected pregnant macaques and evaluated ZIKV burden in the maternal-fetal interface and fetus shortly after maternal infection. The pattern of infection observed suggests that ZIKV vertically transmits through the fetal membranes, not the placenta. This finding is significant because it challenges the assumption that vertical transmission occurs exclusively across the placenta. By including the fetal membranes in our models of vertical transmission, we can more accurately determine which pathogens can be vertically transmitted. Ultimately, this study demonstrates that fetal membranes are an essential barrier to pathogens that warrant further investigation.

pathology↗

bioRxiv2022

Ablation of Tmem65 Causes Lethal Mitochondrial Encephalomyopathy in Mouse

Transmembrane protein 65 (TMEM65) depletion in a patient carrying a homozygous variant in the Tmem65 splice site resulted in severe mitochondrial encephalomyopathy, indicating the clinical importance of TMEM65. However, the function of TMEM65 remains unknown. Here, we generated a TMEM65 reporter mouse as well as whole-body and tissue-specific Tmem65 knockout (KO) mice to investigate the localization and function of TMEM65. We show that TMEM65 is localized to mitochondria in heart, skeletal muscle, and throughout the brain. Both whole-body and nervous system-specific Tmem65 KO result in severe growth retardation and sudden death following seizures ~3 weeks after birth, indicating TMEM65 is indispensable for normal brain function. In addition, we find that skeletal muscle-specific Tmem65 KO leads to progressive, adult-onset myopathy preceded by elevated mitochondrial calcium levels despite unaltered expression of known mitochondrial or cellular calcium handling proteins. Consistently, we demonstrate that ablation of TMEM65 results in a loss of sodium-dependent mitochondrial calcium export. Finally, we show that blocking mitochondrial calcium entry through removal of the mitochondrial calcium uniporter (MCU) rescues the early lethality of whole-body TMEM65 ablation. Our data not only reveal the essential role of TMEM65 in mammalian physiology, but also suggest modulating mitochondrial calcium may offer a potential therapeutical approach to address defects associated with TMEM65 misexpression.

physiology↗

bioRxiv2022

Hypoxia shapes the immune landscape in lung injury promoting inflammation persistence

Acute Respiratory Distress Syndrome (ARDS), an often-fatal complication of pulmonary or systemic inflammation, has no cure. Hypoxemia is a defining feature, yet its impact on inflammation is often neglected. Patients with ARDS are monocytopenic early in the onset of the disease. Endotoxin or Streptococcus pneumoniae acute lung injury (ALI) in the context of hypoxia replicates this finding, through hypoxia-driven suppression of type I interferon signalling. This results in failed lung monocyte-derived interstitial macrophages (IM) niche expansion and unchecked neutrophilic inflammation. Administration of colony stimulating factor 1 (CSF1) rescues the monocytopenia, alters the circulating classical monocyte phenotype in hypoxic endotoxin-driven ALI and enables lung IM population expansion, thus limiting lung injury in endotoxin- and virally-induced hypoxic ALI. Hypoxia directly alters immune dynamics to the detriment of the host and manipulation of this aberrant response offers new therapeutic strategies for ARDS.

cell biology↗

bioRxiv2020

Nano-Emulsion Adjuvant Enhances Cross-presentation by Lipid Body Formation Independent of Glycolysis

Here, we report that a carbomer-based adjuvant, Adjuplex(R) (ADJ), stimulated robust CD8 T-cell responses to subunit antigens by modulating multiple steps in the cytosolic pathway of cross-presentation, and afforded effective immunity against virus and intracellular bacteria. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs and NLRP3-driven caspase 1 activity. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, induction of ROS and lipid bodies (LBs) and alterations in LB composition mediated by ADJ were critical for DC cross-presentation. These findings challenge the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have implications in the rational development of novel adjuvants.

immunology↗

bioRxiv2020

The complete genome sequence of the Staphylococcus bacteriophage Metroid

Phages infecting bacteria of the genus Staphylococcus play an important role in their hosts ecology and evolution. On one hand, horizontal gene transfer from phage can encourage the rapid adaptation of pathogenic Staphylococcus enabling them to escape host immunity or access novel environments. On the other hand, lytic phages are promising agents for the treatment of bacterial infections, especially those resistant to antibiotics. As part of an ongoing effort to gain novel insights into bacteriophage diversity, we characterized the complete genome of the Staphylococcus bacteriophage Metroid, a cluster C phage with a genome size of 151kb, encompassing 254 predicted protein-coding genes as well as 4 tRNAs. A comparative genomic analysis highlights strong similarities - including a conservation of the lysis cassette - with other Staphylococcus cluster C1 bacteriophages, several of which were previously characterized for therapeutic applications.

genomics↗

bioRxiv2019

Progressive shifts in the gut microbiome reflect prediabetes and diabetes development in a treatment-naive Mexican cohort

Type 2 diabetes (T2D) is a global epidemic that affects more than 8% of the worlds population and is a leading cause of death in Mexico. Diet and lifestyle are known to contribute to the onset of T2D. However, the role of the gut microbiome in T2D progression remains uncertain. Associations between microbiome composition and diabetes are confounded by medication use, diet, and obesity. Here we present data on a treatment-naive cohort of 405 Mexican individuals across varying stages of T2D severity. Associations between gut bacteria and more than 200 clinical variables revealed a defined set of bacterial genera that were consistent biomarkers of T2D prevalence and risk. Specifically, gradual increases in blood glucose levels, beta cell dysfunction, and the accumulation of measured T2D risk factors was correlated with the relative abundances of four bacterial genera. In a cohort of 25 individuals, T2D treatment - predominantly metformin - reliably returned the microbiome to the normoglycemic community state. Deep clinical characterization allowed us to broadly control for confounding variables, indicating that these microbiome patterns were independent of common T2D comorbidities, like obesity or cardiovascular disease. Thus, our work provides the first solid evidence for a direct link between the gut microbiome and T2D in a critically high-risk population. Whether or not these T2D-associated changes in the gut contribute to the etiology of T2D or its comorbidities remains to be seen.

systems biology↗