Biology subjects

Rodriguez, S.

Publications and source records attributed to Rodriguez, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Comprehensive Assessment of Initial Adaptation of ESBL Positive ST131 Escherichia coli to Carbapenem Exposure

BackgroundIt remains unclear how high-risk Escherichia coli lineages, like sequence type (ST) 131, initially adapt to carbapenem exposure in their progression to becoming carbapenem resistant. MethodsCarbapenem mutation frequency was measured in multiple subclades of extended-spectrum {beta}-lactamase (ESBL) positive ST131 clinical isolates using a fluctuation assay followed by whole genome sequencing (WGS) characterization. Genomic, transcriptomic, and porin analyses of ST131 C2/H30Rx isolate, MB1860, under prolonged, increasing carbapenem exposure was performed using two distinct experimental evolutionary platforms to measure fast vs. slow adaptation. ResultsAll thirteen ESBL positive ST131 strains selected from a diverse (n=184) ST131 bacteremia cohort had detectable ertapenem (ETP) mutational frequencies with a statistically positive correlation between initial ESBL gene copy number and mutation frequency (r = 0.87, P-value <1e-5). WGS analysis of mutants showed initial response to ETP exposure resulted in significant increases in ESBL gene copy numbers or mutations in outer membrane porin (Omp) encoding genes in the absence of ESBL gene amplification with subclade specific associations. In both experimental evolutionary platforms, MB1860 responded to initial ETP exposure by increasing blaCTX-M-15 copy numbers via modular, insertion sequence 26 (IS26) mediated pseudocompound transposons (PCTns). Transposase activity driven by PCTn upregulation was a conserved expression signal in both experimental evolutionary platforms. Stable mutations in Omp encoding genes were detected only after prolonged increasing carbapenem exposure consistent with clinical observations. ConclusionsESBL gene amplification is a conserved response to initial carbapenem exposure, especially within the high-risk ST131 C2/H30Rx subclade. Targeting such amplification could assist with mitigating carbapenem resistance development.

microbiology↗

bioRxiv2024

TYK2 as a novel therapeutic target in Alzheimer Disease

Neuroinflammation is a pathological feature of many neurodegenerative diseases, including Alzheimers disease (AD)1,2 and amyotrophic lateral sclerosis (ALS)3, raising the possibility of common therapeutic targets. We previously established that cytoplasmic double-stranded RNA (cdsRNA) is spatially coincident with cytoplasmic pTDP-43 inclusions in neurons of patients with C9ORF72-mediated ALS4. CdsRNA triggers a type-I interferon (IFN-I)-based innate immune response in human neural cells, resulting in their death 4. Here, we report that cdsRNA is also spatially coincident with pTDP-43 cytoplasmic inclusions in brain cells of patients with AD pathology and that type-I interferon response genes are significantly upregulated in brain regions affected by AD. We updated our machine-learning pipeline DRIAD-SP (Drug Repurposing In Alzheimers Disease with Systems Pharmacology) to incorporate cryptic exon (CE) detection as a proxy of pTDP-43 inclusions and demonstrated that the FDA-approved JAK inhibitors baricitinib and ruxolitinib that block interferon signaling show a protective signal only in cortical brain regions expressing multiple CEs. Furthermore, the JAK family member TYK2 was a top hit in a CRISPR screen of cdsRNA-mediated death in differentiated human neural cells. The selective TYK2 inhibitor deucravacitinib, an FDA-approved drug for psoriasis, rescued toxicity elicited by cdsRNA. Finally, we identified CCL2, CXCL10, and IL-6 as candidate predictive biomarkers for cdsRNA-related neurodegenerative diseases. Together, we find parallel neuroinflammatory mechanisms between TDP-43 associated-AD and ALS and nominate TYK2 as a possible disease-modifying target of these incurable neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Highly pathogenic avian influenza H5N1 virus infections in pinnipeds and seabirds in Uruguay: a paradigm shift to virus transmission in South America

The highly pathogenic avian influenza viruses of the clade 2.3.4.4b have caused unprecedented deaths in South American wild birds, poultry, and marine mammals. In September 2023, pinnipeds and seabirds appeared dead on the Uruguayan Atlantic coast. Sixteen influenza virus strains were characterized by real-time reverse transcription PCR and genome sequencing in samples from sea lions (Otaria flavescens), fur seals (Arctocephalus australis), and terns (Sterna hirundinacea). Phylogenetic and ancestral reconstruction analysis showed that these strains have pinnipeds as the most likely ancestral host, representing a recent introduction of the clade 2.3.4.4b in Uruguay. The Uruguayan and closely related strains from Peru (sea lions) and Chile (sea lions and a human case) carry mammalian adaptative residues 591K and 701N in the viral polymerase basic protein 2 (PB2). Our findings suggest that the clade 2.3.4.4b strains in South America may have spread from mammals to mammals and seabirds, revealing a new transmission route.

microbiology↗

bioRxiv2023

Activity of Antibiotics against Burkholderia cepacia complex in Artificial Sputum Medium

Burkholderia cepacia complex (Bcc) is a collection of intrinsically drug-resistant gram-negative bacteria that cause life-threatening pulmonary disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in people with Bcc infections, likely due in part to the significant differences between in vitro testing conditions and the environment in which Bcc grow in the lungs of people with CF. We tested the activity of six commonly used antibiotics against two clinical Bcc strains grown to high density in an artificial sputum medium in order to assess their activity in conditions mimicking those found in vivo. There were major discrepancies between standard susceptibility results and activity in our model, with some antibiotics, including ceftazidime, showing minimal activity despite low MICs, while others, notably tobramycin, were more active in high-density growth conditions than in standard assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.

microbiology↗

bioRxiv2023

Neuroprotective Effects of Naltrexone in a Mouse Model of Post-Traumatic Epilepsy

Traumatic Brain Injury (TBI) induces neuroinflammatory responses that can initiate epileptogenesis, which develops into epilepsy. Recently, we identified the anti-convulsive effects of naltrexone, a mu-opioid receptor (MOR) antagonist. While blocking opioid receptors can reduce inflammation, it is unclear if post-TBI epileptogenesis can be prevented by blocking MORs. Here, we tested if naltrexone prevents neuroinflammation and epileptogenesis post-TBI. TBI was induced by a modified Marmarau Weight-Drop (WD) method applied to four-week-old C57BL/6J male mice. Mice were given the pro-convulsant pentylenetetrazol (PTZ) on day two post-injury while telemetry-monitored mice received PTZ on day five. Naltrexone/vehicle treatment started two hours after PTZ. Integrated EEG-video (vEEG) recorded interictal events and spontaneous seizures for three months. Molecular, histological and neuroimaging techniques were used to evaluate neuroinflammation and neurodegeneration both acutely and chronically. Peripheral immune responses were assessed through serum chemokine/cytokine measurements. We observed increases in MOR expression, nitro-oxidative stress, mRNA expression of inflammatory cytokines, microgliosis, neurodegeneration, and white matter damage in the neocortex of TBI mice. vEEG revealed increased interictal events in TBI mice, with 71% developing epilepsy. Naltrexone ameliorated neuroinflammation and neurodegeneration, reduced interictal events and prevented epilepsy, illustrating that naltrexone is a promising drug to prevent TBI-associated neuroinflammation and epileptogenesis in post-traumatic epilepsy.

neuroscience↗

bioRxiv2023

Tetracycline transactivator overexpression in keratinocytes triggers a TRPV1 primary sensory neuron-dependent neuropathic itch

Mouse models that combine tetracycline-controlled gene expression systems and conditional genetic activation can tightly regulate transgene expression in discrete cell types and tissues. However, the commonly used Tet-Off variant, tetracycline transactivator (tTA), when overexpressed and fully active, can lead to developmental lethality, disease, or more subtle behavioral phenotypes. Here we describe a profound itch phenotype in mice expressing a genetically encoded tTA that is conditionally activated within the Phox2a lineage. Phox2a; tTA mice develop intense, localized scratching and regional skin lesions that can be controlled by the tTA inhibitor, doxycycline. As gabapentin, but not morphine, relieved the scratching, we consider this phenotype to result from chronic neuropathic itch, not pain. In contrast to the Phox2a lineage, mice with tTA activated within the Phox2b lineage, which has many similar areas of recombination within the nervous system, did not recapitulate the scratching phenotype. In Phox2a-Cre mice, but not Phox2b-Cre, intense Cre-dependent reporter expression was found in skin keratinocytes which formed the area at which skin lesions developed. Most interestingly, repeated topical application of the DREADD agonist, CNO, which chronically induced Gi signaling in Phox2a-keratinocytes, completely reversed the localized scratching and skin lesions. Furthermore, ablation of TRPV1-expressing, primary afferent neurons reduced the scratching with a time course comparable to that produced by Gi-DREADD inhibition. These temporal properties suggest that the neuropathic itch condition arises not only from localized keratinocyte activation of peripheral nerves but also from a persistent, gabapentin-sensitive state of central sensitization.

neuroscience↗

bioRxiv2023

The nucleolar aberrancies that drive ribosome impairment induced by RNA binding proteins are hallmarks of aging

The nucleolus is a dynamic structure where ribosome subunits are produced. Indeed, nucleoli respond to any change in cellular homeostasis by altering the rate of ribosome biogenesis, thus working as a stress sensor. Therefore, an imbalance in ribosome biogenesis promotes changes in morphology and function and can evoke a nucleolar stress response. Changes in the structure and composition of nucleoli impair ribosome biogenesis and have been described as nucleolar stress, a mechanism related to aging and cancer. Here, we show the role of the RNA binding protein Hnrnpk in nucleolar dynamics and ribosome function. Hnrnpk is a ribonucleoprotein in charge of escorting nascent transcripts to its processing and nuclear export to ribosomes. When Hnrnpk is overexpressed, the nucleolus is altered and shows stress-like phenotype, with accumulation and delocalization of components such as Ncl, driving ribosome biogenesis impairment and halting protein translation. Nucleolin haploinsufficiency is correlated with enlarged nucleoli, increased ribosome components and translation and induces a reduction in lifespan. Thus, gain of Ncl generated by Hnrnpk overexpression can cause ribosome biogenesis defects associated with ribosome impairment leading to ribosomopathies and bone marrow failure syndrome. Aging and bone marrow failure share common biological hallmarks. Indeed, Hnrnpk overexpression and nucleolar stress trigger cell cycle arrest and senescence of the cells, a feature of both processes. Together, these findings support the idea that nucleolar abnormalities contribute to ribosome impairment, thus triggering the onset of hematopoiesis and the aging process. Here, we decipher a novel master regulator of this mechanism: Hnrnpk.

molecular biology↗

bioRxiv2023

Hypermetabolism in mice carrying a near complete human chromosome 21

The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are due to sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health.

physiology↗

bioRxiv2022

Genetic risk for Multiple Sclerosis originated in Pastoralist Steppe populations

Multiple sclerosis (MS) is a modern neuro-inflammatory and -degenerative disease, which is most prevalent in Northern Europe. Whilst it is known that inherited risk to MS is located within or within close proximity to immune genes, it is unknown when, where and how this genetic risk originated 1. By using the largest ancient genome dataset from the Stone Age 2, along with new Medieval and post-Medieval genomes, we show that many of the genetic risk variants for MS rose to higher frequency among pastoralists located on the Pontic Steppe, and were brought into Europe by the Yamnaya-related migration approximately 5,000 years ago. We further show that these MS-associated immunogenetic variants underwent positive selection both within the Steppe population, and later in Europe, likely driven by pathogenic challenges coinciding with dietary, lifestyle, and population density changes. This study highlights the critical importance of this period as a determinant of modern immune responses and its subsequent impact on the risk of developing MS in a changing environment.

evolutionary biology↗

bioRxiv2022

Robust and Versatile Arrayed Libraries for Human Genome-Wide CRISPR Activation, Deletion and Silencing

Arrayed CRISPR libraries extend the scope of gene-perturbation screens but require large numbers of efficacious sgRNA-expressing vectors. Using a newly invented liquid-phase plasmid cloning methodology, we constructed genome-wide arrayed libraries for human gene ablation (19,936 plasmids), activation, and epigenetic silencing (22,442 plasmids). At least 76% of each plasmid preparation encoded an intact array of 4 non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. We achieved perturbation efficacies of 75-99%, 76-92% and up to 10,000x in deletion, silencing and activation experiments, respectively. Upon conversion into massively parallel lentiviral vectors, an arrayed activation screen of 1,634 human transcription factors yielded 11 novel regulators of the cellular prion protein PrPC. Furthermore, a screen using a pooled version of the ablation library identified 5 novel modifiers of autophagy that went undetected with either of two 1sgRNA libraries. The CRISPR libraries described here represent a powerful resource for the targeted perturbation of human protein-coding genes.

genomics↗

bioRxiv2022

Myeloid-biased HSC require Semaphorin 4A from the bone marrow niche for self-renewal under stress and life-long persistence

Tissue stem cells are hierarchically organized. Those that are most primitive serve as key drivers of regenerative response but the signals that selectively preserve their functional integrity are largely unknown. Here, we identify a secreted factor, Semaphorin 4A (Sema4A), as a specific regulator of myeloid-biased hematopoietic stem cells (myHSC), which are positioned at the top of the HSC hierarchy. Lack of Sema4A leads to exaggerated myHSC (but not downstream "balanced" HSC) proliferation after acute inflammatory stress, indicating that Sema4A enforces myHSC quiescence. Strikingly, aged Sema4A knock-out myHSC expand but almost completely lose reconstitution capacity. The effect of Sema4A is non cell-autonomous, since upon transplantation into Sema4A-deficient environment, wild-type myHSC excessively proliferate but fail to engraft long-term. Sema4A constrains inflammatory signaling in myHSC and acts via a surface receptor Plexin-D1. Our data support a model whereby the most primitive tissue stem cells critically rely on a dedicated signal from the niche for self-renewal and life-long persistence.

cell biology↗

bioRxiv2021

Evidence of positive and negative selection associated with DNA methylation

Signatures of negative selection are pervasive amongst complex traits and diseases. However, it is unclear whether such signatures exist for DNA methylation (DNAm) that has been proposed to have a functional role in disease. We estimate polygenicity, SNP-based heritability and model the joint distribution of effect size and minor allele frequency (MAF) to estimate a selection coefficient (S) for 2000 heritable DNAm sites in 1774 individuals from the Avon Longitudinal Study of Parents and Children. Additionally, we estimate S for meta stable epi alleles and DNAm sites associated with aging and mortality, birthweight and body mass index. Quantification of MAF-dependent genetic architectures estimated from genotype and DNAm reveal evidence of positive (S > 0) and negative selection (S < 0) and confirm previous evidence of negative selection for birthweight. Evidence of both negative and positive selection highlights the role of DNAm as an intermediary in multiple biological pathways with competing function.

genetics↗

bioRxiv2021

Transcriptomics reveals how minocycline-colistin synergy overcomes antibiotic resistance in multidrug-resistant Klebsiella pneumoniae

Multidrug resistant gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using RNA-Seq, we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline. IMPORTANCEFew treatment options exist for multidrug resistant gram-negative bacteria. Synergistic antimicrobial combinations (in which two individually inactive drugs regain activity when used in combination) can overcome resistance. In susceptible bacteria, minocycline acts inside the cell by preventing protein synthesis, while colistin disrupts the outer membrane. Even in bacteria resistant to both drugs, the combination of the two is active. To understand this activity, we studied Klebsiella pneumoniae isolates resistant to both drugs. We extracted messenger RNA during treatment with each drug alone and with the combination, then performed RNA sequencing to analyze gene expression levels. Bacteria treated with the combination had an expression profile similar to bacteria treated with minocycline alone. Our findings suggest that synergy results from low-level, subinhibitory membrane disruption by colistin, which permits sufficient accumulation of minocycline in the cell to overcome resistance. Understanding this pathway will facilitate further development of synergistic antimicrobial combination regimens.

microbiology↗

bioRxiv2021

Profiling the in vitro and in vivo activity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action

Streptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked {beta}-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940s, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50 and MIC90 for S-F and S-D were 2 and 4 {micro}M, and 0.25 and 0.5 {micro}M, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. There was >10-fold in vitro selectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operon E. coli identify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, gram-negative pathogens. IMPORTANCEStreptothricins are a historic class of antibiotics discovered by Waksman and colleagues in the 1940s. Toxicities associated with the streptothricin natural product mixture, also known as nourseothricin, discouraged further development. However, we found that a component of nourseothricin, streptothricin-F, retained potent activity against contemporary carbapenem-resistant Enterobacterales with significant selectivity in in vitro and in vivo assays. This included demonstration of rapid bactericidal activity in vitro and substantial therapeutic effect in the murine thigh model against a pandrug-resistant Klebsiella pneumoniae isolate at non-toxic concentrations. Through resistance mutation analysis, we identified helix 34 of 16S rRNA in the prokaryotic ribosome, and specifically bases C1054 and A1196, as critical for streptothricins activity. The mechanism of action is distinct from other known translation inhibitors. Based on promising and unique activity, we believe the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens.

microbiology↗

bioRxiv2021

Single calcium channel nanodomains drive presynaptic calcium entry at individual lamprey reticulospinal presynaptic terminals

Synchronous neurotransmission is central to efficient information transfer in neural circuits, requiring precise coupling between action potentials, Ca2+ entry and neurotransmitter release. However, determinations of Ca2+ requirements for release, which may originate from entry through single voltage-gated Ca2+ channels, remain largely unexplored in simple active zone synapses common in the nervous system. Understanding these requirements is key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurological and neuropsychiatric disorders. Here, we present single channel analysis of evoked active zone Ca2+ entry, using cell-attached patch clamp and lattice light sheet microscopy over active zones at single central lamprey reticulospinal presynaptic terminals. Our findings show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising subtypes N-type (CaV2.2), P/Q-type (CaV2.1) and R-type (CaV2.3), available to gate neurotransmitter release. Significantly, of this pool only 1-6 (mean of 4) channels open upon depolarization. High temporal fidelity lattice light sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations between action potentials and stochastic variation of precise locations of Ca2+ entry within the active zone. Further, Ca2+ channel numbers at each active zone correlate to the number of presynaptic primed synaptic vesicles. Together, our findings indicate 1:1 association of Ca2+ channels with primed vesicles, suggesting Ca2+ entry via as few as one channel may trigger neurotransmitter release.

neuroscience↗

bioRxiv2021

PDE9 Inhibition Activates PPARa to Stimulate Mitochondrial Fat Metabolism and Reduce Cardiometabolic Syndrome

Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show inhibiting cyclic-GMP selective phosphodiesterase-9A (PDE9-I) suppresses established diet-induced obesity and CMS in ovariectomized female and male mice. PDE9-I reduces abdominal, hepatic, and myocardial fat accumulation, stimulates mitochondrial activity in brown and white fat, and improves CMS, without altering activity or food intake. PDE9 localizes to mitochondria, and its inhibition stimulates lipolysis and mitochondrial respiration coupled to PPAR-dependent gene regulation. PPAR upregulation is required for PDE9-I metabolic efficacy and is absent in non-ovariectomized females that also display no metabolic benefits from PDE9-I. The latter is compatible with estrogen receptor- altering PPAR chromatin binding identified by ChIPSeq. In humans with heart failure and preserved ejection fraction, myocardial expression of PPARA and its regulated genes is reduced versus control. These findings support testing PDE9-I to treat obesity/CMS in men and postmenopausal women. SummaryOral inhibition of phosphodiesterase type 9 stimulates mitochondrial fat metabolism and lipolysis, reducing central obesity without changing appetite

physiology↗

bioRxiv2021

Genome-wide analysis of mitochondrial DNA copy number reveals multiple loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses were used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 x 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 x 10-8) and mtDNA replication (p = 1.2 x 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 x 10-4).

genetics↗

bioRxiv2020

Generation of a SARS-CoV-2 Replicon as a Model System to Dissect Virus Replication and Antiviral Inhibition

SARS-CoV-2 research and antiviral discovery are hampered by the lack of a cell-based virus replication system that can be readily adopted without biosafety level 3 (BSL-3) restrictions. Here, the construction of a non-infectious SARS-CoV-2 reporter replicon and its application in deciphering viral replication mechanisms and evaluating SARS-CoV-2 inhibitors are presented. The replicon genome is replication competent but does not produce progeny virions. Its replication can be inhibited by RdRp mutations or by known SARS-CoV-2 antiviral compounds. Using this system, a high-throughput antiviral assay has also been developed. Significant differences in potencies of several SARS-CoV-2 inhibitors in different cell lines were observed, which highlights the challenges of discovering antivirals capable of inhibiting viral replication in vivo and the importance of testing compounds in multiple cell culture models. The generation of a SARS-CoV-2 replicon provides a powerful platform to expand the global research effort to combat COVID-19.

microbiology↗