Biology subjects

Stone, N. E.

Publications and source records attributed to Stone, N. E..

7 recordsLinked to original sources

bioRxiv2026

Clinical and molecular characterization of an outbreak of leptospirosis in dogs from Los Angeles County, California, USA, 2021

BackgroundIn 2021, the Los Angeles County (LAC) Department of Public Health suspected a leptospirosis outbreak in LAC affecting over 200 client-owned dogs. ObjectiveTo characterize the outbreak and describe microbiologic findings, risk factors, diagnostic test performance, and outcomes in dogs diagnosed with leptospirosis at two specialty practices. MethodsLeptospira culture isolates from four cases were subjected to serotyping and whole genome sequencing (WGS); WGS was also performed on one enriched genome isolate. After the outbreak, data were gathered on 59 cases through record review and compared to the background hospital population (controls, n=15,536). ResultsAll isolates were Leptospira interrogans serovar Canicola, but each was distinct based on WGS. Cases clustered in space and in time. Cases evaluated during the outbreak peak had increased odds of exposure to indoor congregate facilities (ICFs). None of 47 dogs with known leptospirosis vaccination history were completely vaccinated. Leptospira real-time PCR on blood and urine and initial serologic testing using the microscopic agglutination test and point-of-care tests were positive in 15/56 (27%), 49/54 (91%) dogs, 22/29 (76%), and 27/35 (77%) dogs respectively. Fifty-four (92%) of 59 dogs survived to discharge; some remained azotemic. No associated human cases were identified. Conclusions and Clinical ImportanceL. interrogans serovar Canicola was associated with a leptospirosis outbreak in unvaccinated dogs from LAC, which had public health implications given widespread dog ownership rates. Data analysis suggested multiple infection sources, including ICFs. Urine PCR was the most sensitive diagnostic test. Such outbreaks might be prevented through more widespread vaccination.

microbiology↗

bioRxiv2026

High-throughput Genome Wide CRISPR Knock Out mechanical sort identifies genes driving metastatic cancer cell softening

Cell mechanics can serve as an important biomarker for cell state and phenotype, such as metastatic ability. While some molecular mechanisms underlying cell mechanical properties have been investigated through targeted analyses, a genome-wide study of human genes and gene networks that modulate cell biophysical properties has not been attempted. In this work, we combined a microfluidic stiffness-based sorting device with a genome-scale CRISPR knockout (GeCKO) screen in order to investigate the effect of individual gene knockouts on cell stiffening and cell softening across the entire protein-coding genome. We processed approximately 150 million Cas9-expressing ovarian cancer cells that had been transduced with a library of 76,000 single guide RNAs (sgRNAs) against the 19,000 protein-coding genes in the genome. The cells were sorted into 5 mechanical subsets. We identified 7 gene knockouts that were significantly depleted in the softer subsets and over 700 gene knockouts that were significantly enriched in the stiffer subsets. Of these significant genes of interest, we selected 3 genes that were highly expressed in our ovarian cancer cell line with greater than 100-fold enrichment in the stiff outlet and resulted in significant changes in ovarian cancer patient survival. These genes, PIK3R4, CCDC88A, and GSK3B, when knocked out result in a significant and predicted increase in cell stiffness. This study is the first to explore the relation between human gene expression and cell mechanics at the genome-scale to generate datasets at the intersection between cell genotype, mechanotype, and phenotype for metastatic cancer cells. The method could also be applied to study the effect of genes on other biophysical cell processes as well as for identifying pathways for the control of cellular mechanics across many cell types.

bioengineering↗

bioRxiv2024

Host population structure and rare dispersal events drive leptospirosis transmission patterns among Rattus norvegicus in Boston, Massachusetts, US

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, such as rats, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus, is an important reservoir of leptospirosis in urban settings. We investigated leptospirosis among brown rats in Boston, Massachusetts and hypothesized that rat dispersal in this urban setting influences the movement, persistence, and diversity of Leptospira. We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira. We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira isolates obtained from frozen and fresh tissue from some of the 59 Leptospira-positive rat kidneys. When isolates were not obtained, we attempted Leptospira genomic DNA capture and enrichment, which yielded 14 additional Leptospira genomes from rats. We also generated an enriched Leptospira genome from a 2018 human case in Boston. We found evidence of high genetic structure and limited dispersal among rat populations that is likely influenced by major roads and/or other unknown dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats, with specific clades tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and movement of leptospirosis in this urban rat community is driven by rat dispersal. Finally, our genomic analyses of the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other urban settings.

molecular biology↗

bioRxiv2024

Device-free isolation of photoreceptor cells from patient iPSC-derived retinal organoids

Autologous photoreceptor cell replacement therapy shows great promise for treating patients with multiple forms of inherited retinal degenerative blindness. Specifically, in disorders such as retinitis pigmentosa and Stargardts disease, selective death of photoreceptor cells results in irreversible blindness. Induced pluripotent stem cell (iPSC) derived retinal organoids, which faithfully recapitulate the structure of the neural retina, are an ideal source of photoreceptor cells required for these therapies. However, in addition to photoreceptor cells, retinal organoids also contain many other retinal cell types. Therefore, approaches for isolating fate committed photoreceptors from dissociated retinal organoids are desirable to produce photoreceptor cell replacement therapies. In this work, we present a partial dissociation strategy, which leverages the high level of organization found in retinal organoids to enable selective enrichment of photoreceptor cells without the use of specialized equipment or reagents such as antibody labels. Given that this technique can be performed with only standard plasticware and cGMP compliant reagents, it is an ideal candidate for use in the preparation of clinical cell therapies.

bioengineering↗

bioRxiv2021

Stenoparib, an inhibitor of cellular poly (ADP-ribose) polymerases (PARPs), blocks in vitro replication of SARS-CoV-2 variants

We recently published a preliminary assessment of the activity of a poly (ADP-ribose) polymerase (PARP) inhibitor, stenoparib, also known as 2X-121, which inhibits viral replication by affecting pathways of the host. Here we show that stenoparib effectively inhibits a SARS-CoV-2 wt (BavPat1/2020) strain and four additional variant strains; alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) and gamma (P.1) in vitro, with 50% effective concentration (EC50) estimates of 4.1 M, 8.5 M, 24.1 M, 8.2 M and 13.6 M, respectively. A separate experiment focusing on a combination of 10 M stenoparib and 0.5 M remdesivir, an antiviral drug, resulted in over 80% inhibition of the alpha (B.1.1.7) variant, which is substantially greater than the effect achieved with either drug alone, suggesting at least additive effects from combining the different mechanisms of activity of stenoparib and remdesivir.

microbiology↗

bioRxiv2021

The K18-hACE2 Transgenic Mouse Model Recapitulates Non-Severe and Severe COVID-19 in Response to Infectious Dose of SARS-CoV-2 Virus

A comprehensive analysis and characterization of a SARS-CoV-2 infection model that mimics non-severe and severe COVID-19 in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2x103 and 2x104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lungs, liver, and kidney, while lower doses (2x101 and 2x102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this humanized hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in biopsy samples from COVID-19 patients. Finally, the mice that recovered after infection with a low dose of virus also survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human biopsy samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. ImportanceThe pandemic of COVID-19 has reached 112,589,814 cases and caused 2,493,795 deaths worldwide as of February 23, 2021, has raised an urgent need for development of novel drugs and therapeutics to prevent the spread and pathogenesis of SARS-CoV-2. To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 virus to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of anti-viral drugs and therapeutics.

microbiology↗

bioRxiv2021

The Effect of Minnelide against SARS-CoV-2 in a Murine Model

Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of coronavirus disease 2019, COVID-19, and the current COVID-19 pandemic. Even as more vaccine candidates are released, more treatment options are critically needed. Here, we investigated the use of Minnelide, a water soluble pro-drug with anti-inflammatory properties, for the treatment of COVID-19. To do this, k18-hACE2 mice were infected with SARS-CoV-2 or given PBS control intranasally. The next day mice were either treated daily with low dose (0.0025mg/day) or high dose Minnelide (0.005mg/day), or given vehicle control intraperitoneal. Mice were weighed daily, and sacrificed at day 6 and 10 post-infection to analyze viral burden, cytokine response, and pathology. We observed a reduction in viral load in the lungs of Minnelide-treated mice infected with SARS-CoV-2 at day 10 post-infection compared to day 6 post-infection. All SARS-CoV-2 infected non-treated mice were moribund six days post-infection while treatment with Minnelide extended survival for both low (60% survival) and high (100% survival) dose treated mice ten days post-infection. Interestingly, cytokine analysis demonstrated a significant reduction in IL-6 (lung and heart) and D-dimer (serum) in high dose treated SARS-CoV-2 infected mice compared to mice infected with SARS-CoV-2 alone at day 6 post-infection. Additionally, histology analysis revealed that Minnelide treatment significantly improved lung pathology ten days post-infection with SARS-CoV-2 with all the mice exhibiting normal lung tissue with thin alveolar septa and no inflammatory cells. Overall, our study exhibits potential for the use of Minnelide to improve survival in COVID-19 patients.

microbiology↗