Biology subjects

Suh, J.

Publications and source records attributed to Suh, J..

23 records · Page 2Linked to original sources

bioRxiv2021

Machine Learning Identification of Capsid Mutations to Improve AAV Production Fitness

The adeno-associated virus (AAV) holds great potential for gene therapy efforts by providing a viable vector. However, current efforts are constrained by a lack of AAV variants that exhibit specific tropisms or immunogenicity and a lack of sustainable industrial projection. Departing from experimental approaches to addressing these issues, we built a model to predict residue mutations to improve AAV production fitness. Our model leverages the evolutionary paradigm and microenvironment characteristics by analyzing structural AAV data without needing domain knowledge or experimental fitness data for AAV as inputs. When testing our model's predictions for AAV2 residue mutations, we found a threefold increase in the percent of mutations yielding variants with better production fitness than wild type compared to random mutations, achieving a p-value of 7.46x10-12. Given these results, our machine learning approach of using structural data to approximate fitness data has the potential to accelerate AAV development.

bioinformatics↗

bioRxiv2021

Phage Cocktails can Prevent the Evolution of Phage-Resistant Enterococcus

Antibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy is often deployed as a cocktail of phages, but there is little understanding of how to most effectively combine phages. Here we utilized a collection of 20 Enterococcus phages to test principles of phage cocktail design and determine the phenotypic effects of evolving phage resistance in Enterococcus isolates that were susceptible or resistant to antibiotics (e.g., Vancomycin Resistant Enterococcus (VRE)). We tested the ability of each phage to clear Enterococcus host cultures and prevent the emergence of phage resistant Enterococcus. We found that some phages which were ineffective individually were effective at clearing the bacterial culture when used in cocktails. To understand the dynamics within phage cocktails, we used qPCR to track which phages increased in abundance in each cocktail, and saw dynamics ranging from one dominant phage to even phage growth. Further, we isolated several phage-resistant mutants to test for altered Vancomycin sensitivity. We found that mutants tended to have no change or slightly increased resistance to Vancomycin. By demonstrating the efficacy of phage cocktails in suppressing growth of antibiotic susceptible and VRE clinical isolates when exposed to phages, this work will help to inform cocktail design for future phage therapy applications. IMPORTANCEAntibiotic resistant Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy could be an alternative to antibiotics and has shown promise in in vitro and in early clinical studies. Phage therapy in the form of cocktails is often suggested, with similar goals as the combination therapy that has been successful in the treatment of HIV infection, but there is little understanding about how to combine phages most effectively. Here we utilized a collection of 20 Enterococcus phages to test whether several phage cocktails could prevent the host from evolving resistance to therapy and to determine whether evolving resistance to phages affected host susceptibility to antibiotics. We showed that cocktails of two or three unrelated phages often prevented the growth of phage-resistant mutants, when the same phages applied individually were not able to.

microbiology↗

bioRxiv2019

Reversible loss of Hippocampal Function in a Mouse Model of Demyelination/Remyelination

Demyelination of axons in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and other demyelinating diseases. Cycles of demyelination, followed by remyelination, appear in the majority of MS patients, and are associated with the onset and quiescence of disease-related symptoms, respectively. Previous studies have shown in human patients and animal models that vast demyelination is accompanied by wide-scale changes to brain activity, but details of this process are poorly understood. We use electrophysiological recordings and nonlinear imaging of fluorescence from genetically-encoded calcium indicators to monitor the activity of hippocampal neurons during demyelination and remyelination processes over a period of 100 days. We find in vitro that synaptic transmission in CA1 neurons is diminished, and in vivo both CA1 and dentate gyrus (DG) neuronal firing rates are substantially reduced during demyelination and partially recover after a short remyelination period. This new approach allows monitoring how synaptic transmission changes, induced by cuprizone diet, are affecting neuronal activity, and can potentially be used to study the effects of therapeutic interventions in protecting the functionality of CNS neurons.

neuroscience↗

bioRxiv2019

Prevalence of antimicrobial resistance genes and integrons in commensal Gram-negative bacteria in a college community

Although the human intestinal microbiome has been shown to harbor antimicrobial drug-resistance genes (ARG), the prevalence of such genes in a healthy population and their impact on extraintestinal infections that occur in that community are not well established. This study sought to identify ARG prevalence and their mobile elements in the intestines of a healthy community population at a California university, and compared these genes to those found in uropathogenic Escherichia coli isolated from patients with community-acquired urinary tract infection (CA-UTI). We isolated Gram-negative bacteria (GNB) from fecal samples of healthy volunteers and screened them by polymerase chain reaction (PCR) for ARG encoding resistance against ampicillin (AMP), trimethoprim-sulfamethoxazole (TMP-SMX), gentamicin (GENT), and colistin (COL). We found antimicrobial resistant GNB from 85 (83%) of 102 non-redundant rectal swab samples. Sixty-seven (66%) of these samples contained {beta}-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA, blaOXY), dihydrofolate reductase (DHFR) genes (dhfr-A17, dhfr-A7, dhfr-A5, dhfr-A21, dhfr-A1, dhfr-A15, and dhfr-B3), and aminoglycoside resistance genes (aadA5, aadA1, and aadB). Integron sequences were found in 37 fecal samples. These genes were found in 11 different GNB species isolated from the fecal samples. The same ARG were found in E. coli strains isolated from patients with CA-UTI examined at the college outpatient health clinic. The high prevalence of clinically-common ARG and integrons harbored by GNB in the intestine of a healthy population suggest that human intestines may serve as a major reservoir of these mobile ARG that appear in E. coli strains causing extraintestinal infections in the same community.\n\nImportanceIncreasing frequency of antimicrobial resistance (AMR) in human pathogenic bacteria has compromised our ability to treat infections. Since mobile antibiotic resistance genes (ARG) are readily exchanged between different species of bacteria through horizontal gene transfer, there is interest in investigating sources of these genes. The normal intestinal flora has been shown to contain a wide variety of ARG, which may have been introduced via food-containing AMR bacteria. We sought to assess the prevalence of ARG carriage in the intestines of a healthy population and determine if these ARG are found in E. coli strains that cause community-acquired urinary tract infection (CA-UTI) in the same community. Our findings indicate that the human intestine may serve as an important reservoir as well as a site in which ARG are transferred into E. coli that cause UTI. Further research is needed to reduce ARG carriage and devise new strategies to prevent AMR infections.

microbiology↗

bioRxiv2018

A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9 mediated somatic recombination

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single \"workhorse\" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras G12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion (\"indel\") and frameshift mutations of targeted tumor suppressor alleles. By using a single \"workhorse\" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we have created a facile autochthonous platform for interrogation of the PDAC genome.

cancer biology↗