Biology subjects

Grimes, S.

Publications and source records attributed to Grimes, S..

4 recordsLinked to original sources

bioRxiv2023

PubNavigator as an undergraduate teaching tool

The ability to use data and evidence as the basis for scientific reasoning is a critical skill that undergraduate students must learn to successfully transition into science careers. There is a need for pedagogical tools that promote the development of science process skills and reduce barriers to implementation in the classroom. PubNavigator (www.pubnavigator.com) is a science communication platform that shares the findings of primary literature using accessible language and features biographical information about the authors. This research assesses the utility of PubNavigator as an undergraduate teaching resource. We analyzed responses to an assignment prompting discussion of PubNavigator articles in an asynchronous online discussion forum in an introductory biology course. We conducted thematic analysis guided by the evidence-based reasoning framework to identify evidence of science reasoning and interactive enagement. We found evidence that the PubNavigator assignment elicited engagement in scientific reasoning. Students who demonstrated higher levels of engagement with the assignment also demonstrated higher levels of science reasoning in their discussion of the articles. Our findings support PubNavigator as an undergraduate teaching tool that can be used to promote science reasoning and engagement in introductory biology. We provide recommendations for implementation to instructors interested in integrating PubNavigator activities into their courses.

scientific communication and education↗

bioRxiv2022

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for both this phage and a cos system, reveal unexpected mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

biochemistry↗

bioRxiv2019

Single cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment

PurposeThe tumor microenvironment (TME) consists of a heterogenous cellular milieu that can influence cancer cell behavior. The characteristics of the cellular TME have a dramatic impact on treatments such as immunotherapy. These features can be revealed with single-cell RNA sequencing (scRNA-seq). We hypothesized that single cell gene expression studies of gastric cancer (GC) together with paired normal tissue and peripheral blood mononuclear cells (PBMCs) would identify critical elements of cellular dysregulation not apparent with other approaches.\n\nMethodsSingle cell gene expression studies were conducted on seven patients with GC and one patient with intestinal metaplasia. We sequenced 56,167 cells comprising GC (32,407 cells), paired normal tissue (18,657 cells) and PBMCs (5,103 cells). Protein expression of genes of interest was validated by multiplex immunofluorescence.\n\nResultsTumor epithelium had copy number alterations and a distinct gene expression program compared to normal with intra-tumor heterogeneity. The GC TME was significantly enriched for stromal cells, macrophages, dendritic cells (DCs) and Tregs. TME-exclusive stromal cells expressed extracellular matrix components distinct from normal tissue. Macrophages were transcriptionally heterogenous and did not conform to a binary M1/M2 paradigm. Gene expression program of tumor DCs was unique from PBMC DCs. TME-specific cytotoxic T cells comprised of two exhausted heterogenous subsets. Helper, cytotoxic T, Treg and NK cells expressed multiple immune checkpoint or costimulatory molecules. Receptor-ligand analysis revealed TME-exclusive inter-cellular communication.\n\nConclusionsSingle cell gene expression studies revealed widespread reprogramming across multiple cellular elements in the milieu of the GC TME. Cellular remodeling was delineated by changes in cell numbers, transcriptional states and inter-cellular interactions. This characterization facilitates understanding of tumor biology and enables the identification of novel molecular targets including for cancer immunotherapy.\n\nSTATEMENT OF TRANSLATIONAL RELEVANCEWe leveraged the power of single-cell genomics to characterize the heterogenous cell types and states in the tumor microenvironment (TME). By profiling thousands of single cells from surgical resections of gastric cancer together with paired normal mucosa and peripheral blood mononuclear cells (PBMCs), we determined the deviations in the TME from physiological conditions. Our analysis revealed a cellular reprogramming of the TME compared to normal mucosa in immune and stromal lineages. We detected transcriptional heterogeneity within macrophages and a TME-specific gene expression program in dendritic cells. Cytotoxic T cells in the TME had heterogenous profiles of exhaustion and expression of multiple immune checkpoint and costimulatory molecules. We constructed a receptor-ligand based inter-cellular communications network that was exclusive to tumor tissue. These discoveries provide information at a highly granular cellular resolution enabling advances in cancer biology, biomarker discovery and identification of treatment targets such as for immunotherapy.

cancer biology↗

bioRxiv2017

A Robust Targeted Sequencing Approach For Low Input And Variable Quality DNA From Clinical Samples

Next-generation deep sequencing of gene panels is being adopted as a diagnostic test to identify actionable mutations in cancer patient samples. However, clinical samples, such as formalin-fixed, paraffin-embedded specimens, frequently provide low quantities of degraded, poor quality DNA. To overcome these issues, many sequencing assays rely on extensive PCR amplification leading to an accumulation of bias and artifacts. Thus, there is a need for a targeted sequencing assay that performs well with DNA of low quality and quantity without relying on extensive PCR amplification. We evaluate the performance of a targeted sequencing assay based on Oligonucleotide Selective Sequencing, which permits the enrichment of genes and regions of interest and the identification of sequence variants from low amounts of damaged DNA. This assay utilizes a repair process adapted to clinical FFPE samples, followed by adaptor ligation to single stranded DNA and a primer-based capture technique. Our approach generates sequence libraries of high fidelity with reduced reliance on extensive PCR amplification - this facilitates the accurate assessment of copy number alterations in addition to delivering accurate SNV and indel detection. We apply this method to capture and sequence the exons of a panel of 130 cancer-related genes, from which we obtain high read coverage uniformity across the targeted regions at starting input DNA amounts as low as 10 ng per sample. We further demonstrate the performance of this assay using a series of reference DNA samples, and by identifying sequence variants in DNA from matched clinical samples originating from different tissue types.

genomics↗