Creation and evaluation of a self-directed, first-year intervention for pre-health undergraduates: setting students up for success.

University-established modalities to help undergraduate students navigate the path to medical school are often implemented toward the end of college or following graduation. This imposes cost and time burdens that may be contribute to the high rate of premedical attrition, especially for students who are members of a marginalized community. In the fall 2022 semester, an asynchronous, self-directed pre-health module was offered to biology majors at the University of North Carolina at Chapel Hill enrolled in a required introductory biology research skills course. The objective of the five-lesson intervention was to enhance student understanding of the path to becoming a successful applicant early in their college career. The module aimed to increase the accessibility of pre-health advising and was designed to be easily shared and adapted across various learning management systems. A pre- and post-module survey was administered to assess changes in students' perceived understanding of and confidence for success on the pre-health track following completion of the course.

A basement membrane discovery pipeline uncovers network complexity, regulators, and human disease associations.

Basement membranes (BMs) are ubiquitous extracellular matrices whose composition remains elusive, limiting our understanding of BM regulation and function. By developing a bioinformatic and in vivo discovery pipeline, we define a network of 222 human proteins and their animal orthologs localized to BMs. Network analysis and screening in C. elegans and zebrafish uncovered BM regulators, including ADAMTS, ROBO, and TGFß. More than 100 BM network genes associate with human phenotypes, and by screening 63,039 genomes from families with rare disorders, we found loss-of-function variants in LAMA5, MPZL2, and MATN2 and show that they regulate BM composition and function. This cross-disciplinary study establishes the immense complexity of BMs and their impact on in human health.

A team-based learning model using clinical vignettes in an advanced undergraduate pre-health professional physiology course facilitated by medical students.

There is evidence that demonstrates that teaching preclinical and clinical material can have numerous benefits for both students and teachers, with the majority of literature focusing on peer medical student teaching. There is a dearth of literature exploring the benefit of medical students teaching undergraduate, pre-health professional students and using clinical cases in this setting. We explore our implementation of a team-based learning curriculum built around clinical cases to teach advanced physiology and introduce pathology, pharmacology, and interprofessional collaboration for pre-health students. This course was entirely taught by medical students. Course evaluations and future implications are discussed.

Wild Caught Nematode Identification and Early Embryo Development: An accessible undergraduate research experience.

Ample evidence suggests that participation in undergraduate research in community college is critical for stimulating interest and retention in STEM careers. Guided skill development and practice in a collaborative lab setting allows students to be competitive when applying to future research opportunities. The goals of this undergraduate research experience (URE) was for student-driven discovery with unknown outcomes including: introduction to primary literature, developmental biology, developing hypotheses, learning worm maintenance, microscopy, PCR, and sequencing analysis. The use of C. elegans and wild caught nematodes facilitated an exciting and affordable project that can be built on in future UREs.

Comprehensive Endogenous Tagging of Basement Membrane Components Reveals Dynamic Movement within the Matrix Scaffolding.

Basement membranes (BMs) are supramolecular matrices built on laminin and type IV collagen networks that provide structural and signaling support to tissues. BM complexity, however, has hindered an understanding of its formation, dynamics, and regulation. Using genome editing, we tagged 29 BM matrix components and receptors in C. elegans with mNeonGreen. Here, we report a common template that initiates BM formation, which rapidly diversifies during tissue differentiation. Through photobleaching studies, we show that BMs are not static-surprisingly, many matrix proteins move within the laminin and collagen scaffoldings. Finally, quantitative imaging, conditional knockdown, and optical highlighting indicate that papilin, a poorly studied glycoprotein, is the most abundant component in the gonadal BM, where it facilitates type IV collagen removal during BM expansion and tissue growth. Together, this work introduces methods for holistic investigation of BM regulation and reveals that BMs are highly dynamic and capable of rapid change to support tissues.

α-Integrins dictate distinct modes of type IV collagen recruitment to basement membranes.

Basement membranes (BMs) are cell-associated extracellular matrices that support tissue integrity, signaling, and barrier properties. Type IV collagen is critical for BM function, yet how it is directed into BMs in vivo is unclear. Through live-cell imaging of endogenous localization, conditional knockdown, and misexpression experiments, we uncovered distinct mechanisms of integrin-mediated collagen recruitment to Caenorhabditis elegans postembryonic gonadal and pharyngeal BMs. The putative laminin-binding αINA-1/ßPAT-3 integrin was selectively activated in the gonad and recruited laminin, which directed moderate collagen incorporation. In contrast, the putative Arg-Gly-Asp (RGD)-binding αPAT-2/ßPAT-3 integrin was activated in the pharynx and recruited high levels of collagen in an apparently laminin-independent manner. Through an RNAi screen, we further identified the small GTPase RAP-3 (Rap1) as a pharyngeal-specific PAT-2/PAT-3 activator that modulates collagen levels. Together, these studies demonstrate that tissues can use distinct mechanisms to direct collagen incorporation into BMs to precisely control collagen levels and construct diverse BMs.

Adaptive F-Actin Polymerization and Localized ATP Production Drive Basement Membrane Invasion in the Absence of MMPs.

Matrix metalloproteinases (MMPs) are associated with decreased patient prognosis but have failed as anti-invasive drug targets despite promoting cancer cell invasion. Through time-lapse imaging, optical highlighting, and combined genetic removal of the five MMPs expressed during anchor cell (AC) invasion in C. elegans, we find that MMPs hasten invasion by degrading basement membrane (BM). Though irregular and delayed, AC invasion persists in MMP- animals via adaptive enrichment of the Arp2/3 complex at the invasive cell membrane, which drives formation of an F-actin-rich protrusion that physically breaches and displaces BM. Using a large-scale RNAi synergistic screen and a genetically encoded ATP FRET sensor, we discover that mitochondria enrich within the protrusion and provide localized ATP that fuels F-actin network growth. Thus, without MMPs, an invasive cell can alter its BM-breaching tactics, suggesting that targeting adaptive mechanisms will be necessary to mitigate BM invasion in human pathologies.

A Scalable CURE Using a CRISPR/Cas9 Fluorescent Protein Knock-In Strategy in Caenorhabditis elegans.

Genome editing with CRISPR/Cas9 technology has advanced from the lab bench to clinical application with multiple trials underway. This article introduces a course-based undergraduate experience (CURE) combining CRISPR/Cas9 genome editing (using a modified two-plasmid system) and the animal model Caenorhabditis elegans. This CURE is designed to be a scalable, semester-long laboratory that will introduce the students to literature searches, molecular biology, experiment planning, microscopy, CRISPR bioethics discussion, and scientific writing. Here, students challenged themselves to endogenously tag the C. elegans gene zmp-4, a matrix metalloproteinase enzyme, with a fluorescent protein marker and successfully generated a new worm strain. The knock-in was confirmed with genotyping and imaging and will be available for use by the entire worm community.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain.

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Combining Oncolytic Virotherapy with p53 Tumor Suppressor Gene Therapy.

Oncolytic virus (OV) therapy utilizes replication-competent viruses to kill cancer cells, leaving non-malignant cells unharmed. With the first U.S. Food and Drug Administration-approved OV, dozens of clinical trials ongoing, and an abundance of translational research in the field, OV therapy is poised to be one of the leading treatments for cancer. A number of recombinant OVs expressing a transgene for p53 (TP53) or another p53 family member (TP63 or TP73) were engineered with the goal of generating more potent OVs that function synergistically with host immunity and/or other therapies to reduce or eliminate tumor burden. Such transgenes have proven effective at improving OV therapies, and basic research has shown mechanisms of p53-mediated enhancement of OV therapy, provided optimized p53 transgenes, explored drug-OV combinational treatments, and challenged canonical roles for p53 in virus-host interactions and tumor suppression. This review summarizes studies combining p53 gene therapy with replication-competent OV therapy, reviews preclinical and clinical studies with replication-deficient gene therapy vectors expressing p53 transgene, examines how wild-type p53 and p53 modifications affect OV replication and anti-tumor effects of OV therapy, and explores future directions for rational design of OV therapy combined with p53 gene therapy.

Boundary cells restrict dystroglycan trafficking to control basement membrane sliding during tissue remodeling.

Epithelial cells and their underlying basement membranes (BMs) slide along each other to renew epithelia, shape organs, and enlarge BM openings. How BM sliding is controlled, however, is poorly understood. Using genetic and live cell imaging approaches during uterine-vulval attachment in C. elegans, we have discovered that the invasive uterine anchor cell activates Notch signaling in neighboring uterine cells at the boundary of the BM gap through which it invades to promote BM sliding. Through an RNAi screen, we found that Notch activation upregulates expression of ctg-1, which encodes a Sec14-GOLD protein, a member of the Sec14 phosphatidylinositol-transfer protein superfamily that is implicated in vesicle trafficking. Through photobleaching, targeted knockdown, and cell-specific rescue, our results suggest that CTG-1 restricts BM adhesion receptor DGN-1 (dystroglycan) trafficking to the cell-BM interface, which promotes BM sliding. Together, these studies reveal a new morphogenetic signaling pathway that controls BM sliding to remodel tissues.

Novel biomarkers of resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) based recombinant viruses (such as VSV-ΔM51) are effective oncolytic viruses (OVs) against a majority of pancreatic ductal adenocarcinoma (PDAC) cell lines. However, some PDAC cell lines are highly resistant to VSV-ΔM51. We recently showed that treatment of VSV-resistant PDAC cells with ruxolitinib (JAK1/2 inhibitor) or TPCA-1 (IKK-ß inhibitor) breaks their resistance to VSV-ΔM51. Here we compared the global effect of ruxolitinib or TPCA-1 treatment on cellular gene expression in PDAC cell lines highly resistant to VSV-ΔM51. Our study identified a distinct subset of 22 interferon-stimulated genes (ISGs) downregulated by both ruxolitinib and TPCA-1. Further RNA and protein analyses demonstrated that 4 of these genes (MX1, EPSTI1, XAF1, and GBP1) are constitutively co-expressed in VSV-resistant, but not in VSV-permissive PDACs, thus serving as potential biomarkers to predict OV therapy success. Moreover, shRNA-mediated knockdown of one of such ISG, MX1, showed a positive effect on VSV-ΔM51 replication in resistant PDAC cells, suggesting that at least some of the identified ISGs contribute to resistance of PDACs to VSV-ΔM51. As certain oncogene and tumor suppressor gene variants are often associated with increased tropism of OVs to cancer cells, we also analyzed genomic DNA in a set of PDAC cell lines for frequently occurring cancer associated mutations. While no clear correlation was found between such mutations and resistance of PDACs to VSV-ΔM51, the analysis generated valuable genotypic data for future studies.

A new front in cell invasion: The invadopodial membrane.

Invadopodia are F-actin-rich membrane protrusions that breach basement membrane barriers during cell invasion. Since their discovery more than 30 years ago, invadopodia have been extensively investigated in cancer cells in vitro, where great advances in understanding their composition, formation, cytoskeletal regulation, and control of the matrix metalloproteinase MT1-MMP trafficking have been made. In contrast, few studies examining invadopodia have been conducted in vivo, leaving their physiological regulation unclear. Recent live-cell imaging and gene perturbation studies in C. elegans have revealed that invadopodia are formed with a unique invadopodial membrane, defined by its specialized lipid and associated protein composition, which is rapidly recycled through the endolysosome. Here, we provide evidence that the invadopodial membrane is conserved and discuss its possible functions in traversing basement membrane barriers. Discovery and examination of the invadopodial membrane has important implications in understanding the regulation, assembly, and function of invadopodia in both normal and disease settings.

An unexpected inhibition of antiviral signaling by virus-encoded tumor suppressor p53 in pancreatic cancer cells.

Virus-encoded tumor suppressor p53 transgene expression has been successfully used in vesicular stomatitis virus (VSV) and other oncolytic viruses (OVs) to enhance their anticancer activities. However, p53 is also known to inhibit virus replication via enhanced type I interferon (IFN) antiviral responses. To examine whether p53 transgenes enhance antiviral signaling in human pancreatic ductal adenocarcinoma (PDAC) cells, we engineered novel VSV recombinants encoding human p53 or the previously described chimeric p53-CC, which contains the coiled-coil (CC) domain from breakpoint cluster region (BCR) protein and evades the dominant-negative activities of endogenously expressed mutant p53. Contrary to an expected enhancement of antiviral signaling by p53, our global analysis of gene expression in PDAC cells showed that both p53 and p53-CC dramatically inhibited type I IFN responses. Our data suggest that this occurs through p53-mediated inhibition of the NF-κB pathway. Importantly, VSV-encoded p53 or p53-CC did not inhibit antiviral signaling in non-malignant human pancreatic ductal cells, which retained their resistance to all tested VSV recombinants. To the best of our knowledge, this is the first report of p53-mediated inhibition of antiviral signaling, and it suggests that OV-encoded p53 can simultaneously produce anticancer activities while assisting, rather than inhibiting, virus replication in cancer cells.

Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.

Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.

Recombinant adeno-associated virus vectors in the treatment of rare diseases.

INTRODUCTION: An estimated 25 million Americans are living with rare diseases. Adeno-associated virus (AAV)-mediated gene therapy is an emerging therapeutic option for the more than 7,000 identified rare diseases. This paper highlights the benefits of AAV therapy compared to conventional small molecules, discusses current pre-clinical and clinical applications of AAV-mediated gene therapy, and offers insights into cutting edge research that will shape the future of AAV for broad therapeutic use. AREAS COVERED: In this review the biology of AAV and our ability to generate disease-specific variants is summarized. Limitations of current therapy are reviewed, with an emphasis on immune detection of virus, viral tropism and tissue targeting, and limitations of gene expression. Information for this review was found using PubMed and clinicaltrials.gov. EXPERT OPINION: Currently the scope of clinical trials of AAV gene therapy is concentrated in an array of phase I/II safety trials with less than two dozen rare diseases featured. Pre-clinical, translational studies are expanding in number as developments within the last decade have made generation of improved AAV vectors available to more researchers. Further, one bottleneck that is being overcome is the availability of disease models, which will allow for improved preclinical testing and advancement of AAV to more clinical applications.

Oncolytic vesicular stomatitis virus in an immunocompetent model of MUC1-positive or MUC1-null pancreatic ductal adenocarcinoma.

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.

Understanding and altering cell tropism of vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV's broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV's neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications.