Investigating Phage- Host Protein Interactions

Mycobacteriophages are viruses that infect bacteria in the genus Mycobacterium. The Mycobacterium genus is home to more than 100 identified species including the human pathogens that cause tuberculosis and leprosy. There is evidence that when mycobacteriophages initiate lytic growth – the phage development pathway that results in the immediate production and release of new phage particles concomitant with host cell death – key metabolic machinery of the host cell is strategically seized and redirected for phage replication. Further, the extreme genetic diversity evident in mycobacteriophage genomes suggests this class of viruses use multiple means to carry out this act of cellular piracy. However, the process by which even a single mycobacteriophage hijacks a host cell is unknown. In an effort to understand this process, we have identified multiple mycobacteriophage genes that are cytotoxic when expressed individually in the host M. smegmatis. We hypothesized that at least some of these cytotoxic effects are due to specific phage-host protein-protein interactions. The focus of this work is the cytotoxic VIX80 gene product from mycobacteriophage Vix. Vix gp80 was analyzed using biochemical techniques to find interacting host cell proteins. Vix gp80 was expressed in E. coli and used in a magnetic bead pull-down assay to isolate M. smegmatis proteins that bound to the phage gene product. M. smegmatis Elongation Factor-Tu was isolated using this technique and identified following protein analysis. The interaction of Vix gp80 and EF-Tu will be verified using additional biochemical methods and the yeast two-hybrid system for detecting physical interactions in vivo. A procedure for a two hybrid-system positive control, necessary for functional testing, is being developed and refined

Investigating Mycobacteriophage-Host Protein Interactions

Mycobacteriophages are viruses that infect bacterial cells of the genus Mycobacterium. They possess a multitude of unfamiliar or novel genes – genes encoding protein sequences that do not resemble any previously studied proteins – and thus encode products with functions not readily predicted. We hypothesized that some of those genes encode products that interfere with the normal metabolism of the host cell, possibly through specific phage-host protein-protein interactions, and thus have a role in enabling phage infection. Further, we predicted that those gene products when expressed alone in host cells would still be toxic and impair cell growth. We have investigated unfamiliar genes in two genetically distinct mycobacteriophages, Pumpkin and Vix, and have identified 4 single genes (Pumpkin_115, Pumpkin_119, Pumpkin_142, Vix_80) and several small genomic regions (Pumpkin gene segment 130-133 and Vix gene segments 65-66, 68-72, 87-88) that are cytotoxic to M. smegmatis. We are taking a multi-prong approach to further identify the specific functions associated with these genes and their products and to determine their roles in the infection process: 1) we have identified mutants of M. smegmatis that are resistant to the expression of those genes, 2) we are using E. coli- expressed phage genes to screen for interacting host proteins, 3) we are collecting microscopy data that could identity phage interruption of normal cellular function, and 4) we are in the process of deleting these genes from the phage genome to determine the effect on infection. 24-hour expression of individual cytotoxic phage genes in M. smegmatis resulted in a significant increase in mean host cell length and some subtle effects on cell shape. Ongoing analysis of the mutants has identified a common mechanism of resistance to distinct phage gene expression, while protein-protein interaction studies have not yet identified a potential host target involved in translation

Co‐Locating Obstetrics and Addiction Medicine Clinics to Improve Attendance in Services for Pregnant People with Opioid Use Disorder

Objective Pregnant people receiving treatment for opioid use disorders (OUD) are at significant risk of return to use during the postpartum period. Recently, practice groups and other national organizations have called for the co‐location of addiction medicine and obstetric care to reduce the burden on pregnant and postpartum people with OUD associated with engaging in treatment. This paper examines the effectiveness of co‐locating services in retaining pregnant people with OUD in care following childbirth. Methods A records review of pregnant people receiving medication for OUD between 2012 and 2017 in stand‐alone addiction medicine clinic (n = 23) and from 2017 to 2021 following the creation of an integrated addiction medicine‐obstetric care clinic (n = 67) was conducted to compared rates of attendance in both obstetric and addiction medicine services. Results Findings from this study suggest that individuals receiving services in a co‐located clinic had significantly fewer missed appointments during the postpartum period relative to individuals who sought care at separate addiction medicine and obstetric care clinics. Conclusions Results from this study support the potential for co‐locating clinics to reduce barriers to accessing obstetric and addiction medicine services, as well as support continued attendance in care across a vulnerable period

The Role of Delay Discounting in the Generation of Stressful Life Events Across Adolescence

Hammen\u27s (1991) model of stress generation suggests that depressed individuals are more likely to behave in ways that bring about greater exposure to negative life events. More recent research suggests that adolescents with other types of psychological vulnerabilities, including those more likely to make impulsive choices, may also be predisposed to experience greater increases in stress over time. The current study examined whether delay discounting (DD), defined as the tendency to prefer smaller but immediately available rewards relative to larger, delayed rewards, predicts the generation of negative life events across adolescence and whether this is due to the association between DD and depressive symptoms. Participants (n = 213, M(age) = 15, range 12-17) completed self-report measures of depressive symptoms and negative life events, as well as a behavioral measure assessing DD annually over four years. Results of latent growth models suggest that both independent and dependent negative life events increased across adolescence. Consistent with a stress generation framework, DD predicted the growth in dependent, but not independent, negative life events over this time period, controlling for baseline levels of depressive symptoms. Further exploratory analyses suggest that DD was associated with increases in depressive symptomology across adolescence, but that the relation between DD and changes in independent negative life events was not better accounted for by increases in depressive symptoms over time. Taken together, these findings suggest the importance of DD in predicting youths\u27 exposure to dependent negative life events and point to potential avenues for clinical intervention