Characterization of the Mycobacteriophage Ukulele Integration System; Identification of Integration Site Attp and the Role of the Integrase in Lysogeny Regulation

Mycobacteriophage (phage) are a group of viruses that infect bacteria in the genus Mycobacterium. Two phage lifestyles are lytic and temperate. Lytic phage only carry out the lytic life cycle, resulting in host cell lysis. Temperate phage are capable of completing both lytic and lysogenic life cycles. During the lysogenic life cycle, a phage-encoded integrase facilitates integration at sites attP in the phage genome and attB in the host to form a lysogen. The cluster E mycobacteriophage integration system is poorly understood. Ukulele, a lysogenic cluster E phage, is being used to identify the Cluster E attP and characterize lysogeny regulation. A putative attP containing sequence was identified in the Ukulele genome by computational analysis. To confirm the presence of attP, this sequence will be inserted into a plasmid and transferred into integrase expressing M. smegmatis (pST-KT-int). Cells will be screened for plasmid integrated into the genome. To characterize the role of the integrase in lysogeny regulation, we will determine the impact of integrase expression levels on induction event frequency in M. smegmatis (pST-KT-int) – Ukulele lysogens

Survey of Federal Whistleblower and Anti-Retaliation Laws

This report provides an overview of federal whistleblower and anti-retaliation laws. In general, these laws protect employees who report misconduct by their employers or who engage in various protected activities, such as participating in an investigation or filing a complaint. In recent years, Congress has expanded employee protections for a variety of private-sector workers. Eleven of the forty laws reviewed in this report were enacted after 1999. Among these laws are the Sarbanes-Oxley Act, the FDA Food Safety Modernization Act, and the Dodd-Frank Wall Street Reform and Consumer Protection Act. The report focuses on key aspects of the federal whistleblower and anti-retaliation laws. For each law, the report summarizes the activities that are protected, how the law’s protections are enforced, whether the law provides a private right of action, the remedies prescribed by the law, and the year the law’s whistleblower or anti-retaliation provisions were adopted and amended. With regard to amendment dates, the report identifies only dates associated with substantive amendments. For enactments after 2001, the report provides information on congressional sponsorship and votes

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-Induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain. This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN

Northern Bobwhite Demographics and Resource Selection Are Explained by Prescribed Fire with Grazing and Woody Cover in Southwest Missouri

Understanding the effects of landscape management on northern bobwhite (Colinus virginianus; hereafter, bobwhite) population growth requires information about seasonal- and stage-specific demographic parameters linked across the annual cycle. We review results to date from 3 years (2016–2018) of an intensive field study evaluating drivers of bobwhite population dynamics and resource selection during the breeding and non-breeding season in southwest Missouri, USA using data from adult and juvenile bobwhite fitted with radio-transmitters. Land cover of our study sites ranged from large blocks of native grasslands maintained with prescribed fire and grazing to more traditional management resulting in small patches of grasslands interspersed with food plots, disked idle areas, and woody cover. During the breeding season, relative probability of selection by broods increased in relation to proportion of native grass managed by grazing and burning and proportion of cropland. Brood survival was also greatest on native grasslands burned and grazed within the past 2 growing seasons. During the fall and winter, relative probability of selection by adults increased as woody edge density increased. Fall and winter survival increased as distance from trees increased and decreased as distance to shrubs increased. Our integrated population model indicated that the number of young hatched per female and adult breeding season survival were greatest on sites with the most native grassland managed by prescribed fire with grazing. However, non-breeding season survival was greater on sites with more agriculture or food plots and woody cover. Abundance declined across all sites from 2016 to 2019. Our work suggests that native grasslands managed by prescribed fire with grazing can provide quail habitat superior to traditional management that strived for a mixture of agriculture, woody cover, and grassland. The combination of conservation grazing and fire in native grasslands interspersed with shrubs may provide the greatest chance for bobwhite populations to persist in southwest Missouri and similar landscapes

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nn-ITN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)(4) spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nn-ITN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.Peer reviewe

EWI-2 Inhibits Cell-Cell Fusion at the HIV-1 Virological Presynapse.

Cell-to-cell transfer of virus particles at the Env-dependent virological synapse (VS) is a highly efficient mode of HIV-1 transmission. While cell-cell fusion could be triggered at the VS, leading to the formation of syncytia and preventing exponential growth of the infected cell population, this is strongly inhibited by both viral (Gag) and host (ezrin and tetraspanins) proteins. Here, we identify EWI-2, a protein that was previously shown to associate with ezrin and tetraspanins, as a host factor that contributes to the inhibition of Env-mediated cell-cell fusion. Using quantitative fluorescence microscopy, shRNA knockdowns, and cell-cell fusion assays, we show that EWI-2 accumulates at the presynaptic terminal (i.e., the producer cell side of the VS), where it contributes to the fusion-preventing activities of the other viral and cellular components. We also find that EWI-2, like tetraspanins, is downregulated upon HIV-1 infection, most likely by Vpu. Despite the strong inhibition of fusion at the VS, T cell-based syncytia do form in vivo and in physiologically relevant culture systems, but they remain small. In regard to that, we demonstrate that EWI-2 and CD81 levels are restored on the surface of syncytia, where they (presumably) continue to act as fusion inhibitors. This study documents a new role for EWI-2 as an inhibitor of HIV-1-induced cell-cell fusion and provides novel insight into how syncytia are prevented from fusing indefinitely

Longitudinal Association of Maternal Attempt to Lose Weight During the Postpartum Period and Child Obesity at Age 3 Years

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93678/1/oby.2011.25.pd

Salmonella Strains Isolated from Galápagos Iguanas Show Spatial Structuring of Serovar and Genomic Diversity

It is thought that dispersal limitation primarily structures host-associated bacterial populations because host distributions inherently limit transmission opportunities. However, enteric bacteria may disperse great distances during food-borne outbreaks. It is unclear if such rapid long-distance dispersal events happen regularly in natural systems or if these events represent an anthropogenic exception. We characterized Salmonella enterica isolates from the feces of free-living Galápagos land and marine iguanas from five sites on four islands using serotyping and genomic fingerprinting. Each site hosted unique and nearly exclusive serovar assemblages. Genomic fingerprint analysis offered a more complex model of S. enterica biogeography, with evidence of both unique strain pools and of spatial population structuring along a geographic gradient. These findings suggest that even relatively generalist enteric bacteria may be strongly dispersal limited in a natural system with strong barriers, such as oceanic divides. Yet, these differing results seen on two typing methods also suggests that genomic variation is less dispersal limited, allowing for different ecological processes to shape biogeographical patterns of the core and flexible portions of this bacterial species' genome

Regulation of host fusion inhibitory proteins at the virological synapse and upon syncytia formation

Cell-to-cell transmission at the HIV-1 virological synapse (VS) is an efficient mode of spread. While the VS typically resolves with cell separation, it can result in cell-cell fusion, forming a multinucleated infected cell (syncytium). Fusion is largely prevented at the VS by the viral protein Gag and host proteins ezrin, tetraspanins, and EWI-2. We are now determining how these host proteins work together and are temporally regulated to prevent excessive HIV-1-induced syncytia formation using targeted manipulations of each protein and kinetic analysis of their recruitment to the HIV-1 VS. Further, CD81 and EWI-2 are downregulated from the surface of mononucleated infected cells but partially restored on syncytia. We show that the increased level of surface protein is the result of an influx of protein from the (previously) uninfected cell that fused with an infected cell to form a syncytium. We predict that increased levels of fusion inhibitory proteins in syncytia makes them less fusogenic than mononucleated infected cells, possibly explaining why syncytia do not fuse indefinitely. We plan to determine whether syncytia maintain this altered surface profile or if these proteins are downregulated over time. Together, these studies will increase our understanding of how host proteins regulate HIV-1-induced cell-cell fusion

Expression of MHC II in DRG neurons attenuates paclitaxel-induced cold hypersensitivity in male and female mice.

Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts cancer survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4+ T cells are activated, and the extent cytokines released by CD4+ T cells target DRG neurons are unknown. Here, we are the first to detect major histocompatibility complex II (MHCII) protein in mouse DRG neurons and to find CD4+ T cells breaching the satellite glial cell barrier to be in close proximity to neurons, together suggesting CD4+ T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male and female mouse DRG but increased after PTX in small nociceptive neurons in only female DRG. Reducing one copy of MHCII in small nociceptive neurons decreased anti-inflammatory IL-10 and IL-4 producing CD4+ T cells in naïve male DRG and increased their hypersensitivity to cold. Administration of PTX to male and female mice that lacked one copy of MHCII in nociceptive neurons decreased anti-inflammatory CD4+ T cells in the DRG and increased the severity of PTX-induced cold hypersensitivity. Collectively, our results demonstrate expression of MHCII protein in mouse DRG neurons, which modulates cytokine producing CD4+ T cells in the DRG and attenuates cold hypersensitivity during homeostasis and after PTX treatment