A Dynamic Culture System to Support the In Vitro Growth and Maturation of Ovarian Follicles

Ovarian follicle growth is a prolonged process that involves progressive development of the follicle unit through specific histologically defined stages of development. Groups around the world have begun ovarian cryopreservation programs for young girls and women undergoing potentially sterilizing surgery or chemotherapy with the hope that follicles can be isolated from these tissues and grown in vitro at a later date. Though follicles derived from mice can be grown up to maturity using conventional culture techniques, scientists have been unsuccessful with the in vitro development of follicles from species that have larger follicles. The objective of this study was to develop a culture system that could better support the growth and maturation of these larger follicles. The aims of the study focused on maintaining structural integrity through a suspension culture technique, providing three-dimensional support by utilizing an alginate microencapsulation technique, and creating a unique oxygen environment that more closely mimicked the oxygen levels of the native ovary. The suspension culture technique was found to eliminate follicle flattening that occurred with larger follicles on flat surfaces in a static culture. The alginate microencapsulation technique was shown to improve the support of three-dimensional growth of preantral follicles; but requires the inclusion of FSH in the scaffold in order to maintain the growth rate of unencapsulated follicles. Finally, by implementing a dynamic oxygen protocol based on the unique oxygen environment of the ovary, both the yield and quality of the oocytes derived from in vitro cultured preantral follicles were significantly improved when compared to oocytes from follicles cultured at the traditional ambient oxygen levels. In addition, these oocytes were not only able to undergo parthenogenetic activation, but were also fertilized through intracytoplasmic sperm injection. A subsequent gene expression analysis uncovered that follicles grown in a high oxygen environment possessed more differentially expressed genes compared to an in vivo control than did follicles cultured in a low oxygen environment. Furthermore, these differentially expressed genes were found to regulate several key processes that contribute to proper follicular development. These findings have contributed to the development of a novel culture system that has enhanced the in vitro support of follicle and oocyte maturation

US Cosmic Visions: New Ideas in Dark Matter 2017: Community Report

This white paper summarizes the workshop "U.S. Cosmic Visions: New Ideas in Dark Matter" held at University of Maryland on March 23-25, 2017.Comment: 102 pages + reference

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

The Critical Role of Notch Ligand Delta-like 1 in the Pathogenesis of Influenza A Virus (H1N1) Infection

Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4+and CD8+T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection

Host Genetic Variation Impacts SARS-CoV-2 Vaccination Response in the Diversity Outbred Mouse Population

The COVID-19 pandemic led to the rapid and worldwide development of highly effective vaccines against SARS-CoV-2. However, there is significant individual-to-individual variation in vaccine efficacy due to factors including viral variants, host age, immune status, environmental and host genetic factors. Understanding those determinants driving this variation may inform the development of more broadly protective vaccine strategies. While host genetic factors are known to impact vaccine efficacy for respiratory pathogens such as influenza and tuberculosis, the impact of host genetic variation on vaccine efficacy against COVID-19 is not well understood. To model the impact of host genetic variation on SARS-CoV-2 vaccine efficacy, while controlling for the impact of non-genetic factors, we used the Diversity Outbred (DO) mouse model. We found that DO mice immunized against SARS-CoV-2 exhibited high levels of variation in vaccine-induced neutralizing antibody responses. While the majority of the vaccinated mice were protected from virus-induced disease, similar to human populations, we observed vaccine breakthrough in a subset of mice. Importantly, we found that this variation in neutralizing antibody, virus-induced disease, and viral titer is heritable, indicating that the DO serves as a useful model system for studying the contribution of genetic variation of both vaccines and disease outcomes

SARS-CoV Pathogenesis Is Regulated by a STAT1 Dependent but a Type I, II and III Interferon Receptor Independent Mechanism

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1−/− mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1−/− mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation

Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analyses

<p>Abstract</p> <p>Background</p> <p>Pneumonia and myocarditis are the most commonly reported diseases due to <it>Histophilus somni</it>, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in <it>H. somni </it>using traditional methods. Analyses of the genome sequences of several <it>Pasteurellaceae </it>species have provided insights into their biology and evolution. In view of the economic and ecological importance of <it>H. somni</it>, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the <it>Pasteurellaceae</it>.</p> <p>Results</p> <p>The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the <it>Pasteurellaceae</it>, several <it>H. somni </it>genes that may encode proteins involved in virulence (<it>e.g</it>., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor.</p> <p>Conclusions</p> <p>Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two <it>H. somni </it>strains.</p

Dynamic Oxygen Enhances Oocyte Maturation in Long-Term Follicle Culture

Traditionally, follicles have been grown in standard incubators with atmospheric oxygen concentration. However, preantral follicles exist in the avascular cortex of the ovary. This study examines the effectiveness of an oxygen delivery protocol that more closely mimics the in vivo environment of the ovary on oocyte viability, maturation, parthenogenetic activation, and fertilization from in vitro cultured rat preantral follicles. Of 54 oocytes cultured in the dynamic oxygen environment, 35 were viable while only 22 of 50 oocytes cultured within an ambient oxygen concentration remained viable (p < 0.05). Germinal vesicle breakdown was observed in 56% of oocytes from the dynamic oxygen group compared to 30% of oocytes from the ambient oxygen group (p < 0.05). Parthenogenetic activation was observed in a significant number of oocytes from the dynamic oxygen group, while none of the oocytes from the ambient oxygen group activated (p < 0.05). However, the proportions of oocytes from the dynamic oxygen group that remained viable underwent germinal vesicle breakdown, and activated were still significantly less than those from the in vivo control group (p < 0.05). Fertilization of the oocytes from the dynamic oxygen group was confirmed through a successful trial of intracytoplasmic sperm injection

Point-of-care urine tenofovir test predicts future HIV pre-exposure prophylaxis discontinuation among young users

Young men who have sex with men and transgender women (YMSM/TGW) have disproportionately high HIV incidence and lower PrEP adherence. Point-of-care (POC) urine tenofovir (TFV) rapid assay (UTRA) testing permits real-time monitoring for nonadherence within clinical settings. We performed UTRA testing among PrEP users to examine the relationship between low PrEP adherence and future PrEP discontinuation, and the accuracy of POC testing compared to gold-standard liquid chromatography tandem mass spectrometry (LC/MS/MS).BACKGROUNDYoung men who have sex with men and transgender women (YMSM/TGW) have disproportionately high HIV incidence and lower PrEP adherence. Point-of-care (POC) urine tenofovir (TFV) rapid assay (UTRA) testing permits real-time monitoring for nonadherence within clinical settings. We performed UTRA testing among PrEP users to examine the relationship between low PrEP adherence and future PrEP discontinuation, and the accuracy of POC testing compared to gold-standard liquid chromatography tandem mass spectrometry (LC/MS/MS).YMSM/TGW participants (n = 100) were recruited during a daily PrEP visit. Logistic regression models analyzed the relationship between the primary predictor of urine POC assay results (cutoff 1,500 ng/mL) and the primary outcome of PrEP discontinuation, defined as no PrEP follow-up or prescription within 120 days.METHODSYMSM/TGW participants (n = 100) were recruited during a daily PrEP visit. Logistic regression models analyzed the relationship between the primary predictor of urine POC assay results (cutoff 1,500 ng/mL) and the primary outcome of PrEP discontinuation, defined as no PrEP follow-up or prescription within 120 days.Overall, 19% of participants had low urine TFV and 21% discontinued PrEP, while 11% of participants self-reported low PrEP adherence (< 4 pills per week), which was only 43% sensitive/84% specific in predicting low TFV levels and was not associated with PrEP discontinuation. Low urine TFV level predicted PrEP discontinuation (AOR 6.1; 95% CI: 1.4-11; p = 0.005) and was 71% sensitive/90% specific for discontinuation after 120 days. Compared to LC/MS/MS, UTRA testing had a 98% positive and 100% negative predictive value.RESULTSOverall, 19% of participants had low urine TFV and 21% discontinued PrEP, while 11% of participants self-reported low PrEP adherence (< 4 pills per week), which was only 43% sensitive/84% specific in predicting low TFV levels and was not associated with PrEP discontinuation. Low urine TFV level predicted PrEP discontinuation (AOR 6.1; 95% CI: 1.4-11; p = 0.005) and was 71% sensitive/90% specific for discontinuation after 120 days. Compared to LC/MS/MS, UTRA testing had a 98% positive and 100% negative predictive value.In a sample of YMSM/TGW on daily PrEP, POC UTRA testing predicted PrEP discontinuation more accurately than self-reported adherence, with high predictive values compared to LC/MS/MS. UTRA testing may be a clinical tool for directing preventive interventions towards those likelier to discontinue PrEP despite ongoing HIV vulnerability.CONCLUSIONSIn a sample of YMSM/TGW on daily PrEP, POC UTRA testing predicted PrEP discontinuation more accurately than self-reported adherence, with high predictive values compared to LC/MS/MS. UTRA testing may be a clinical tool for directing preventive interventions towards those likelier to discontinue PrEP despite ongoing HIV vulnerability