Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus.

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation.

Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.

Novel human butyrylcholinesterase variants: toward organophosphonate detoxication.

Human butyrylcholinesterase (hBChE) is currently being developed as a detoxication enzyme for stoichiometric binding and/or catalytic hydrolysis of organophosphates. Herein, we describe the use of a molecular evolution method to develop novel hBChE variants with increased resistance to stereochemically defined nerve agent model compounds of soman, sarin, and cyclosarin. Novel hBChE variants (Y332S, D340H, and Y332S/D340H) were identified with an increased resistance to nerve agent model compounds that retained robust intrinsic catalytic efficiency. Molecular dynamics simulations of these variants revealed insights into the mechanism by which these structural changes conferred nerve agent model compound resistance.

Hyperexcitability and translational phenotypes in a preclinical mouse model of SYNGAP1-Related Intellectual Disability.

Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability, motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicting the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated primary neurons from Syngap1+/- mice displayed increased network firing activity, greater bursts, and shorter inter-burst intervals between peaks by employing high density microelectrode arrays (HD-MEA). Our work bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.

Phage-specific immunity impairs efficacy of bacteriophage targeting Vancomycin Resistant Enterococcus in a murine model.

Bacteriophage therapy is a promising approach to address antimicrobial infections though questions remain regarding the impact of the immune response on clinical effectiveness. Here, we develop a mouse model to assess phage treatment using a cocktail of five phages from the Myoviridae and Siphoviridae families that target Vancomycin-Resistant Enterococcus gut colonization. Phage treatment significantly reduces fecal bacterial loads of Vancomycin-Resistant Enterococcus. We also characterize immune responses elicited following administration of the phage cocktail. While minimal innate responses are observed after phage administration, two rounds of treatment induces phage-specific neutralizing antibodies and accelerate phage clearance from tissues. Interestingly, the myophages in our cocktail induce a more robust neutralizing antibody response than the siphophages. This anti-phage immunity reduces the effectiveness of the phage cocktail in our murine model. Collectively, this study shows phage-specific immune responses may be an important consideration in the development of phage cocktails for therapeutic use.

Intensive glycaemic targets in overweight and obese individuals with gestational diabetes mellitus: clinical trial protocol for the iGDM study.

INTRODUCTION: The prevalence of both obesity and gestational diabetes mellitus (GDM) has increased, and each is associated with adverse perinatal outcomes including fetal overgrowth, neonatal morbidity, hypertensive disorders of pregnancy and caesarean delivery. Women with GDM who are also overweight or obese have higher rates of pregnancy complications when compared with normal-weight women with GDM, which may occur in part due to suboptimal glycaemic control. The current recommendations for glycaemic targets in pregnant women with diabetes are based on limited evidence and exceed the mean fasting (70.9±7.8 mg/dL) and 1-hour postprandial (108.9±12.9 mg/dL) glucose values in pregnant individuals without diabetes. Our prior work demonstrated that the use of intensive (fasting <90 mg/dL and 1-hour postprandial <120 mg/dL) compared with standard (fasting <95 mg/dL and 1-hour postprandial <140 mg/dL) glycaemic targets resulted in improved glycaemic control without increasing the risk for hypoglycaemia in pregnant individuals with GDM, but the impact of intensive glycaemic targets on perinatal outcomes is unknown. METHODS AND ANALYSIS: The Intensive Glycemic Targets in Overweight and Obese Women with Gestational Diabetes Mellitus: A Multicenter Randomized Trial (iGDM Trial) is a large, pragmatic randomised clinical trial designed to investigate the impact of intensive versus standard glycaemic targets on perinatal outcomes in women with GDM who are overweight and obese. During the 5-year project period, a multidisciplinary team of investigators from five medical centres representing regions of the USA with high rates of obesity will randomise 828 overweight and obese women with GDM to either intensive or standard glycaemic targets. We will test the central hypothesis that intensive glycaemic targets will result in lower rates of neonatal composite morbidity including large for gestational age birth weight, neonatal hypoglycaemia, respiratory distress syndrome and need for phototherapy when compared with standard glycaemic targets using the intention-to-treat approach to analysis. ETHICS AND DISSEMINATION: The Institutional Review Board (IRB) at Indiana University School of Medicine approved this study (IRB# 11435; initial approval date 25 August 2021). We will submit the results of the trial for publication in peer-reviewed journals and presentations at international scientific meetings. TRIAL REGISTRATION NUMBER: NCT05124808.

Characterization of multiple myeloma vesicles by label-free relative quantitation.

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell-to-cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label-free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label-free spectral counting applied to determine relative protein abundances in EVs.

Analysis of long-range chromatin interactions using Chromosome Conformation Capture.

Chromosome Conformation Capture, or 3C, is a pioneering method for investigating the three-dimensional structure of chromatin. 3C is used to analyze long-range looping interactions between any pair of selected genomic loci. Most 3C studies focus on defined genomic regions of interest that can be up to several hundred Kb in size. The method has become widely adopted and has been modified to increase throughput to allow unbiased genome-wide analysis. These large-scale adaptations are presented in other articles in this issue of Methods. Here we describe the 3C procedure in detail, including the appropriate use of the technology, the experimental set-up, an optimized protocol and troubleshooting guide, and considerations for data analysis. The protocol described here contains previously unpublished improvements, which save time and reduce labor. We pay special attention to primer design, appropriate controls and data analysis. We include notes and discussion based on our extensive experience to help researchers understand the principles of 3C-based techniques and to avoid common pitfalls and mistakes. This paper represents a complete resource and detailed guide for anyone who desires to perform 3C.

The role of the minor colonization factor CS14 in adherence to intestinal cell models by geographically diverse ETEC isolates.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low- to middle-income countries. ETEC adheres to small intestinal epithelia via colonization factors (CFs) and secretes heat-stable toxin and/or heat-labile toxin, causing dysregulated ion transport and water secretion. There are over 30 CFs identified, including major CFs associated with moderate-to-severe diarrhea (MSD) and minor CFs for which a role in pathogenesis is less clear. The Global Enteric Multicenter Study identified CS14, a class 5a fimbriae, as the only minor CF significantly associated with MSD and was recommended for inclusion in ETEC vaccines. Despite detection of CS14 in ETEC isolates, the sequence conservation of the CS14 operon, its role in adherence, and functional cross-reactivity to other class 5a fimbriae like CFA/I and CS4 are not understood. Sequence analysis determined that the CS14 operon is >99.9% identical among seven geographically diverse isolates with expanded sequence analysis demonstrating SNPs exclusively in the gene encoding the tip adhesin CsuD. Western blots and electron microscopy demonstrated that CS14 expression required the growth of isolates on CFA agar with the iron chelator deferoxamine mesylate. CS14 expression resulted in significantly increased adherence to cultured intestinal cells and human enteroids. Anti-CS14 antibodies and anti-CS4 antibodies, but not anti-CFA/I antibodies, inhibited the adherence of a subset of ETEC isolates, demonstrating CS14-specific inhibition with partial cross-reactivity within the class 5a fimbrial family. These data provide support for CS14 as an important fimbrial CF and its consideration as a vaccine antigen in future strategies. IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) infection causes profuse watery diarrhea in adults and children in low- to middle-income countries and is a leading cause of traveler's diarrhea. Despite increased use of rehydration therapies, young children especially can suffer long-term effects including gastrointestinal dysfunction as well as stunting and malnutrition. As there is no licensed vaccine for ETEC, there remains a need to identify and understand specific antigens for inclusion in vaccine strategies. This study investigated one adhesin named CS14. This adhesin is expressed on the bacterial surface of ETEC isolates and was recently recognized for its significant association with diarrheal disease. We demonstrated that CS14 plays a role in bacterial adhesion to human target cells, a critical first step in the disease process, and that adherence could be blocked by CS14-specific antibodies. This work will significantly impact the ETEC field by supporting inclusion of CS14 as an antigen for ETEC vaccines.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1 +/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1 +/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1R-ID, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene.

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements.

The role of CFA/I in adherence and toxin delivery by ETEC expressing multiple colonization factors in the human enteroid model.

Enterotoxigenic Escherichia coli (ETEC) is a primary causative agent of diarrhea in travelers and young children in low-to-middle-income countries (LMICs). ETEC adhere to intestinal epithelia via colonization factors (CFs) and secrete heat-stable toxin (ST) and/or heat-labile toxin (LT), causing dysregulated cellular ion transport and water secretion. ETEC isolates often harbor genes encoding more than one CF that are targets as vaccine antigens. CFA/I is a major CF that is associated with ETEC that causes moderate-to-severe diarrhea and plays an important role in pathogenesis. The Global Enteric Multicenter Study finding that 78% of CFA/I-expressing ETEC also encode the minor CF CS21 prompted investigation of the combined role of these two CFs. Western blots and electron microscopy demonstrated growth media-dependent and strain-dependent differences in CFA/I and CS21 expression. The critical role of CFA/I in adherence by ETEC strains expressing CFA/I and CS21 was demonstrated using the human enteroid model and a series of CFA/I- and CS21-specific mutants. Furthermore, only anti-CFA/I antibodies inhibited adherence by global ETEC isolates expressing CFA/I and CS21. Delivery of ST and resulting cGMP secretion was measured in supernatants from infected enteroid monolayers, and strain-specific ST delivery and time-dependent cGMP production was observed. Interestingly, cGMP levels were similar across wildtype and CF-deficient strains, reflecting a limitation of this static aerobic infection model. Despite adherence by ETEC and delivery of ST, the enteroid monolayer integrity was not disrupted, as shown by the lack of decrease in transepithelial electrical resistance and the lack of IL-8 cytokines produced during infection. Taken together, these data demonstrate that targeting CFA/I in global clinical CFA/I-CS21 strains is sufficient for adherence inhibition, supporting a vaccine strategy that focuses on blocking major CFs. In addition, the human enteroid model has significant utility for the study of ETEC pathogenesis and evaluation of vaccine-induced functional antibody responses.

Hidden Costs of Multiple-Dose Products: Quantifying Ipratropium Inhaler Wastage in the Hospital Setting.

BACKGROUND: Previous studies have quantified wastage involving drugs that are available in multiple-dose formats. Ipratropium bromide by metered dose inhaler (MDI) is commonly used in hospitals, and may be contributing to waste of pharmaceutical and financial resources. OBJECTIVES: The primary objective was to quantify the number of patients in the authors' health authority with waste of at least 1 ipratropium MDI. Secondary outcomes were the total number of wasted inhalers, the total number of wasted doses, the cost of wasted inhalers, the cost of wasted doses, and possible factors or explanations for inhaler wastage. METHODS: A retrospective chart review was conducted for patients with an order for ipratropium by MDI in 2019 at one of the acute care sites within the health authority (predefined sample size 336). The number of inhalers dispensed was compared with doses received to determine the number of inhalers wasted. Each patient's electronic chart was audited for possible factors and explanations for wasting of inhalers. RESULTS: Of the 336 patients, 79 (24%) had wastage of at least 1 inhaler. In total, 34% (98/290) of all inhalers dispensed and 87% (50 693/58 000) of all doses dispensed were wasted. The total cost of wasted inhalers for the sample population was $2156. The most common reason for inhaler wastage was no doses being administered after an inhaler was dispensed; the second most common reason was dispensing of an extra inhaler associated with a change in directions for use. CONCLUSIONS: The use of multiple-dose MDI products in hospitals can lead to wastage of drugs and financial resources. Procedures need to be implemented to aid pharmacy and nursing staff in ensuring the most efficient use of these products. Evaluations of pilot methods to mitigate this waste are encouraged.

CONTEXTE: Des études antérieures ont quantifié le gaspillage de médicaments disponibles dans des formats multidoses. Le bromure d'ipratropium administré par inhalateur-doseur (ID) est communément utilisé dans les hôpitaux et pourrait entraîner un gaspillage des ressources pharmaceutiques et financières. OBJECTIFS: L'objectif principal consistait à quantifier le nombre de patients relevant de l'autorité sanitaire des auteurs, qui étaient source d'un gaspillage d'au moins un ID d'ipratropium. Les résultats secondaires visaient à déterminer le nombre total d'inhalateurs et de doses gaspillés, le coût associé au gaspillage des uns et des autres, ainsi que les facteurs pouvant expliquer cette situation. MÉTHODES: Les dossiers des patients ayant reçu une prescription d'ipratropium administrée par ID en 2019 dans l'un des sites de soins intensifs de l'autorité sanitaire ont fait l'objet d'un examen rétrospectif (taille de l'échantillon prédéfinie : 336). Une comparaison entre le nombre d'inhalateurs distribués et les doses reçues a permis de déterminer le nombre d'inhalateurs gaspillés. La vérification de chaque dossier électronique des patients a révélé les facteurs et les explications possibles du gaspillage des inhalateurs. RÉSULTATS: Sur les 336 patients, on a noté un gaspillage d'au moins un inhalateur tous les 79 patients (24 %). Au total, le gaspillage se montait à 34 % (98/290) de tous les inhalateurs distribués et à 87 % (50 693/58 000) de toutes les doses distribuées. Le coût total des inhalateurs distribués à l'échantillon de population se montait à 2156 $. La raison du gaspillage la plus fréquente était l'absence de doses administrées après la distribution d'un inhalateur; la deuxième raison concernait la distribution d'un inhalateur supplémentaire associée à une modification des instructions relatives à son utilisation. CONCLUSIONS: L'utilisation de produits ID multidoses dans les hôpitaux peut entraîner un gaspillage de médicaments et de ressources financières. Des procédures doivent être mises en place pour aider les membres du personnel des pharmacies et le personnel infirmier à utiliser plus efficacement ces produits. Il serait indiqué de procéder à des évaluations de méthodes pilotes pour atténuer ce gaspillage.

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens.

Enteric bacterial pathogens cause significant morbidity and mortality globally. Studies in tissue culture and animal models shaped our initial understanding of these host-pathogen interactions. However, intrinsic shortcomings in these models limit their application, especially in translational applications like drug screening and vaccine development. Human intestinal enteroid and organoid models overcome some limitations of existing models and advance the study of enteric pathogens. In this review, we detail the use of human enteroids and organoids to investigate the pathogenesis of invasive bacteria Shigella, Listeria, and Salmonella, and noninvasive bacteria pathogenic Escherichia coli, Clostridium difficile, and Vibrio cholerae. We highlight how these studies confirm previously identified mechanisms and, importantly, reveal novel ones. We also discuss the challenges for model advancement, including platform engineering to integrate environmental conditions, innate immune cells and the resident microbiome, and the potential for pre-clinical testing of recently developed antimicrobial drugs and vaccines.

Effectiveness of Auditory Measures for Detecting Hidden Hearing Loss and/or Cochlear Synaptopathy: A Systematic Review.

Standard audiometric evaluations are not sensitive enough to identify hidden hearing loss (HHL) and/or cochlear synaptopathy (CS). Patients with either of these conditions frequently present with difficulty understanding speech in noise or other complaints such as tinnitus. The purpose of this systematic review is to identify articles in peer-reviewed journals that assessed the sensitivity of audiologic measures for detecting HHL and/or CS, and which showed potential for use in a clinical test battery for these disorders. A reference librarian submitted specific boolean terminology to MEDLINE, Embase, and Web of Science. The authors used a consensus approach with specially designed score sheets for the selection of titles, abstracts, and then articles for inclusion in the systematic review and for quality assessment. Fifteen articles were included in the systematic review. Seven articles involved humans; seven involved animals, and one study used both humans and animals. Results showed that pure-tone audiometry to 20 kHz, otoacoustic emissions, electrocochleography, auditory brainstem response (ABR), electrophysiological tests, speech recognition in noise with and without temporal distortion, interviews, and self-report measures have been used to assess HHL and/or CS. For HHL, ultra-high-frequency audiometry may help identify persons with sensory hair cell loss that does not show up on standard audiograms. Promising nonbehavioral measures for CS included ABR wave I amplitude, the summating potential-to-action potential ratio, and speech recognition in noise with and without temporal distortion. Self-report questionnaires also may help identify auditory dysfunction in persons with normal hearing.

Complement Component C1q Programs a Pro-Efferocytic Phenotype while Limiting TNFα Production in Primary Mouse and Human Macrophages.

Deficiency in complement component C1q is associated with an inability to clear apoptotic cells (efferocytosis) and aberrant inflammation in lupus, and identification of the pathways involved in these processes should reveal important regulatory mechanisms in lupus and other autoimmune or inflammatory diseases. In this study, C1q-dependent regulation of TNFα/IL-6 expression and efferocytosis was investigated using primary mouse bone marrow-derived macrophages and human monocyte-derived macrophages. C1q downregulated LPS-dependent TNFα production in mouse and human macrophages. While prolonged stimulation with C1q (18 h) was required to elicit a dampening of TNFα production from mouse macrophages, the human macrophages responded to C1q with immediate downregulation of TNFα. IL-6 production was unchanged in mouse and upregulated by human macrophages following prolonged stimulation with C1q. Our previous studies indicated that C1q programmed enhanced efferocytosis in mouse macrophages by enhancing expression of Mer tyrosine kinase and its ligand Gas6, a receptor-ligand pair that also inhibits proinflammatory signaling. Here, we demonstrated that C1q-dependent programming of human macrophage efferocytosis required protein synthesis; however, neither Mer nor the related receptor Axl was upregulated in human cells. In addition, while the C1q-collagen-like tails are sufficient for promoting C1q-dependent phagocytosis of antibody-coated targets, the C1q-tails failed to program enhanced efferocytosis or dampen TNFα production. These data further elucidate the mechanisms by which C1q regulates proinflammatory signaling and efferocytosis in macrophages, functions that are likely to influence the progression of autoimmunity and chronic inflammation.

Host Factors Alter Effects of Angiopoietin-Like Protein 8 on Glucose Homeostasis in Diabetic Mice.

Recovery of functional beta cell mass offers a biological cure for type 1 diabetes. However, beta cell mass is difficult to regain once lost since the proliferation rate of beta cells after youth is very low. Angiopoietin like-protein 8 (ANGPTL8), a peptide that has a role in the regulation of lipoprotein lipase activity, was reported to increase beta cell proliferation in mice in 2013. Subsequent studies of human ANGPTL8 for short term (3 to 8 days) in non-diabetic mice showed little or no increase in beta cell proliferation. Here, we examined the effect of ANGPTL8 on glucose homeostasis in models that have not been examined previously. We expressed mouse ANGPTL8 using adenovirus in 2 mouse models of diabetes (streptozotocin and Non-Obese Diabetic (NOD) mice) over 2 weeks. Also, we tested ANGPTL8 in NOD mice deficient in leukocyte 12-lipoxygenase (12LO), an enzyme that contributes to insulitis and loss of beta cell function in NOD, in an effort to determine whether 12LO deficiency alters the response to ANGPTL8. Adenovirus-mediated expression of ANGPTL8 lowered blood glucose levels in streptozotocin treated mice without an increase in beta cell proliferation or serum insulin concentration. While ANGPTL8 did not reverse hyperglycemia in overtly hyperglycemic NOD mice or alter glucose homeostasis of non-diabetic NOD mice, ANGPTL8 reduced blood glucose levels in 12LOKO NOD mice. However, the lower glucose levels in 12LOKO NOD were not associated with higher serum insulin levels or beta cell proliferation. In summary, while mouse ANGPTL8 does not increase beta cell proliferation in NOD mice or streptozotocin treated mice in agreement with studies in non-diabetic mice, it lowers blood glucose levels in multiple low-dose streptozotocin induced diabetes and 12LO deficiency indicating that host factors influence the impact of ANGPTL8 on glucose homeostasis.

Supercritical fluid extraction provides an enhancement to the immune response for orally-delivered hepatitis B surface antigen.

The hepatitis B virus continues to be a major pathogen worldwide despite the availability of an effective parenteral vaccine for over 20 years. Orally-delivered subunit vaccines produced in maize may help to alleviate the disease burden by providing a low-cost, heat-stable alternative to the parenteral vaccine. Oral subunit vaccination has been an elusive goal due to the large amounts of antigen required to induce an immunologic response when administered through the digestive tract. Here we show that high levels of HBsAg were obtained in maize grain, the grain was formed into edible wafers, and wafers were fed to mice at a concentration of approximately 300 µg/g. When these wafers were made with supercritical fluid extraction (SFE)-treated maize material, robust IgG and IgA responses in sera were observed that were comparable to the injected commercial vaccine (Recombivax(®)). In addition, all mice administered SFE wafers showed high secretory IgA titers in fecal material whereas Recombivax(®) treated mice showed no detectable titer. Increased salivary IgA titers were also detected in SFE-fed mice but not in Recombivax(®) treated mice. Wafers made from hexane-treated or full fat maize material induced immunologic responses, but fecal titers were attenuated relative to those produced by SFE-treated wafers. These responses demonstrate the feasibility of using a two-dose oral vaccine booster in the absence of an adjuvant to induce immunologic responses in both sera and at mucosal surfaces, and highlight the potential limitations of using an exclusively parenteral dosing regime.

In vitro inhibition of cytochrome P450-mediated reactions by gemfibrozil, erythromycin, ciprofloxacin and fluoxetine in fish liver microsomes.

Inhibition of mammalian cytochrome P450 enzymes (CYPs) is well characterized; major hepatic CYPs can be inhibited by drugs and other environmental contaminants. CYP function and inhibition has not yet been well established in fish yet these studies are important for several reasons. First, such studies will provide functional information for non-mammalian CYPs. Second, specific inhibitors can be used as a diagnostic tool for studying CYP-mediated reactions. Lastly, pharmaceutical mixtures are found in the aquatic environment and adverse effects associated with drug-drug interactions, including CYP inhibition by pharmaceuticals may be of concern. Using liver microsomes from untreated and ß-naphthoflavone (BNF)-treated rainbow trout, eight fluorescent CYP-mediated catalytic assays were used to assess in vitro CYP inhibition by four pharmaceuticals: fluoxetine, ciprofloxacin, gemfibrozil and erythromycin. Expressed zebrafish CYP1 proteins (CYP1A, CYP1B1, CYP1C1 and CYP1C2) were assessed for inhibition with selected substrates. All pharmaceuticals decreased the metabolism of a number of substrates. Fluoxetine was the strongest and most broad inhibitor of CYP-mediated reactions in liver microsomes. Zebrafish CYP1s were strongly inhibited by erythromycin and fluoxetine. Although the pharmaceuticals are selective CYP inhibitors in mammals, inhibition across a number of substrates suggests they are broad inhibitors in fish. These data demonstrate that in vitro hepatic CYP inhibition by pharmaceuticals is possible in fish and the patterns seen here are different than what would be expected based on CYP inhibition in mammals.

Assessment of cytochrome P450 fluorometric substrates with rainbow trout and killifish exposed to dexamethasone, pregnenolone-16alpha-carbonitrile, rifampicin, and beta-naphthoflavone.

Cytochrome P450s (CYPs) are important xenobiotic metabolizing proteins. While their functions are well understood in mammals, CYP function in non-mammalian vertebrate systems is much less defined, with function often inferred from mammalian data, assuming similar function across vertebrate species. In this study, we investigate whether in vivo treatment with known mammalian CYP inducers can alter the in vitro catalytic activity of fish microsomes using eleven fluorescent CYP-mediated substrates. We investigate the basal metabolism and induction potential for hepatic CYPs in two fish species, rainbow trout (Oncorhynchus mykiss) and killifish (Fundulus heteroclitus). Species differences were found in the baseline metabolism of these substrates. Killifish have significantly higher metabolic rates for all tested substrates except 7-benzyloxyquinoline and 7-benzyloxy-4-trifluoromethylcoumarin (both mammalian CYP3A substrates); significant differences were also seen between male and female killifish. Treatment with dexamethasone, pregnenolone-16alpha-carbonitrile, and rifampicin did not cause broad, measurable CYP induction in either fish species. In trout, dexamethasone (100 mg kg(-1)) significantly induced 3-cyano-7-ethoxycoumarin metabolism and rifampicin (100 mg kg(-1)) induced the dealkylation of 7-methoxyresorufin, although both were highly variable. Female killifish exposed to pregnenolone-16alpha-carbonitrile (100 mg kg(-1)) showed significantly higher metabolism of 7-pentoxyresorufin. Overall, dexamethasone, pregnenolone-16alpha-carbonitrile and rifampicin did not appear to consistently increase CYP activity in fish. Trout treated with 10 or 50 mg kg(-1) beta-naphthoflavone (BNF), a CYP1A inducer, showed significantly induced activity across almost all substrates tested, exceptions being 7-benzyloxyquinoline, 7-benzyloxy-4-trifluoromethylcoumarin and dibenzylfluorescein. 7-Methoxy-4-(aminomethyl)coumarin, a typical CYP2D substrate in mammals, was not metabolized by untreated fish liver microsomes; however, treatment with BNF significantly induced the metabolism of this substrate in trout. Induced substrate metabolism in BNF-treated microsomes was only correlated across selective substrates, suggesting that BNF induces multiple CYPs in fish liver. These include the known BNF inducible CYP1s plus a number of as yet unidentified fish CYPs. Overall, many of these catalytic assays could be valuable tools for identification of the function of specific CYP subfamilies and individual isoforms in fish.