An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV.Abbreviations: BV, B virus (Macacine herpesvirus 1); EU, ELISA units; g, glycoprotein; HSV, herpes simplex virus; tELISA, titration ELISA; UN, uninfected; WBA, western blot analysisB virus (BV; Macacine herpesvirus 1) is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. The virus occurs naturally in macaques (Macaca spp.) and causes a lethal zoonotic infection in 80% of untreated humans. Because biomedical professionals working with macaques, their cells, or tissues are at risk for becoming infected with BV, it is important to know the status of macaques involved in potential BV exposures. Although cases of BV infection after encounters between tourists and macaques have not been reported, any event that involves direct or fomite-associated contact with macaques has inherent risks. Identification of zoonotic BV infection through the detection of antibodies enables timely antiviral intervention, which is critical to reduce or prevent morbidity and mortality. Similarly rapid detection is important to maintain the biointegrity of SPF captive macaque colonies. The identification of BV in clinical specimens is achieved by using cell culture, PCR, or antibody detection methods. Because BV is shed only rarely from peripheral sites, the identification of BV infection in monkeys and humans currently is based on antibody detection (serology).^14,23,28In our laboratory, current serological diagnosis for B virus infections has been based on 2 principal tests: a titration-based (that is, traditional) ELISA (tELISA) as a screening test and western blot analysis (WBA) as a confirmatory test. Each test uses quality-controlled BV antigens that are prepared from lysates of infected cells.^20,22,23 Because BV is the only simplex virus in the Alphaherpesvirinae subfamily that is known to infect macaques,^14,28 antibodies interacting with BV antigens are used to indicate BV infection and not an infection due to a crossreacting virus. In practice, tELISA has identified numerous BV antibody-positive sera, the majority of which are low-titer sera from SPF colonies, which fail to be confirmed by WBA, and therefore, are classified as false positives.²³ We, therefore, searched for other approaches that could be used for confirmation of tELISA results. One reasonable option was the use of BV recombinant proteins as antigens. Numerous investigators have used recombinant-based assays for routine diagnosis of infections with viruses, including cytomegalovirus,³⁶ Epstein–Barr,⁶ herpes simplex (HSV1 and HSV2)^{2,3,17,31,32,34} Crimean–Congo hemorrhagic fever,¹⁰ HIV,³⁶ dengue,^5,11,27 hepatitis C,²⁴ hepatitis B,⁸ West Nile,²⁶ influenza,¹⁶ Ebola, and Marburg³³ viruses.Screening for the presence of serum IgG molecules against an array of defined and purified recombinant antigens has distinct advantages over assays that use the entire complement of viral antigens that are present in virus-infected cells. This is particularly true for pathogens that require BSL4 laboratories.^28,33 The pattern of reactivity obtained against each individual recombinant protein may have diagnostic value, by enabling identification of the stage of infection and the prediction of the prognosis of the disease.^3,4,18 However, using a single or only a few recombinant proteins as ELISA antigens can lead to a false-negative result if the antibody repertoire produced after BV infection reacts with other antigenic determinants that are not represented by the particular recombinant antigens used in the test.^{3,18,28,31,34}Several laboratories have examined the efficacy of using a single BV recombinant antigen (that is, glycoprotein D [gD]) for diagnosing BV infections in macaques^25,37 and humans,¹⁵ and we previously reported the diagnostic potential of an ELISA that incorporated several recombinant BV antigens.²⁸ We chose 4 recombinant BV glycoproteins as candidate antigens: peptides corresponding to the full-length extracellular domain of gB, gC, and gD and the membrane-associated segment of gG (gGm). Among these antigens, gGm was the most BV-specific, because it failed to crossreact with antibodies induced by HSV1 and HSV2. To validate the use of the recombinant BV antigens for the purpose of BV antibody detection, a panel of antibody-negative (n = 40) and antibody-positive (n = 75) macaque sera that were confirmed to be positive by tELISA and WBA were tested against the panel of the 4 B virus recombinant antigens, all of which showed fairly high sensitivity for detecting antibodies to BV.²⁸Here, we examine the performance of the recombinant-based ELISA (rELISA) for BV detection by using numerous (>1000) macaque sera, which have a broad range of antibody titers as determined by tELISA. Because manual ELISA to identify antibodies against an array of antigens are too laborious to be cost-effective, we adapted a previously described high-throughput automated single-antigen ELISA performed in 384-well plates to detect antibodies in macaque sera to multiple BV antigens.²³ This assay format has been adapted to include antigens from other alphaherpesviruses²³ and can be easily modified further for other viruses. We then compared the performance of the rELISA with that of whole-virus tELISA and WBA. The main goal of this study was to determine whether the 384-well rELISA is an effective alternative to WBA as a confirmatory assay for tELISA.     

Proteomic characterization of histotroph during the preimplantation phase of the estrous cycle in cattle

Uterine secretions, or histotroph, are a critical component for early embryo survival, functioning as the sole supply of vitamins, minerals, enzymes, and other myriad of nutrients required by the developing conceptus before implantation. Histotroph is therefore a promising source for biomarkers of uterine function and for enhancing our understanding of the environment supporting early embryo development and survival. Utilizing label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics, we characterized the uterine proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We identified 300 proteins on Day 7 and 510 proteins on Day 13 including 281 proteins shared between days. Five proteins were more abundant (P < 0.05) on Day 7 compared with Day 13 and included novel histotroph proteins cytokeratin 10 and stathmin. Twenty-nine proteins were more abundant (P < 0.05) including 13 unique on Day 13 compared with Day 7 and included previously identified legumain, metalloprotease inhibitor-2, and novel histotroph proteins chromogranin A and pyridoxal kinase. Functional analysis of the 34 differentially expressed proteins (including 14 novel to histotroph) revealed distinct biological roles putatively involved in early pregnancy, including remodelling of the uterine environment in preparation for implantation; nutrient metabolism; embryo growth, development and protection; maintenance of uterine health; and maternal immune modulation. This study is the first reported LC-MS/MS based global proteomic characterization of the uterine environment in any domesticated species before implantation and provides novel information on the temporal alterations in histotroph composition during critical stages for early embryo development and uterine function during the early establishment of pregnancy.     

Morphology and innervation of the buccal glands of the Southern Hemisphere lamprey, Geotria australis.

The buccal glands of adults of the Southern Hemisphere lamprey Geotria australis consist of a pair of small, bean-shaped, hollow sacs, embedded within the basilaris muscle in the region below the eyes and to either side of the piston cartilage. Each gland, which is lined by a simple columnar epithelium and surrounded by an incomplete layer of skeletal muscle, discharges its contents into the oral cavity via a long, narrow duct. In downstream migrating young adults, the epithelial cells are low columnar, intermediate in electron density, and contain dark-staining inclusions and numerous lipid-like droplets. After saltwater acclimation, the epithelial cells become taller and the numbers of dark-staining inclusions increase whereas those of lipid-like droplets decline. By the end of the marine phase, the epithelium is more folded and now also contains dark and light cells. The ultrastructure of the epithelium shows the characteristics of both apocrine and merocrine secretion. Although intra-epithelial nerve endings were not observed, axons and occasional neurons are present in the lamina propria. Since the skeletal muscle capsule is also well innervated and contains neurons, a local feed-back mechanism may regulate the release of buccal gland fluid by monitoring the luminal pressure. Contractions of the skeletal muscle capsule and movements of the basilaris muscle during feeding would presumably assist the movement of secretion along the duct. The secretion possesses anticoagulating and haemolytic properties.     

Perspective taking and decision-making in educational game play: A mixed-methods study

Video games have the potential to be contexts for moral learning. We investigated whether Quandary, a video game designed to promote ethical thinking and moral considerations for decision-making, would help promote positive skills such as perspective taking and empathy in adolescents. We examined the effect of playing Quandary on 131 middle school students on self-reported measures of moral thinking via mixed-method randomized control trials. In addition, we conducted qualitative analyses of one-on-one participant interviews and short-answer responses to capture experiences and reflections from playing Quandary, as well as the depth in which students across conditions responded to the interview questions. We found that short-term quantitative indicators did not show change across conditions; however, qualitative analyses revealed thematic responses that are consistent with the core components of the Quandary game, and that students in the Quandary condition showed a greater depth of response to interview questions. This work is a first step in exploring the potential for virtual game play on children’s social, emotional, and cognitive development.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.     

Synthesis and antibacterial activity of 7-(1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl) quinolones

A novel series of 7-(1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl) quinolones has been designed and synthesized in which the heterocyclic side chain is attached to the quinolone core through a carbon–carbon linkage. The antibacterial activity of the compounds was determined against a panel of Gram-positive and Gram-negative pathogens. Compounds 1b and 1e, bearing an 8-methoxy group as well as unsubstituted and (3S)-methyl substituted 1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-7-yl side chains, respectively, demonstrated notable activity against ciprofloxacin-resistant clinical isolates of Streptococcus pneumoniae.