Development of a real-time QPCR assay for the detection of RV2 lineage-specific rhadinoviruses in macaques and baboons

BACKGROUND: Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR) assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2) lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF 59/60 intergenic region which were highly conserved between the macaque RV2 rhadinoviruses, rhesus rhadinovirus (RRV) and Macaca nemestrina rhadinovirus-2 (MneRV2). These primers showed little similarity to the corresponding sequences of the macaque RV1 rhadinoviruses, retroperitoneal fibromatosis herpesvirus Macaca nemestrina (RFHVMn) and Macaca mulatta (RFHVMm). To determine viral loads per cell, an additional TaqMan QPCR assay was developed to detect the single copy cellular oncostatin M gene. RESULTS: We show that the RV2 QPCR assay is linear from less than 2 to more than 300,000 copies using MneRV2 DNA, and is non-reactive with RFHVMn DNA up to 1 billion DNA templates per reaction. RV2 loads ranging from 6 to 2,300 viral genome equivalent copies per 10(6 )cells were detected in PBMC from randomly sampled macaques from the Washington National Primate Research Center. Screening tissue from other primate species, including another macaque, Macaca fascicularis, and a baboon, Papio cynocephalus, revealed the presence of novel rhadinoviruses, MfaRV2 and PcyRV2, respectively. Sequence comparison and phylogenetic analysis confirmed their inclusion within the RV2 lineage of KSHV-like rhadinoviruses. CONCLUSIONS: We describe a QPCR assay which provides a quick and sensitive method for quantitating rhadinoviruses belonging to the RV2 lineage of KSHV-like rhadinoviruses found in a variety of macaque species commonly used for biomedical research. While this assay broadly detects different RV2 rhadinovirus species, it is unreactive with RV1 rhadinovirus species which commonly co-infect the same primate hosts. We also show that this QPCR assay can be used to identify novel RV2 rhadinoviruses in different primate species

Nutritional adequacy and dietary disparities in an adult Caribbean population of African descent with a high burden of diabetes and cardiovascular disease.

The Caribbean island of Barbados has a high burden of diabetes and cardiovascular disease. Dietary habits were last described in 2005. A representative population-based sample (n = 363, aged 25-64 years) provided two nonconsecutive 24-hr dietary recalls in this cross-sectional study. Mean daily nutrient intakes were compared with the Dietary Guidelines for Americans. Subgroup differences by age, sex, and educational level were examined using logistic regression. High sugar intakes exist for both sexes with 24% (95% CIs 18.9, 30.0) consuming less than the recommended <10% of energy from added sugars (men 22%; 15.0, 31.6; women 26%; 18.9, 33.7). Sugar-sweetened beverages provide 43% (42.2%, 44.4%) of total sugar intake. Inadequate dietary fiber intakes (men 21 g, 18.2, 22.8; women 18 g, 16.7, 18.9) exist across all age groups. Inadequate micronutrient intake was found in women for calcium, folate, thiamine, zinc, and iron. Older persons (aged 45-64 years) were more likely to report adequacy of dietary fiber (OR = 2.7, 1.5, 4.8) and iron (OR = 3.0, 1.7, 5.3) than younger persons (aged 25-44). Older persons (aged 45-64 years) were less likely to have an adequate supply of riboflavin (OR = 0.4, 0.2, 0.6) than younger persons. Men were more likely to have adequate intakes of iron (OR = 13.0, 6.1, 28.2), folate (OR = 2.4, 1.3, 4.6), and thiamine (OR = 3.0, 1.5, 5.0) than women. Education was not associated with nutrient intake. The Barbadian diet is characterized by high sugar intakes and inadequate dietary fiber; a nutrient profile associated with an increased risk of obesity, type 2 diabetes, and related noncommunicable diseases

Ultra-processed food consumption in Barbados: evidence from a nationally representative, cross-sectional study.

Our objective was to describe, for the first time in an English-speaking Caribbean country, the contribution of ultra-processed foods (UPFs) to nutrients linked to non-communicable disease. Using a cross-sectional study design, dietary data were collected from two non-consecutive 24-h dietary recalls. Recorded food items were then classified according to their degree of processing by the NOVA system. The present study took place in Barbados (2012-13). A representative population-based sample of 364 adult Barbadians (161 males and 203 females) aged 25-64 years participated in the study. UPFs represented 40⋅5 % (838 kcal/d; 95 % CI 791, 885) of mean energy intake. Sugar-sweetened beverages made the largest contribution to energy within the UPF category. Younger persons (25-44 years) consumed a significantly higher proportion of calories from UPF (NOVA group 4) compared with older persons (45-64 years). The mean energy shares of UPF ranged from 22⋅0 to 58⋅9 % for those in the lowest tertile to highest tertile. Within each tertile, the energy contribution was significantly higher in the younger age group (25-44 years) compared with the older (45-64 years). One-quarter of persons consume ≥50 % of their daily calories from UPF, this being significantly higher in younger persons. The ultra-processed diet fraction contained about six times the mean of free sugars and about 0⋅8 times the dietary fibre of the non-ultra-processed fraction (NOVA groups 1-3). Targeted interventions to decrease the consumption of UPF especially in younger persons is thus of high priority to improve the diet quality of Barbadians.This work was supported by the Ministry of Health and Wellness of the Government of Barbados

Field evaluation of two rapid diagnostic tests for Neisseria meningitidis serogroup A during the 2006 outbreak in Niger.

The Pastorex((R)) (BioRad) rapid agglutination test is one of the main rapid diagnostic tests (RDTs) for meningococcal disease currently in use in the "meningitis belt". Earlier evaluations, performed after heating and centrifugation of cerebrospinal fluid (CSF) samples, under good laboratory conditions, showed high sensitivity and specificity. However, during an epidemic, the test may be used without prior sample preparation. Recently a new, easy-to-use dipstick RDT for meningococcal disease detection on CSF was developed by the Centre de Recherche Médicale et Sanitaire in Niger and the Pasteur Institute in France. We estimate diagnostic accuracy in the field during the 2006 outbreak of Neisseria meningitidis serogroup A in Maradi, Niger, for the dipstick RDT and Pastorex((R)) on unprepared CSF, (a) by comparing each test's sensitivity and specificity with previously reported values; and (b) by comparing results for each test on paired samples, using McNemar's test. We also (c) estimate diagnostic accuracy of the dipstick RDT on diluted whole blood. We tested unprepared CSF and diluted whole blood from 126 patients with suspected meningococcal disease presenting at four health posts. (a) Pastorex((R)) sensitivity (69%; 95%CI 57-79) was significantly lower than found previously for prepared CSF samples [87% (81-91); or 88% (85-91)], as was specificity [81% (95%CI 68-91) vs 93% (90-95); or 93% (87-96)]. Sensitivity of the dipstick RDT [89% (95%CI 80-95)] was similar to previously reported values for ideal laboratory conditions [89% (84-93) and 94% (90-96)]. Specificity, at 62% (95%CI 48-75), was significantly lower than found previously [94% (92-96) and 97% (94-99)]. (b) McNemar's test for the dipstick RDT vs Pastorex((R)) was statistically significant (p<0.001). (c) The dipstick RDT did not perform satisfactorily on diluted whole blood (sensitivity 73%; specificity 57%).Sensitivity and specificity of Pastorex((R)) without prior CSF preparation were poorer than previously reported results from prepared samples; therefore we caution against using this test during an epidemic if sample preparation is not possible. For the dipstick RDT, sensitivity was similar to, while specificity was not as high as previously reported during a more stable context. Further studies are needed to evaluate its field performance, especially for different populations and other serogroups

Towards Abolition: Undoing the Colonized Curriculum

Racial injustice has traditionally been observed from the viewpoint of its impact and outcomes. Subsequently, educators and policy makers have generally focused on outcomes; unequal opportunity structures, disparities in educational achievement, the school-to-prison pipeline, disproportional health indicators, incarceration rates, and harsher punishment in school and judicial systems, are just a few of the contexts by which this nation’s racialized roots can be measured for present day mistreatment and disparate outcomes for minoritized populations. As policy makers and educators look to the impact of racial injustice, a true ontological vantage would reveal the cause as well as the perpetuation of these outcomes. As the current COVID-19 pandemic continues, and with increased interest in online learning, it is vital that teachers and professors seek new pedagogy and tools to teach about racism. Our study examined whether a virtual 1-hour presentation on white humanists influences students’ understanding of racial justice. Our research demonstrates that a colonized curriculum impacts student’s outlook on the world and themselves. Inversely, when we expose students to humanists throughout history, we are able to show that white people have a legacy and responsibility to fight for racial justice. This provides students with alternative models – beyond those that perpetuate white supremacy

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxinand model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses

Paid parental leave evaluation: Phase 2 report

Since 1 January 2011, most Australian families in which a mother was in paid employment before the birth or adoption of a child have been eligible for the new Australian Government funded Paid Parental Leave (PPL) scheme.2 The scheme provides eligible parents with up to 18 weeks of Parental Leave Pay (PLP), paid at the rate of the National Minimum Wage, following the birth or recent adoption of a child. The PPL scheme brings Australia into line with all other Organisation for Economic Cooperation and Development (OECD) countries, except the United States, in having a national scheme for paid leave available to mothers following childbirth. This report describes the results of an evaluation of the initial operation of the scheme

The ORF59 DNA polymerase processivity factor homologs of Old World primate RV2 rhadinoviruses are highly conserved nuclear antigens expressed in differentiated epithelium in infected macaques

Background ORF59 DNA polymerase processivity factor of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus (KSHV), is required for efficient copying of the genome during virus replication. KSHV ORF59 is antigenic in the infected host and is used as a marker for virus activation and replication. Results We cloned, sequenced and expressed the genes encoding related ORF59 proteins from the RV1 rhadinovirus homologs of KSHV from chimpanzee (PtrRV1) and three species of macaques (RFHVMm, RFHVMn and RFHVMf), and have compared them with ORF59 proteins obtained from members of the more distantly-related RV2 rhadinovirus lineage infecting the same non-human primate species (PtrRV2, RRV, MneRV2, and MfaRV2, respectively). We found that ORF59 homologs of the RV1 and RV2 Old World primate rhadinoviruses are highly conserved with distinct phylogenetic clustering of the two rhadinovirus lineages. RV1 and RV2 ORF59 C-terminal domains exhibit a strong lineage-specific conservation. Rabbit antiserum was developed against a C-terminal polypeptide that is highly conserved between the macaque RV2 ORF59 sequences. This anti-serum showed strong reactivity towards ORF59 encoded by the macaque RV2 rhadinoviruses, RRV (rhesus) and MneRV2 (pig-tail), with no cross reaction to human or macaque RV1 ORF59 proteins. Using this antiserum and RT-qPCR, we determined that RRV ORF59 is expressed early after permissive infection of both rhesus primary fetal fibroblasts and African green monkey kidney epithelial cells (Vero) in vitro. RRV- and MneRV2-infected foci showed strong nuclear expression of ORF59 that correlated with production of infectious progeny virus. Immunohistochemical studies of an MneRV2-infected macaque revealed strong nuclear expression of ORF59 in infected cells within the differentiating layer of epidermis corroborating previous observations that differentiated epithelial cells are permissive for replication of KSHV-like rhadinoviruses Conclusion The ORF59 DNA polymerase processivity factor homologs of the Old World primate RV1 and RV2 rhadinovirus lineages are phylogenetically distinct yet demonstrate similar expression and localization characteristics that correlate with their use as lineage-specific markers for permissive infection and virus replication. These studies will aid in the characterization of virus activation from latency to the replicative state, an important step for understanding the biology and transmission of rhadinoviruses, such as KSHV

Effects of Relaxation and Deep-Breathing on High School Students: ACT Prep

The purpose of this study was to relieve test anxiety in high school juniors preparing to take the ACT: a high-stakes, college admissions, standardized test. Participants included 81 eleventh grade students (25 males, 56 females) from a Midwestern public high school. Results demonstrated that relaxation training in the experimental group significantly lowered student’s perceptions of test anxiety from pre-test to post-test. Further research regarding systematic deep breathing and relaxation techniques is needed