Insect-Specific Flavivirus Replication in Mammalian Cells Is Inhibited by Physiological Temperature and the Zinc-Finger Antiviral Protein

The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and insect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The zinc-finger antiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 °C. In ZAP-expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP’s antiviral function, did. Importantly, at 34 °C, both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection. This provides new insights into flavivirus evolution and the mechanisms associated with host switching.     

What Do Medical Students Do and Want When Caring for “Difficult Patients”?

Phenomenon: Medical students, like physicians, experience negative emotions such as frustration when interacting with some patients, and many of these interactions occur for the first time during clinical clerkships. Students receive preclinical training in the social and behavioral sciences, often including learning about “difficult patient” interactions, yet little is known about their desire for training during clinical education. We explored students’ strategies in these difficult clinical interactions, whether they felt prepared by the curriculum, and what support they would have liked. These data inform proposed strategies for supporting clinical learning. Approach: We interviewed 4th-year students about interactions with patients toward whom they felt negative emotions and sought to identify strategies and supports needed in these interactions. Interviews ended when theoretical sufficiency was achieved. We used qualitative content analysis to organize strategies into themes about areas benefiting from curricular supports. We mapped students’ desired curricular support examples to cognitive apprenticeship teaching methods—modeling, coaching, reflection, scaffolding, exploration, and articulation—and aligned them with traditional pedagogical techniques. Findings: We interviewed 26 medical students (44 volunteered/180 invited). Their strategies formed five themes: finding empathy (with a subtheme of focusing on social determinants of health), using learned communication approaches, anticipating challenging interactions, seeking support, and considering it an opportunity for more responsibility. Students described ideal clinical teaching, including postinteraction debriefs with an emphasis on validating their emotional reactions and challenges. Students mentioned all cognitive apprenticeship teaching methods, most prominently modeling (observing supervisors in such interactions) and supported oral reflection. They also identified a need for faculty and resident development to enact these teaching methods. Insights: Although students use some learned strategies in interactions in which they feel negative emotions toward patients, they desire more preparation and support during their clinical rotations. Their desires map to traditional pedagogical techniques and to methods of cognitive apprenticeship. Our findings point to the need to use these techniques to enhance clinical learning for students who experience emotionally challenging patient interactions.     

Adenoviral gene transfer of mutant phospholamban rescues contractile dysfunction in failing rabbit myocytes with relatively preserved SERCA function

In heart failure (HF) a main factor in reduced contractility is reduced SR Ca2+ content and reversed force-frequency response (FFR), ie, from positive to negative. Our arrhythmogenic rabbit HF model exhibits decreased contractility mainly due to an increase in Na/Ca exchange (NCX) activity (with only modest decrease in SR Ca2+-ATPase (SERCA) function), similar to many end-stage HF patients. Here we test whether phospholamban (PLB) inhibition using a dominant-negative mutant PLB adenovirus (K3E/R14E, AdPLB-dn, with beta-galactosidase adenovirus as control) could enhance SERCA function and restore Ca2+ transients and positive FFR in ventricular myocytes from these HF rabbits. HF myocytes infected with AdPLB-dn (versus control) had enhanced Ca2+ transient amplitude (2.0+/-0.1 versus 1.6+/-0.05 F/Fo at 0.5 Hz, P<0.05) and had a positive FFR, whereas acutely isolated HF myocytes or those infected with Adbetagal had negative FFR. Ca2+ transients declined faster in AdPLB-dn versus Adbetagal myocytes (RT50%: 317+/-29 versus 551+/-90 ms at 0.5 Hz, P<0.05) and had an increased SR Ca2+ load (3.5+/-0.3 versus 2.6+/-0.2 F/Fo at 0.5 Hz, P<0.05), indicative of increased SERCA function. Furthermore, this restoration of function was not due to changes in NCX or SERCA expression. Thus, increasing SERCA activity in failing myocytes by AdPLB-dn gene transfer reversed the contractile dysfunction (and restored positive FFR) by increasing SR Ca2+ load. This approach could enhance contractile function in failing hearts of various etiologies, even here where reduced SERCA activity is not the main dysfunction.     

Associations Between Macrolevel Economic Factors and Weight Distributions in Low- and Middle-Income Countries: A Multilevel Analysis of 200?000 Adults in 40 Countries

Objectives. We examined associations between macrolevel economic factors hypothesized to drive changes in distributions of weight and body mass index (BMI) in a representative sample of 200 796 men and women from 40 low- and middle-income countries.Methods. We used meta-regressions to describe ecological associations between macrolevel factors and mean BMIs across countries. Multilevel regression was used to assess the relation between macrolevel economic characteristics and individual odds of underweight and overweight relative to normal weight.Results. In multilevel analyses adjusting for individual-level characteristics, a 1–standard-deviation increase in trade liberalization was associated with 13% (95% confidence interval [CI] = 0.76, 0.99), 17% (95% CI = 0.71, 0.96), 13% (95% CI = 0.76, 1.00), and 14% (95% CI = 0.75, 0.99) lower odds of underweight relative to normal weight among rural men, rural women, urban men, and urban women, respectively. Economic development was consistently associated with higher odds of overweight relative to normal weight. Among rural men, a 1–standard-deviation increase in foreign direct investment was associated with 17% (95% CI = 1.02, 1.35) higher odds of overweight relative to normal weight.Conclusions. Macrolevel economic factors may be implicated in global shifts in epidemiological patterns of weight.Cardiovascular diseases are among the leading causes of death in low- and middle-income countries (LMICs),1 where mortality from such diseases has been increasing and is expected to continue doing so until 2030.2 In parallel to this trend, there has been an increase in average body mass index (BMI; defined as weight in kilograms divided by the square of height in meters) in most regions of the world.3 With population-based studies indicating a U- or J-shaped relation between BMI and cardiovascular disease mortality,4,5 these shifts in BMI may increase the proportion of the population at greatest risk for cardiovascular diseases. As such, increases in BMI may contribute to escalating cardiovascular disease mortality in LMICs,6 highlighting the need for understanding BMI patterns and predictors.Comparative longitudinal data that can be used to monitor BMI changes (often expressed according to prevalence of underweight, overweight, and obesity) across LMICs are scant; however, existing data suggest that the prevalence of underweight has decreased, the prevalence of overweight and obesity has increased, and, in general, there is a greater burden of overweight than underweight in most LMICs, particularly in urban areas.7–9 Shifts in the key determinants of weight, including diet and physical activity, are hypothesized to influence these patterns.3 Major changes in global dietary consumption have increased per capita food intake in LMICs, as well as the proportion of people’s daily diet derived from energy-dense and fatty foods.3,10–12 Although cross-national and longitudinal data on physical activity are limited, available evidence suggests that forms of transportation, employment, and leisure activities have become more sedentary and may contribute to changing patterns of weight at the population level.13,14Macrolevel economic factors, including economic development, urbanization, foreign investment, and trade liberalization, are hypothesized to drive shifting patterns of dietary composition, physical activity, and other determinants of nutritional outcomes.3,15 Economic growth and attendant increases in per capita income, for example, are associated with increased consumption of energy-dense foods,16 and recent cross-national analyses suggest that economic development is associated with a faster rate of growth in the prevalence of overweight among lower-income groups in LMICs.17,18 Urbanization is hypothesized to increase access to processed diets, reduce opportunities for physical activity, and expose residents to food marketing, thereby promoting a more sedentary lifestyle associated with less energy expenditure and greater caloric intake.15The influx of foreign direct investment (FDI), defined as investments by an enterprise in one country intended to acquire a lasting management interest in an enterprise operating in a foreign economy, represents one mechanism through which transnational corporations enter into new markets. FDI inflows are, along with greater openness to trade,19 hypothesized to be a key element in reshaping the global market for food, particularly in LMICs, by threatening traditional modes of agricultural production and facilitating the processing, distribution, and marketing of lower-cost, energy-dense food.20,21Despite the potential role that these macrolevel economic factors may play in shaping the epidemiological pattern of diet, behavior, and weight in LMICs, few empirical studies have investigated the relation between contextual factors and individual weight. A limited number of ecological studies have been conducted,9,22 but their results cannot be used to draw inferences about health at the individual level. Furthermore, the social patterning of diet and physical activity according to area of residence (urban or rural) and gender suggests that the macrolevel factors posited to drive changes in weight may have distinct implications for particular groups of individuals,23,24 and ecological studies cannot assess whether associations between macrolevel economic characteristics and weight vary according to such individual-level characteristics.We used data from a sample of approximately 200 000 adults from 40 LMICs to describe the ecological associations between macrolevel economic factors hypothesized to drive changes in determinants of weight (i.e., economic development, urbanization, FDI, trade liberalization) and average BMIs across countries and examine the association between macrolevel characteristics and the probability at the individual level of underweight and overweight or obesity relative to normal weight. We also assessed cross-level interactions of macrolevel factors with gender and area of residence.     

OraSure InteliSwab™ Rapid Antigen Test Performance with the SARS-CoV-2 Variants of Concern—Alpha,Beta, Gamma,Delta, and Omicron

The emergence of SARS-CoV-2 in the human population and the resulting COVID-19 pandemic have led to the development of various diagnostic tests. The OraSure InteliSwab^™ COVID-19 Rapid Test is a recently developed and FDA emergency use-authorized rapid antigen-detecting test that functions as a lateral flow device targeting the nucleocapsid protein. Due to SARS-CoV-2 evolution, there is a need to evaluate the sensitivity of rapid antigen-detecting tests for new variants, especially variants of concern such as Omicron. In this study, the sensitivity of the OraSure InteliSwab^™ Test was investigated using cultured strains of the known variants of concern (VOCs, Alpha, Beta, Gamma, Delta, and Omicron) and the ancestral lineage (lineage A). Based on dilution series in cell culture medium, an approximate limit of detection for each variant was determined. The OraSure InteliSwab^™ Test showed an overall comparable performance using recombinant nucleocapsid protein and different cultured variants, with recorded limits of detection ranging between 3.77 × 10⁵ and 9.13 × 10⁵ RNA copies/mL. Finally, the sensitivity was evaluated using oropharyngeal swabs from Syrian golden hamsters inoculated with the six VOCs. Ultimately, the OraSure InteliSwab^™ COVID-19 Rapid Test showed no decrease in sensitivity between the ancestral SARS-CoV-2 strain and any VOCs including Omicron.     

Extended cycle combined hormonal contraception

Extended cycle contraception is a safe and acceptable form of contraception and may be more efficacious than cyclic regimens, especially for users of oral contraceptive pills. Most extended cycle regimens result in fewer scheduled bleeding episodes, an outcome desired by many women. They may also result in more unscheduled bleeding and/or spotting episodes that decrease with time. Women who use extended cycle contraception experience fewer menstrual symptoms, particularly headache. Some women have medical conditions or menstrual symptoms that make extended cycle contraception a preferred method. Although we do not have enough data to recommend one particular extended cycle regimen over another, the use of these new regimens will provide women with more options, and almost certainly will improve the acceptability and efficacy of hormonal contraception. In this article, the authors focus on extended cycle combined hormonal contraceptive regimens and summarize their acceptability, efficacy, and safety. They also argue that extended cycle combined hormonal contraceptive may have increased efficacy compared with traditional cyclic combined hormonal contraceptive.     

Jet nebulization of prostaglandin E1 during neonatal mechanical ventilation: Stability, emitted dose and aerosol particle size

BACKGROUND: We have previously reported the safety of aerosolized PGE1 in neonatal hypoxemic respiratory failure. The aim of this study is to characterize the physicochemical properties of PGE1 solution, stability, emitted dose and the aerodynamic particle size distribution (APSD) of PGE1 aerosol in a neonatal ventilator circuit. METHODS: PGE1 was diluted in normal saline and physicochemical properties of the solution characterized. Chemical stability and emitted dose were evaluated during jet nebulization in a neonatal conventional (CMV) or high frequency (HFV) ventilator circuit by a high performance liquid chromatography-mass spectrometry method. The APSD of the PGE1 aerosol was evaluated with a 6-stage cascade impactor during CMV. RESULTS: PGE1 solution in normal saline had a low viscosity (0.9818 cP) and surface tension (60.8 mN/m) making it suitable for aerosolization. Little or no degradation of PGE1 was observed in samples from aerosol condensates, the PGE1 solution infused over 24h, or the residual solution in the nebulizer. The emitted dose of PGE1 following jet nebulization was 32-40% during CMV and 0.1% during HFV. The PGE1 aerosol had a mass median aerodynamic diameter of 1.4 microm and geometric S.D. of 2.9 with 90% of particles being <4.0 microm in size. CONCLUSION: Nebulization of PGE1 during neonatal CMV or HFV is efficient and results in rapid nebulization without altering the chemical structure. On the basis of the physicochemical properties of PGE1 solution and the APSD of the PGE1 aerosol, one can predict predominantly alveolar deposition of aerosolized PGE1.     

Typical but not "atypical" antipsychotic effects on startle gating deficits in prepubertal rats

RATIONALE: Dopamine (DA) agonists and NMDA antagonists disrupt sensorimotor gating in rats, as measured by a loss of prepulse inhibition of the startle reflex. These effects are used in predictive models for antipsychotic efficacy: clinically "typical" and "atypical" antipsychotics restore PPI in adult rats treated with DA agonists such as apomorphine (APO), while clinically "atypical" antipsychotics restore PPI in rats treated with NMDA antagonists such as phencyclidine (PCP). We previously reported that the PPI disruptive effects of both APO and PCP are evident in 16- to 18-day-old rat pups, suggesting that the brain substrates for these effects are functional very early in development. OBJECTIVE: In the present study we assessed the developmental patterns of antipsychotic effects in these measures. METHODS: The PPI-disruptive effects of APO and PCP, and their antagonism by the typical antipsychotic haloperidol, and the atypical antipsychotic quetiapine, were assessed across development in Sprague-Dawley rats. RESULTS: Similar to the pattern seen in adults, both haloperidol and quetiapine opposed APO-induced PPI deficits in 16- to 19-day-old rat pups. However, the "atypical" antipsychotic quetiapine did not oppose PCP-induced PPI deficits in pups or prepubertal (45 day) adolescents, but did oppose these PCP effects in postpubertal rats. CONCLUSIONS: While brain substrates mediating the PPI-disruptive effects of DA agonists and NMDA antagonists are functional early in development, some physiological event associated with puberty is a necessary condition for the "atypical antipsychotic profile" in this predictive model.     

Inhibition of spontaneous acetylcholine secretion by 2-chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction

1. Previous studies have reported discrepancies in the potencies of A(1) adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A(1) receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2. CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 microM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 microM) produced highly statistically-significant reductions in spontaneous ACh release. 3. In the presence of the non-selective protein kinase inhibitor, H7 (50 microM), the potency of CADO was increased such that 1 microM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4. Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 microM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 microM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5. The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A(1) adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition.