Human cytomegalovirus UL38 protein blocks apoptosis

Apoptosis is an innate cellular defense response to viral infection. The slow-replicating human cytomegalovirus (HCMV) blocks premature death of host cells prior to completion of the infection cycle. In this study, we report that the HCMV UL38 gene encodes a cell death inhibitory protein. A mutant virus lacking the pUL38 coding sequence, ADdlUL38, grew poorly in human fibroblasts, failed to accumulate viral DNA to wild-type levels, and induced excessive death of infected cells. Cells expressing pUL38 were resistant to cell death upon infection and effectively supported the growth of ADdlUL38. Cells infected with the pUL38-deficient virus showed morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, vesicle release, and chromatin condensation and fragmentation. The proteolytic cleavage of two key enzymes involved in apoptosis, namely, caspase 3 and poly(ADP-ribose) polymerase, was activated upon ADdlUL38 infection, and the cleavage was blocked in cells expressing pUL38. The pan-caspase inhibitor Z-VAD-FMK largely restored the growth of ADdlUL38 in normal fibroblasts, indicating that the defective growth of the mutant virus mainly resulted from premature death of host cells. Furthermore, cells expressing pUL38 were resistant to cell death induced by a mutant adenovirus lacking the antiapoptotic E1B-19K protein or by thapsigargin, which disrupts calcium homeostasis in the endoplasmic reticulum. Taken together, these results indicate that the HCMV protein pUL38 suppresses apoptosis, blocking premature death of host cells to facilitate efficient virus replication.     

Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ~23 to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.     

Efficacy model for antibody-mediated pre-erythrocytic malaria vaccines

Antibodies to the pre-erythrocytic antigens, circumsporozoite protein (CSP), thrombospondin-related adhesive protein (TRAP) and liver-stage antigen 1, have been measured in field studies of semi-immune adults and shown to correlate with protection from Plasmodium falciparum infection. A mathematical model is formulated to estimate the probability of sporozoite infection as a function of antibody titres to multiple pre-erythrocytic antigens. The variation in antibody titres from field data was used to estimate the relationship between the probability of P. falciparum infection per infectious mosquito bite and antibody titre. Using this relationship, we predict the effect of vaccinations that boost baseline CSP or TRAP antibody titres. Assuming the estimated relationship applies to vaccine-induced antibody titres, then single-component CSP or TRAP antibody-mediated pre-erythrocytic vaccines are likely to provide partial protection from infection, with vaccine efficacy of approximately 50 per cent depending on the magnitude of the vaccine-induced boost to antibody titres. It is possible that the addition of a TRAP component to a CSP-based vaccine such as RTS,S would provide an increase in infection-blocking efficacy of approximately 25 per cent should the problem of immunological interference between antigens be overcome.     

Origin and development of the atrioventricular myocardial lineage: insight into the development of accessory pathways

Defects originating from the atrioventricular canal region are part of a wide spectrum of congenital cardiovascular malformations that frequently affect newborns. These defects include partial or complete atrioventricular septal defects, atrioventricular valve defects, and arrhythmias, such as atrioventricular re-entry tachycardia, atrioventricular nodal block, and ventricular preexcitation. Insight into the cellular origin of the atrioventricular canal myocardium and the molecular mechanisms that control its development will aid in the understanding of the etiology of the atrioventricular defects. This review discusses current knowledge concerning the origin and fate of the atrioventricular canal myocardium, the molecular mechanisms that determine its specification and differentiation, and its role in the development of certain malformations such as those that underlie ventricular preexcitation.     

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse. The adult null mice exhibited increased cardiac mass, larger cardiomyocytes, and ejection fractions that were 9% higher by magnetic resonance imaging compared with wild-type animals. Notably, this occurred in the absence of hypertension. Total Na-K-ATPase activity was 50% lower in the phospholemman-deficient hearts. Expression (per unit of membrane protein) of total Na-K-ATPase was only slightly diminished, but expression of the minor alpha(2)-isoform, which has been specifically implicated in the control of contractility, was reduced by 60%. The absence of phospholemman thus results in a complex response, including a surprisingly large reduction in intrinsic Na-K-ATPase activity, changes in Na-K-ATPase isoform expression, increase in ejection fraction, and increase in cardiac mass. We hypothesize that a primary effect of phospholemman is to modulate the Na-K-ATPase and that its reduced activity initiates compensatory responses.     

Enhanced susceptibility of staggerer (RORalphasg/sg) mice to lipopolysaccharide-induced lung inflammation

The retinoid-related orphan receptor alpha (RORalpha), a member of the ROR subfamily of nuclear receptors, has been implicated in the control of a number of physiological processes, including the regulation of several immune functions. To study the potential role of RORalpha in the regulation of innate immune responses in vivo, we analyzed the induction of airway inflammation in response to lipopolysaccharide (LPS) challenge in wild-type and staggerer (RORalpha(sg/sg)) mice, a natural mutant strain lacking RORalpha expression. Examination of hematoxylin and eosin-stained lung sections showed that RORalpha(sg/sg) mice displayed a higher degree of LPS-induced inflammation than wild-type mice. Bronchoalveolar lavage (BAL) was performed at 3, 16, and 24 h after LPS exposure to monitor the increase in inflammatory cells and the level of several cytokines/chemokines. The increased susceptibility of RORalpha(sg/sg) mice to LPS-induced airway inflammation correlated with a higher number of total cells and neutrophils in BAL fluids from LPS-treated RORalpha(sg/sg) mice compared with those from LPS-treated wild-type mice. In addition, IL-1beta, IL-6, and macrophage inflammatory protein-2 were appreciably more elevated in BAL fluids from LPS-treated RORalpha(sg/sg) mice compared with those from LPS-treated wild-type mice. The enhanced susceptibility of RORalpha(sg/sg) mice appeared not to be due to a repression of IkappaBalpha expression. Our observations indicate that RORalpha(sg/sg) mice are more susceptible to LPS-induced airway inflammation and are in agreement with the hypothesis that RORalpha functions as a negative regulator of LPS-induced inflammatory responses.     

Triglyceride modulation by acifran analogs: activity towards the niacin high and low affinity G protein-coupled receptors HM74A and HM74

Niacin is known to exert profound beneficial effects on cholesterol levels in humans, although its use is somewhat hampered by the gram quantities necessary to exert effects and the prevalence of compliance-limiting skin flushing side effects that occur. Recently, two G protein-coupled receptors (GPCRs) for niacin were identified and characterized as high (HM74A; GPR109A) and low (HM74; GPR109B) affinity receptors based on the binding affinities of niacin. These receptors also bind acifran (AY-25,712), which is known to modulate lipid levels like niacin, with similar affinities. Twelve analogs of acifran were chemically synthesized. One analogue demonstrated a dose-dependent decrease in serum triglycerides in rats within 3h of oral administration. Next, the acifran analogs were assessed for their activity towards the high and low affinity niacin receptors expressed in CHO-K1 cells. Constructs expressing HM74A or HM74 were stably transfected into CHO-K1 cells and shown to elicit phosphorylation of p42 and p44 mitogen-activated protein kinase (ERK1/ERK2) phosphorylation upon addition of niacin or acifran. The presence of functionally coupled GPCRs was further confirmed using Pertussis toxin, which completely inhibited the ability of either niacin or acifran to elicit phospho-ERK1/ERK2. The EC(50) of p-ERK1/ERK2 for niacin for the high and low affinity receptors was 47nM and indeterminate (i.e., >100microM), respectively, while the EC(50) for acifran was 160 and 316nM, respectively. Two chemical analogs of acifran demonstrated robust phosphorylation of ERK1/ERK2. Collectively, these data suggest that the synthesis of acifran analogs may be a suitable path for developing improved HM74A agonists.     

Robust assessment of associations between weather and eastern wild turkey nest success

Temperature and precipitation have been identified as factors that potentially influence eastern wild turkey (Meleagris gallopavo silvestris) reproduction, but robust analyses testing the relationship between weather parameters and turkey nest success are lacking. Therefore, we assessed how weather influenced turkey daily nest survival using 8 years of data collected from 715 nests across the southeastern United States. We also conducted exploratory analyses investigating if weather conditions during or prior to nesting best predicted nest success. We then assessed the possible implications of climate change through 2041–2060 for future eastern wild turkey daily nest survival and nest success for variables determined significant in analyses. During incubation, positive anomalies of minimum daily temperature were associated with greater daily nest survival. Precipitation during nesting was not a good predictor of daily nest survival. Exploratory analyses unexpectedly indicated that weather conditions in January prior to incubation were more important to nest success than weather conditions during incubation. In January, negative anomalies of minimum temperature and greater average daily precipitation were associated with greater nest success. Projections of future nest success or daily nest survival based on these relationships with the predictive covariates, and informed by climate models, suggest that nest success may increase as January precipitation increases and that daily nest survival may increase as temperature during incubation increases. These positive associations could be offset by a negative association between nest success and the expected increases in January minimum average temperature. Additional research is needed to investigate causes of these relationships and assess the implications of climate change for eastern wild turkey poult survival.     

Survival and cause-specific mortality of male wild turkeys across the southeastern United States

Estimating survival and cause-specific mortality of male eastern wild turkeys (Meleagris gallopavo silvestris) is important for understanding population dynamics and implementing appropriate harvest management. To better understand age-specific estimates of annual survival and harvest rates, we captured and marked male wild turkeys with leg bands (n = 311) or bands and transmitters (n = 549) in Georgia, Louisiana, North Carolina, and South Carolina, USA, during 2014–2022. We fitted time to event models to data from radio-marked birds to estimate cause-specific mortality and annual survival. We used band recovery models incorporating both band recovery and telemetry data to further investigate harvest rates and survival. Annual survival from known-fate models in hunted populations was 0.54 (95% CI = 0.49–0.59) for adults and 0.86 (95% CI = 0.81–0.92) for juveniles. Cause-specific mortality analysis produced an annual harvest estimate of 0.29 (95% CI = 0.24–0.33) for adults and 0.02 (95% CI = 0.01–0.03) for juveniles, whereas predation was 0.15 (95% CI = 0.10–0.20) and 0.12 (95% CI = 0.08–0.17), respectively. Annual survival for adult males in a non-hunted population was 0.83 (95% CI = 0.72–0.97). Survival rate was negatively correlated with harvest rate, indicating harvest was an additive mortality source. Annual survival from band recovery models was 0.40 (95% CI = 0.37–0.44) for adults and 0.88 (95% CI = 0.81– 0.93) for juveniles, whereas annual harvest estimates were 0.24 (95% CI = 0.23–0.25) for adults and 0.04 (95% CI = 0.03–0.05) for juveniles. Both models suggested no differences in annual survival across years or among study areas, which included privately owned and public properties. Harvest was an additive mortality source for male wild turkeys, suggesting that managers interested in increasing annual survival of adult males could consider ways of reducing harvest rates.