Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections

Astroviruses (AstVs) are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP) can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific.     

Methane oxidation coupled to oxygenic photosynthesis in anoxic waters

Freshwater lakes represent large methane sources that, in contrast to the Ocean, significantly contribute to non-anthropogenic methane emissions to the atmosphere. Particularly mixed lakes are major methane emitters, while permanently and seasonally stratified lakes with anoxic bottom waters are often characterized by strongly reduced methane emissions. The causes for this reduced methane flux from anoxic lake waters are not fully understood. Here we identified the microorganisms and processes responsible for the near complete consumption of methane in the anoxic waters of a permanently stratified lake, Lago di Cadagno. Interestingly, known anaerobic methanotrophs could not be detected in these waters. Instead, we found abundant gamma-proteobacterial aerobic methane-oxidizing bacteria active in the anoxic waters. In vitro incubations revealed that, among all the tested potential electron acceptors, only the addition of oxygen enhanced the rates of methane oxidation. An equally pronounced stimulation was also observed when the anoxic water samples were incubated in the light. Our combined results from molecular, biogeochemical and single-cell analyses indicate that methane removal at the anoxic chemocline of Lago di Cadagno is due to true aerobic oxidation of methane fuelled by in situ oxygen production by photosynthetic algae. A similar mechanism could be active in seasonally stratified lakes and marine basins such as the Black Sea, where light penetrates to the anoxic chemocline. Given the widespread occurrence of seasonally stratified anoxic lakes, aerobic methane oxidation coupled to oxygenic photosynthesis might have an important but so far neglected role in methane emissions from lakes.     

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.     

Histone H4 histidine phosphorylation: kinases, phosphatases, liver regeneration and cancer

Phosphorylation of histone H4 on one or both of its two histidine residues has been known to occur in liver cells for nearly 40?years and has been associated with proliferation of hepatocytes during regeneration of the liver following mechanical damage. More recently, large increases in histone H4 histidine kinase activity have been found to occur associated with proliferation and differentiation of liver progenitor cells following chemical damage that prevents hepatocyte proliferation. In addition, it has been shown this histone H4 histidine kinase activity is elevated nearly 100-fold in human foetal liver and several hundredfold in hepatocellular carcinoma tissue compared with normal adult liver. In the present paper, we review what is currently known about histone H4 histidine phosphorylation, the kinase(s) responsible and the phosphatases capable of catalysing its dephosphorylation, and briefly summarize the techniques used to detect and measure the histidine phosphorylation of histone H4 and the corresponding kinase activity.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Alpha helical trans-membrane proteins: Enhanced prediction using a Bayesian approach

Membrane proteins, which constitute approximately 20% of most genomes, are poorly tractable targets for experimental structure determination, thus analysis by prediction and modelling makes an important contribution to their on-going study. Membrane proteins form two main classes: alpha helical and beta barrel trans-membrane proteins. By using a method based on Bayesian Networks, which provides a flexible and powerful framework for statistical inference, we addressed alpha-helical topology prediction. This method has accuracies of 77.4% for prokaryotic proteins and 61.4% for eukaryotic proteins. The method described here represents an important advance in the computational determination of membrane protein topology and offers a useful, and complementary, tool for the analysis of membrane proteins for a range of applications.     

Combining algorithms to predict bacterial protein sub-cellular location: Parallel versus concurrent implementations

We describe a novel and potentially important tool for candidate subunit vaccine selection through in silico reverse-vaccinology. A set of Bayesian networks able to make individual predictions for specific subcellular locations is implemented in three pipelines with different architectures: a parallel implementation with a confidence level-based decision engine and two serial implementations with a hierarchical decision structure, one initially rooted by prediction between membrane types and another rooted by soluble versus membrane prediction. The parallel pipeline outperformed the serial pipeline, but took twice as long to execute. The soluble-rooted serial pipeline outperformed the membrane-rooted predictor. Assessment using genomic test sets was more equivocal, as many more predictions are made by the parallel pipeline, yet the serial pipeline identifies 22 more of the 74 proteins of known location.     

LIPPRED: A web server for accurate prediction of lipoprotein signal sequences and cleavage sites

Bacterial lipoproteins have many important functions and represent a class of possible vaccine candidates. The prediction of lipoproteins from sequence is thus an important task for computational vaccinology. Na?ve-Bayesian networks were trained to identify SpaseII cleavage sites and their preceding signal sequences using a set of 199 distinct lipoprotein sequences. A comprehensive range of sequence models was used to identify the best model for lipoprotein signal sequences. The best performing sequence model was found to be 10-residues in length, including the conserved cysteine lipid attachment site and the nine residues prior to it. The sensitivity of prediction for LipPred was 0.979, while the specificity was 0.742. Here, we describe LipPred, a web server for lipoprotein prediction; available at the URL: http://www.jenner.ac.uk/LipPred/. LipPred is the most accurate method available for the detection of SpaseIIcleaved lipoprotein signal sequences and the prediction of their cleavage sites.     

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by ¹H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by ³¹P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.     

Kinetics of an enzyme-catalyzed reaction measured by electrospray ionization mass spectrometry using a simple rapid mixing attachment

Mass spectrometry offers a potential means of measuring virtually all enzyme-catalyzed reactions by simultaneously measuring the concentrations of substrates, products, and intermediates where there are differences in mass between them. To perform these measurements the reaction mixture must be aged for different times and then ionized. Electrospray ionization mass spectrometry provides the most direct means of measuring these reactions. Here we describe a simple reaction mixing and ageing attachment for an electrospray ionization mass spectrometer, built from commercially available components. We have employed this device to measure the kinetics of a model reaction, namely the hydrolysis of N2-(carbobenzyloxy)-L-lysine-p-nitrophenyl ester-catalyzed by trypsin. In this way we were able to measure the kinetics of substrate depletion, product formation, and changes in both free enzyme and acyl-enzyme intermediate concentration in the approach to steady state. With this device we were able to measure reaction times down to about 640 ms.