Reduced alphabet for protein folding prediction

What are the key building blocks that would have been needed to construct complex protein folds? This is an important issue for understanding protein folding mechanism and guiding de novo protein design. Twenty naturally occurring amino acids and eight secondary structures consist of a 28‐letter alphabet to determine folding kinetics and mechanism. Here we predict folding kinetic rates of proteins from many reduced alphabets. We find that a reduced alphabet of 10 letters achieves good correlation with folding rates, close to the one achieved by full 28‐letter alphabet. Many other reduced alphabets are not significantly correlated to folding rates. The finding suggests that not all amino acids and secondary structures are equally important for protein folding. The foldable sequence of a protein could be designed using at least 10 folding units, which can either promote or inhibit protein folding. Reducing alphabet cardinality without losing key folding kinetic information opens the door to potentially faster machine learning and data mining applications in protein structure prediction, sequence alignment and protein design. Proteins 2015; 83:631–639. © 2015 Wiley Periodicals, Inc.     

An integrated approach to assessing nitroso-redox balance in systemic inflammation

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.     

Effects of enzyme loading and β-glucosidase supplementation on enzymatic hydrolysis of switchgrass processed by leading pretreatment technologies

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25 mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO₂, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.     

Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B₃ (CVB₃), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC₅₀ values of 0.42 ± 0.18 μM and 0.60 ± 0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.     

Phylogeography and genetic structure of the red-legged partridge (Alectoris rufa): more evidence for refugia within the Iberian glacial refugium

The Pleistocene climatic oscillations promoted the diversification in avian species during the last glacial period. The red‐legged partridge (Alectoris rufa, Family Phasianidae) has a large natural distribution extending from the Mediterranean to humid temperate zones. However, the genetic structure for this species is unknown. The present study investigates the phylogeography, genetic structure and demographic history of A. rufa across its distribution, employing both mitochondrial DNA control region sequences and nuclear microsatellite loci. Our results propose that this species was greatly affected by Pleistocene glaciations. The mismatch analyses suggest that the current populations resulted from post‐glacial expansion and subsequent differentiation resulting in five diagnosable genetic clusters: Southwestern, Central‐eastern, Northwestern, Balearic and French and Italian. Further, we found evidence of three glacial refugia within the currently recognized Iberian glacial refugium. The intraspecific structure revealed by both maternal and biparental phylogeographic analyses was not resolved in the phylogenetic analyses. Based on all considerations, we recommended that five management units be recognized.     

Purinergic mechanisms in gliovascular coupling

Regional elevations in cerebral blood flow (CBF) often occur in response to localized increases in cerebral neuronal activity. An ever expanding literature has linked this neurovascular coupling process to specific signaling pathways involving neuronal synapses, astrocytes and cerebral arteries and arterioles. Collectively, these structures are termed the "neurovascular unit" (NVU). Astrocytes are thought to be the cornerstone of the NVU. Thus, not only do astrocytes "detect" increased synaptic activity, they can transmit that information to proximal and remote astrocytic sites often through a Ca(2+)- and ATP-related signaling process. At the vascular end of the NVU, a Ca(2+)-dependent formation and release of vasodilators, or substances linked to vasodilation, can occur. The latter category includes ATP, which upon its appearance in the extracellular compartment, can be rapidly converted to the potent vasodilator, adenosine, via the action of ecto-nucleotidases. In the present review, we give consideration to experimental model-specific variations in purinergic influences on gliovascular signaling mechanisms, focusing on the cerebral cortex. In that discussion, we compare findings obtained using in vitro (rodent brain slice) models and multiple in vivo models (2-photon imaging; somatosensory stimulation-evoked cortical hyperemia; and sciatic nerve stimulation-evoked pial arteriolar dilation). Additional attention is given to the importance of upstream (remote) vasodilation; the key role played by extracellular ATP hydrolysis (via ecto-nucleotidases) in gliovascular coupling; and interactions among multiple signaling pathways.     

Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Genetic diversity and differentiation of central European freshwater pearl mussel (Margaritifera margaritifera L.) populations: implications for conservation and management

Despite the fact that mollusc species play an important role in many aquatic ecosystems, little is known about their biodiversity and conservation genetics. Freshwater pearl mussel (Margaritifera margaritifera L.) populations are seriously declining all over Europe and a variety of conservation programs are being established to support the remaining endangered central European populations. In order to provide guidelines for conservation strategies and management programs, we investigated the genetic structure of 24 freshwater pearl mussel populations originating from five major central European drainages including Elbe, Danube, Rhine, Maas and Weser, representing the last and most important populations in this area. We present a nondestructive sampling method of haemolymph for DNA analyses, which is applicable for endangered bivalves. The analyses of nine microsatellite loci with different levels of polymorphism revealed a high degree of fragmented population structure and very different levels of genetic diversity within populations. These patterns can be explained by historical and demographic effects and have been enforced by anthropogenic activities. Even within drainages, distinct conservation units were detected, as revealed from high F(ST) values, private alleles and genetic distance measures. Populations sampled close to contact zones between main drainage systems showed lowest levels of correct assignment to present-day drainage systems. Populations with high priority for conservation should not only be selected by means of census population size and geographical distance to other populations. Instead, detailed genetic analyses are mandatory for revealing differentiation and diversity parameters, which should be combined with ecological criteria for sustainable conservation and recovery programs.