Effect of gamma radiation on the activity of hemocytes and on the course of Schistosoma mansoni infection in resistant Biomphalaria tenagophila snails

High doses of gamma radiation (10 Krad) in Biomphalaria tenagophila snails (Taim strain), which have been found to be resistant to Schistosoma mansoni, were not sufficient to impair their resistance to the parasite. The number of hemocytes, as well as their phagocytic activity, were not affected by irradiation, thus showing resemblance with mammal macrophages, which are resistant to gamma irradiation also.     

New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.     

Artificial Evolution by Viability Rather than Competition

Evolutionary algorithms are widespread heuristic methods inspired by natural evolution to solve difficult problems for which analytical approaches are not suitable. In many domains experimenters are not only interested in discovering optimal solutions, but also in finding the largest number of different solutions satisfying minimal requirements. However, the formulation of an effective performance measure describing these requirements, also known as fitness function, represents a major challenge. The difficulty of combining and weighting multiple problem objectives and constraints of possibly varying nature and scale into a single fitness function often leads to unsatisfactory solutions. Furthermore, selective reproduction of the fittest solutions, which is inspired by competition-based selection in nature, leads to loss of diversity within the evolving population and premature convergence of the algorithm, hindering the discovery of many different solutions.Here we present an alternative abstraction of artificial evolution, which does not require the formulation of a composite fitness function. Inspired from viability theory in dynamical systems, natural evolution and ethology, the proposed method puts emphasis on the elimination of individuals that do not meet a set of changing criteria, which are defined on the problem objectives and constraints.Experimental results show that the proposed method maintains higher diversity in the evolving population and generates more unique solutions when compared to classical competition-based evolutionary algorithms. Our findings suggest that incorporating viability principles into evolutionary algorithms can significantly improve the applicability and effectiveness of evolutionary methods to numerous complex problems of science and engineering, ranging from protein structure prediction to aircraft wing design.     

THDP17 Decreases Ammonia Production through Glutaminase Inhibition. A New Drug for Hepatic Encephalopathy Therapy

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing V_max and K_m values of 384.62 µmol min⁻¹, 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.     

Candida Sepsis: a New Entity?

Despite progress in the field of biological sciences, a definitive diagnosis of Candida sepsis remains an elusive target. Candida, which is frequently found in non-immunocompromised patients in intensive care units, causes severe sepsis, septic shock, and multiple-organ failure in a similar fashion as bacteria. Despite its imprecision, the blood and/or sterile-site culture is still the gold standard for diagnosis, although new biological markers are becoming available for earlier and more accurate diagnosis of invasive candidiasis. Mortality remains high due to a number of factors, and early initiation of appropriate antifungal therapy is a critical factor in positive outcomes. Echinocandins are the drug of choice for severe Candida infections in hospitalized patients. Antifungal stewardship programs may decrease the likelihood of resistance strains, and preventive measures are becoming available to lower the rate of fungal infections in intensive care units.     

Detection of Atomic Scale Changes in the Free Volume Void Size of Three-Dimensional Colorectal Cancer Cell Culture Using Positron Annihilation Lifetime Spectroscopy

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.     

Analysis of ECs and related compounds in plasma: artifactual isomerization and ex vivo enzymatic generation of 2-MGs

The analysis of peripheral endocannabinoids (ECs) is a good biomarker of the EC system. Their concentrations, from clinical studies, strongly depend on sample collection and time processing conditions taking place in clinical and laboratory settings. The analysis of 2-monoacylglycerols (MGs) (i.e., 2-arachidonoylglycerol or 2-oleoylglycerol) is a particularly challenging issue because of their ex vivo formation and chemical isomerization that occur after blood sample collection. We provide evidence that their ex vivo formation can be minimized by adding Orlistat, an enzymatic lipase inhibitor, to plasma. Taking into consideration the low cost of Orlistat, we recommend its addition to plasma collecting tubes while maintaining sample cold chain until storage. We have validated a method for the determination of the EC profile of a range of MGs and N-acylethanolamides in plasma that preserves the original isomer ratio of MGs. Nevertheless, the chemical isomerization of 2-MGs can only be avoided by an immediate processing and analysis of samples due to their instability during conservation. We believe that this new methodology can aid in the harmonization of the measurement of ECs and related compounds in clinical samples.     

Use of Limited Proteolysis and Mutagenesis To Identify Folding Domains and Sequence Motifs Critical for Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase Activity

Triacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids.