Cranberry and Recurrent Cystitis: More than Marketing?

Epidemiologic studies indicate that millions of people suffer from recurrent cystitis, a pathology requiring antibiotic prophylaxis and entailing high social costs. Cranberry is a traditional folk remedy for cystitis and, which, in the form of a variety of products and formulations has over several decades undergone extensive evaluation for the management of urinary tract infections (UTI). The aim of this retrospective study is to summarize and review the most relevant and recent preclinical and clinical studies on cranberries for the treatment of UTIs. The scientific literature selected for this review was identified by searches of Medline via PubMed. A variety of recent experimental evidence has shed light on the mechanism underlying the anti-adhesive properties of proanthrocyanidins, their structure–activity relationships, and pharmacokinetics. Analysis of clinical studies and evaluation of the cranberry efficacy/safety ratio in the prevention of UTIs strongly support the use of cranberry in the prophylaxis of recurrent UTIs in young and middle-aged women. However, evidence of its clinical use among other patients remains controversial.     

Protein thermostability in extremophiles

Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification. Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such as electrostatic (hydrogen bonds and ion-pairs) and hydrophobic interactions. Proteins produced by thermo and hyperthermophilic microorganisms, growing between 45 and 110 degrees C are in general more resistant to thermal and chemical denaturation than their mesophilic counterparts. The observed structural resistance may reflect a restriction on the flexibility of these proteins, which, while allowing them to be functionally competent at elevated temperatures, renders them unusually rigid at mesophilic temperatures (10-45 degrees C). The increased rigidity at mesophilic temperatures may find a structural determinant in increased compactness. In thermophilic proteins a number of amino acids are often exchanged. These exchanges with some strategic placement of proline in beta-turns give rise to a stabilization of the protein. Mutagenesis experiments have confirmed this statement. From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds and salt bridges. Thermostability appears also related to a better packing within buried regions. Despite these generalisations, no universal rules can be found in these proteins to achieve thermostability.     

Acid-induced disassembly of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus occurs below pH 2.0

The stability of the hexameric glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus at low pH values has been studied by activity assay, spectroscopic methods, size-exclusion chromatography and ultracentrifugation analysis. The enzyme is exceptionally stable and at pH 2.0 its hexameric assembly is preserved despite the changes observed in its tertiary structure. Below pH 1.7 dissociation into monomers starts and is accompanied by a progressive loss of tertiary interactions. Dissociation intermediate(s) were not detectable. At pH 2.0 the addition of NaCl causes the same structural changes observed upon further addition of protons. The monomeric state of the enzyme at pH 1.0 shows a significant content of native secondary structure and can be unfolded by guanidinium chloride. The role of electrostatic interactions in the high stability of the enzyme structure at low pH values is discussed.     

On the flame height definition for upward flame spread

Flame height is defined by the experimentalists as the average position of the luminous flame and, consequently is not directly linked with a quantitative value of a physical parameter. To determine flame heights from both numerical and theoretical results, a more quantifiable criterion is needed to define flame heights and must be in agreement with the experiments to allow comparisons. For wall flames, steady wall flame experiments revealed that flame height may be defined by a threshold value on the wall heat flux. From steady wall flame measurements, three definitions of flame height from wall heat flux are retained: the first is based on the continuous flame while the two others are based on threshold values of 4 kW/m² and 10 kW/m². These definitions are applied to determine flame heights from a two-dimensional time-dependent CFD model used to describe flame spread on a vertical slab of PMMA. Results show that the predicted flame heights are consistent with the available data of the literature. Defining flame height by threshold values on the wall heat flux of 4 and 10 kW/m² allows the correlation of the wall heat flux in terms of (x−x_p)/(x_fl−x_p), which is the dimensionless characteristic length scale for upward flame spread. It is also found that the continuous flame is not a characteristic length for the heat transfer to the unburnt fuel and is not really appropriate to define flame height in upward flame spread.     

Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.     

Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability

The glutamate dehydrogenase gene from the hyperthermophilicarchaeon Pyrococcus furiosus has been functionally expressedin Escherichia coli under the control of the X, PL promoter.The P.furiosus glutamate dehydrogenase amounted to 20% of thetotal E.coli cell protein, and the vast majority consisted ofhexamers. Following activation by heat treatment, an enzymecould be purified from E.coli that was indistinguishable fromthe glutamate dehydrogenase purified from P.furiosus. Hybridgenes, that consisted of the coding regions for the homologousglutamate dehydrogenases from P.furiosus and the mesophilicbacterium Clostridium difficile, were constructed and successfullyexpressed in E.coli. One of the resulting hybrid proteins, containingthe glutamate binding domain of the C.difficile enzyme and thecofactor binding domain of the P.furiosus enzyme, did not showa detectable activity. In contrast, the complementary hybridcontaining the P.furiosus glutamate and the C.difficile cofactorbinding domain was a catalytically active hexamer that showeda reduced substrate affinity but maintained efficient cofactorbinding with the specificity found in the Clostridium symbiosumenzyme. Compared with the C.difficile glutamate dehydrogenase,the archaeal-bacterial hybrid is slightly more thermoactive,less thermostable but much more stable towards guanidinium chloride-inducedinactivation and denaturation     

Analysis and Interpretation of the Impact of Missense Variants in Cancer

Large scale genome sequencing allowed the identification of a massive number of genetic variations, whose impact on human health is still unknown. In this review we analyze, by an in silico-based strategy, the impact of missense variants on cancer-related genes, whose effect on protein stability and function was experimentally determined. We collected a set of 164 variants from 11 proteins to analyze the impact of missense mutations at structural and functional levels, and to assess the performance of state-of-the-art methods (FoldX and Meta-SNP) for predicting protein stability change and pathogenicity. The result of our analysis shows that a combination of experimental data on protein stability and in silico pathogenicity predictions allowed the identification of a subset of variants with a high probability of having a deleterious phenotypic effect, as confirmed by the significant enrichment of the subset in variants annotated in the COSMIC database as putative cancer-driving variants. Our analysis suggests that the integration of experimental and computational approaches may contribute to evaluate the risk for complex disorders and develop more effective treatment strategies.