The effects of an ideal beta-turn on beta-2 microglobulin fold stability

Beta-2 microglobulin (β2m) is the light chain of Class I major histocompatibility complex (MHC-I) complex. β2m is an intrinsically amyloidogenic protein capable of forming amyloid fibrils in vitro and in vivo. β2m displays the typical immunoglobulin-like fold with a disulphide bridge (Cys25-Cys80) cross-linking the two β-sheets. Engineering of the loop comprised between β-strands D and E has shown that mutations in this region affect protein structure, fold stability, folding kinetics and amyloid aggregation properties. Such overall effects have been related to the DE loop backbone structure, which presents a strained conformation in the wild-type (wt) protein, and a type I β-turn in the W60G mutant. Here, we report a biophysical and structural characterization of the K58P-W60G β2m mutant, where a Pro residue has been introduced in the type I β-turn i + 1 position. The K58P-W60G mutant shows improved chemical and temperature stability and faster folding relative to wt β2m. The crystal structure (1.25 ? resolution) shows that the Cys25-Cys80 disulphide bridge is unexpectedly severed, in agreement with electrospray ionization-mass spectrometry (ESI-MS) spectra that indicate that a fraction of the purified protein lacks the internal disulphide bond. These observations suggest a stabilizing role for Pro58, and stress a crucial role for the DE loop in determining β2m biophysical properties.     

Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa

Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.     

Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding,genotyping, and disease diagnostics from fungal and oomycete samples

The ubiquity, high diversity and often‐cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one‐step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single‐copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe‐based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol‐chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol‐chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68–71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.     

Quantile regression for longitudinal data using the asymmetric Laplace distribution

In longitudinal studies, measurements of the same individuals are taken repeatedly through time. Often, the primary goal is to characterize the change in response over time and the factors that influence change. Factors can affect not only the location but also more generally the shape of the distribution of the response over time. To make inference about the shape of a population distribution, the widely popular mixed-effects regression, for example, would be inadequate, if the distribution is not approximately Gaussian. We propose a novel linear model for quantile regression (QR) that includes random effects in order to account for the dependence between serial observations on the same subject. The notion of QR is synonymous with robust analysis of the conditional distribution of the response variable. We present a likelihood-based approach to the estimation of the regression quantiles that uses the asymmetric Laplace density. In a simulation study, the proposed method had an advantage in terms of mean squared error of the QR estimator, when compared with the approach that considers penalized fixed effects. Following our strategy, a nearly optimal degree of shrinkage of the individual effects is automatically selected by the data and their likelihood. Also, our model appears to be a robust alternative to the mean regression with random effects when the location parameter of the conditional distribution of the response is of interest. We apply our model to a real data set which consists of self-reported amount of labor pain measurements taken on women repeatedly over time, whose distribution is characterized by skewness, and the significance of the parameters is evaluated by the likelihood ratio statistic.     

Antiinflammatory Effect of Phytosterols in Experimental Murine Colitis Model: Prevention,Induction, Remission Study

Phytosterols, besides hypocholesterolemic effect, present anti-inflammatory properties. Little information is available about their efficacy in Inflammatory Bowel Disease (IBD). Therefore, we have evaluated the effect of a mixture of phytosterols on prevention/induction/remission in a murine experimental model of colitis. Phytosterols were administered x os before, during and after colitis induction with Dextran Sodium Sulfate (DSS) in mice. Disease Activity Index (DAI), colon length, histopathology score, ¹⁸F-FDG microPET, oxidative stress in the intestinal tissue (ileum and colon) and gallbladder ileum and colon spontaneous and carbachol (CCh) induced motility, plasma lipids and plasma, liver and biliary bile acids (BA) were evaluated. A similar longitudinal study was performed in a DSS colitis control group. Mice treated with DSS developed severe colitis as shown by DAI, colon length, histopathology score, ¹⁸F-FDG microPET, oxidative stress. Both spontaneous and induced ileal and colonic motility were severely disturbed. The same was observed with gallbladder. DSS colitis resulted in an increase in plasma cholesterol, and a modification of the BA pattern. Phytosterols feeding did not prevent colitis onset but significantly reduced the severity of the disease and improved clinical and histological remission. It had strong antioxidant effects, almost restored colon, ileal and gallbladder motility. Plasmatic levels of cholesterol were also reduced. DSS induced a modification in the BA pattern consistent with an increase in the intestinal BA deconjugating bacteria, prevented by phytosterols. Phytosterols seem a potential nutraceutical tool for gastrointestinal inflammatory diseases, combining metabolic systematic and local anti-inflammatory effects.     

A Self-Reported Screening Tool for Detecting Community-Dwelling Older Persons with Frailty Syndrome in the Absence of Mobility Disability: The FiND Questionnaire

Background

The “frailty syndrome” (a geriatric multidimensional condition characterized by decreased reserve and diminished resistance to stressors) represents a promising target of preventive interventions against disability in elders. Available screening tools for the identification of frailty in the absence of disability present major limitations. In particular, they have to be administered by a trained assessor, require special equipment, and/or do not discriminate between frail and disabled individuals. Aim of this study is to verify the agreement of a novel self-reported questionnaire (the “Frail Non-Disabled” [FiND] instrument) designed for detecting non-mobility disabled frail older persons with results from reference tools.

Methodology/Principal Findings

Data are from 45 community-dwelling individuals aged ≥60 years. Participants were asked to complete the FiND questionnaire separately exploring the frailty and disability domains. Then, a blinded assessor objectively measured the frailty status (using the phenotype proposed by Fried and colleagues) and mobility disability (using the 400-meter walk test). Cohen''s kappa coefficients were calculated to determine the agreement between the FiND questionnaire with the reference instruments. Mean age of participants (women 62.2%) was 72.5 (standard deviation 8.2) years. Seven (15.6%) participants presented mobility disability as being unable to complete the 400-meter walk test. According to the frailty phenotype criteria, 25 (55.6%) participants were pre-frail or frail, and 13 (28.9%) were robust. Overall, a substantial agreement of the instrument with the reference tools (kappa = 0.748, quadratic weighted kappa = 0.836, both p values<0.001) was reported with only 7 (15.6%) participants incorrectly categorized. The agreement between results of the FiND disability domain and the 400-meter walk test was excellent (kappa = 0.920, p<0.001).

Conclusions/Significance

The FiND questionnaire presents a very good capacity to correctly identify frail older persons without mobility disability living in the community. This screening tool may represent an opportunity for diffusing awareness about frailty and disability and supporting specific preventive campaigns.     

Interferon signaling suppresses the unfolded protein response and induces cell death in hepatocytes accumulating hepatitis B surface antigen

Virus infection, such as hepatitis B virus (HBV), occasionally causes endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) is counteractive machinery to ER stress, and the failure of UPR to cope with ER stress results in cell death. Mechanisms that regulate the balance between ER stress and UPR are poorly understood. Type 1 and type 2 interferons have been implicated in hepatic flares during chronic HBV infection. Here, we examined the interplay between ER stress, UPR, and IFNs using transgenic mice that express hepatitis B surface antigen (HBsAg) (HBs-Tg mice) and humanized-liver chimeric mice infected with HBV. IFNα causes severe and moderate liver injury in HBs-Tg mice and HBV infected chimeric mice, respectively. The degree of liver injury is directly correlated with HBsAg levels in the liver, and reduction of HBsAg in the transgenic mice alleviates IFNα mediated liver injury. Analyses of total gene expression and UPR biomarkers’ protein expression in the liver revealed that UPR is induced in HBs-Tg mice and HBV infected chimeric mice, indicating that HBsAg accumulation causes ER stress. Notably, IFNα administration transiently suppressed UPR biomarkers before liver injury without affecting intrahepatic HBsAg levels. Furthermore, UPR upregulation by glucose-regulated protein 78 (GRP78) suppression or low dose tunicamycin alleviated IFNα mediated liver injury. These results suggest that IFNα induces ER stress-associated cell death by reducing UPR. IFNγ uses the same mechanism to exert cytotoxicity to HBsAg accumulating hepatocytes. Collectively, our data reveal a previously unknown mechanism of IFN-mediated cell death. This study also identifies UPR as a potential target for regulating ER stress-associated cell death.     

Effect of temperature on the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases

A study on the response of the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases to temperature variations has been carried out. The thermodynamic properties of the heat inactivation process and of the reaction with the substrates glutamate and α-ketoglutarate have been investigated. The heat inactivation of nuclear glutamate dehydrogenase proceeds at a faster rate than that of the mitochondrial enzyme in the temperature range 40–51 °C; the enthalpy of activation of the inactivation process is higher and the entropy is almost double, compared to the values of mitochondrial glutamate dehydrogenase. The effect of temperature on the maximal velocity shows that, with both glutamate and α-ketoglutarate, the enthalpy of activation with nuclear glutamate dehydrogenase is double and the decrease in entropy almost half of the values of the mitochondrial enzyme. The variation of the apparent K_m with temperature shows a decrease of the affinity of both enzymes for glutamate, with no major difference in the thermodynamic properties of the reaction. With α-ketoglutarate, on the other hand, the affinity of nuclear glutamate dehydrogenase decreased, whereas that of the mitochondrial enzyme increased with temperature. The process is therefore exothermic with the former enzyme, endothermic with the latter; furthermore, it occurs with a decrease in enthropy with nuclear glutamate dehydrogenase, but with a large increase with the mitochondrial enzyme. The studies on the effect of temperature on the activity were carried out in the range 20–44 °C.