Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

Laparoscopic radical nephrectomy: morcellate or leave intact? Leave intact

At specialized centers worldwide, laparoscopic radical nephrectomy is now routine practice for management of indicated patients with localized renal cell carcinoma. Although the various intraoperative technical steps and maneuvers have been standardized for both the retroperitoneal and the transperitoneal laparoscopic approaches, controversy persists regarding the method of extraction of the nephrectomized cancerous specimen. Opinion is divided between intact extraction and morcellation. Clearly, the larger skin incision used for intact extraction is a cosmetic compromise. However, available data suggest that patient morbidity is not adversely impacted to any significant degree. Intact specimen extraction does build confidence into this procedure by respecting scientifically established oncologic guidelines, and it provides a specimen identical to that obtained in open surgery. This facilitates accurate pathologic staging, including assessment of surgical margins, which allows formulation of individualized, patient-specific follow-up protocols. For these reasons, intact extraction for renal cell cancer is preferred at our institution and multiple others worldwide.     

Association of Tumor Necrosis Factor-α and Myeloperoxidase enzyme with Severe Asthma: A comparative study

Background:Tumor necrosis factor-alpha (TNF-α) may stimulate airway hyperresponsiveness in asthma, which is also affected by neutrophils activity. The latter can be determined indirectly by evaluating myeloperoxidase (MPO) activity. The insufficient studies that investigated the combined association of serum TNF-α and MPO with asthma was objective of this study.Methods:A case-control study included 110-asthmatics besides 92-controls. All participants underwent venous sampling for TNF-α and MPO immunoassays. A percentage of predicted "forced expiratory volume in one second (FEV1%)", and the "peak expiratory flow rate (PEF/L)" of all participants were verified. The statistical analyses had done using SPSS V-25. The accuracy, specificity, sensitivity, and significance of both biomarkers to distinguish asthma examined "under the ROC-curves".Results:High TNF-α levels observed among the controls(p-0.006), opposing the higher MPO levels among the patients(p-0.00). There were nonsignificant variations of two biomarkers between the treatment groups and nonsignificant correlations of MPO with FEV1 and PEF. There was a significant correlation of MPO with the TNF-α levels of all participants. The TNF-α showed lower sensitivity, specificity, and accuracy to diagnose asthma. There were no MPO differences according to asthma levels. The TNF-α was higher among the severe asthmatics significantly.DiscussionTNF-α may be a contributory particle for neutrophilic inflammation of severe asthma. MPO levels were significantly higher among asthmatics, whereas TNF-α levels were lower. TNF-α levels were higher among those with severe compared to mild/moderate asthma. The MPO level has a significant predictive capacity compared to TNF-α for distinguishing asthma from healthy subjects.Key Words: Asthma, Inflammation, MPO, Neutrophils, TNF-α     

Rhus coriaria var. zebaria (Anacaridaceae), a new variety from Iraq

Rhus coriaria var. zebaria from Duhok/Aqrae, Kurdistan, Iraq is described as a variety new to science. Field expeditions started in spring 2011, continued during 2012 and 2013 with the aim of collecting sumac specimens from different physiographic regions of Kurdistan, Iraq. A total of ten plants for each variety were sampled. An equal number of tagged herbarium specimens were prepared. The holotype was designated and deposited together with other herbarium specimens in the herbarium of the Faculty of Agriculture and Forestry, Univ. of Duhok. Morphological characters of the leaf, inflorescence, flowers, infructescence, and fruit were studied. The pH value of the pericarp in addition to the hilum length was measured. Results were compared with Rhus coriaria L. var. coriaria. The most distinctive feature of the new variety is the dull whitish brown color of the drupe pericarp of var. zebaria as compared to the reddish brown color of the drupe pericarp of var. coriaria. The whitish color of the drupes of var. zebaria, in combination with their larger size, represents diagnostic characters for distinguishing this variety. In contrast, most leaf characters are of little or no taxonomic value in this species. Only shape, apex and marginal teeths of the leaflets show minor differences for separating the two varieties. Female and male inflorescences and flowers characters show no significant differences. Both hilum length and pH value characters may be useful for taxonomic application if mean values are taken in consideration.     

Sex-specific signaling through Toll-Like Receptors 2 and 4 contributes to survival outcome of Coxsackievirus B3 infection in C57Bl/6 mice

Background

Coxsackievirus B3 (CVB3) induces myocarditis, an inflammatory heart disease, which affects men more than women. Toll-like receptor (TLR) signaling has been shown to determine the severity of CVB3-induced myocarditis. No direct role for signaling through TLR2 had been shown in myocarditis although published studies show that cardiac myosin is an endogenous TLR2 ligand and stimulates pro-inflammatory cytokine expression by dendritic cells in vitro. The goal of this study is to determine which TLRs show differential expression in CVB3 infected mice corresponding to male susceptibility and female resistance in this disease.

Methods

Male and female C57Bl/6 mice were infected with 10² PFU CVB3 and killed on day 3 or 6 post infection. Hearts were evaluated for virus titer, myocardial inflammation, and TLR mRNA expression by PCR array and microarray analysis. Splenic lymphocytes only were evaluated by flow cytometry for the number of TLR+/CD3+, TLR+/CD4+, TLR+F4/80+ and TLR+/CD11c+ subpopulations and the mean fluorescence intensity to assess upregulation of TLR expression on these cells. Mice were additionally treated with PAM3CSK4 (TLR2 agonist) or ultrapure LPS (TLR4 agonist) on the same day as CVB3 infection or 3 days post infection to confirm their role in myocarditis susceptibility.

Results

Despite equivalent viral titers, male C57Bl/6 mice develop more severe myocarditis than females by day 6 after infection. Microarray analysis shows a differential expression of TLR2 at day 3 with female mice having higher levels of TLR2 gene expression compared to males. Disease severity correlates to greater TLR4 protein expression on splenic lymphocytes in male mice 3 days after infection while resistance in females correlates to preferential TLR2 expression, especially in spleen lymphocytes. Treating male mice with PAM reduced mortality from 55% in control CVB3 infected animals to 10%. Treating female mice with LPS increased mortality from 0% in control infected animals to 60%.

Conclusion

CVB3 infection causes an up-regulation of TLR2 in female and of TLR4 in male mice and this differential expression between the sexes contributes to disease resistance of females and susceptibility of males. While previous reports demonstrated a pathogenic role for TLR4 this is the first report that TLR2 is preferentially up-regulated in CVB3 infected female mice or that signaling through this TLR directly causes myocarditis resistance.

     

Mutations in c4orf26, encoding a Peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.     

Serum Myeloperoxidase as a Biomarker of Asthma Severity Among Adults: A Case Control Study

Background:The contribution of neutrophils is still indistinct in the inflammatory response of bronchial asthma (BAs). Myeloperoxidase (MPO) is an enzyme released from the primary azurophilic granules of the neutrophils. The study aimed to evaluate the levels of serum MPO as a biomarker for the assessment of the level of asthma control. Methods:The study participants included 94 asthmatic patients and 86 healthy controls. The identification of asthma severity had assessed using the ''''Global Initiative for Asthma guidelines''''. Asthmatic adults had divided into three groups: Good (n= 22), partial (n= 30), and poor control (n= 44). Also, patients have been divided again into two groups (treated and untreated) for BAs.Results:The predicted FEV1% and the peak expiratory flow (PEF/L) of all participants had verified by spirometry. The mean patients’ age was 31.9±15.1 year, with a predominance of females. The mean asthma duration was 10.5±8.6 years. Mean spirometric parameters (FEV1 and PEF) were significantly lower among the patients (0.00). Significant higher MPO levels had observed among BAs patients (p-0.00). The MPO levels have not differed significantly with asthma levels and had significant differences with the history of treatment. There was a nonsignificant negative correlation between the mean MPO levels and the spirometry variables among the patients. ROC curves revealed a sensitivity, specificity, accuracy for MPO (80.9%, 72.1%, and 84.3%), respectively to predict asthmatic severity.Conclusion:There were significantly higher MPO levels compared to healthy controls. Levels of serum MPO had a non-significant positive correlation with levels of asthma control, but a negative non-significant correlation with spirometric results. Key Words: Asthma, And Neutrophils, FEV1, MPO, PEF     

Bacillus subtilis Bactofilins Are Essential for Flagellar Hook- and Filament Assembly and Dynamically Localize into Structures of Less than 100 nm Diameter underneath the Cell Membrane

Bactofilins are a widely conserved protein family implicated in cell shape maintenance and in bacterial motility. We show that the bactofilins BacE and BacF from Bacillus subtilis are essential for motility. The proteins are required for the establishment of flagellar hook- and filament structures, but apparently not for the formation of basal bodies. Functional YFP fusions to BacE and to BacF localize as discrete assemblies at the B. subtilis cell membrane, and have a diameter of 60 to 70 nm. BacF assemblies are relatively static, and partially colocalize with flagellar basal bodies, while BacE assemblies are fewer per cell than those of BacF and are highly mobile. Tracking of BacE foci showed that the assemblies arrest at a single point for a few hundred milliseconds, showing that a putative interaction with flagellar structures would be transient and fast. When overexpressed or expressed in a heterologous cell system, bactofilins can form filamentous structures, and also form multimers as purified proteins. Our data reveal a propensity for bactofilins to form filaments, however, in B. subtilis cells, bactofilins assemble into defined size assemblies that show a dynamic localization pattern and play a role in flagellar assembly.     

Detecting Selection in Population Trees: The Lewontin and Krakauer Test Extended

Detecting genetic signatures of selection is of great interest for many research issues. Common approaches to separate selective from neutral processes focus on the variance of F_ST across loci, as does the original Lewontin and Krakauer (LK) test. Modern developments aim to minimize the false positive rate and to increase the power, by accounting for complex demographic structures. Another stimulating goal is to develop straightforward parametric and computationally tractable tests to deal with massive SNP data sets. Here, we propose an extension of the original LK statistic (T_LK), named T_F–LK, that uses a phylogenetic estimation of the population''s kinship () matrix, thus accounting for historical branching and heterogeneity of genetic drift. Using forward simulations of single-nucleotide polymorphisms (SNPs) data under neutrality and selection, we confirm the relative robustness of the LK statistic (T_LK) to complex demographic history but we show that T_F–LK is more powerful in most cases. This new statistic outperforms also a multinomial-Dirichlet-based model [estimation with Markov chain Monte Carlo (MCMC)], when historical branching occurs. Overall, T_F–LK detects 15–35% more selected SNPs than T_LK for low type I errors (P < 0.001). Also, simulations show that T_LK and T_F–LK follow a chi-square distribution provided the ancestral allele frequencies are not too extreme, suggesting the possible use of the chi-square distribution for evaluating significance. The empirical distribution of T_F–LK can be derived using simulations conditioned on the estimated matrix. We apply this new test to pig breeds SNP data and pinpoint outliers using T_F–LK, otherwise undetected using the less powerful T_LK statistic. This new test represents one solution for compromise between advanced SNP genetic data acquisition and outlier analyses.THE development of methods aiming at detecting molecular signatures of selection is one of the major concerns of modern population genetics. Broadly, such methods can be classified into four groups: methods focusing on (i) the interspecific comparison of gene substitution patterns, (ii) the frequency spectrum and models of selective sweeps, (iii) linkage disequilibrium (LD) and haplotype structure, and (iv) patterns of genetic differentiation among populations (for a review see Nielsen 2005). Tests based on the comparison of polymorphism and divergence at the species level inform on mostly ancient selective processes. Population-based approaches, however, are designed to pinpoint modern processes of local adaptation and speciation occurring among populations within a species. Such approaches also become crucial in the fields of agronomical and biomedical sciences, for instance, to pinpoint possible interesting (QTL) regions and disease susceptibility genes. Especially, human, livestock, and cultivated plants genetics may benefit from such methods while whole-genome single-nucleotide polymorphisms (SNPs) genotyping technologies are becoming routinely available (e.g., Barreiro et al. 2008; Flori et al. 2009).In the population genomic era (Luikart et al. 2003), identifying genes under selection or neutral markers influenced by nearby selected genes is a task in itself for quantifying the role of selection in the evolutionary history of species. Conversely, the accurate inference of demographic parameters such as effective population sizes, migration rates, and divergence times between populations relies on the use of neutral marker data sets. One approach of detecting loci under selection (outliers) with population genetic data is based on the genetic differentiation between loci influenced only by neutral processes (genetic drift, mutation, migration) and loci influenced by selection.Lewontin and Krakauer''s (LK) test for the heterogeneity of the inbreeding coefficient (F) across loci was the first to be developed with regard to this concept (Lewontin and Krakauer 1973). The LK test was immediately subject to criticisms (Nei and Maruyama 1975; Lewontin and Krakauer 1975; Robertson, 1975a,b; Tsakas and Krimbas 1976; Nei and Chakravarti 1977; Nei et al. 1977). Indeed, its assumptions are likely to be violated due to loci with high mutation rate, variation of F due to unequal effective population size (N_e) among demes, and correlation of allele frequencies among demes due to historical branching. The robustness of the LK test to the effects of demography was tested through coalescent simulations by Beaumont and Nichols (1996). They tested the influence of different models of population structure on the joint distribution of F_ST (i.e., the inbreeding coefficient F) and heterozygosity (H_e). The F_ST distribution under an infinite-island model is inflated for low H_e values under both the infinite-allele model (IAM) and the stepwise mutation model (SMM) (Beaumont and Nichols 1996). This tendency becomes, however, more marked when strong differences in effective size N_e and gene flow among demes occur, that is, when allele frequencies are correlated among local demes. This suggests an excess of false significant loci when one assumes an infinite-island model as a null hypothesis, while correlations of gene frequencies substantially occur. However, the F_ST distribution shows robustness properties for high H_e values (typical from microsatellite markers). Therefore, Beaumont and Nichols (1996) suggested the possibility of detecting outliers by using the distribution of neutral F_ST conditionally on H_e under the infinite-island model of symmetric migration, with mutation.The problem of accounting for correlations of allele frequencies among subpopulations was discussed by Robertson (1975a), who showed how these correlations inflated the variance of the LK test. Different approaches were taken to cope with the problem. It was, for instance, proposed to restrict the analysis to pairwise comparisons (Tsakas and Krimbas 1976; Vitalis et al. 2001). However, as pointed out by Beaumont (2005), reducing the number of populations to be compared to many pairwise comparisons raises the problem of nonindependence in multiple testing and may reduce the power to detect outliers. Another way was to assume that subpopulation allele frequencies are correlated through a common migrant gene pool, that is, the ancestral population in a star-like population divergence. In this case, subpopulations evolve with an unequal number of migrants coming from the migrant pool and/or to different amounts of genetic drift. This demographic scenario can be explicitly modeled using the multinomial-Dirichlet likelihood approach (Balding 2003). This multinomial-Dirichlet likelihood (or Beta-binomial for biallelic markers such as SNPs) was implemented by Beaumont and Balding (2004) and subsequently by Foll and Gaggiotti (2008), Gautier et al. (2009), Guo et al. (2009), and Riebler et al. (2010), in a Bayesian hierarchical model in which the F_ST is decomposed into two components: a locus-specific (α) effect and a population-specific (β) effect. This Bayesian statistical model together with prior assumptions on α and β was implemented in a Markov chain Monte Carlo (MCMC) algorithm. A substantial improvement made by Foll and Gaggiotti (2008) was to use a reverse-jumping (RJ)-MCMC to simultaneously estimate the posterior distribution of a model with selection (with α and β) and of a model without selection (with β only). More recently, Excoffier et al. (2009) addressed the issue of accounting for “heterogeneous affinities between sampled populations”—in other words, accounting for migrant genes that do not necessarily originate from the same pool—by using a hierarchically structured population model. They showed by simulations that the false positive rate is lower under a hierarchically structured population model than under a simple island model, for the IAM and the SMM applicable to microsatellite markers and for a SNP mutation model. Excoffier et al.(2009) thus proposed to extend the Beaumont and Nichols (1996) method to a hierarchically structured population model.Nowadays, a computational challenge is to analyze data sets with increasing numbers of markers and populations, under complex demographic histories, in a reasonable amount of time. This is especially the case in agronomical and biomedical sciences with the increasingly used biallelic SNP markers. A question arises as to whether F_ST-based methods would be sufficiently powerful to detect outliers with SNP markers. Indeed, for low H_e values, the inflation of the F_ST distribution under the infinite-island model accentuates dramatically when assuming a mutation model typical for SNPs (simulations of Eveno et al. 2008). Excoffier et al. (2009) corroborated these results and also indicated that the F_ST distribution is generally broader under a model of hierarchically structured populations when using SNP markers. In addition, as the authors pinpoint, although the hierarchical island model is more conservative than the island model, an excess of false positives can be obtained “if the underlying genetic structure is more complex …, for instance in case of complex demographic histories, involving population splits, range expansion, bottleneck or admixture events” (Excoffier et al. 2009, p. 12). The Bayesian hierarchical models developed by Beaumont and Balding (2004) and Foll and Gaggiotti (2008) effectively account for strong effective size and migration rate variation among subpopulations, but they still impose a star-like demographic model in which the current populations share a common migrant pool and are not supposed to have undergone historical branching. More practically, MCMC-based methods might suffer from a computational time requirement when analyzing large marker data sets such as SNP chips data sets. Therefore, the development of simple parametric tests potentially dealing with a summary of the population tree, including historical branching as well as population size variation, remains an alternative solution to achieve a good compromise between advanced genetic data acquisition and outlier analyses.In this article, we describe an extension of the original parametric LK test for biallelic markers that deals with complex population trees through a statistic that takes into account the kinship (or coancestry) matrix between populations, under pure drift with no migration. The statistics of the classical test (T_LK) and its extension (T_F–LK) are expected to follow a chi-square distribution with (n – 1) d.f., where n is the number of populations studied. Through forward simulations of neutral SNPs data under increasingly complex demographic histories, we obtained the empirical distribution of both statistics and showed that they follow a chi-square distribution provided the ancestral allele frequencies are not too extreme. These results also emphasize the robustness of these statistics to variation in demographic histories. Forward simulations of the same demographic models but including selection in one population allowed us to evaluate the power of both statistics to detect selection. We show that the extension of the LK test is more powerful at detecting outliers than the classical LK test for complex demographic histories. A comparison with one of the MCMC methods for multinomial-Dirichlet models (Foll and Gaggiotti 2008) also revealed substantial additional power. We apply this new statistical test to a data set of SNP markers in known genes of the pig genome, taking advantage of the availability of microsatellite markers for the estimation of the kinship matrix. This new parametric test can help to screen large marker data sets and large numbers of populations for outliers in a reasonable amount of time, although we recommend to simulate the empirical distribution of the T_F–LK statistics conditionally on the estimated kinship matrix.     

Cholera toxin B subunits assemble into pentamers--proposition of a fly-casting mechanism

The cholera toxin B pentamer (CtxB(5)), which belongs to the AB(5) toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB(5) and the related heat labile enterotoxin LTB(5) have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.