Phylogeny and host-specificity of European seed beetles (Coleoptera, Bruchidae), new insights from molecular and ecological data

We used partial sequences of three mitochondrial genes (12S rRNA, cytochrome b, and cytochrome c oxidase subunit I) to reconstruct the phylogeny of European seed beetles (Bruchidae) belonging to the genera Bruchus Linnaeus and Bruchidius Schilsky. Adult beetles examined in this study were obtained from larvae bred from seeds directly collected in the field. Parsimony, maximum likelihood, and Bayesian inference were used to infer phylogenetic relationships among species. Both genera, Bruchidius and Bruchus, formed monophyletic groups in all analyses. Our results were partially in discrepancy with existing taxonomic groups (Borowiec, 1987). Critical analysis of relationships among taxa, and exhaustive review of host-plants data highlight the very high level of specialization of these seed beetles. Phylogenetically related insects were associated with host-plants belonging to the same botanical tribes.     

An algorithm for detecting directional and non-directional positive selection,neutrality and negative selection in protein coding DNA sequences

Positive selection or adaptive evolution is thought to be responsible, at least some of the time, for the rapid accumulation of advantageous changes in protein-coding genes. The origin of new enzymatic functions, erection of barriers to heterospecific fertilization, and evasion of host response by pathogens, among other things, are thought to be instances of adaptive evolution. Detecting positive selection in protein-coding genes is fraught with difficulties. Saturation for sequence change, codon usage bias, ephemeral selection events and differential selective pressures on amino acids all contribute to the problem. A number of solutions have been proposed with varying degrees of success, however they suffer from limitations of not being accurate enough or being prohibitively computationally intensive. We have developed a character-based method of identifying lineages that undergo positive selection. In our method we assess the possibility that for each internal branch of a phylogenetic tree an event occurred that subsequently gave rise to a greater number of replacement substitutions than might be expected. We classify these replacement substitutions into two categories – whether they subsequently became invariable or changed again in at least one descendent lineage. The former situation indicates that the new character state is under strong selection to preserve its new identity (directional selection), while the latter situation indicates that there is a persistent pressure to change identity (non-directional selection). The method is fast and accurate, easy to implement, sensitive to short-lived selection events and robust with respect to sampling density and proportion of sites under the influence of positive selection.     

A multiple regression method for analysing stage-frequency data

A linear regression method that allows survival rates to vary from stage to stage is described for the analysis of stage-frequency data. It has advantages over previously suggested methods since the calculations are not iterative, and it is not necessary to have independent estimates of stage durations, numbers entering stages, or the rate of entry to stage 1. Simulation is proposed to determine standard errors for estimates of population parameters, and to assess the goodness of fit of models.     

The influence of anisotropy on brain injury prediction

Traumatic Brain Injury (TBI) occurs when a mechanical insult produces damage to the brain and disrupts its normal function. Numerical head models are often used as tools to analyze TBIs and to measure injury based on mechanical parameters. However, the reliability of such models depends on the incorporation of an appropriate level of structural detail and accurate representation of the material behavior. Since recent studies have shown that several brain regions are characterized by a marked anisotropy, constitutive equations should account for the orientation-dependence within the brain. Nevertheless, in most of the current models brain tissue is considered as completely isotropic. To study the influence of the anisotropy on the mechanical response of the brain, a head model that incorporates the orientation of neural fibers is used and compared with a fully isotropic model. A simulation of a concussive impact based on a sport accident illustrates that significantly lowered strains in the axonal direction as well as increased maximum principal strains are detected for anisotropic regions of the brain. Thus, the orientation-dependence strongly affects the response of the brain tissue. When anisotropy of the whole brain is taken into account, deformation spreads out and white matter is particularly affected. The introduction of local axonal orientations and fiber distribution into the material model is crucial to reliably address the strains occurring during an impact and should be considered in numerical head models for potentially more accurate predictions of brain injury.     

Improving the accuracy of chloride measurements through participation in regular external quality assessment programme

BackgroundA chloride test is an integral part of a basic metabolic panel that is essential for the assessment of a patient’s acid-base and electrolyte status. While many methods are available commercially for the routine measurement of chloride, there is a need to address the accuracy and variability among the measurement results, especially with the prevalence of patients seeking treatment across different healthcare providers for alternative opinions.MethodA method based on sector field inductively coupled plasma isotope dilution mass spectrometry (SF-ICP-IDMS) was developed for the measurement of chloride in human serum. The SF-ICP-IDMS method was then used to assign the target values in the Health Sciences Authority (HSA) External Quality Assessment (EQA) Programme to evaluate the results of chloride test from participating clinical laboratories.ResultsThe accuracy of the measurements was evaluated by comparing the results with the certified values of Electrolytes in Frozen Human Serum Certified Reference Materials (SRM 956c and SRM 956d) from the National Institute of Standards and Technology (NIST) at different chloride concentration levels. Over a five-year period from 2014–2018, the number of clinical laboratories which participated in the EQA Programme increased from 23 to 33. Comparison of robust means from the laboratories’ results with our assigned target values revealed a reduction in relative deviation over time. The relationship between the deviation of each brand of clinical analysers and the chloride levels was established, where a larger deviation was uncovered at low chloride concentration. The SF-ICP-IDMS method was further demonstrated to be comparable with methods used by other metrology institutes in an international comparison organised by HSA under the auspice of the Consultative Committee for Amount of Substance – Metrology in Chemistry and Biology (CCQM).ConclusionThe use of metrologically traceable assigned target values enabled the study of method biasness from a small pool of dataset in each of the four brands of clinical analysers in HSA EQA Programme. This work underscores the need to improve the accuracy of chloride measurements by regular participation in an accuracy-based EQA Programme.     

Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.     

iTRAQ as a method for optimization: Enhancing peptide recovery after gel fractionation

At the dawn of a new era in label‐free quantitation on high‐resolution MS instruments, classical methods such as iTRAQ continue to provide very useful insights in comparative proteomics. The potential to multiplex samples makes this reporter‐based labeling technique highly suited for method optimization as demonstrated here by a set of standard series. Instead of studying ratios of annotated proteins, we propose an alternative method, based on the analysis of the average reporter ratios of all the spectra from a sample or a large distinct subset herein. This strategy circumvents the bias, associated with the annotation and iTRAQ quantitation, leading to increased adequacy in measuring yield differences between workflows. As gel electrophoresis prior to MS analysis is highly beneficial, for example, as a fractionation step, the approach was applied to evaluate the influence of several parameters of the established in‐gel digestion protocol. We quantified the negative effect of SYPRO Ruby staining and the positive effect of gel fixation prior to digestion on peptide yield. Finally, we emphasize the benefits of adding CaCl₂ and ACN to a tryptic in‐gel digest, resulting in an up to tenfold enhanced peptide recovery and fewer trypsin missed cleavages.     

Development and evaluation of an off‐the‐slide genotyping technique for identifying Giardia cysts and Cryptosporidium oocysts directly from US EPA Method 1623 slides

Aims

This study developed and systematically evaluated performance and limit of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts.

Methods and Results

Slide standards containing flow‐sorted (oo)cysts were used to evaluate the off‐the‐slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide.

Conclusions

This study successfully developed and evaluated recovery rates and limits of detection of an off‐the‐slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide.

Significance and Impact of the Study

This off‐the‐slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions.     

Collaborative study report: Evaluation of the ATCC experimental mycoplasma reference strains panel prepared for comparison of NAT-based and conventional mycoplasma detection methods

The main goal of this collaborative study was to evaluate the experimental panel of cryopreserved mycoplasma reference strains recently prepared by the American Type Culture Collection (ATCC^®) in order to assess the viability and dispersion of cells in the mycoplasma stocks by measuring the ratio between the number of genomic copies (GC) and the number of colony forming units (CFU) in the reference preparations. The employment of microbial reference cultures with low GC/CFU ratios is critical for unbiased and reliable comparison of mycoplasma testing methods based on different methodological approaches, i.e., Nucleic Acid Testing (NAT) and compendial culture-based techniques. The experimental panel included ten different mycoplasma species known to represent potential human and animal pathogens as well as common contaminants of mammalian and avian cell substrates used in research, development, and manufacture of biological products. Fifteen laboratories with expertise in field of mycoplasma titration and quantification of mycoplasmal genomic DNA participated in the study conducted from February to October of 2012. The results of this study demonstrated the feasibility of preparing highly viable and dispersed (possessing low GC/CFU ratios) frozen stocks of mycoplasma reference materials, required for reliable comparison of NAT-based and conventional mycoplasma detection methods.     

Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed Tryptophan Fluorescence

Magnesium is the most abundant divalent cation present in the cell, and an abnormal Mg²⁺ homeostasis is associated with several diseases in humans. However, among ion channels, the mechanisms of intracellular regulation and transport of Mg²⁺ are poorly understood. MgtE is a homodimeric Mg²⁺-selective channel and is negatively regulated by high intracellular Mg²⁺ concentration where the cytoplasmic domain of MgtE acts as a Mg²⁺ sensor. Most of the previous biophysical studies on MgtE have been carried out in detergent micelles and the information regarding gating-related structural dynamics of MgtE in physiologically-relevant membrane environment is scarce. In this work, we monitored the changes in gating-related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in micelles and membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain site-specific information on the gating-related structural dynamics of MgtE in membrane-mimetic systems. Our results indicate that Mg²⁺-induced gating might involve the possibility of a ‘conformational wave’ from the cytosolic N-domain to transmembrane domain of MgtE. Although MgtE is responsive to Mg²⁺-induced gating in both micelles and membranes, the organization and dynamics of MgtE is substantially altered in physiologically important phospholipid membranes compared to micelles. This is accompanied by significant changes in hydration dynamics and conformational heterogeneity. Overall, our results highlight the importance of lipid-protein interactions and are relevant for understanding gating mechanism of magnesium channels in general, and MgtE in particular.