Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.     

Sex ratio adjustments in common terns: influence of mate condition and maternal experience

Adaptive sex allocation has frequently been studied in sexually size dimorphic species, but far less is known about patterns of sex allocation in species without pronounced sexual size dimorphism. Parental optimal investment can be predicted under circumstances in which sons and daughters differ in costs and/or fitness returns. In common terns Sterna hirundo, previous studies suggest that sons are the more costly sex to produce and rear. We investigated whether hatching and fledging sex ratio and sex‐specific chick mortality correlated with the ecological environment (laying date, clutch size, hatching order and year quality) and parental traits (condition, arrival date, experience and breeding success), over seven consecutive years. Population‐wide sex ratios and sex‐specific mortality did not differ from parity, but clutch size, mass of the father, maternal breeding experience and to some extent year quality correlated with hatching sex ratio. The proportion of sons tended to increase in productive years and when the father was heavier, suggesting the possibility that females invest more in sons when the environmental and the partner conditions are good. The proportion of daughters increased with clutch size and maternal breeding experience, suggesting a decline in breeding performance or a resources balance solved by producing more of the cheaper sex. No clear patterns of sex‐specific mortality were found, neither global nor related to parental traits. Our results suggest lines for future studies on adaptive sex allocation in sexually nearly monomorphic species, where adjustment of sex ratio related to parental factors and differential allocation between the offspring may also occur.     

Systematic Identification and Characterization of Novel Human Skin-Associated Genes Encoding Membrane and Secreted Proteins

Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics.     

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

BackgroundIn vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE).AimTo investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers.MethodsPast and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed.ResultsIVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001).ConclusionsOur results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.     

Affective Computing and the Impact of Gender and Age

Affective computing aims at the detection of users’ mental states, in particular, emotions and dispositions during human-computer interactions. Detection can be achieved by measuring multimodal signals, namely, speech, facial expressions and/or psychobiology. Over the past years, one major approach was to identify the best features for each signal using different classification methods. Although this is of high priority, other subject-specific variables should not be neglected. In our study, we analyzed the effect of gender, age, personality and gender roles on the extracted psychobiological features (derived from skin conductance level, facial electromyography and heart rate variability) as well as the influence on the classification results. In an experimental human-computer interaction, five different affective states with picture material from the International Affective Picture System and ULM pictures were induced. A total of 127 subjects participated in the study. Among all potentially influencing variables (gender has been reported to be influential), age was the only variable that correlated significantly with psychobiological responses. In summary, the conducted classification processes resulted in 20% classification accuracy differences according to age and gender, especially when comparing the neutral condition with four other affective states. We suggest taking age and gender specifically into account for future studies in affective computing, as these may lead to an improvement of emotion recognition accuracy.     

Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

Introduction

Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens.

Materials and Methods

Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens.

Results

Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin.

Discussion

Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin.     

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

Despite recent advances in high‐throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club‐legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired‐end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild‐caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high‐throughput sequencing and graph‐based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.     

Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173

Background and Aims

The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn²⁺ and Cu²⁺ concentration during hemodialysis.

Methods and Results

Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn²⁺ or Cu²⁺ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn²⁺ and Cu²⁺ concentrations after dialysis.

Conclusions

Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.