A Technology for Developing Synbodies with Antibacterial Activity

The rise in antibiotic resistance has led to an increased research focus on discovery of new antibacterial candidates. While broad-spectrum antibiotics are widely pursued, there is evidence that resistance arises in part from the wide spread use of these antibiotics. Our group has developed a system to produce protein affinity agents, called synbodies, which have high affinity and specificity for their target. In this report, we describe the adaptation of this system to produce new antibacterial candidates towards a target bacterium. The system functions by screening target bacteria against an array of 10,000 random sequence peptides and, using a combination of membrane labeling and intracellular dyes, we identified peptides with target specific binding or killing functions. Binding and lytic peptides were identified in this manner and in vitro tests confirmed the activity of the lead peptides. A peptide with antibacterial activity was linked to a peptide specifically binding Staphylococcus aureus to create a synbody with increased antibacterial activity. Subsequent tests showed that this peptide could block S. aureus induced killing of HEK293 cells in a co-culture experiment. These results demonstrate the feasibility of using the synbody system to discover new antibacterial candidate agents.     

Bacterial Communities of Three Saline Meromictic Lakes in Central Asia

Meromictic lakes located in landlocked steppes of central Asia (~2500 km inland) have unique geophysiochemical characteristics compared to other meromictic lakes. To characterize their bacteria and elucidate relationships between those bacteria and surrounding environments, water samples were collected from three saline meromictic lakes (Lakes Shira, Shunet and Oigon) in the border between Siberia and the West Mongolia, near the center of Asia. Based on in-depth tag pyrosequencing, bacterial communities were highly variable and dissimilar among lakes and between oxic and anoxic layers within individual lakes. Proteobacteria, Bacteroidetes, Cyanobacteria, Actinobacteria and Firmicutes were the most abundant phyla, whereas three genera of purple sulfur bacteria (a novel genus, Thiocapsa and Halochromatium) were predominant bacterial components in the anoxic layer of Lake Shira (~20.6% of relative abundance), Lake Shunet (~27.1%) and Lake Oigon (~9.25%), respectively. However, few known green sulfur bacteria were detected. Notably, 3.94% of all sequencing reads were classified into 19 candidate divisions, which was especially high (23.12%) in the anoxic layer of Lake Shunet. Furthermore, several hydro-parameters (temperature, pH, dissolved oxygen, H₂S and salinity) were associated (P< 0.05) with variations in dominant bacterial groups. In conclusion, based on highly variable bacterial composition in water layers or lakes, we inferred that the meromictic ecosystem was characterized by high diversity and heterogenous niches.     

Genetic Determinants of Thrombin Generation and Their Relation to Venous Thrombosis: Results from the GAIT-2 Project

Background

Venous thromboembolism (VTE) is a common disease where known genetic risk factors explain only a small portion of the genetic variance. Then, the analysis of intermediate phenotypes, such as thrombin generation assay, can be used to identify novel genetic risk factors that contribute to VTE.

Objectives

To investigate the genetic basis of distinct quantitative phenotypes of thrombin generation and its relationship to the risk of VTE.

Patients/Methods

Lag time, thrombin peak and endogenous thrombin potential (ETP) were measured in the families of the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project. This sample consisted of 935 individuals in 35 extended families selected through a proband with idiopathic thrombophilia. We performed also genome wide association studies (GWAS) with thrombin generation phenotypes.

Results

The results showed that 67% of the variation in the risk of VTE is attributable to genetic factors. The heritabilities of lag time, thrombin peak and ETP were 49%, 54% and 52%, respectively. More importantly, we demonstrated also the existence of positive genetic correlations between thrombin peak or ETP and the risk of VTE. Moreover, the major genetic determinant of thrombin generation was the F2 gene. However, other suggestive signals were observed.

Conclusions

The thrombin generation phenotypes are strongly genetically determined. The thrombin peak and ETP are significantly genetically correlated with the risk of VTE. In addition, F2 was identified as a major determinant of thrombin generation. We reported suggestive signals that might increase our knowledge to explain the variability of this important phenotype. Validation and functional studies are required to confirm GWAS results.     

Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species

Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S‐adenosylmethionine, which, after transferring its methyl group, is converted to S‐adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging.     

CORAL: Building up QSAR models for the chromosome aberration test

A high level of chromosomal aberrations in peripheral blood lymphocytes may be an early marker of cancer risk, but data on risk of specific cancers and types of chromosomal aberrations are limited. Consequently, the development of predictive models for chromosomal aberrations test is important task. Majority of models for chromosomal aberrations test are so-called knowledge-based rules system. The CORAL software (http://www.insilico.eu/coral, abbreviation of “CORrelation And Logic”) is an alternative for knowledge-based rules system. In contrast to knowledge-based rules system, the CORAL software gives possibility to estimate the influence upon the predictive potential of a model of different molecular alerts as well as different splits into the training set and validation set. This possibility is not available for the approaches based on the knowledge-based rules system. Quantitative Structure–Activity Relationships (QSAR) for chromosome aberration test are established for five random splits into the training, calibration, and validation sets. The QSAR approach is based on representation of the molecular structure by simplified molecular input-line entry system (SMILES) without data on physicochemical and/or biochemical parameters. In spite of this limitation, the statistical quality of these models is quite good.     

Spinal cord tissue affects sprouting from aortic fragments in ex vivo co‐culture

It is a well‐known fact, that there is a close interconnection between vascular and neural structures in both embryonic development and postnatal life. Different models have been employed to dissect the mechanisms of these interactions, ranging from in vitro systems (e.g., co‐culture of neural and endothelial cells) to in vivo imaging of central neural system recovery in laboratory animals after artificially induced trauma. Nevertheless, most of these models have serious limitations. Here, we describe an ex vivo model, representing an organotypic co‐culture of aortic fragments (AF) with longitudinal slices of mouse neonatal spinal cord (SC) or dorsal root ganglia (DRG). The samples were co‐cultured in a medium adapted for SC tissue and lacking any pro‐angiogenic or neurotrophic growth factors. It was found, that cultivation of AFs in the SC injury zone (transversal dissection of a SC slice) resulted in the initiation of active aortic sprouting. Remarkably, the endothelial cells exiting the AFs never invaded the SC tissue, concentrating in a nearby area (negative taxis). In contrast, the DRGs, while also promoting the sprouting, were a target of active endothelial CD31⁺ cell invasion (positive taxis). Thus, the tissues of both central and peripheral nervous systems have a prominent positive effect on aortic sprouting, while the vector of endothelial cell expansion is strictly nervous‐tissue‐type dependent. The ex vivo AF co‐culture with SC or DRG appeared to be a useful and promising model for a further endeavor into the mechanisms driving the complex interactions between neural and endothelial tissues.     

Real-time technique for conversion of skin temperature into skin blood flow: human skin as a low-pass filter for thermal waves

Monitoring of skin blood flow oscillations related with mechanical activity of vessels is a very useful modality during diagnosis of peripheral hemodynamic disorders. In this study, we developed a new model and technique for real-time conversion of skin temperature into skin blood flow oscillations, and vice versa. The technique is based on the analogy between the thermal properties of the human skin and electrical properties of the special low-pass filter. Analytical and approximated impulse response functions for the low- and high-pass filters are presented. The general algorithm for the reversible conversion of temperature into blood flow is described. The proposed technique was verified using simulated or experimental data of cold stress, deep inspiratory gasp, and post-occlusive reactive hyperaemia tests. The implementation of the described technique will enable to turn a temperature sensor into a blood flow sensor.     

Dynamic evaluation of blood flow microcirculation by combined use of the laser Doppler flowmetry and high‐speed videocapillaroscopy methods

The dynamic light scattering methods are widely used in biomedical diagnostics involving evaluation of blood flow. However, there exist some difficulties in quantitative interpretation of backscattered light signals from the viewpoint of diagnostic information. This study considers the application of the high‐speed videocapillaroscopy (VCS) method that provides the direct measurement of the red blood cells (RBCs) velocity into a capillary. The VCS signal presents true oscillation nature of backscattered light caused by moving RBCs. Thus, the VCS signal can be assigned as a reference one with respect to more complicated signals like in laser Doppler flowmetry (LDF). An essential correlation between blood flow velocity oscillations in a separate human capillary and the integral perfusion estimate obtained by the LDF method has been found. The observation of blood flow by the VCS method during upper arm occlusion has shown emergence of the reverse blood flow effect in capillaries that corresponds to the biological zero signal in the LDF. The reverse blood flow effect has to be taken into account in interpretation of LDF signals.      

Phenotypic plasticity closely linked to climate at origin and resulting in increased mortality under warming and frost stress in a common grass

Phenotypic plasticity is important for species responses to global change and species coexistence. Phenotypic plasticity differs among species and traits and changes across environments. Here, we investigated phenotypic plasticity of the widespread grass Arrhenatherum elatius in response to winter warming and frost stress by comparing phenotypic plasticity of 11 geographically and environmentally distinct populations of this species to phenotypic plasticity of populations of different species originating from a single environment. The variation in phenotypic plasticity was similar for populations of a single species from different locations compared to populations of functionally and taxonomically diverse species from one environment for the studied traits (leaf biomass production and root integrity after frost) across three indices of phenotypic plasticity (RDPI, PIN, slope of reaction norm). Phenotypic plasticity was not associated with neutral genetic diversity but closely linked to the climate of the populations’ origin. Populations originating from warmer and more variable climates showed higher phenotypic plasticity. This indicates that phenotypic plasticity can itself be considered as a trait subject to local adaptation to climate. Finally, our data emphasize that high phenotypic plasticity is not per se positive for adaptation to climate change, as differences in stress responses are resulting in high phenotypic plasticity as expressed by common plasticity indices, which is likely to be related to increased mortality under stress in more plastic populations.