Search for genetic markers of climatic adaptation in populations of North Eurasia

Genetic diversity of native populations of North Eurasia is investigated using a panel of genetic markers of candidate genes for cold climate adaptation. A high level of within- and between-population variability is detected. Comparative analysis of data on North Eurasian populations combined with data on worldwide populations from the 1000 Genomes and HDGP projects reveals correlations of genetic diversity in candidate genes for cold climate adaptation with key climate parameters, as well as the increase of genetic diversity in markers of this group of genes with the increase of latitude, that is, as modern humans migrated out of Africa. Using the method of searching for extreme empirical values of the coefficient of genetic diversity, signals of directional selection for markers of six genes adaptive to cold (MYOF, LONP2, IFNL4, MKL1, SLC2A12, and CPT1A) are found. The data are discussed in framework of the hypothesis of decanalization of genome–phenome relationships under the pressure of natural selection during human settlement throughout the world.     

Development of a URA5 integrative cassette for gene disruption in the Candida guilliermondii ATCC 6260 strain

We designed an efficient transformation system for Candida guilliermondii based on a ura5 ATCC 6260 derived recipient strain and a URA5 recyclable selection marker. This “URA5 blaster” disruption system represents a powerful tool to study the function of a large pallet of genes in this yeast of clinical and biotechnological interest.     

The pathway and spatial scale for MscS inactivation

The mechanosensitive channel of small conductance (MscS) is a bacterial tension-driven osmolyte release valve with homologues in many walled eukaryotic organisms. When stimulated by steps of tension in excised patches, Escherichia coli MscS exhibits transient opening followed by reversible adaptation and then complete inactivation. Here, we study properties of the inactivation transition, which renders MscS nonconductive and tension insensitive. Using special pressure protocols we demonstrate that adaptation and inactivation are sequential processes with opposite tension dependencies. In contrast to many eukaryotic channels, which inactivate from the open state, MscS inactivates primarily from the closed state because full openings by preconditioning pulses do not influence the degree of inactivation, and saturating tensions keeping channels open prevent inactivation. The easily opened A98S mutant lacks inactivation completely, whereas the L111S mutant with a right-shifted activation curve inactivates silently before reaching the threshold for opening. This suggests that opening and inactivation are two independent tension-driven pathways, both starting from the closed state. Analysis of tension dependencies for inactivation and recovery rates estimated the in-plane expansion (ΔA) associated with inactivation as 8.5 nm(2), which is about half of the area change for opening. Given that the interhelical contact between the outer TM1-TM2 pairs and the core TM3s is the force-transmitting path from the periphery to the gate, the determined ΔA now can be used as a constraining parameter for the models of the inactivated state in which the lipid-facing TM1-TM2 pairs are displaced and uncoupled from the gate.     

Calcium-Activated Potassium Current Modulates Ventricular Repolarization in Chronic Heart Failure

The role of I_KCa in cardiac repolarization remains controversial and varies across species. The relevance of the current as a therapeutic target is therefore undefined. We examined the cellular electrophysiologic effects of I_KCa blockade in controls, chronic heart failure (HF) and HF with sustained atrial fibrillation. We used perforated patch action potential recordings to maintain intrinsic calcium cycling. The I_KCa blocker (apamin 100 nM) was used to examine the role of the current in atrial and ventricular myocytes. A canine tachypacing induced model of HF (1 and 4 months, n = 5 per group) was used, and compared to a group of 4 month HF with 6 weeks of superimposed atrial fibrillation (n = 7). A group of age-matched canine controls were used (n = 8). Human atrial and ventricular myocytes were isolated from explanted end-stage failing hearts which were obtained from transplant recipients, and studied in parallel. Atrial myocyte action potentials were unchanged by I_KCa blockade in all of the groups studied. I_KCa blockade did not affect ventricular myocyte repolarization in controls. HF caused prolongation of ventricular myocyte action potential repolarization. I_KCa blockade caused further prolongation of ventricular repolarization in HF and also caused repolarization instability and early afterdepolarizations. SK2 and SK3 expression in the atria and SK3 in the ventricle were increased in canine heart failure. We conclude that during HF, I_KCa blockade in ventricular myocytes results in cellular arrhythmias. Furthermore, our data suggest an important role for I_KCa in the maintenance of ventricular repolarization stability during chronic heart failure. Our findings suggest that novel antiarrhythmic therapies should have safety and efficacy evaluated in both atria and ventricles.     

The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.     

Variations of Dynamic Contrast-Enhanced Magnetic Resonance Imaging in Evaluation of Breast Cancer Therapy Response: A Multicenter Data Analysis Challenge

Pharmacokinetic analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) time-course data allows estimation of quantitative parameters such as K^trans (rate constant for plasma/interstitium contrast agent transfer), v_e (extravascular extracellular volume fraction), and v_p (plasma volume fraction). A plethora of factors in DCE-MRI data acquisition and analysis can affect accuracy and precision of these parameters and, consequently, the utility of quantitative DCE-MRI for assessing therapy response. In this multicenter data analysis challenge, DCE-MRI data acquired at one center from 10 patients with breast cancer before and after the first cycle of neoadjuvant chemotherapy were shared and processed with 12 software tools based on the Tofts model (TM), extended TM, and Shutter-Speed model. Inputs of tumor region of interest definition, pre-contrast T₁, and arterial input function were controlled to focus on the variations in parameter value and response prediction capability caused by differences in models and associated algorithms. Considerable parameter variations were observed with the within-subject coefficient of variation (wCV) values for K^trans and v_p being as high as 0.59 and 0.82, respectively. Parameter agreement improved when only algorithms based on the same model were compared, e.g., the K^trans intraclass correlation coefficient increased to as high as 0.84. Agreement in parameter percentage change was much better than that in absolute parameter value, e.g., the pairwise concordance correlation coefficient improved from 0.047 (for K^trans) to 0.92 (for K^trans percentage change) in comparing two TM algorithms. Nearly all algorithms provided good to excellent (univariate logistic regression c-statistic value ranging from 0.8 to 1.0) early prediction of therapy response using the metrics of mean tumor K^trans and k_ep (= K^trans/v_e, intravasation rate constant) after the first therapy cycle and the corresponding percentage changes. The results suggest that the interalgorithm parameter variations are largely systematic, which are not likely to significantly affect the utility of DCE-MRI for assessment of therapy response.