Obesity Affects Myocardial Vasoreactivity and Coronary Flow Response to Insulin

Objective: Obesity is associated with increased risk for cardiovascular diseases and peripheral endothelial dysfunction. We examined whether myocardial vasoreactivity and coronary‐flow response to insulin stimulation are altered in obesity. Research Methods and Procedures: Myocardial blood flow was quantitated in 10 obese men (body mass index, 33.6 ± 1.9 kg/m²) and 10 healthy matched non‐obese men (body mass index, 24.2 ± 1.9 kg/m²), using positron emission tomography and oxygen‐15‐labeled water. The measurements were performed basally and during adenosine infusion (140 μg/kg per minute), with or without simultaneous physiological (1 mU/kg per minute) and supraphysiological (5 mU/kg per minute) hyperinsulinemia. Results: Basal myocardial blood flow was not significantly different between obese and non‐obese subjects. Adenosine‐stimulated flow was blunted in obese (3.2 ± 0.6 mL/g per minute) when compared with non‐obese subjects (4.0 ± 1.1 mL/g per minute, p < 0.05). Simultaneous physiological hyperinsulinemia increased adenosine‐stimulated myocardial flow significantly in both groups (to 4.03 ± 1.24 and 4.85 ± 1.04 mL/g per minute in obese and non‐obese men, respectively; p < 0.05 vs. adenosine). Supraphysiological hyperinsulinemia further enhanced the adenosine‐stimulated flow in non‐obese subjects (to 5.56 ± 0.98 mL/g per minute; p < 0.05) but not in obese subjects. Discussion: Young obese, healthy men have reduced myocardial vasoreactivity, which may represent an early precursor of future coronary artery disease. Additionally, insulin‐induced enhancement of myocardial blood flow is blunted in obesity. Thus, endothelial dysfunction seems to also characterize myocardial vasculature of obese subjects.     

Selection for novel,acid-tolerant <Emphasis Type="Italic">Desulfovibrio</Emphasis> spp. from a closed Transbaikal mine site in a temporal pH-gradient bioreactor

Almost all the known isolates of acidophilic or acid-tolerant sulphate-reducing bacteria (SRB) belong to the spore-forming genus Desulfosporosinus in the Firmicutes. The objective of this study was to isolate acidophilic/acid-tolerant members of the genus Desulfovibrio belonging to deltaproteobacterial SRB. The sample material originated from microbial mat biomass submerged in mine water and was enriched for sulphate reducers by cultivation in anaerobic medium with lactate as an electron donor. A stirred tank bioreactor with the same medium composition was inoculated with the sulphidogenic enrichment. The bioreactor was operated with a temporal pH gradient, changing daily, from an initial pH of 7.3 to a final pH of 3.7. Among the bacteria in the bioreactor culture, Desulfovibrio was the only SRB group retrieved from the bioreactor consortium as observed by 16S rRNA-targeted denaturing gradient gel electrophoresis. Moderately acidophilic/acid-tolerant isolates belonged to Desulfovibrio aerotolerans-Desulfovibrio carbinophilus-Desulfovibrio magneticus and Desulfovibrio idahonensis-Desulfovibrio mexicanus clades within the genus Desulfovibrio. A moderately acidophilic strain, Desulfovibrio sp. VK (pH optimum 5.7) and acid-tolerant Desulfovibrio sp. ED (pH optimum 6.6) dominated in the bioreactor consortium at different time points and were isolated in pure culture.     

High-temperature cultivation and 5′ mRNA optimization are key factors for the efficient overexpression of thermostable Deinococcus geothermalis purine nucleoside phosphorylase in Escherichia coli

Overexpression of genes from thermophiles in Escherichia coli is an attractive approach towards the large-scale production of thermostable biocatalysts. However, various factors can challenge efficient heterologous protein expression – one example is the formation of stable 5′ mRNA secondary structures that can impede an efficient translation initiation.In this work, we describe the expression optimization of purine nucleoside phosphorylase from the thermophilic microbe Deinococcus geothermalis in E. coli. Poor expression levels caused by stable secondary 5′ mRNA structure formation were addressed by two different approaches: (i) increasing the cultivation temperature above the range used typically for recombinant protein expression and (ii) optimizing the 5′ mRNA sequence for reduced secondary structures in the translation initiation region.The increase of the cultivation temperature from 30 °C to 42 °C allowed a more than 10-fold increase of activity per cell and optimizing the 5′ mRNA gene sequence further increased the activity per cell 1.7-fold at 42 °C. Thus, the combination of high-temperature cultivation and 5′ sequence optimization is described as an effective approach to overcome poor expression levels resulting from stable secondary 5′ mRNA structure formation. We suggest that this method is especially suitable for improving the expression of proteins derived from thermophiles in E. coli.     

Multiomics characterization implicates PTK7 in ovarian cancer EMT and cell plasticity and offers strategies for therapeutic intervention

Most patients with ovarian cancer (OC) are diagnosed at a late stage when there are very few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or an oncogenic addiction that can be targeted pharmacologically, unlike other types of cancer. Here, we identified protein tyrosine kinase 7 (PTK7) as a potential new therapeutic target in OC following a multiomics approach using genetic and pharmacological interventions. We performed proteomics analyses upon PTK7 knockdown in OC cells and identified novel downstream effectors such as synuclein-γ (SNCG), SALL2, and PP1γ, and these findings were corroborated in ex vivo primary samples using PTK7 monoclonal antibody cofetuzumab. Our phosphoproteomics analyses demonstrated that PTK7 modulates cell adhesion and Rho-GTPase signaling to sustain epithelial-mesenchymal transition (EMT) and cell plasticity, which was confirmed by high-content image analysis of 3D models. Furthermore, using high-throughput drug sensitivity testing (525 drugs) we show that targeting PTK7 exhibited synergistic activity with chemotherapeutic agent paclitaxel, CHK1/2 inhibitor prexasertib, and PLK1 inhibitor GSK461364, among others, in OC cells and ex vivo primary samples. Taken together, our study provides unique insight into the function of PTK7, which helps to define its role in mediating aberrant Wnt signaling in ovarian cancer.Subject terms: Cancer, Cell signalling     

Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts

The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653-658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [3H]cholesterol. The presence of HDL3 in the medium (100 micrograms/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [3H]cholesterol efflux from the plasma membranes to HDL3. In addition, we also observed that the degradation of sphingomyelin in the HDL3 particles (complete degradation) did not change the apparent rate of [3H]cholesterol transfer from HDL3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.     

Two-year cyclicity in recruitment of a fish population is driven by an inter-stage effect

A 25-year time series for a vendace (Coregonus albula (L.)) population was analysed with mathematical models to reveal the factors that regulate its recruitment. The fitted recruitment model was an age-structured model incorporating the compensatory effects of the spawning population and the abundance of the previous year class. Wind forcing index was also added as an example of an external source of recruitment variability. The auto-correlation analysis revealed a tendency for 2-year generation cyclicity in recruitment. The compensatory effects of both spawning biomass and previous year class abundance on recruitment had to be incorporated into the model to remove cyclicity from residuals. Wind forcing during the larval and early juvenile periods negatively affected recruitment. Re-estimating the parameters of the Cushing recruitment function and the effect of previous year-class from simulated data revealed that the time series structure and measurement errors induce strong biases in parameters exaggerating the density independent population growth rate parameter and the amount of compensation. The negative effect of previous year class was also exaggerated but less severely. Simulations with the artificially perturbed deterministic model skeleton revealed a tendency for cyclicity in recruitment. The model typically generated dampening oscillations, but the dynamics appeared as limit cycles when assuming high mortality, a low level of compensation by spawning biomass and a considerable negative effect of the previous year-class. No single ultimate mechanism for inter-stage effects causing cyclicity can be presently designated despite the rather extensive studies on vendace population dynamics. Several of the suggested mechanisms may be operating in concert.     

Robust regression for periodicity detection in non-uniformly sampled time-course gene expression data

Background  

In practice many biological time series measurements, including gene microarrays, are conducted at time points that seem to be interesting in the biologist's opinion and not necessarily at fixed time intervals. In many circumstances we are interested in finding targets that are expressed periodically. To tackle the problems of uneven sampling and unknown type of noise in periodicity detection, we propose to use robust regression.