Thermodynamic activity‐based intrinsic enzyme kinetic sheds light on enzyme–solvent interactions

The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme–solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert‐butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity‐based kinetic modeling might be a suitable tool to initially curtail the type of enzyme–solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96–103, 2017     

Whole blood pro-inflammatory cytokines and adhesion molecules post-lipopolysaccharides exposure in hyperbaric conditions

Hyperbaric oxygen (HBO) is a therapeutic intervention with applications in a large variety of diseases, including traumatic injuries and acute or chronic infections. The presence of pro-inflammatory cytokines regulates certain factors including adhesion molecules, which play a significant role in HBO effects. We have investigated the effect of HBO on pro-inflammatory cytokine release [tumor necrosis factor-alpha (TNF-alpha), interleukin 6 and 8 (IL-6 and IL-8)], and the regulation of adhesion molecules [soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular adhesion molecule (sVCAM)] after lipopolysaccharide (LPS) stimulation in 16 healthy individuals, originating from an urban area. A total number of 64 samples were treated, divided into four groups: Group A: not stimulated with LPS and not exposed to HBO. Group B: stimulated with LPS and not exposed to HBO. Group C: not stimulated with LPS and exposed to HBO. Group D: stimulated with LPS and exposed to HBO. The LPS stimulation dose was 100 pg\ml for 0.1 ml whole blood diluted 1:10. After incubation, samples were exposed to HBO with 100% O2 at 2.4 atmospheres absolute (ATA) for 90 min. TNF-alpha, IL-6, IL-8 and sICAM-1, sVCAM levels were determined in culture supernatant, with ELISA. We observed an enhanced effect of LPS stimulation following exposure to HBO, which caused an increase in cytokine production (TNF-alpha, IL-6, IL-8), a reduction in sICAM, and no change to sVCAM, while their levels without stimulation remained almost invariable. The decrease in sICAM levels could be related to the increased levels of IL-8, as the production of this chemokine is involved in the regulation of adhesion molecules.     

Enantiomeric separation and determination of absolute stereochemistry of asymmetric molecules in drug discovery: building chiral technology toolboxes

The application of Chiral Technology, or the (extensive) use of techniques or tools for the determination of absolute stereochemistry and the enantiomeric or chiral separation of racemic small molecule potential lead compounds, has been critical to successfully discovering and developing chiral drugs in the pharmaceutical industry. This has been due to the rapid increase over the past 10-15 years in potential drug candidates containing one or more asymmetric centers. Based on the experiences of one pharmaceutical company, a summary of the establishment of a Chiral Technology toolbox, including the implementation of known tools as well as the design, development, and implementation of new Chiral Technology tools, is provided.     

Factors affecting the establishment and growth of annual legumes in semi-arid mediterranean grasslands

Legumes are an important component of mediterranean grasslands with a significant ecological and economic role. The aim of this study was to investigate the factors that affect their establishment and growth and how they survive in a highly variable and unpredictable environment. The research was carried out in a grassland characterised by a semi-arid mediterranean climate and located on a calcareous substrate at about 150 m a.s.l., in Macedonia, northern Greece. It was dominated by annual legumes such as Hippocrepis multisiliquosa, Medicago disciformis, Medicago minima, Onobrychis aequindentata, Trifolium angustifolium, Trifolium campestre and Trifolium scabrum. It was subjected to the following treatments for four consecutive years: prescribed burning, irrigation, digging, cutting, P fertilization and control. Total legume density was measured in late autumn and in the following spring each year, while total legume biomass was measured only in spring. Dominant legume species densities and biomasses were measured only in spring in the last 3 years. Also, monthly precipitation and air temperature were recorded in a nearby weather station. A great reduction of both legume density and biomass occurred at the third growing season due to adverse weather conditions. Among treatments, P fertilization affected the positively annual legume density and biomass. The other treatments such as burning, irrigation, digging and cutting influenced positively or negatively annual legume density and biomass depending on the climatic characteristics of the particular growing season involved. It is concluded that in semi-arid mediterranean grasslands with cold winters, weather conditions strongly interact with human disturbances in affecting establishment and growth of annual legumes.     

Elevated expression of DecR1 impairs ErbB2/Neu-induced mammary tumor development

Tumor cells utilize glucose as a primary energy source and require ongoing lipid biosynthesis for growth. Expression of DecR1, an auxiliary enzyme in the fatty acid beta-oxidation pathway, is significantly diminished in numerous spontaneous mammary tumor models and in primary human breast cancer. Moreover, ectopic expression of DecR1 in ErbB2/Neu-induced mammary tumor cells is sufficient to reduce levels of ErbB2/Neu expression and impair mammary tumor outgrowth. This correlates with a decreased proliferative index and reduced rates of de novo fatty acid synthesis in DecR1-expressing breast cancer cells. Although DecR1 expression does not affect glucose uptake in ErbB2/Neu-transformed cells, sustained expression of DecR1 protects mammary tumor cells from apoptotic cell death following glucose withdrawal. Moreover, expression of catalytically impaired DecR1 mutants in Neu-transformed breast cancer cells restored Neu expression levels and increased mammary tumorigenesis in vivo. These results argue that DecR1 is sufficient to limit breast cancer cell proliferation through its ability to limit the extent of oncogene expression and reduce steady-state levels of de novo fatty acid synthesis. Furthermore, DecR1-mediated suppression of tumorigenesis can be uncoupled from its effects on Neu expression. Thus, while downregulation of Neu expression may contribute to DecR1-mediated tumor suppression in certain cell types, this is not an obligate event in all Neu-transformed breast cancer cells.     

Sequential pulses of apical epithelial secretion and endocytosis drive airway maturation in Drosophila

The development of air-filled respiratory organs is crucial for survival at birth. We used a combination of live imaging and genetic analysis to dissect respiratory organ maturation in the embryonic Drosophila trachea. We found that tracheal tube maturation entails three precise epithelial transitions. Initially, a secretion burst deposits proteins into the lumen. Solid luminal material is then rapidly cleared from the tubes, and shortly thereafter liquid is removed. To elucidate the cellular mechanisms behind these transitions, we identified gas-filling-deficient mutants showing narrow or protein-clogged tubes. These mutations either disrupt endoplasmatic reticulum-to-Golgi vesicle transport or endocytosis. First, Sar1 is required for protein secretion, luminal matrix assembly, and diametric tube expansion. Subsequently, a sharp pulse of Rab5-dependent endocytic activity rapidly internalizes and clears luminal contents. The coordination of luminal matrix secretion and endocytosis may be a general mechanism in tubular organ morphogenesis and maturation.     

Is maximum stimulation intensity required in the assessment of muscle activation capacity?

Voluntary activation assessment using the interpolation twitch technique (ITT) has almost invariably been done using maximal stimulation intensity, i.e., an intensity beyond which no additional joint moment or external force is produced by increasing further the intensity of stimulation. The aim of the study was to identify the minimum stimulation intensity at which percutaneous ITT yields valid results. Maximal stimulation intensity and the force produced at that intensity were identified for the quadriceps muscle using percutaneous electrodes in eight active men. The stimulation intensities producing 10–90% (in 10% increments) of that force were determined and subsequently applied during isometric contractions at 90% of maximum voluntary contraction (MVC) via twitch doublets. Muscle activation was calculated with the ITT and pain scores were obtained for each stimulation intensity and compared to the respective values at maximum stimulation intensity. Muscle activation at maximal stimulation intensity was 91.6 (2.5)%. The lowest stimulation intensity yielding comparable muscle activation results to maximal stimulation was 50% (88.8 (3.9)%, p < 0.05). Pain score at maximal stimulation intensity was 6.6 (1.5) cm and it was significantly reduced at 60% stimulation intensity (3.7 (1.5) cm, p < 0.05) compared to maximal stimulation intensity. Submaximal stimulation can produce valid ITT results while reducing the discomfort obtained by the subjects, widening the assessment of ITT to situations where discomfort may otherwise impede maximal electrostimulation.     

Seasonal aerobic performance variations in elite soccer players

The purpose of this study was to examine the seasonal changes in body composition and aerobic performance in elite soccer players. Twelve elite professional soccer players (aged 25 6 5 years, weight 75.7 6 5.3 kg, height 1.79 6 0.06 m) were measured for body fat (%), maximum oxygen consumption (VO2max), running velocity at VO2max (VO2max), running velocity at a fixed blood lactate concentration of 4 mmol · L21 (v-4 mM) at the start of the preseason period, at the beginning of the competitive period, and at midseason. VO2max, v-4 mM, and vVO2max increased significantly (p , 0.05) by 4.5, 10.5, and 7.8,respectively, after the preseason period. Thereafter, the aerobic performance parameters remained relatively constant, with no significant changes throughout the competitive period. The results of this study suggest that moderate improvements were observed in VO2max, and the %VO2max at 4 v-4 mM, whereas higher improvements were observed in VO2max and v-4 mmol · L21 after the preseason training period. On the other hand, during the competitive period, aerobic performance remained unchanged.In addition, this study suggests that heart rate, lactate, vVO2, and VO2max are useful and practical predictors that help monitor aerobic performance changes during a soccer season.     

Autophagy in endometrial carcinomas and prognostic relevance of 'stone-like' structures (SLS): what is destined for the atypical endometrial hyperplasia?

Autophagy, as an intracellular adaptation mechanism for oxygen and nutrient deprivation, is associated with tumor cell survival and aggressiveness. This was reaffirmed in a series of 360 endometrial carcinomas, using a standard immunohistochemical technique and the LC3A antibody, capable of recognizing both the soluble (LC3A-I) and the membrane-bound form (LC3A-II) of the protein. LC3A reactivity was recognized in three basic patterns-diffuse cytoplasmic, cytoplasmic/juxta-nuclear, and the so-called "stone-like" structures (SLS). The latter has emerged as the: hallmark of autophagic activity, being detected exclusively in endometrial carcinomas and their immediate precursor lesions, namely the atypical hyperplasias, albeit in small numbers. Other forms of hyperplasia without cytological atypia and normal endometrial tissues expressed only cytoplasmic staining patterns. High SLS counts, presumed to reflect excessive levels of autophagic activity, were associated with tumors of extremely poor prognosis. In contrast, a basal level of autophagic activity, as exemplified by the diffuse cytoplasmic and the cytoplasmic/juxta-nuclear patterns, had no impact on prognosis. Survival, according to tumor cell types, showed that serous papillary, clear cell and the high-grade endometrioid carcinomas had the worst prognosis compared to low-grade endometrioid carcinomas, but interestingly, within this tumor group, those having high-SLS counts had a much worse survival rate than those that did not. It is concluded that an assessment of autophagic activity, particularly in the form of SLS, is useful for evaluating tumor aggressiveness and, in the absence or an excess of SLS, it may also prove valid for differentiating grade 1 endometrioid adenocarcinomas from their precursor lesions.