Microcolonial Fungi on Rocks: A Life in Constant Drought?

Black microcolonial fungi (MCF) and black yeasts are among the most stress-resistant eukaryotic organisms known on Earth. They mainly inhabit bare rock surfaces in hot and cold deserts of all regions of the Earth, but some of them have a close phylogenetic relation to human pathogenic black fungi which makes them important model organisms also with respect to clinical mycology. The environment of those fungi is especially characterized by extreme changes from humidity to long periods of desiccation and extreme temperature differences. A key to the understanding of MCF ecology is the question about metabolic activity versus dormancy in the natural environments. In this study, the time lag from the desiccated state to rehydration and full metabolic activity and growth was measured and defined in accordance with simulated environmental conditions. The ability to survive after desiccation and the speed of rehydration as well as changes of the whole cell protein pattern are demonstrated. Whereas both mesophilic strains—Exophiala jeanselmei and Knufia perforans (=Coniosporium perforans)—show a clear reaction toward desiccation by production of small proteins, Cryomyces antarcticus—the extremotolerant MCF—does not show any response to desiccation but seems just to down-regulate its metabolism. Data on intracellular sugar suggest that both trehalose and mannitol might play a cell protective role in those fungi.     

Factors Affecting Work Ability in Day and Shift‐Working Nurses

Satisfactory work ability is sustained and promoted by good physical and mental health and by favorable working conditions. This study examined whether favorable and rewarding work‐related factors increased the work ability among European nurses. The study sample was drawn from the Nurses' Early Exit Study and consisted of 7,516 nursing staff from seven European countries working in state‐owned and private hospitals. In all, 10.8% were day, 4.2% were permanent night, 20.9% were shift without night shift, and 64.1% were shift workers with night shifts. Participants were administered a composite questionnaire at baseline (Time 0) and 1 yr later (Time 1). The Work Ability Index (WAI) at Time 1 was used as the outcome measure, while work schedule, sleep, rewards (esteem and career), satisfaction with pay, work involvement and motivation, and satisfaction with working hours at Time 0 were included as potential determinants of work ability. Univariate and multivariate analyses were conducted after adjusting for a number of confounders (i.e., country, age, sex, type of employment, family status, and other job opportunities in the same area). Work schedule was not related to Time 1 changes in WAI. Higher sleep quality and quantity and more favorable psychosocial factors significantly increased work ability levels. Higher sleep quality and quantity did not mediate the effect of work schedule on work ability. No relevant interaction effects on work ability were observed between work schedule and the other factors considered at Time 0. As a whole, sleep and satisfaction with working time were gradually reduced from day work to permanent night work. However, scores on work involvement, motivation, and satisfaction with pay and rewards were the highest in permanent night workers and the lowest in rotating shift workers that included night shifts.     

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.     

Tyrosinase and Layer-by-Layer supported tyrosinases in the synthesis of lipophilic catechols with antiinfluenza activity

Catechol derivatives with lipophilic properties have been selectively synthesized by tyrosinase in high yield avoiding long and tedious protection/deprotection steps usually required in traditional procedures. The synthesis was effective also with immobilized tyrosinase able to perform for more runs. The novel catechols were evaluated against influenza A virus, that continue to represent a severe threat worldwide. A significant antiviral activity was observed in derivatives characterized by antioxidant activity and long carbon alkyl side-chains, suggesting the possibility of a new inhibition mechanism based on both redox and lipophilic properties.     

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium <Emphasis Type="Italic">Thalassospira</Emphasis> sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.     

Chemoattractant action and molecular signaling pathways of Kit ligand on mouse primordial germ cells

Using a Transwell chamber as migration assay for mouse primordial germ cells (PGCs), we show here that these cells posses directional migration in the absence of somatic cell and defined matrix support and in response to a Kit ligand (KL) gradient or medium conditioned by Aorta/Gonad/Mesonephros and gonadal ridges. Other putative PGC chemoattractants such as SDF1 and TGFbeta did not exert any attractive action on PGCs. The chemoattractant activity of KL and conditioned medium was also evidenced by their ability to stimulate actin reorganization in PGCs. In the aim to identify downstream signaling pathways governing KL chemoattraction on PGCs, we demonstrated that in such cells KL rapidly (5 min) increased autophosphorylation of its receptor c-Kit and caused phosphorylation of the serine-threonine kinase AKT through the action of PI3K. 740Y-P peptide, a direct activator of PI3 kinase, stimulated PGC migration at levels similar to those elicited by KL. LY294002 (a specific inhibitor of PI3K) abolished KL-dependent PGC migration or the chemoattractant activity of the conditioned medium and inhibited AKT phosphorylation; Src kinase inhibitors PP2 and SU6656, caused significant reduction of the KL-dependent PGC migration and AKT phosphorylation, while U0126, a selective inhibitor of the MEK/ERK protein kinase cascade, reduced PGC migration and AKT phosphorylation at lesser extent. SU6656 completely abolished the chemoattractant activity of the conditioned medium. Finally, SB202190 (a p38 inhibitor) and rapamycin (mTOR inhibitor) did not affect PGC migration. In addition, to demonstrate that somatic cells are not essential for PGC motility and directional migration, we evidenced a novel role for KL as PGC chemoattractant and for PI3K/AKT and Src kinase, as players involved in the activation of the PGC migratory machinery and likely important for their directional movement towards the gonadal ridges.     

Growth hormone and ghrelin secretion in severely obese women before and after bariatric surgery

Objective: The objective was to evaluate ghrelin and growth hormone (GH) interactions and responses to a growth hormone‐releasing hormone (GHRH)/arginine test in severe obesity before and after surgically‐induced weight loss. Research Methods and Procedures: Our study population included 11 severely obese women 39 ± 12 years of age, with a mean BMI of 48.6 ± 2.4 kg/m², re‐studied in a phase of stabilized body weight, with a BMI of 33.4 ± 1.2 kg/m², 18 months after having successfully undergone biliopancreatic diversion (BPD). A GHRH/arginine test was performed before and 18 months after BPD to evaluate ghrelin and GH interactions. Active ghrelin, measured by radioimmunoassay (RIA), and GH, measured by chemiluminescence assay, were assayed before and after the GHRH/arginine test. Results: Fasting serum GH levels and GH area under the curve (AUC) significantly increased from 0.2 ± 0.05 ng/mL to 1 ± 0.3 ng/mL (p < 0.05) and from 514.76 ± 98.7 ng/mL for 120 minutes to 1957.3 ± 665.1 ng/mL for 120 minutes after bariatric surgery (p < 0.05), respectively. Although no significant change in fasting ghrelin levels was observed (573 ± 77.9 before BPD vs. 574.1 ± 32.7 after BPD), ghrelin AUC significantly increased from ?3253.9 ± 2180.9 pg/mL for 120 minutes to 1142.3 ± 916.4 pg/mL for 120 minutes after BPD (p < 0.05). Fasting serum insulin‐like growth factor (IGF)‐1 concentration did not change significantly (133.6 ± 9.9 ng/mL before vs. 153.3 ± 25.2 ng/mL after BPD). Discussion: Our study demonstrates that the mechanisms involved in ghrelin and GH secretion after the secretagogue stimulus (GHRH/arginine) are consistent with patterns observed in other populations.