Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC)

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism’s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air–liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro.     

Physiology and Bioenergetics of [NiFe]-Hydrogenase 2-Catalyzed H2-Consuming and H2-Producing Reactions in Escherichia coli

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H₂ to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H₂ evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H₂ evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H₂-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H₂ oxidation or H₂ evolution in whole cells, reversible H₂-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H₂ evolution mode. Our findings suggest that Hyd-2 switches between H₂-consuming and H₂-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H₂ evolution from glycerol requires reverse electron transport.     

The Intracellular Bacteria Chlamydia Hijack Peroxisomes and Utilize Their Enzymatic Capacity to Produce Bacteria-Specific Phospholipids

Chlamydia trachomatis is an obligate intracellular pathogen responsible for loss of eyesight through trachoma and for millions of cases annually of sexually transmitted diseases. The bacteria develop within a membrane-bounded inclusion. They lack enzymes for several biosynthetic pathways, including those to make some phospholipids, and exploit their host to compensate. Three-dimensional fluorescence microscopy demonstrates that small organelles of the host, peroxisomes, are translocated into the Chlamydia inclusion and are found adjacent to the bacteria. In cells deficient for peroxisome biogenesis the bacteria are able to multiply and give rise to infectious progeny, demonstrating that peroxisomes are not essential for bacterial development in vitro. Mass spectrometry-based lipidomics reveal the presence in C. trachomatis of plasmalogens, ether phospholipids whose synthesis begins in peroxisomes and have never been described in aerobic bacteria before. Some of the bacterial plasmalogens are novel structures containing bacteria-specific odd-chain fatty acids; they are not made in uninfected cells nor in peroxisome-deficient cells. Their biosynthesis is thus accomplished by the metabolic collaboration of peroxisomes and bacteria.     

Rapid disease development in scrapie-infected mice deficient for CD40 ligand

The inhibition of CD40-CD40L interaction-mediated signalling was suggested as a therapeutic strategy for the treatment of Alzheimer's disease. Conversely, CD40-deficient neurons were reported to be more vulnerable to stress associated with ageing as well as nerve growth factor-beta and serum withdrawal. We studied the scrapie infection of CD40L-deficient (CD40L(-/-)) mice to see whether ablation of the CD40L gene would be beneficial or detrimental in this model of a neurodegenerative amyloidosis. CD40L(-/-) mice died on average 40 days earlier than wild-type control mice and exhibited a more pronounced vacuolation of the neuropil and an increased microglia activation. The experimental model indicates that a deficiency for CD40L is highly detrimental in prion diseases and reinforces the neuroprotective function of intact CD40-CD40L interactions. The stimulation of neuroprotective pathways may represent a possibility to delay therapeutically the disease onset in prion infections of the central nervous system.     

Sex- and Time-Dependent Patterns in Risk Factors of End-Stage Renal Disease: A Large Austrian Cohort with up to 20 Years of Follow-Up

Objective

We investigated the association between metabolic factors and End-Stage Renal Disease (ESRD) and quantified the magnitude of their influence dependent on sex and time of exposure up to 20 years.

Material and Methods

A prospective cohort study was conducted to determine risk factors for the development of ESRD. From 1988 to 2005 185,341 persons (53.9% women) participated in the “Vorarlberg Health Monitoring and Promotion Programme” (VHM&PP). Data on body mass index (BMI), fasting blood glucose (FBG), systolic (BPsys) and diastolic (BPdia) blood pressure, total cholesterol (TC), triglycerides (TG), gamma-glutamyltransferase (GGT) and smoking status were collected. Data of the population-based VHM&PP were merged with the Austrian Dialysis and Transplant Registry. Cox proportional hazards models were applied to calculate hazard ratios (HRs) for ESRD, stratified by sex and 5-year time intervals.

Results

During a mean follow-up of 17.5 years 403 patients (39.1% women) developed ESRD. Significant risk factors were: BMI (per 1 kg/m²) HR 1.04 (95% CI 1.01–1.06), FBG (per 1 mmol/L) HR 1.09 (1.05–1.12), BPsys (per 5 mmHg) HR 1.10 (1.07–1.14), BPdia (per 5 mmHg) HR 1.09 (1.03–1.15), TG (per 1 mmol/L) HR 1.07 (1.02–1.13), TC (per 1 mmol/L) HR 1.22 (1.13–1.32). We observed a sex-specific risk pattern with an increased ESRD risk for men for increasing TG and smoking, and for women for increasing BMI and GGT. In time interval analyses BPsys and TC were associated with early ESRD onset, whereas BMI, FBG, BPdia and GGT were associated with later onset.

Conclusions

Anthropometric and metabolic factors are differentially associated with the long-term risk for ESRD in a sex- and time-dependent manner. Consideration of these patterns in preventive and therapeutic strategies could have an impact on ESRD incidence.     

Egg sex ratio and paternal traits: using within-individual comparisons

Empirical studies of sex ratios in birds have been limited dueto difficulties in determining offspring sex. Since molecularsexing techniques removed this constraint, the last 5 yearshas seen a great increase in studies of clutch sex ratio manipulationby female birds. Typically these studies investigate variationin clutch sex ratios across individuals in relation to environmentalcharacteristics or parental traits, and often they find no relationships. In this study we also found that clutch sex ratiosdid not vary in relation to a number of biological and environmentalfactors for 238 great tit Parus major nests. However, interestingsex ratio biases were revealed when variation in clutch sexratios was analyzed within individual females breeding in successiveyears. There was a significant positive relationship betweenthe change in sex ratio of a female's clutch from one yearto the next and the relative body condition of her partner.Females mating with males of higher body condition in yearx + 1 produced relatively male-biased sex ratios, and the oppositewas true for females mated with lower condition males. Within-individualanalysis also allowed investigations of sex ratio in relationto partner change. There was no change in sex ratios of femalespairing with the same male; however, females pairing with anew male produced clutches significantly more female biased. Comparisons of clutch sex ratios within individuals may be apowerful method for detecting sex ratio variation, and perhapsfemale birds may indeed manipulate egg sex but require personalcontextual experience for such decisions.     

A cost of cryptic female choice in the yellow dung fly

Female dung flies Scathophaga stercoraria (L.) store sperm from several males in three or four spermathecae. Selection on the number of spermathecae was successful and the morphological intermediate stages in the evolution from three to four spermathecae are illustrated. The genetic quality of a male from a female's perspective depends on an interaction between their genotypes and the microhabitat in which the offspring will grow. Females influence the paternity pattern of their offspring, and do this differently in different microhabitats. Females with four spermathecae are better able to influence paternity than are those with three spermathecae. However, there must be a cost to building and maintaining an extra spermatheca. We estimate, using the animal model on pedigree data, that this cost is approximately five eggs per clutch, i.e. around 8% of the mean clutch size. This is a substantial cost and such costs should not be ignored in discussions of the benefits to females of assessing the genetic qualities of their mating partners. We suggest that the number of spermathecae in the study population is stable because the relative benefits in quality of offspring through cryptic female choice is balanced by the costs in total numbers of offspring.