Transfection of Sertoli cells with androgen receptor alters gene expression without androgen stimulation.

BACKGROUND: Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is involved in various cell biological processes such as sex differentiation. To study the functional mechanisms of androgen action, cell culture systems and AR-transfected cell lines are needed. Transfection of AR into cell lines and subsequent gene expression analysis after androgen treatment is well established to investigate the molecular biology of target cells. However, it remains unclear how the transfection with AR itself can modulate the gene expression even without androgen stimulation. Therefore, we transfected Ar-deficient rat Sertoli cells 93RS2 by electroporation using a full length human AR. RESULTS: Transfection success was confirmed by Western Blotting, immunofluorescence and RT-PCR. AR transfection-related gene expression alterations were detected with microarray-based genome-wide expression profiling of transfected and non-transfected 93RS2 cells without androgen stimulation. Microarray analysis revealed 672 differentially regulated genes with 200 up- and 472 down-regulated genes. These genes could be assigned to four major biological categories (development, hormone response, immune response and metabolism). Microarray results were confirmed by quantitative RT-PCR analysis for 22 candidate genes. CONCLUSION: We conclude from our data, that the transfection of Ar-deficient Sertoli cells with AR has a measurable effect on gene expression even without androgen stimulation and cause Sertoli cell damage. Studies using AR-transfected cells, subsequently stimulated, should consider alterations in AR-dependent gene expression as off-target effects of the AR transfection itself.

Reference genes for the developing mouse lung under consideration of biological, technical and experimental confounders.

For gene expression analysis, the raw data obtained from RT-qPCR are preferably normalized to reference genes, which should be constantly expressed regardless of experimental conditions. Selection of reference genes is particularly challenging for the developing lung because of the complex transcriptional and epigenetic regulation of genes during organ maturation and injury repair. To date, there are only limited experimental data addressing reliable reference genes for this biological circumstance. In this study, we evaluated reference genes for the lung in neonatal C57BL/6 mice under consideration of biological, technical and experimental conditions. For that, we thoroughly selected candidates from commonly used reference genes side-by-side with novel ones by analyzing publicly available microarray datasets. We performed RT-qPCR of the selected candidate genes and analyzed their expression variability using GeNorm and Normfinder. Cell-specific expression of the candidate genes was analyzed using our own single-cell RNA-sequencing data from the developing mouse lung. Depending on the investigated conditions, i.e., developmental stages, sex, RNA quality, experimental condition (hyperoxia) and cell types, distinct candidate genes demonstrated stable expression confirming their eligibility as reliable reference genes. Our results provide valuable information for the selection of proper reference genes in studies investigating the neonatal mouse lung.

Does heart surgery change the capacity of α1-antitrypsin to inhibit the ATP-induced release of monocytic interleukin-1ß? A preliminary study.

Heart surgery involving cardiopulmonary bypass induces systemic inflammation that is, at least in part, caused by extracellular ATP originating from damaged cells and by proteases secreted by activated neutrophils. The anti-protease α1-antitrypsin (AAT) forms complexes with several proteases including neutrophil elastase, resulting in a mutual loss of activity. We demonstrated recently that AAT inhibits the ATP-induced release of the pro-inflammatory cytokine interleukin-1ß by human monocytes by a mechanism involving activation of metabotropic functions at nicotinic acetylcholine receptors. Interleukin-1ß importantly contributes to the pathogenesis of sterile inflammatory response syndrome. Thus, AAT might function as an endogenous safeguard against life-threatening systemic inflammation. In this preliminary study, we test the hypothesis that during cardiopulmonary bypass, AAT is inactivated as an anti- protease and as an inhibitor of ATP-induced interleukin-1ß release. AAT was affinity-purified from the blood plasma of patients before, during and after surgery. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with ATP in the presence or absence of patient AAT to test for its inhibitory effect on interleukin-1ß release. Anti-protease activity was investigated via complex formation with neutrophil elastase. The capacity of patient AAT to inhibit the ATP-induced release of interleukin-1ß might be slightly reduced in response to heart surgery and complex formation of patient AAT with neutrophil elastase was unimpaired. We conclude that surgery involving cardiopulmonary bypass does not markedly reduce the anti-inflammatory and the anti-protease activity of AAT. The question if AAT augmentation therapy during heart surgery is suited to attenuate postoperative inflammation warrants further studies in vivo.

Persistent hypomagnesemia following cisplatin chemotherapy in patients with ovarian cancer.

The development of acute hypomagnesemia following cisplatin administration is a well-recognized complication associated with the use of this chemotherapeutic agent. However, there is limited information available in the medical literature concerning how long this abnormality may persist following the discontinuation of cisplatin. Of 13 patients with ovarian cancer who had a baseline serum magnesium determination obtained prior to the initiation of a second-line cisplatin-based chemotherapy regimen, 9 (69%) were found to be hypomagnesemic (serum magnesium less than 1.4 mg/l), including 3 patients with serum magnesium values less than 1.0 mequiv/l. The median cisplatin-free interval for these 9 patients was 19 months (range 6-40 months). We conclude that persistent, and possibly permanent, hypomagnesemia is common following cisplatin chemotherapy.

Fluid balance in critically ill patients. Should we really rely on it?

BACKGROUND: The computation of fluid balances (FBs) by subtracting fluid outputs from inputs is a common critical care practice. Limited information exists about the accuracy and consistency of nurse-registered cumulative FBs and regarding the value of suggested corrections for non-measurable losses. METHODS: From 147 ICU patients, we prospectively evaluated the cumulative FBs and their relationship to changes in body weight (BW). Standardised measurements of BW were performed on admission and discharge. FB charts were accurately reviewed and arithmetic errors corrected. Net cumulative FBs and adjusted cumulative FBs (considering sensible or insensible fluid losses/fever/liquid faeces) were analysed for all patients and 3 subgroups (cardiac-cerebral, septic, and others). Agreement between FBs and BW changes was calculated according to the defined subgroups and confounding variables. RESULTS: Cumulative FBs were inaccurate in 49 cases (33%) with errors ranging from -3606 mL to +2020 mL. The total (average daily) difference between measured BW and FBs (mean ± SD) was 0.185±1.874 kg (0.101±1.020 kg). Correlation (r(2)) and Bland-Altman agreement was poor between BW changes and net cumulative FBs (0.552 and -1.26±5.41 kg) and slightly better between BW changes and adjusted cumulative FBs (0.714 and +0.18±3.68 kg). Standard deviations of the average daily differences between BW changes and FBs were always >1 L. Correction of the net FBs as suggested in the literature were not useful. New multiple regression models only modestly improved correlation. CONCLUSION: For a large portion of patients nurse-registered cumulative FBs are neither accurate nor do they agree with standardised BW measurements. Patient care and clinical decision-making should be based on more objective techniques.

Genetic and ecological divergence of a monophyletic cichlid species pair under fully sympatric conditions in Lake Ejagham, Cameroon.

Although there is mounting evidence that speciation can occur under sympatric conditions, unambiguous examples from nature are rare and it is almost always possible to propose alternative allopatric or parapatric scenarios. To identify an unequivocal case of sympatric speciation it is, therefore, necessary to analyse natural settings where recent monophyletic species flocks have evolved within a small and confined spatial range. We have studied such a case with a cichlid species flock that comprises five Tilapia forms endemic to a tiny lake (Lake Ejagham with a surface area of approximately 0.49 km2) in Western Cameroon. Analysis of mitochondrial D-Loop sequences shows that the flock is very young (approximately 10(4) years) and has originated from an adjacent riverine founder population. We have focused our study on a particular pair of forms within the lake that currently appears to be in the process of speciation. This pair is characterized by an unique breeding colouration and specific morphological aspects, which can serve as synapomorphic characters to prove monophyly. It has differentiated into a large inshore and a small pelagic form, apparently as a response to differential utilization of food resources. Still, breeding and brood care occurs in overlapping areas, both in time and space. Analysis of nuclear gene flow on the basis of microsatellite polymorphisms shows a highly restricted gene flow between the forms, suggesting reproductive isolation between them. This reproductive isolation is apparently achieved by size assortative mating, although occasional mixed pairs can be observed. Our findings are congruent with recent theoretical models for sympatric speciation, which show that differential ecological adaptations in combination with assortative mating could easily lead to speciation in sympatry.