Cryptorchidism in mice with an androgen receptor ablation in gubernaculum testis

Androgens play a critical role in the development of the male reproductive system, including the positioning of the gonads. It is not clear, however, which developmental processes are influenced by androgens and what are the target tissues and cells mediating androgen signaling during testicular descent. Using a Cre-loxP approach, we have produced male mice (GU-ARKO) with conditional inactivation of the androgen receptor (Ar) gene in the gubernacular ligament connecting the epididymis to the caudal abdominal wall. The GU-ARKO males had normal testosterone levels but developed cryptorchidism with the testes located in a suprascrotal position. Although initially subfertile, the GU-ARKO males became sterile with age. We have shown that during development, the mutant gubernaculum failed to undergo eversion, a process giving rise to the processus vaginalis, a peritoneal outpouching inside the scrotum. As a result, the cremasteric sac did not form properly, and the testes remained in the low abdominal position. Abnormal development of the cremaster muscles in the GU-ARKO males suggested the participation of androgens in myogenic differentiation; however, males with conditional AR inactivation in the striated or smooth muscle cells had a normal testicular descent. Gene expression analysis showed that AR deficiency in GU-ARKO males led to the misexpression of genes involved in muscle differentiation, cell signaling, and extracellular space remodeling. We therefore conclude that AR signaling in gubernacular cells is required for gubernaculum eversion and outgrowth. The GU-ARKO mice provide a valuable model of isolated cryptorchidism, one of the most common birth defects in newborn boys.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

EFFECTS OF MORINGA OLEIFERA SEED EXTRACT ON RUMEN FERMENTATION IN VITRO

Moringa oleifera is a pantropical tree of the family Moringaceae. A previously undescribed property of an aqueous extract from the seeds of this plant is the modulation of ruminal fermentation patterns, especially protein degradation, as demonstrated in a short-term batch incubation system. Gas, short chain fatty acids (SCFA) and cellulolytic enzyme activities were determined as general fermentation parameters. A dot blot assay able to directly detect true protein in rumen fluid samples was used to quantify protein degradation. For complex substrates the interpretation of protein degradation profiles was amended by polyacrylamide gel electrophoresis (PAGE) of the samples. When incubated with pure carbohydrates at a concentration of 1 mg ml^–1, the extract reduced microbial degradation of the model protein, bovine serum albumin (BSA), such that its concentration was at least 40% above the control after 12 h of incubation. Total protein degradation was thus delayed by approximately 9 h. When fermented along with wheat straw, leaf protein (Rubisco) was almost entirely protected during 12 h of fermentation. The degradation of soy proteins was retarded by at least 4- 6 h, depending on the protein band. There were strong side effects on the fermentation of pure cellulose (SCFA yield -60% after 12 h), whereas cellobiose and starch fermentation were less affected (-18 and -8%, respectively). When the complex substrates were fermented, SCFA yield was reduced by approximately 30% after 12 h. In our work we clearly demonstrate the efficacy of the new substance, which is neither a tannin nor a saponin, in an in vitro system, using pure as well as complex substrates. The properties shown in vitro for the crude extract suggest that it could have a positive effect on the protein metabolism of ruminants under intensive management and that negative side effects can be overcome by an optimized dosage. If the chemical nature of the active substance and its mechanism of action can be clarified, it may provide an alternative to replace critical synthetic feed additives (such as antibiotics) for high yielding dairy cows.     

Correlative light and electron microscopy imaging of autophagy in a zebrafish infection model

High-resolution imaging of autophagy has been used intensively in cell culture studies, but so far it has been difficult to visualize this process in detail in whole animal models. In this study we present a versatile method for high-resolution imaging of microbial infection in zebrafish larvae by injecting pathogens into the tail fin. This allows visualization of autophagic compartments by light and electron microscopy, which makes it possible to correlate images acquired by the 2 techniques. Using this method we have studied the autophagy response against Mycobacterium marinum infection. We show that mycobacteria during the progress of infection are frequently associated with GFP-Lc3-positive vesicles, and that 2 types of GFP-Lc3-positive vesicles were observed. The majority of these vesicles were approximately 1 μm in size and in close vicinity of bacteria, and a smaller number of GFP-Lc3-positive vesicles was larger in size and were observed to contain bacteria. Quantitative data showed that these larger vesicles occurred significantly more in leukocytes than in other cell types, and that approximately 70% of these vesicles were positive for a lysosomal marker. Using electron microscopy, it was found that approximately 5% of intracellular bacteria were present in autophagic vacuoles and that the remaining intracellular bacteria were present in phagosomes, lysosomes, free inside the cytoplasm or occurred as large aggregates. Based on correlation of light and electron microscopy images, it was shown that GFP-Lc3-positive vesicles displayed autophagic morphology. This study provides a new approach for injection of pathogens into the tail fin, which allows combined light and electron microscopy imaging in vivo and opens new research directions for studying autophagy process related to infectious diseases.     

Neuron‐specific translational control shift ensures proteostatic resilience during ER stress

Proteostasis is essential for cellular survival and particularly important for highly specialised post‐mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R‐like endoplasmic reticulum (ER) kinase (PERK)‐mediated phosphorylation of eukaryotic translation initiation factor 2α (p‐eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type‐specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK‐deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK‐deficient neurons. Haem‐regulated inhibitor (HRI) mediates p‐eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back‐up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.     

The role of mast cells and macrophages in amiodarone induced pulmonary fibrosis and the possible attenuating role of atorvastatin

Amiodarone (AM) is an effective anti-arrhythmic drug. We investigated the role of mast cells and macrophages on AM induced pulmonary fibrosis and the action of atorvastatin on this fibrosis. Rats were allocated into four groups; negative control (1), positive control (2), 30 mg/kg body weight/day AM (3) and AM + 10 mg/kg/day atorvastatin (4). Lungs were harvested and prepared for histology and immunohistochemistry. Hematoxylin and eosin stained sections of group 3 exhibited disorganized lung architecture. We found cellular debris in the lumen of both intrapulmonary bronchi and bronchioles with partial disruption of the thickened epithelial lining and mononuclear cellular infiltration into the lamina propria. We also observed thickening of the epithelial lining and the smooth muscle layer. Congested, dilated and thickened blood capillaries and thickened inter-alveolar septa were observed with mononuclear cellular infiltrates in the lung of group 3. Most alveoli were collapsed, but some dilated ones were detected. In some alveoli, type ?? pneumocytes were increased, while type I cells were decreased. We observed significant increases in the amount of collagen in the thickened inter-alveolar septa, around bronchioles and around blood capillaries in sections from group 3. We found a significant increase in mast cells and alveolar macrophages in group 3 compared to group 1. Mast cells and macrophages appear to play important roles in AM induced pulmonary fibrosis. Atorvastatin appears to attenuate this condition.     

Anti-carbamylated protein antibodies in the pre-symptomatic phase of rheumatoid arthritis,their relationship with multiple anti-citrulline peptide antibodies and association with radiological damage

IntroductionThe presence of a new autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, has been identified in rheumatoid arthritis (RA). The presence of anti-CarP antibodies was evaluated in samples taken from individuals who subsequently developed RA before and after onset of symptoms and related to previously analysed antibodies against citrullinated peptides (ACPA specificities) and anti-CCP2.MethodsA total of 252 individuals, with 423 samples from before onset of symptoms of RA, and 197 population controls were identified as donors to the Medical Biobank of Northern Sweden; 192 of them were also sampled at the time of diagnosis. All samples were analysed for anti-CarP IgG and anti-CCP2 antibodies using ELISAs. Ten different antibody reactivities against citrullinated antigens (ACPA specificities) were analysed using a custom-made microarray based on the ImmunoCAP ISAC system (Phadia).ResultsThe concentration of anti-CarP antibodies was significantly increased in the pre-symptomatic individuals compared with controls (P <0.001) and also increased significantly after disease onset (P <0.001). The sensitivity for anti-CarP antibodies in the pre-symptomatic individuals was 13.9% (95% CI: 11 to 17.6) and 42.2% (95% CI: 35.4 to 49.3) following development of RA. Anti-CarP antibody positivity was found in 5.1% to 13.3% of individuals negative for anti-CCP2 or ACPA specificities. Presence of anti-CarP antibodies was significantly related to radiological destruction at baseline, at 24 months and also to radiological change (P <0.05, all).ConclusionsThe results indicate that anti-CarP antibodies are associated with disease development, even after adjusting for the presence of different ACPA fine specificities, and in anti-CCP2 negative individuals and contribute to the identification of a subset of patients with worse radiological progression of the disease independent of ACPA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0536-2) contains supplementary material, which is available to authorized users.     

Thymol and Carvacrol Prevent Cisplatin‐Induced Nephrotoxicity by Abrogation of Oxidative Stress,Inflammation, and Apoptosis in Rats

The aim of the present study is to assess the possible protective effects of thymol and carvacrol against cisplatin (CP)‐induced nephrotoxicity. A single dose of CP {6 mg/kg, intraperitoneally (i.p.)} injected to male rats revealed significant increases in serum urea, creatinine, and tumor necrosis factor alpha levels. It also increased kidney contents of malondialdehyde and caspase‐3 activity with significant reduction in serum albumin, kidney content of reduced glutathione as well as catalase, and superoxide dismutase activity as compared to that of the control group. In contrast, administration of thymol {20 mg/kg, orally (p.o.)} and/or carvacrol (15 mg/kg, p.o.) for 14 days before CP injection and for 7 days after CP administration restored the kidney function and examined oxidative stress parameters. In conclusion, thymol was more effective nephroprotective than carvacrol. Moreover, a combination of thymol and carvacrol had a synergistic nephroprotective effect that might be attributed to antioxidant, anti‐inflammatory, and antiapoptotic activities.