CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis.

Mononuclear phagocytes (MPs) play an active role in the immunological homeostasis of the urogenital tract. In the epididymis, a finely tuned balance between tolerance to antigenic sperm and immune activation is required to maintain epididymal function while protecting sperm against pathogens and stressors. We previously characterized a subset of resident MPs that express the CX3CR1 receptor, emphasizing their role in antigen sampling and processing during sperm maturation and storage in the murine epididymis. Bacteria-associated epididymitis is the most common cause of intrascrotal inflammation and frequently leads to reproductive complications. Here, we examined whether the lack of functional CX3CR1 in homozygous mice (CX3CR1EGFP/EGFP, KO) alters the ability of MPs to initiate immune responses during epididymitis induced by LPS intravasal-epididymal injection. Confocal microscopy revealed that CX3CR1-deficient MPs located in the initial segments of the epididymis displayed fewer luminal-reaching membrane projections and impaired antigen capture activity. Moreover, flow cytometry showed a reduction of epididymal KO MPs with a monocytic phenotype under physiological conditions. In contrast, flow cytometry revealed an increase in the abundance of MPs with a monocytic signature in the distal epididymal segments after an LPS challenge. This was accompanied by the accumulation of CD103+ cells in the interstitium, and the prevention or attenuation of epithelial damage in the KO epididymis during epididymitis. Additionally, CX3CR1 deletion induced downregulation of Gja1 (connexin 43) expression in KO MPs. Together, our study provides evidence that MPs are gatekeepers of the immunological blood-epididymis barrier and reveal the role of the CX3CR1 receptor in epididymal mucosal homeostasis by inducing MP luminal protrusions and by regulating the monocyte population in the epididymis at steady state as well as upon infection. We also uncover the interaction between MPs and CD103+ dendritic cells, presumably through connexin 43, that enhance immune responses during epididymitis. Our study may lead to new diagnostics and therapies for male infertility and epididymitis by identifying immune mechanisms in the epididymis.

Sperm acquire epididymis-derived proteins through epididymosomes.

STUDY QUESTION: Are epididymosomes implicated in protein transfer from the epididymis to spermatozoa? SUMMARY ANSWER: We characterized the contribution of epididymal secretions to the sperm proteome and demonstrated that sperm acquire epididymal proteins through epididymosomes. WHAT IS KNOWN ALREADY: Testicular sperm are immature cells unable to fertilize an oocyte. After leaving the testis, sperm transit along the epididymis to acquire motility and fertilizing abilities. It is well known that marked changes in the sperm proteome profile occur during epididymal maturation. Since the sperm is a transcriptional and translational inert cell, previous studies have shown that sperm incorporate proteins, RNA and lipids from extracellular vesicles (EVs), released by epithelial cells lining the male reproductive tract. STUDY DESIGN, SIZE, DURATION: We examined the contribution of the epididymis to the post-testicular maturation of spermatozoa, via the production of EVs named epididymosomes, released by epididymal epithelial cells. An integrative analysis using both human and mouse data was performed to identify sperm proteins with a potential epididymis-derived origin. Testes and epididymides from adult humans (n = 9) and adult mice (n = 3) were used to experimentally validate the tissue localization of four selected proteins using high-resolution confocal microscopy. Mouse epididymal sperm were co-incubated with carboxyfluorescein succinimidyl ester (CFSE)-labeled epididymosomes (n = 4 mice), and visualized using high-resolution confocal microscopy. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult (12-week-old) C57BL/CBAF1 wild-type male mice and adult humans were used for validation purposes. Testes and epididymides from both mice and humans were obtained and processed for immunofluorescence. Mouse epididymal sperm and mouse epididymosomes were obtained from the epididymal cauda segment. Fluorescent epididymosomes were obtained after labeling the epididymal vesicles with CFSE dye followed by epididymosome isolation using a density cushion. Immunofluorescence was performed following co-incubation of sperm with epididymosomes in vitro. High-resolution confocal microscopy and 3D image reconstruction were used to visualize protein localization and sperm-epididymosomes interactions. MAIN RESULTS AND THE ROLE OF CHANCE: Through in silico analysis, we first identified 25 sperm proteins with a putative epididymal origin that were conserved in both human and mouse spermatozoa. From those, the epididymal origin of four sperm proteins (SLC27A2, EDDM3B, KRT19 and WFDC8) was validated by high-resolution confocal microscopy. SLC27A2, EDDM3B, KRT19 and WFDC8 were all detected in epithelial cells lining the human and mouse epididymis, and absent from human and mouse seminiferous tubules. We found region-specific expression patterns of these proteins throughout the mouse epididymides. In addition, while EDDM3B, KRT19 and WFDC8 were detected in both epididymal principal and clear cells (CCs), SLC27A2 was exclusively expressed in CCs. Finally, we showed that CFSE-fluorescently labeled epididymosomes interact with sperm in vitro and about 12-36% of the epididymosomes contain the targeted sperm proteins with an epididymal origin. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The human and mouse sample size was limited and our results were descriptive. The analyses of epididymal sperm and epididymosomes were solely performed in the mouse model due to the difficulties in obtaining epididymal luminal fluid human samples. Alternatively, human ejaculated sperm and seminal EVs could not be used because ejaculated sperm have already contacted with the fluids secreted by the male accessory sex glands, and seminal EVs contain other EVs in addition to epididymosomes, such as the abundant prostate-derived EVs. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that epididymosomes are capable of providing spermatozoa with a new set of epididymis-derived proteins that could modulate the sperm proteome and, subsequently, participate in the post-testicular maturation of sperm cells. Additionally, our data provide further evidence of the novel role of epididymal CCs in epididymosome production. Identifying mechanisms by which sperm mature to acquire their fertilization potential would, ultimately, lead to a better understanding of male reproductive health and may help to identify potential therapeutic strategies to improve male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER 'una manera de hacer Europa' PI13/00699 and PI16/00346 to R.O.; and Sara Borrell Postdoctoral Fellowship, Acción Estratégica en Salud, CD17/00109 to J.C.), by National Institutes of Health (grants HD040793 and HD069623 to S.B., grant HD104672-01 to M.A.B.), by the Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306 to F.B.), by a Lalor Foundation Fellowship (to F.B. and M.A.B.), by the Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016-2020, SLT002/16/00337 to M.J.), by Fundació Universitària Agustí Pedro i Pons (to F.B.), and by the American Society for Biochemistry and Molecular Biology (PROLAB Award from ASBMB/IUBMB/PABMB to F.B.). Confocal microscopy and transmission electron microscopy was performed in the Microscopy Core facility of the Massachusetts General Hospital (MGH) Center for Systems Biology/Program in Membrane Biology which receives support from Boston Area Diabetes and Endocrinology Research Center (BADERC) award DK57521 and Center for the Study of Inflammatory Bowel Disease grant DK43351. The Zeiss LSM800 microscope was acquired using an NIH Shared Instrumentation Grant S10-OD-021577-01. The authors have no conflicts of interest to declare.

From initial segment to cauda: a regional characterization of mouse epididymal CD11c+ mononuclear phagocytes based on immune phenotype and function.

Successful sperm maturation and storage rely on a unique immunological balance that protects the male reproductive organs from invading pathogens and spermatozoa from a destructive autoimmune response. We previously characterized one subset of mononuclear phagocytes (MPs) in the murine epididymis, CX3CR1+ cells, emphasizing their different functional properties. This population partially overlaps with another subset of understudied heterogeneous MPs, the CD11c+ cells. In the present study, we analyzed the CD11c+ MPs for their immune phenotype, morphology, and antigen capturing and presenting abilities. Epididymides from CD11c-EYFP mice, which express enhanced yellow fluorescent protein (EYFP) in CD11c+ MPs, were divided into initial segment (IS), caput/corpus, and cauda regions. Flow cytometry analysis showed that CD11c+ MPs with a macrophage phenotype (CD64+ and F4/80+) were the most abundant in the IS, whereas those with a dendritic cell signature [CD64- major histocompatibility complex class II (MHCII)+] were more frequent in the cauda. Immunofluorescence revealed morphological and phenotypic differences between CD11c+ MPs in the regions examined. To assess the ability of CD11c+ cells to take up antigens, CD11c-EYFP mice were injected intravenously with ovalbumin. In the IS, MPs expressing macrophage markers were most active in taking up the antigens. A functional antigen-presenting coculture study was performed, whereby CD4+ T cells were activated after ovalbumin presentation by CD11c+ epididymal MPs. The results demonstrated that CD11c+ MPs in all regions were capable of capturing and presenting antigens. Together, this study defines a marked regional variation in epididymal antigen-presenting cells that could help us understand fertility and contraception but also has larger implications in inflammation and disease pathology.

The complete mitochondrial genome of the hermaphroditic freshwater mussel Anodonta cygnea (Bivalvia: Unionidae): in silico analyses of sex-specific ORFs across order Unionoida.

BACKGROUND: Doubly uniparental inheritance (DUI) of mitochondrial DNA in bivalves is a fascinating exception to strictly maternal inheritance as practiced by all other animals. Recent work on DUI suggests that there may be unique regions of the mitochondrial genomes that play a role in sex determination and/or sexual development in freshwater mussels (order Unionoida). In this study, one complete mitochondrial genome of the hermaphroditic swan mussel, Anodonta cygnea, is sequenced and compared to the complete mitochondrial genome of the gonochoric duck mussel, Anodonta anatina. An in silico assessment of novel proteins found within freshwater bivalve species (known as F-, H-, and M-open reading frames or ORFs) is conducted, with special attention to putative transmembrane domains (TMs), signal peptides (SPs), signal cleavage sites (SCS), subcellular localization, and potential control regions. Characteristics of TMs are also examined across freshwater mussel lineages. RESULTS: In silico analyses suggests the presence of SPs and SCSs and provides some insight into possible function(s) of these novel ORFs. The assessed confidence in these structures and functions was highly variable, possibly due to the novelty of these proteins. The number and topology of putative TMs appear to be maintained among both F- and H-ORFs, however, this is not the case for M-ORFs. There does not appear to be a typical control region in H-type mitochondrial DNA, especially given the loss of tandem repeats in unassigned regions when compared to F-type mtDNA. CONCLUSION: In silico analyses provides a useful tool to discover patterns in DUI and to navigate further in situ analyses related to DUI in freshwater mussels. In situ analysis will be necessary to further explore the intracellular localizations and possible role of these open reading frames in the process of sex determination in freshwater mussel.

Early replication dynamics of sex-linked mitochondrial DNAs in the doubly uniparental inheritance species Ruditapes philippinarum (Bivalvia Veneridae).

Mitochondrial homoplasmy, which is maintained by strictly maternal inheritance and a series of bottlenecks, is thought to be an adaptive condition for metazoans. Doubly uniparental inheritance (DUI) is a unique mode of mitochondrial transmission found in bivalve species, in which two distinct mitochondrial genome (mtDNA) lines are present, one inherited through eggs (F) and one through sperm (M). During development, the two lines segregate in a sex- and tissue-specific manner: females lose M during embryogenesis, whereas males actively segregate it in the germ line. These two pivotal events are still poorly characterized. Here we investigated mtDNA replication dynamics during embryogenesis and pre-adulthood of the venerid Ruditapes philippinarum using real-time quantitative PCR. We found that both mtDNAs do not detectably replicate during early embryogenesis, and that the M line might be lost from females around 24 h of age. A rise in mtDNA copy number was observed before the first reproductive season in both sexes, with the M mitochondrial genome replicating more than the F in males, and we associate these boosts to the early phase of gonad production. As evidence indicates that DUI relies on the same molecular machine of mitochondrial maternal inheritance that is common in most animals, our data are relevant not only to DUI but also to shed light on how differential segregations of mtDNA variants, in the same nuclear background, may be controlled during development.

Schmallenberg virus: experimental infection in goats and bucks.

BACKGROUND: Schmallenberg virus (SBV) is an emerging Orthobunyavirus of ruminant livestock species currently circulating in Europe. SBV causes a subclinical or mild disease in adult animals but vertical transmission to pregnant dams may lead to severe malformations in the offspring. Data on the onset of clinical signs, viremia and seroconversion in experimentally infected adult animals are available for cattle and sheep but are still lacking for goats. For a better understanding of the pathogenesis of SBV infection in adult ruminants, we carried out experimental infections in adult goats. Our specific objectives were: (i) to record clinical signs, viremia and seroconversion; (ii) to monitor viral excretion in the semen of infected bucks; (iii) to determine in which tissues SBV replication took place and virus-induced lesions developed. RESULTS: Four goats and two bucks were inoculated with SBV. Virus inoculation was followed by a short viremic phase lasting 3 to 4 days and a seroconversion occurring between days 7 and 14 pi in all animals. The inoculated goats did not display any clinical signs, gross lesions or histological lesions. Viral genomic RNA was found in one ovary but could not be detected in other organs. SBV RNA was not found in the semen samples collected from two inoculated bucks. CONCLUSIONS: In the four goats and two bucks, the kinetics of viremia and seroconversion appeared similar to those previously described for sheep and cattle. Our limited set of data provides no evidence of viral excretion in buck semen.

Spontaneous obturator internus muscle haematoma: a new unpublished cause of ilioipelvic pain in haemophilia.

Obturator muscles haematoma are rarely reported. The most often reported cases are primary pyomyositis or posttraumatic haematomas occurring during pelvic fractures. We firstly report herein two cases of spontaneous obturator internus haematoma (OIH) in two haemophiliacs with inhibitor. Clinical data and imaging of two patients treated in our clinic are reported here according to previously defined criteria of OIH in posttraumatic situation. Both patients were children suffering from severe and moderate haemophilia A, respectively, with an inhibitor at the time of the event. The clinical feature was marked by an iliopelvic pain letting discussing hip haemarthrosis, appendicitis or iliopsoas haematoma. For both patients ultrasonography (US) failed to provide the diagnosis. Careful and repeated clinical examinations eventually lead to suspect obturator haematoma which was confirmed by abdominopelvic computed tomography (CT) and magnetic resonance imaging (MRI). Respectively, high dose of FVIII or rFVIIa regimen allowed a rapid control of the muscular bleeding in the low and high responder inhibitor patients. Spontaneous OIH may be added to the differential diagnosis of iliopelvic pain in severe forms of haemophilia. US still often performed at first in such case remains unhelpful; abdominopelvic CT or MRI should be performed to discriminate among different diagnoses, including OIH which stays probably undiagnosed.

Elective subtotal colectomy with ileosigmoid anastomosis for colon cancer preserves bowel function and quality of life.

AIM: We report on our experience of elective subtotal colectomy and ileosigmoid anastomosis for colon cancer with focus on postoperative results, function and quality of life. METHOD: Between 1998 and 2011, 106 consecutive patients with colonic malignancy underwent this procedure electively. Function and quality of life (EORTC QLQ-C30) were evaluated retrospectively with questionnaires sent to all patients free of recurrence. RESULTS: There were 62 men and 44 women (mean age 63 years). Postoperative mortality and morbidity rates were 1.9 and 26.4%, respectively. Persistent ileus was the main early complication (16%). After a mean follow-up of 67 ± 36 months, 50 (78.1) out of 64 patients have been evaluated for function and quality of life. The mean number of bowel movements per 24 h was 3 ± 2 and significantly lower when the length of the remaining sigmoid colon was more than 15 cm (P = 0.049). Compared with a European reference population for EORTC QLQ-C30 results, our patients had significantly more diarrhoea (26 vs 3, P = 0.0002) but less pain (10 vs 25, P < 0.0001) and better global quality of life (77 vs 62, P < 0.0001). CONCLUSION: Elective subtotal colectomy for colon cancer is safe and associated with good function and quality of life. Ileosigmoid anastomosis should be discussed when extended colectomy is required, providing the rectosigmoid junction and its vascular supply can be oncologically preserved. For tumours located in the transverse colon or at the splenic flexure, this procedure may be the best surgical option.

The contribution of inflammatory astrocytes to BBB impairments in a brain-chip model of Parkinson's disease.

Astrocyte dysfunction has previously been linked to multiple neurodegenerative disorders including Parkinson's disease (PD). Among their many roles, astrocytes are mediators of the brain immune response, and astrocyte reactivity is a pathological feature of PD. They are also involved in the formation and maintenance of the blood-brain barrier (BBB), but barrier integrity is compromised in people with PD. This study focuses on an unexplored area of PD pathogenesis by characterizing the interplay between astrocytes, inflammation and BBB integrity, and by combining patient-derived induced pluripotent stem cells with microfluidic technologies to generate a 3D human BBB chip. Here we report that astrocytes derived from female donors harboring the PD-related LRRK2 G2019S mutation are pro-inflammatory and fail to support the formation of a functional capillary in vitro. We show that inhibition of MEK1/2 signaling attenuates the inflammatory profile of mutant astrocytes and rescues BBB formation, providing insights into mechanisms regulating barrier integrity in PD. Lastly, we confirm that vascular changes are also observed in the human postmortem substantia nigra of both males and females with PD.

Primary aneurysmal bone cysts in children: percutaneous sclerotherapy with absolute alcohol and proposal of a vascular classification.

BACKGROUND: Percutaneous sclerotherapy is an effective treatment for aneurysmal bone cysts (ABCs). OBJECTIVE: The purpose of this study was to demonstrate the safety and efficacy of sclerotherapy with absolute alcohol and to propose a vascular classification of ABCs based on a retrospective review. MATERIALS AND METHODS: This was a review of children treated with absolute alcohol sclerotherapy for ABC at a single institution from January 1995 until November 2009. Treatment response was evaluated radiographically and clinically. Cyst fluid was classified as clear, partially bloody, or bloody. Presence of any venous drainage of the cyst was assessed by injection of contrast medium into the cyst cavity. RESULTS: Twenty-nine children with ages ranging from 2 to 16 years were included. Treatment response was good in 17 (59%), partial in 9 (31%), and poor in 3 (10%) children. Venous drainage was absent in six out of seven clear-fluid cysts, which we classified as lymphatic. Drainage was present in all seven bloody-fluid cysts, which we classified as venous. In seven partially bloody-fluid cysts, venous drainage was seen in three. CONCLUSION: Sclerotherapy with absolute alcohol is a safe and effective treatment of ABC. We propose classifying ABC as lymphatic or venous and suggest considering ABC intraosseous slow-flow vascular malformations.

SATB2-associated syndrome: characterization of skeletal features and of bone fragility in a prospective cohort of 19 patients.

BACKGROUND: Individuals with pathogenic variants in SATB2 display intellectual disability, speech and behavioral disorders, dental abnormalities and often features of Pierre Robin sequence. SATB2 encodes a transcription factor thought to play a role in bone remodeling. The primary aim of our study was to systematically review the skeletal manifestations of SATB2-associated syndrome. For this purpose, we performed a non-interventional, multicenter cohort study, from 2017 to 2018. We included 19 patients, 9 females and 10 males ranging in age from 2 to 19 years-old. The following data were collected prospectively for each patient: clinical data, bone markers and calcium and phosphate metabolism parameters, skeletal X-rays and bone mineral density. RESULTS: Digitiform impressions were present in 8/14 patients (57%). Vertebral compression fractures affected 6/17 patients (35%). Skeletal demineralization (16/17, 94%) and cortical thinning of vertebrae (15/17) were the most frequent radiological features at the spine. Long bones were generally demineralized (18/19). The distal phalanges were short, thick and abnormally shaped. C-telopeptide (CTX) and Alkaline phosphatase levels were in the upper normal values and osteocalcin and serum procollagen type 1 amino-terminal propeptide (P1NP) were both increased. Vitamin D insufficiency was frequent (66.7%). CONCLUSION: We conclude that SATB2 pathogenic variants are responsible for skeletal demineralization and osteoporosis. We found increased levels of bone formation markers, supporting the key role of SATB2 in osteoblast differentiation. These results support the need for bone evaluation in children and adult patients with SATB2-associated syndrome (SAS).

Acidification of the male reproductive tract by a proton pumping (H+)-ATPase.

An acidic luminal pH (ref. 1-3) is involved in sperm maturation, and in maintaining sperm in an immotile state in the epididymis and vas deferens (2,4-6). Neutralization by prostatic fluid is one of a complex series of events that triggers sperm motility (2,7,8). Failure of the acidification mechanism might, therefore, result in poor sperm maturation, premature motility and infertility. We have shown that a vacuolar (H+)-ATPase is expressed at high levels on the luminal plasma membrane of specialized cells in the epididymis (9), which closely resemble acid-secreting kidney intercalated cells (10,11). We now show that similar cells are also present in the vas deferens, and that a bafilomycin-sensitive proton flux can be detected using a noninvasive proton-selective vibrating probe. Up to 80% of the net proton secretion in the vas deferens is inhibited by bafilomycin, consistent with a major role of a vacuolar-type (H+)-ATPase in this process. This acidification mechanism is a potential target for novel strategies aimed at modulating the acidification capacity of parts of the male reproductive tract and, therefore, in regulating male fertility.

Epithelial dynamics in the epididymis: role in the maturation, protection, and storage of spermatozoa.

Epithelial cells line the lumen of tubular organs and are key players in their respective functions. They establish a unique luminal environment by providing a protective barrier and by performing vectorial transport of ions, nutrients, solutes, proteins, and water. Complex intercellular communication networks, specific for each organ, ensure their interaction with adjacent epithelial and non-epithelial cells, allowing them to respond to and modulate their immediate environment. In the epididymis, several epithelial cell types work in a concerted manner to establish a luminal acidic milieu that is essential for the post-testicular maturation and storage of spermatozoa. The epididymis also prevents autoimmune responses against auto-antigenic spermatozoa, while ensuring protection against ascending and blood pathogens. This is achieved by a network of immune cells that are in close contact and interact with epithelial cells. This review highlights the coordinated interactions between spermatozoa, basal cells, principal cells, narrow cells, clear cells, and immune cells that contribute to the maturation, protection, selection, and storage of spermatozoa in the lumen of the epididymis.

Revisiting structure/functions of the human epididymis.

BACKGROUND: The epididymis is the hallmark of all vertebrate species practicing internal fertilization. While the functions of the epididymis are well documented in laboratory rodents and some domestic animals, the structure and functions of the epididymis in humans remain poorly documented. OBJECTIVES: Using human tissues obtained with the collaboration of our local organ transplantation program, the histology, cell types, and three-dimensional organization of the excurrent duct were investigated. Microarrays were performed to determine the gene expression pattern along the human epididymis. MATERIALS AND METHODS: The histology of longitudinal sections of the proximal epididymis was described, and immunohistochemistry using specific antibodies was used to characterize cell types of the efferent duct and caput epididymis epithelia. The epididymis was divided into eight segments permitting gene profiling by microarray and gene ontology analysis. RESULTS: The proximal region of the human epididymis is formed exclusively by efferent ducts. These ducts form a complex histological structure particularly at the junction of the efferent ducts and caput epididymis. The efferent ducts exhibit a specific cellular signature when compared with the adjacent epididymis tubule. Efferent duct gene expression is not segmented and is dedicated to cilium differentiation and movement. The gene expression pattern of the caput segment is homogeneous and specialized in defense and immune responses and fertilization. DISCUSSION: In murine species, the epididymis is segmented into the initial segment, caput, corpus, and cauda regions, whereas in humans, the proximal region is formed by efferent ducts. The caput tubules have their own histological organization with a well-defined gene expression pattern. The distal corpus and cauda epididymis are distinct by a limited number of differentially expressed genes. CONCLUSIONS: Knowledge of epididymis functions and structure obtained using laboratory species should be extrapolated to humans with caution.

Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells.

In collecting duct principal cells, aquaporin 2 (AQP2) is shuttled from intracellular vesicles to the plasma membrane upon vasopressin (VP) stimulation. VP activates adenylyl cyclase, increases intracellular cAMP, activating protein kinase A (PKA) to phosphorylate AQP2 on the COOH-terminal residue, serine 256. Using rat kidney slices and LLC-PK1 cells stably expressing AQP2 (LLC-AQP2 cells), we now show that AQP2 trafficking can be stimulated by cAMP-independent pathways. In these systems, the nitric oxide (NO) donors sodium nitroprusside (SNP) and NONOate and the NO synthase substrate L-arginine mimicked the effect of VP, stimulating relocation of AQP2 from cytoplasmic vesicles to the plasma membrane. Unlike VP, these other agents did not increase intracellular cAMP. However, SNP increased intracellular cGMP, and exogenous cGMP stimulated AQP2-membrane insertion. Atrial natriuretic factor, which signals via cGMP, also stimulated AQP2 translocation. The VP and SNP effects were blocked by the kinase inhibitor H89. SNP did not stimulate membrane insertion of AQP2 in LLC-PK1 cells expressing the phosphorylation-deficient mutant 256SerAla-AQP2, indicating that phosphorylation of Ser256 is required for signaling. Both PKA and cGMP-dependent protein kinase G phosphorylated AQP2 on this COOH-terminal residue in vitro. These results demonstrate a novel, cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion in renal epithelial cells.

A survey of modelling methods for high-fidelity wind farm simulations using large eddy simulation.

Large eddy simulations (LES) of wind farms have the capability to provide valuable and detailed information about the dynamics of wind turbine wakes. For this reason, their use within the wind energy research community is on the rise, spurring the development of new models and methods. This review surveys the most common schemes available to model the rotor, atmospheric conditions and terrain effects within current state-of-the-art LES codes, of which an overview is provided. A summary of the experimental research data available for validation of LES codes within the context of single and multiple wake situations is also supplied. Some typical results for wind turbine and wind farm flows are presented to illustrate best practices for carrying out high-fidelity LES of wind farms under various atmospheric and terrain conditions.This article is part of the themed issue 'Wind energy in complex terrains'.

Is there a future for cyclo-oxygenase inhibitors in Alzheimer's disease?

Several epidemiological studies have indicated that the long-term use of NSAIDs, most of which are cyclo-oxygenase (COX) inhibitors, may reduce the risk of Alzheimer's disease. For this reason, anti-inflammatory COX-inhibiting NSAIDs have received increased attention in experimental and therapeutic trials for Alzheimer's disease. However, several recent efforts attempting to demonstrate a therapeutic effect of NSAIDs in Alzheimer's disease have largely failed. Clinicians and scientists currently believe that this lack of success may be attributable to two key problems: (i) clinical trials of NSAIDs have been conducted in patients with late-stage Alzheimer's disease, wherein advanced neurodegeneration may be refractory to anti-inflammatory drug treatment; and (ii) it is not known which of the large family of NSAIDs (i.e. COX-1, COX-2 or mixed inhibitors) is most efficacious in preventing Alzheimer's disease. The wide list of putative functions for COX in the brain, and the significant functional heterogeneity of NSAIDs, which appear to influence the beta-amyloid (Abeta) neuropathology associated with Alzheimer's disease via both COX-dependent and COX-independent pathways, complicate the interpretation of the mechanisms through which COX-inhibiting NSAIDs may beneficially influence Alzheimer's disease. As discussed in this review, for patients at high risk of developing Alzheimer's disease (e.g. those with mild cognitive impairment), preventative treatment with COX-inhibiting NSAIDs may ultimately represent a viable strategy in the management of clinical Alzheimer's disease. However, the recent evidence showing an increased risk of major cardiovascular events among patients treated with certain COX-1 and COX-2 inhibitors leaves many questions unanswered. We suggest that further investigation into the physiological role(s) of COXs in normal health and in disease conditions, and the identification of safer and better tolerated COX inhibitors, will provide renewed impetus to the application of anti-inflammatory strategies for the prevention and treatment of Alzheimer's disease.

Recombinant pICln forms highly cation-selective channels when reconstituted into artificial and biological membranes.

pICln has been proposed to be the swelling-activated anion channel responsible for ICl, swell, or a channel regulator. We tested the anion channel hypothesis by reconstituting recombinant pICln into artificial and biological membranes. Single channels were observed when pICln was reconstituted into planar lipid bilayers. In the presence of symmetrical 300 mM KCl, the channels had a high open probability and a slope conductance of 48 pS, and were outwardly rectifying. Reduction of trans KCl to 50 mM shifted the reversal potential by -31.2 +/- 0.06 mV, demonstrating that the channel is at least seven times more selective for cations than for anions. Consistent with this finding, channel conductance was unaffected by substitution of Cl- with glutamate, but was undetectable when K+ was replaced by N-methyl-D-glucamine. Reconstitution of pICln into liposomes increased 86Rb+ uptake by three- to fourfold, but had no effect on 36Cl- uptake. Phosphorylation of pICln with casein kinase II or mutation of G54, G56, and G58 to alanine decreased channel open probability and 86Rb+ uptake. When added to the external medium bathing Sf9 cells, pICln inserted into the plasma membrane and increased cell cation permeability. Taken together, these observations demonstrate that channel activity is due to pICln and not minor contaminant proteins. However, these findings do not support the hypothesis that pICln is the anion-selective ICl, swell channel. The observed cation channel activity may reflect an as yet to be defined physiological function of pICln, or may be a consequence of in vitro reconstitution of purified, recombinant protein.

Basolateral distribution of caveolin-1 in the kidney. Absence from H+-atpase-coated endocytic vesicles in intercalated cells.

In kidney epithelial cells, a variety of physiological processes are dependent on the active recycling of membrane proteins between intracellular vesicles and the cell surface. Although clathrin-mediated endocytosis occurs in several renal cell types, endocytosis can also occur by non-clathrin-coated vesicles, including pinocytotic structures known as caveolae that contain a novel coat protein, caveolin. Exo- and endocytosis of a vacuolar H+-ATPase in intercalated cells also occurs via specialized "coated" vesicles that do not contain clathrin. The aim of this study was to localize caveolin in the kidney and, in addition, to determine whether it could be a component of the H+-ATPase recycling process. Using an antibody against the alpha- and beta-isoforms of caveolin-1, our immunocytochemical data show a marked heterogeneity in the cellular expression of this isoform of caveolin in kidney. In contrast, caveolin-3 was not detectable in renal epithelial cells. Caveolin-1 was abundant in endothelial cells and smooth muscle cells and was present in the parietal cells of Bowman's capsule. Distal tubule cells, connecting tubule cells, and collecting duct principal cells exhibited marked punctate basolateral staining, corresponding to the presence of caveolae detected by electron microscopy, whereas all intercalated cells were negative in both cortex and medulla. These data indicate that although caveolin-1 may participate in basolateral events in some kidney epithelial cell types, it does not appear to be involved in the regulated recycling of H+-ATPase in intercalated cells. Therefore, these cells recycle H+-ATPase by a mechanism that involves neither clathrin nor caveolin-1.