Mammalian Clusterin associated protein 1 is an evolutionarily conserved protein required for ciliogenesis

BACKGROUND: Clusterin associated protein 1 (CLUAP1) was initially characterized as a protein that interacts with clusterin, and whose gene is frequently upregulated in colon cancer. Although the consequences of these observations remain unclear, research of CLUAP1 homologs in C. elegans and zebrafish indicates that it is needed for cilia assembly and maintenance in these models. To begin evaluating whether Cluap1 has an evolutionarily conserved role in cilia in mammalian systems and to explore the association of Cluap1 with disease pathogenesis and developmental abnormalities, we generated Cluap1 mutant mice. METHODS: Cluap1 mutant embryos were generated and examined for gross morphological and anatomical defects using light microscopy. Reverse transcription PCR, β-galactosidase staining assays, and immunofluorescence analysis were used to determine the expression of the gene and localization of the protein in vivo and in cultured cell lines. We also used immunofluorescence analysis and qRT-PCR to examine defects in the Sonic hedgehog signaling pathway in mutant embryos. RESULTS: Cluap1 mutant embryos die in mid-gestation, indicating that it is necessary for proper development. Mutant phenotypes include a failure of embryonic turning, an enlarged pericardial sac, and defects in neural tube development. Consistent with the diverse phenotypes, Cluap1 is widely expressed. Furthermore, the Cluap1 protein localizes to primary cilia, and mutant embryos were found to lack cilia at embryonic day 9.5. The phenotypes observed in Cluap1 mutant mice are indicative of defects in Sonic hedgehog signaling. This was confirmed by analyzing hedgehog signaling activity in Cluap1 mutants, which revealed that the pathway is repressed. CONCLUSIONS: These data indicate that the function of Cluap1 is evolutionarily conserved with regard to ciliogenesis. Further, the results implicate mammalian Cluap1 as a key regulator of hedgehog signaling and as an intraflagellar transport B complex protein. Future studies on mammalian Cluap1 utilizing this mouse model may provide insights into the role for Cluap1 in intraflagellar transport and the association with colon cancer and cystic kidney disorders

Coiled-coil domain containing 42 (Ccdc42) is necessary for proper sperm development and male fertility in the mouse

Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans

A CreER Mouse to Study Melanin Concentrating Hormone Signaling in the Developing Brain

The neuropeptide, melanin concentrating hormone (MCH), and its G protein‐coupled receptor, melanin concentrating hormone receptor 1 (Mchr1), are expressed centrally in adult rodents. MCH signaling has been implicated in diverse behaviors such as feeding, sleep, anxiety, as well as addiction and reward. While a model utilizing the Mchr1 promoter to drive constitutive expression of Cre recombinase (Mchr1‐Cre) exists, there is a need for an inducible Mchr1‐Cre to determine the roles for this signaling pathway in neural development and adult neuronal function. Here, we generated a BAC transgenic mouse where the Mchr1 promotor drives expression of tamoxifen inducible CreER recombinase. Many aspects of the Mchr1‐Cre expression pattern are recapitulated by the Mchr1‐CreER model, though there are also notable differences. Most strikingly, compared to the constitutive model, the new Mchr1‐CreER model shows strong expression in adult animals in hypothalamic brain regions involved in feeding behavior but diminished expression in regions involved in reward, such as the nucleus accumbens. The inducible Mchr1‐CreER allele will help reveal the potential for Mchr1 signaling to impact neural development and subsequent behavioral phenotypes, as well as contribute to the understanding of the MCH signaling pathway in terminally differentiated adult neurons and the diverse behaviors that it influences

Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity.

Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin-melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3-5% of all severe obesity cases in humans1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons 6 , organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation 7 . We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans

An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue

BACKGROUND: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them. RESULTS: To facilitate analyses of mammalian cilia function we generated an inducible Cilia(GFP) mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration. CONCLUSIONS: The ability to visualize cilia in live samples within the Cilia(GFP) mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes

Osmotic dehydration of peaches as a function of temperature and concentration of sucrose syrup

The production of dried peach by osmotic dehydration is an alternative for using the excess of peach production. The influence of the temperature and concentration of the sucrose syrup in osmotic dehydration of peaches was evaluated. Variations in physical and chemical properties, and osmotic dehydration parameters (weight loss and water loss; solids incorporation) were investigated. An experimental central composite design was employed ranging the temperature (30 to 50 ºC) and concentration (45 to 65 °Brix), keeping the syrup:fruit mass ratio 4:1, process time 4 h, and format pieces (halves). The degree of acceptance was used in the sensory analysis, evaluating the following characteristics: appearance, taste, texture, colour and overall quality. The results were modelled using the Statistica program (v. 6.0) employing the Response Surface Methodology. The following mathematical models resulted significant (p < 0.05) and predictive: dimensionless soluble solids content and colour parameter L*, water loss and weight loss parameters. The temperature was the prevalent effect. The process conditions from 50 to 54.1 °C and from 55 to 65 °Brix presented more water loss and better sensory performances.A desidratação osmótica pela qual passa a produção de fruta, é uma alternativa para o aproveitamento dos excedentes da persicultura. Neste trabalho se avaliou a influência da temperatura e da concentração do xarope de sacarose na desidratação osmótica de pêssegos, e se investigaram variações de propriedades físicas, químicas e dos parâmetros da desidratação osmótica (perda de massa e de água; incorporação de sólidos). O delineamento central composto rotacional foi utilizado variando-se a temperatura (30 a 50 ºC) e a concentração (45 a 65 °Brix) da solução osmótica e se fixando a razão mássica xarope:fruta 4:1, tempo de processo 4 h, e formato dos pedaços (metades). Na análise sensorial empregou-se o método de aceitação avaliando-se os atributos aparência, sabor, textura, cor e qualidade geral. Modelaram-se os resultados com o programa Statistica (v 6.0), através da Metodologia de Superfície de Resposta. Os seguintes modelos matemáticos resultaram significativos (p < 0,05) e preditivos: adimensionais dos teores de sólidos solúveis e do parâmetro L* de cor, parâmetros perda de água e perda de massa. A temperatura é o efeito preponderante. As condições de processo de 50 a 54,1 ° C e 55 a 65 °Brix, proporcionaram maiores perdas de água e melhores desempenhos sensoriais.16116

Q fever endocarditis masquerading as Mixed cryoglobulinemia type II. A case report and review of the literature

BACKGROUND: The clinical manifestations of Q fever endocarditis are protean in nature. Mixed cryoglobulinemia type II is rarely a facet of the presenting clinical manifestations of Q fever endocarditis. CASE PRESENTATION: We report a case of a 65-year-old pensioner with such an association and review the literature. As transesophageal echocardiograms are usually normal and blood cultures are usually negative in Q fever endocarditis, many of the manifestations (fever, rash, glomerulonephritis/evidence of renal disease, low serum C4 complement component, presence of mixed type II cryoglobulin, constitutional symptoms as arthralgias and fatigue) can be attributed to Mixed cryoglobulinemia type II per se. The use of Classic Duke Endocarditis Service criteria does not always suffice for the diagnosis of Q fever. CONCLUSION: The application of the modified criteria proposed by Fournier et al for the improvement of the diagnosis of Q fever endocarditis will help to reach the diagnosis earlier and thus reduce the high mortality of the disease. We would like to stress the importance of ruling out the diagnosis of Q fever endocarditis in cases of mixed type II cryoglobulinemia

Two-stage revision for prosthetic joint infection: predictors of outcome and the role of reimplantation microbiology

We describe rates of success for two-stage revision of prosthetic joint infection (PJI), including data on reimplantation microbiology. Methods: We retrospectively collected data from all the cases of PJI that were managed with two-stage revision over a 4 year period. Patients were managed with an antibiotic-free period before reimplantation, in order to confirm, clinically and microbiologically, that infection was successfully treated. Results: One hundred and fifty-two cases were identified. The overall success rate (i.e. retention of the prosthesis over 5.75 years of follow-up) was 83%, but was 89 % for first revisions and 73 % for re-revisions [hazard ratio2.9, 95 % confidence interval (CI) 1.2–7.4, P0.023]. Reimplantation microbiology was frequently positive (14%), but did not predict outcome (hazard ratio1.3, 95 % CI 0.4–3.7, P0.6). Furthermore, most unplanned debridements following the first stage were carried out before antibiotics were stopped (25 versus 2 debridements). Conclusions: We did not identify evidence supporting the use of an antibiotic-free period before reimplantation and routine reimplantation microbiology. Re-revision was associated with a significantly worse outcome

Chronic Fluid Flow Is an Environmental Modifier of Renal Epithelial Function

Although solitary or sensory cilia are present in most cells of the body and their existence has been known since the sixties, very little is been known about their functions. One suspected function is fluid flow sensing- physical bending of cilia produces an influx of Ca++, which can then result in a variety of activated signaling pathways. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a progressive disease, typically appearing in the 5th decade of life and is one of the most common monogenetic inherited human diseases, affecting approximately 600,000 people in the United States. Because ADPKD is a slowly progressing disease, I asked how fluid flow may act, via the primary cilium, to alter epithelial physiology during the course of cell turnover. I performed an experiment to determine under what conditions fluid flow can result in a change of function of renal epithelial tissue. A wildtype epithelial cell line derived the cortical collecting duct of a heterozygous offspring of the Immortomouse (Charles River Laboratory) was selected as our model system. Gentle orbital shaking was used to induce physiologically relevant fluid flow, and periodic measurements of the transepithelial Sodium current were performed. At the conclusion of the experiment, mechanosensitive proteins of interest were visualized by immunostaining. I found that fluid flow, in itself, modifies the transepithelial sodium current, cell proliferation, and the actin cytoskeleton. These results significantly impact the understanding of both the mechanosensation function of primary cilia as well as the understanding of ADPKD disease progression