MHM assay: molecular sexing based on the sex-specific methylation pattern of the MHM region in chickens

Bird sex determination using molecular methods has proved to be a valuable tool in different studies. Although it is possible to sex most birds by coupling the CHD assay with others available methods, no sex-determining gene like SRY in mammalians has been identified in birds. The male hypermethylated (MHM) region on the Z chromosome has been found to be hypermethylated in males and hypomethylated in females in birds of the order Galliformes. We analyzed the DNA from feathers of 50 adult chickens to verify the methylation pattern of the MHM region by PCR and the restriction enzyme HpaII (a method named MHM assay). The results, visualized in agarose gel, were compared with PCR amplification of the CHD-Z and CHD-W genes (polyacrylamide gel) and with the birds` phenotype. All males (25) showed hypermethylation of the MHM region, and all females (25) showed hypomethylation. The sexing by MHM assay was in according with phenotype and CHD sexing. To our knowledge, this is the first study that uses the MHM region for sexing birds. Although the real role of the MHM region in the sex determination is still unclear, this could be a universal marker for sexing birds and may be involved in sex determination by its influence on transcriptional processes. The MHM assay could be a good alternative for CHD assay in developmental studies.

Polymorphisms of Lewis and Secretor genes are related to breast cancer and metastasis in axillary lymph nodes

ABH and Lewis antigen expression has been associated with cancer development and prognosis, tumor differentiation, and metastasis. Considering that invasive ductal breast carcinoma (IDC) presents multiple molecular alterations, the aim of the present study was to determine whether the polymorphism of ABO, Lewis, and Secretor genes, as well as ABO phenotyping, could be associated with tumor differentiation and lymph nodes metastasis. Seventy-six women with IDC and 78 healthy female blood donors were submitted to ABO phenotyping/genotyping and Lewis and Secretor genotyping. Phenotyping was performed by hemagglutination and genotyping by the polymerase chain reaction with sequence-specific primers. ABO, Lewis, and Secretor genes were classified by individual single nucleotide polymorphism at sites 59, 1067, 202, and 314 of the Lewis gene, 428 of the Secretor gene, and 261 (O1 allele), 526 (O2 and B allele), and 703 (B allele). No association was found between breast cancer and ABO antigen expression (P = 0.9323) or genotype (P = 0.9356). Lewis-negative genotype was associated with IDC (P = 0.0126) but not with anatomoclinical parameters. Nonsecretor genotype was associated with axillary lymph node metastasis (P = 0.0149). In conclusion, Lewis and Secretor genotyping could be useful to predict respectively breast cancer susceptibility and axillary lymph nodes metastasis.

Effect of image analysis software on neurofunctional activation during processing of emotional human faces

Functional brain imaging techniques such as functional MRI (fMRI) that allow the in vivo investigation of the human brain have been exponentially employed to address the neurophysiological substrates of emotional processing. Despite the growing number of fMRI studies in the field, when taken separately these individual imaging studies demonstrate contrasting findings and variable pictures, and are unable to definitively characterize the neural networks underlying each specific emotional condition. Different imaging packages, as well as the statistical approaches for image processing and analysis, probably have a detrimental role by increasing the heterogeneity of findings. In particular, it is unclear to what extent the observed neurofunctional response of the brain cortex during emotional processing depends on the fMRI package used in the analysis. In this pilot study, we performed a double analysis of an fMRI dataset using emotional faces. The Statistical Parametric Mapping (SPM) version 2.6 (Wellcome Department of Cognitive Neurology, London, UK) and the XBAM 3.4 (Brain Imaging Analysis Unit, Institute of Psychiatry, Kings College London, UK) programs, which use parametric and non-parametric analysis, respectively, were used to assess our results. Both packages revealed that processing of emotional faces was associated with an increased activation in the brain`s visual areas (occipital, fusiform and lingual gyri), in the cerebellum, in the parietal cortex, in the cingulate cortex (anterior and posterior cingulate), and in the dorsolateral and ventrolateral prefrontal cortex. However, blood oxygenation level-dependent (BOLD) response in the temporal regions, insula and putamen was evident in the XBAM analysis but not in the SPM analysis. Overall, SPM and XBAM analyses revealed comparable whole-group brain responses. Further Studies are needed to explore the between-group compatibility of the different imaging packages in other cognitive and emotional processing domains. (C) 2009 Elsevier Ltd. All rights reserved.

Significance of topoisomerase III beta expression in breast ductal carcinomas: strong associations with disease-specific survival and metastasis

Topoisomerases are ubiquitous nuclear enzymes that regulate DNA structure in eukaryotic cells. The role of topoisomerase III beta, the newest member of the topoisomerase family, in the clinical outcome of breast cancer is still poorly understood. This study aims to investigate the immunoexpression of topoisomerase III beta in breast cancer and its relationships with clinicopathologic features and immunohistochemical markers of prognostic significance in breast pathology. Using tissue microarrays containing 171 cases of primary invasive breast cancer, we analyzed the immunoexpression of topoisomerase III beta, estrogen receptor, progesterone receptor, HER-2, BRCA-1, p53, and Ki67. Immunostaining for topoisomerase III beta was found in 33.9% of breast carcinomas, and immunopositivity was correlated with distant metastasis (P = .036) and death (P = .006). Decreased expression of topoisomerase III beta correlated with low expression of Ki67 (P < .001) and negativity for HER-2 (P < .001), BRCA-1 (P = .001), and p53 (P < .001). In the multivariate analysis, topoisomerase Hip expression was a significant predictor of survival (hazard ratio, 3.006 [95% confidence interval, 1.582-5.715]; P = .001). In conclusion, topoisomerase III beta expression can be a useful marker in assessing the prognosis of patients with breast cancer and is an independent predictor of survival. (C) 2010 Elsevier Inc. All rights reserved.

Determination of temperature variation during the individual steps of the production of hospital diets of modified consistency

Background & aim: Many disease outbreaks of food origin are caused by foods prepared in Food Service and Nutrition Units of hospitals, affecting hospitalized patients who, in most cases, are immunocompromised and therefore at a higher risk of severe worsening of their clinical status. The aim of this study was to determine the variations in temperature and the time-temperature factor of hospital diets. Methods: The time and temperature for the preparation of 4 diets of modified consistency were determined on 5 nonconsecutive days in a hospital Diet and Nutrition Unit at the end of preparation and during the maintenance period, portioning and distribution at 3 sites, i.e., the first, the middle and the last to receive the diets. Results and discussion: All foods reached an adequate temperature at the end of cooking, but temperature varied significantly from the maintenance period to the final distribution, characterizing critical periods for microorganism proliferation. During holding, temperatures that presented a risk were reached by 16.7% of the meats and 59% of the salads of the general diet, by 16.7% of the garnishes in the bland diet and by 20% of the meats and garnishes in the viscous diet. The same occurred at the end of distribution for 100% of the hot samples and of the salads and for 61% of the desserts. None of the preparations remained at risk temperature for a time exceeding that established by law. Conclusion: The exposure to inadequate temperature did not last long enough to pose risks to the patient.

Mineralocorticoid Receptor Mutations Differentially Affect Individual Gene Expression Profiles in Pseudohypoaldosteronism Type 1

Context: Type 1 pseudohypoaldosteronism (PHA1), a primary form of mineralocorticoid resistance, isdueto inactivating mutations of the NR3C2 gene, coding for the mineralocorticoid receptor (MR). Objective: The objective of the study was to assess whether different NR3C2 mutations have distinct effects on the pattern of MR-dependent transcriptional regulation of aldosterone-regulated genes. Design and Methods: Four MR mutations affecting residues in the ligand binding domain, identified in families with PHA1, were tested. MR proteins generated by site-directed mutagenesis were analyzed for their binding to aldosterone and were transiently transfected into renal cells to explore the functional effects on the transcriptional activity of the receptors by cis-trans-cotrans-activation assays and by measuring the induction of endogenous gene transcription. Results: Binding assays showed very low or absent aldosterone binding for mutants MR(877Pro), MR(848Pro), and MR(947stop) and decreased affinity for aldosterone of MR(843Pro). Compared with wildtype MR, the mutations p.Leu843Pro and p.Leu877Pro displayed half-maximal aldosterone-dependent transactivation of reporter genes driven by mouse mammary tumor virus or glucocorticoid response element-2 dependent promoters, whereas MR(848Pro) and MR(947stop) nearly or completely lost transcriptional activity. Although MR(848Pro) and MR(947stop) were also incapable of inducing aldosterone-dependent gene expression ofendogenoussgk1, GILZ, NDRG2, and SCNN1A, MR(843Pro) retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR(877Pro) negatively affected the expression of sgk1, NDRG2, and SCNN1A. Conclusions: Our data demonstrate that MR mutations differentially affect individual gene expression in a promoter-dependent manner. Investigation of differential gene expression profiles in PHA1 may allow a better understanding of the molecular substrate of phenotypic variability and to elucidate pathogenic mechanisms underlying the disease. (J Clin Endocrinol Metab 96: E519-E527, 2011)

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children`s growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781. (Blood. 2009; 114: 3216-3226)

Keyhole sign: how specific is it for the diagnosis of posterior urethral valves?

Objectives Posterior urethral valves (PUV) are the most common cause of renal impairment in boys during early childhood. Although antenatal suspicion of this pathology has become quite common in recent years, prenatal diagnosis remains challenging. The aim of this study was to evaluate the predictive value of different ultrasound criteria currently used to diagnose PUV. Methods We reviewed the antenatal and postnatal files of 54 male patients referred to our center from 2000 to 2006 after detection of fetal bilateral hydronephrosis. The following ultrasound criteria were evaluated in relation to the postnatal diagnosis of P U V: amniotic fluid volume, bladder wall thickness, bladder dilatation and the presence of the `keyhole sign`. Results Forty-two fetuses (77.8%) were suspected to have PUV on prenatal examination. Out of these, 29 (69.0%) had PUV confirmed postnatally. The sensitivity and specificity of the antenatal diagnosis of PUV were 94% and 43%, respectively. Increased bladder wall thickness and bladder dilatation were highly associated with the diagnosis of PUV (P < 0.001). However, a thick-walled bladder was observed in 39.1% and a dilated bladder in 47.8% of the infants with a postnatal diagnosis other than PUV. The presence of the keyhole sign was not found to predict a diagnosis of PUV (P = 0.27). Conclusion In this series the use of classical prenatal ultrasound signs to diagnose PUV showed high sensitivity but low specificity. The best diagnostic indicators were increased bladder wall thickness and dilatation of the bladder. The keyhole sign was not found to be a reliable predictor of PUV. Copyright (C) 2009 ISUOG. Published by John Wiley & Sons, Ltd.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

Protective effects of carvedilol on systemic vascular damage induced by angiotensin II: Organ-specific effects independent of antihypertensive effects

Background: The protective effect of carvedilol on multiple organ damage induced by angiotensin II (Ang II) remains unclear. The aim of this study was to evaluate the protective effect of carvedilol on the heart, liver, and kidney in rats infused with Ang II. Material/Methods: Wistar rats were randomly distributed into three groups: control (no treatment), continuously infused with Ang II (150 eta g/min for 72 hr), and treated with Ang II + carvedilol (90 mg/kg/d). Histological sections of the myocardium, kidney, and liver were analyzed for the presence of necrosis. Results: Ang II induced arterial hypertension which was not affected by carvedilol treatment (tail-cuff blood pressures, control: 125 +/- 13.6, Ang II: 163 +/- 27.3, Ang II + CV: 178 +/- 39.8 mmHg, p<0.05). Also, there were perivascular inflammation and necrosis in the myocardium, kidney, and hepatocytes necrosis around the terminal vein. Carvedilol treatment fully prevented damage to the heart and kidney and attenuated liver lesions induced by the Ang II infusion. Conclusions: The protective effect of carvedilol on perivascular damage induced by Ang II infusion depended on the target organ. The prevention of heart damage occurred independently of the antihypertensive effects of carvedilol.

Modeling the Dynamics of Viral Evolution Considering Competition Within Individual Hosts and at Population Level: The Effects of Treatment

We consider two viral strains competing against each other within individual hosts (at cellular level) and at population level (for infecting hosts) by studying two cases. In the first case, the strains do not mutate into each other. In this case, we found that each individual in the population can be infected by only one strain and that co-existence in the population is possible only when the strain that has the greater basic intracellular reproduction number, R (0c) , has the smaller population number R (0p) . Treatment against the one strain shifts the population equilibrium toward the other strain in a complicated way (see Appendix B). In the second case, we assume that the strain that has the greater intracellular number R (0c) can mutate into the other strain. In this case, individual hosts can be simultaneously infected by both strains (co-existence within the host). Treatment shifts the prevalence of the two strains within the hosts, depending on the mortality induced by the treatment, which is, in turn, dependent upon the doses given to each individual. The relative proportions of the strains at the population level, under treatment, depend both on the relative proportions within the hosts (which is determined by the dosage of treatment) and on the number of individuals treated per unit time, that is, the rate of treatment. Implications for cases of real diseases are briefly discussed.

Food aversion: A critical balance between allergen-specific IgE levels and taste preference

Animals sensitized to allergens change their feeding behavior and avoid drinking the otherwise preferred sweetened solutions containing the allergens. This phenomenon, known as food aversion, appears to be mediated by allergen-specific IgE antibodies. Here we investigated food aversion in BALB/c and C57BL/6 mice, which differ in their allergic responses to the allergen ovalbumin as well as in their preference for sweet taste. BALB/c mice present higher levels of IgE and a natural lower preference for sweet flavors when compared to C57BL/6 mice. Specifically, we studied a conflicting situation in which animals simultaneously experienced the aversive contact with the allergen and the attractive sweet taste of increasing concentrations of sucrose. We found that BALB/c mice were more prone to develop food aversion than C57BL/6 mice and that this aversive behavior could be abolished in both strains by increasing the palatability of the solution containing the allergen. In both strains food aversion was positively correlated with the levels of allergen-specific IgE antibodies and inversely correlated with their preference for sucrose sweetened solutions. (C) 2009 Elsevier Inc. All rights reserved.

Effects of individual housing on behavior and resistance to Ehrlich tumor growth in mice

This study analyzed in Balb/C mice the effects of individual housing on behavior, serum corticosterone and resistance to Ehrlich tumor growth. Mice (60 days old) were individually (IH) or grouped housed (G) (10-12 animals/cage) for 14-21 days. The 1st day of the housing condition was considered experimental day 1 (ED1). Results showed that on ED21, IH mice, when compared to G mice, presented no differences on corticosterone serum levels when kept undisturbed; however, an increased level of this hormone was observed in IH mice after an immobilization stress challenge. An increased time spent in the plus-maze closed arms and a decreased time in the open arms were also observed in IH mice. When compared to G animals, after inoculation with 105 Ehrlich tumor cells on ED1, IH mice presented an increase in volume of ascitic fluid and number of tumor cells. The survival time of IH mice was also shorter than that measured in G animals. Furthermore, IH mice injected with a different number of tumor cells on ED1 always presented increased Ehrlich tumor cells than G group. Interestingly. these effects were not observed when the tumor cells injection was done on ED4. These results suggest that individual-housing conditions induce an altered immune-endocrine response and, at the same time, decrease animals` resistance to Ehrlich tumor growth. It is proposed that the neural link between the behavioral and immunological changes observed after the stress of individual housing might involve the activation of the HPA axis. (C) 2008 Elsevier Inc. All rights reserved.

Maned wolf survival rate in central Brazil

Although many carnivores are of conservation concern, most are poorly studied. The maned wolf Chrysocyon brachyurus Illiger, 1811 is the largest South American canid with a broad distribution; however, the largest portion of its range is in the Brazilian Cerrado savannah, where due to intensive agricultural expansion, it is threatened by habitat loss. Maned wolf population trends are virtually unknown. We analyzed radio telemetry data from a 13-year study in Emas National Park, central Brazil, with Burnham`s live recapture/dead recovery models in the program MARK to obtain the first analytically sound estimate of the apparent maned wolf survival rate. We constructed 16 candidate models including variation in survival rate and resighting probability associated with an individual`s sex or age and year of study. Apparent adult survival rate throughout the study ranged from 0.28 (se=0.08) to 0.97 (se=0.06). There was no evidence for sex specificity but strong support for time variation. Model weights supported an age effect and the subadult survival rate was 0.63 (se=0.15). Results indicate similar life patterns for male and female maned wolves and similar mortality risks for adults and subadults in the study area. The observed temporal fluctuations of adult survival rate are important for population dynamics as they decrease average population growth rates. Population dynamics are central for conservation planning and our results are an important step towards a better understanding of the maned wolf`s ecology.

Specific infiltration of langerin-positive dendritic cells in EBV-infected tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma

We report here the existence of a novel subset of langerin (CD207)-positive, immature dendritic cells (DCs) (CD83(neg)) abundantly infiltrating Epstein Barr virus (EBV)-infected areas in tonsil, Hodgkin lymphoma and nasopharyngeal carcinoma. These CD207(+) DCs differ from conventional epidermal Langerhans cells in their lack of CD1a and CCR6 and their unusual tissue localization. CD207(+) DC infiltration strongly correlates with EBV infection because it was neither detected in EBV negative specimens nor in tissues infected with other human viruses. These immature DCs might represent good candidates for induction of the EBV-specific immune response.