European ancestry and polymorphisms in DNA repair genes modify the risk of melanoma: A case-control study in a high UV index region in Brazil

Background: UV radiation is the major environmental factor related to development of cutaneous melanoma. Besides sun exposure and the influence of latitude, some host characteristics such as skin phototype and hair and eye color are also risk factors for melanoma. Polymorphisms in DNA repair genes could be good candidates for susceptibility genes, mainly in geographical regions exposed to high solar radiation. Objective: Evaluate the role of host characteristic.; and DNA repair polymorphism in melanoma risk in Brazil. Methods: We carried out a hospital-based case-control study in Brazil to evaluate the contribution of host factors and polymorphisms in DNA repair to melanoma risk. A total of 412 patients (202 with melanoma and 210 controls) were analyzed regarding host characteristics for melanoma risk as well as for 11 polymorphisms in DNA repair genes. Results: We found an association of host characteristics with melanoma development, such as eye and hair color, fair skin, history of pigmented lesions removed, sunburns in childhood and adolescence, and also European ancestry. Regarding DNA repair gene polymorphisms, we found protection for the XPG 1104 His/His genotype (OR 0.32; 95% CI 0.13-0.75), and increased risk for three polymorphisms in the XPC gene (PAT+; IV-6A and 939Gln), which represent a haplotype for XPC. Melanoma risk was higher in individuals carrying the complete XPC haplotype than each individual polymorphism (OR 3.64; 95% CI 1.77-7.48). Conclusions: Our data indicate that the host factors European ancestry and XPC polymorphisms contributed to melanoma risk in a region exposed to high sun radiation. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Better CD4+T Cell Recovery in Brazilian HIV-Infected Individuals Under HAART Due to Cumulative Carriage of SDF-1-3`A, CCR2-V64I, CCR5-D32 and CCR5-Promoter 59029A/G Polymorphisms

Polymorphisms of chemokines and chemokine-receptors genes have been shown to influence the rate of progression to AIDS; however, their influence on response to HAART remains unclear. We investigated the frequency of the SDF-1-3`A, CCR2-64I, CCR5-D32 and CCR5-Promoter-59029-A/G polymorphisms in Brazilian HIV-1-infected and uninfected individuals and their influence on CD4+ T-cell evolution HIV-1 infected individuals before and during HAART. Polymorphism detection was done in a transversal study of 200 HIV-1-infected and 82 uninfected individuals. The rate of CD4+ T cell increase or decrease was studied in a cohort of 155 HIV-1 infected individuals on pre and post-HAART. Polymorphisms were determined by PCR associated with RFLP. The rate of CD4+ T-cell decline or increase was also determined. HIV-1 infected and uninfected subjects showed, respectively, frequencies of 0.193 and 0.220 for SDF-1-3`A, of 0.140 and 0.110 for CCR2-V64I, of 0.038 and 0.055 for CCR5-D32, and of 0.442 and 0.390 for CCR5-P-59029-A/G. HIV-1-infected subjects carrying one, two or three of these four polymorphisms showed better CD4+ T-cell recovery than HIV-1-infected subjects carrying the four wild-type alleles (+2.7, +1.6, +3.5, and -0.9 lymphocytes/mu l/month, respectively). Regression logistic analysis showed that the CCR5-D32/CCR2-V64I association was predictor of positive CD4+ T cell slope after HAART. The distribution of polymorphisms did not differ between HIV-1-infected and uninfected individuals, but differed from more homogenous ethnic groups probably reflecting the miscegenation of the Brazilian population. We add further evidence of the role of these polymorphisms by showing that the CD4 gain was influenced by carriage of one or more of the polymorphisms studied here. These results highlight the possibility that these genetic traits can be useful to identify patients at risk for faster progression to AIDS or therapeutic failure.

A novel WT1 heterozygous nonsense mutation (p.K248X) causing a mild and slightly progressive nephropathy in a 46,XY patient with Denys-Drash syndrome

WT1 mutations have been described in a variety of syndromes, including Denys-Drash syndrome (DDS), which is characterized by predisposition to Wilms` tumor, genital abnormalities and development of early nephropathy. The most frequent WT1 defects in DDS are missense mutations located in exons 8-9. Our aim is to report a novel WT1 mutation in a 46,XY patient with a DDS variant, who presented a mild nephropathy with a late onset diagnosed during adolescence. He had ambiguous genitalia at birth. At 4 months of age he underwent nephrectomy (Wilms` tumor) followed by chemotherapy. Ambiguous genitalia were corrected and bilateral gonadectomy was performed. Sequencing of WT1 identified a novel heterozygous mutation (c.742A > T) in exon 4 that generates a premature stop codon (p.K248X). Interestingly, this patient has an unusual DDS nephropathy progression, which reinforces that patients carrying WT1 mutations should have the renal function carefully monitored due to the possibility of late-onset nephropathy.

Total Pancreatectomy: Porcine Model for Inducing Diabetes - Anatomical Assessment and Surgical Aspects

Background: The swine is an essential model for carrying out preclinical research and for teaching complex surgical procedures. There is a lack of experimental models describing anatomical and surgical aspects of total pancreatectomy in the pig. Materials and Methods: The experiments were performed on 10 white male swine weighing 27-33 kg. The animals were premedicated with midazolam (0.4 mg/kg, i.m.) and ketamine (4 mg/kg, i.m.). Anesthesia was induced with propofol (1-2 mg/kg, i.v.) and was maintained with propofol and fentanyl (0.3 mg and 0.1 mu g/kg/min, respectively, i.v.). The surgical period ranged from 44 to 77 min. The pancreas anatomy, and the main arterial, venous and pancreatic duct anatomy were assessed. Results: The pancreas anatomy was composed of 3 lobes, the `splenic`, `duodenal` and `connecting` lobe which is attached to the anterior portion of the portal vein. The splenic artery and the junction of the splenic vein and portal vein were divided. The left gastric artery was dissected and separated from its origin at the splenic artery. The head of the pancreas is disposed in a C shape. The pancreas was dissected and liberated from the right portion of the portal vein and the infrahepatic vena cava. The pancreas was separated from the duodenum preserving the pancreaticoduodenal artery, then we performed the total pancreatectomy preserving the duodenum, common bile duct and spleen. Conclusion: Total pancreatectomy with duodenum, bile duct and spleen preservation in the pig is feasible and an important instrument for research purposes and teaching surgical technique. Copyright (C) 2010 S. Karger AG, Basel

trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3)center dot H(2)O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py = pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600 nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2 h of incubation. The complex with concentrations lower than 1 x 10(-4) M did not show toxicity in B16-F 10 murine cells. The complex in solution is toxic at higher concentrations (> 1 x 10(-3) M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by radiation with light only. (c) 2007 Elsevier Inc. All rights reserved.

Dissemination of bla(KPC-2) by the Spread of Klebsiella pneumoniae Clonal Complex 258 Clones (ST258, ST11, ST437) and Plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae Species in Brazil

This article reports the spread of bla(KPC-2) in the Sao Paulo and Rio de Janeiro states, facilitated by globally spread K. pneumoniae clonal complex 258 (CC258) clones (ST258, ST11, and ST437) and a diversity of plasmids (IncFII, IncN, and IncL/M, two untypeable plasmids carrying Tn4401a or Tn4401b) successfully disseminated among species of the Enterobacteriaceae (Enterobacter cloacae, Serratia marcescens, and Citrobacter freundii). It also constitutes the first description of sequence type 258 (ST258) in Brazil, which was associated with a nosocomial hospital outbreak in Ribeirao Preto city.

Identification of genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in the palA gene

To identify genes differentially expressed in a strain of the mold Aspergillus nidulans carrying a loss-of-function mutation in palA, a gene in the pH-responsive signal transduction pathway, suppression subtractive hybridization was performed between RNA isolated from the biA1 and biA1 palA1 strains grown under limiting inorganic phosphate at pH 5.0. We have identified several genes upregulated in the biA1 palA1 mutant strain that play important roles in mitotic fidelity, stress responses, enzyme secretion, signal transduction mechanisms, development, genome stability, phosphate sensing, and transcriptional regulation among others. The upregulation of eight of these transcripts was also validated by Northern blot. Moreover, we show that a loss of function mutation in the palA gene drastically reduced the neutral sugar content of the acid phosphatase PacA secreted by the fungus A. nidulans grown at pH 5.0 compared with a control strain.

Obesity due to Melanocortin 4 Receptor (MC4R) Deficiency Is Associated with Increased Linear Growth and Final Height, Fasting Hyperinsulinemia, and Incompletely Suppressed Growth Hormone Secretion

Context: Melanocortin receptor 4 (MC4R) deficiency is characterized by increased linear growth greater than expected for the degree of obesity. Objective: The objective of the investigation was to study the somatotroph axis in obese MC4R-deficient patients and equally obese controls. Patients and Methods: We obtained anthropometric measurements and insulin concentrations in 153 MC4R-deficient subjects and 1392 controls matched for age and severity of obesity. We measured fasting IGF-I, IGF-II, IGF binding protein (IGFBP)-1, IGFBP-3, and acid-labile subunit levels in a subset of 33 MC4R-deficient patients and 36 control subjects. We examined pulsatile GH secretion in six adult MC4R-deficient subjects and six obese controls. Results: Height so score was significantly greater in MC4R-deficient children under 5 yr of age compared with controls (mean +/- SEM: 2.3 +/- 0.06 vs. 1.8 +/- 0.04, P < 0.001), an effect that persisted throughout childhood. Final height (cm) was greater in MC4R-deficient men (mean +/- SEM 173 +/- 2.5 vs. 168 +/- 2.1, P < 0.001) and women (mean 165 +/- 2.1 vs. 158 +/- 1.9, P < 0.001). Fasting IGF-I, IGF-II, acid-labile subunit, and IGFBP-3 concentrations were similar in the two groups. GH levels were markedly suppressed in obese controls, but pulsatile GH secretion was retained in MC4R deficiency. The mean maximal GH secretion rate per burst (P < 0.05) and mass per burst (P < 0.05) were increased in MC4R deficiency, consistent with increased pulsatile and total GH secretion. Fasting insulin levels were markedly elevated in MC4R-deficient children. Conclusions: In MC4R deficiency, increased linear growth in childhood leads to increased adult final height, greater than predicted by obesity alone. GH pulsatility is maintained in MC4R deficiency, a finding consistent with animal studies, suggesting a role for MC4R in controlling hypothalamic somatostatinergic tone. Fasting insulin levels are significantly higher in children carrying MC4R mutations. Both of these factors may contribute to the accelerated growth phenotype characteristic of MC4R deficiency. (J Clin Endocrinol Metab 96: E181-E188, 2011)

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio=3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information gained through observation of pigmentation phenotype.

Combined effect of cyclosporine and sirolimus on improving the longevity of recombinant adenovirus-mediated transgene expression in the retina

Objectives: To reevaluate the longevity and intraocular safety of recombinant adenovirus (rAd)-mediated gene delivery after subretinal injection, and to prolong transgene expression through the combination of 2 synergistic immunosuppressants. Methods: An rAd vector carrying green fluorescent protein (GFP) gene was delivered subretinally in the rat eye. The GFP expression was monitored in real time by fundus fluorescent photography. Intraocular safety was examined by observation of changes of retinal pigmentation, cell infiltration in virus-contacted area, immunophenotyping for CD4(+) and CD8(+) cytotoxic T lymphocytes, and CD68(+) macrophages, histologic findings, and dark-adapted electroretinography. Two synergistic immunosuppressants, cyclosporine and sirolimus, were used alone or in combination to prolong transgene expression by temporary immunosuppression. Results: The GFP expression peaked on day 4, dramatically decreased on day 10, and was not detectable on day 14. The decreased GFP expression was coincident with cell infiltration in virus-contacted area. Immunostaining showed that the infiltrating cells were CD4(+) and CD8(+) cytotoxic T lymphocytes and CD68(+) macrophages. Clumped retinal pigmentation and decreased b wave of dark-adapted electroretinogram were observed at 3 to 4 weeks after injection. Histologic examination confirmed rAd-induced retinal degeneration. Transient immunosuppression by cyclosporine and sirolimus, either alone or in combination, improved transgene expression, with the combination being the most efficient. The combined immunosuppression attenuated but did not retard the rAd-induced retinal damage. Conclusions: Transgene expression mediated by rAd after subretinal delivery is short-term and toxic to the retina. Combination of cyclosporine and sirolimus may act as an immunosuppressive adjunct to prolong rAd-mediated gene transfer. Clinical Relevance: The intraocular safety of rAd should be carefully considered before clinical trials are performed.

Postural control of the trunk in response to lateral support surface translations during trunk movement and loading

The postural response to translation of the support surface may be influenced by the performance of an ongoing voluntary task. This study was designed to test this proposal by applying lateral perturbations while subjects handled a load in the frontal plane. Measurements were made of medio-lateral displacement of the centre of pressure, angular displacement of the trunk and thigh in the frontal plane and intra-abdominal pressure. Subjects were translated randomly to the left and right in a variety of conditions that involved standing either quietly or with a 5 kg load in their left hand, which they were required either to hold statically or to lift or lower. The results indicate that when the perturbation occurred towards the loaded left side the subjects were able to return their centre of pressure, trunk and thigh rapidly and accurately to the initial position. However, when the perturbation occurred towards the right (away from the load) this correction was delayed and associated with multiple changes in direction of movement, suggesting decreased efficiency of the postural response. This reduced efficiency can be explained by a conflict between the motor commands for the ongoing voluntary task and the postural response, and/or by the mechanical effect of the asymmetrical addition of load to the trunk.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY 1002/3A4. which express respective human P450 enzymes and NADPH-cytochrome P350 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA 1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double me promoter and the other, pOA 102, carrying O-AT and umuClacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 135 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 1-Amino-1,4-dimethyl-5H-pyrido[4.3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B-1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta -Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs. (C) 2001 Elsevier Science B.V. All rights reserved.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Relationship between oviposition preference and offspring performance in Australian Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

We investigated the oviposition preference and larval performance of Helicoverpa armigera under laboratory conditions to determine if the oviposition preference of individual females on maize, cowpea and cotton correlates with offspring performance on the leaves of the same host plants. The host-plant preference hierarchy of females did not correlate with their offspring performance. Female moths chose host plants that contributed less to their offspring fitness. Plant effects accounted for the largest amount of variation in offspring performance, while the effects of female (family) was low. The offspring of most females (80%, n = 10) were broadly similar, but 20% (two out of 10), showed marked difference in their offspring performance across the host-plant species. Similarly, there was no relation between larval feeding preference and performance. However, like most laboratory experiments, our experi-mental design does not allow the evaluation of ecological factors (for example, natural enemies, host abundance, etc.) that can play an important role in larval performance in the field. Overall, the results highlight the importance of carrying out preference performance analysis on the individual or family level, rather than pooling individuals to obtain average population data.

Pathogenic specialisation within Colletotrichum trifolii in Australia, and lucerne cultivar reactions to all known Australian pathotypes

Anthracnose and crown rot, caused by Colletotrichum trifolii, are serious diseases of lucerne (Medicago saliva L.) in humid regions of the world. A race survey was conducted by inoculating individual lucerne clones (genotypes) with C. trifolii isolates collected from a range of Medicago hosts, locations, and years in south-eastern Queensland. This survey revealed for the first time in Australia the presence of race 2 (virulence on anthracnose resistance gene An I) and the first world report of race 4 (virulence on An(2)). A collection of North American race I and race 2 C. trifolii isolates, when inoculated onto the Australian differential clones, gave responses that were in agreement with their North American reactions. A RAPD analysis was conducted on 9 Australian C. trifolii isolates including races 1, 2, and 4; two C. destructivum and one C. gloeosporioides isolate were included as known outliers. For the C. trifolii isolates, 94.6% similarity was found regardless of host origin or race, compared with 2.2% similarity between this group and the C. gloeosporioides and C. destructivum isolates, confirming that the new races belong to C. trifolii. Currently, it is hypothesised that only plants carrying genes An, and An2 are resistant to the 3 races. Of 22 cultivars screened against the 3 races, only UQL-1, Hallmark, and Pioneer 54Q53 had >30% of plants resistant to the 3 races in separate screenings. The research highlights the need to find new sources of resistance to C. trifolii in lucerne.