Microquantification of proteins with low chromophore content

The advantages of the intrinsic fluorescence of the tryptophan and the absorbance of the methionine residues of the 18 kDa-hsp - a recombinant protein from Mycobacterium leprae - was exploited here to develop a sensitive and low costs method for protein assaying. They presented linearity between 3 and 1000 g of protein. The correlations between intrinsic fluorescence or absorbance at 230 nm and protein contents were both superiors to 0.99. These methods can be extended to others proteins with low aromatic residue contents.     

Candidemia by Species of the Candida parapsilosis Complex in Children’s Hospital: Prevalence, Biofilm Production and Antifungal Susceptibility

Opportunistic infections are an increasingly common problem in hospitals, and the yeast Candida parapsilosis has emerged as an important nosocomial pathogen. The aims of this study were to determine and compare (i) the prevalence rate among C. parapsilosis complex organisms isolated from blood in a public children’s hospital in São Paulo state, (ii) the ability of the complex C. parapsilosis species identified to produce biofilm and (iii) the antifungal susceptibility profiles. Forty-nine (49) specimens of isolated blood yeast were analyzed, previously identified as C. parapsilosis by conventional methods. After the molecular analysis, the isolates were characterized as C. parapsilosis sensu stricto (83.7 %), C. orthopsilosis (10.2 %) and C. metapsilosis (6.1 %). All species were able to form biofilm. The species with the highest biofilm production was C. parapsilosis sensu stricto, followed by C. orthopsilosis and further by C. metapsilosis. All of the strains have demonstrated similar susceptibility to fluconazole, caspofungin, voriconazole, cetoconazole and 5-flucytosine. Only one strain of C. parapsilosis was resistant to amphotericin B. Regarding itraconazole, 66.6 and 43.9 % isolates of C. metapsilosis and C. parapsilosis, respectively, have demonstrated to be susceptible dose-dependent, with one isolate of the latter species resistant to the drug. Candida parapsilosis sensu stricto has demonstrated to be the less susceptible, mainly to amphotericin B, caspofungin and “azoles” such as fluconazole. Therefore, C. metapsilosis and C. orthopsilosis are still involved in a restricted number of infections, but these data have become essential for there are very few studies of these species in Latin America.     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.     

Cytomegalovirus Infection in Inflammatory Bowel Disease Is Not Associated with Worsening of Intestinal Inflammatory Activity

Background

Cytomegalovirus is highly prevalent virus and usually occurs in immunocompromised patients. The pathophysiology and treatment of inflammatory bowel disease often induce a state of immunosuppression. Because this, there are still doubts and controversies about the relationship between inflammatory bowel disease and cytomegalovirus.

Aim

Evaluate the frequency of cytomegalovirus in patients with inflammatory bowel disease and identify correlations.

Methods

Patients with inflammatory bowel disease underwent an interview, review of records and collection of blood and fecal samples. The search for cytomegalovirus was performed by IgG and IgM blood serology, by real-time PCR in the blood and by qualitative PCR in feces. Results were correlated with red blood cell levels, C-reactive protein levels, erythrocyte sedimentation rates and fecal calprotectin levels for each patient.

Results

Among the 400 eligible patients, 249 had Crohn''s disease, and 151 had ulcerative colitis. In the group of Crohn''s disease, 67 of the patients had moderate or severe disease, but 126 patients presented with active disease, based on the evaluation of the fecal calprotectin. In patients with ulcerative colitis, only 21 patients had moderate disease, but 76 patients presented with active disease, based on the evaluation of the fecal calprotectin. A large majority of patients had positive CMV IgG. Overall, 10 patients had positive CMV IgM, and 9 patients had a positive qualitative detection of CMV DNA by PCR in the feces. All 400 patients returned negative results after the quantitative detection of CMV DNA in blood by real-time PCR. Analyzing the 19 patients with active infections, we only found that such an association occurred with the use of combined therapy (anti-TNF-alpha + azathioprine)

Conclusion

The findings show that latent cytomegalovirus infections are frequent and active cytomegalovirus infection is rare. We did not find any association between an active infection of CMV and inflammatory bowel disease activity.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.     

Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

Allograft Tissue for Use in Valve Replacement

Homograft or allograft tissue has been available for use as replacement for diseased valves or reconstruction of major vessels for decades. However, with respect to replacement of diseased valvular tissue the search for the ideal valve still continues. In this review we will discuss the clinical indications, surgical techniques, and outcome of aortic homografts.     

Association between IGF2 and CYP21 gene polymorphisms and characteristics of economic interest in Nellore cattle

We analyzed two single nucleotide polymorphisms (SNPs) of the IGF2 and CYP21 genes in Nellore cattle participating in the Brazilian Animal Breeding Program. The SNPs were found in exon 6 of the IGF2 (insulin-like growth factor 2) gene (RFLP/MboII) as well as in the promoter region of the CYP21 (steroid 21-hydroxylase) gene (RFLP/HpaII) of these animals. The TC heterozygotes were significantly more frequent than CC and TT homozygotes in the RFLP/MboII polymorphism. The T allele was significantly more frequent than the C allele in RFLP/HpaII polymorphism. This population was found to be in Hardy-Weinberg equilibrium for these SNPs. Association of these polymorphisms with expected progeny differences of reproductive and productive traits was investigated, but proved to be significant only for DP550 (expected progeny differenced for weight at 365 days - IGF2 - RFLP/MboII) and DP450 (expected progeny differenced for weight at 450 days - CYP21 - RFLP/HpaII). This is the first study on the occurrence of these two polymorphisms in this Zebu breed of cattle. A total of 147 Nellore animals participating in the Breeding Program of the Nellore Breed (PMGRN) under the management of the National Association of Breeders and Researchers (ANCP) in the city of Ribeir?o Preto were analyzed.     

Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.