Detecting multiple DNA human profile from a mosquito blood meal.

Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

Trace samples of human blood in mosquitoes as a forensic investigation tool.

Investigations of any type of crime invariably starts at the crime scene by collecting evidence. Thus, the purpose of this research was to collect and analyze an entomological trace from an environment that is similar to those of indoor crime scenes. Hematophagous mosquitoes were collected from two residential units; saliva of volunteers that were residents in the units was also collected for genetic analysis as reference samples. We examined the allele frequencies of 15 short tandem repeat loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) and amelogenin. A total of 26 female hematophagous mosquitoes were identified as Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus; we were able to obtain 11 forensically valid genetic profiles, with a minimum of 0.028203 ng/µL of human DNA. Thus, the results of this study showed that it was possible to correlate human genetic information from mosquitoes with the volunteer reference samples, which validates the use of this information as forensic evidence. Furthermore, we observed mixed genetic profiles from one mosquito. Therefore, it is clearly important to collect these insects indoors where crimes were committed, because it may be possible to find intact genetic profiles of suspects in the blood found in the digestive tract of hematophagous mosquitoes for later comparison to identify an offender and/or exclude suspects.

Interleukin-6 gene -174G>C and -636G>C promoter polymorphisms and prostate cancer risk.

Prostate cancer (PCa) is one of the most commonly diagnosed internal malignancies affecting men. Due to the important roles of IL-6 in different physiological and pathophysiological processes, IL-6 polymorphisms may modulate PCa risk. IL-6 -174 G>C (rs 1800795, also designated -236 G>C) and -636 G>C (rs 1800796, also designated -572 G>C) promoter polymorphisms have been implicated in PCa susceptibility, albeit still controversial. A literature search using PubMed and Highwire databases was conducted, resulting in eight case-control studies concerning the IL-6 -174 G>C polymorphism (11,613 PCa cases and 13,992 controls) and four case-control publications regarding the IL-6 -636 G>C polymorphism (1,941 PCa cases and 3,357 controls). In order to derive a more precise estimation, a meta-analysis based upon these selected case-control studies was performed. There was no significant association between IL-6 -174 G>C polymorphism and PCa increased risk. Nevertheless, the presence of allele C and the CC genotype were statistically significantly associated with decreased PCa risk in the overall analysis for IL-6 -636 G>C polymorphism. Additional studies in larger samples and analyses of functional repercussions of these SNPs in prostate tumor cells are necessary to validate these findings.

Association of the IL-10 polymorphisms and periodontitis: a meta-analysis.

No clear consensus has been reached regarding the association of IL-10 polymorphisms and periodontitis. Therefore, we performed a meta-analysis of case-control studies and a systemic review in an effort to systematically summarize the existing knowledge. Studies were identified by searching PubMed database until December 2011. IL-10 -1082 (-1087) A>G, -819 (-824) C>T and -592 (-597) C>A polymorphisms were included in the present meta-analysis. We calculated the specific odds ratios along with their 95 % confidence intervals to compare the distribution of alleles and genotypes between cases and controls. An additive "per-allele" model (major allele vs. minor allele) was performed, and dominant and recessive models were also considered. The random-effects model was applied for the analysis. Cumulative analysis was also performed. Heterogeneity and publication bias were assessed. Nine case-control studies involving 841 periodontitis cases (644 chronic periodontitis and 197 aggressive periodontitis cases) and 748 controls were included. We found statistically significant association of IL-10 -819 (-824) C>T and IL-10 -592 (-597) C>A polymorphisms in Caucasians. The IL-10 -819 (-824) T and -592 (-597) A alleles may confer a relative increase in the risk for chronic periodontitis in Caucasians. Future studies may be important to reinforce these findings.

Effect of anatomical origin and cell passage number on the stemness and osteogenic differentiation potential of canine adipose-derived stem cells.

Mesenchymal stem cells have a great potential for application in cell based therapies, such as tissue engineering. Adipose derived stem cells have shown the capacity to differentiate into several lineages, and have been isolated in many animal species. Dog is a very relevant animal model to study several human diseases and simultaneously an important subject in veterinary medicine. Thus, in this study we assessed the potential of canine adipose tissue derived stem cells (cASCs) to differentiate into the osteogenic and chondrogenic lineages by performing specific histological stainings, and studied the cell passaging effect on the cASCs stemness and osteogenic potential. We also evaluated the effect of the anatomical origin of the adipose tissue, namely from abdominal subcutaneous layer and from greater omentum. The stemness and osteogenic differentiation was followed by real time RT-PCR analysis of typical markers of mesenchymal stem cells (MSCs) and osteoblasts. The results obtained revealed that cASCs exhibit a progressively decreased expression of the MSCs markers along passages and also a decreased osteogenic differentiation potential. In the author's knowledge, this work presents the first data about the MSCs markers profile and osteogenic potential of cASCs along cellular expansion. Moreover, the obtained data showed that the anatomical origin of the adipose tissue has an evident effect in the differentiation potential of the ASCs. Due to the observed resemblances with the human ASCs, we conclude that canine ASCs can be used as a model cells in tissue engineering research envisioning human applications.

Natural occurrence of the Fusarium solani on Tityus stigmurus (Thorell, 1876) (Scorpiones: Buthidae).

Members of the Fusarium solani species complex are agents of human mycoses, also affecting plants and other animals. Nevertheless, this fungus has not been reported on scorpions. Ten specimens of Tityus stigmurus collected in the field and showing their surface covered by white mycelia were used to assess fungus presence in the animal after its death. Identification of the fungi was based upon the cultural and morphological characteristics. The fungus was isolated from chelicerae and intersegmental regions. Infected individuals had their behaviour modified by reducing feeding and locomotion. None of the infected individuals survived. It is likely that this fungus may have a role in the regulation of field scorpion populations.

Immune defense mechanisms of Culex quinquefasciatus (Diptera: Culicidae) against Candida albicans infection.

Mosquitoes have an efficient defense system against infection. The cellular immune defense mechanism initiated by the mosquito Culex quinquefasciatus infected with the fungus Candida albicans was investigated in this study. Differences in the hemocyte counts in hemolymph perfused from uninoculated, saline-inoculated, and C. albicans-infected mosquitoes were compared using a light microscope. Phagocytosis was also investigated using electron microscopy. Four types of hemocytes were identified in control mosquitoes: prohemocytes (9.8%), plasmatocytes (38.8%), granular cells (44.2%), and oenocytoids (7.3%). Between 3 and 18 h postinoculation the total hemocyte count was significantly higher in infected, compared to uninfected, mosquitoes. Differential hemocyte counts from infected mosquitoes at 3, 6, and 18 h after inoculation showed that the relative proportion of plasmatocytes (48.6, 50.7, 45%) was higher and, concomitantly, the proportion of granular cells was lower (38, 36.8, 35%, respectively). Yeast cells were phagocytosed and limited growth was observed within the plasmatocytes. Melanized nodules were found attached to different insect tissues at 24 to 72 h following infection. These results suggest that phagocytosis, followed by nodule formation, was capable of clearing the hemolymph of yeast cells.

[First report of Aedes albopictus in areas of Mata Atlantica, Recife, PE, Brazil]. / Primeiro registro de Aedes albopictus em área da Mata Atlântica, Recife, PE, Brasil.

This is the first report of the presence of Aedes albopictus in the native rain forest, near the urban area of Recife (State of Pernambuco, Brazil). Adult female mosquitoes were collected using human bait. Mosquitoes in aquatic stages were looked for in treeholes, bamboos, bromeliads and old tires. The existence of Ae. albopictus in the metropolitan area of Recife poses a potential risk for the interaction of this mosquito species with the urban human population.

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti.

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2. 4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.

Seven novel mutations in the APC gene of Portuguese families with familial adenomatous polyposis: correlation with phenotype.

Germ-line mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP). In the present study, we have used the protein truncation test to screen for mutations in exon 15 and exons 1-14 of the APC gene and denaturing gradient gel electrophoresis to analyze exons 1-14. We have studied nine unrelated FAP kindreds, eight with the classical phenotype and one with an atypical phenotype, with several family members exhibiting fewer than 50 colonic polyps. The combined use of these two methodologies allowed the identification of seven novel mutations, with two unrelated families sharing the same mutation. All mutations were chain terminating: six resulted from small deletions, one from a small insertion, and one was a point mutation, resulting in a premature stop codon. Seven mutations were located in exon 15 of the APC gene, one was in exon 10, and the remaining one, which corresponded to the kindred with an atypical phenotype, was located in exon 4.

Validation and simplification of Bethesda guidelines for identifying apparently sporadic forms of colorectal carcinoma with microsatellite instability.

BACKGROUND: Colorectal tumors with microsatellite instability (MSI) that do not comply with previously defined clinical criteria may be found in recently diagnosed hereditary nonpolyposis colorectal carcinoma families. Until recently, the indications for MSI testing were not clearly established. The objective of the current study was to validate the recently published Bethesda guidelines for MSI testing in a series of patients with apparently sporadic forms of colorectal carcinoma (CRC). METHODS: Sixty-two patients with so-called sporadic CRC were included in the current study. MSI was analyzed at seven poly(CA) repeat sequences and at one poly(A) locus. RESULTS: Nine of 62 patients (14.5%) had tumors exhibiting MSI at > or = 2 loci and 7 patients (11%) had MSI at > or = 3 loci. Patients with MSI positive tumors were younger (P < 0.05), and their tumors more frequently were right-sided (P < 0.02) and more often exhibited a mucinous component (P < 0.05). The Bethesda guidelines were positive in 18% (11 of 62) of patients. The sensitivity of these guidelines in identifying tumors with MSI at > or = 3 loci was 43% and the positive predictive value (PPV) was 27% (3 of 11 cases). Other variables were considered as alternative criteria to identify CRCs with MSI: age < 45 years and/or a right-sided tumor with a mucinous component. Using these 2 criteria alone, sensitivity increased to 85% and PPV to 46%. CONCLUSIONS: In this study group, the use of three clinical criteria as sole indicators for MSI testing in patients with apparently sporadic forms of CRC were significantly more discriminating compared with the Bethesda guidelines, in addition to being substantially easier.

Microsatellite instability in non-neoplastic mucosa of patients with ulcerative colitis: effect of folate supplementation.

OBJECTIVE: Microsatellite instability (MSI) detected in non-neoplastic mucosa of patients with ulcerative colitis has been ascribed to an excess of DNA damage associated with chronic inflammation. Folate deficiency, commonly found in patients with long-standing disease, could further contribute to this defect because folate is essential for DNA replication and repair. We evaluated MSI in the colonic mucosa of 26 patients with ulcerative colitis for >10 yr and 10 patients with Crohn's colitis and correlated MSI with folate status. METHODS: DNA was amplified using primers directed at nine different loci. Folate concentrations in serum, whole blood, and colonic mucosa were determined using the Lactobacillus casei assay. RESULTS: MSI was found in 3/23 patients (13%) with ulcerative colitis and in none of the patients with Crohn's colitis. All three patients with MSI had inactive histological disease, whereas all patients with active disease were negative for MSI (p = 0.08). Serum, whole blood, and colonic concentrations of folate were 30-50% lower in patients with MSI (p > 0.05), and folate supplements had been administered less frequently during the past 5-yr (p = 0.06). One of the patients with MSI was randomized to receive folate 5 mg/d for 6 months, and a clear change in MSI pattern was observed in three of six markers. CONCLUSIONS: A defect in DNA repair associated with a low folate status may be one additional cause for patients with ulcerative colitis exhibiting MSI in non-neoplastic mucosa.

Full sequence and characterization of two insect defensins: immune peptides from the mosquito Aedes aegypti.

We report the complete amino acid sequence and biological activity of two immune peptides, from the yellow fever mosquito Aedes aegypti, that are induced in response to infection. Both peptides display biological activity against the Gram positive microbe Micrococcus luteus and substantial sequence homology to insect defensins, small heat-stable, antibiotic peptides previously described from several non-vector insects. These mosquito peptides, designated Ae. aegypti defensins A and B, are isoforms. Defensin B is the most abundant antibacterial peptide in this species whereas defensin A is much less abundant and carries two amino acid substitutions compared to defensin B, making it more basic in character. Apparent convergence between isoforms from Ae. aegypti and the fleshfly Phormia terranovae is discussed. The synergistic activity previously described between Ae. aegypti immune haemolymph and lysozyme is not caused by these peptides because synergy occurred only at concentrations far outside the physiological range seen in Ae. aegypti.

Concomitant malaria (Plasmodium gallinaceum) and filaria (Brugia pahangi) infections in Aedes aegypti: effect on parasite development.

Mixed infections with malarial (Plasmodium gallinaceum) and filarial (Brugia pahangi) parasites were carried out in 8 trials with filaria susceptible (REFM) and filaria refractory (REP-RR) Aedes aegypti strains. A secondary infection with B. pahangi microfilariae (mff) by intrathoracic inoculation, reduced the development rate of a pre-existing P. gallinaceum infection. The level of reduction ranged from 9.5 to 49% in REFM and from 50 to 90% in REP-RR. An immune response against oocysts was seen as melanization in mosquitoes with a double infection in the strain refractory to B. pahangi (REP-RR) and a reduction in oocyst size in both mosquito strains. Melanization was not observed in mosquitoes infected only with P. gallinaceum. This may indicate that activation of the prophenoloxidase (PPO) cascade in response to mff in the haemolymph can also be addressed against oocysts in the midgut. No significant difference in the number of filarial parasites recovered was observed when comparing groups with a single or double infection. Retardation in development of filaria larvae was observed in mosquitoes with double infection (REFM strain), together with melanization and a higher rate of abnormal development. Nutritional deficiency caused by superinfection might also be responsible for the delay in filarial development and reduced oocyst size.

Genetic variability at the esterase-6 locus in natural populations of Drosophila simulans in relation to environmental heterogeneity.

The allozymic variation at at the esterase-6 locus examined in fourteen samples of natural populations of Drosophila simulans collected in two different localities. The samples were collected from different species of fruits in the two localities and from banana baits located some meters apart from the fruits. The analysis was done by sequential electrophoresis, using varied gel concentrations and buffers. By this technique, we detected 27 alleles at the esterase-6 locus, where only five alleles were detected by the usual method. The results obtained suggest that habitat choice alone may not be a selective factor, but may be selective in combination with other environmental factors.