mtDNAoffice: A software to assign human mtDNA macro haplogroups through automated analysis of the protein coding region

We describe a fast, automated process to determine distances between mtDNA sequences allowing their subsequent clustering and haplogroup assignment that may increase the speed of data analysis and avoid human errors.In order to avoid complexities/ambiguities resulting from recurrence and insertion/deletion phenomena and thus improving evolutionary signal-to-noise ratio, protein coding sequences were compared using a vectorial representation method, and the corresponding genetic distance matrix was used for the construction of a neighbor-joining/UPGMA tree or an MDS graphic, which generally agrees with the consensus mtDNA phylogeny.mtDNAoffice software, detailed instructions and example files are freely available on the web at http://www.portugene.com/SupMat/setupmtDNAoffice.rar.     

Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response

A Leishmania (Viannia) braziliensis (LB) promastigote cDNA library in λgt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Esherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzi- or L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.     

Evaluation of the acarofauna of the domiciliary ecosystem in Juiz de Fora, State of Minas Gerais, Brazil.

From August 1999 to January 2000, samples of house dust were collected from 160 domiciles in the city of Juiz de Fora, State of Minas Gerais, Brazil. In 36 of these domiciles kitchen samples were obtained. Prevalence rate was 77.5%, varying according to the geographical sector. There were found 2,278 specimens of mites, with 1,530 (67.2%) in the adult stage and 748 (32.8%) in immature forms. The main species found were Dermatophagoides pteronyssinus, D. farinae, Euroglyphus maynei, Blomia tropicalis and Tyrophagus putrescentiae. In a minor incidence we found Lepidoglyphus destructor, Suidasia pontificiae, Chortoglyphus arcuatus, Cheyletus malaccensis, C. fortis, Ker bakeri, Cheletonella vespertilionis, C. caucasica and others. C. vespertilionis and C. caucasica were identified for the first time in the domiciliary ecosystem and in Brazil. The abundance rate and the infestation intensity were analyzed. There was a varied correlation between climatic conditions and positive domiciles and number of mites. The difference between the number of positive domiciles in the urban area and in the expanding urban area was significant and so was the difference between samples from the domiciles compared to those from the kitchens.     

Antileishmanial Screening,Cytotoxicity, and Chemical Composition of Essential Oils: A Special Focus on Piper callosum Essential Oil

Leishmania amazonensis is the etiological agent of tegumentary leishmaniasis, a disease characterized by the emergence of cutaneous and mucocutaneous ulcerated lesions that can evolve into severe destruction of skin tissue. Treatment of the disease is often accompanied by high toxicity and variable efficacy. Essential oils stand out for having diverse pharmacological properties. Here, we screened a panel of fourteen essential oils for their anti-L. amazonensis activity, cytotoxicity, and chemical profile. Lippia sidoides (LSEO) and Piper callosum (PCEO) oils displayed the best anti-promastigote and anti-amastigote activities with IC₅₀ of 31 and 21 μg/ml, respectively. PCEO was the safest oil with a desirable selectivity index >10. In addition, PCEO showed no cytotoxicity against the VERO line and erythrocytes. PCEO-treated amastigotes displayed mitochondrial membrane depolarization and high levels of intracellular ROS. Safrole (54.72 %) was the main component of PCEO. The results described here highlight the use of essential oils to combat tegumentary leishmaniasis.     

Operational stability of high initial activity protease catalysts in organic solvents

The first studies on the operational stability of cross-linked enzyme crystals (CLECs) in organic media are described. Although these catalysts display high initial specific activity, they inactivate rapidly, losing more than 50% of the initial activity within the first 4 h under continuous flow. Furthermore, the inactivation is not reversible when returned to an aqueous medium. The same rapid inactivation occurs with adsorbed protease preparations that show similar high initial specific activity (propanol-rinsed enzyme preparations (PREPs) of subtilisin and alpha-chymotrypsin).     

Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Anti-Inflammatory Properties and Chemical Characterization of the Essential Oils of Four Citrus Species

Citrus fruits have potential health-promoting properties and their essential oils have long been used in several applications. Due to biological effects described to some citrus species in this study our objectives were to analyze and compare the phytochemical composition and evaluate the anti-inflammatory effect of essential oils (EO) obtained from four different Citrus species. Mice were treated with EO obtained from C. limon, C. latifolia, C. aurantifolia or C. limonia (10 to 100 mg/kg, p.o.) and their anti-inflammatory effects were evaluated in chemical induced inflammation (formalin-induced licking response) and carrageenan-induced inflammation in the subcutaneous air pouch model. A possible antinociceptive effect was evaluated in the hot plate model. Phytochemical analyses indicated the presence of geranial, limonene, γ-terpinene and others. EOs from C. limon, C. aurantifolia and C. limonia exhibited anti-inflammatory effects by reducing cell migration, cytokine production and protein extravasation induced by carrageenan. These effects were also obtained with similar amounts of pure limonene. It was also observed that C. aurantifolia induced myelotoxicity in mice. Anti-inflammatory effect of C. limon and C. limonia is probably due to their large quantities of limonene, while the myelotoxicity observed with C. aurantifolia is most likely due to the high concentration of citral. Our results indicate that these EOs from C. limon, C. aurantifolia and C. limonia have a significant anti-inflammatory effect; however, care should be taken with C. aurantifolia.     

A genetic historical sketch of European Gypsies: The perspective from autosomal markers

In this study, 123 unrelated Portuguese Gypsies were analyzed for 15 highly polymorphic autosomal short tandem repeats (STRs). Average gene diversity across the 15 markers was 76.7%, which is lower than that observed in the non‐Gypsy Portuguese population. Subsets of STRs were used to perform comparisons with other Gypsy and corresponding host populations. Interestingly, diversity reduction in Gypsy groups compared to their non‐Gypsy surrounding populations apparently varied according to an East‐West gradient, which parallels their dispersion in Europe as well as a decrease in complexity of their internal structure. Analysis of genetic distances revealed that the average level of genetic differentiation between Gypsy groups was much larger than that observed between the corresponding non‐Gypsy populations. The high rate of heterogeneity among Gypsies can be explained by strong genetic drift and limited intergroup gene flow. However, when genetic relationships were addressed through principal component analysis, all Gypsy populations clustered together and was clearly distinguished from other populations, a pattern that suggests their common origin. Concerning the putative ancestral genetic component, admixture analysis did not reveal strong Indian ancestry in the current Gypsy gene pools, in contrast to the high admixture estimates for either Europeans or Western Asians. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.     

Betaine–homocysteine methyltransferase 742G>A polymorphism and risk of down syndrome offspring in a Brazilian population

Down syndrome (DS) is the most common form of mental retardation of genetic etiology. Several polymorphisms in genes involved with the folic acid cycle have been associated to the risk of bearing a DS child; however, the results are controversial. Betaine-homocysteine methyltransferase (BHMT) is a key enzyme of folate pathway, and catalyzes the remethylation of homocysteine into methionine. Recent studies suggest that the polymorphism BHMT 742G>A may be associated with a decreased risk of having a DS child. We herein investigate the association of this polymorphism with the occurrence of DS in a Brazilian population. We have genotyped 94 mothers of DS infants (DSM) and 134 control mothers (CM) for this polymorphism through PCR–RFLP, and found significant differences for both BHMT 742G>A genotype (P = 0.04) and allele (P = 0.03) frequencies between DSM and CM. The observed genotypic frequencies were GG = 0.45; GA = 0.45 and AA = 0.10 in CM, and GG = 0.54; GA = 0.38 and AA = 0.02 in DSM. Allelic frequencies were G = 0.68 and A = 0.32 in CM and G = 0.78 and A = 0.22 in DSM. The presence of the mutant BHMT 742 A allele decreases 40 % the risk of bearing a DS child (OR = 0.61; 95 % CI: 0.40–0.93; P = 0.03), and the risk is diminished up to >80 % in association with the homozygous genotype (OR = 0.17; 95 % CI: 0.04–0.80; P = 0.01). Our results indicate that women harboring the single nucleotide polymorphism BHMT 742G>A have a decreased risk of a DS pregnancy, and further studies are necessary to confirm this protective effect.