Development of microsatellite markers for the neotropical tree species Dipteryx alata (Fabaceae)

? Premise of the study: Microsatellite markers were developed for the population genetic analyses of the neotropical tree Dipteryx alata (Fabaceae). ? Methods and Results: Microsatellites were developed from a genomic shotgun library. Polymorphism at each microsatellite loci was analyzed based on 94 individuals from three populations. Eight loci amplified successfully and presented one to 10 alleles, and expected heterozygosities ranged from 0.097 to 0.862. Four loci also amplified in Pterodon emarginatus and presented similar polymorphism. ? Conclusion: The eight microsatellite primer pairs are potentially suitable for population genetic studies and successfully amplified in another Fabaceae species.     

Rapid pre-screening of cervical smears as a method of internal quality control in a cervical screening programme

Objective:  To evaluate the performance of rapid pre-screening (RPS) as a method of internal quality control in the cytopathological examination of cervical smears for cervical cancer screening.
Methods:  The sample consisted of 6135 cervical smears submitted to RPS and routine screening (RS) methods. The smears classified as negative in RPS and RS were considered final diagnoses, and were not, therefore, submitted to any additional review. The smears identified as suspect or unsatisfactory according to RPS were analysed separately by two different cytologists irrespective of the diagnosis reached in RS. Smears considered abnormal or unsatisfactory at RS were also reviewed. When both cytologists issued concordant diagnoses, this was considered the final diagnosis. Discordant results were analysed by a third cytologist and a consensus meeting was held to define the final diagnosis.
Results:  Taking abnormalities detected by RS as the denominator, RPS had a sensitivity of 63.0% for the detection of all abnormal smears and 96.7% for high grade squamous intraepithelial lesion (HSIL). When compared with the final diagnosis, sensitivity of RPS for all abnormal smears was 74.9% and for HSIL 95.0%. Of the 529 abnormal smears confirmed in the final diagnosis, 2.15% were detected only by the RPS.
Conclusion:  RPS is an effective alternative method of internal quality control with high sensitivity for the detection of more severe lesions. It also permits monitoring of the laboratory rate of false-negative results, and allows constant evaluation of the performance both of the pre-screening and RS cytologists.     

Prediction of Antimicrobial Activity of Synthetic Peptides by a Decision Tree Model

Antimicrobial resistance is a persistent problem in the public health sphere. However, recent attempts to find effective substitutes to combat infections have been directed at identifying natural antimicrobial peptides in order to circumvent resistance to commercial antibiotics. This study describes the development of synthetic peptides with antimicrobial activity, created in silico by site-directed mutation modeling using wild-type peptides as scaffolds for these mutations. Fragments of antimicrobial peptides were used for modeling with molecular modeling computational tools. To analyze these peptides, a decision tree model, which indicated the action range of peptides on the types of microorganisms on which they can exercise biological activity, was created. The decision tree model was processed using physicochemistry properties from known antimicrobial peptides available at the Antimicrobial Peptide Database (APD). The two most promising peptides were synthesized, and antimicrobial assays showed inhibitory activity against Gram-positive and Gram-negative bacteria. Colossomin C and colossomin D were the most inhibitory peptides at 5 μg/ml against Staphylococcus aureus and Escherichia coli. The methods described in this work and the results obtained are useful for the identification and development of new compounds with antimicrobial activity through the use of computational tools.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.     

Reproducibility of Positive Results for the Detection of Serum Galactomannan by Platelia™ Aspergillus EIA

Galactomannan (GM) was recently included in consensus guidelines as an indirect mycological criterion for the diagnosis of invasive aspergillosis. Currently, there is an enzyme immunoassay available to detect GM in biological samples, the Platelia? Aspergillus EIA. In this study, the reproducibility of positive results obtained using this assay was evaluated using serum samples from neutropenic patients. A trend toward lower values was observed, and 55 %(27/49) of positive results were negative after retesting. A low reproducibility of positive results for the detection of GM in serum was observed.     

First record of chemical signals from the queen during the oviposition process in stingless bees

In stingless bees, the cell provisioning and oviposition process consists of several integrated behavioral sequences and several stereotyped queen–worker interactions. This study aims to demonstrate that chemical signals originating from the queen may contribute as cues for the sequence of the oviposition process in Melipona marginata. For this, we analyzed the cell before and after queen laying, and compared them with the cuticular hydrocarbons of the queen’s abdomen, using a gas-chromatography and mass spectrometry system.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

Ticks (Acari: Ixodidae) of the state of Amazonas,Brazil

The tick fauna of Brazil is currently composed by 72 species. The state of Amazonas is the largest of Brazil, with an area of ≈ 19% of the Brazilian land. Besides its vast geographic area, only 19 tick species have been reported for Amazonas. Herein, lots containing ticks from the state of Amazonas were examined in three major tick collections from Brazil. A total of 5933 tick specimens were examined and recorded, comprising 2693 males, 1247 females, 1509 nymphs, and 484 larvae. These ticks were identified into the following 22 species: Amblyomma cajennense sensu lato, Amblyomma calcaratum, Amblyomma coelebs, Amblyomma dissimile, Amblyomma dubitatum, Amblyomma geayi, Amblyomma goeldii, Amblyomma humerale, Amblyomma latepunctatun, Amblyomma longirostre, Amblyomma naponense, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma rotundatum, Amblyomma scalpturatum, Amblyomma varium, Dermacentor nitens, Haemaphysalis juxtakochi, Ixodes cf. Ixodes fuscipes, Ixodes luciae, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato. Ticks were collected from 17 (27.4%) out of the 62 municipalities that currently compose the state of Amazonas. The following four species are reported for the first time in the state of Amazonas: A. coelebs, A. dubitatum, H. juxtakochi, and Ixodes cf. I. fuscipes. The only tick species previously reported for Amazonas and not found in the present study is Amblyomma parvum. This study provides a great expansion of geographical and host records of ticks for the state of Amazonas, which is now considered to have a tick fauna composed by 23 species. It is noteworthy that we report 1391 Amblyomma nymphs that were identified to 13 different species.     

Cytotoxic Effect of Prodigiosin,Natural Red Pigment,Isolated from Serratia marcescens UFPEDA 398

Prodigiosin is a secondary metabolite, with red pigmentation, produced by Serratia marcescens. Red pigment is a natural alkaloid whose chemical structure has three pyrrole rings. Prodigiosin has been described for several biological activities, including antitumor, inducing apotosis in T and B lymphocytes. This work aimed to evaluate the cytotoxic activity of prodigiosin in NCHI-292, HEp-2, MCF-7 and HL-60 tumor cell lines. The red pigment was isolated from Serratia marcescens UFPEDA 398 biomass whose fractions were previously separated by column chromatography, purified, identified and further characterized by GC–MS and compared with the computerized library of m/z values. The pigment corresponded to prodigiosin with maximum absorption at 534 nm, molecular weight 323 and structural formula C20H25N3O. During the prodigiosin purification process a purple absorbance fraction at 272.65 nm was also observed. Significant cytotoxic effects of prodigiosin were evidenced for NCHI-292, Hep-2, MCF-7 and HL-60 tumor cell lines. The isolated purple fraction had no cytotoxic effect (IC50 11.3 µg/mL) when compared to prodigiosin (IC50 3.4 µg/mL) for the tumor cell lines studied. The MCF-7 strain was slightly more pigment resistant (IC50 5.1 µg/mL). Therefore, further studies will be needed to elucidate the antitumor mechanisms of prodigiosin action against tumor strains from flow cytometry tests. However, although these data are preliminary, it was evidenced that prodigiosin showed cytotoxic activity in tumor cell lines suggesting promising antitumor properties. In this sense, future studies on the cytotoxic and genotoxic effects of prodigiosin produced by S. marcecsens UFPEDA 398 are suggested.