Development of a droplet digital PCR assay for quantification of the proviral load of bovine leukemia virus.

Droplet digital PCR (ddPCR) is a highly sensitive tool developed for the detection and quantification of short-sequence variants-a tool that offers unparalleled precision enabling measurement of smaller-fold changes. We describe here the use of ddPCR for the detection of Bovine leukemia virus (BLV) DNA provirus. Serum samples and whole blood from experimentally infected sheep and naturally infected cattle were analyzed through ddPCR to detect the BLV gp51 gene, and then compared with serologic and molecular tests. The ddPCR assay was significantly more accurate and sensitive than AGID, ELISA, nested PCR, and quantitative PCR. The limit of detection of ddPCR was 3.3 copies/µL, detecting positive experimentally infected sheep beginning at 6 d post-infection. The ddPCR methodology offers a promising tool for evaluating the BLV proviral load, particularly for the detection of low viral loads.

Alterations on growth and cell organization of Giardia intestinalis trophozoites after treatment with KH-TFMDI, a novel class III histone deacetylase inhibitor.

Giardia trophozoites have developed resistance mechanisms to currently available compounds, leading to treatment failures. In this context, the development of new additional agents is mandatory. Sirtuins, which are class III NAD+-dependent histone deacetylases, have been considered important targets for the development of new anti-parasitic drugs. Here, we evaluated the activity of KH-TFMDI, a novel 3-arylideneindolin-2-one-type sirtuin inhibitor, on G. intestinalis trophozoites. This compound decreased the trophozoite growth presenting an IC50 value lower than nicotinamide, a moderately active inhibitor of yeast and human sirtuins. Light and electron microscopy analysis showed the presence of multinucleated cell clusters suggesting that the cytokinesis could be compromised in treated trophozoites. Cell rounding, concomitantly with the folding of the ventro-lateral flange and flagella internalization, was also observed. These cells eventually died by a mechanism which lead to DNA/nuclear damage, formation of multi-lamellar bodies and annexin V binding on the parasite surface. Taken together, these data show that KH-TFMDI has significant effects against G. intestinalis trophozoites proliferation and structural organization and suggest that histone deacetylation pathway should be explored on this protozoon as target for chemotherapy.

Are your results valid? Cellular authentication a need from the past, an emergency on the present.

The cultures of immortalized cells have been established in the 50s and become popular as a biological model for in vitro assays. The success and popularization brought side effects. Still, in the 60 years emerge the first cases of misidentification/contamination of cell line. Because of that, the scientific community has been oriented to authenticate their lines before performing assays. The use of cells with incorrect identification or contamination has been identified as responsible for an increasing number of unmatched results and a waste of resources. For this reason, we implemented the Cell Line Authentication Service at Brazilian Metrology Institute (Inmetro), open to Brazilian scientific community and society in general. From 2012 to 2014 were conducted 111 cell line authentication test, of which 13.8% had some problem. Here are the description and discussion of these data and simple guidelines to minimize the risk of contamination and misidentification, and invite the scientific community to maintain an alert system to avoid spending unnecessary resources and produce unreliable data.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Mycalina: another crack in the Poecilosclerida framework.

This is the first phylogenetic analysis integrating both morphological and molecular data of the sponge suborder Mycalina (Poecilosclerida), which was erected in 1994. A cladistic analysis of morphology supported the monophyly of Cladorhizidae (including Euchelipluma), Guitarridae (excluding Euchelipluma), Isodictyidae, Latrunculiidae, and Podospongiidae but rejected monophyly for Desmacellidae, Esperiopsidae, Hamacanthidae, and Mycalidae. Analyses of partial 16S and partial 28S rRNA datasets combined, as well as that of a complete 18S rDNA dataset, suggest that Mycalina is not monophyletic; Biemnidae is only distantly related to other poecilosclerids; Merlia and Desmacella branch near the base of a diverse Poecilosclerida clade; Mycalidae is monophyletic (excluding Mycale [Anomomycale] titubans in 18S); and Esperiopsidae and Isodictyidae form a clade. Analyses of the two molecular datasets differed on the monophyly of Podospongiidae and about the relationship of Podospongiidae to Isodictyidae + Esperiopsidae.