Chiral bioanalytical methods in bioequivalence studies of intravenous vs. oral formulations of ibuprofen

According to the Ibuprofen Product-Specific Bioequivalence Guidance of the European Medicines Agency, achiral bioanalytical methods are considered acceptable for demonstration of bioequivalence of ibuprofen-containing products. The aim of this investigation is to compare the bioequivalence outcomes obtained with individual R and S ibuprofen enantiomers and the sum of both enantiomers from bioequivalence studies in which new intravenous ibuprofen products were compared with oral ibuprofen products. Bioequivalence was assessed for S and R enantiomers of ibuprofen and the sum of both enantiomers, which was calculated to represent the results that would have been obtained with an achiral assay. The infusion rates of 15, 20, and 30 minutes modify the maximum concentration (C_max) of the intravenous administrations. In contrast, the time when the maximum concentration is observed (T_max) was insensitive to detect differences in input rate within this range of infusion times. The eutomer S-ibuprofen is the least sensitive analyte to detect differences in input rate; therefore, the regulatory acceptance of achiral bioanalytical methods for ibuprofen bioequivalence studies is justified because the sum of both enantiomers is more discriminative than the chiral methods where only the eutomer is used for regulatory decisions.     

Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro

Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell–cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4⁺ cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4⁺ cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env⁺ cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4⁺ cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell–cell fusion can be performed by flow cytometry.     

Male Lineages in Brazil: Intercontinental Admixture and Stratification of the European Background

The non-recombining nature of the Y chromosome and the well-established phylogeny of Y-specific Single Nucleotide Polymorphisms (Y-SNPs) make them useful for defining haplogroups with high geographical specificity; therefore, they are more apt than the Y-STRs to detect population stratification in admixed populations from diverse continental origins. Different Y-SNP typing strategies have been described to address issues of population history and movements within geographic territories of interest. In this study, we investigated a set of 41 Y-SNPs in 1217 unrelated males from the five Brazilian geopolitical regions, aiming to disclose the genetic structure of male lineages in the country. A population comparison based on pairwise F_ST genetic distances did not reveal statistically significant differences in haplogroup frequency distributions among populations from the different regions. The genetic differences observed among regions were, however, consistent with the colonization history of the country. The sample from the Northern region presented the highest Native American ancestry (8.4%), whereas the more pronounced African contribution could be observed in the Northeastern population (15.1%). The Central-Western and Southern samples showed the higher European contributions (95.7% and 93.6%, respectively). The Southeastern region presented significant European (86.1%) and African (12.0%) contributions. The subtyping of the most frequent European lineage in Brazil (R1b1a-M269) allowed differences in the genetic European background of the five Brazilian regions to be investigated for the first time.     

Turnip remains from Byzantine Sparta

Charred parenchymous tissue was recovered from Byzantine levels at the site of Sparta, Greece. On examination, using morphological and histological characteristics it was identified as being turnip,Brassica campestris ssp.rapifera (Metzg.) Sinsk. This discovery is of great significance in the study of the site, and also of Byzantine economy.     

Abordaje del anciano en el servicio de urgencias de un hospital de agudos

Elderly people use often and in an appropriate way the Emergency Department (ED). However, we don’t dispose of evidences that demonstrate utility of a specific model of geriatric assessment (GA) applied in ED. Nowadays; GA in ED should be used being adapted to the environment and with some clear objectives that allow to carry out a multidimensional diagnosis and to establish therapeutic priorities. GA contributes benefits in the continuity of care on the part of the Primary Attention when elderly people are discharged from an ED.     

Muscarinic cholinoceptor activation by pilocarpine triggers apoptosis in human skin fibroblast cells

The aim of the present work was to examine the role of muscarinic acetylcholine receptors (mAChRs) on apoptosis in human skin fibroblast cells. Neonatal human skin fibroblast cultures were stimulated with pilocarpine in the presence or absence of specific antagonists. Pilocarpine stimulates apoptosis, total inositol phosphates (InsP) accumulation and nitric oxide synthase (NOS) activity. All these effects were inhibited by atropine, mustard hydrochloride (4‐DAMP) and pirenzepine, indicating that M₁ and M₃ mAChRs are implicated in pilocarpine action. Pilocarpine apoptotic action is accompanied by caspase‐3 and JNK activation. The intracellular pathway leading to pilocarpine‐induced biological effects involved phospholipase C, calcium/calmodulin and extracellular calcium as U‐73122, W‐7, verapamil, BAPTA and BAPTA‐AM blocked pilocarpine effects. L ‐NMMA, a NOS inhibitor, had no effect, indicating that the enzyme does not participate in the apoptosis phenomenon. These results may contribute to a better understanding of the modulatory role of the parasympathetic muscarinic system on the apoptotic human skin fibroblast process. J. Cell. Physiol. 222: 640–647, 2010. © 2009 Wiley‐Liss, Inc.     

Kinetochore individualization in meiosis I is required for centromeric cohesin removal in meiosis II

Partitioning of the genome in meiosis occurs through two highly specialized cell divisions, named meiosis I and meiosis II. Step‐wise cohesin removal is required for chromosome segregation in meiosis I, and sister chromatid segregation in meiosis II. In meiosis I, mono‐oriented sister kinetochores appear as fused together when examined by high‐resolution confocal microscopy, whereas they are clearly separated in meiosis II, when attachments are bipolar. It has been proposed that bipolar tension applied by the spindle is responsible for the physical separation of sister kinetochores, removal of cohesin protection, and chromatid separation in meiosis II. We show here that this is not the case, and initial separation of sister kinetochores occurs already in anaphase I independently of bipolar spindle forces applied on sister kinetochores, in mouse oocytes. This kinetochore individualization depends on separase cleavage activity. Crucially, without kinetochore individualization in meiosis I, bivalents when present in meiosis II oocytes separate into chromosomes and not sister chromatids. This shows that whether centromeric cohesin is removed or not is determined by the kinetochore structure prior to meiosis II.     

Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer,multi-species spread and geographical clustering

The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.