Monopolin subunit Csm1 associates with MIND complex to establish monopolar attachment of sister kinetochores at meiosis I

Sexually reproducing organisms halve their cellular ploidy during gametogenesis by undergoing a specialized form of cell division known as meiosis. During meiosis, a single round of DNA replication is followed by two rounds of nuclear divisions (referred to as meiosis I and II). While sister kinetochores bind to microtubules emanating from opposite spindle poles during mitosis, they bind to microtubules originating from the same spindle pole during meiosis I. This phenomenon is referred to as mono-orientation and is essential for setting up the reductional mode of chromosome segregation during meiosis I. In budding yeast, mono-orientation depends on a four component protein complex referred to as monopolin which consists of two nucleolar proteins Csm1 and Lrs4, meiosis-specific protein Mam1 of unknown function and casein kinase Hrr25. Monopolin complex binds to kinetochores during meiosis I and prevents bipolar attachments. Although monopolin associates with kinetochores during meiosis I, its binding site(s) on the kinetochore is not known and its mechanism of action has not been established. By carrying out an imaging-based screen we have found that the MIND complex, a component of the central kinetochore, is required for monopolin association with kinetochores during meiosis. Furthermore, we demonstrate that interaction of monopolin subunit Csm1 with the N-terminal domain of MIND complex subunit Dsn1, is essential for both the association of monopolin with kinetochores and for monopolar attachment of sister kinetochores during meiosis I. As such this provides the first functional evidence for a monopolin-binding site at the kinetochore

Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Background The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration

Determination of total and available fractions of PAHs by SPME in oily wastewaters : overcoming interference from NAPL and NOM

Background, aim, and scope Polycyclic aromatic hydrocarbons (PAHs) are often found in oily wastewaters. Their presence is usually the result of human activities and has a negative effect on the environment. One important step in addressing this problem is to evaluate the effectiveness of PAH removal by biological processes since these are the most cost-effective treatments known today. Many techniques are presently available for PAH determination in wastewaters. Solid phase microextracion (SPME) is known to be one of the most effective techniques for this purpose. When analyzing complex matrices with substances such as natural organic matter (NOM) and non-aqueous phase liquids (NAPL), it is important to differentiate the free dissolved PAH from matrix-bonded PAH. PAHs associated with the bonded fraction are less susceptible to biological treatment. The present study concerns the development of a simple and suitable methodology for the determination of the freely dissolved and the total fraction of PAHs present in oily wastewaters. The methodology was then applied to an oily wastewater from a fuel station retention basin. Material and methods Headspace SPME was used for analyzing PAH since the presence of a complex or dirty matrix in direct contact with the fiber may damage it. Four model PAHs—anthracene, fluorene, phenanthrene, and pyrene—were analyzed by GC-MS. Negligible depletion SPME technique was used to determine the free fraction. Total PAH was determined by enhancing the mass transfer from the bonded phase to the freely dissolved phase by temperature optimization and the use of the method of standard additions. The PAH absorption kinetics were determined in order to define the optimal sampling conditions for this method. The fitting of the experimental data to a mathematical model was accomplished using Berkeley Madonna software. Humic acid and silicon oil were used as model NOM and NAPL, respectively, to study the effect of these compounds on the decrease of SPME response. Then, the method was evaluated with wastewater from a fuel station spill retention basin. Results The SPME kinetic parameters—k 1 (uptake rate), k 2 (desorption rate), and K SPME (partition coefficient)—were determined from experimental data modeling. The determination of the free fraction required 15-min sampling to ensure that PAH depletion from sample was below 1%. For total PAH, a 30-min extraction at 100°C ensured the maximum signal response in the GC-MS. For the determination of free and total PAHs, extractions were performed before reaching the SPME equilibrium. The wastewater used in this study had no free fraction of the analyzed PAHs. However, the four studied PAHs were found when the method for total PAH was used. Discussion The addition of NOM and NAPL dramatically decreased the efficiency of the SPME. This decrease was the result of a greater partition of the PAHs to the NAPL and NOM phases. This fact was also observed in the analysis of the fuel station spill retention basin, where no free PAH was measured. However, using the method of standard addition for the determination of total PAH, it was possible to quantify all four PAHs. Conclusions The method developed in the present study was found to be adequate to differentiate between free and total PAH present in oily wastewater. It was determined that the presence of NOM and NAPL had a negative effect on SPME efficiency. Recommendations and perspectives The presence of binding substances had a great influence on SPME kinetics. Therefore, it is of extreme importance to determine their degree of interference when analyzing oily wastewaters or results can otherwise be erroneous. Other factors influencing the total PAH determinations should be considered in further studies.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/ 18816/2004, POCI/AMB/61044/200

Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles

Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD) human (HAd) and canine (CAV-2) adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV) effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS). With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways - but in opposite directions - suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer

Pathways for horizontal gene transfer in bacteria revealed by a global map of their plasmids

Plasmids can mediate horizontal gene transfer of antibiotic resistance, virulence genes, and other adaptive factors across bacterial populations. Here, we analyze genomic composition and pairwise sequence identity for over 10,000 reference plasmids to obtain a global map of the prokaryotic plasmidome. Plasmids in this map organize into discrete clusters, which we call plasmid taxonomic units (PTUs), with high average nucleotide identity between its members. We identify 83 PTUs in the order Enterobacterales, 28 of them corresponding to previously described archetypes. Furthermore, we develop an automated algorithm for PTU identification, and validate its performance using stochastic blockmodeling. The algorithm reveals a total of 276 PTUs in the bacterial domain. Each PTU exhibits a characteristic host distribution, organized into a six-grade scale (I-VI), ranging from plasmids restricted to a single host species (grade I) to plasmids able to colonize species from different phyla (grade VI). More than 60% of the plasmids in the global map are in groups with host ranges beyond the species barrier.This work was funded by grant BFU2017-86378-P from the Spanish MINEC

The mediating role of shared flow and perceived emotional synchrony on compassion for others in a mindful-dancing program

While there is a growing understanding of the relationship between mindfulness and compassion, this largely relates to the form of mindfulness employed in first-generation mindfulness-based interventions such as Mindfulness-Based Stress Reduction. Consequently, there is limited knowledge of the relationship between mindfulness and compassion in respect of the type of mindfulness employed in second-generation mindfulness-based interventions (SG-MBIs), including those that employ the principle of working harmoniously as a “secular sangha.” Understanding this relationship is important because research indicates that perceived emotional synchrony (PES) and shared flow—that often arise during participation in harmonized group contemplative activities—can enhance outcomes relating to compassion, subjective well-being, and group identity fusion. This pilot study analyzed the effects of participation in a mindful-dancing SG-MBI on compassion and investigated the mediating role of shared flow and PES. A total of 130 participants were enrolled into the study that followed a quasi-experimental design with an intervention and control group. Results confirmed the salutary effect of participating in a collective mindful-dancing program, and demonstrated that shared flow and PES fully meditated the effects of collective mindfulness on the kindness and common humanity dimensions of compassion. Further research is warranted to explore whether collective mindfulness approaches, such as mindful dancing, may be a means of enhancing compassion and subjective well-being outcomes due to the mediating role of PES and shared flow.N/

Initial Commitment to Pre-Exposure Prophylaxis and Circumcision for HIV Prevention amongst Indian Truck Drivers

Studies of HIV prevention interventions such as pre-exposure prophylaxis (PREP) and circumcision in India are limited. The present study sought to investigate Indian truck-drivers initial commitment to PREP and circumcision utilizing the AIDS Risk Reduction Model. Ninety truck-drivers completed an in-depth qualitative interview and provided a blood sample for HIV and HSV-2 testing. Truck-drivers exhibited low levels of initial commitment towards PREP and even lower for circumcision. However, potential leverage points for increasing commitment were realized in fear of infecting family rather than self, self-perceptions of risk, and for PREP focusing on cultural beliefs towards medication and physicians. Cost was a major barrier to both HIV prevention interventions. Despite these barriers, our findings suggest that the ARRM may be useful in identifying several leverage points that may be used by peers, health care providers and public health field workers to enhance initial commitment to novel HIV prevention interventions in India

A Novel Extracytoplasmic Function (ECF) Sigma Factor Regulates Virulence in Pseudomonas aeruginosa

Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS systems sense signals from outside the cell and transmit them into the cytoplasm. They generally consist of a TonB-dependent outer membrane receptor, a sigma factor regulator (or anti-sigma factor) in the cytoplasmic membrane, and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa CSS systems are involved in the regulation of iron uptake, we have identified a novel system involved in the regulation of virulence. This CSS system, which has been designated PUMA3, has a number of unusual characteristics. The most obvious difference is the receptor component which is considerably smaller than that of other CSS outer membrane receptors and lacks a β-barrel domain. Homology modeling of PA0674 shows that this receptor is predicted to be a bilobal protein, with an N-terminal domain that resembles the N-terminal periplasmic signaling domain of CSS receptors, and a C-terminal domain that resembles the periplasmic C-terminal domains of the TolA/TonB proteins. Furthermore, the sigma factor regulator both inhibits the function of the ECF sigma factor and is required for its activity. By microarray analysis we show that PUMA3 regulates the expression of a number of genes encoding potential virulence factors, including a two-partner secretion (TPS) system. Using zebrafish (Danio rerio) embryos as a host we have demonstrated that the P. aeruginosa PUMA3-induced strain is more virulent than the wild-type. PUMA3 represents the first CSS system dedicated to the transcriptional activation of virulence functions in a human pathogen

Genetic Differentiation of the Western Capercaillie Highlights the Importance of South-Eastern Europe for Understanding the Species Phylogeography

The Western Capercaillie (Tetrao urogallus L.) is a grouse species of open boreal or high altitude forests of Eurasia. It is endangered throughout most mountain range habitat areas in Europe. Two major genetically identifiable lineages of Western Capercaillie have been described to date: the southern lineage at the species' southernmost range of distribution in Europe, and the boreal lineage. We address the question of genetic differentiation of capercaillie populations from the Rhodope and Rila Mountains in Bulgaria, across the Dinaric Mountains to the Slovenian Alps. The two lineages' contact zone and resulting conservation strategies in this so-far understudied area of distribution have not been previously determined. The results of analysis of mitochondrial DNA control region sequences of 319 samples from the studied populations show that Alpine populations were composed exclusively of boreal lineage; Dinaric populations of both, but predominantly (96%) of boreal lineage; and Rhodope-Rila populations predominantly (>90%) of southern lineage individuals. The Bulgarian mountains were identified as the core area of the southern lineage, and the Dinaric Mountains as the western contact zone between both lineages in the Balkans. Bulgarian populations appeared genetically distinct from Alpine and Dinaric populations and exhibited characteristics of a long-term stationary population, suggesting that they should be considered as a glacial relict and probably a distinct subspecies. Although all of the studied populations suffered a decline in the past, the significantly lower level of genetic diversity when compared with the neighbouring Alpine and Bulgarian populations suggests that the isolated Dinaric capercaillie is particularly vulnerable to continuing population decline. The results are discussed in the context of conservation of the species in the Balkans, its principal threats and legal protection status. Potential conservation strategies should consider the existence of the two lineages and their vulnerable Dinaric contact zone and support the specificities of the populations