Rapid isolation of mycoviral double-stranded RNA from Botrytis cinerea and Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>In most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA.</p> <p>Results</p> <p>A rapid method to isolate dsRNA from a virus-infected filamentous fungus, <it>Botrytis cinerea</it>, and from a killer strain of <it>Saccharomyces cerevisiae </it>using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing.</p> <p>Conclusions</p> <p>The method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.</p

Denaturing Gradient Gel Electrophoresis (DGGE) as a Powerful Novel Alternative for Differentiation of Epizootic ISA Virus Variants

Infectious Salmon Anemia is a devastating disease critically affecting world-wide salmon production. Chile has been particularly stricken by this disease which in all cases has been directly related with its causative agent, a novel orthomyxovirus which presents specific and distinctive infective features. Among these, two molecular markers have been directly associated with pathogenicity in two of the eight RNA sub genomic coding units of the virus: an insertion hot spot region present in viral segment 5 and a Highly Polymorphic Region (HPR) located in viral segment 6. Here we report the successful adaptation of a PCR-dependent denaturing gel electrophoresis technique (DGGE), which enables differentiation of selected reported HPR epizootic variants detected in Chile. At the same time, the technique allows us to distinguish one nucleotide differences in sequences associated with the intriguing, and still not well-understood, insertion events which tend to occur on RNA Segment 5. Thus, the versatility of the technique opens new opportunities for improved understanding of the complex biology of all ISA variants as well as possible applications to other highly variable pathogens

Anterior Closing-Wedge Osteotomy for Posterior Slope Correction With Tibial Tubercle Preservation

An excessive posterior tibial slope has been identified as a potential risk factor for anterior cruciate ligament tears. Anterior closing-wedge osteotomy decreases the posterior slope and can eliminate this risk factor in patients with recurrent instability and greater than 12° posterior slope. We will describe an anterior closing-wedge osteotomy technique performed at the tibial tubercle (TT), in which the TT is not detached to preserve the extensor mechanism attachment. A vertical cut is performed in the sagittal plane just posterior to the TT, leaving a distal cortical hinge. Two proximal parallel K-wires and 2 distal parallel K-wires convergent to the proximal ones are inserted from the anterior cortex on both sides of the tubercle toward the tibial posterior cortex at the posterior cruciate ligament's tibial insertion. Proximal and distal cuts are performed to remove the bone wedge. Reduction is achieved by gentle knee extension. Fixation is completed with 2 staples placed medially and laterally to the TT

Additional file 2: Table S1. of Draft genome sequence of Janthinobacterium lividum strain MTR reveals its mechanism of capnophilic behavior

The table shows the ANI values among the strain analyzed. ANI values were calculated according to Goris et al. 2007 (Goris J., Konstantinidis K., Klappenbach J., Coenye T., Vandamme P., Tiedje J. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 2007 57(Pt 1):81–91), using web application (http://enve-omics.ce.gatech.edu/ani/) (Figueras M., Beaz-Hidalgo R., Hossain M., Liles M. Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis. Genome Announc. 2014 2(6). pii: e00927-14. doi: 10.1128/genomeA.00927-14 ). (DOC 235 kb

Melatonin drives apoptosis in head and neck cancer by increasing mitochondrial ROS generated via reverse electron transport.

The oncostatic effects of melatonin correlate with increased reactive oxygen species (ROS) levels, but how melatonin induces this ROS generation is unknown. In the present study, we aimed to elucidate the two seemingly opposing actions of melatonin regarding its relationship with free radicals. We analyzed the effects of melatonin on head and neck squamous cell carcinoma cell lines (Cal-27 and SCC-9), which were treated with 0.5 or 1 mM melatonin. We further examined the potential effects of melatonin to induce ROS and apoptosis in Cal-27 xenograft mice. Here we report that melatonin mediates apoptosis in head and neck cancer by driving mitochondrial reverse electron transport (RET) to induce ROS production. Melatonin-induced changes in tumoral metabolism led to increased mitochondrial activity, which, in turn, induced ROS-dependent mitochondrial uncoupling. Interestingly, mitochondrial complex inhibitors, including rotenone, abolished the ROS elevation indicating that melatonin increased ROS generation via RET. Melatonin also increased membrane potential and CoQ10 H2 /CoQ10 ratio to elevate mitochondrial ROS production, which are essential conditions for RET. We found that genetic manipulation of cancer cells with alternative oxidase, which transfers electrons from QH2 to oxygen, inhibited melatonin-induced ROS generation, and apoptosis. RET restored the melatonin-induced oncostatic effect, highlighting the importance of RET as the site of ROS production. These results illustrate that RET and ROS production are crucial factors in melatonin's effects in cancer cells and establish the dual effect of melatonin in protecting normal cells and inducing apoptosis in cancer cells

Guidelines for the use of flow cytometry and cell sorting in immunological studies

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis