In vivo distribution and behavior of paramagnetic dinitrosyl dithiolato iron complex in the abdomen of mouse

It has been shown that a dinitrosyl dithiolato iron complex is formed under physiological conditions and that it functions as an NO transporter. In the present study, a diglutathionyl dinitrosyl iron complex [DNIC-(GS)₂] was injected into mice and its abdominal distribution and behavior were examined by using electron paramagnetic resonance (EPR) spectroscopy. The X-band EPR signal intensity of the blood, liver, kidney, and spleen decreased with time but signals from the liver and kidney were readily detectable even 24h after the injection. The time courses of signal intensity were quite similar when the agent was administered via intravenous and subcutaneous injection routes, suggesting that DNIC-(GS)₂ can penetrate readily and rapidly through the membranes. Real-time detection of DNIC-(GS)₂ in the upper abdomen of the living mice was performed by employing an in vivo EPR spectroscopy. These results suggest that DNIC-(GS)₂, an endogenous NO carrier, has an excellent membrane permeability and has a relatively high affinity for the liver and kidney.     

Imaging mass spectrometry at cellular length scales

Imaging mass spectrometry (IMS) allows the direct investigation of both the identity and the spatial distribution of the molecular content directly in tissue sections, single cells and many other biological surfaces. In this protocol, we present the steps required to retrieve the molecular information from tissue sections using matrix-enhanced (ME) and metal-assisted (MetA) secondary ion mass spectrometry (SIMS) as well as matrix-assisted laser desorption/ionization (MALDI) IMS. These techniques require specific sample preparation steps directed at optimal signal intensity with minimal redistribution or modification of the sample analytes. After careful sample preparation, different IMS methods offer a unique discovery tool in, for example, the investigation of (i) drug transport and uptake, (ii) biological processing steps and (iii) biomarker distributions. To extract the relevant information from the huge datasets produced by IMS, new bioinformatics approaches have been developed. The duration of the protocol is highly dependent on sample size and technique used, but on average takes approximately 5 h.     

A new species of fern, genus Danaea (Marattiales: Marattiaceae) endemic to Costa Rica

Danaea tuomistoana A. Rojas (Marattiaceae) is described and illustrated as a new species endemic to Costa Rica; it differs from D. crispa Endres et Richb. by a truncate blade, and by the pinnae, which are 2/5-1/2 incised, cuneated at the base and with a flat margin. The range of D. erecta H. Tuomisto & R.C. Moran in Costa Rica is expanded with a new locality in Puntarenas (8 degrees 57'15" N-82 degrees 50'1" W, 1,500-1,580 masl).     

Recent advances in understanding sex differences in cardiac repolarization

A number of gender differences exist in the human electrocardiogram (ECG): the P-wave and P-R intervals are slightly longer in men than in women, whilst women have higher resting heart rates than do men, but a longer rate-corrected QT (QT_C) interval. Women with the LQT1 and LQT2 variants of congenital long-QT syndrome (LQTS) are at greater risk of adverse cardiac events. Similarly, many drugs associated with acquired LQTS have a greater risk of inducing torsades de pointes (TdP) arrhythmia in women than in men. There are also male:female differences in Brugada syndrome, early repolarisation syndrome and sudden cardiac death. The differences in the ECG between men and women, and in particular those relating to the QT interval, have been explored experimentally and provide evidence of differences in the processes underlying ventricular repolarization. The data available from rabbit, canine, rat, mouse and guinea pig models are reviewed and highlight involvement of male:female differences in Ca and K currents, although the possible involvement of rapid and persistent Na current and Na–Ca exchange currents cannot yet be excluded. The mechanisms underlying observed differences remain to be elucidated fully, but are likely to involve the influence of gonadal steroids. With respect to the QT interval and risk of TdP, a range of evidence implicates a protective role of testosterone in male hearts, possibly by both genomic and non-genomic pathways. Evidence regarding oestrogen and progesterone is less unequivocal, although the interplay between these two hormones may influence both repolarization and pro-arrhythmic risk.     

A novel method for human hematopoietic stem/progenitor cell isolation from umbilical cord blood based on immunoaffinity aqueous two-phase partitioning

A novel cell separation process based on immunoaffinity aqueous two phase systems is presented to isolate and purify CD34⁺ stem/progenitor cells directly from the whole umbilical cord blood (UCB). A system, composed of polyethylene glycol and dextran, was evaluated for the selective recovery of CD34⁺ cells from UCB. A monoclonal antibody against the CD34 surface antigen was used for the direct partitioning of CD34⁺ cells in UCB to the PEG-rich phase. The initial population of CD34⁺ cells (0.2% of the initial sample) was enriched to values up to 42% in a single partitioning step, while the majority of contaminant cells were partitioned to the dextran-rich phase (1.37 × 10⁻² < K_P < 2.76 × 10⁻²). This novel selection method allowed a recovery yield of 95% of CD34⁺ cells with a purification factor of 245 and is expected to pave a new way to purify hematopoietic stem/progenitor cells for use in a variety of clinical settings.     

The role of curcumin on intestinal oxidative stress,cell proliferation and apoptosis after ischemia/reperfusion injury in rats

The aim of this study was to demonstrate the role of curcumin on oxidative stress, cell proliferation and apoptosis in the rat intestinal mucosa after ischemia/reperfusion (I/R). A total of 30 male Wistar albino rats were divided into three groups: sham, I/R and I/R+ curcumin; each group contain 10 animals. Sham group animals underwent laparotomy without I/R injury. After I/R groups animals underwent laparotomy, 1 h of superior mesenteric artery ligation were followed by 1 h of reperfusion. In the curcumin group, 3 days before I/R, curcumin (100 mg/kg) was administered by gastric gavage. All animals were sacrificed at the end of reperfusion and intestinal tissues samples were obtained for biochemical and histopathological investigation in all groups. Curcumin treatment significantly decreased the elevated tissue malondialdehyde levels and increased of reduced superoxide dismutase, and glutathione peroxidase enzyme activities in intestinal tissues samples. I/R caused severe histopathological injury including mucosal erosions and villous congestion and hemorrhage. Curcumin treatment significantly attenuated the severity of intestinal I/R injury, with inhibiting of I/R-induced apoptosis and cell proliferation. These results suggest that curcumin treatment has a protective effect against intestinal damage induced by intestinal I/R. This protective effect is possibly due to its ability to inhibit I/R-induced oxidative stress, apoptosis and cell proliferation.     

Cell- and gene-therapy approaches to inner ear repair

Sensorineural hearing loss is the most common sensory disorder in humans. It is primarily due to the degeneration of highly specialised mechanosensory cells in the cochlea, the so-called hair cells. Hearing problems can also be caused or further aggravated by the death of auditory sensory neurons that convey the information from the hair cells to the brain stem. Despite the discovery of stem/progenitor cells in the mammalian cochlea, no regeneration of either damaged hair cells or auditory neurons has been observed in mammals, in contrast to what is seen in avians and non-mammalian vertebrates. The reasons for this divergence have not yet been elucidated, although loss of stem cells and/or loss of their phenotypic plasticity in adult mammals have been put forward as possible explanations. Given the high incidence of this disorder and its economic and social implications, a considerable number of research lines have been set up aimed towards the regeneration of cochlear sensory cell types. This review summarizes the various routes that have been explored, ranging from the genetic modification of endogenous cells remaining in the inner ear in order to promote their transdifferentiation, to the implantation of exogenous stem or progenitor cells and their subsequent differentiation within the host tissue. Prophylactic treatments to fight against progressive sensory cell degeneration in the inner ear are also discussed.     

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions

Background

Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs.

Methodology/Principal Findings

We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters.

Conclusions/Significance

In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.     

Radiolabeling rhesus monkey CD34+ hematopoietic and mesenchymal stem cells with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for microPET imaging

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34+ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34+ hematopoietic cells were radiolabeled with 0 to 40 microCi/mL 64Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34+ cells radiolabeled with 20 microCi/mL and rhMSCs radiolabeled with 10 microCi/mL 64Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34+ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 microCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50x10(4) CD34+ cells (1.1 pCi/cell) and 6.25x10(3) rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34+ hematopoietic cells and rhMSCs can be safely radiolabeled with 64Cu-PTSM without adverse cellular effects.     

The mechanical stability of ubiquitin is linkage dependent

Ubiquitin chains are formed through the action of a set of enzymes that covalently link ubiquitin either through peptide bonds or through isopeptide bonds between their C terminus and any of four lysine residues. These naturally occurring polyproteins allow one to study the mechanical stability of a protein, when force is applied through different linkages. Here we used single-molecule force spectroscopy techniques to examine the mechanical stability of N-C-linked and Lys48-C-linked ubiquitin chains. We combined these experiments with steered molecular dynamics (SMD) simulations and found that the mechanical stability and unfolding pathway of ubiquitin strongly depend on the linkage through which the mechanical force is applied to the protein. Hence, a protein that is otherwise very stable may be easily unfolded by a relatively weak mechanical force applied through the right linkage. This may be a widespread mechanism in biological systems.