A genetic map of tetraploid <Emphasis Type="Italic">Paspalum notatum</Emphasis> Flügge (bahiagrass) based on single-dose molecular markers

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F₁ family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.     

Morpho-functional characterization of the goldfish (Carassius auratus L.) heart

Using morphological and physiological approaches we provided, for the first time, a structural and functional characterization of Carassius auratus L. heart. Besides to the classical four chambers, i.e. sinus venosus, atrium, ventricle, bulbus, we described two distinct structures corresponding to the atrio-ventricular (AV) region and the conus arteriosus. The atrium is very large and highly trabeculated; the ventricle shows an outer compacta, vascularized by coronary vessels, and an inner spongiosa; the bulbus wall is characterized by a high elastin/collagen ratio, which makes it extremely compliant. Immunolocalization revealed a strong expression of activated eNOS-like isoforms both at coronary endothelium and, to a lesser extent, in the myocardiocytes and the endocardial endothelium (EE). The structural design of the heart appears to comply with its mechanical function. Using an in vitro working heart preparation, cardiac performance was evaluated at different filling and afterload pressures. The hearts were very sensitive to filling pressure increases. Maximum Stroke volume (SV=1.08 ± 0.09 mL/kg body mass) was obtained with an input pressure of 0.4 kPa. The heart was not able to sustain afterload increases, values higher than 1.5 kPa impairing its performance. These morpho-functional features are consistent with a volume pump mechanical performance.     

Autophagy Inhibition Favors Survival of Rubrospinal Neurons After Spinal Cord Hemisection

Spinal cord injuries (SCIs) are devastating conditions of the central nervous system (CNS) for which there are no restorative therapies. Neuronal death at the primary lesion site and in remote regions that are functionally connected to it is one of the major contributors to neurological deficits following SCI.Disruption of autophagic flux induces neuronal death in many CNS injuries, but its mechanism and relationship with remote cell death after SCI are unknown. We examined the function and effects of the modulation of autophagy on the fate of axotomized rubrospinal neurons in a rat model of spinal cord dorsal hemisection (SCH) at the cervical level. Following SCH, we observed an accumulation of LC3-positive autophagosomes (APs) in the axotomized neurons 1 and 5 days after injury. Furthermore, this accumulation was not attributed to greater initiation of autophagy but was caused by a decrease in AP clearance, as demonstrated by the build-up of p62, a widely used marker of the induction of autophagy. In axotomized rubrospinal neurons, the disruption of autophagic flux correlated strongly with remote neuronal death and worse functional recovery. Inhibition of AP biogenesis by 3-methyladenine (3-MA) significantly attenuated remote degeneration and improved spontaneous functional recovery, consistent with the detrimental effects of autophagy in remote damage after SCH. Collectively, our results demonstrate that autophagic flux is blocked in axotomized neurons on SCI and that the inhibition of AP formation improves their survival. Thus, autophagy is a promising target for the development of therapeutic interventions in the treatment of SCIs.     

PicoNewton-millisecond force steps reveal the transition kinetics and mechanism of the double-stranded DNA elongation

We study the kinetics of the overstretching transition in λ-phage double-stranded (ds) DNA from the basic conformation (B state) to the 1.7-times longer and partially unwound conformation (S state), using the dual-laser optical tweezers under force-clamp conditions at 25°C. The unprecedented resolution of our piezo servo-system, which can impose millisecond force steps of 0.5–2 pN, reveals the exponential character of the elongation kinetics and allows us to test the two-state nature of the B-S transition mechanism. By analyzing the load-dependence of the rate constant of the elongation, we find that the elementary elongation step is 5.85 nm, indicating a cooperativity of ∼25 basepairs. This mechanism increases the free energy for the elementary reaction to ∼94 k_BT, accounting for the stability of the basic conformation of DNA, and explains why ds-DNA can remain in equilibrium as it overstretches.     

Metabotropic Glutamate Receptor 1 Expression and Its Polymorphic Variants Associate with Breast Cancer Phenotypes

Several epidemiological studies have suggested a link between melanoma and breast cancer. Metabotropic glutamate receptor 1 (GRM1), which is involved in many cellular processes including proliferation and differentiation, has been implicated in melanomagenesis, with ectopic expression of GRM1 causing malignant transformation of melanocytes. This study was undertaken to evaluate GRM1 expression and polymorphic variants in GRM1 for associations with breast cancer phenotypes. Three single nucleotide polymorphisms (SNPs) in GRM1 were evaluated for associations with breast cancer clinicopathologic variables. GRM1 expression was evaluated in human normal and cancerous breast tissue and for in vitro response to hormonal manipulation. Genotyping was performed on genomic DNA from over 1,000 breast cancer patients. Rs6923492 and rs362962 genotypes associated with age at diagnosis that was highly dependent upon the breast cancer molecular phenotype. The rs362962 TT genotype also associated with risk of estrogen receptor or progesterone receptor positive breast cancer. In vitro analysis showed increased GRM1 expression in breast cancer cells treated with estrogen or the combination of estrogen and progesterone, but reduced GRM1 expression with tamoxifen treatment. Evaluation of GRM1 expression in human breast tumor specimens demonstrated significant correlations between GRM1 staining with tissue type and molecular features. Furthermore, analysis of gene expression data from primary breast tumors showed that high GRM1 expression correlated with a shorter distant metastasis-free survival as compared to low GRM1 expression in tamoxifen-treated patients. Additionally, induced knockdown of GRM1 in an estrogen receptor positive breast cancer cell line correlated with reduced cell proliferation. Taken together, these findings suggest a functional role for GRM1 in breast cancer.