Monoterpene glycoside biosynthesis in detached grape berries grown in vitro

A procedure for the culture in vitro of isolated small berries of Vitis vinifera L. cv. Muscat of Alexandria in a Murashige and Skoog basal medium supplemented with N⁶-benzyladenine and indoleacetic acid is described. Berries developed well in culture during 60 days and tripled in size, but remained green and smaller than normal berries grown in vivo. Some callus formed on the distal end of the berry, and where major skin damage occurred, callus emerged from the cracked berries. In order to examine their biosynthetic competency, berries which were previously cultured in vitro for 60 days were incubated for 48 h in a Murashige and Skoog medium containing a [¹⁴C]-labelled water-soluble fraction. This fraction was isolated from grape berries located adjacent to a leaf that had been exposed to gaseous ¹⁴CO₂ in full sunlight for 5 h. The berries were then recultured for 48 h after which a glycosidic fraction was isolated on a C18 reversed phase column and further separated by thin layer chromatography (TLC). The major labelled band corresponded to the geranyl-β-rutinoside marker, indicating that grape berries have the ability to synthesize monoterpene glycosides. This band also consisted of other monoterpene glycosides as revealed by the gas chromatography-mass spectrometry (GC-MS) analysis of their aglycones (released by enzymatic hydrolysis).     

Quantitative analysis of Escherichia coli metabolic phenotypes within the context of phenotypic phase planes

In silico models of Escherichia coli metabolism have been developed to predict metabolic behavior and propose experimentally testable hypotheses. However, a thorough assessment of the metabolic phenotype requires well-designed experimentation and reproducible experimental techniques. A method for the quantitative analysis of E. coli metabolism in vivo within the framework of in silico phenotypic phase plane analysis is presented. Using this approach, we have quantitatively studied E. coli metabolism in various environmental conditions and nutritional media. Our experimental methodology, in combination with steady-state metabolic models, can be used to study biological properties and evaluate the metabolic capabilities of microbes.     

The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks

Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.     

Antenatal Dexamethasone after Asphyxia Increases Neural Injury in Preterm Fetal Sheep

Background and Purpose

Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain.

Methods

Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach.

Results

Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290±76 vs 484±98 neurons/mm², mean±SEM, P<0.05) and basal ganglia (putamen, 538±112 vs 814±34 neurons/mm², P<0.05) compared to asphyxia-saline, and with greater loss of both total (913±77 vs 1201±75/mm², P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66±8 vs 114±12/mm², P<0.05, vs sham controls 165±10/mm², P<0.001). This was associated with transient hyperglycemia (peak 3.5±0.2 vs. 1.4±0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum −1.5±1.2 dB vs. −5.0±1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures.

Conclusions

In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.     

Genetic and molecular characterization of the human Osteosarcoma 3AB‐OS cancer stem cell line: A possible model for studying osteosarcoma origin and stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second‐line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB‐OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB‐OS cells have hypertriploid karyotype with 71–82 chromosomes. By comparing 3AB‐OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let‐7/98 and miR‐29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets. J. Cell. Physiol. 228: 1189–1201, 2013. © 2012 Wiley Periodicals, Inc.     

Soil nutrients trump intraspecific effects on understory plant communities

Understanding the links between intraspecific genetic variation and patterns of diversity in associated communities has been the primary focus of community genetics or ‘genes-to-ecosystem’ research in ecology. While other ecological factors, such as the abiotic environment, have well-documented influences on communities, the relative contributions of genetic variation versus the environment to species interactions remains poorly explored. In this study, we use a common garden experiment to study a coastal dune plant community dominated by the shrub, Baccharis pilularis, which displays a morphological dimorphism in plant architecture. We found the differences in the understory plant community between erect and prostrate morphs of Baccharis to be statistically significant, but small relative to the impacts of nutrient additions (NPK and C additions), for the richness, cover, and biomass of the understory plant community. There were no significant interactions between Baccharis morphology and nutrient-addition treatments, suggesting the influence of nutrient addition was consistent between erect and prostrate morphs. Moreover, we found no difference in overall plant community composition between Baccharis morphs, while NPK additions led to shifts in understory community composition compared to unfertilized shrubs. In sum, our results indicate that nutrients are the more important factor governing understory plant community structure in a coastal dunes ecosystem followed by intraspecific variation in dominant shrub architecture. Our results address a growing call to understand the extended consequences of intraspecific variation across heterogeneous environments in terrestrial ecosystems.     

Retinal Cell Death Induced by TRPV1 Activation Involves NMDA Signaling and Upregulation of Nitric Oxide Synthases

The activation of the transient receptor potential vanilloid type 1 channel (TRPV1) has been correlated with oxidative and nitrosative stress and cell death in the nervous system. Our previous results indicate that TRPV1 activation in the adult retina can lead to constitutive and inducible nitric oxide synthase-dependent protein nitration and apoptosis. In this report, we have investigated the potential effects of TRPV1 channel activation on nitric oxide synthase (NOS) expression and function, and the putative participation of ionotropic glutamate receptors in retinal TRPV1-induced protein nitration, lipid peroxidation, and DNA fragmentation. Intravitreal injections of the classical TRPV1 agonist capsaicin up-regulated the protein expression of the inducible and endothelial NOS isoforms. Using 4,5-diaminofluorescein diacetate for nitric oxide (NO) imaging, we found that capsaicin also increased the production of NO in retinal blood vessels. Processes and perikarya of TRPV1-expressing neurons in the inner nuclear layer of the retina were found in the vicinity of nNOS-positive neurons, but those two proteins did not colocalize. Retinal explants exposed to capsaicin presented high protein nitration, lipid peroxidation, and cell death, which were observed in the inner nuclear and plexiform layers and in ganglion cells. This effect was partially blocked by AP-5, a NMDA glutamate receptor antagonist, but not by CNQX, an AMPA/kainate receptor antagonist. These data support a potential role for TRPV1 channels in physiopathological retinal processes mediated by NO, which at least in part involve glutamate release.     

A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT‐causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III‐specific subunit), Trs120p and Trs130p (TRAPP II‐specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol‐to‐vacuole (cvt) pathway as well as non‐selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed .     

Hemodynamical analysis of MHD two phase blood flow through a curved permeable artery having variable viscosity with heat and mass transfer

Biomechanics and Modeling in Mechanobiology - A numerical investigation of MHD blood flow through a stenosed permeable curved artery has been done in this study. Blood flow is considered in...