Synthesis and Conformational Analysis of A Novel Type of Spin Labelled Bicyclonucleoside Based on A Tetrahydrofurano[2,3-c]Pyrrolidine Sugar Skeleton

Abstract

Bicyclonucleosides bearing a 5-deoxy-5-N-hydroxyamino-3,N ⁵-(1,1-ethano)-β-o-furanosyl sugar moiety (15–18) have been prepared by glycosidation of the corresponding bicyclosugars obtained via an intramolecular reverse Cope elimination. The configuration of the asymmetric carbon of the 1,1-ethano bridge is the most important factor directing the conformation of the N-hydroxypyrrolidine ring and its invertomers ratio as shown by variable temperature H NMR experiments.     

Effect of reduced oxygen tension on reactive oxygen species production and activity of antioxidant enzymes in swine granulosa cells

During follicle growth swine granulosa cells are physiologically exposed to a progressive oxygen shortage. It has already been shown that hypoxia stimulates angiogenesis through an increase of VEGF production, however, despite considerable progress in the understanding of the final events induced by cellular hypoxia, the signal transduction pathway remains elusive. Recent evidence suggest a role for Reactive Oxygen Species (ROS) as hypoxia signal transducer. Granulosa cells were isolated from pig follicles (> 5 mm) and cultured for 18 h in normoxic (19% O2), hypoxic (5% O2) or anoxic (1% O2) conditions. Following the incubation ROS (O2- and H2O2) production and the activity of scavenging enzymes (SOD, catalase and peroxidase) were determined. It was apparent from our data that ROS generation was reduced by hypoxia. On the contrary, SOD and peroxidase, but not catalase, increased their activity. Further studies are needed to verify whether ROS are involved in signalling hypoxia.     

Effect of niacin supplementation on rumen fermentation characteristics and nutrient flow at the duodenum in lactating dairy cows fed a diet with a negative rumen nitrogen balance

The aim of the present experiment was to ascertain if a daily niacin supplementation of 6 g/cow to lactating dairy cow diets can compensate for the decrease in rumen microbial fermentation due to a negative rumen nitrogen balance (RNB). A total of nine ruminally and duodenally fistulated lactating multiparous German Holstein cows was used. The diets consisted of 10 kg dry matter (DM) maize silage and 7 kg DM concentrate and differed as follows: (i) Diet RNB- (n = 6) with energy and utilisable crude protein (CP) at the duodenum (uCP) according to the average requirement of the animals, but with a negative RNB (-0.41 g N/MJ metabolisable energy [ME]); (ii) Diet RNB0 (n = 7) with energy, uCP, and RNB (0.08 g N/MJ ME) according to the average requirement of the animals; and (iii) Diet NA (nicotinic acid; n = 5), which was the same diet as RNB-, but supplemented with 6 g niacin/d. The negative RNB affected the rumen fermentation pattern and reduced ammonia content in rumen fluid and the daily duodenal flows of microbial CP (MP) and uCP. Niacin supplementation increased the apparent ruminal digestibility of neutral detergent fibre. The efficiency of microbial protein synthesis per unit of rumen degradable CP was higher, whereby the amount of MP reaching the duodenum was unaffected by niacin supplementation. The number of protozoa in rumen fluid was higher in NA treatment. The results indicated a more efficient use of rumen degradable N due to changes in the microbial population in the rumen when niacin was supplemented to diets deficient in RNB for lactating dairy cows.     

Short term exposure of Lemna minor and Lemna gibba to mercury, cadmium and chromium

The effects of mercury (Hg), cadmium (Cd) and chromium (Cr) in concentrations ranging from 0.02 to 20 mg L^?1 applied for 24 h were assessed in Lemna minor and Lemna gibba by measuring changes in protein concentration, ascorbic acid, phenolics, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), the activity of guaiacol peroxidase (G-POX) and catalase (CAT). Ascorbic acid, phenolics, catalase and guaiacol peroxidase played a key role in the antioxidative response of L. gibba. Inadequate activity of antioxidant enzymes in the L. minor resulted in MDA and H₂O₂ accumulation. In both used species, Hg treatment decreased protein content and increased CAT and G-POX activity, but decreased MDA and H₂O₂ levels. Cadmium and chromium had opposite impacts on two used Lemna species on almost all observed parameters. Enhanced antioxidative responses of L. gibba to lower concentrations of Hg, Cd and Cr indicated greater abiotic stress tolerance than L. minor.     

Gender role behavior in children with XY karyotype and disorders of sex development

Children exhibit gender-typical preferences in play, toys, activities and interests, and playmates. Several studies suggest that high concentrations of pre- and postnatal androgens contribute to male-typical behavior development, whereas female-typical behavior develops in the absence of high androgens levels. This study aims to explore the consequences of hypoandrogenization on gender-typical behavior in children who have an XY karyotype and disorder of sex development (DSD). Participants included 33 children (ages 2-12 years) with an XY karyotype and DSD; 21 reared as girls and 12 reared as boys. Children's preferred activities and interests and playmate preferences were assessed with parent report questionnaires, a structured free-play task, and choice of a toy to keep as a gift. Participant's responses were compared to those of children recruited in a pre-school and elementary school survey (N=166). In this study, the degree of hypoandrogenization as indicated by genital stage and diagnosis showed a significant relationship to nearly all of the gender-related behaviors assessed, supporting the hypothesis that masculinization of gender role behavior is a function of prenatal androgen exposure. Despite the fact that children with partial androgen effects reared as girls showed increased "boyish" behaviors, they did not show increased signs of gender identity confusion or instability on a group level. We conclude that androgen exposure plays a decisive role in the development of gender-typical behavior in children with XY karyotype and DSD conditions.     

The effects of reduced oxygen tension on swine granulosa cell

Follicular growth is characterized by an augmented vascularization, possibly driven by a fall in the oxygen supply. The present study was undertaken to investigate the effects of hypoxia on swine granulosa cells. At first, we quantified oxygen partial pressure (pO₂) in follicular fluid from different size follicles; the granulosa cells collected from large follicles (>5 mm) were subjected for 18 h to normoxia (19% O₂), partial (5% O₂) or total hypoxia (1% O₂). The effects of these conditions were tested on the main parameters of granulosa cell function, steroidogenesis and cell proliferation, and on vascular endothelial growth factor (VEGF), nitric oxide (NO) and superoxide anion (O₂⁻) production. Oxygen tension in follicular fluid was negatively related to follicular size, pointing out a gradual reduction during follicular growth. Severe hypoxic conditions determined a reduction of both 17β estradiol and progesterone production, while partial hypoxia did not seem to affect them. Hypoxia increased VEGF as well as O₂⁻ production in swine granulosa cells without impairing cell growth; in addition, it decreased NO output.

We may conclude that physiological hypoxia could play a pivotal role in the follicular angiogenic process stimulating VEGF synthesis by granulosa cells. ROS are possibly involved in hypoxic signalling.     

AAV exploits subcellular stress associated with inflammation, endoplasmic reticulum expansion, and misfolded proteins in models of cystic fibrosis

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.     

Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM? sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM? within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.