Antimicrobial and antiviral activity-guided fractionation from Scutia buxifolia Reissek extracts

Antimicrobial and antiviral activities of the fractions from Scutia buxifolia stem bark and leaves were evaluated. Best antimicrobial results occurred with the ethyl acetate (EA) and n-butanolic (NB) fractions from the leaves against Micrococcus sp. (minimal inhibitory concentration—MIC = 62.5 μg/ml), and NB fraction from stem bark and leaves against Klebsiella pneumoniae and Enterococcus faecalis (MIC = 62.5 μg/ml). The most active fractions were selected and fractioned into silica column to perform an in vitro antibiofilm assay, which evidenced subfractions EA2 and EA3 as the more active against Candida albicans (biofilm inhibitory concentration—BIC = 582 ± 0.01 μg/ml) and Staphylococcus aureus (BIC = 360 ± 0.007 μg/ml), respectively. The NB (selectivity index—SI = 25.78) and the EA (SI = 15.97) fractions from the stem bark, and the EA (SI = 14.13) fraction from the leaves exhibited a potential antiviral activity towards Herpes Simplex Virus type 1 whereas EA2 and EA3 subfractions from leaves (SI = 12.59 and 10.06, respectively), and NB2 subfraction from stem bark (SI = 12.34) maintained this good activity. Phenolic acids and flavonoids (gallic acid, chlorogenic acid, caffeic acid, rutin, isoquercitrin, quercitrin and quercetin) were identified by HPLC and may be partially responsible for the antimicrobial and antiherpes activities observed. The results obtained in this study showed that Scutia buxifolia has antibiofilm and anti-herpetic activities and that these properties are reported for the first time for this species.     

Contribution of genetic and epigenetic mechanisms to Wnt pathway activity in prevalent skeletal disorders

We reported previously that the expression of Wnt-related genes is lower in osteoporotic hip fractures than in osteoarthritis. We aimed to confirm those results by analyzing β-catenin levels and explored potential genetic and epigenetic mechanisms involved.     

3 Origins and evolution of the translation machinery

The protein synthesis machinery largely evolved prior to the last common ancestor and hence its study can provide insight to early events in the origin of life, including the transition from the hypothetical RNA world to living systems as we know them. By utilizing information from primary sequences, atomic resolution structures, and functional properties of the various components, it is possible to identify timing relationships (Hsiao et al., 2009; Fox, 2010). Taken together, these timing events are used to develop a preliminary time line for major evolutionary events leading to the modern protein synthesis machinery. It has been argued that a key initial event was the hybridization of two or more RNAs that created the peptidyl transferase center, (PTC), of the ribosome (Agmon et al. 2005). The PTC, left side of figure, contains a characteristic cavity/pore that serves as the entrance to the exit tunnel and is thought to be essential to the catalysis (Fox et al., 2012). This cavity is distinct from typical RNA pores (right side of figure) in that the nitrogenous bases face towards the lumen of the pore and thus are available for hydrogen bonding interactions. In typical RNA pores, the bases carefully avoid the lumen region. In support of Agmon et al. 2005), it is argued that this key difference reflects the fact the pore was created by an early hybridization event rather than normal RNA folding.     

Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei

Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSA ^nT-nG , which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSA ^nG , which drives nuclear EGFP accumulation. Conditions were established for analyzing marked nuclei by flow cytometry on the basis of red–green fluorescence and ploidy, with a particular focus on liver nuclei. Hydrodynamic delivery of a Cre-expression plasmid was used to time-stamp arbitrary hepatocytes for lineage tracing. The distinct green fluorescence of nuclei from Cre-exposed lineages facilitated analyses of ploidy transitions within clones. To assess developmental transitions in liver nuclei, ROSA ^nT-nG was combined with the hepatocyte-specific AlbCre transgene, facilitating discrimination between hepatocyte and nonhepatocyte nuclei. Nuclei extracted from postnatal day 2 (P2) livers were 41 % green and 59 % red and reached a stable level of 84 % green by P22. Until P20, green nuclei were >98 % diploid (2N); at P40 they were ~56 % 2N, 43 % 4N, and <1 % 8N; and by P70 they reached a stable distribution of ~46 % 2N, 45 % 4N, and 9 % 8N. In conclusion, ROSA ^nT-nG will facilitate in vivo and ex vivo studies on liver and will likely be valuable for studies on tissues like muscle, kidney, or brain in which cells are refractory to whole-cell flow cytometry, or like trophectoderm derivatives or cancers in which cells undergo ploidy transitions.     

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.     

A new simple fluorimetric method to assay cytosolic ATP content: application to durum wheat seedlings to assess modulation of mitochondrial potassium channel and uncoupling protein activity under hyperosmotic stress

The assays commonly used to determine ATP content in biological samples generally measure total cellular ATP content, but not the different subcellular pools. In this study a new simple method for measuring ATP content in a cytosol-enriched fraction (CEF) was developed, based on a rapid cytosolic ATP extraction (by an isotonic grinding medium that preserves organelle integrity) and its detection monitoring the NADPH fluorescence generated via hexokinase/glucose-6-phosphate dehydrogenase coupled reactions. Four protocols, differing for timing of NADPH generation and for either the presence or absence of some inhibitors of ATP and NADPH metabolism, were compared by determining CEF-ATP, as well as total ATP, in durum wheat (Triticum durum Desf.) etiolated seedlings. The best protocol was the one adopting both simultaneous NADPH generation and use of inhibitors during tissue homogenization. This protocol also showed higher performance than the classical trichloroacetic acid extraction. Using the new method, CEF-ATP content was assessed in control, salt- and osmotic-stressed seedlings, resulting 2.68 ± 0.04, 1.69 ± 0.12 (?40%) and 1.35 ± 0.16 (?50%) μmol/g dry weight, respectively. Finally, the effects of this stress-dependent decrease of cytosolic ATP were evaluated with respect to a possible modulation of two mitochondrial energy-dissipating systems, the uncoupling protein (PUCP) and the K⁺ channel (PmitoK_ATP), both inhibited by cytosolic ATP. Experiments carried out at different physiological ATP concentrations suggest that the decreased cytosolic ATP content occurring under hyperosmotic stress may contribute to attenuate inhibition of PmitoK_ATP, thus promoting its activity (up to about 90%), but not of PUCP, that appears to lose ATP sensitivity under stress condition.