Three members of the Arabidopsis glycosyltransferase family 8 are xylan glucuronosyltransferases

Xylan is a major component of the plant cell wall and the most abundant noncellulosic component in the secondary cell walls that constitute the largest part of plant biomass. Dicot glucuronoxylan consists of a linear backbone of β(1,4)-linked xylose residues substituted with α(1,2)-linked glucuronic acid (GlcA). Although several genes have been implicated in xylan synthesis through mutant analyses, the biochemical mechanisms responsible for synthesizing xylan are largely unknown. Here, we show evidence for biochemical activity of GUX1 (for GlcA substitution of xylan 1), a member of Glycosyltransferase Family 8 in Arabidopsis (Arabidopsis thaliana) that is responsible for adding the glucuronosyl substitutions onto the xylan backbone. GUX1 has characteristics typical of Golgi-localized glycosyltransferases and a K(m) for UDP-GlcA of 165 μm. GUX1 strongly favors xylohexaose as an acceptor over shorter xylooligosaccharides, and with xylohexaose as an acceptor, GlcA is almost exclusively added to the fifth xylose residue from the nonreducing end. We also show that several related proteins, GUX2 to GUX5 and Plant Glycogenin-like Starch Initiation Protein6, are Golgi localized and that only two of these proteins, GUX2 and GUX4, have activity as xylan α-glucuronosyltransferases.     

Rapid conversion of spirostans into furostan skeletons at room temperature

We report a facile protocol to obtain 22-substituted furostans and pseudosapogenins in high yields from (25R)- and (25S)-sapogenins. This method involves the treatment of the sapogenin with acetic-trifluoroacetic mixed anhydride and BF(3)·OEt(2) at room temperature, followed by the addition of a nucleophile (H(2)O, MeOH or KSeCN). In the case of 22-hydroxyfurostans, they can be transformed to pseudosapogenins by treatment with p-toluensulfonic acid.     

N-Methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides as a novel class of cannabinoid receptors agonists with low CNS penetration

Cannabinoid CB(1) receptor agonists exhibit potent analgesic effects in rodents and humans, but their clinical utility as analgesic drugs is often limited by centrally mediated side effects. We report herein the preparation of N-methyl-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamides as a novel class of hCB(1)/hCB(2) dual agonists with attractive physicochemical properties. More specifically, (R)-N,9-dimethyl-N-(4-(methylamino)-4-oxobutyl)-3-(tetrahydro-2H-pyran-4-yl)-2,3,4,9-tetrahydro-1H-carbazole-6-carboxamide, displayed an extremely low level of CNS penetration (Rat Cbr/Cplasma=0.005 or 0.5%) and was devoid of CNS side effects during pharmaco-dynamic testing.     

Sleep fragmentation and motor restlessness in a Drosophila model of Restless Legs Syndrome

Restless Legs Syndrome (RLS), first chronicled by Willis in 1672 and described in more detail by Ekbom in 1945, is a prevalent sensorimotor neurological disorder (5%-10% in the population) with a circadian predilection for the evening and night. Characteristic clinical features also include a compelling urge to move during periods of rest, relief with movement, involuntary movements in sleep (viz., periodic leg movements of sleep), and fragmented sleep. Although the pathophysiology of RLS is unknown, dopaminergic neurotransmission and deficits in iron availability modulate expressivity. Genome-wide association studies have identified a polymorphism in an intronic region of the BTBD9 gene on chromosome 6 that confers substantial risk for RLS. Here, we report that loss of the Drosophila homolog CG1826 (dBTBD9) appreciably disrupts sleep with concomitant increases in waking and motor activity. We further show that BTBD9 regulates brain dopamine levels in flies and controls iron homeostasis through the iron regulatory protein-2 in human cell lines. To our knowledge, this represents the first reverse genetic analysis of a "novel" or heretofore poorly understood gene implicated in an exceedingly common and complex sleep disorder and the development of an RLS animal model that closely recapitulates all disease phenotypes.     

The effects of inbreeding, genetic dissimilarity and phenotype on male reproductive success in a dioecious plant

Pollen fate can strongly affect the genetic structure of populations with restricted gene flow and significant inbreeding risk. We established an experimental population of inbred and outbred Silene latifolia plants to evaluate the effects of (i) inbreeding depression, (ii) phenotypic variation and (iii) relatedness between mates on male fitness under natural pollination. Paternity analysis revealed that outbred males sired significantly more offspring than inbred males. Independently of the effects of inbreeding, male fitness depended on several male traits, including a sexually dimorphic (flower number) and a gametophytic trait (in vitro pollen germination rate). In addition, full-sib matings were less frequent than randomly expected. Thus, inbreeding, phenotype and genetic dissimilarity simultaneously affect male fitness in this animal-pollinated plant. While inbreeding depression might threaten population persistence, the deficiency of effective matings between sibs and the higher fitness of outbred males will reduce its occurrence and counter genetic erosion.     

High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.     

Downregulation of RKIP is associated with poor outcome and malignant progression in gliomas

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients.     

Universal sequence replication, reversible polymerization and early functional biopolymers: a model for the initiation of prebiotic sequence evolution

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities.     

Love me tender: an Omics window on the bovine meat tenderness network

Meat tenderness prediction is a challenging task, especially in Maremmana, an Italian autochtonous and highly appreciated beef breed. In the present study we reported an integrated proteomics, phosphoproteomics and metabolomics overview of meat tenderness in longissimus dorsi from 15 male Maremmana individuals, through the discrimination of tender and tough groups via standard meat tenderness indicators (WBS, MFI(4 h), MFI(10 days), sarcomere length) and their correlation with results from Omics analyses. Results revealed that the tender meat group was characterized by higher levels of glycolytic enzymes, which were less phosphorylated and overall more active (lactate accumulation was higher in the tender group), than in tough counterparts. Additionally, we could observe a higher level of oxidative stress in the tender group. No proteomics nor phosphoproteomics result hinted at the widely accepted role of calpains and cathepsins, except for the indication of calcium homeostasis dysregulation. Nevertheless, myofibrillar degradation was monitored and related to structural protein fragmentations. Fragmentation of structural proteins and activities of glycolytic enzymes were inversely related to their phosphorylation levels, suggesting that PTMs might add further levels of complexity in the frame of meat tenderness.