Adaptation of the Carrez procedure for the purification of ferulic and p-coumaric acids released from lignocellulosic biomass prior to LC/MS analysis

The objective of this study was to adapt and improve an environmentally friendly and fast routine method for the analysis of ferulic and p-coumaric acids released from grass cell-walls by alkaline hydrolysis. This methodological development was performed on maize samples selected for their contrasted contents in ferulic and p-coumaric acids as a consequence of their different maturity stages (from stage of 7th leaf with visible ligule to stage of silage harvest). We demonstrate that the Carrez method is an efficient substitute to the common solvent-consuming extraction by ethyl acetate for the preparation of samples suitable for HPLC-ESI-MS analysis. We prove that it is possible to replace methanol by ethanol in the Carrez step and at last we propose a scale reduction of this procedure that offer a first step towards high throughput determinations. The new method leads to a solvent consumption reduced by a factor 100 and only requires ethanol as organic solvent.     

Liquid facets-related (lqfR) is required for egg chamber morphogenesis during Drosophila oogenesis

Clathrin interactor 1 [CLINT1] (also called enthoprotin/EpsinR) is an Epsin N-terminal homology (ENTH) domain-containing adaptor protein that functions in anterograde and retrograde clathrin-mediated trafficking between the trans-Golgi network and the endosome. Removal of both Saccharomyces cerevisiae homologs, Ent3p and Ent5p, result in yeast that are viable, but that display a cold-sensitive growth phenotype and mistrafficking of various vacuolar proteins. Similarly, either knock-down or overexpression of vertebrate CLINT1 in cell culture causes mistrafficking of proteins. Here, we have characterized Drosophila CLINT1, liquid-facets Related (lqfR). LqfR is ubiquitously expressed throughout development and is localized to the Golgi and endosome. Strong hypomorphic mutants generated by imprecise P-element excision exhibit extra macrochaetae, rough eyes and are female sterile. Although essentially no eggs are laid, the ovaries do contain late-stage egg chambers that exhibit abnormal morphology. Germline clones reveal that LqfR expression in the somatic follicle cells is sufficient to rescue the oogenesis defects. Clones of mutant lqfR follicle cells have a decreased cell size consistent with a downregulation of Akt1. We find that while total Akt1 levels are increased there is also a significant decrease in activated phosphorylated Akt1. Taken together, these results show that LqfR function is required to regulate follicle cell size and signaling during Drosophila oogenesis.     

The β-glucan receptor Dectin-1 activates the integrin Mac-1 in neutrophils via Vav protein signaling to promote Candida albicans clearance

Resistance to fungal infections is attributed to engagement of host pattern-recognition receptors, notably the β-glucan receptor Dectin-1 and the integrin Mac-1, which induce phagocytosis and antifungal immunity. However, the mechanisms by which these receptors coordinate fungal clearance are unknown. We show that upon ligand binding, Dectin-1 activates Mac-1 to also recognize fungal components, and this stepwise process is critical for neutrophil cytotoxic responses. Both Mac-1 activation and Dectin-1- and Mac-1-induced neutrophil effector functions require Vav1 and Vav3, exchange factors for RhoGTPases. Mac-1- or Vav1,3-deficient mice have increased susceptibility to systemic candidiasis that is not due to impaired neutrophil recruitment but defective intracellular killing of C.?albicans yeast forms, and Mac-1 or Vav1,3 reconstitution in hematopoietic cells restores resistance. Our results demonstrate that antifungal immunity depends on Dectin-1-induced activation of Mac-1 functions that is coordinated by Vav proteins, a pathway that may localize cytotoxic responses of circulating neutrophils to infected tissues.     

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.     

Lytopylus Förster (Hymenoptera, Braconidae, Agathidinae) species from Costa Rica, with an emphasis on specimens reared from caterpillars in Area de Conservación Guanacaste

Twelve species of Costa Rican Lytopylus are treated; these include all species reared from Lepidoptera caterpillars in Area de Conservación Guanacaste, Costa Rica, over 32 years of caterpillar inventory, as well as two species recorded in the literature as occurring in Costa Rica. Ten new species are described, i.e., Lytopylus bradzlotnicki, Lytopylus colleenhitchcockae, Lytopylus gregburtoni, Lytopylus jessicadimauroae, Lytopylus jessiehillae, Lytopylus mingfangi, Lytopylus rebeccashapleyae, Lytopylus robpringlei, Lytopylus sandraberriosae, Lytopylus vaughntani. The following species are transferred to Lytopylus: Metriosoma flavicalcar Enderlein 1920 to Lytopylus flavicalcarcomb. n.; Bassus macadamiae Brice?o and Sharkey 2000 to Lytopylus macadamiaecomb. n.; Metriosoma bicarinatum Enderlein 1920 to Lytopylus bicarinatumcomb. n.; Metriosoma brasiliense Enderlein 1920 to Lytopylus brasiliensecomb. n.; Bassus tayrona Campos 2007 to Lytopylus tayronacomb. n.; Microdus femoratus Cameron 1887 to Lytopylus femoratuscomb. n.; Microdus melanocephalus Cameron 1887 to Lytopylus melanocephaluscomb. n.; Bassus pastranai Blanchard 1952 to Lytopylus pastranaicomb. n.; Agathis nigrobalteata Cameron 1911 to Lytopylus nigrobalteatuscomb. n. Two keys to species of Lytopylus are presented, one interactive and the other static.     

Dicer-labile PEG conjugates for siRNA delivery

Poly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3'-sense or 5'-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3'-sense or 5'-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3'-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3'-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.     

Quantitative Muscle MRI as an Assessment Tool for Monitoring Disease Progression in LGMD2I: A Multicentre Longitudinal Study

Background

Outcome measures for clinical trials in neuromuscular diseases are typically based on physical assessments which are dependent on patient effort, combine the effort of different muscle groups, and may not be sensitive to progression over short trial periods in slow-progressing diseases. We hypothesised that quantitative fat imaging by MRI (Dixon technique) could provide more discriminating quantitative, patient-independent measurements of the progress of muscle fat replacement within individual muscle groups.

Objective

To determine whether quantitative fat imaging could measure disease progression in a cohort of limb-girdle muscular dystrophy 2I (LGMD2I) patients over a 12 month period.

Methods

32 adult patients (17 male;15 female) from 4 European tertiary referral centres with the homozygous c.826C>A mutation in the fukutin-related protein gene (FKRP) completed baseline and follow up measurements 12 months later. Quantitative fat imaging was performed and muscle fat fraction change was compared with (i) muscle strength and function assessed using standardized physical tests and (ii) standard T1-weighted MRI graded on a 6 point scale.

Results

There was a significant increase in muscle fat fraction in 9 of the 14 muscles analyzed using the quantitative MRI technique from baseline to 12 months follow up. Changes were not seen in the conventional longitudinal physical assessments or in qualitative scoring of the T₁w images.

Conclusions

Quantitative muscle MRI, using the Dixon technique, could be used as an important longitudinal outcome measure to assess muscle pathology and monitor therapeutic efficacy in patients with LGMD2I.