Postsynaptic effects of crotoxin and of its isolated subunits.

Crotoxin is a potent neurotoxin from the venom of Crotalus durissus terrificus. It is composed of two subunits: a basic phospholipase A2 with low toxicity (component B) and an acidic protein seemingly devoid of intrinsic biological activity (component A). Crotoxin and its isolated phospholipase subunit block the depolarisation caused by cholinergic agonists on the isolated electroplaque from Electrophorus electricus. The other component, which is inactive when applied alone, enhances the pharmacological activity of the phospholipase when the two components are used together. Crotoxin also blocks the increase of 22Na+ efflux caused by carbamylcholine from excitable microsacs prepared from Torpedo marmorata electric organ. Crotoxin therefore acts postsynaptically, but does not interfere with the binding of alpha-toxin from Naja nigricollis to the nicotinic cholinergic receptor site. Instead, like local anesthetics, it stabilizes a desensitized form of the acetylcholine receptor characterized by its high affinity for agonists. The phospholipase component B binds in a non-saturable manner to receptor-rich membranes. In contrast, component A does not bind to acetylcholine receptor-rich membranes, but completely prevents the non-saturable binding of component B. When the two components are applied together, a saturable binding of the latter is observed with the acetylcholine receptor-rich membranes.     

Global gene expression profile of Orientia tsutsugamushi

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria‐infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.     

Responses of flowering phenology of snowbed plants to an experimentally imposed extreme advanced snowmelt

In snowbed habitats, characterized by a long-lasting snow cover, the timing of snowmelt can be included among the major factors controlling plant phenology. Nevertheless, only a few ecological studies have tested the responses of flowering phenology of species growing in very late snow-free habitats to an advanced snowmelt (AS) date. The aim of this study was to determine the impacts of an extremely earlier melt-out of snow on flowering phenology of vascular plant species inhabiting an alpine snowbed. The study was conducted in the high Gavia Valley (Italy, 2,700 m a.s.l.). On 30th May 2012, we removed manually the snow cover and set up an experiment with 5 AS and 5 control plots. Phenological observations of the most abundant vascular species were conducted every 4–6 days. Moreover, we calculated cumulative soil temperature and recorded the mortality of reproductive structures of three species. For several species flowering occurred earlier, and the prefloration period was extended in the AS treatment in comparison with the control. For the majority of species, cumulative soil temperatures in the AS treatment and the control were comparable, confirming that temperature exerts the main control on the flowering of the species inhabiting snowbeds. Earlier flowering species resulted more affected by an AS date in comparison with later flowering species. The mortality of reproductive structures did not increase in the AS treatments in comparison with the control suggesting that few and weak frost events in late spring do not affect the survival of reproductive structures of the species studied.     

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

Introducing a New Breed of Wine Yeast: Interspecific Hybridisation between a Commercial Saccharomyces cerevisiae Wine Yeast and Saccharomyces mikatae

Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.     

Economic Recession and Emergence of an HIV-1 Outbreak among Drug Injectors in Athens Metropolitan Area: A Longitudinal Study

Background

During 2011, a dramatic increase (1600%) of reported HIV-1 infections among injecting drug users (IDUs) was noted in Athens, Greece. We herein assess the potential causal pathways associated with this outbreak.

Methods

Our study employed high resolution HIV-1 phylogenetic and phylogeographic analyses. We examined also longitudinal data of ecological variables such as the annual growth of gross domestic product (GDP) of Greece in association with HIV-1 and HCV sentinel prevalence in IDUs, unemployment and homelessness rates and HIV transmission networks in Athens IDUs before and during economic recession (2008–2012).

Results

IDU isolates sampled in 2011 and 2012 suggested transmission networks in 94.6% and 92.7% of the cases in striking contrast with the sporadic networking (5%) during 1998–2009. The geographic origin of most HIV-1 isolates was consistent with the recently documented migratory waves in Greece. The decline in GDP was inversely correlated with annual prevalence rates of HIV and HCV and with unemployment and homelessness rates in IDUs (all p<0.001). The slope of anti-HCV prevalence in the sentinel populations of IDUs and in “new” drug injectors was found 120 and 1.9-fold (p = 0.007, p = 0.08 respectively) higher in 2008–2012 (economic recession) compared with 2002–2006. The median (25th, 75th) size of transmission networks were 34 (12, 58) and 2 (2, 2) (p = 0.057) in 2008–2012 and 1998–2007, respectively. The coverage of harm reduction services was low throughout the study period.

Conclusions

Scaling-up harm reduction services and addressing social and structural factors related to the current economic crisis should be urgently considered in environments where HIV-1 outbreaks may occur.     

Low Dose Infliximab for Prevention of Postoperative Recurrence of Crohn’s Disease: Long Term Follow-Up and Impact of Infliximab Trough Levels and Antibodies to Infliximab

Objective

In patients with postoperative recurrence of Crohn’s disease endoscopic and clinical remission can be maintained for up to 1 year with low infliximab doses (3 mg/Kg). However, in theory low-dose infliximab treated patients could develop subtherapeutic trough levels, infiximab antibodies, and might loose response to therapy. To verify this hypothesis infliximab pharmacokinetics and clinical/endoscopic response were checked in a group of patients treated in the long term with low infliximab doses.

Design

Infliximab antibodies, infliximab levels, highly-sensitive CRP and fecal calprotectin were measured during the 8-week interval in 5 consecutive patients in clinical (Crohn’s Disease Activity Index < 150) and endoscopic (Rutgeerts scores 0–1) remission after one year of therapy with infliximab 3 mg/Kg. For comparison with reported standards, infliximab pharmacokinetics and inflammatory parameters were also tested in 6 Crohn’s disease patients who did not undergo surgery and who were in clinical remission while on infliximab 5 mg/Kg. Patients on low infliximab dose also underwent colonoscopy after 18 additional months of therapy.

Results

Highly sensitive CRP and fecal calprotectin increased in all patients during the 8-week interval. Infliximab trough levels were lower in patients treated with the low dose compared to controls (mean±SE: 2.0±0.3 vs 4.75±0.83 μg/mL respectively p<0.05). Infliximab antibodies were present in two of the subjects treated with low infliximab dose and in none of the controls. However, in low dose-treated patients after 18 additional months of therapy endoscopy continued to show mucosal remission and none of them developed clinical recurrence or side effects.

Conclusions

Patients treated with low infliximab doses had lower trough levels compared to patients treated with 5 mg/Kg and some developed antibodies to infliximab. However, low infliximab doses sustained clinical and endoscopic remission for a total of 30 months of treatment.     

Complex regulation of the TRPM8 cold receptor channel: role of arachidonic acid release following M3 muscarinic receptor stimulation

Cold/menthol-activated TRPM8 (transient receptor potential channel melastatin member 8) is primarily expressed in sensory neurons, where it constitutes the principal receptor of environmental innocuous cold. TRPM8 has been shown to be regulated by multiple influences such as phosphorylation, pH, Ca(2+), and lipid messengers. One such messenger is arachidonic acid (AA), which has been shown to inhibit TRPM8 channel activity. However, the physiological pathways mediating the inhibitory effect of AA on TRPM8 still remain unknown. Here, we demonstrate that TRPM8 is regulated via M3 muscarinic acetylcholine receptor-coupled signaling cascade based on the activation of cytosolic phospholipase A2 (cPLA2) and cPLA2-catalyzed derivation of AA. Stimulation of M3 receptors heterologously co-expressed with TRPM8 in HEK-293 cells by nonselective muscarinic agonist, oxotremorine methiodide (Oxo-M), caused inhibition of TRPM8-mediated membrane current, which could be mimicked by AA and antagonized by pharmacological or siRNA-mediated cPLA2 silencing. Our results demonstrate the intracellular functional link between M3 receptor and TRPM8 channel via cPLA2/AA and suggest a novel physiological mechanism of arachidonate-mediated regulation of TRPM8 channel activity through muscarinic receptors. We also summarize the existing TRPM8 regulations and discuss their physiological and pathological significance.