Endosomal Fusion upon SNARE Knockdown is Maintained by Residual SNARE Activity and Enhanced Docking

SNARE proteins mediate membrane fusion in the secretory pathway of eukaryotic cells. Genetic deletion and siRNA-based knockdown have been instrumental in assigning given SNAREs to defined intracellular transport steps. However, SNARE depletion occasionally results in barely detectable phenotypes. To understand how cells cope with SNARE loss, we have knocked down several SNAREs functioning in early endosome fusion. Surprisingly, knockdown of syntaxin 13, syntaxin 6 and vti1a, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. We found that the residual SNARE levels (typically ∼10%) were sufficient for a substantial amount of SNARE–SNARE interactions. Conversely, in wild-type cells, most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the knockdown organelles exhibited enhanced docking. We conclude that SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery.     

Effect of Compositional Factors against the Thermal Oxidative Deterioration of Novel Food Emulsions

This work attempts to determine any relationship between certain endogenous parameters and the oxidative deterioration of protein-stabilized oil-in-water emulsions. The contribution of compositional factors (e.g., type and amount of emulsifier, fat phase, etc.) is further elucidated. Among 10% cottonseed o/w emulsions prepared by 1% emulsifier (Tween, sodium caseinate, or whey protein), lipid autoxidation (at 40°C) was much faster in the Tween emulsion than in the protein ones, with whey protein presenting a clear antioxidant effect. Increase in protein concentration (0.5–2% w/w) led to a decrease in droplet size but an increase in oxidative stability, in terms of conjugated diene hydroperoxides formation at 232 nm. The type of lipid phase significantly affected the rate of thermal oxidation at 60°C. In the most oxidatively vulnerable sunflower-oil-based emulsions, an increase in fat content (10–40%) resulted in a reduction of oxidative deterioration. By selecting a more concentrated emulsion (20% o/w, 2% emulsifier), in order to structurally approach real novel food products, any influence of the composition of the emulsifier (combination of Tween and sodium caseinate preparation) was subsequently tested. An increase in protein proportion in the emulsifier was found to inhibit proportionally the oxidative instability of the emulsions, as evaluated by the determination of both primary (conjugated diene and lipid hydroperoxides) and secondary [thiobarbituric acid-reactive substances (TBARS)] oxidation products.     

Overcoming physiologic barriers to cancer treatment by molecularly targeting the tumor microenvironment

It is widely recognized that the vasculature of the tumor is inadequate to meet the demands of the growing mass. The malformed vasculature is at least in part responsible for regions of the tumor that are hypoxic, acidotic, and exposed to increased interstitial fluid pressure. These unique aspects of the tumor microenvironment have been shown to act as barriers to conventional chemotherapy or radiation-based therapies. It now seems that while the vasculature initiates these tumor-specific conditions, the cells within the tumor respond to these stresses and add to the unique solid tumor physiology. Gene expression changes have been reported in the tumor for vascular endothelial growth factor, carbonic anhydrase IX, and pyruvate dehydrogenase kinase 1. The activity of these gene products then influences the tumor physiology through alterations in vascular permeability and interstitial fluid pressure, extracellular acidosis, and mitochondrial oxygen consumption and hypoxia, respectively. Novel molecular strategies designed to interfere with the activities of these gene products are being devised as ways to overcome the physiologic barriers in the tumor to standard anticancer therapies.     

Melatonin does not prevent the protection of ischemic preconditioning in vivo despite its antioxidant effect against oxidative stress

Free radicals are involved in the protective mechanism of preconditioning (PC), whereas antioxidant compounds abolish this benefit. Melatonin is a hormone with antioxidant properties. The aim of our study was to evaluate the effect of melatonin on infarct size in ischemic preconditioning in vivo. We randomly divided 33 male rabbits into four groups and subjected them to 30 min of myocardial ischemia and 3 h of reperfusion with the following prior interventions: (i) no intervention, (ii) iv melatonin at a total dose of 50 mg/kg, (iii) PC with two cycles of 5 min ischemia and 10 min reperfusion, and (iv) combined melatonin and PC. In a second series of experiments, another antioxidant agent N-acetylcysteine (NAC) was used in a control and in a PC group. Myocardial infarct size was determined and blood samples were drawn at different time points for the determination of lipid peroxidation products, total superoxide dismutase (SOD) activity, and (1)H-NMR spectra to evaluate the changes in the metabolic profile. Melatonin showed no effect on myocardial infarct size in the group of sustained ischemia (42.9 +/- 3.6% vs 47.4 +/- 4.9%) and it did not attenuate the reduction of myocardial infarct size in the PC group (13.6 +/- 2.4% vs 14.0 +/- 1.7%). A similar effect was found in NAC-treated groups (44.8 +/- 3.4% vs 14.3 +/- 1.3%). Lipid peroxidation product levels were significantly elevated in the control and PC groups, whereas melatonin decreased them in both groups. The SOD activity was enhanced in the PC group compared to controls; melatonin kept SOD activity unchanged during ischemia/reperfusion and enhanced its activity when it was combined with PC. Melatonin did not change the metabolic profile of the control and PC groups. Melatonin does not prevent the beneficial effect of ischemic PC on infarct size despite its antioxidant properties.     

<Emphasis Type="Italic">Narcissus</Emphasis> bulblet formation <Emphasis Type="Italic">in vitro</Emphasis>: effects of carbohydrate type and osmolarity of the culture medium

Shoot clump cultures of Narcissus cultivars St. Keverne and Hawera were used to investigate the effects of culture medium carbon supply, type of carbohydrate and osmolarity on in vitro bulblet development. Increasing the medium osmolarity using mannitol or sorbitol, which did not act as substrates for growth, failed to stimulate bulblet formation with either cultivar. An exception to this was a relatively small increase in total bulblet dry weight per culture, in the cultivar Hawera only, caused by adding 30 g l ^–1 sorbitol in combination with 30 g l^–1 sucrose. Simultaneously increasing the medium osmolarity and carbon supply using the metabolisable carbohydrate sources, sucrose, glucose, fructose or an equimolar mixture of glucose and fructose stimulated bulblet production, total dry matter accumulation and partitioning into bulblets. At comparable levels of carbon supply up to 19.0 g l^–1, bulblet development of both cultivars was similar with monosaccharide and sucrose media. This indicates that substrate supply is more important for bulblet development than osmolarity of the culture medium. The cultivar Hawera also showed similar responses to monosaccharide and sucrose media supplying 37.9 g C l^–1, despite the high osmolarity of monosaccharide media (c. 650 m Osm kg^–1, equivalent to –1.6 MPa, compared to 380 m Osm kg^–1 for sucrose medium). However in St. Keverne total dry matter accumulation and dry weight per bulblet were further stimulated only by increasing the sucrose supply from 19.0 to 37.9 g C l^–1, not by increasing the monosaccharide supply. Implications of the findings for Narcissus micropropagation are discussed.     

Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine,as pharmacological post-conditioning agent

Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop compounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of 3.8 μmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs 26.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could be used as mimics or enhancers of PostC.     

Time-Resolved Human Kinome RNAi Screen Identifies a Network Regulating Mitotic-Events as Early Regulators of Cell Proliferation

Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening.     

Prevention of Malaria Resurgence in Greece through the Association of Mass Drug Administration (MDA) to Immigrants from Malaria-Endemic Regions and Standard Control Measures

Greece was declared malaria-free in 1974 after a long antimalarial fight. In 2011–2012, an outbreak of P. vivax malaria was reported in Evrotas, an agricultural area in Southern Greece, where a large number of immigrants from endemic countries live and work. A total of 46 locally acquired and 38 imported malaria cases were detected. Despite a significant decrease of the number of malaria cases in 2012, a mass drug administration (MDA) program was considered as an additional measure to prevent reestablishment of the disease in the area. During 2013 and 2014, a combination of 3-day chloroquine and 14-day primaquine treatment was administered under direct observation to immigrants living in the epicenter of the 2011 outbreak in Evrotas. Adverse events were managed and recorded on a daily basis. The control measures implemented since 2011 continued during the period of 2013–2014 as a part of a national integrated malaria control program that included active case detection (ACD), vector control measures and community education. The MDA program was started prior to the transmission periods (from May to December). One thousand ninety four (1094) immigrants successfully completed the treatment, corresponding to 87.3% coverage of the target population. A total of 688 adverse events were recorded in 397 (36.2%, 95% C.I.: 33.4–39.1) persons, the vast majority minor, predominantly dizziness and headache for chloroquine (284 events) and abdominal pain (85 events) for primaquine. A single case of primaquine-induced hemolysis was recorded in a person whose initial G6PD test proved incorrect. No malaria cases were recorded in Evrotas, Laconia, in 2013 and 2014, though three locally acquired malaria cases were recorded in other regions of Greece in 2013. Preventive antimalarial MDA to a high-risk population in a low transmission setting appears to have synergized with the usual antimalarial activities to achieve malaria elimination. This study suggests that judicious use of MDA can be a useful addition to the antimalarial armamentarium in areas threatened with the reintroduction of the disease.