RNA-mediated gene silencing of ToxB in Pyrenophora tritici-repentis

The fungus Pyrenophora tritici-repentis causes tan spot, a wheat leaf disease of worldwide importance. The pathogen produces three host-selective toxins, including Ptr ToxB, which causes chlorophyll degradation and foliar chlorosis on toxin-sensitive wheat genotypes. The ToxB gene, which codes for Ptr ToxB, was silenced in a wild-type race 5 isolate of the fungus thorough a sense- and antisense-mediated silencing mechanism. Toxin production by the silenced strains was evaluated in culture filtrates of the fungus via Western blotting analysis, and plant bioassays were conducted to test the virulence of the transformants in planta. The chlorosis-inducing ability of the silenced strains was correlated with the quantity of Ptr ToxB, and transformants in which toxin production was strongly decreased also caused very little disease on toxin-sensitive wheat genotypes. Cytological analysis of the infection process revealed that, in addition to a reduced capacity to induce chlorosis, the silenced strains with the greatest decrease in the levels of Ptr ToxB produced significantly fewer appressoria than the wild-type isolate, 12 and 24 h after inoculation onto wheat leaves. The results provide strong support for the suggestion that the amount of Ptr ToxB protein produced by fungal isolates plays a significant role in the quantitative variation in the virulence of P. tritici-repentis.     

Cytochrome allelic variants and clopidogrel metabolism in cardiovascular diseases therapy

Clopidogrel and aspirin are among the most prescribed dual antiplatelet therapies to treat the acute coronary syndrome and heart attacks. However, their potential clinical impacts are a subject of intense debates. The therapeutic efficiency of clopidogrel is controlled by the actions of hepatic cytochrome P450 (CYPs) enzymes and impacted by individual genetic variations. Inter-individual polymorphisms in CYPs enzymes affect the metabolism of clopidogrel into its active metabolites and, therefore, modify its turnover and clinical outcome. So far, clinical trials fail to confirm higher or lower adverse cardiovascular effects in patients treated with combinations of clopidogrel and proton pump inhibitors, compared with clopidogrel alone. Such inconclusive findings may be due to genetic variations in the cytochromes CYP2C19 and CYP3A4/5. To investigate potential interactions/effects of these cytochromes and their allele variants on the treatment of acute coronary syndrome with clopidogrel alone or in combination with proton pump inhibitors, we analyze recent literature and discuss the potential impact of the cytochrome allelic variants on cardiovascular events and stent thrombosis treated with clopidogrel. The diversity of CYP2C19 polymorphisms and prevalence span within various ethnic groups, subpopulations and demographic areas are also debated.     

Evaluation of a novel commercial quaternary ammonium compound for eradication of Mycobacteria,HCV and HBV in Egypt

Endoscopes are a common source of outbreaks of healthcare-associated infections. It is therefore important to identify high-level disinfectants capable of eliminating or killing all vegetative bacteria, mycobacteria, and viruses. Aldehydebased disinfectants are most commonly used in clinical practice but resistance has recently been detected and side effects associated with these disinfectants are well documented. In this study, we evaluated Virusolve+® EDS, a novel quaternary ammonium compound formulation supplied by Amity international, against Mycobacterium bovis (ATCC-27289), hepatitis C virus (HCV)-positive serum and hepatitis B surface antigen-positive serum. We also compared its efficacy against Cidex® (glutaraldehyde 2%), an aldehyde-based disinfectant. M. bovis showed no growth after 10 weeks with either Virusolve+® or Cidex®. Virusolve+® achieved a 10⁴- fold reduction in the initial 10⁶ HCV load under clean conditions (without red blood cells) for 20 min, whereas Cidex® achieved this reduction under clean and dirty conditions (without and with red blood cells, respectively) after both 10 and 20 min. Both Virusolve+® and Cidex® were able to eradicate hepatitis B virus (HBV) infectivity under clean conditions after 10 and 20 min, whereas under dirty conditions they were only able to eradicate virus infectivity after 20 min. Virusolve+® EDS when compared with Cidex® showed equal mycobactericidal activity completely eradicating M. bovis. However, both showed comparable virucidal activity against HBV, which was more effective under clean conditions, emphasizing the importance of the cleaning step in endoscope reprocessing. Cidex® was more effective at eradicating HCV under dirty conditions after a short contact time.     

Actin-filament stochastic dynamics mediated by ADF/cofilin

BACKGROUND: The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, we combined evanescent-wave microscopy with a biomimetic system and followed the behavior of single actin filaments in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. RESULTS: In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. CONCLUSIONS: Our proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells.     

Chemodiversity and Antiproliferative Activity of the Essential Oil of Schinus molle Growing in Jordan

The leaves and unripe and fully‐grown fruits of Schinus molle were collected from three geographical regions of Jordan: Amman (the Mediterranean), Madaba (Irano‐Turanean), and Sahab (Saharo‐Arabian). The hydrodistilled volatile oils of fresh and dried leaves and fruits were analyzed by gas chromatography‐mass spectrometry (GC/MS). The actual composition of the emitted volatiles was determined using Solid Phase Micro‐Extraction (SPME). α‐ and β‐Phellandrenes were the major components in all the analyzed samples. Quantitative differences were observed in the obtained essential oils (0.62–5.25 %). Additionally, cluster analysis was performed. Biologically, the antiproliferative activity of the essential oil, ethanol, and water extracts of the fruits and leaves was screened on Caco2, HCT116, MCF7, and T47D cell lines. The essential oil and ethanol extracts exhibited a dose‐dependent inhibition of cell growth with IC₅₀ ranging between 21 and 65 μg/mL. The water extract did not exhibit any antiproliferative activity against the investigated cell lines.     

Mathematical models and simulations of cellular processes based on actin filaments

Actin filaments help to maintain the physical integrity of cells and participate in many processes that produce cellular movements. Studies of the processes that depend on actin filaments have progressed to the point where mathematical models and computer simulations are an essential part of the experimental toolkit. These quantitative models integrate knowledge about the structures of the key proteins and the rate and equilibrium constants for the reactions for comparison with a growing body of quantitative measurements of dynamic processes in live cells. Models and simulations are essential because it is impossible to appreciate by intuition alone the properties that emerge from a network of coupled reactions, particularly when the system contains many components, and force is one of the parameters.     

Purine and phosphoribosylpyrophosphate synthesis in differentiating murine virus-induced erythroleukemic cells in vitro.

Phosphoribosylpyrophosphate synthetase activity was determined in Friend virus-inducted erythroleukemic cells in culture, stimulated to differentiate in the presence of dimethylsulfoxide. The activity of phosphoribosylpyrophosphate synthetase did not decrease in cells which had acquired the specialized function of hemoglobin synthesis, nor was the phosphoribosylpyrophosphate content of untreated erythroleukemic cells significantly different from that of cultures exposed to dimethylsulfoxide for 96 hours. However, the rate of the early steps of de novo purine biosynthesis as measured by the incorporation of [1-14C] glycine and [1-14C] formate into formyglycinamide ribonucleotide, was significantly lower in differentiating cell cultures. The addition of glutamine or ammonia increased glycine incorporation of control cultures, but failed to do so in treated cultures. In the course of the normal development of erythrocytes in vivo, phosphoribosylpyrophosphate synthetase activity is preserved, while the capacity to synthesize purines de novo is lost, as is the activity of the phosphoribosyl-l-amine synthesizing enzymes. Our present study suggests that the rate of de novo purine biosynthesis in this erythroleukemic cell line is not limited by the availability of phosphoribosylphrophosphate, but rather by a decrease in the phosphoribosyl-l-amine synthesizing enzymes. These findings provide further evidence that during dimethylsulfoxide-stimulated erythroid maturation, the same regulatory mechanisms are operative as in normal cellular development, and that ammonia-dependent purine biosynthesis is subject to the same regulatory mechanisms as is glutamine-dependent biosynthesis.     

Genetically engineered neural stem cells (NSCs) therapy for neurological diseases; state-of-the-art

Neural stem cells (NSCs) are multipotent stem cells with remarkable self-renewal potential and also unique competencies to differentiate into neurons, astrocytes, and oligodendrocytes (ODCs) and improve the cellular microenvironment. In addition, NSCs secret diversity of mediators, including neurotrophic factors (e.g., BDNF, NGF, GDNF, CNTF, and NT-3), pro-angiogenic mediators (e.g., FGF-2 and VEGF), and anti-inflammatory biomolecules. Thereby, NSCs transplantation has become a reasonable and effective treatment for various neurodegenerative disorders by their capacity to induce neurogenesis and vasculogenesis and dampen neuroinflammation and oxidative stress. Nonetheless, various drawbacks such as lower migration and survival and less differential capacity to a particular cell lineage concerning the disease pathogenesis hinder their application. Thus, genetic engineering of NSCs before transplantation is recently regarded as an innovative strategy to bypass these hurdles. Indeed, genetically modified NSCs could bring about more favored therapeutic influences post-transplantation in vivo, making them an excellent option for neurological disease therapy. This review for the first time offers a comprehensive review of the therapeutic capability of genetically modified NSCs rather than naïve NSCs in neurological disease beyond brain tumors and sheds light on the recent progress and prospect in this context.     

Capsaicin pretreatment attenuates LPS-induced hypothermia through TRPV1-independent mechanisms in chicken

It has been demonstrated that chicken TRPV1 (transient receptor potential vanilloid of subtype-1) is insensitive to capsaicin (CAP), and therefore, a chicken model is suitable to analyze the CAP-sensitive TRPV1-independent pathway. We elucidated here the possible involvement of the pathway in hypothermia induced by bacterial endotoxin (lipopolysaccharide, LPS) in chickens. Chicks were pretreated with CAP (10 mg/kg, iv) at 1, 2 and 3 days of age to desensitize them towards the CAP-sensitive pathway. An intravenous injection of LPS in 4-day-old chicks caused progressive hypothermia, ending with collapse and 78% mortality within 12 h after injection. The CAP pretreatment rescued the LPS-induced endotoxin shock and hypothermia in chicks. LPS-induced iNOS expression as well as NO production in liver and lung was suppressed by CAP pretreatment. CAP pretreatment also attenuated hypothermia due to exposure of chicks to cold ambient temperature. These findings suggest that a CAP-sensitive TRPV1-independent pathway may be involved in pathophysiological hypothermic reactions through the mediation of NO in chickens.     

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.