Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.     

The Effects of Oral Consumption of Selenium Nanoparticles on Chemotactic and Respiratory Burst Activities of Neutrophils in Comparison with Sodium Selenite in Sheep

The present study was designed to compare the effects of nano-selenium and of sodium selenite on the chemotactic and respiratory burst activities of neutrophils in sheep. Fifteen sheep were randomly divided into three groups. Groups 1 and 2 received selenium nanoparticles (1 mg/kg) or sodium selenite (1 mg/kg) orally, respectively, for ten consecutive days, and the third group was considered as the control. To determine the chemotactic and respiratory burst activities of the neutrophils, the leading front assay and the NBT test were used on heparinized blood samples that were collected at different intervals (days 0, 10th, 20th, and 30th). The results obtained showed that the chemotactic activities in groups 1 and 2 increased significantly on the 10th, 20th, and 30th day, compared to day 0, and on the 20th day in comparison with the 10th day, while in group 2, there was a significant decrease on the 30th day compared to the 20th day. The chemotactic activities in group 1 were significantly higher than in group 2 on the 10th day and in the control group on the 10th, 20th, and 30th day, but the chemotactic activities in group 2 were significantly higher than those in the control group only on the 20th day. On the 30th day into the experiment, the respiratory bursts in groups 1 and 2 were significantly stronger in comparison with those at day 0. Overall, nano-selenium increased the chemotactic and respiratory burst activities more significantly than sodium selenite, which is suggestive of a stronger stimulatory effect of the Se nanoparticles on intracellular activities.     

A protective coat of microorganisms on macroalgae: inhibitory effects of bacterial biofilms and epibiotic microbial assemblages on barnacle attachment

Effects of epibiotic bacteria associated with macroalgae on barnacle larval attachment were investigated. Eight bacterial isolates obtained from samples of three macroalga species were cultured as monospecies bacterial films and tested for their activity against barnacle (Amphibalanus improvisus) attachment in field experiments (Western Baltic Sea). Furthermore, natural biofilm communities associated with the surface of the local brown alga, Fucus vesiculosus, which were exposed to different temperatures (5, 15 and 20?°C), were harvested and subsequently tested. Generally, monospecies bacterial biofilms, as well as natural microbial assemblages, inhibited barnacle attachment by 20-67%. denaturing gradient gel electrophoresis fingerprints showed that temperature treatment shifted the bacterial community composition and weakened the repellent effects at 20?°C. Repellent effects were absent when settlement pressure of cyprids was high. Nonviable bacteria tended to repel cyprids when compared to the unfilmed surfaces. We conclude that biofilms can have a repellent effect benefiting the host by preventing heavy fouling on its surface. However, severe settlement pressure, as well as stressful temperature, may reduce the protective effects of the alga's biofilm. Our results add to the notion that the performance of F.?vesiculosus may be reduced by multiple stressors in the course of global warming.     

Orexins cause epileptic activity

Orexins have been implicated in the regulation of sleep-wake cycle, energy homeostasis, drinking behavior, analgesia, attention, learning and memory but their effects on epileptic activity are controversial. We investigated whether intracortical injections of orexin A (100pmol) and B (100pmol) cause epileptic activity in rats. We observed epileptic seizure findings on these two groups rats. Orexin A and B also significantly increased total EEG power spectrum. Our findings indicate that orexins cause epileptic activity.     

Colonic microbiome is altered in alcoholism

Several studies indicate the importance of colonic microbiota in metabolic and inflammatory disorders and importance of diet on microbiota composition. The effects of alcohol, one of the prominent components of diet, on colonic bacterial composition is largely unknown. Mounting evidence suggests that gut-derived bacterial endotoxins are cofactors for alcohol-induced tissue injury and organ failure like alcoholic liver disease (ALD) that only occur in a subset of alcoholics. We hypothesized that chronic alcohol consumption results in alterations of the gut microbiome in a subgroup of alcoholics, and this may be responsible for the observed inflammatory state and endotoxemia in alcoholics. Thus we interrogated the mucosa-associated colonic microbiome in 48 alcoholics with and without ALD as well as 18 healthy subjects. Colonic biopsy samples from subjects were analyzed for microbiota composition using length heterogeneity PCR fingerprinting and multitag pyrosequencing. A subgroup of alcoholics have an altered colonic microbiome (dysbiosis). The alcoholics with dysbiosis had lower median abundances of Bacteroidetes and higher ones of Proteobacteria. The observed alterations appear to correlate with high levels of serum endotoxin in a subset of the samples. Network topology analysis indicated that alcohol use is correlated with decreased connectivity of the microbial network, and this alteration is seen even after an extended period of sobriety. We show that the colonic mucosa-associated bacterial microbiome is altered in a subset of alcoholics. The altered microbiota composition is persistent and correlates with endotoxemia in a subgroup of alcoholics.     

Activation of alternative pathways of angiogenesis and involvement of stem cells following anti-angiogenesis treatment in glioma

Malignant gliomas are hypervascular tumors that are highly resistant to all the currently available multimodal treatments. Therefore, anti-angiogenic therapies targeting VEGF or VEGF receptors (VEGFRs) were designed and thought to be an effective tool for controlling the growth of malignant gliomas. However, recent results of early clinical trials using humanized monoclonal antibodies against VEGF (Bevacizumab), as well as small-molecule tyrosine kinase inhibitors that target different VEGF receptors (VEGFRs) (Vatalanib, Vandetanib, Sunitinib, Sorafenib, etc) alone or in combination with other therapeutic agents demonstrated differing outcomes, with the majority of reports indicating that glioma developed resistance to the employed anti-angiogenic treatments. It has been noted that continued anti-angiogenic therapy targeting only the VEGF-VEGFR system might affect pro-angiogenic factors other than VEGF, such as basic fibroblast growth factor (bFGF), stromal derived factor 1 (SDF-1) and Tie-2. These factors may in turn stimulate angiogenesis by mobilizing bone marrow derived precursor cells, such as endothelial progenitor cells (EPCs), which are known to promote angiogenesis and vasculogenesis. In this short review, the current antiangiogenic treatments, possible mechanisms of activation of alternative pathways of angiogenesis, and possible involvement of bone marrow derived progenitor cells in the failure of anti-angiogenic treatments are discussed.     

Association of the ACE-II genotype with the risk of nephrotic syndrome in Pakistani children

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR = 6.755; C.I = 3-14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.     

Detection of Hepatozoon sp. in a Persian leopard (Panthera pardus ciscaucasica)

A free-ranging, adult, male Persian leopard (Panthera pardus ciscaucasica) was found at Geloul-Sarani protected zone, province of North-Khorasan, Iran and transported to the Ferdowsi University of Mashhad Veterinary Teaching Hospital. The leopard had normal temperature and respiratory and cardiac frequency, but was significantly dehydrated and had elevated capillary perfusion. The animal also was cachectic, with pale mucus membranes, third-eyelid protrusion, and bilaterally enlarged submandibular lymph nodes. The leopard was stabilized by intensive fluid and electrolyte therapy and hospitalized. In 2 days, the leopard had improved clinically but had severe ataxia and head pressing. Blood smears revealed gamonts of Hepatozoon sp. within some neutrophils. Hematologic and plasma chemistry abnormalities included moderate anemia, leukocytosis, hypocholestrolemia, and hypophosphatemia. In radiographic evaluations, no sign of periosteal reactions or new bone formation was seen on the skull, spine, long bones, pelvis, or vertebrae. The leopard was treated successfully with Tazocin and clindamycin for 1 mo. This is the first detection of a Hepatozoon sp. in wild Felidae in Iran. Because most Iranian wild felids and canids are endangered, knowing whether Hepatozoon infection represents a threat for these animals is important.     

Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.