DNA vector-based RNA interference to study gene function in cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.     

Phytochemical profiles and antioxidant potential of four Arctic vascular plants from Svalbard

Environmental stress in the Arctic region leads to damage in plant membranes as a result of oxidation processes. To withstand these stress conditions, plants are expected to produce antioxidants that differ from phenolics. Here, we investigated the chemical composition and antioxidative activities of four Arctic flowering plant species (Dryas octopetala, Carex rupestris, Silene uralensis and Deschampsia alpina.) through in vitro measurements of the free radical scavenging activities (FRS), inhibition of lipid peroxidation (ILP) and trolox equivalent antioxidant capacities (TEAC). D. octopetala exhibited the highest ILP (76.45?%) and FRS (86.58?%) activities. The TEAC values were higher than those of the Trolox vitamin E standard in all four species. Overall, the antioxidative activity was highest in D. octopetala, followed by C. rupestris, S. uralensis and D. alpina. Electrospray ionization tandem mass spectrometric (ESI–MS/MS) analysis of methanolic extracts of these plants revealed the presence of organoselenides, linear alkylbenzenesulfonates (LAS) and oligosaccharides, some of which are reported as antioxidants in the literature. Hence, it is likely that the antioxidant activities exhibited by these plants are not only related to the production of phenolics. This is the first report of the antioxidant potential of four Arctic flowering plants and the presence of selenides in D. octopetala and S. uralensis, and the production of LAS in C. rupestris. Our findings suggest that these plants can be used as nutraceutical sources of selenium and as biomarkers for environmental pollution.     

Onychomycosis by <Emphasis Type="Italic">Scytalidium dimidiatum</Emphasis> in green tea leaf pluckers: report of two cases

Two cases of onychomycosis in green tea leaf pluckers caused by Scytalidium dimidiatum, a dematiaceous non-dermatophytic mold has been desribed. Onychomycosis caused by Scytalidium dimidiatum is clinically indistinguishable from that caused by dermatophytes and responds poorly to antifungals. The clinical presentations in the cases were of distal subungual type with associated melanonychia involving thumbnails. These cases have unique importance, because besides disfigurement, this has adversely affected the livelihood of the patient and poor response to antifungal dissuaded them from treatment compliance. To the best of our knowledge, this is the first report of onychomycosis caused by Scytalidium dimidiatum in green tea leaf pluckers.     

When the shoe doesn’t fit: applying conservation unit concepts to western painted turtles at their northern periphery

As biodiversity continues to be lost at an alarming rate, strategies for prioritizing populations for conservation have become increasingly important. Maintaining intraspecific genetic diversity is of particular importance for preserving evolutionary history and the potential for future adaptation. In order to effectively protect this diversity, units below the species level need to be defined. However, delineation of such units is subject to many challenges, with no one strategy applying universally across taxa. In this study we carried out the first genetic assessment of the western painted turtle (Chrysemys picta bellii) at its northern periphery in British Columbia (BC), Canada, using mitochondrial DNA haplotypic and microsatellite genotypic data to examine population structure and demographic history. We compared the application of evolutionarily significant unit and management unit criteria with Canadian designatable unit guidelines to determine appropriate conservation units. Our results show that BC western painted turtles form a single evolutionarily significant unit, with each occupied site constituting a separate management unit. In contrast, there is evidence for six discrete designatable units. Patterns of genetic variation in BC western painted turtles indicate that the conservation of each region is important to maintaining regional diversity and evolutionary novelty in this widespread species.     

Deficiency of p110δ Isoform of the Phosphoinositide 3 Kinase Leads to Enhanced Resistance to Leishmania donovani

Background

Visceral leishmaniasis is the most clinically relevant and dangerous form of human leishmaniasis. Most traditional drugs for treatment of leishmaniasis are toxic, possess many adverse reactions and drug resistance is emerging. Therefore, there is urgent need for identification of new therapeutic targets. Recently, we found that mice with an inactivating knock-in mutation in the p110δ isoform of pi3k, (p110δ^d910a) are hyper resistant to L. major, develop minimal cutaneous lesion and rapidly clear their parasite. Here, we investigated whether pi3k signaling also regulates resistance to L. donovani, one of the causative agents of visceral leishmaniasis.

Methodology/Principal Findings

WT and p110δ^D910A mice (on a BALB/c background) were infected with L. donovani. At different time points, parasite burden and granuloma formation were assessed. T and B cell responses in the liver and spleen were determined. In addition, Tregs were expanded in vivo and its impact on resistance was assessed. We found that p110δ^D910A mice had significantly reduced splenomegaly and hepatomegaly and these organs harbored significantly fewer parasites than those of WT mice. Interestingly, infected p110δ^D910A mice liver contains fewer and less organized granulomas than their infected WT counterparts. Cells from p110δ^D910A mice were significantly impaired in their ability to produce cytokines compared to WT mice. The percentage and absolute numbers of Tregs in infected p110δ^D910A mice were lower than those in WT mice throughout the course of infection. In vivo expansion of Tregs in infected p110δ^D910A mice abolished their enhanced resistance to L. donovani infection.

Conclusions/Significance

Our results indicate that the enhanced resistance of p110δ^D910A mice to L. donovani infection is due to impaired activities of Tregs. They further show that resistance to Leishmania in the absence of p110δ signaling is independent of parasite species, suggesting that targeting the PI3K signaling pathway may be useful for treatment of both visceral and cutaneous leishmaniasis.     

Conundrum of pathogenesis of diabetic cardiomyopathy: role of vascular endothelial dysfunction, reactive oxygen species, and mitochondria

Diabetic cardiomyopathy and heart failure have been recognized as the leading causes of mortality among diabetics. Diabetic cardiomyopathy has been characterized primarily by the manifestation of left ventricular dysfunction that is independent of coronary artery disease and hypertension among the patients affected by diabetes mellitus. A complex array of contributing factors including the hypertrophy of left ventricle, alterations of metabolism, microvascular pathology, insulin resistance, fibrosis, apoptotic cell death, and oxidative stress have been implicated in the pathogenesis of diabetic cardiomyopathy. Nevertheless, the exact mechanisms underlying the pathogenesis of diabetic cardiomyopathy are yet to be established. The critical involvement of multifarious factors including the vascular endothelial dysfunction, microangiopathy, reactive oxygen species (ROS), oxidative stress, mitochondrial dysfunction has been identified in the mechanism of pathogenesis of diabetic cardiomyopathy. Although it is difficult to establish how each factor contributes to disease, the involvement of ROS and mitochondrial dysfunction are emerging as front-runners in the mechanism of pathogenesis of diabetic cardiomyopathy. This review highlights the role of vascular endothelial dysfunction, ROS, oxidative stress, and mitochondriopathy in the pathogenesis of diabetic cardiomyopathy. Furthermore, the review emphasizes that the puzzle has to be solved to firmly establish the mitochondrial and/or ROS mechanism(s) by identifying their most critical molecular players involved at both spatial and temporal levels in diabetic cardiomyopathy as targets for specific and effective pharmacological/therapeutic interventions.     

Distribution and Diversity of Planktonic Fungi in the West Pacific Warm Pool

Fungi contribute substantially to biogeochemical cycles of terrestrial and marine habitats by decomposing matter and recycling nutrients. Yet, the diversity of their planktonic forms in the open ocean is poorly described. In this study, culture-independent and molecular approaches were applied to investigate fungal diversity and abundance derived from samples collected from a broad swath of the Pacific Warm Pool across major environmental gradients Our results revealed that planktonic fungi were molecularly diverse and their diversity patterns were related to major phytoplankton taxa and various nutrients including nitrate, nitrite, orthophosphate and silicic acid. Over 400 fungal phylotypes were recovered across this region and nearly half of them grouped into two major fungal lineages of Ascomycota and Basidiomycota, whose abundance varied among stations. These results suggest that planktonic fungi are a diverse and integral component of the marine microbial community and should be included in future marine microbial ecosystem models.     

Enhanced attraction of sand fly vectors of Leishmania infantum to dogs infected with zoonotic visceral leishmaniasis

BackgroundThe sand fly Phlebotomus perniciosus is the main vector of Leishmania infantum, etiological agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin. Dogs are the main reservoir host of this disease. The main objective of this study was to determine, under both laboratory and field conditions, if dogs infected with L. infantum, were more attractive to female P. perniciosus than uninfected dogs.Methodology/Principal findingsWe carried out a series of host choice experiments and found that infected dogs were significantly more attractive to P. perniciosus than uninfected dogs in the laboratory as well as in the field. Significantly more P. perniciosus fed on infected dogs than on uninfected dogs. However, the fecundity of P. perniciosus fed on infected dogs was adversely impacted compared to uninfected dogs by lowering the number of laid eggs. Phlebotomus perfiliewi, the second most abundant sand fly species in the field site and a competent vector of L. infantum had similar trends of attractivity as P. perniciosus toward infected dogs under field conditions.ConclusionsThe results strongly suggest that L. infantum causes physiological changes in the reservoir host which lead to the host becoming more attractive to both male and female P. perniciosus. These changes are likely to improve the chance of successful transmission because of increased contact with infected hosts and therefore, infected dogs should be particularly targeted in the control of zoonotic visceral leishmaniasis in North Africa.