A Simple and Non-destructive Method of Direct-PCR for Plant Systems

To date, PCR is a fundamental tool for most of the research concerning plant diversity analysis, marker-assisted selection, genetic purity testing, disease diagnostics, and transgene analysis. In all of these analyses, good-quality DNA serves as a template for amplification of target sequences. Extraction of good-quality DNA requires many steps, making the whole process time consuming, tedious, labor intensive, and expensive due to costlier and toxic chemicals. To overcome these preparatory steps from PCR-based DNA amplification, we have developed a direct-PCR amplification method for plants without isolating DNA. The method is unique and beneficial over some previously described methods of direct-PCR which fail due to inefficient amplification of target DNA in the presence of PCR inhibitors and crop specificity. Moreover, such methods are non-specific and, being destructive, cannot be replicated; one cannot completely rely on them due to lack of reproducibility. This method was streamlined from our earlier observation that alcohol-desiccated tissues maintain intact DNA for a long time. This method is specific, rapid, and, being non-destructive, allows replication, giving advantages over existing methods. The method was tested over a wide range of plant species and found very effective and quick in generating data. The method was successfully used to test the genetic purity of pearl millet hybrid (RHB-127) and its restorer (RIB 3135-18) and CMS line (ICMA 93333A). Our method is especially important for developing inexpensive and high-throughput non-invasive genetic analyses.     

Omnitemporal choreographies of all five STIM/Orai and IP3Rs underlie the complexity of mammalian Ca2+ signaling

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A novel nest‐monitoring camera system using a Raspberry Pi micro‐computer

The utility, availability, cost‐effectiveness, and reliability of prefabricated video systems designed to monitor wildlife have lagged behind the unique and varied needs of many researchers. Many systems are limited by inflexible video settings, lack of adequate data storage, and cannot be programmed by the user. More sophisticated systems can be cost prohibitive, and the literature describing remote wildlife video monitoring has, for the most part, not incorporated advances in camera and computer technology. Here, we present details of a pilot study to design and construct a lower cost (US $340) nest camera system to record the behavior of Acorn Woodpeckers (Melanerpes formicivorus) in artificial tree cavity nests. This system incorporates a Raspberry Pi micro‐computer, Pi NoIR infrared camera, a wireless adapter to transmit video over the Internet, and Deka rechargeable gel batteries for power. We programmed the system to motion‐sense, to record exclusively during daylight hours, and to automatically upload videos to the cloud over wireless Internet. The Raspberry Pi micro‐computer does not require advanced programming or electrical engineering skills to build and configure and, because it is programmable, provides unprecedented flexibility for field researchers who wish to configure the system to the specific needs of their study.     

RNA interference mediated knockdown of juvenile hormone esterase gene in Bemisia tabaci (Gennadius): Effects on adults and their progeny

Juvenile hormone is responsible for regulating metamorphosis and reproduction in insects. Analysis of key elements of juvenile hormone regulation would enhance the understanding of this complex mechanism. Juvenile hormone esterase plays an important role in maintaining juvenile hormone titres in insects. In this study, effects of knockdown of juvenile hormone esterase gene (jhe) in Bemisia tabaci were studied using RNA interference (RNAi) technique. dsRNA corresponding to two conserved regions of jhe gene, substrate binding pocket site (jhe1), catalytic triad site (jhe2), green fluorescent protein gene (gfp) as control were synthesized. dsRNAs incorporated in artificial diet (20% sucrose solution) @ 2.5, 1.0, 0.5 and 0.1 μg/μl were fed to adult whiteflies for 48 h, followed by shifting whiteflies to live plants for next generation biology study. Based on qRT-PCR analyses, reduced jhe gene expression was observed in adult whiteflies after dsRNA feeding @ 2.5 and 1.0 μg/μl. jhe gene knockdown affects the survival and reproduction of whiteflies adversely in a dose-dependent manner. Moreover, oral feeding of dsRNA to adult whiteflies @ 2.5 and 1.0 μg/μl showed adverse effects on next generation of whitefly viz., lower egg hatchability and shortened egg incubation period. Minimum number of viable eggs (1.04 and 1.80 eggs/female) were observed when whiteflies were fed with highest concentration of dsjhe1 and dsjhe2 as compared to control (16.58 eggs/female). These data suggest that jhe gene acts as a major biological player in whitefly and its progeny and further indicate to be potential target for managing whitefly population.     

Acceleration of epithelial cell syndecan-1 shedding by anthrax hemolytic virulence factors

Background  

It has been recently reported that major pathogens Staphylococcus aureus and Pseudomonas aeruginosa accelerate a normal process of cell surface syndecan-1 (Synd1) ectodomain shedding as a mechanism of host damage due to the production of shedding-inducing virulence factors. We tested if acceleration of Synd1 shedding takes place in vitro upon treatment of epithelial cells with B. anthracis hemolysins, as well as in vivo during anthrax infection in mice.     

Visualization and Quantification of Intraperitoneal Tumors by In Vivo Computed Tomography Using Negative Contrast Enhancement Strategy in a Mouse Model of Ovarian Cancer

Small animal computed tomography (CT) has poor intrinsic soft tissue contrast, limiting evaluation of intra-abdominal structures. Using standard intravascular-extracellular intravenous contrast (IE-IV) alone is theoretically limited by long acquisition times of traditional small animal scanners that may result in equilibration. We assessed whether a negative contrast strategy of enhancing normal tissue surrounding tumor, instead of the tumor itself, can visualize and quantify intraperitoneal (IP) cancer in a mouse model. Two and a half weeks after IP injection of Hey A8 cells, four groups of three animals each were administered serial dilutions of IV Fenestra LC (RES-IV), oral Gastroview, and IP Optiray 320. Another group of three animals was administered IV Optiray 320 (IE-IV), oral Gastroview, and IP Optiray 320 in successive combinations. Both groups were imaged by CT. Tumor and organ Hounsfield units were measured, and visualization was assessed. With increasing contrast amount, the Hounsfield unit of organs generally increased, whereas that of tumor remained essentially stable. The visualization of abdominal organs and tumor also generally increased with increasing contrast amount. Visualization of tumor and its margins adjacent to liver, spleen, and stomach was significantly better on administering RES-IV. However, for tumor adjacent to bladder, both IE-IV and RES-IV were equivalent. In vivo CT-derived tumor weights correlated highly with ex vivo tumor weights (r = 0.96, P < .0001, n = 15). Thus, CT using negative contrast enhancement strategy allows visualization and quantification of IP tumors. Such a strategy will also enable anatomic localization of functional signal for combination/molecular imaging.     

Suppression of Tie-1 in endothelial cells in vitro induces a change in the genome-wide expression profile reflecting an inflammatory function

Tie-1 is an endothelial specific receptor tyrosine kinase that is upregulated in diseases such as atherosclerosis and rheumatoid arthritis. We recently demonstrated that Tie-1 induced a proinflammatory response when overexpressed in endothelial cells. Here, we used a complementary approach and suppressed endogenous Tie-1 expression in endothelial cells to examine its function by microarray analysis. Tie-1 appeared to govern expression of many genes involved in inflammation. Expression knockdown of Tie-1 significantly reduced endothelial conditioned medium ability to stimulate MCP-1 production in U937 cells. Collectively, our results support the notion that Tie-1 has an inflammatory function in endothelial cells.     

Evaluation of drying methods on nutritional constituents and antioxidant activities of Chlorella vulgaris cultivated in an outdoor open raceway pond

Chlorella vulgaris is known for its protein, growth factor, and nutritional constituents. Chlorella vulgaris was cultivated in a 1000-L outdoor open raceway pond with a maximum volumetric productivity of 130 mg L^-1 day^-1. The harvested biomass was dried through different methods, viz., sun drying (30 °C), oven drying (60 °C), lyophilization (?110 °C), drum drying (120 °C), and spray drying (100–150 °C). The effect of the drying method on proximate composition, pigments (chlorophyll, carotenoids), bioactive compounds (total phenolic content, flavonoid content), vitamin B₁₂, antioxidant properties (ferric reducing antioxidant power, DPPH, and total antioxidant activity), and the color quality of C. vulgaris biomass was evaluated. Surface characterization by scanning electron microscopy (SEM) and functional group characterization through Fourier transform infrared spectroscopy were also performed. The biomass dried through lyophilization and sun drying retained maximum bioactive compounds and antioxidant activities. In contrast, drum drying resulted in a loss of nutrients, viz., protein (up to 44%), lipid (up to 41%), vitamin B₁₂ (up to 40%), total phenolic content (> 50%), total flavonoid content (> 50%), and antioxidant activity (> 50%). Oven drying led to a loss of 30% in total flavonoid content and 17% in ferric reducing antioxidant power. SEM showed the destruction of cell wall integrity in the drum-dried sample and porous structure in the spray-dried sample. This study suggests that drying methods affect the nutrients and bioactive compounds of C. vulgaris biomass, and therefore a drying method should be selected carefully depending on the end use of the biomass.