Heterologous expression,characterization and evaluation of the matrix protein from Newcastle disease virus as a target for antiviral therapies

Newcastle disease virus (NDV) is an infectious agent of a large variety of birds, including chicken, which poses a real threat to the agriculture industry. Matrix (M) proteins of NDV and many other viruses perform critical functions during viral assembly and budding from the host cell. M-proteins are well conserved and therefore are potential targets for antiviral therapies. To validate this, we expressed the NDV M-protein in its native form in Saccharomyces cerevisiae and in inclusion bodies in Escherichia coli. Proper refolding of the recombinant protein produced in E. coli was verified using circular dichroism and infrared spectroscopies and electron microscopy. Immunization of chickens with the NDV M-protein elicited significant serum antibody titers. However, the antibodies conferred little protection against the ND following lethal viral challenges. We conclude that the M-protein is not exposed on the surface of the host cell or the virus at any stage during its life cycle. We discuss how the conserved M-protein can further be exploited as an antiviral drug target.     

Common Oncogenic Mutations Are Infrequent in Oral Squamous Cell Carcinoma of Asian Origin

Objectives

The frequency of common oncogenic mutations and TP53 was determined in Asian oral squamous cell carcinoma (OSCC).

Materials and Methods

The OncoCarta^™ panel v1.0 assay was used to characterize oncogenic mutations. In addition, exons 4-11 of the TP53 gene were sequenced. Statistical analyses were conducted to identify associations between mutations and selected clinico-pathological characteristics and risk habits.

Results

Oncogenic mutations were detected in PIK3CA (5.7%) and HRAS (2.4%). Mutations in TP53 were observed in 27.7% (31/112) of the OSCC specimens. Oncogenic mutations were found more frequently in non-smokers (p = 0.049) and TP53 truncating mutations were more common in patients with no risk habits (p = 0.019). Patients with mutations had worse overall survival compared to those with absence of mutations; and patients who harbored DNA binding domain (DBD) and L2/L3/LSH mutations showed a worse survival probability compared to those patients with wild type TP53. The majority of the oncogenic and TP53 mutations were G:C > A:T and A:T > G:C base transitions, regardless of the different risk habits.

Conclusion

Hotspot oncogenic mutations which are frequently present in common solid tumors are exceedingly rare in OSCC. Despite differences in risk habit exposure, the mutation frequency of PIK3CA and HRAS in Asian OSCC were similar to that reported in OSCC among Caucasians, whereas TP53 mutations rates were significantly lower. The lack of actionable hotspot mutations argue strongly for the need to comprehensively characterize gene mutations associated with OSCC for the development of new diagnostic and therapeutic tools.     

Condition‐dependent expression of pre‐ and postcopulatory sexual traits in guppies

Female choice can impose persistent directional selection on male sexually selected traits, yet such traits often exhibit high levels of phenotypic variation. One explanation for this paradox is that if sexually selected traits are costly, only the fittest males are able to acquire and allocate the resources required for their expression. Furthermore, because male condition is dependent on resource allocation, condition dependence in sexual traits is expected to underlie trade‐offs between reproduction and other life‐history functions. In this study we test these ideas by experimentally manipulating diet quality (carotenoid levels) and quantity in the guppy (Poecilia reticulata), a livebearing freshwater fish that is an important model for understanding relationships between pre‐ and post‐copulatory sexually selected traits. Specifically, we test for condition dependence in the expression of pre‐ and postcopulatory sexual traits (behavior, ornamentation, sperm traits) and determine whether diet manipulation mediates relationships among these traits. Consistent with prior work we found a significant effect of diet quantity on the expression of both pre‐ and postcopulatory male traits; diet‐restricted males performed fewer sexual behaviors and exhibited significant reductions in color ornamentation, sperm quality, sperm number, and sperm length than those fed ad libitum. However, contrary to our expectations, we found no significant effect of carotenoid manipulation on the expression of any of these traits, and no evidence for a trade‐off in resource allocation between pre‐ and postcopulatory episodes of sexual selection. Our results further underscore the sensitivity of behavioral, ornamental, and ejaculate traits to dietary stress, and highlight the important role of condition dependence in maintaining the high variability in male sexual traits.     

Computational fluid dynamics as a tool for testing functional and ecological hypotheses in fossil taxa

Computational fluid dynamics is a method for simulating fluid flows that has been widely used in engineering for decades, and which also has applications for studying function and ecology in fossil taxa. However, despite the possible benefits of this approach, computational fluid dynamics has been used only rarely in palaeontology to date. The theoretical basis underlying the technique is outlined and the main steps involved in carrying out computer simulations of fluid flows are detailed. I also describe previous studies that have applied the method to fossils and discuss their potential for informing future research directions in palaeontology. Computational fluid dynamics can enable large‐scale comparative analyses, as well as exacting tests of hypotheses related to the function and ecology of ancient organisms. In this way, it could transform our understanding of many extinct fossil groups.     

Purification of immature neuronal cells from neural stem cell progeny

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

Culture of rotifers with reference to some physico-chemical properties of water in nursery pond

Rotifers were cultured with five different organic and inorganic fertilizers in nursery ponds. Of the fertilizers used, mustard oil cake gave significantly (p < 0.01) higher production of rotifers than that of mohua oil cake followed by cow-dung, wheat bran, mixture of NPK and control. The higher production of rotifers was directly related with the higher doses of fertilizers. Among the rotifer species identified, the abundance of Brachionus caudatus and B. forficula were significantly (p < 0.01) higher than others. Available N, available P, exchangeable K and exchangeable Ca and exchangeable Mg were generally higher in ponds where organic fertilizers were used. Proximate composition of rotifers varied depending on the kinds of fertilizers. The multiple correlations of physico-chemical properties were highly significant (p < 0.01) with the growth and production of B. caudatus (R = 0.995), B. forficula (R = 0.932), Trichocerca capucina (R = 0.917), B. patulus (R = 0.901) and B. angularis (R = 0.892) and simply significant (p < 0.05) in the case of Keratella tropica (R = 0.880), Hexarthra intermadia (R = 0.875), B. calyciflorus (R = 0.864) and Filinia spp. (R = 0.856) contributing 91.20%, 86.86%, 84.09%, 81.18%, 79.57%, 77.44%, 76.56%, 74.65% and 73.27% of total effect of water properties on the growth of these species, respectively. The residual effect of nine different physico-chemical properties of water on the production of rotifers was 78.92% which indicates that these properties of water had only 21.08% influence on the production of rotifers.     

Carbohydrate-based micelle clusters which enhance hydrophobic drug bioavailability by up to 1 order of magnitude

Amphiphilic chitosan-based polymers (Mw < 20 kDa) self-assemble in aqueous media at low micromolar concentrations to give previously unknown micellar clusters of 100-300 nm in size. Micellar clusters comprise smaller 10-30 nm aggregates, and the nanopolarity/drug incorporation efficiency of their hydrophobic domains can be tailored by varying the degree of lipidic derivatization and molecular weight of the carbohydrate. The extent of drug incorporation by these novel micellar clusters is 1 order of magnitude higher than is seen with triblock copolymers, with molar polymer/drug ratios of 1:48 to 1:67. On intravenous injection, the pharmacodynamic activity of a carbohydrate propofol formulation is increased by 1 order of magnitude when compared to a commercial emulsion formulation, and on topical ocular application of a carbohydrate prednisolone formulation, initial drug aqueous humor levels are similar to those found with a 10-fold dose of prednisolone suspension.     

Design of High Speed and Low Offset Dynamic Latch Comparator in 0.18 μm CMOS Process

The cross-coupled circuit mechanism based dynamic latch comparator is presented in this research. The comparator is designed using differential input stages with regenerative S-R latch to achieve lower offset, lower power, higher speed and higher resolution. In order to decrease circuit complexity, a comparator should maintain power, speed, resolution and offset-voltage properly. Simulations show that this novel dynamic latch comparator designed in 0.18 µm CMOS technology achieves 3.44 mV resolution with 8 bit precision at a frequency of 50 MHz while dissipating 158.5 µW from 1.8 V supply and 88.05 µA average current. Moreover, the proposed design propagates as fast as 4.2 nS with energy efficiency of 0.7 fJ/conversion-step. Additionally, the core circuit layout only occupies 0.008 mm².