Protection of Shrimp Penaeus monodon from WSSV Infection Using Antisense Constructs

White spot syndrome caused by white spot syndrome virus (WSSV) is one of the most threatening diseases of shrimp culture industry. Previous studies have successfully demonstrated the use of DNA- and RNA-based vaccines to protect WSSV infection in shrimp. In the present study, we have explored the protective efficacy of antisense constructs directed against WSSV proteins, VP24, and VP28, thymidylate synthase (TS), and ribonucleotide reductase-2 (RR2) under the control of endogenous shrimp histone-3 (H3) or penaedin (Pn) promoter. Several antisense constructs were generated by inserting VP24 (pH3–VP24, pPn–VP24), VP28 (pH3–VP28, pPn–VP28), TS (pH3–TS, pPn–TS), and RR2 (pH3–RR2) in antisense orientation. These constructs were tested for their protective potential in WSSV infected cell cultures, and their effect on reduction of the viral load was assessed. A robust reduction in WSSV copy number was observed upon transfection of antisense constructs in hemocyte cultures derived from Penaeus monodon and Scylla serrata. When tested in vivo, antisense constructs offered a strong protection in WSSV challenged P. monodon. Constructs expressing antisense VP24 and VP28 provided the best protection (up to 90 % survivability) with a corresponding decrease in the viral load. Our work demonstrates that shrimp treated with antisense constructs present an efficient control strategy for combating WSSV infection in shrimp aquaculture.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage

The DNA repair enzyme human uracil DNA glycosylase (UNG) scans short stretches of genomic DNA and captures rare uracil bases as they transiently emerge from the DNA duplex via spontaneous base pair breathing motions. The process of DNA scanning requires that the enzyme transiently loosen its grip on DNA to allow stochastic movement along the DNA contour, while engaging extrahelical bases requires motions on a more rapid timescale. Here, we use NMR dynamic measurements to show that free UNG has no intrinsic dynamic properties in the millisecond to microsecond and subnanosecond time regimes, and that the act of binding to nontarget DNA reshapes the dynamic landscape to allow productive millisecond motions for scanning and damage recognition. These results suggest that DNA structure and the spontaneous dynamics of base pairs may drive the evolution of a protein sequence that is tuned to respond to this dynamic regime.     

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background  

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.     

Injectable Lipid Emulsions—Advancements,Opportunities and Challenges

Injectable lipid emulsions, for decades, have been clinically used as an energy source for hospitalized patients by providing essential fatty acids and vitamins. Recent interest in utilizing lipid emulsions for delivering lipid soluble therapeutic agents, intravenously, has been continuously growing due to the biocompatible nature of the lipid-based delivery systems. Advancements in the area of novel lipids (olive oil and fish oil) have opened a new area for future clinical application of lipid-based injectable delivery systems that may provide a better safety profile over traditionally used long- and medium-chain triglycerides to critically ill patients. Formulation components and process parameters play critical role in the success of lipid injectable emulsions as drug delivery vehicles and hence need to be well integrated in the formulation development strategies. Physico-chemical properties of active therapeutic agents significantly impact pharmacokinetics and tissue disposition following intravenous administration of drug-containing lipid emulsion and hence need special attention while selecting such delivery vehicles. In summary, this review provides a broad overview of recent advancements in the field of novel lipids, opportunities for intravenous drug delivery, and challenges associated with injectable lipid emulsions.     

The binding between p67 and eukaryotic initiation factor 2 plays important roles in the protection of eIF2alpha from phosphorylation by kinases

Phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 is the major regulatory step in the initiation of protein synthesis in mammals. P67, a cellular glycoprotein, protects phosphorylation of eIF2alpha from kinases. Previously, we reported that the D6/2 mutant of p67 has higher levels of protection of eIF2alpha phosphorylation (POEP) activity. In this study, we report that the D6/2 mutant and its double mutants containing second-site alanine substitutions at the five conserved amino acid residues (D251, D262, H331, E364, and E459) show increased POEP activity in serum-starved rat tumor hepatoma cells. Serum-restoration to those cells did not abolish their increased POEP activity except the D6/2+H331A double mutant. The latter mutant shows slight inhibition of POEP activity during serum starvation and this inhibition increased significantly during serum restoration. KRC-7 cells constitutively expressing the D6/2 mutant showed slightly decreased levels of PKR phosphorylation and significantly low level of phosphorylation of ERKs 1 and 2. The D6/2 mutant also showed increased binding with eIF2alpha and eIF2gamma and almost similar binding with ERKs 1 and 2 as compared to wild type p67. Altogether, our data demonstrate that the increased binding of the D6/2 mutant with the subunits of eIF2 may be in part the cause for its high POEP activity.     

Phosphatidylinositol 3-kinase/Akt signaling stimulates colonic mucosal cell survival during aging

Although aging is shown to be associated with decreased apoptosis and increased survival of cells in the colonic mucosa of Fischer 344 rats, the regulatory mechanisms are poorly understood. The current investigation examines the involvement of phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway in mediating the events of colonic mucosal cell survival during aging. We have observed that aging is associated with activation of PI3K/Akt signaling, as evidenced by the higher levels of phosphorylated forms of p85, the regulatory subunit of PI3K and of Akt in the proximal and distal colonic mucosa, of aged (21-23 mo) than in young (4-7 mo) rats. These increases are accompanied by a concomitant rise in phosphorylation of proapoptotic protein Bad, which is sequestered by the 14-3-3 family of proteins following phosphorylation by Akt, resulting in a reduction in nonphosphorylated Bad. The amount of antiapoptotic Bcl-xL bound to nonphosporylated Bad in the colonic mucosa is found to be substantially lower in aged than in young rats, resulting in a marked rise in the unbound/free form of Bcl-xL in the aging colon. The age-related activation of PI3K and the reduction in caspase-3 activity could be reversed by wortmannin, a specific inhibitor of PI3K. Increased levels of Bcl-xL and phosphorylated forms of Akt and Bad and reduction in caspase-3 activity were observed throughout the entire length of the colonic crypt of aged rats. We conclude that the constitutive activation of the PI3K/Akt-signaling pathway is partly responsible for the age-related increase in colonic mucosal cell survival. This is evident throughout the entire length of the colonic crypt.     

Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in the air-breathing catfish, Clarias gariepinus

We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T(3) levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.