Integration of Bioagent and Bioproduct for the Management of Root-Knot Nematode, Meloidogyne Incognita on Eggplant

Efficacy of bioagent (Paecilomyces lilacinus) and the bioproduct (Radiant) in various combinations was assessed on the reproduction of Meloidogyne incognita on eggplant. The influence of P. lilacinus and Radiant was determined on egg hatching and second stage juvenile (J2) mortality under in vitro conditions. The concentrations of 1% and 100% of Radiant and P. lilacinus respectively both alone and in combined application caused significant mortality and reduction in egg hatching at all time intervals. The interaction of P. lilacinus and Radiant was determined individually, concomitantly, and sequentially on reproduction of M. incognita on eggplant under greenhouse. The reproduction of M. incognita was significantly reduced in the concomitant treatment consisting of both P. lilacinus and Radiant followed by sequential and individual treatment of Radiant and the plant growth parameters incresed significantly. Our findings suggest that P. lilacinus and Radiant have the ability to regulate nematode population and may serve as nematicides. Keywords: P. lilacinus, Radiant, Egg hatching, Mortality, Concomitant, Sequentia

Modulation of Mouse Coagulation Gene Transcription following Acute In Vivo Delivery of Synthetic Small Interfering RNAs Targeting HNF4α and C/EBPα

Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo

Determination of ROS Scavenging, Antibacterial and Antifungal Potential of Methanolic Extract of <i>Otostegia limbata</i> (Benth.) Boiss.

Wide spectrum medicinal significance augments plant utilization as the primary source of significant pharmaceutical agents. In vitro investigation of antioxidant and antimicrobial activity highlights the therapeutic potential of Otostegia limbata. Methanol extract of the plant (MEP) shows considerable dose dependent antioxidant ability at six concentrations (7.81 µg/mL to 250 µg/mL) in 2.2-diphenyl-1-picrylhydrazyl (DPPH) assay, phosphomolybdate assay (PMA) and reducing power assay (RPA). The plant capability to scavenge free radicals in the mixture ranged from 37.89% to 63.50% in a concentration-dependent manner. MEP was active against five tested bacterial strains in the agar-well diffusion method. Staphylococcus aureus, gram-positive bacteria was found to be most susceptible followed by S. epidermidis with 18.80 mm and 17.47 mm mean zone of inhibition. The mean inhibition zone against gram-negative strains Klebsiella pneumonia, Pseudomonas spp. and Escherichia coli were 15.07 mm, 14.73 mm, and 12.17 mm. MEP revealed potential against Alternaria spp. and Aspergillus terreus fungal strains evaluated through agar-tube dilution assay. Aspergillus terreus was more sensitive than Alternaria spp. with an average 78.45% and 68.0% inhibition. These findings can serve as a benchmark for forthcoming scrutiny such as bioactive components discovery and drug development

Determination of ROS Scavenging, Antibacterial and Antifungal Potential of Methanolic Extract of Otostegia limbata (Benth.) Boiss.

Wide spectrum medicinal significance augments plant utilization as the primary source of significant pharmaceutical agents. In vitro investigation of antioxidant and antimicrobial activity highlights the therapeutic potential of Otostegia limbata. Methanol extract of the plant (MEP) shows considerable dose dependent antioxidant ability at six concentrations (7.81 µg/mL to 250 µg/mL) in 2.2-diphenyl-1-picrylhydrazyl (DPPH) assay, phosphomolybdate assay (PMA) and reducing power assay (RPA). The plant capability to scavenge free radicals in the mixture ranged from 37.89% to 63.50% in a concentration-dependent manner. MEP was active against five tested bacterial strains in the agar-well diffusion method. Staphylococcus aureus, gram-positive bacteria was found to be most susceptible followed by S. epidermidis with 18.80 mm and 17.47 mm mean zone of inhibition. The mean inhibition zone against gram-negative strains Klebsiella pneumonia, Pseudomonas spp. and Escherichia coli were 15.07 mm, 14.73 mm, and 12.17 mm. MEP revealed potential against Alternaria spp. and Aspergillus terreus fungal strains evaluated through agar-tube dilution assay. Aspergillus terreus was more sensitive than Alternaria spp. with an average 78.45% and 68.0% inhibition. These findings can serve as a benchmark for forthcoming scrutiny such as bioactive components discovery and drug development

Extraction, Separation and Purification of Bioactive Anticancer Components from <i>Peganum harmala</i> against Six Cancer Cell Lines Using Spectroscopic Techniques

Conventional cancer treatments normally involve chemotherapy or a combination of radio- and chemotherapy. However, the adverse effects of synthetic medicines encouraged the exploration of novel therapeutic medications of a bio-friendly nature. In an effort to explore anticancer compounds from natural resources, crude extract of Peganum harmala (seeds) was fractionated on the basis of polarity, and the fractions were further tested for anticancer activity. Brine shrimp lethality assays and potato disc antitumor assays were used to test each fraction for cytotoxic and antitumor potential. The ethyl acetate fraction was found to be most potent, with LC50 and IC50 values of 34.25 µg/mL and 38.58 µg/mL, respectively. Further activity-guided fractionation led to the isolation of the bioactive compound PH-HM-10 which was identified and characterized by Mass Spectroscopy (MS), Infrared Spectroscopy (IR), Proton Nuclear Magnetic Resonance Spectroscopy (1HNMR), Carbon Nuclear Magnetic Resonance Spectroscopy (13CNMR) and Heteronuclear Single Quantum Correlation (HSQC). Anticancer aspects in the isolated compound were determined against six human cancer cell lines with a maximum anticancer effect (IC50 = 36.99 µg/mL) against the tested human myeloid leukemia (HL-60) cell line, followed by the human lung adenocarcinoma epithelial cell line (A549) and the breast cancer cell line (MCF-7) with an IC50 of 63.5 µg/mL and 85.9 µg/mL, respectively). The findings of the current study suggest that the isolated compound (Pegaharmine E) is significantly active against the tested cancer cell lines and can be further investigated to develop future novel anticancer chemotherapeutic agents

Orexin-A Regulates Follicular Growth, Proliferation, Cell Cycle and Apoptosis in Mouse Primary Granulosa Cells via the AKT/ERK Signaling Pathway

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility

Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease

Patients with von Willebrand disease (VWD) are often heterozygous for a missense mutation in the von Willebrand factor (VWF) gene. Investigating the pathogenic features of VWF mutations in cells directly derived from patients has been challenging. Here, we have used blood outgrowth endothelial cells (BOECs) isolated from human peripheral blood to analyze the storage and secretion of VWF. BOECs showed full endothelial characteristics and responded to Weibel-Palade body (WPB) secretagogues except desmopressin. We examined BOECs derived from a single subject heterozygous for a type 2N mutation (p.Arg854Gln) and from 4 patients with type 1 VWD who were, respectively, heterozygous for p.Ser1285Pro, p.Leu1307Pro, p.Tyr1584Cys, and p.Cys2693Tyr. Compared with normal BOECs, BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr showed morphologically abnormal WPB and retention of VWF in the endoplasmic reticulum, whereas BOECs heterozygous for p.Arg854Gln or p.Tyr1584Cys showed normal WPB. The agonist-induced exocytosis of WPB from BOECs and formation of VWF strings on BOECs heterozygous for p.Ser1285Pro, p.Leu1307Pro, or p.Cys2693Tyr, but not for p.Arg854Gln or p.Tyr1584Cys, were reduced. In conclusion, VWD phenotype can be recapitulated in BOECs, and thus BOECs provide a feasible bona fide cell model to study the pathogenic effects of VWF mutations. (Blood. 2013;121(14):2762-2772

Overview of gene transcript levels in mouse livers following C/EBPα knockdown.

<p>Livers from siCEBPα injected animals (hatched bars) and siNEG injected animals as controls (open bars) were subjected to control and coagulation gene transcript levels by QPCR. Data are presented for mice two days post siRNA injection. β-actin was used as internal control for quantification and normalization. The ΔCt values of the individual samples were related to the mean ΔCt of the siNEG group. Data are expressed as mean with error bars representing the difference between 2 POWER of upper and lower range of the mean ΔΔCt (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038104#pone-0038104-g001" target="_blank">figure 1</a> legends). Data were statistically analysed using Mann Whitney Rank Sum test. <i>P</i>-values<0.05 were regarded as statistically significant. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. On the x-axis the coagulation genes are ranked according to the magnitude of effects observed in the siRNA injected animals.</p

Plasma analysis of siHNF4α and siC/EPBα injected mice.

<p>Data are represented as mean ± SEM with the group injected with negative siRNA set as a reference.</p>*<p><i>P</i><0.05 and</p>**<p><i>P</i><0.01 versus mice injected with control siNEG. Prothrombine time (PT), activated partial thromboplastine time (aPTT), Factor activity (F11), Factor 12 activity (F12), Fibrinogen antigen (Fbg) and circulating liver enzymes alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosfatase (ALP) and plasma total billirubine (Tbil). N.D. = not determined. Values for uninjected control mice: billirubin 8.3±0.2 µmol/L, ALT<20 U/L, AST 28±3 U/L, ALP 96±6 U/L.</p