Biorational management of maize fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) using Bacillus thuringiensis (Berliner) enriched with chemical additives

An invasive pest, fall armyworm, Spodoptera frugiperda (J.E.Smith) (Lepidoptera: Noctuidae) attacks maize at every stage of development, from seedling emergence up to cob formation. Early instar larvae were seen mostly on leaves of maize with characteristics pin or shot hole symptoms. Later instar larvae were confined to deep whorls, leaving typically ragged like appearance and fed on the reproductive stage of the crop especially tassels and developing cobs resulting in quality and quantity loss of maize produce. The effect of commercially available Bacillus thuringiensis subsp. kurstaki product, Dipel® against the second instar larvae of Fall Armyworm (FAW )was not promising under laboratory conditions. Hence, an effort was made to add an adjuvant along with B. thuringiensis to increase the virulence of commercially available B. thuringiensis.The Laboratory bioassays with B. thuringiensis and seven chemical additives ( T1- Bt + Boric acid, T2- Bt + Zinc oxide, T3- Bt + Sodium nitrate, T4- Bt + Peptone, T5- Bt + Urea, T6- Bt + EDTA, T7- Bt + Citric acid & T8-  Bt alone T9- Control) were tested against second instar larvae of Spodoptera frugiperda larvae. The results showed that B. thuringiensis plus sodium nitrate (T3) promoted maximum mortality 82.2 per cent with a minimum LC50 value of 54.620 mg/l. Sodium nitrate boosted B. thuringiensis activity at a concentration of 0.05 per cent by 2.128-fold than B. thuringiensis alone. Overall, sodium nitrate improved the efficacy of B. thuringiensis spray at the maximum level followed by boric acid, urea, EDTA and peptone

Effect of ageing on in vitro true seed and in vivo drupe germination and its dormancy mechanism in teak (Tectona grandis Linn.f)

The germination percentage of teak seed is generally very poor due to its higher percentage of empty seed and poor seed viability. The viable seeds exhibit protracted germination behaviour due to their inherent seed dormancy and other physiochemical characteristics. Hence establishing a teak nursery for largescale plantation activities is a challenging task. This study was undertaken to study the effect of ageing on in vitro true seed and in vivo drupe germination and its dormancy mechanism in teak. Fresh, one-year and two-year stored drupes were used to represent different levels of ageing. Under in vivo conditions, poor drupe germination was observed in fresh drupes (3%) and germination percentage was increased when the drupes were subjected to ageing for one year (17%) or two years (32%). When true seeds separated from fresh drupes and germinated under in vitro conditions, enhanced germination (58.3%) was observed. Biochemical analysis showed that indole-3- acetic acid, indole butyric acid, abscisic acid and coumarin are not present in fresh, one year and two-year-old true seeds. The gibberellic acid was increased with an increase in ageing, but the GA3 did not influence the germination percent under in vitro conditions. Scanning electron microscope (SEM) image of fresh teak true seed showed that embryo tip was shrivelled, whereas one and two-year-old true seed embryo tip bulged; this was confirmed that one and two-year-old true seed embryos were matured and satisfied the after-ripening requirement. Nursery studies revealed that one and two-year-old drupes recorded the highest germination compared to fresh drupes.                

Enhancer of Zeste Homolog 2 Induces Pulmonary Artery Smooth Muscle Cell Proliferation

Pulmonary Arterial Hypertension (PAH) is a progressively devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. The proliferation of cancer cells is mediated by increased expression of Enhancer of Zeste Homolog 2 (EZH2), a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes. However, the role of EZH2 in PAH has not been studied. In this study, it is hypothesized that EZH2 could play a role in the proliferation of PASMCs.In the present study, the expression patterns of EZH2 were investigated in normal and hypertensive mouse PASMCs. The effects of EZH2 overexpression on the proliferation of human PASMCs were tested. PASMCs were transfected with EZH2 or GFP using nucleofector system. After transfection, the cells were incubated for 48 hours at 37°C. Proliferation and cell cycle analysis were performed using flow cytometry. Apoptosis of PASMCs was determined using annexin V staining and cell migration was tested by wound healing assay.EZH2 protein expression in mouse PASMCs were correlated with an increase in right ventricular systolic pressure and Right Ventricular Hypertrophy (RVH). The overexpression of EZH2 in human PASMCs enhances proliferation, migration, and decrease in the rate of apoptosis when compared to GFP-transfected cells. In the G2/M phase of the EZH2 transfected cells, there was a 3.5 fold increase in proliferation, while there was a significant decrease in the rate of apoptosis of PASMCs, when compared to control.These findings suggest that EZH2 plays a role in the migration and proliferation of PASMCs, which is a major hallmark in PAH. It also suggests that EZH2 could play a role in the development of PAH and can serve as a potential target for new therapies for PAH

Antimicrobial peptide tilapia hepcidin (TH) 2-3 mediated modulation of immune responses in LPS stimulated mouse macrophages, and TH1-5 mediated antiviral function against IPNV in CHSE-214 cells

抗菌胜肽（antimicrobial peptide, AMP）在對抗各種病原體的非特異性宿主防禦機制上，發揮著重要作用。 除了直接的抗菌功能外，它們還參與了對抗病原菌感染之免疫反應基因的調節。鐵調素為一種重要的抗菌胜肽。直到最近，羅非魚鐵調素2-3（TH2-3）2-3及羅非魚鐵調素1-5(TH1–5)才複製到羅非魚上。羅非魚鐵調素2-3(TH2-3)的表達已由細菌內毒素脂多醣(lipopolysachcharride, LPS）上調，羅非魚鐵調素1-5則由聚肌苷酸胞苷酸(Poly I：Poly C）所激活，這是一種模擬合成的雙鏈核糖核酸（double stranded RNA, dsRNA），產自大部分病毒和被稱為類鐸受體（toll like receptor, TLR）3激活素生命週期中的複製過程。它顯示了細菌和病毒介導了TH2-3及1-5的誘導。此外，最近也報導了有關TH2-3的抗菌功能。本研究旨在探討TH2-3和TH1-5分子方面個別的抗菌和抗病毒功能。本文第一部分簡要回顧了魚類的抗菌肽，並介紹了羅非魚鐵調素。 本論文報告第二部分是探討TH2-3對細菌內毒素脂多醣 (LPS)刺激小鼠巨噬細胞的前炎性細胞因子的介導調控。研究表明，TH2-3對腫瘤壞死因子(TNF)-α和其他炎性細胞因子。 第三部分簡要地探討了在巨噬細胞株(RAW264.7)中，TH2 – 3、蛋白激酶C(PKC)激活劑、和佛波12肉荳蔻 - 13 -醋酸（PMA）對蛋白激酶C（PKC）相關蛋白質的差分調節。以40或80微克/毫升TH2 - 3處理，並沒影響細胞的形態。但以120微克/毫升TH2- 3處理所產生的形態變化，和以PMA在巨噬細胞株(RAW264.7)處理後的變化相似。 此外，脂多醣(LPS)所刺激巨噬細胞株中的腫瘤壞死因子-α啟動子活性和細胞表面COX - 2的表達，明顯的是由TH2-3而不是由PMA所抑制。 根據蛋白質印跡(Western blotting) 對蛋白激酶C成員的表達顯示，蛋白激酶C –μ、磷酸-PKCμ在絲氨酸（第）-744和P -蛋白質激酶Cδ的表達是被PMA所激活，但卻為TH2- 3所抑制。此外，在S – 916的磷-蛋白激酶C是由TH2-3活化並為PMA所抑制。整體而言，PMA和TH2-3對蛋白激酶C亞型(isoforms)的微調可能是經由對形態學變化的影響和調節巨噬細胞株(RAW264.7)的腫瘤壞死因子(TNF)-α。 本論文第四部分報告TH1 - 5對傳染性胰臟壞死病毒（IPNV）在鮭魚胚胎細胞(CHSE)214的抗病毒功能。 這部分所呈現細胞的生存和斑塊的檢測結果表明TH1 - 5對 IPNV的抗病毒性質。 實時聚合酵素連鎖反應(PCR)技術的研究表明，TH1 – 52對白細胞介素、膜聯蛋白(annexin)、以及其他對病毒作出反應的基因表達具調節作用。第五部分介紹了這項研究的未來前景。這項調查顯示，TH2 - 3及Th1 – 5對宿主細胞在LPS和IPNV感染分別具抗菌和免疫調節功能。此外， TH2- 3對脂多醣(LPS) 誘導的前炎性細胞因子產生抑制的潛在機制也被探索。Antimicrobial peptides (AMPs) play a vital role in non-specific innate host defense mechanism against various pathogens. In addition to direct antimicrobial function, they are also involved in the modulation of immune responsive genes against pathogen infection. Hepcidin is one of the important AMPs known for its antimicrobial and iron homeostasis function. The tilapia hepcidin (TH) 2-3 and TH1-5 were recently cloned in tilapia fish. The expression of TH2-3 has been shown to be up-regulated by the bacterial endotoxin lipopolysachcharride (LPS), and the TH1-5 is activated by Polyinosinic-polycytidylic acid (poly I:poly C), a synthetic analog of double-stranded RNA (dsRNA) that is produced during the replication of many viruses is known as a toll-like receptor (TLR)3 activator. It shows the bacterial and virus mediated induction of TH2-3 and TH1-5. In addition, the antibacterial function of TH2-3 has been reported recently. The present study aims to investigate the molecular aspects of the antibacterial and the antiviral functions of TH2-3 and TH1-5, respectively. The first part of this thesis briefly reviews the fish antimicrobial peptides, and introduces the tilapia hepcidins. The second part examines the TH2-3-mediated regulation of proinflammatory cytokines in bacterial endotoxin lipopolysaccharide (LPS)-stimulated mouse macrophages. The study shows the inhibition of TNF-α, interleukin (IL)-1α, IL-1β, IL-6, and cyclooxygenase (COX)-2, by TH2-3. Studies with the COX-2-specific inhibitor, melaxicam, COX-2-overexpressing cells and the estimate of intracellular cAMP demonstrates that the inhibition of COX-2 is among the mechanisms through which TH2-3 controls TNF-α secretion. In addition, the presence of TH2-3 in LPS-stimulated cells had suppressed the induction of pERK1/2, and prevented the LPS-stimulated nuclear accumulation of NF-κB family proteins of p65, NF-κB2 and c-Rel. Altogether, TH2-3 inhibits TNF-α, and other proinflammatory cytokines through COX-2-, PDE4D-, and pERK1/2-dependent mechanisms. The third part briefly investigates the differential modulation of protein kinase C (PKC)-associated proteins by TH2-3, and the PKC activator, phorbol 12-myristate 13-acetate (PMA), in RAW264.7 cells. Treatment with TH2-3 at 40 or 80 μg/ml did not affect the cell morphology, but TH2-3 at 120 μg/ml produced morphological changes similar to those after treatment with PMA in RAW264.7 cells. In addition the TNF-α promoter activity and cell surface COX-2 expression in LPS-stimulated RAW264.7 cells, was significantly suppressed by TH2-3, but not by PMA. The expression of PKC member proteins according to western blotting demonstrated that the expression of PKC-μ, phosphorylated (p)-PKCμ at serine(S)-744, and p-PKCδ were activated by PMA, but were suppressed by TH2-3. In addition, p-PKC at S-916 was activated by TH2-3 and inhibited by PMA. In conclusion, the differential regulation of PKC isoforms by PMA and TH2-3 may influence morphological changes and regulation of TNF-α in RAW264.7 cells. The fourth part of the thesis reports the antiviral functions of TH1-5 against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells. The cell survival and plaque assays results of this part demonstrate the antiviral property of TH1-5 against IPNV. Real-time PCR studies showed the modulation of interleukin, annexin, and other viral-responsive gene expressions by TH1-5. The fifth part describes the future prospects of this investigation. This investigation demonstrates the antimicrobial and immunomodulatory functions of TH2-3 and TH1-5 against LPS and IPNV infections in the host cells respectively. Furthermore, the underlying signaling mechanism for the suppression of LPS-induced pro-inflammatory cytokines by TH2-3 also explored.  Page No. Acknowledgement……………………………………………………………… i Abstract………………………………………………………………………… ii List of Tables…………………………………………………………………… ix List of Figures…………………………………………………………………… x List of Appendix………………………………………………………………… xiv Part 1: Bird's eye view of fish antimicrobial peptides and tilapia hepcidins… 1 Abstract 2 Introduction 3 Fish antimicrobial peptides (AMPs) 4 Antibacterial function of fish AMPs 5 Antiviral, antifungal, and antiparasitic function of fish AMPs 6 Immunomodulatory function of fish AMPs 6 AMP as antitumor agents 7 AMPs as a vaccine adjuvant 8 AMP in the preparation of Inactivated vaccines 8 Other aspects of fish AMPs 8 Tilapia hepcidins 9 Part 2: The tilapia hepcidin 2-3 peptide modulates lipopolysaccharide (LPS)-induced cytokines and inhibits tumor necrosis factor (TNF)-α through cyclooxygenase (COX)-2 and phosphodiesterase (PDE)4D……… 16 Abstract……………………………………………………………………… 17 Introduction…………………………………………………………………. 18 Materials and Methods……………………………………………………. 22 Results………………………………………………………………………. 30 Discussion…………………………………………………………………… 38 Part 3: The antimicrobial peptide, tilapia hepcidin 2-3 (TH2-3), and PMA differentially regulate the protein kinase C (PKC) isoforms, TNF-α and COX-2, in mouse RAW264.7 macrophage cells………………………… 62 Abstract…………………………………………………………………… 63 Introduction……………………………………………………………….. 64 Materials and Methods……………………………………………………. 67 Results…………………………………………………………………….. 72 Discussion………………………………………………………………… 76 Part 4: Antiviral function of tilapia hepecidin 1-5 and its modulation on immune-related genes expression against infectious pancreatic necrosis virus (IPNV) in Chinook salmon embryo (CHSE)-214 cells……………….… 89 Abstract…………………………………………………………………… 90 Introduction……………………………………………………………….. 91 Materials and Methods……………………………………………………. 93 Results…………………………………………………………………….. 97 Discussion………………………………………………………………… 101 Part 5: Future prospects……………………………………………………… 114 References……………………………………………………………………… 118 Appendix………………………………………………………………………... 139 Resume…………………………………………………………………………... 18

Enhanced Control of Bladder-Associated Tumors Using Shrimp Anti-Lipopolysaccharide Factor (SALF) Antimicrobial Peptide as a Cancer Vaccine Adjuvant in Mice

Shrimp anti-lipopolysaccharide factor (SALF) is an antimicrobial peptide with reported anticancer activities, such as suppression of tumor progression. In this study, we prepared a potential cancer vaccine comprised of SALF in conjunction with the cell lysate of inactivated murine bladder carcinoma cells (MBT-2), and evaluated its efficacy in a mouse tumor model. Our study shows that SALF added to cell culture media inhibits growth progression of MBT-2, and that SALF together with inactivated MBT-2 lysate elevates the level of inflammasome activity, and modulates the levels of IL-1β, MCP-1, IL-6, IL-12, and TNF-α in mouse macrophages. Immunization of 7, 14, and 21 day-old mice with the vaccine prevented growth of MBT-2 cell-mediated tumors. The vaccine was found to enhance expression of T-cell, cytotoxic T cells, and NK cells in the immunized mice groups. Recruitment of macrophages, T-helper cells, and NK cells was enhanced, but levels of VEGF were decreased in immunized mice. This report provides empirical evidence that our SALF as vaccine adjuvant enhances antitumor immunity in mice

Inflammasome Inhibition Suppresses Alveolar Cell Permeability Through Retention of Neuregulin-1 (NRG-1)

Background: Neuregulin (NRG)-1-human epidermal receptor (HER)-2 signaling pathway is a key regulator of IL-1β-mediated pulmonary inflammation and epithelial permeability. The inflammasome is a newly discovered molecular platform required for caspase-1 activation and maturation of IL-1β. However, the role of the inflammasome in NRG-1-HER2 signaling-mediated alveolar cell permeability is unknown. Methods: The inflammasome was activated or inhibited in THP-1 cells; supernatants from these cells were added to A549 cells and human small airway epithelial cells (HSAEC). The protein expression of NRG-1 and phospho-HER2 (pHER2) were measured by Western blot analysis and epithelial permeability was measured using Lucifer yellow dye. Results: Results reveal that alveolar permeability in A549 cells and HSAEC is increased when treated with supernatants of inflammasome-activated THP-1 cells. Alveolar permeability is significantly suppressed when treated with supernatant of inflammasome-inhibited THP-1 cells. Inflammasome-mediated permeability is decreased when A549 cells and HSAEC are pretreated with IL-1β receptor antagonist (IL-1βRA). In addition, HER2 kinase inhibitor AG825 or NRG-1 inhibitor TAPI inhibits inflammasome-mediated permeability in A549 cells and HSAEC demonstrating critical roles of IL-1β, NRG-1, and HER2 in inflammasome-mediated alveolar permeability. Conclusion: These findings suggest that inflammasome-induced alveolar cell permeability is mediated by NRG-1/HER2 signaling through IL-1β regulation

Dietary supplementation of recombinant antimicrobial peptide Epinephelus lanceolatus piscidin improves growth performance and immune response in Gallus gallus domesticus.

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 μg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals