METHODS: TEM, SEM and ATP efflux assay were used to evaluate the effect of hybrid peptides on the integrity of the pneumococcal cell wall/membrane. DNA retardation assay was assessed to measure the impact of hybrid peptides on the migration of genomic DNA through the agarose gel. In vitro synergistic effect was checked using the chequerboard assay. ICR male mice were used to evaluate the in vivo toxicity and antibacterial activity of the hybrid peptides in a standalone form and in combination with ceftriaxone.

RESULTS: The results obtained from TEM and SEM indicated that the hybrid peptides caused significant morphological alterations in Streptococcus pneumoniae and disrupting the integrity of the cell wall/membrane. The rapid release of ATP from pneumococcal cells after one hour of incubation proposing that the antibacterial action for the hybrid peptides is based on membrane permeabilization and damage. The DNA retardation assay revealed that at 62.5 µg/ml all the hybrid peptides were capable of binding and preventing the pneumococcal genomic DNA from migrating through the agarose gel. In vitro synergy was observed when pneumococcal cells treated with combinations of hybrid peptides with each other and with conventional drugs erythromycin and ceftriaxone. The in vivo therapeutic efficacy results revealed that the hybrid peptide RN7-IN8 at 20 mg/kg could improve the survival rate of pneumococcal bacteremia infected mice, as 50% of the infected mice survived up to seven days post-infection. In vivo antibacterial efficacy of the hybrid peptide RN7-IN8 was signficantly improved when combined with the standard antibiotic ceftriaxone at (20 mg/kg + 20 mg/kg) as 100% of the infected mice survived up to seven days post-infection.

MATERIALS AND METHODS: In this experimental study, female ICR mice (6-8 weeks old) were superovulated and cohabited with fertile males for 24 hours. Afterwards, their ovi- ducts were excised and embryos harvested. Embryos at the 2-cell stage were catego- rized as EC embryos, while zygotes with two pronuclei were categorized as LC embryos. Embryos were cultured in M16 medium supplemented with 3% bovine serum albumin (BSA) in a humidified 5% CO2atmosphere. Control embryos were cultured until the blastocyst stage without vitrification. Experimental embryos at the 2-cell stage were vitri- fied for one hour using 40% v/v ethylene glycol, 18% w/v Ficoll-70 and 0.5 M sucrose as the cryoprotectant. We recorded the numbers of surviving embryos from the control and experimental groups and their development until the blastocyst stage. Results were analyzed using the chi-square test.

RESULTS: A significantly higher proportion of EC embryos (96.7%) from the control group developed to the blastocyst stage compared with LC embryos (57.5%, P<0.0001). Similarly, in the experimental group, a significantly higher percentage of vitrified EC embryos (69.4%) reached the blastocyst stage compared to vitrified LC embryos (27.1%, P<0.0001).

AIM: The present study was conducted to investigate the possible mechanism of actions underlying the systemic antinociception activity of the essential oil of Zingiber zerumbet (EOZZ) in chemical-induced nociception tests in mice.

MATERIALS AND METHODS: Acetic acid-induced abdominal constriction, capsaicin-, glutamate- and phorbol 12-myristate 13-acetate-induced paw licking tests in mice were employed in the study. In all experiments, EOZZ was administered systemically at the doses of 50, 100, 200 and 300 mg/kg.

RESULTS: It was shown that EOZZ given to mice via intraperitoneal and oral routes at 50, 100, 200 and 300 mg/kg produced significant dose dependent antinociception when assessed using acetic acid-induced abdominal writing test with calculated mean ID(50) values of 88.84 mg/kg (80.88-97.57 mg/kg) and 118.8 mg/kg (102.5-137.8 mg/kg), respectively. Likewise, intraperitoneal administration of EOZZ at similar doses produced significant dose dependent inhibition of neurogenic pain induced by intraplantar injection of capsaicin (1.6 μg/paw), glutamate (10 μmol/paw) and phorbol 12-myristate 13-acetate (1.6μg/paw) with calculated mean ID(50) of 128.8 mg/kg (118.6-139.9 mg/kg), 124.8 mg/kg (111.4-139.7 mg/kg) and 40.29 (35.39-45.86) mg/kg, respectively. It was also demonstrated that pretreatment with l-arginine (100mg/kg, i.p.), a nitric oxide precursor significantly reversed antinociception produced by EOZZ suggesting the involvement of l-arginine/nitric oxide pathway. In addition, methylene blue (20mg/kg, i.p.) significantly enhanced antinociception produced by EOZZ. Administration of glibenclamide (10mg/kg, i.p.), an ATP-sensitive K(+) channel antagonist significantly reversed antinociceptive activity induced by EOZZ.

AIM OF THE STUDY: The molecular mechanisms of the anti-inflammatory properties of M. accedens are not yet understood. Therefore, we examined those mechanisms using a methanol extract of M. accedens (Ma-ME) and determined the target molecule in macrophages.

MATERIALS AND METHODS: We evaluated the anti-inflammatory effects of Ma-ME in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and in an HCl/EtOH-triggered gastritis model in mice. To investigate the anti-inflammatory activity, we performed a nitric oxide (NO) production assay and ELISA assay for prostaglandin E2 (PGE2). RT-PCR, luciferase gene reporter assays, western blotting analyses, and a cellular thermal shift assay (CETSA) were conducted to identify the mechanism and target molecule of Ma-ME. The phytochemical composition of Ma-ME was analyzed by HPLC and LC-MS/MS.

RESULTS: Ma-ME suppressed the production of NO and PGE2 and the mRNA expression of proinflammatory genes (iNOS, IL-1β, and COX-2) in LPS-stimulated RAW264.7 cells without cytotoxicity. Ma-ME inhibited NF-κB activation by suppressing signaling molecules such as IκBα, Akt, Src, and Syk. Moreover, the CETSA assay revealed that Ma-ME binds to Syk, the most upstream molecule in the NF-κB signal pathway. Oral administration of Ma-ME not only alleviated inflammatory lesions, but also reduced the gene expression of IL-1β and p-Syk in mice with HCl/EtOH-induced gastritis. HPLC and LC-MS/MS analyses confirmed that Ma-ME contains various anti-inflammatory flavonoids, including quercetin, daidzein, and nevadensin.

MATERIALS AND METHODS: In the initiation phase, the mice received a single dose of 100µl/100 µg DMBA (group I-V) or 100µl acetone (group VI) topically on the dorsal shaved skin area followed by the promotion phase involving treatment with the respective test solutions (100 µl of acetone, 10 mg/kg curcumin or MEMM (30, 100 and 300mg/kg)) for 30 min followed by the topical application of tumour promoter (100µl croton oil). Tumors were examined weekly and the experiment lasted for 15 weeks.

RESULTS: MEMM and curcumin significantly (p<0.05) reduced the tumour burden, tumour incidence and tumour volume, which were further supported by the histopathological findings.

MAIN METHODS: A pull-down assay was performed to identify the binding partner of the L-SP40 peptide. Co-immunoprecipitation and co-localization assays with the L-SP40 peptide were employed to confirm the receptor partner in RD cells. The outcomes were validated using receptor knockdown and antibody blocking assays. The L-SP40 peptide was further evaluated for the protection of neonatal mice against lethal challenge by mouse-adapted EV-A71.

KEY FINDINGS: The L-SP40 peptide was found to interact and co-localize with nucleolin, the key attachment receptor of Enteroviruses A species, as demonstrated in the pull-down, co-immunoprecipitation and co-localization assays. Knockdown of nucleolin from RD cells led to a significant reduction of 3.5 logs of viral titer of EV-A71. The L-SP40 peptide demonstrated 80% protection of neonatal mice against lethal challenge by the mouse-adapted virus with a drastic reduction in the viral loads in the blood (~4.5 logs), skeletal muscles (1.5 logs) and brain stem (1.5 logs).