A series of triclosan mimic diphenyl ether derivatives have been synthesized and evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv. The binding mode of the compounds at the active site of enoyl-acyl carrier protein reductase of M. tuberculosis has been explored. Among them, compound 10b was found to possess antitubercular activity (minimum inhibitory concentration =12.5 µg/mL) comparable to triclosan. All the synthesized compounds exhibited low levels of cytotoxicity against Vero and HepG2 cell lines, and three compounds 10a, 10b, and 10c had a selectivity index more than 10. Compound 10b was also evaluated for log P, pKa, human liver microsomal stability, and % protein binding, in order to probe its druglikeness. Based on the antitubercular activity and druglikeness profile, it may be concluded that compound 10b could be a lead for future development of antitubercular drugs.
The 2-amino-5-(3/4-fluorostyryl)acetophenones were prepared and reacted with benzaldehyde derivatives to afford the corresponding 5-styryl-2-aminochalcone hybrids. The trans geometry of the styryl and α,β-unsaturated carbonyl arms, and the presence of NH…O intramolecular hydrogen bond were validated using 1H-NMR and X-ray data. The 2-amino-5-styrylacetophenones and their 5-styryl-2-aminochalcone derivatives were screened in vitro for their capability to inhibit α-glucosidase and/or α-amylase activities. Their antioxidant properties were evaluated in vitro through the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) free radical scavenging assays. Kinetic studies of the most active derivatives from each series against α-glucosidase and/or α-amylase activities have been performed supported by molecular docking studies to determine plausible protein-ligand interactions on a molecular level. The key aspects of the pharmacokinetics of these compounds, i.e., absorption, distribution, metabolism, and excretion have also been simulated at theoretical level. The most active compounds from each series, namely, 2a and 3e, were evaluated for cytotoxicity against the normal monkey kidney cells (Vero cells) and the adenocarcinomic human epithelial (A549) cell line to establish their safety profile at least in vitro.
The aim of this study is to prepare a model for the production of Newcastle disease virus (NDV) lentogenic F strain using cell culture in bioreactor for live attenuated vaccine preparation. In this study, firstly we investigated the growth of Vero cells in several culture media. The maximum cell number was yielded by culture of Vero cells in Dulbecco's Modified Eagle Medium (DMEM) which was 1.93 x 10(6) cells/ml. Secondly Vero cells were grown in two-litre stirred tank bioreactor by using several commercial microcarriers. We achieved the maximum cell concentration about 7.95 x 10(5) cells/ml when using Cytodex 1. Later we produced Newcastle Disease virus in stirred tank bioreactor based on the design developed using Taguchi L4 method. Results reveal that higher multiplicity of infection (MOI) and size of cell inoculums can yield higher virus titer. Finally, virus samples were purified using high-speed centrifugation based on 3( * *)(3-1) Fractional Factorial Design. Statistical analysis showed that the maximum virus titer can be achieved at virus sample concentration of 58.45% (v/v), centrifugation speed of 13729 rpm, and centrifugation time of 4 hours. As a conclusion, high yield of virus titer could be achieved through optimization of cell culture in bioreactor and separation by high-speed centrifugation.
During 2005, 764 children were brought to a large children's hospital in Ho Chi Minh City, Vietnam, with a diagnosis of hand, foot, and mouth disease. All enrolled children had specimens (vesicle fluid, stool, throat swab) collected for enterovirus isolation by cell culture. An enterovirus was isolated from 411 (53.8%) of the specimens: 173 (42.1%) isolates were identified as human enterovirus 71 (HEV71) and 214 (52.1%) as coxsackievirus A16. Of the identified HEV71 infections, 51 (29.5%) were complicated by acute neurologic disease and 3 (1.7%) were fatal. HEV71 was isolated throughout the year, with a period of higher prevalence in October-November. Phylogenetic analysis of 23 HEV71 isolates showed that during the first half of 2005, viruses belonging to 3 subgenogroups, C1, C4, and a previously undescribed subgenogroup, C5, cocirculated in southern Vietnam. In the second half of the year, viruses belonging to subgenogroup C5 predominated during a period of higher HEV71 activity.
The use of nontoxic biological compounds in the synthesis of nanomaterials is an economic and eco-friendly approach. The present work was undertaken to develop zinc oxide nanoparticles (ZnO-NPs) by a green method using simple precursor from the solution consisting of zinc acetate and the flower extract of Anchusa italica (A. italica). Effect of annealing temperature on structural and antimicrobial properties was investigated. The crystalline structure of ZnO-NPs was shown using X-ray diffraction (XRD) analysis. Transmission electron microscopy (TEM) results showed that ZnO-NPs are hexagonal in shapes with mean particle size of ~8 and ~14nm at 100°C and 200°C annealing temperatures respectively. The optical band gap was increased from 3.27eV to 3.30eV with the decreasing of the particle size. The antimicrobial activity of ZnO-NPs towards Gram positive (Bacillus megaterium and Stapphylococcus aureus) and Gram negative (Escherichia coli and Salmonella typhimurium) pathogens decreased with the increasing of the heat treating temperature. In vitro cytotoxicity studies on Vero cells, a dose dependent toxicity with non-toxic effect of concentration below 142μg/mL was shown. The results indicated that A. italica is an appropriate reaction media to prepare ZnO-NPs for cosmetic and bio-medical productions.
Dengue virus (DENV) is considered to be the most important arthropod-borne viral disease and causes more than 100 million human infections annually. To further characterize primary DENV infection in vivo, rhesus macaques were infected with DENV-1, DENV-2, DENV-3, or DENV-4 and clinical parameters, as well as specificity and longevity of serologic responses, were assessed. Overt clinical symptoms were not present after infection. However, abnormalities in blood biochemical parameters consistent with heart, kidney, and liver damage were observed, and changes in plasma fibrinogen, D-dimers, and protein C indicated systemic activation of the blood coagulation pathway. Significant homotypic and heterotypic serum immunoglobulins were present in all animals, and IgG persisted for at least 390 days. Serum neutralizing antibody responses were highly serotype specific by day 120. However, some heterotypic neutralizing activity was noted in infected animals. Identification of serotype-specific host responses may help elucidate mechanisms that mediate severe DENV disease after reinfection.
Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
A new resveratrol dimer, acuminatol (1), was isolated along with five known compounds from the acetone extract of the stem bark of Shorea acuminata. Their structures and stereochemistry were determined by spectroscopic methods, which included the extensive use of 2D NMR techniques. All isolated compounds were evaluated for their antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (RSA) and the β-carotene-linoleic acid (BCLA) assays, and compared with those of the standards of ascorbic acid (AscA) and butylated hydroxytoluene (BHT). All compounds tested exhibited good to moderate antioxidant activity in the DPPH assay (IC₅₀s 0.84 to 10.06 mM) and displayed strong inhibition of β-carotene oxidation (IC₅₀s 0.10 to 0.22 mM). The isolated compounds were evaluated on the Vero cell line and were found to be non-cytotoxic with LC₅₀ values between 161 to 830 µM.
Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 µM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.
Thymoquinone (TQ), the active constituent of Nigella sativa or black cumin exhibited cytotoxic effects in several cancer cell lines. In this study, the cytotoxicity of TQ in human cervical squamous carcinoma cells (SiHa) was investigated. TQ was cytotoxic towards SiHa cells with IC50 values of 10.67 ± 0.12 and 9.33 ± 0.19 μg/mL as determined by MTT assay and trypan blue dye exclusion test, respectively, after 72 h of incubation. TQ was more cytotoxic towards SiHa cells compared to cisplatin. Interestingly, TQ was less cytotoxic towards the normal cells (3T3-L1 and Vero). Cell cycle analysis performed by flowcytometer showed a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Apoptosis induction by TQ was further confirmed by Annexin V/PI and AO/PI staining. Significant elevation of p53 and down-regulation of the anti-apoptotic Bcl-2 protein was found in the treated cells, without any changes in the expression of the pro-apoptotic Bax protein. In conclusion, thymoquinone from N. sativa was more potent than cisplatin in elimination of SiHa cells via apoptosis with down-regulation of Bcl-2 protein.
Syzygium aqueum, a species in the Myrtaceae family, commonly called the water jambu is native to Malaysia and Indonesia. It is well documented as a medicinal plant, and various parts of the tree have been used in traditional medicine, for instance as an antibiotic. In this study, we show S. aqueum leaf extracts to have a significant composition of phenolic compounds, protective activity against free radicals as well as low pro-oxidant capability. Its ethanolic extract, in particular, is characterized by its excellent radical scavenging activity of EC(50) of 133 μg mL(-1) 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 65 μg mL(-1) 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 71 μg mL(-1) (Galvinoxyl), low pro-oxidant capabilities and a phenolic content of 585-670 mg GAE g(-1) extract. The extract also displayed other activities, deeming it an ideal cosmetic ingredient. A substantial tyrosinase inhibition activity with an IC(50) of about 60 μg mL(-1) was observed. In addition, the extract was also found to have anti-cellulite activity tested for its ability to cause 98% activation of lipolysis of adipocytes (fat cells) at a concentration of 25 μg mL(-1). In addition, the extract was not cytotoxic to Vero cell lines up to a concentration of 600 μg mL(-1). Although various parts of this plant have been used in traditional medicine, this is the first time it has been shown to have cosmeceutical properties. Therefore, the use of this extract, alone or in combination with other active principles, is of interest to the cosmetic industry.
Xanthorrhizol is a natural sesquiterpenoid compound isolated from the rhizome of Curcuma xanthorrhizza Roxb (Zingerberaceae). Recent studies of xanthorrhizol in cell cultures strongly support the role of xanthorrhizol as an antiproliferative agent. In our study, we tested the antiproliferative effect of xanthorrhizol using different breast cancer cell lines. The invasive breast cancer cell line, MDA-MB-231, was then selected for further investigations. Treatment with xanthorrhizol caused 50% growth inhibition on MDA-MB-231 cells at 8.67 +/- 0.79 microg/ml as determined by sulforhodamine B (SRB) assay. Hoechst 33258 nuclear staining assay showed the rate of apoptosis of MDA-MB-231 cells to increase in response to xanthorrhizol treatment. Immunofluorescence staining using antibody MitoCapture and fluorescein isothiocyanate (FITC)-labeled cytochrome c revealed the possibility of altered mitochondrial transmembrane potential and the release of cytochrome c respectively. This was further confirmed by Western-blotting, where cytochrome c was showed to migrate from mitochondrial fraction to the cytosol fraction of treated MDA-MB-231 cells. Caspase activity assay showed the involvement of caspase-3 and caspase-9, but not caspase-6 or caspase-8 in MDA-MB-231 apoptotic cell death. Subsequently, cleavage of PARP-1 protein is suggested. These data suggest treatment with xanthorrhizol modulates MDA-MB-231 cell apoptosis through the mitochondria-mediated pathway subsequent to the disruption of mitochondrial transmembrane potential, release of cytochrome c, activation of caspase-3 and caspase-9, and the modulation of PARP-1 protein.
Xanthorrhizol is a sesquiterpenoid compound extracted from the rhizome of Curcuma xanthorrhiza. This study investigated the antiproliferative effect and the mechanism of action of xanthorrhizol on human hepatoma cells, HepG2, and the mode of cell death. An antiproliferative assay using methylene blue staining revealed that xanthorrhizol inhibited the proliferation of the HepG2 cells with a 50% inhibition of cell growth (IC50) value of 4.17 +/- 0.053 microg/ml. The antiproliferative activity of xanthorrhizol was due to apoptosis induced in the HepG2 cells and not necrosis, which was confirmed by the Tdt-mediated dUTP nick end labeling (TUNEL) assay. The xanthorrhizol-treated HepG2 cells showed typical apoptotic morphology such as DNA fragmentation, cell shrinkage and elongated lamellipodia. The apoptosis mediated by xanthorrhizol in the HepG2 cells was associated with the activation of tumor suppressor p53 and down-regulation of antiapoptotic Bcl-2 protein expression, but not Bax. The levels of Bcl-2 protein expression decreased 24-h after treatment with xanthorrhizol and remained lower than controls throughout the experiment, resulting in a shift in the Bax to Bcl-2 ratio thus favouring apoptosis. The processing of the initiator procaspase-9 was detected. Caspase-3 was also found to be activated, but not caspase-7. Xanthorrhizol exerts antiproliferative effects on HepG2 cells by inducing apoptosis via the mitochondrial pathway.
In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.
Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.
The common physical and chemical methods for controlling rat pest are less than satisfactory and inhumane. Immunocontraception approach has been considered more humane and it can be accomplished by inducing the relevant host immune response that block further development of reproductive gametes. ZP3 proteins are known to play very important role during sperm-ovum fertilization. It is a self-antigen and only localized in female ovaries. Therefore, an immunization with ZP3 protein elsewhere will induce a generalize host immune response against ZP3 protein. This study employed rat ZP3 (rZP3) gene prepared from its cDNA of Rattus rattus diardii. It was delivered and expressed in vivo by naked plamid DNA (DrZP3) or recombinant ZP3-Adenovirus (Ad-rZP3). Expression studies in vitro with DrZP3 or Ad-ZP3 showed rZP3 proteins were successfully expressed in Vero cells. Hyperimmune serum against rZP3 that were prepared by immunizing several rats with purified rZP3-pichia yeast fusion protein showed it blocked sperms from binding DrZP3-transfected Vero cells. Female Sprague Dawley rats immunized with DrZP3 demonstrated a long-term effect for significant reduction of fertility up to 92.6%. Ovaries from rats immunized with DrZP3 were severely atrophied with disappearance of primordial follicles from ovarian cortex with an increased in the amount of oocyte-free cell clusters. Female rats immunized with Ad-rZP3 demonstrated 27% reduction of fertility. The infertility induced by Ad-rZP3 is comparatively low and ineffective. This could be due to a strong host immune response that suppresses the recombinant virus itself resulted in minimum rZP3 protein presentation to the host immune system. As a result, low antibody titers produced against rZP3 is insufficient to block oocytes from maturity and fertilization. Therefore, immunization with DrZP3 for immunocontraception is more effective than Ad-rZP3 recombinant adenovirus. It is proposed to explore further on the use of adenovirus or other alternative viruses to deliver ZP3 protein and for the development of enhanced expression of rZP3 in target host.
Natural polymer guar gum has one of the highest viscosities in water solution and hence, these are significantly used in pharmaceutical applications. Guar gum inter-connected micelles as a new carrier has been developed for poor water soluble rifampicin drug. The hydrogel inter-connected micelle core was formulated as a hydrophilic inner and hydrophobic outer core by using guar gum/chitosan/polycaprolactone and the carrier interaction with rifampicin was confirmed by FT-IR. The morphological observations were carried out through TEM, SEM and AFM analysis. The encapsulation efficiency and in-vitro drug release behavior of prepared hydrogel based micelle system was analyzed by UV-vis spectrometry. The anti-bacterial activity against K. pneumoniae and S. aureus was studied by observing their ruptured surface by SEM. The cytotoxicity study reveals that the pure polymeric system has no toxic effect whereas drug loaded ones showed superior activity against THP-1 cells. From the cell apoptosis analyses, the apoptosis was carried out in a time dependent manner. The cell uptake behavior was also observed in THP-1 cells which indicate that the hydrogel based micelle system is an excellent material for the mucoadhesive on intracellular alveolar macrophage treatment.
The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.