Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.
Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus that has caused multiple unprecedented and re-emerging outbreaks in both tropical and temperate countries. Despite ongoing research efforts, the underlying factors involved in facilitating CHIKV replication during early infection remains ill-characterized. The present study serves to identify host proteins modulated in response to early CHIKV infection using a proteomics approach.
Doxycycline is an antibiotic derived from tetracycline that possesses antimicrobial and anti-inflammatory activities. Antiviral activity of doxycycline against dengue virus has been reported previously; however, its anti-dengue properties need further investigation. This study was conducted to determine the potential activity of doxycycline against dengue virus replication in vitro. Doxycycline inhibited the dengue virus serine protease (DENV2 NS2B-NS3pro) with an IC50 value of 52.3 ± 6.2 μM at 37 °C (normal human temperature) and 26.7 ± 5.3 μM at 40 °C (high fever temperature). The antiviral activity of doxycycline was first tested at different concentrations against DENV2 using a plaque-formation assay. The virus titter decreased significantly after applying doxycycline at levels lower than its 50 % cytotoxic concentration (CC50, 100 μM), showing concentration-dependent inhibition with a 50 % effective concentration (EC50) of approximately 50 μM. Doxycycline significantly inhibited viral entry and post-infection replication of the four dengue serotypes, with serotype-specific inhibition (high activity against DENV2 and DENV4 compared to DENV1 and DENV3). Collectively, these findings underline the need for further experimental and clinical studies on doxycycline, utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue virus infection.
Dengue virus infects millions of people worldwide and there is no vaccine or anti-dengue therapeutic available. Screening large numbers of medicinal plants for anti-dengue activities is an alternative strategy in order to find the potent therapeutic compounds. Therefore, this study was designed to identify anti-dengue activities in nineteen medicinal plant extracts that are used in traditional medicine. Local medicinal plants Vernonia cinerea, Hemigraphis reptans, Hedyotis auricularia, Laurentia longiflora, Tridax procumbers and Senna angustifolia were used in this study. The highest inhibitory activates against dengue NS2B-NS3pro was observed in ethanolic extract of S. angustifolia leaves, methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems. These findings were further verified by in vitro viral inhibition assay. Methanolic extract of V. cinerea leaves, ethanol extract of T. procumbens stems and at less extent ethanolic extract of S. angustifolia leaves were able to maintain the normal morphology of DENV2-infected Vero cells without causing much cytopathic effects (CPE). The percentage of viral inhibition of V. cinerea and T. procumbens extracts were significantly higher than S. angustifolia extract as measured by plaque formation assay and RT-qPCR. In conclusion, The outcome of this study showed that the methanolic extract of V. cinerea leaves and ethanol extract of T. procumbens stems possessed high inhibitory activates against dengue virus that worth more investigation.
Density, viscosity and ionic conductivity data sets of deep eutectic solvents (DESs) formed by tetrabutylammonium bromide (TBABr) paired with ethlyene glycol, 1,3-propanediol, 1,5-pentanediol and glycerol hydrogen bond donors (HBDs) are reported. The properties of DES were measured at temperatures between 303 K and 333 K for HBD percentages of 66.7% to 90%. The effects of HBDs under different temperature and percentages are systematically analyzed. As expected, the measured density and viscosity of the studied DESs decreased with an increase in temperature, while ionic conductivity increases with temperature. In general, DESs made of TBABr and glycerol showed the highest density and viscosity and the lowest ionic conductivity when compared to other DESs. The presence of an extra hydroxyl group on glycerol in a DES affected the properties of the DES.
Chikungunya virus (CHIKV) outbreaks have led to a serious economic burden, as the available treatment strategies can only alleviate disease symptoms, and no effective therapeutics or vaccines are currently available for human use. Here, we report the use of a new cost-effective approach involving production of a recombinant antiviral peptide-fusion protein that is scalable for the treatment of CHIKV infection. A peptide-fusion recombinant protein LATA-PAP1-THAN that was generated by joining Latarcin (LATA) peptide with the N-terminus of the PAP1 antiviral protein, and the Thanatin (THAN) peptide to the C-terminus, was produced in Escherichia coli as inclusion bodies. The antiviral LATA-PAP1-THAN protein showed 89.0% reduction of viral plaque formation compared with PAP1 (46.0%), LATA (67.0%) or THAN (79.3%) peptides alone. The LATA-PAP1-THAN protein reduced the viral RNA load that was 0.89-fold compared with the untreated control cells. We also showed that PAP1 resulted in 0.44-fold reduction, and THAN and LATA resulting in 0.78-fold and 0.73-fold reductions, respectively. The LATA-PAP1-THAN protein inhibited CHIKV replication in the Vero cells at an EC50 of 11.2μg/ml, which is approximately half of the EC50 of PAP1 (23.7μg/ml) and protected the CHIKV-infected mice at the dose of 0.75mg/ml. We concluded that production of antiviral peptide-fusion protein in E. coli as inclusion bodies could accentuate antiviral activities, enhance cellular internalisation, and could reduce product toxicity to host cells and is scalable to epidemic response quantities.
A novel and potent series of ene-amides featuring azetidines has been developed as FabI inhibitors active against drug resistant Gram-positive pathogens particularly staphylococcal organisms. Most of the compounds from the series possessed excellent biochemical inhibition of Staphylococcus aureus FabI enzyme and whole cell activity against clinically relevant MRSA, MSSA and MRSE organisms which are responsible for significant morbidity and mortality in community as well as hospital settings. The binding mode of one of the leads, AEA16, in Escherichia coli FabI enzyme was determined unambiguously using X-ray crystallography. The lead compounds displayed good metabolic stability in mice liver microsomes and pharmacokinetic profile in mice. The in vivo efficacy of lead AEA16 has been demonstrated in a lethal murine systemic infection model.
It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells.
Brain segmentation in magnetic resonance images (MRI) is an important stage in clinical studies for different issues such as diagnosis, analysis, 3-D visualizations for treatment and surgical planning. MR Image segmentation remains a challenging problem in spite of different existing artifacts such as noise, bias field, partial volume effects and complexity of the images. Some of the automatic brain segmentation techniques are complex and some of them are not sufficiently accurate for certain applications. The goal of this paper is proposing an algorithm that is more accurate and less complex).
Platelet rich plasma clot- releasate (PRCR) shows significant influence on tissue regeneration in clinical trials. Although, the mechanism of PRCR effect on fibroblast differentiation has been studied on 2D culture system, a detailed investigation is needed to establish the role of PRCR in cell seeded in 3D scaffolds. Therefore, a study was conducted to evaluate the influence of PRCR in fibroblasts (DFB) differentiation and extracellular matrix formation on both 3D and 2D culture systems. Cell viability was measured using MTT assay and DFB differentiation was evaluated by determining the expression levels of nucleostamin and alpha smooth muscle actin (α-SMA), using indirect immunostaining and Western blotting. The expression levels of extracellular matrix genes (collagen-I, collagen-III, fibronectin and laminin) and focal adhesion formation gene (integrin beta-1) were measured using Real-time PCR. The PRCR at 10% showed significant effect on cells viability compared with 5% and 20% in both culture environments. The decrease in the expression levels of nucleostamin and the increase in α-SMA signify the DFB differentiation to myofibroblast-like cells that was prominently greater in 3D compared to 2D culture. In 3D culture systems, the total collage production, expression levels of the extracellular matrix gene and the focal adhesion gene were increased significantly compared to 2D culture. In conclusion, 3D culture environments enhances the proliferative and differentiation effects of PRCR on DFB, thereby potentially increases the efficacy of DFB for future tissue engineering clinical application.
Plasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.
: The distinctive properties of graphene, characterized by its high carrier mobility and biocompatibility, have stimulated extreme scientific interest as a promising nanomaterial for future nanoelectronic applications. In particular, graphene-based transistors have been developed rapidly and are considered as an option for DNA sensing applications. Recent findings in the field of DNA biosensors have led to a renewed interest in the identification of genetic risk factors associated with complex human diseases for diagnosis of cancers or hereditary diseases. In this paper, an analytical model of graphene-based solution gated field effect transistors (SGFET) is proposed to constitute an important step towards development of DNA biosensors with high sensitivity and selectivity. Inspired by this fact, a novel strategy for a DNA sensor model with capability of single-nucleotide polymorphism detection is proposed and extensively explained. First of all, graphene-based DNA sensor model is optimized using particle swarm optimization algorithm. Based on the sensing mechanism of DNA sensors, detective parameters (Ids and Vgmin) are suggested to facilitate the decision making process. Finally, the behaviour of graphene-based SGFET is predicted in the presence of single-nucleotide polymorphism with an accuracy of more than 98% which guarantees the reliability of the optimized model for any application of the graphene-based DNA sensor. It is expected to achieve the rapid, quick and economical detection of DNA hybridization which could speed up the realization of the next generation of the homecare sensor system.
Viruses belonging to the Flaviviridae family primarily spread through arthropod vectors, and are the major causes of illness and death around the globe. The Flaviviridae family consists of 3 genera which include the Flavivirus genus (type species, yellow fever virus) as the largest genus, the Hepacivirus (type species, hepatitis C virus) and the Pestivirus (type species, bovine virus diarrhea). The flaviviruses (Flavivirus genus) are small RNA viruses transmitted by mosquitoes and ticks that take over host cell machinery in order to propagate. However, hepaciviruses and pestiviruses are not antropod-borne. Despite the extensive research and public health concern associated with flavivirus diseases, to date, there is no specific treatment available for any flavivirus infections, though commercially available vaccines for yellow fever, Japanese encephalitis and tick-born encephalitis exist. Due to the global threat of viral pandemics, there is an urgent need for new drugs. In many countries, patients with severe cases of flavivirus infections are treated only by supportive care, which includes intravenous fluids, hospitalization, respiratory support, and prevention of secondary infections. This review discusses the strategies used towards the discovery of antiviral drugs, focusing on rational drug design against Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Yellow Fever virus (YFV) and Hepatitis C virus (HCV). Only modified peptidic, nonpeptidic, natural compounds and fragment-based inhibitors (typically of mass less than 300 Da) against structural and non-structural proteins are discussed.
In the efforts to find an anti-viral treatment for dengue, a simple tryptophan fluorescence-screening assay aimed at identifying dengue domain III envelope (EIII) protein inhibitors was developed. Residue Trp391 of EIII was used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residue upon binding to a peptide. The analysis was based on the electron excitation at 280 nm and fluorescence emission at 300-400 nm of EIII, followed by quenching of fluorescence in the presence of potential peptidic inhibitors coded DS36wt, DS36opt, DN58wt and DN58opt. The present study found that the fluorescence of the recombinant EIII was quenched following the binding of DS36opt, DN58wt and DN58opt in a concentration-dependent manner. Since the λmax for emission remained unchanged, the effect was not due to a change in the environment of the tryptophan side chain. In contrast, a minimal fluorescence-quenching effect of DS36wt at 20 and 40 µM suggested that the DS36wt does not have any binding ability to EIII. This was supported by a simple native-page gel retardation assay that showed a band shift of EIII domain when incubated with DS36opt, DN58wt and DN58opt but not with DS36wt. We thus developed a low-cost and convenient spectrophotometric binding assay for the analysis of EIII-peptide interactions in a drug screening application.
Although there have been considerable advances in the study of dengue virus, no vaccines or anti-dengue drugs are currently available for humans. Therefore, new approaches are necessary for the development of potent anti-dengue drugs. Natural antimicrobial peptides (AMPs) with potent antiviral activities are potential hits-to-leads for antiviral drug discovery. We performed this study to identify and characterise the inhibitory potential of the latarcin peptide (Ltc 1, SMWSGMWRRKLKKLRNALKKKLKGE) against dengue virus replication in infected cells.
Dengue virus (DENV) broadly disseminates in tropical and sub-tropical countries and there are no vaccine or anti-dengue drugs available. DENV outbreaks cause serious economic burden due to infection complications that requires special medical care and hospitalization. This study presents a new strategy for inexpensive production of anti-DENV peptide-fusion protein to prevent and/or treat DENV infection. Antiviral cationic peptides protegrin-1 (PG1) and plectasin (PLSN) were fused with MAP30 protein to produce recombinant antiviral peptide-fusion protein (PG1-MAP30-PLSN) as inclusion bodies in E. coli. High yield production of PG1-MAP30-PLSN protein was achieved by solubilization of inclusion bodies in alkaline buffer followed by the application of appropriate refolding techniques. Antiviral PG1-MAP30-PLSN protein considerably inhibited DENV protease (NS2B-NS3pro) with half-maximal inhibitory concentration (IC50) 0.5±0.1 μM. The real-time proliferation assay (RTCA) and the end-point proliferation assay (MTT assay) showed that the maximal-nontoxic dose of the peptide-fusion protein against Vero cells is approximately 0.67±0.2 μM. The cell-based assays showed considerable inhibition of the peptide-fusion protein against binding and proliferating stages of DENV2 into the target cells. The peptide-fusion protein protected DENV2-challeged mice with 100% of survival at the dose of 50 mg/kg. In conclusion, producing recombinant antiviral peptide-fusion protein by combining short antiviral peptide with a central protein owning similar activity could be useful to minimize the overall cost of short peptide production and take advantage of its synergistic antiviral activities.
Carbon, in its variety of allotropes, especially graphene and carbon nanotubes (CNTs), holds great potential for applications in variety of sensors because of dangling π-bonds that can react with chemical elements. In spite of their excellent features, carbon nanotubes (CNTs) and graphene have not been fully exploited in the development of the nanoelectronic industry mainly because of poor understanding of the band structure of these allotropes. A mathematical model is proposed with a clear purpose to acquire an analytical understanding of the field-effect-transistor (FET) based gas detection mechanism. The conductance change in the CNT/graphene channel resulting from the chemical reaction between the gas and channel surface molecules is emphasized. NH3 has been used as the prototype gas to be detected by the nanosensor and the corresponding current-voltage (I-V) characteristics of the FET-based sensor are studied. A graphene-based gas sensor model is also developed. The results from graphene and CNT models are compared with the experimental data. A satisfactory agreement, within the uncertainties of the experiments, is obtained. Graphene-based gas sensor exhibits higher conductivity compared to that of CNT-based counterpart for similar ambient conditions.
In this paper, we propose an easy-to-implement passive liquid valve (PLV) for the microfluidic compact-disc (CD). This valve can be implemented by introducing venting chambers to control the air flow of the source and destination chambers. The PLV mechanism is based on equalizing the main forces acting on the microfluidic CD (i.e., the centrifugal and capillary forces) to control the burst frequency of the source chamber liquid. For a better understanding of the physics behind the proposed PLV, an analytical model is described. Moreover, three parameters that control the effectiveness of the proposed valve, i.e., the liquid height, liquid density, and venting chamber position with respect to the CD center, are tested experimentally. To demonstrate the ability of the proposed PLV valve, microfluidic liquid switching and liquid metering are performed. In addition, a Bradford assay is performed to measure the protein concentration and evaluated in comparison to the benchtop procedure. The result shows that the proposed valve can be implemented in any microfluidic process that requires simplicity and accuracy. Moreover, the developed valve increases the flexibility of the centrifugal CD platform for passive control of the liquid flow without the need for an external force or trigger.
Dengue is the current leading cause of death among children in several Latin American and Asian countries. Due to poverty in areas where the disease is prevalent and the high cost of conventional diagnostic systems, low cost devices are needed to reduce the burden caused by dengue infection. Centrifugal microfluidic platforms are an alternative solution to reduce costs and increase the availability of a rapid diagnostic system. The rate of chemical reactions in such devices often depends on the efficiency of the mixing techniques employed in their microfluidic networks. This paper introduces a micromixer that operates by the expansion and contraction of a microballoon to produce a consistent periodical 3D reciprocating flow. We established that microballoons reduced mixing time of 12 μl liquids from 170 min, for diffusional mixing, to less than 23 s. We have also tested the effect of the microballoon mixers on the detection of the dengue virus. The results indicate that employing a microballoon mixer enhances the detection sensitivity of the dengue virus by nearly one order of magnitude compared to the conventional ELISA method.