Synthetic peptide optimization improves the inhibition of dengue NS2B-NS3 protease and dengue replication in vitro.

Dengue virus (DENV) infection is one of the most widely-spread flavivirus infections with no effective antiviral drugs available. Peptide inhibitors have been considered as one of the best drug candidates due to their high specificity, selectivity in their interactions and minimum side effects. In this study, we employed computational studies using YASARA, HADDOCK server and PyMOL software to generate short and linear peptides based on a reference peptide, CP5-46A, to block DENV NS2B-NS3 protease. The inhibition potencies of the peptides were evaluated using in-house DENV2 serine protease and fluorogenic peptide substrates. In vitro analyses were performed to determine the peptides cytotoxicity and the inhibitory effects against DENV2 replication in WRL-68 cells. Our computational analyses revealed that the docking energy of AYA3, a 16 amino acid (aa) (-81.2 ± 10.6 kcal/mol) and AYA9, a 15 aa peptide (-83.8 ± 6.8 kcal/mol) to DENV NS2B-NS3 protease were much lower than the reference peptide (46 aa; -70.9 ± 7.8 kcal/mol) and the standard protease inhibitor, aprotinin (58 aa; -48.2 ± 10.6 kcal/mol). Both peptides showed significant inhibition against DENV2 NS2B-NS3 protease activity with IC50 values of 24 µM and 23 µM, respectively. AYA3 and AYA9 peptides also demonstrated approximately 68% and 83% of viral plaque reduction without significantly affecting cell viability at 50 µM concentration. In short, we generated short linear peptides with lower cytotoxic effect and substantial antiviral activities against DENV2. Further studies are required to investigate the inhibitory effects of these peptides in vivo. Keywords: peptide inhibitors; dengue virus; NS2B-NS3 protease; plaque reduction.

Psychometric properties of the Malay version of motivation scales in drug treatment.

INTRODUCTION: In recognition of the role of motivation in drug use treatment, patient motivational screening instruments are needed for strategic planning and treatment. The aims of this study were to evaluate the reliability and validity of the Malay version of the Treatment Motivation Scale, and to compare the motivational levels of patients receiving substance abuse treatment with different modalities (inpatient vs. outpatient). The motivational scale consists of three scales: problem recognition, desire for help and treatment readiness. METHOD: A convenience sample of 102 patients was recruited from four Cure and Care Service Centres in Malaysia. RESULTS: Principal component analysis with varimax rotation supported two-factor solutions for each subscale: problem recognition, desire for help and treatment readiness, which accounted for 63.5%, 62.7% and 49.1% of the variances, respectively. The Cronbach's alpha coefficients were acceptable for the overall measures (24 items: ∝ = 0.89), the problem recognition scale (10 items; ∝ = 0.89), desire for help (6 items; ∝ = 0.64) and treatment readiness scale (8 items; ∝ = 0.60). The results also indicated significant motivational differences for different modalities, with inpatients having significantly higher motivational scores in each scale compared to outpatients. CONCLUSION: The present study pointed towards the favourable psychometric properties of a motivation for treatment scale, which can be a useful instrument for clinical applications of drug use changes and treatment.

Missed opportunities for earlier HIV-testing in patients with HIV infection referred to a tertiary hospital, a cross-sectional study.

INTRODUCTION: In Malaysia, the prevalence of missed opportunities for HIV-testing is unknown. Missed opportunities have been linked to late diagnosis of HIV and poorer outcome for patients. We describe missed opportunities for earlier HIV-testing in newly-HIV-diagnosed patients. METHODS: Cross sectional study. Adult patients diagnosed with HIV infection and had at least one medical encounter in a primary healthcare setting during three years prior to diagnosis were included. We collected data on sociodemographic characteristics, patient characteristics at diagnosis, HIV-related conditions and whether they were subjected to risk assessment and offered HIV testing during the three years prior to HIV diagnosis. RESULTS: 65 newly HIV-diagnosed patients (male: 92.3%; Malays: 52.4%; single: 66.7%; heterosexual: 41%; homosexual 24.6%; CD4 <350 at diagnosis: 63%). 93.8% were unaware of their HIV status at diagnosis. Up to 56.9% had presented with HIV-related conditions at a primary healthcare facility during the three years prior to diagnosis. Slightly more than half were had risk assessment done and only 33.8% were offered HIV-testing. CONCLUSIONS: Missed opportunities for HIV-testing was unacceptably high with insufficient risk assessment and offering of HIV-testing. Risk assessment must be promoted and primary care physicians must be trained to recognize HIV-related conditions that will prompt them to offer HIVtesting.

Disruption of pocket protein dream complexes by E7 proteins of different types of human papillomaviruses.

It has been shown that the E7 protein of the high-risk HPV-16 transforms cells in vitro and binds pRB, p107 and p130, so called pocket proteins associated in cells with DREAM proteins, while that of the low-risk HPV-6 does not transform cells and binds p130 but not pRB or p107. These facts may indicate that p130 is essential for the HPV life cycle. To gain further insight into the relationship between HPV E7 proteins and pocket protein-DREAM complexes, E7 proteins of HPVs of various risk categories were expressed via appropriate vectors in T98G cells and the levels of various pocket proteins either total or associated with DREAM were analyzed. The obtained results demonstrated that high-risk HPV-16, HPV-18 and HPV-33, low-risk HPV-1 and HPV-11, and cutaneous HPV-48 disrupted pocket protein-DREAM complexes in T98G cells to a similar extent.

Comparison of serum specific IgE with skin prick test in the diagnosis of allergy in Malaysia.

We compared a newer serum specific IgE (SSIgE) test with skin prick testing (SPT) in the diagnosis of allergy in Malaysia. Ninety newly diagnosed allergic patients were enrolled for both tests. Using SPT as a clinical gold standard, the sensitivity, specificity, positive, and negative predictive values (PPV, NPV) were calculated for SSIgE for each of the common allergens tested. The highest positive results for both SPT and SSIgE were for house dust mite and cat. Compared to SPT, SSIgE showed better sensitivity but poorer specificity, low PPV and good NPV in all the allergens tested. Significant positive correlation was seen between the diameter of wheal and flare of SPT and the SSIgE results.

Evaluation of neutralizing antibodies produced by papaya mosaic virus nanoparticles fused to the E2EP3 peptide epitope of Chikungunya envelope.

Chikungunya virus (CHIKV) infection is the cause of acute symptoms and chronic symmetrical polyarthritis associated with long-term morbidity and mortality. Currently, there is no available licensed vaccine or particularly useful drug for human use against CHIKV infection. This study was conducted to evaluate the efficacy of antibodies produced by papaya mosaic virus (PapMV) nanoparticles fused to E2EP3 peptide of CHIKV envelope as a recombinant CHIKV vaccine. PapMV, PapMV-C- E2EP3, and E2EP3-N-PapMV were produced in E. coli with an approximate size of 27 to 30 kDa. ICR mice (5 to 6 weeks of age) were injected subcutaneously with 25 micrograms of vaccine construct, and ELISA measured the titer of CHIKV specific IgG antibodies. The results showed that both recombinant proteins E2EP3-N-PapMV and PapMVC-E2EP3 were able to induce IgG antibodies production in immunized mice against CHIKV while immunization with recombinant PapMV showed no IgG antibodies induction. The neutralizing activity of the antibodies generated by either E2EP3-N-PapMV or PapMV-C-E2EP3 exhibited similar inhibition to CHIKV replication in Vero cells using the cells based antibody neutralizing assay and analyzed by plaque formation assay. This study showed the effectiveness of nanoparticles vaccine generated by fusing epitope peptide of CHIKV envelope to papaya mosaic virus envelope in inducing a robust immune response in mice against CHIKV. The data showed that levels of neutralizing antibodies correlate with a protective immune response CHIKV replication.

Antiviral and virucidal activities of sulphated polysaccharides against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a member of the family Flaviviridae, causes severe neurological disorders in humans. JEV infections represent one of the most widely spread mosquito-borne diseases, and therefore, it has been considered as an endemic disease. An effective antiviral drug is still unavailable to treat JEV, and current drugs only provide supportive treatment to alleviate the symptoms and stabilize patients' conditions. This study was designed to evaluate the antiviral activity of the sulphated polysaccharides "Carrageenan," a linear sulphated polysaccharide that is extracted from red edible seaweeds against JEV replication in vitro. Viral inactivation, attachment, and post-infection assays were used to determine the mode of inhibition of Carrageenan. Virus titters after each application were evaluated by plaque formation assay. MTT assay was used to determine the 50% cytotoxic concentration (CC50), and ELISA-like cell-based assay and immunostaining and immunostaining techniques were used to evaluate the 50% effective concentration (EC50). This study showed that Carrageenan inhibited JEV at an EC50 of 15 µg/mL in a dose-dependent manner with CC50 more than 200 µg/mL in healthy human liver cells (WRL68). The mode of inhibition assay showed that the antiviral effects of Carrageenan are mainly due to their ability to inhibit the early stages of virus infection such as the viral attachment and the cellular entry stages. Our investigation showed that Carrageenan could be considered as a potent antiviral agent to JEV infection. Further experimental and clinical studies are needed to investigate the potential applications of Carrageenan for clinical intervention against JEV infection.

Discovery of small molecule inhibitors against the NS3/4A serine protease of Hepatitis C virus genotype 3 via highthroughput virtual screening and in vitro evaluations.

The hepatitis C virus (HCV) consists of eight genotypes and 90 subtypes, with genotype (GT) 3 being the second most common globally and is linked to higher incidences of steatosis and rapid development of fibrosis and cirrhosis. The NS3/4A serine protease, a heterodimer complex of two HCV non-structural proteins, is an effective target for pharmaceutical intervention due to its essential roles in processing HCV polyproteins and inhibiting innate immunity. This study combines structure-based virtual screening (SBVS) of predefined compound libraries, pharmacokinetic prediction (ADME/T) and in vitro evaluation to identify potential low molecular weight (<500 Dalton) inhibitors of the NS3/4A serine protease (GT3). In silico screening of ZINC and PubChem libraries yielded five selected compounds as potential candidates. Dose-dependent inhibition of the NS3/4A serine protease and HCV replication in HuH-7.5 cells revealed that compound A (PubChem ID No. 16672637) exhibited inhibition towards HCV GT3 with an IC50 of 106.7µM and EC50 of 25.8µM, respectively. Thus, compound A may be developed as a potent, low molecular weight drug against the HCV NS3/4A serine protease of GT3.

The synthetic molecules YK51 and YK73 attenuate replication of dengue virus serotype 2.

Dengue virus infection has been posing alarming economic and social burden on affected nations. It is estimated that 50-100 million dengue infections occur annually with over 2.5 billion people at risk for endemic transmission. In the effort to develop effective antiviral agents, we previously reported potential antiviral activities from selected array of natural products and compounds against dengue virus serotype 2 (DV2). In this study, we report the synthesis of two efficacious novel compounds, YK51 and YK73, and their activities against DV2 replication. Both compounds were chemically synthesised from nicotinic acid using a modified method for the synthesis of dihydropyridine. The products were tested with cell-based assays against DV2 followed by a serine protease assay. As a result, both YK51 and YK73 exhibited intriguing antiviral properties with EC50 of 3.2 and 2.4 µM, respectively. In addition, YK51 and YK73 were found to attenuate the synthesis of intracellular viral RNA and protect the switching of non-classic mechanism of protein translation. These compounds demonstrated inhibitory properties toward the activity of DV2 serine protease in a dose dependent manner. These findings demonstrate that both YK51 and YK73 serve as DV2 serine protease inhibitors that abrogate viral RNA synthesis and translation. Further investigation on these compounds to corroborate its therapeutic properties towards dengue is warranted.

A Unified Framework for Brain Segmentation in MR Images.

Brain MRI segmentation is an important issue for discovering the brain structure and diagnosis of subtle anatomical changes in different brain diseases. However, due to several artifacts brain tissue segmentation remains a challenging task. The aim of this paper is to improve the automatic segmentation of brain into gray matter, white matter, and cerebrospinal fluid in magnetic resonance images (MRI). We proposed an automatic hybrid image segmentation method that integrates the modified statistical expectation-maximization (EM) method and the spatial information combined with support vector machine (SVM). The combined method has more accurate results than what can be achieved with its individual techniques that is demonstrated through experiments on both real data and simulated images. Experiments are carried out on both synthetic and real MRI. The results of proposed technique are evaluated against manual segmentation results and other methods based on real T1-weighted scans from Internet Brain Segmentation Repository (IBSR) and simulated images from BrainWeb. The Kappa index is calculated to assess the performance of the proposed framework relative to the ground truth and expert segmentations. The results demonstrate that the proposed combined method has satisfactory results on both simulated MRI and real brain datasets.

Temperature regulation with neural networks and alternative control schemes.

In this article, we compare the neuro-control algorithm to three other control algorithms: fuzzy logic control, generalized predictive control, and proportional-plus-integral control. Each of these four algorithms is implemented on a water bath temperature control system. The four systems are compared through experimental studies under identical conditions with respect to set-point regulation, the effect of unknown load disturbances, large parameter variation, and variable deadtime in the system. It is found that the neurocontrol system compares well with the other three control systems and offers encouraging advantages. From the results of the experimental studies, however, the best characteristics of each of these different classes of control systems may be combined for realizing a more efficient and intelligent control scheme.

Tuning of a neuro-fuzzy controller by genetic algorithm.

Due to their powerful optimization property, genetic algorithms (GAs) are currently being investigated for the development of adaptive or self-tuning fuzzy logic control systems. This paper presents a neuro-fuzzy logic controller (NFLC) where all of its parameters can be tuned simultaneously by GA. The structure of the controller is based on the radial basis function neural network (RBF) with Gaussian membership functions. The NFLC tuned by GA can somewhat eliminate laborious design steps such as manual tuning of the membership functions and selection of the fuzzy rules. The GA implementation incorporates dynamic crossover and mutation probabilistic rates for faster convergence. A flexible position coding strategy of the NFLC parameters is also implemented to obtain near optimal solutions. The performance of the proposed controller is compared with a conventional fuzzy controller and a PID controller tuned by GA. Simulation results show that the proposed controller offers encouraging advantages and has better performance.

Screening of antiviral activities in medicinal plants extracts against dengue virus using dengue NS2B-NS3 protease assay.

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.

Three dimensional optical imaging of blood volume and oxygenation in the neonatal brain.

Optical methods provide a means of monitoring cerebral oxygenation in newborn infants at risk of brain injury. A 32-channel optical imaging system has been developed with the aim of reconstructing three-dimensional images of regional blood volume and oxygenation. Full image data sets were acquired from 14 out of 24 infants studied; successful images have been reconstructed in 8 of these infants. Regional variations in cerebral blood volume and tissue oxygen saturation are present in healthy preterm infants. In an infant with a large unilateral intraventricular haemorrhage, a corresponding region of low oxygen saturation was detected. These results suggest that optical tomography may provide an appropriate technique for investigating regional cerebral haemodynamics and oxygenation at the cotside.

Isolation of Bifidobacteria infantis and its antagonistic activity against ETEC 0157 and Salmonella typhimurium S-285 in weaning foods.

Probiotic organism Bifidobacteria was isolated from the faeces of breast-fed infants at Universiti Putra Malaysia. Trypticase phytone peptone yeast extract agar (TPY) was used as a selective media for the isolation. Morphological examination of the isolates indicated that Bifidobacteria was Gram-positive rods in nature, curved with characteristics of V and Y shapes. The organisms were non-catalase producing, non-nitrate reducing, non-motile, had an absence of indole and were unable to liquify gelatin. The ratios of acetic and lactic acids were determined using high performance liquid chromatography (HPLC). Using carbohydrate fermentation profile test API-CH-50 kits, 20 Bifidobacteria strains had been identified: they were the species of Bifidobacteria infantis and two different sub-species, mainly infantis and lacentis. Based on a wide zone of inhibition, three suitable strains of B. infantis, Bifi-11, Bifi-19 and Bifi-20, were tested in weaning foods for antimicrobial activity towards two human pathogens: E. coli-0157 (World Health Organization) and Salmonella typhimurium S-285. The pH, titratable acidity of weaning foods and total colony count for Bifidobacteria, enteropathogenic Escherichia coli and S. typhimurium were recorded at 3-h intervals for 30 h. It was found that after 9 h of incubation of weaning foods, the pH declined to < 3.6 from pH 6.0, whereas titratable acidity increased from 0.026 to 0.08%. It was indicated that Bifidobacteria inhibited E. coli better than did S typhimurium due to low pH. After 24 h of incubation, approximately 98% of E. coli was inhibited by Bifidobacteria. It is suggested that the inhibitory effect of Bifidobacteria strains in weaning foods towards the growth of enteropathogenic E. coli and S. typhimurium was solely due to low pH and the production of volatile acid components by the organism.

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro.

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

Effect of ragi and LXXX-lactate-producing cultures on enteric pathogens in a rice-based weaning food.

The use of the Southeast-Asian starter culture ragi in enhancing the safety of a rice-based model weaning food is described and compared with the use of diastatic malt extract. Ragi was shown to be an effective saccharifying agent convenient for use in weaning-food preparation on a domestic scale. Saccharification and fermentation with ragi alone produced some antimicrobial effect against the three enteric bacterial pathogens tested but this was much improved when ragi was used in conjunction with the LXXX-lactate-producing Lactococcus lactis or Lactobacillus bavaricus. The latter showed the greatest inhibition of pathogens, reducing viable numbers by more than a factor of 10(4) within 4 h. The antibacterial effects observed correlated with the total acid produced (ragi alone giving 0.2%; ragi with Lc. lactis giving 0.3% and ragi with Lb. bavaricus giving 0.4%). The proportion of the physiological LXXX-lactate isomer was highest in Lc. lactis fermentations (>99% compared with 80% with Lb. bavaricus). There was no evidence of any pronounced antimicrobial effect due to the nisin produced during fermentation by Lc. lactis (150 IU/g). Whereas bacteriocin production may play little role in pathogen control, it may be desirable as a way of preventing fermentations conducted non-aseptically from becoming dominated by lactic acid bacteria producing unacceptable amounts of DXXX-lactate.