Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Elevation of Cleaved p18 Bax Levels Associated with the Kinetics of Neuronal Cell Death during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

Deciphering critical amino acid residues to modify and enhance the binding affinity of ankyrin scaffold specific to capsid protein of human immunodeficiency virus type 1.

BACKGROUND: AnkGAG1D4 is an artificial ankyrin repeat protein which recognizes the capsid protein (CA) of the human immunodeficiency virus type 1 (HIV-1) and exhibits the intracellular antiviral activity on the viral assembly process. Improving the binding affinity of AnkGAG1D4 would potentially enhance the AnkGAG1D4-mediated antiviral activity. OBJECTIVE: To augment the affinity of AnkGAG1D4 scaffold towards its CA target, through computational predictions and experimental designs. METHOD: Three dimensional structure of the binary complex formed by AnkGAG1D4 docked to the CA was used as a model for van der Waals (vdW) binding energy calculation. The results generated a simple guideline to select the amino acids for modifications. Following the predictions, modified AnkGAG1D4 proteins were produced and further evaluated for their CA-binding activity, using ELISA-modified method and bio-layer interferometry (BLI). RESULTS: Tyrosine at position 56 (Y56) in AnkGAG1D4 was experimentally identified as the most critical residue for CA binding. Rational substitutions of this residue diminished the binding affinity. However, vdW calculation preconized to substitute serine for tyrosine at position 45. Remarkably, the affinity for the viral CA was significantly enhanced in AnkGAG1D4-S45Y mutant, with no alteration of the target specificity. CONCLUSIONS: The S-to-Y mutation at position 45, based on the prediction of interacting amino acids and on vdW binding energy calculation, resulted in a significant enhancement of the affinity of AnkGAG1D4 ankyrin for its CA target. AnkGAG1D4-S45Y mutant represented the starting point for further construction of variants with even higher affinity towards the viral CA, and higher therapeutic potential in the future.

AnkPlex: algorithmic structure for refinement of near-native ankyrin-protein docking.

BACKGROUND: Computational analysis of protein-protein interaction provided the crucial information to increase the binding affinity without a change in basic conformation. Several docking programs were used to predict the near-native poses of the protein-protein complex in 10 top-rankings. The universal criteria for discriminating the near-native pose are not available since there are several classes of recognition protein. Currently, the explicit criteria for identifying the near-native pose of ankyrin-protein complexes (APKs) have not been reported yet. RESULTS: In this study, we established an ensemble computational model for discriminating the near-native docking pose of APKs named "AnkPlex". A dataset of APKs was generated from seven X-ray APKs, which consisted of 3 internal domains, using the reliable docking tool ZDOCK. The dataset was composed of 669 and 44,334 near-native and non-near-native poses, respectively, and it was used to generate eleven informative features. Subsequently, a re-scoring rank was generated by AnkPlex using a combination of a decision tree algorithm and logistic regression. AnkPlex achieved superior efficiency with ≥1 near-native complexes in the 10 top-rankings for nine X-ray complexes compared to ZDOCK, which only obtained six X-ray complexes. In addition, feature analysis demonstrated that the van der Waals feature was the dominant near-native pose out of the potential ankyrin-protein docking poses. CONCLUSION: The AnkPlex model achieved a success at predicting near-native docking poses and led to the discovery of informative characteristics that could further improve our understanding of the ankyrin-protein complex. Our computational study could be useful for predicting the near-native poses of binding proteins and desired targets, especially for ankyrin-protein complexes. The AnkPlex web server is freely accessible at http://ankplex.ams.cmu.ac.th .

Antiviral effect of melatonin on Japanese encephalitis virus infection involves inhibition of neuronal apoptosis and neuroinflammation in SH-SY5Y cells.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes high mortality rates in humans and it is the most clinically important and common cause of viral encephalitis in Asia. To date, there is no specific treatment for JEV infection. Melatonin, a neurotropic hormone, is reported to be effective in combating various bacterial and viral infections. However, the effects of melatonin on JEV infection have not yet been studied. The investigation tested the antiviral effects of melatonin against JEV infection and elucidated the possible molecular mechanisms of inhibition. Melatonin inhibited the viral production in JEV-infected SH-SY5Y cells in a time- and dose-dependent manner. Time-of-addition assays demonstrated a potent inhibitory effect of melatonin at the post-entry stage of viral replication. Molecular docking analysis revealed that melatonin negatively affected viral replication by interfering with physiological function and/or enzymatic activity of both JEV nonstructural 3 (NS3) and NS5 protein, suggesting a possible underlying mechanism of JEV replication inhibition. Moreover, treatment with melatonin reduced neuronal apoptosis and inhibited neuroinflammation induced by JEV infection. The present findings reveal a new property of melatonin as a potential molecule for the further development of anti-JEV agents and treatment of JEV infection.

Nano-Delivery System of Ethanolic Extract of Propolis Targeting Mycobacterium tuberculosis via Aptamer-Modified-Niosomes.

Tuberculosis (TB) therapy requires long-course multidrug regimens leading to the emergence of drug-resistant TB and increased public health burden worldwide. As the treatment strategy is more challenging, seeking a potent non-antibiotic agent has been raised. Propolis serve as a natural source of bioactive molecules. It has been evidenced to eliminate various microbial pathogens including Mycobacterium tuberculosis (Mtb). In this study, we fabricated the niosome-based drug delivery platform for ethanolic extract of propolis (EEP) using thin film hydration method with Ag85A aptamer surface modification (Apt-PEGNio/EEP) to target Mtb. Physicochemical characterization of PEGNio/EEP indicated approximately -20 mV of zeta potential, 180 nm of spherical nanoparticles, 80% of entrapment efficiency, and the sustained release profile. The Apt-PEGNio/EEP and PEGNio/EEP showed no difference in these characteristics. The chemical composition in the nanostructure was confirmed by Fourier transform infrared spectrometry. Apt-PEGNio/EEP showed specific binding to Mycobacterium expressing Ag85 membrane-bound protein by confocal laser scanning microscope. It strongly inhibited Mtb in vitro and exhibited non-toxicity on alveolar macrophages. These findings indicate that the Apt-PEGNio/EEP acts as an antimycobacterial nanoparticle and might be a promising innovative targeted treatment. Further application of this smart nano-delivery system will lead to effective TB management.

Seroprevalence of anti-SARS-CoV-2 antibodies in Thai adults during the first three epidemic waves.

This study determined the presence of anti-SARS-CoV-2 antibodies in 4964 individuals, comprising 300 coronavirus disease-19 (COVID-19) prepandemic serum samples, 142 COVID-19 patients, 2113 individuals at risk due to their occupations, 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, and 553 Thai citizens returning after spending extended periods of time in countries with a high disease prevalence. We recruited participants between May 2020 and May 2021, which spanned the first two epidemic waves and part of the third wave of the COVID-19 outbreaks in Thailand. Their sera were tested in a microneutralization and a chemiluminescence immunoassay for IgG against the N protein. Furthermore, we performed an immunofluorescence assay to resolve discordant results between the two assays. None of the prepandemic sera contained anti-SARS-CoV-2 antibodies, while antibodies developed in 88% (15 of 17) of the COVID-19 patients at 8-14 days and in 94-100% of the patients between 15 and 60 days after disease onset. Neutralizing antibodies persisted for at least 8 months, longer than IgG antibodies. Of the 2113 individuals at risk due to their occupation, none of the health providers, airport officers, or public transport drivers were seropositive, while antibodies were present in 0.44% of entertainment workers. Among the 1856 individuals at risk due to sharing workplaces or communities with COVID-19 patients, seropositivity was present in 1.9, 1.5, and 7.5% of the Bangkok residents during the three epidemic waves, respectively, and in 1.3% of the Chiang Mai people during the first epidemic wave. The antibody prevalence varied between 6.5 and 47.0% in 553 Thai people returning from high-risk countries. This serosurveillance study found a low infection rate of SARS-CoV-2 in Thailand before the emergence of the Delta variant in late May 2021. The findings support the Ministry of Public Health's data, which are based on numbers of patients and contact tracing.

Potentiality of Melittin-Loaded Niosomal Vesicles Against Vancomycin-Intermediate Staphylococcus aureus and Staphylococcal Skin Infection.

BACKGROUND: Staphylococcus aureus is an important human pathogen, especially causing skin and soft tissue infections (SSTIs). Over the decades, the infections caused by antibiotic-resistant strains have often become life-threatening. Consequently, exploration and development of competent approaches to combat these serious circumstances are urgently required. METHODS: The antibacterial activity of melittin (Mel) on S. aureus, methicillin-resistant S. aureus (MRSA) and clinical isolates of vancomycin-intermediate S. aureus (VISA) was investigated by minimum inhibitory concentration (MIC) and time-killing assays. The localization of Mel on the bacterial cell was visualized by confocal laser scanning microscopy and its effect on the membrane was indicated based on propidium iodide uptake. The non-ionic surfactant vesicle (NISV) or niosome nanocarrier was established for Mel loading (Mel-loaded NISV) by the thin-film hydration method. Physicochemical and in vitro biological properties of Mel-loaded NISVs were characterized. The cellular uptake of Mel-loaded NISVs was evaluated by holotomography analysis. In addition, an ex vivo study was conducted on a porcine ear skin model to assess the permeation ability of Mel-loaded NISVs and their potential to inhibit bacterial skin infection. RESULTS: The effective inhibitory activity of Mel on skin pathogens was demonstrated. Among the tested strains, VISA was most susceptible to Mel. Regarding to its function, Mel targeted the bacterial cell envelope and disrupted cell membrane integrity. Mel-loaded NISVs were successfully fabricated with a nano-size of 120-200 nm and entrapment efficiency of greater than 90%. Moreover, Mel-loaded NISVs were taken up and accumulated in the intracellular space. Meanwhile, Mel was released and distributed throughout the cytosol and nucleus. Mel-loaded NISVs efficiently inhibited the growth of bacteria, particularly MRSA and VISA. Importantly, they not only penetrated epidermal and dermal skin layers, but also reduced the bacterial growth in infected skin. CONCLUSION: Mel-loaded NISVs have a great potential to exhibit antibacterial activity. Therapeutic application of Mel-loaded NISVs could be further developed as an alternative platform for the treatment of skin infection via dermal and transdermal delivery.

Baculovirus display of single chain antibody (scFv) using a novel signal peptide.

BACKGROUND: Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication. RESULTS: Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6. CONCLUSION: Expression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.

Improved adenovirus type 5 vector-mediated transduction of resistant cells by piggybacking on coxsackie B-adenovirus receptor-pseudotyped baculovirus.

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.

Melittin from Apis florea Venom as a Promising Therapeutic Agent for Skin Cancer Treatment.

Melittin, a major component found in bee venom, is produced by the Apis species of the honey bee. In this study, the effect of melittin derived from Apis florea (Mel-AF), which is a wild honey bee species that is indigenous to Thailand, was investigated against human malignant melanoma (A375) cells. In this study, Mel-AF exhibited considerable potential in the anti-proliferative action of A375 cells. Subsequently, the cellular mechanism of Mel-AF that induced cell death was investigated in terms of apoptosis. As a result, gene and protein expression levels, which indicated the activation of cytochrome-c release and caspase-9 expression, eventually triggered the release of the caspase-3 executioner upon Mel-AF. We then determined that apoptosis-mediated cell death was carried out through the intrinsic mitochondrial pathway. Moreover, advanced abilities, including cell motility and invasion, were significantly suppressed. Mel-AF manipulated the actin arrangement via the trapping of stress fibers that were found underneath the membrane, which resulted in the defective actin cytoskeleton organization. Consequently, the expression of EGFR, a binding protein to F-actin, was also found to be suppressed. This outcome strongly supports the effects of Mel-AF in the inhibition of progressive malignant activity through the disruption of actin cytoskeleton-EGFR interaction and the EGFR signaling system. Thus, the findings of our current study indicate the potential usefulness of Mel-AF in cancer treatments as an apoptosis inducer and a potential actin-targeting agent.

Alterations in Mitochondrial Dynamic-related Genes in the Peripheral Blood of Alzheimer's Disease Patients.

BACKGROUND: Mitochondrial dysfunction is a pathological feature that manifests early in the brains of patients with Alzheimer's Disease (AD). The disruption of mitochondrial dynamics contributes to mitochondrial morphological and functional impairments. Our previous study demonstrated that the expression of genes involved in amyloid beta generation was altered in the peripheral blood of AD patients. OBJECTIVE: The aim of this study was to further investigate the relative levels of mitochondrial genes involved in mitochondrial dynamics, including mitochondrial fission and fusion, and mitophagy in peripheral blood samples from patients with AD compared to healthy controls. METHODS: The mRNA levels were analyzed by real-time polymerase chain reaction. Gene expression profiles were assessed in relation to cognitive performance. RESULTS: Significant changes were observed in the mRNA expression levels of fission-related genes; Fission1 (FIS1) levels in AD subjects were significantly higher than those in healthy controls, whereas Dynamin- related protein 1 (DRP1) expression was significantly lower in AD subjects. The levels of the mitophagy-related genes, PTEN-induced kinase 1 (PINK1) and microtubule-associated protein 1 light chain 3 (LC3), were significantly increased in AD subjects and elderly controls compared to healthy young controls. The mRNA levels of Parkin (PARK2) were significantly decreased in AD. Correlations were found between the expression levels of FIS1, DRP1 and PARK2 and cognitive performance scores. CONCLUSION: Alterations in mitochondrial dynamics in the blood may reflect impairments in mitochondrial functions in the central and peripheral tissues of AD patients. Mitochondrial fission, together with mitophagy gene profiles, might be potential considerations for the future development of blood-based biomarkers for AD.

Unveiling the Properties of Thai Stingless Bee Propolis via Diminishing Cell Wall-Associated Cryptococcal Melanin and Enhancing the Fungicidal Activity of Macrophages.

Cryptococcus neoformans, a life-threatening human yeast pathogen, has the ability to produce melanin, which is one of the common virulence factors contributing to cryptococcal pathogenesis. This virulence factor is closely associated with the cryptococcal cell wall, specifically chitin and chitosan polysaccharides, a complex structure that is essential for maintaining cellular structure and integrity. In this study, we aim to investigate the effects of two stingless bee (SLB) propolis from Tetragonula laeviceps and Tetrigona melanoleuca against cell wall-associated melanin in C. neoformans, and its immune response in RAW 264.7 macrophage. The ethanolic extract of SLB propolis (EEP) has strongly exhibited anti-cryptococcal activity. Moreover, EEP from both sources reduced chitin/chitosan and melanin production against C. neoformans in a dose-dependent manner. Likewise, the mRNA expression level of CDA1, IPC1-PKC1 and LAC1 genes involved in the cryptococcal melanization pathway was significantly decreased at 2 mg/mL in EEP treatment. Additionally, pretreatment with EEP prior to yeast infection dramatically reduced intracellular replication of C. neoformans in RAW 264.7 macrophages in a dose-dependent manner. This study might be a new insight to use a natural powerful source, not only acting to target cell wall-associated molecules, but also being capable to explore a novel strategy by which dysregulation of these molecules leads to promote immunomodulatory activity.

Alterations in the Expression of Amyloid Precursor Protein Cleaving Enzymes mRNA in Alzheimer Peripheral Blood.

BACKGROUND: Alzheimer's disease (AD) is the most common cause of dementia in elderly populations. Changes in the expression of the Amyloid Precursor Protein (APP)-cleaving enzymes directly affect the formation of Amyloid Beta (Aß) plaques, a neuropathological hallmark of AD. OBJECTIVE: We used peripheral blood from AD patients to investigate the expression of genes related to APP-processing [(ß-site APP-cleaving enzyme 1 (BACE1), presenilin1 (PSEN1), and a disintegrin and metalloproteinase family 10 (ADAM10) and 17 (ADAM17)] and the epigenetic genes sirtuin (SIRT)1-3, which regulate Aß production. METHOD: Real-time polymerase chain reactions were performed to determine the specific mRNA levels in plasma. The mRNA levels in AD patients were compared to those in healthy persons and assessed in relation to the subjects' cognitive performance. RESULTS: BACE1 mRNA level in AD subjects was significantly higher than those of healthy controls, whereas ADAM10 level was significantly lower in the AD subjects. The SIRT1 level was significantly decreased, while that of SIRT2 was increased in AD subjects and elderly controls compared to levels in healthy young control. In addition, correlations were found between the expression levels of BACE1, ADAM10 and SIRT1 and cognitive performance scores. Total Aß (Aß40+Aß42) levels and the Aß40/Aß42 ratio were significantly increased in the AD subjects, whereas decrease in plasma Aß42 was found in AD subjects. There was a negative correlation between Aß40 or total Aß and Thai Mental State Examination (TMSE) while there was no correlation between Aß40/Aß42 ratio or Aß42 and TMSE. CONCLUSION: The present findings provide evidence and support for the potential roles of these enzymes that drive Aß synthesis and for epigenetic regulation in AD progression and development, which can possibly be considered peripheral markers of AD.

Remodeling of the Actin Network Associated with the Non-Structural Protein 1 (NS1) of West Nile Virus and Formation of NS1-Containing Tunneling Nanotubes.

The cellular response to the recombinant NS1 protein of West Nile virus (NS1WNV) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1WNV: (i) the exogenous secreted form, sNS1WNV, added to the extracellular milieu; and (ii) the endogenous NS1WNV, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1WNV varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1WNV to the cell culture medium resulted in significant remodeling of the actin filament network in Vero E6 cells. This effect was not observed in SH-SY5Y and U-87MG cells, implying that the cellular uptake of sNS1WNV and actin network remodeling were dependent on cell type. In the three cell types, NS1WNV-expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1WNV proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1WNV and the viral envelope glycoprotein EWNV were also observed in WNV-infected Vero E6 cells.

Current Peptide and Protein Candidates Challenging HIV Therapy beyond the Vaccine Era.

Human immunodeficiency virus (HIV) is a causative agent of acquired immune deficiency syndrome (AIDS). Highly active antiretroviral therapy (HAART) can slow down the replication of HIV-1, leading to an improvement in the survival of HIV-1-infected patients. However, drug toxicities and poor drug administration has led to the emergence of a drug-resistant strain. HIV-1 immunotherapy has been continuously developed, but antibody therapy and HIV vaccines take time to improve its efficiency and have limitations. HIV-1-specific chimeric antigen receptor (CAR)-based immunotherapy founded on neutralizing antibodies is now being developed. In HIV-1 therapy, anti-HIV chimeric antigen receptors showed promising data in the suppression of HIV-1 replication; however, autologous transfusion is still a problem. This has led to the development of effective peptides and proteins for an alternative HIV-1 treatment. In this paper, we provide a comprehensive review of potent anti-HIV-1 peptides and proteins that reveal promising therapeutic activities. The inhibitory mechanisms of each therapeutic molecule in the different stages of the HIV-1 life cycle will be discussed herein.

High-fat diet-induced plasma protein and liver changes in obese rats can be attenuated by melatonin supplementation.

Obesity triggers changes in protein expression in various organs that might participate in the pathogenesis of obesity. Melatonin has been reported to prevent or attenuate such pathological protein changes in several chronic diseases. However, such melatonin effects on plasma proteins have not yet been studied in an obesity model. Using a proteomic approach, we investigated the effect of melatonin on plasma protein profiles after rats were fed a high-fat diet (HFD) to induce obesity. We hypothesized that melatonin would attenuate abnormal protein expression in obese rats. After 10weeks of the HFD, animals displayed increased body weight and fat accumulation as well as increased glucose levels, indicating an obesity-induced prediabetes mellitus-like state. Two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry revealed 12 proteins whose expression was altered in response to the HFD and the melatonin treatment. The altered proteins are related to the development of liver pathology, such as cirrhosis (α1-antiproteinase), thrombosis (fibrinogen, plasminogen), and inflammation (mannose-binding protein A, complement C4, complement factor B), contributing to liver steatosis or hepatic cell death. Melatonin treatment most probably reduced the severity of the HFD-induced obesity by reducing the amplitude of HFD-induced plasma protein changes. In conclusion, we identified several potential biomarkers associated with the progression of obesity and its complications, such as liver damage. Furthermore, our findings reveal melatonin's beneficial effect of attenuating plasma protein changes and liver pathogenesis in obese rats.

Expedient screening for HIV-1 protease inhibitors using a simplified immunochromatographic assay.

A colloidal gold-based immunochromatographic (IC) strip test was developed and validated for the detection of HIV-1 protease (HIV-PR) activity and inhibitory effect of HIV-PR inhibitors (PIs). It is a unique 'two-step' process requiring the combination of proteolysis of HIV-PR and an immunochromatographic reaction. Monoclonal antibodies to the free C-terminus of HIV matrix protein (HIV-MA) conjugated to gold particles and a monoclonal antibody against intact and cleaved forms of the HIV-MA are immobilized on the 'Test'-line of the IC strip. Using lopinavir, a potent HIV protease inhibitor, the IC-strip was optimized to detect inhibitory activity against HIV-protease. At a lopinavir concentration of 1000ng/mL (its suggested minimum effective concentration), a HIV-PRH6 concentration of 6mg/mL and incubation period of 60min were the optimal conditions. A preliminary comparison between a validated high-performance liquid chromatography assay and the IC-strip to semi-quantify HIV protease inhibitor concentrations (lopinavir and atazanavir) demonstrated good agreement. This simplified method is suitable for the rapid screening of novel protease inhibitors for future therapeutic use. Moreover, the IC strip could also be optimized to semi-quantify PIs concentrations in plasma samples.

A drug discovery platform: a simplified immunoassay for analyzing HIV protease activity.

Although numerous methods for the determination of HIV protease (HIV-PR) activity have been described, new high-throughput assays are required for clinical and pharmaceutical applications due to the occurrence of resistant strains. In this study, a simple enzymatic immunoassay to identify HIV-PR activity was developed based on a Ni(2+)-immobilized His(6)-Matrix-Capsid substrate (H(6)MA-CA) is cleaved by HIV protease-His(6) (HIV-PRH(6)) which removes the CA domain and exposes the free C terminus of MA. Following this cleavage, two monoclonal antibodies specific for either the free C-terminal MA or CA epitope are used to quantify the proteolytic activity using a standard ELISA-based system. Specificity for detection of the HIV-PRH(6) activity was confirmed with addition of protease inhibitor (PI), lopinavir. In addition, the assay was able to detect an HIV-PR variant activity indicating that this assay is capable of assessing viral mutation affect HIV-PR activity. The efficacy of commercially available PIs and their 50% inhibitory concentration (IC(50)) were determined. This assay provides a high-throughput method for both validating the efficiency of new drugs in vitro and facilitating the discovery of new PIs. In addition, it could serve as a method for examining the influence of various mutations in HIV-PRs isolated from drug-resistant strains.

Pairwise decomposition of residue interaction energies of single chain Fv with HIV-1 p17 epitope variants.

Computational assisted modeling was carried out to investigate the importance of specific residues in the binding site of scFv. In this study, scFv against HIV-1 epitope at the C-terminal on p17 (scFv anti-p17) was used as a candidate molecule for evaluating the method. The wild-type p17 and its nine natural mutants were docked with scFv anti-p17. Potential mean force (PMF) scores predicted the most favorable binding interaction, and the correlation agreed well with the corresponding activity data from the peptide based ELISA. In the interaction with solvent molecules, the 3D structures of scFv anti-p17 and selected peptide epitopes were further investigated by molecular dynamics (MDs) simulation with the AMBER 9 program. Post-processing of the snapshot at equilibrium was performed to evaluate the binding free energy and pairwise decomposition or residue-based energy calculation of complexes in solution using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) protocol. Our results demonstrated that the specific residues located in the complementary determining regions (CDRs) of scFv anti-p17, MET100, LYS101, ASN169, HIS228, and LEU229, play a crucial role in the effective binding interaction with the absolute relative decomposed energy more than 2.00 kcal/mol in comparison to the original substrate.