Global seroprevalence of Zika virus in asymptomatic individuals: A systematic review.

BACKGROUND: Zika virus (ZIKV) has spread to five of the six World Health Organization (WHO) regions. Given the substantial number of asymptomatic infections and clinical presentations resembling those of other arboviruses, estimating the true burden of ZIKV infections is both challenging and essential. Therefore, we conducted a systematic review and meta-analysis of seroprevalence studies of ZIKV IgG in asymptomatic population to estimate its global impact and distribution. METHODOLOGY/PRINCIPAL FINDINGS: We conducted extensive searches and compiled a collection of articles published from Jan/01/2000, to Jul/31/2023, from Embase, Pubmed, SciELO, and Scopus databases. The random effects model was used to pool prevalences, reported with their 95% confidence interval (CI), a tool to assess the risk of study bias in prevalence studies, and the I2 method for heterogeneity (PROSPERO registration No. CRD42023442227). Eighty-four studies from 49 countries/territories, with a diversity of study designs and serological tests were included. The global seroprevalence of ZIKV was 21.0% (95%CI 16.1%-26.4%). Evidence of IgG antibodies was identified in all WHO regions, except for Europe. Seroprevalence correlated with the epidemics in the Americas (39.9%, 95%CI:30.0-49.9), and in some Western Pacific countries (15.6%, 95%CI:8.2-24.9), as well as with recent and past circulation in Southeast Asia (22.8%, 95%CI:16.5-29.7), particularly in Thailand. Additionally, sustained low circulation was observed in Africa (8.4%, 95%CI:4.8-12.9), except for Gabon (43.7%), and Burkina Faso (22.8%). Although no autochthonous transmission was identified in the Eastern Mediterranean, a seroprevalence of 16.0% was recorded. CONCLUSIONS/SIGNIFICANCE: The study highlights the high heterogeneity and gaps in the distribution of seroprevalence. The implementation of standardized protocols and the development of tests with high specificity are essential for ensuring a valid comparison between studies. Equally crucial are vector surveillance and control methods to reduce the risk of emerging and re-emerging ZIKV outbreaks, whether caused by Ae. aegypti or Ae. albopictus or by the Asian or African ZIKV.

Occurrence and mechanisms of tigecycline resistance in carbapenem- and colistin-resistant Klebsiella pneumoniae in Thailand.

Tigecycline has been regarded as one of the most important last-resort antibiotics for the treatment of infections caused by extensively drug-resistant (XDR) bacteria, particularly carbapenem- and colistin-resistant Klebsiella pneumoniae (C-C-RKP). However, reports on tigecycline resistance have been growing. Overall, ~ 4000 K. pneumoniae clinical isolates were collected over a five-year period (2017-2021), in which 240 isolates of C-C-RKP were investigated. Most of these isolates (91.7%) were resistant to tigecycline. Notably, a high-risk clone of ST16 was predominantly identified, which was associated with the co-harboring of blaNDM-1 and blaOXA-232 genes. Their major mechanism of tigecycline resistance was the overexpression of efflux pump acrB gene and its regulator RamA, which was caused by mutations in RamR (M184V, Y59C, I141T, A28T, C99/C100 insertion), in RamR binding site (PI) of ramA gene (C139T), in MarR (S82G), and/or in AcrR (L154R, R13Q). Interestingly, four isolates of ST147 carried the mutated tet(A) efflux pump gene. To our knowledge, this is the first report on the prevalence and mechanisms of tigecycline resistance in C-C-RKP isolated from Thailand. The high incidence of tigecycline resistance observed among C-C-RKP in this study reflects an ongoing evolution of XDR bacteria against the last-resort antibiotics, which demands urgent action.

Synergistic effect of two antimicrobial peptides, BP203 and MAP-0403 J-2 with conventional antibiotics against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates.

Drug-resistant Enterobacterales infections are a great health concern due to the lack of effective treatments. Consequently, finding novel antimicrobials or combining therapies becomes a crucial approach in addressing this problem. BP203 and MAP-0403 J-2, novel antimicrobial peptides, have exhibited effectiveness against Gram-negative bacteria. In this study, we assessed the in vitro antibacterial activity of BP203 and MAP-0403 J-2, along with their synergistic interaction with conventional antibiotics including colistin, rifampicin, chloramphenicol, ceftazidime, meropenem, and ciprofloxacin against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. The minimal inhibitory concentrations (MIC) of BP203 and MAP-0403 J-2 against tested E. coli isolates were 2-16 and 8-32 µg/mL, respectively. However, for the majority of K. pneumoniae isolates, the MIC of BP203 and MAP-0403 J-2 were >128 µg/mL. Notably, our results demonstrated a synergistic effect when combining BP203 with rifampicin, meropenem, or chloramphenicol, primarily observed in most K. pneumoniae isolates. In contrast, no synergism was evident between BP203 and colistin, chloramphenicol, ceftazidime, rifampicin, or ciprofloxacin when tested against all E. coli isolates. Furthermore, synergistic effects between MAP-0403 J-2 and rifampicin, ceftazidime or colistin were observed against the majority of E. coli isolates. Similarly, the combined effect of MAP-0403 J-2 with rifampicin or chloramphenicol was synergistic in the majority of K. pneumoniae isolates. Importantly, these peptides displayed the stability at high temperatures, across a wide range of pH values, in specific serum concentrations and under physiological salt conditions. Both peptides also showed no significant hemolysis and cytotoxicity against mammalian cells. Our findings suggested that BP203 and MAP-0403 J-2 are promising candidates against colistin-resistant E. coli. Meanwhile, the synergism of these peptides and certain antibiotics could be of great therapeutic value as antimicrobial drugs against infections caused by colistin-resistant E. coli and K. pneumoniae.

Emerging and re-emerging zoonotic viral diseases in Southeast Asia: One Health challenge.

The ongoing significant social, environmental, and economic changes in Southeast Asia (SEA) make the region highly vulnerable to the emergence and re-emergence of zoonotic viral diseases. In the last century, SEA has faced major viral outbreaks with great health and economic impact, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), arboviruses, highly pathogenic avian influenza (H5N1), and Severe Acute Respiratory Syndrome (SARS-CoV); and so far, imported cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Given the recent challenging experiences in addressing emerging zoonotic diseases, it is necessary to redouble efforts to effectively implement the "One Health" initiative in the region, which aims to strengthen the human-animal-plant-environment interface to better prevent, detect and respond to health threats while promoting sustainable development. This review provides an overview of important emerging and re-emerging zoonotic viral diseases in SEA, with emphasis on the main drivers behind their emergency, the epidemiological situation from January 2000 to October 2022, and the importance of One Health to promote improved intervention strategies.

Co-occurrence of mcr-2 and mcr-3 genes on chromosome of multidrug-resistant Escherichia coli isolated from healthy individuals in Thailand.

The aim of this study was to characterize three strains of colistin-resistant E. coli isolated from feces samples of healthy individuals in Thailand. The three strains, namely, SY_EC03, SY_EC07, and SY_EC10 were identified as ST165, ST1602, and ST34. All isolates exhibited multidrug-resistant phenotype, which is mediated by accumulation of various antimicrobial resistance genes. SY_EC03 contained mcr-1.1 while SY_EC07 co-harbored mcr-2.3 and mcr-3.4, and SY_EC10 co-harbored mcr-1.1 and mcr-3.5. Genomic analysis revealed that mcr-1.1 of the two strains were located on IncI2 plasmid with genetic environment of ISApl1-mcr-1.1-PAP2, which is a composite transposon Tn6330 with single-ended. Regarding mcr-2.3, the gene was identified within the composite transposon of ISKpn71-mcr-2.3-ISSpu2-ISKpn71, which was located on a novel mobile genetic element (MGE) that was integrated into the chromosome by phage integrase. For mcr-3.4 and mcr-3.5, the genes were confirmed to locate on the chromosome by S1-PFGE/DNA hybridization. Hence, to the best of our knowledge, this is the first report on co-occurrence of mcr-2 and mcr-3 on chromosome of E. coli. More interestingly, mcr-2 was found to locate on a novel MGE, which had never been described. In addition, we also report the co-occurrence of plasmidic mcr-1.1 and chromosomal mcr-3.5 which is extremely rare. Since all these bacteria were isolated from healthy individuals and the identified STs have been found in a variety of origins, all these clones may serve as reservoir for horizontal and vertical transmission of mcr genes. Strategic action plans to control and prevent the spread of mcr genes are urgently needed.

Emergence of plasmid-mediated colistin resistance mcr-3.5 gene in Citrobacter amalonaticus and Citrobacter sedlakii isolated from healthy individual in Thailand.

Citrobacter spp. are Gram-negative bacteria commonly found in environments and intestinal tracts of humans and animals. They are generally susceptible to third-generation cephalosporins, carbapenems and colistin. However, several antibiotic resistant genes have been increasingly reported in Citrobacter spp., which leads to the postulation that Citrobacter spp. could potentially be a reservoir for spreading of antimicrobial resistant genes. In this study, we characterized two colistin-resistant Citrobacter spp. isolated from the feces of a healthy individual in Thailand. Based on MALDI-TOF and ribosomal multilocus sequence typing, both strains were identified as Citrobacter sedlakii and Citrobacter amalonaticus. Genomic analysis and S1-nuclease pulsed field gel electrophoresis/DNA hybridization revealed that Citrobacter sedlakii and Citrobacter amalonaticus harbored mcr-3.5 gene on pSY_CS01 and pSY_CA01 plasmids, respectively. Both plasmids belonged to IncFII(pCoo) replicon type, contained the same genetic context (Tn3-IS1-ΔTnAs2-mcr-3.5-dgkA-IS91) and exhibited high transferring frequencies ranging from 1.03×10-4 - 4.6×10-4 CFU/recipient cell Escherichia coli J53. Colistin-MICs of transconjugants increased ≥ 16-fold suggesting that mcr-3.5 on these plasmids can be expressed in other species. However, beside mcr, other major antimicrobial resistant determinants in multidrug resistant Enterobacterales were not found in these two isolates. These findings indicate that mcr gene continued to evolve in the absence of antibiotics selective pressure. Our results also support the hypothesis that Citrobacter could be a reservoir for spreading of antimicrobial resistant genes. To the best of our knowledge, this is the first report that discovered human-derived Citrobacter spp. that harbored mcr but no other major antimicrobial resistant determinants. Also, this is the first report that described the presence of mcr gene in C. sedlakii and mcr-3 in C. amalonaticus.

Chikungunya and Zika Viruses: Co-Circulation and the Interplay between Viral Proteins and Host Factors.

Chikungunya and Zika viruses, both transmitted by mosquito vectors, have globally re-emerged over for the last 60 years and resulted in crucial social and economic concerns. Presently, there is no specific antiviral agent or vaccine against these debilitating viruses. Understanding viral-host interactions is needed to develop targeted therapeutics. However, there is presently limited information in this area. In this review, we start with the updated virology and replication cycle of each virus. Transmission by similar mosquito vectors, frequent co-circulation, and occurrence of co-infection are summarized. Finally, the targeted host proteins/factors used by the viruses are discussed. There is an urgent need to better understand the virus-host interactions that will facilitate antiviral drug development and thus reduce the global burden of infections caused by arboviruses.

Urinary PCA3 detection in prostate cancer by magnetic nanoparticles coupled with colorimetric enzyme-linked oligonucleotide assay.

PCA3 is one of the most prostate cancer-specific genes described to date. Of note, PCA3 expression is detectable at high level in the urine of prostate cancer (PCa) patients. Accordingly, PCA3 is an ideal biomarker for PCa diagnosis. Several techniques for the measurement of this biomarker in urine have been developed but there are still some drawbacks. In this study, magnetic nanoparticle-based PCR coupled with streptavidin-horseradish peroxidase and a substrate for colorimetric detection was established as a potential assay for urinary PCA3 detection. The method provided a high specificity for PCA3 gene in LNCaP prostate cancer cell line. Additionally, this technique could detect PCA3 at femtogram level which was approximately 1,000-fold more sensitive than the conventional RT-PCR followed by agarose gel electrophoresis. The effectiveness of the method was assessed by PCA3 detection in clinical specimens. The relative PCA3 expression of PCa patients determined by this assay was significantly greater than that of benign prostatic hyperplasia (BPH) patients and healthy controls. The results of our test were comparable with the results of qRT-PCR. The proposed method is promising to distinguish between cancerous and non-cancerous groups. Altogether, this simple assay is practicable and useful for prostate cancer diagnosis.

Interferon-inducible protein (IFI) 16 regulates Chikungunya and Zika virus infection in human skin fibroblasts.

Chikungunya virus (CHIKV), a re-emerging infectious arbovirus, causes Chikungunya fever that is characterized by fever, skin rash, joint pain, arthralgia and occasionally death. Despite it has been described for 66 years already, neither potential vaccine nor a specific drug is available yet. During CHIKV infection, interferon type I signaling pathway is stimulated and releases hundreds of interferon stimulated genes (ISGs). Our previous study reported that IFI16, a member of ISGs, is up-regulated during CHIKV virus infection and the suppression of the gene resulted in increased virus replication. Furthermore, our group also found that inflammasome activation can inhibit CHIKV infection in human foreskin cells (HFF1). Concomitantly, it has been reported that IFI16 activates the inflammasome to suppress virus infection. Therefore, we have hypothesized that IFI16 could be involved in CHIKV infection. In this study, we confirmed the expression level of IFI16 by Western blotting analysis and found that IFI16 was up-regulated following CHIKV infection in both HFF1 and human embryonic kidney cells. We next investigated its antiviral activity and found that forced expression of IFI16 completely restricted CHIKV infection while endogenous silencing of the gene markedly increased virus replication. Furthermore, we have discovered that IFI16 inhibited CHIKV replication, at least, in cell-to-cell transmission as well as the diffusion step. Interestingly, IFI16 also exerted its antiviral activity against Zika virus (ZIKV) infection, the global threat re-emerging virus can cause microcephaly in humans. Taken together, this study provides the first evidence of an antivirus activity of IFI16 during in vitro arbovirus infection, thus expanding its antiviral spectrum that paves the way to further development of antiviral drugs and vaccines.

Production and Characterization of Recombinant Wild Type Uricase from Indonesian Coelacanth (L. menadoensis) and Improvement of Its Thermostability by In Silico Rational Design and Disulphide Bridges Engineering.

The ideal therapeutic uricase (UOX) is expected to have the following properties; high expression level, high activity, high thermostability, high solubility and low immunogenicity. The latter property is believed to depend largely on sequence identity to the deduced human UOX (dH-UOX). Herein, we explored L. menadoensis uricase (LM-UOX) and found that it has 65% sequence identity to dH-UOX, 68% to the therapeutic chimeric porcine-baboon UOX (PBC) and 70% to the resurrected ancient mammal UOX. To study its biochemical properties, recombinant LM-UOX was produced in E. coli and purified to more than 95% homogeneity. The enzyme had specific activity up to 10.45 unit/mg, which was about 2-fold higher than that of the PBC. One-litre culture yielded purified protein up to 132 mg. Based on homology modelling, we successfully engineered I27C/N289C mutant, which was proven to contain inter-subunit disulphide bridges. The mutant had similar specific activity and production yield to that of wild type (WT) but its thermostability was dramatically improved. Up on storage at -20 °C and 4 °C, the mutant retained ~100% activity for at least 60 days. By keeping at 37 °C, the mutant retained ~100% activity for 15 days, which was 120-fold longer than that of the wild type. Thus, the I27C/N289C mutant has potential to be developed for treatment of hyperuricemia.

Colorimetric detection of PCA3 in urine for prostate cancer diagnosis using thiol-labeled PCR primer and unmodified gold nanoparticles.

BACKGROUND: PCA3, a non-coding RNA, has been approved as a potential urinary biomarker for prostate cancer. However, PCA3 urine tests have some limitations. Therefore, we developed a colorimetric method for PCA3 detection in urine. METHODS: The assay was based on interactions between unmodified gold nanoparticles (AuNPs) and thiolated PCR products. Thiolated PCR products were amplified by RT-PCR using a thiol-labeled primer at the 5' end. Thiolated products of PCA3 bound to the surface of AuNPs and led to the prevention of salt-induced aggregation (red color). In the absence of the PCR products, AuNPs changed their color from red to blue due to the salt-induced aggregation. These changes were detected by the naked eye and spectrophotometer. RESULTS: Our assay was specific for PCA3 in prostate cancer cell lines with a visual detection limit of 31.25 ng/reaction. The absorption ratio 520/640 nm was linear against PCR product concentration (R2 = 0.9798) in the reaction. This method is promising for discrimination of prostate cancer patients from both healthy controls and benign prostatic hyperplasia patients according to their urinary PCA3 expression levels. CONCLUSIONS: This study established a simple, rapid, sensitive and specific assay for PCA3 detection which may be applicable for prostate cancer diagnosis.

Development of sarcosine quantification in urine based on enzyme-coupled colorimetric method for prostate cancer diagnosis.

An enzyme-coupled colorimetric assay for quantification of urinary sarcosine was developed. The proposed method is a specific reaction based on hydrogen peroxide (H2O2) formation via sarcosine oxidase (SOX). The liberated H2O2 reacts with Amplex Red in the presence of horseradish peroxidase (HRP) to produce the red-fluorescent oxidation product, resorufin, which can be measured spectrophotometrically (OD570). The method was performed in the 96-well microtiter plate. Reaction conditions, such as pH and reaction time were optimized. At the optimum conditions, the limit of detection (LOD) and quantification (LOQ) were found to be 0.7 and 1 µM, respectively. A good linearity was revealed with a coefficient of 0.990. The assay showed no significant interference from ascorbic acid, glucose and bilirubin. In addition, it is extremely specific for sarcosine rather than other amino acids. The determination of sarcosine in human urine displayed high accuracy and good reproducibility. This method is promising to differentiate prostate cancer patients from healthy subjects according to urinary sarcosine level. Altogether, this study provides a rapid, simple and specific tool to determine urinary sarcosine which could be useful for prostate cancer diagnosis.

Prevalence of the colistin resistance gene mcr-1 in colistin-resistant Escherichia coli and Klebsiella pneumoniae isolated from humans in Thailand.

OBJECTIVES: Historically, colistin has been considered a last-line therapeutic option against multidrug-resistant Gram-negative bacterial infections. However, chromosomally-encoded and plasmid-mediated colistin resistance is increasingly being reported worldwide. Spread of the plasmid-borne colistin resistance gene mcr-1 is of great concern since it can be transferred between bacteria. The aim of this study was to investigate the prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae collected from human clinical specimens in Thailand during 2014-2017. METHODS: Minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution method for 317 non-duplicate Enterobacteriaceae clinical isolates (37 E. coli and 280 K. pneumoniae). All isolates were screened for the mcr-1 gene by PCR. RESULTS: The colistin MIC50, MIC90 and MIC range for the 37 E. coli isolates were 0.5, 8 and 0.5-32mg/L, respectively. The mcr-1 gene was detected in 11 E. coli isolates (29.7%). Escherichia coli harbouring the mcr-1 gene had a colistin MIC range of 4-32mg/L. The colistin MIC50, MIC90, and MIC range for the 280 K. pneumoniae isolates were 32, >128, and 0.25 to >128mg/L, respectively. The mcr-1 gene was detected in 4 K. pneumoniae isolates (1.4%). Klebsiella pneumoniae harbouring the mcr-1 gene had a colistin MIC range of 4-64mg/L. CONCLUSIONS: This is the first report on the prevalence of the mcr-1 gene in colistin-resistant E. coli and K. pneumoniae isolated from humans in Thailand. These data provide added insight into the mechanism of colistin resistance among Enterobacteriaceae pathogens.

Evaluation of the Rapid Polymyxin NP test for detection of colistin susceptibility in Enterobacteriaceae isolated from Thai patients.

Development and evaluations of the Rapid Polymyxin NP test for detection of colistin resistance in Enterobacteriaceae have been recently reported. In this study, we evaluated the performance of the test using a larger number of Enterobacteriaceae, and a larger proportion of isolates with a colistin MIC close to the breakpoint. Out of 339 isolates, the Rapid Polymyxin NP test detected colistin resistance in 13 isolates of Escherichia coli, 213 isolates of Klebsiella pneumoniae, 9 isolates of Enterobacter aerogenes, and 10 isolates of the other Enterobacteriaceae species. Sensitivity and specificity of the test for detecting colistin resistance were 100% and 95.9%, respectively. Positive predictive value and negative predictive value were 98.3% and 100%, respectively.

Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.

Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions.

Engineering of a novel tri-functional enzyme with MnSOD, catalase and cell-permeable activities.

Cooperative function of superoxide dismutase (SOD) and catalase (CAT), in protection against oxidative stress, is known to be more effective than the action of either single enzyme. Chemical conjugation of the two enzymes resulted in molecules with higher antioxidant activity and therapeutic efficacy. However, chemical methods holds several drawbacks; e.g., loss of enzymatic activity, low homogeneity, time-consuming, and the need of chemical residues removal. Yet, the conjugated enzymes have never been proven to internalize into target cells. In this study, by employing genetic and protein engineering technologies, we reported designing and production of a bi-functional protein with SOD and CAT activities for the first time. To enable cellular internalization, cell penetrating peptide from HIV-1 Tat (TAT) was incorporated. Co-expression of CAT-MnSOD and MnSOD-TAT fusion genes allowed simultaneous self-assembly of the protein sequences into a large protein complex, which is expected to contained one tetrameric structure of CAT, four tetrameric structures of MnSOD and twelve units of TAT. The protein showed cellular internalization and superior protection against paraquat-induced cell death as compared to either complex bi-functional protein without TAT or to native enzymes fused with TAT. This study not only provided an alternative strategy to produce multifunctional protein complex, but also gained an insight into the development of therapeutic agent against oxidative stress-related conditions.

Enhancement of Solubility and Specific Activity of a Cu/Zn Superoxide Dismutase by Co-expression with a Copper Chaperone in Escherichia coli.

BACKGROUND: Human Cu/Zn superoxide dismutase (hSOD1) is an antioxidant enzyme with potential as a therapeutic agent. However, heterologous expression of hSOD1 has remained an issue due to Cu2+ insufficiency at protein active site, leading to low solubility and enzymatic activity. OBJECTIVES: The effect of co-expressed human copper chaperone (hCCS) to enhance the solubility and enzymatic activity of hSOD1 in E. coli was investigated in the presence and absence of Cu2+. MATERIALS AND METHODS: pETDuet-1-hSOD1 and pETDuet-1-hCCS-hSOD1 were constructed and individually transformed into E. coli strain BL21(DE3). The recombinant hSOD1 was expressed and purified using immobilized metal affinity chromatography. The yield and specific activity of hSOD1 in all conditions were studied. RESULTS: Co-expression with hCCS increased hSOD1 solubility at 37°C, but this effect was not observed at 25°C. Notably, the specific activity of hSOD1 was enhanced by 1.5 fold and greater than 3 fold when co-expressed with hCCS at 25°C with and without Cu2+ supplement, respectively. However, the chaperone co-expression did not significantly increase the yield of hSOD1 comparable to the expression of hSOD1 alone. CONCLUSIONS: This study is the first report demonstrating a potential use of hCCS for heterologous production of hSOD1 with high enzymatic activity.

Engineering of chimeric catalase-Angiopep-2 for intracellular protection of brain endothelial cells against oxidative stress.

Blood-brain barrier (BBB) disruption and brain microvascular endothelial cells (BMVECs) death caused by excessive production of hydrogen peroxide (H2O2) have been implicated in several neurological conditions. To overcome this problem, H2O2-degrading enzyme with ability to enter the BMVECs is required. In the present study, genetic fusion of gene encoding human catalase and gene encoding Angiopep-2 (AP2), a brain targeting peptide, was performed. The fusion protein was successfully expressed in Escherichia coli and purified to homogeneity. The protein retained heme content and specific enzymatic activity in the same order of magnitude as that of native enzyme. Study of the BMVECs internalization showed that 0.1µM of the fusion protein can enter the cell within 15min, while internalization of the native protein was not observed at this condition. In addition, treatment of the BMVECs with 20 units of the fusion protein for 30min showed protection against H2O2 up to 5.0mM, whereas this protective effect was not observed from treatment with the native protein. Therefore, construction of chimeric human catalase and AP2 provides an insight into the development of potential therapeutic antioxidant with ability to penetrate the BBB for protection against neurodegenerative disorders.

Engineering of a novel chimera of superoxide dismutase and Vitreoscilla hemoglobin for rapid detoxification of reactive oxygen species.

The genes encoding human manganese superoxide dismutase (MnSOD) and Vitreoscilla hemoglobin (VHb) were fused in-frame to generate a bifunctional enzyme that possessed MnSOD and peroxidase-like activities. At neutral pH, the coupling of the SOD and peroxidase reactions revealed that the bifunctional enzyme exhibited a 2.5 times shorter transient period and a 1.67 times higher reaction rate at steady-state conditions. Furthermore, the catalytic rate of the bifunctional enzyme was not affected as much by the external H2O2 scavenger catalase. This indicates that the bifunctional protein possesses a greater antioxidant capability, which is possibly due to the close proximity between the active site of MnSOD and the heme moiety of VHb. Our findings not only provide insight into the synergistic functions of SOD and peroxidase but also could potentially be used to develop novel therapeutic agents with more efficient O2 carrying capability.

Proteomic alterations of Escherichia coli by paraquat.

Paraquat (PQ; a widely used herbicide) exerts its harmful effect to human, mammals and microorganisms upon intracellular conversion to superoxide radical. Cellular responses against toxic paraquat remain not fully understood, especially on the adaptive metabolic changes as a consequence of oxidative burden. In this study, alterations of metabolic processes of Escherichia coli (E. coli) by paraquat were systematically investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF). In host cells, the first line mechanism was scrutinized by a remarkable induction of endogenous superoxide dismutase (E. coli SOD). The second line involved in the metabolic adaptation and compensation for energy production by up- or down-regulation of the enzymes implicated in glycolysis and tricarboxylic acid cycle. Notably, down-regulation of aconitase enzyme and changes of enzyme isoform from the acidic (pI~5.29) to the higher basidic form (pI~5.59) were detected. Meanwhile, up-regulation of fumarase approximately 4-5 folds were observed. Importantly, overexpression of human manganese superoxide dismutase (human Mn-SOD) in E. coli cells could in turn down-regulate the expression of fumarase enzyme. This observation was not found when the cells expressing human catalase were tested. Other mechanisms such as changes of purine nucleoside phosphorylase and protein transporters (D-ribose-binding protein and oligopeptide binding protein) were also accounted. However, among all the differentially expressed proteins, the fumarase enzyme is evidenced to be a major target responsible for superoxide-generating paraquat, which may further be applied as a potential biomarker for paraquat toxicity in the future.