• Biomolecules & Therapeutics
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• v.31 no.3
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• pp.253-263
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• 2023

The biogenesis and biological roles of extracellular vesicles (EVs) in the progression of liver diseases have attracted considerable attention in recent years. EVs are membrane-bound nanosized vesicles found in different types of body fluids and contain various bioactive materials, including proteins, lipids, nucleic acids, and mitochondrial DNA. Based on their origin and biogenesis, EVs can be classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are the smallest EVs (30-150 nm in diameter), which play a significant role in cell-to-cell communication and epigenetic regulation. Moreover, exosomal content analysis can reveal the functional state of the parental cell. Therefore, exosomes can be applied to various purposes, including disease diagnosis and treatment, drug delivery, cell-free vaccines, and regenerative medicine. However, exosome-related research faces two major limitations: isolation of exosomes with high yield and purity and distinction of exosomes from other EVs (especially microvesicles). No standardized exosome isolation method has been established to date; however, various exosome isolation strategies have been proposed to investigate their biological roles. Exosome-mediated intercellular communications are known to be involved in alcoholic liver disease and nonalcoholic fatty liver disease development. Damaged hepatocytes or nonparenchymal cells release large numbers of exosomes that promote the progression of inflammation and fibrogenesis through interactions with neighboring cells. Exosomes are expected to provide insight on the progression of liver disease. Here, we review the biogenesis of exosomes, exosome isolation techniques, and biological roles of exosomes in alcoholic liver disease and nonalcoholic fatty liver disease.

• Genomics & Informatics
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• v.21 no.3
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• pp.42.1-42.23
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• 2023

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the most deadly infections in humans. The emergence of multidrug-resistant and extensively drug-resistant Mtb strains presents a global challenge. Mtb has shown resistance to many frontline antibiotics, including rifampicin, kanamycin, isoniazid, and capreomycin. The only licensed vaccine, Bacille Calmette-Guerin, does not efficiently protect against adult pulmonary tuberculosis. Therefore, it is urgently necessary to develop new vaccines to prevent infections caused by these strains. We used a subtractive proteomics approach on 23 virulent Mtb strains and identified a conserved membrane protein (MmpL4, NP_214964.1) as both a potential drug target and vaccine candidate. MmpL4 is a non-homologous essential protein in the host and is involved in the pathogen-specific pathway. Furthermore, MmpL4 shows no homology with anti-targets and has limited homology to human gut microflora, potentially reducing the likelihood of adverse effects and cross-reactivity if therapeutics specific to this protein are developed. Subsequently, we constructed a highly soluble, safe, antigenic, and stable multi-subunit vaccine from the MmpL4 protein using immunoinformatics. Molecular dynamics simulations revealed the stability of the vaccine-bound Tolllike receptor-4 complex on a nanosecond scale, and immune simulations indicated strong primary and secondary immune responses in the host. Therefore, our study identifies a new target that could expedite the design of effective therapeutics, and the designed vaccine should be validated. Future directions include an extensive molecular interaction analysis, in silico cloning, wet-lab experiments, and evaluation and comparison of the designed candidate as both a DNA vaccine and protein vaccine.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.14 no.2
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• pp.148-154
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• 2011

Purpose: We aimed to study the distribution of rotavirus genotypes (VP7 and VP4) and disease severity of rotavirus gastroenteritis prevalent in our community. Methods: Stool samples were collected from 156 children who were hospitalized with rotavirus gastroenteritis from December 2007 to June 2008. The disease severity of all patients was scored using the Vesikari scale. After extraction of ds-RNA of the rotavirus, cDNA synthesis using reverse transcription and polymerase chain reaction (RT-PCR) and multiplex PCR was performed. Following this, the final identification of genotypes was performed. Results: Of the 156 samples, VP7(G) and VP4(P) genotypes were identified in 147 (94.2%) and 140 (89.7%) samples, respectively. G1 (116 of 147 samples; 78.9%) and P[8] (137 of 140 samples; 97.9%) were the most prevalent, respectively. Of the 138 samples identified of combination types of VP7 and VP4, G1P[8] (111 samples; 80.4%) was the most prevalent. Other combination types varied with very low distribution rates. 9.4% of genotypes were not included in the new vaccines. The disease severity score was $11.8{\pm}3.3$ ($mean{\pm}2SD$). The distribution of disease severity was mild or moderate in 37.8% and severe in 62.2% of patients. Conclusion: The most prevalent genotype combination of rotavirus was G1P[8] and genotypes not included in the vaccines represented 9.4% in our community. Disease severity distribution of hospitalized children with rotavirus gastroenteritis was higher in the severe than in the mild and moderate categories.

• Biomedical Science Letters
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• v.22 no.2
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• pp.37-45
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• 2016

High risk human papillomavirus (HR-HPV) is major risk factor for uterine cervical cancer. There are approximately 15 types of HR-HPV. Liquid based cytology samples (116 samples) with high grade cervical lesions belonging to cervical intraepithelial neoplasia (CIN) 2, CIN 3, carcinoma in situ (CIS) and squamous cell carcinoma (SCC) were used after histologic confirmation. HR-HPV genotype assay was conducted using DNA chips. The HR-HPV infection rate was 81.9% with SCC samples showing the highest HR-HPV infection rate of 31%. CIN 3, CIS and CIN 2 showed infection rates of 25%, 16.4% and 9.5%, respectively. According to age with HR HPV infection rate, the 30~39 years-old group showed the highest infection rate by 92.3%. According to distribution with HR HPV genotyping, HPV 16 showed the highest infection rate by 42.3% whereas HPV 33 and HPV 58 showed infection rates of 11.7% and 10.8%, respectively. HPV 18 which is the second most common infected HPV genotype in the world showed 3.6%. Of the three most common oncogenic HR-HPV genotypes in CIN 2, we detected HPV 16, 35, 58; CIN 3 was HPV 16, 33, 58; CIS was HPV 16, 58, 33 (35/52); and SCC was HPV 16, 33, and 18 (31/52/58). Among the HPV 18, CIN 2, CIN 3, CIS and SCC showed 0.9%, 0.9%, 0% and 1.8%, respectively. The most often used preventive vaccines for cervical cancers use HPV 16 and HPV 18 as targets. However, results derived from this study suggest that a preventive vaccine against HPV 16 and HPV 18 would not be optimal for populations in this study.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.3
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• pp.1129-1134
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• 2016

Vulva and Vaginal cancers are rare among all gynecological cancers worldwide, including Thailand, and typically affect women in later life. Persistent high risk human papillomavirus (HR-HPV) infection is one of several important causes of cancer development. In this study, we focused on HPV investigation and specific type distribution from Thai women with abnormality lesions and cancers of the vulva and Vaginal. A total of ninety paraffin-embedded samples of vulva and Vaginal abnormalities and cancer cells with histologically confirmed were collected from Thai women, who were diagnosed in 2003-2012 at the National Cancer Institute, Thailand. HPV DNA was detected and genotyped using polymerase chain reaction and enzyme immunoassay with GP5+/bio 6+ consensus specific primers and digoxigenin-labeled specific oligoprobes, respectively. The human ${\beta}$-globin gene was used as an internal control. Overall results represented that HPV frequency was 16/34 (47.1%) and 8/20 (40.0%) samples of vulva with cancer and abnormal cytology lesions, respectively, while, 3/5 (60%) and 16/33 (51.61%) samples of Vaginal cancer and abnormal cytology lesions, respectively, were HPV DNA positive. Single HPV type and multiple HPV type infection could be observed in both type of cancers and abnormal lesion samples in the different histological categorizes. HPV16 was the most frequent type in all cancers and abnormal cytology lesions, whereas HPV 18 was less frequent and could be detected as co-infection with other high risk HPV types. In addition, low risk types such as HPV 6, 11 and 70 could be detected in Vulva cancer and abnormal cytology lesion samples, whereas, all Vaginal cancer samples exhibited only high risk HPV types; HPV 16 and 31. In conclusion, from our results in this study we suggest that women with persistent high risk HPV type infection are at risk of developing vulva and Vaginal cancers and HPV 16 was observed at the highest frequent both of these, similar to the cervical cancer cases. Although the number of samples in this study was limited and might not represent the overall incidence and prevalence in Thai women, but the baseline data are of interest and suggest further study for primary cancer screening and/or developing the efficiency of prophylactic HPV vaccines in Thailand.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.15
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• pp.6769-6772
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• 2015

Background: High risk human papillomaviruses (HPVs) are the leading cause of cervical cancer (CC) and Pap smear screening has not been successful in preventing CC in Tunisia. HPV vaccination that targets HPV16 and 18 offers a new efficient prevention tool. Identification of HPV types in CC is thus essential to determine the impact of HPV vaccine implementation. The aim of this study is to provide specific data from Tunisia. Materials and Methods: A total of 89 histological confirmed paraffin embedded samples isolated from patients with CC diagnosed between 2001 and 2011 were collected from five medical centres from Northern and Southern Tunisia. HPV DNA was detected using a nested PCR (MY09/MY11-GP5+/GP6+) and genotyping was assessed using a reverse blot line hybridisation assay that enables the detection of 32 HPV types. Results: HPV DNA was detected in all samples. Twelve high risk types were detected; HPV16 and/or 18 were predominant, accounting together for 92.1% of all the CC cases (HPV16: 83.1%). Single infections accounted for 48.8% of the cases and were mostly linked to HPV 16 (32.6%) and less frequently to HPV 18 (2.4%). The other high risk HPV single infections were linked to HPV 35 (4.6%), 45 (4.6%), 58 (2.3%) and 59 (2.3%). Multiple infections with mixing of 2 to 4 genotypes predominately featrued HPV16 and/or 18 with HPV 35 and 45 (96.6 %) and less frequently with HPV 59, 40, 66, 73 and 58. There was no statistically significant variation in the relative distribution of HPV types with age. Conclusions: These results strongly indicate that prophylactic HPV vaccines can have a major impact in preventing CC in Tunisia.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.6039-6046
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• 2015

Aims: To investigate effect of metallic nanoparticles, silver (AgNPs) and gold nanoparticles (AuNPs) as antitumor treatment in vitro against human breast cancer cells (MCF-7) and their associated mechanisms. This could provide new class of engineered nanoparticles with desired physicochemical properties and may present newer approaches for therapeutic modalities to breast cancer in women. Materials and Methods: A human breast cancer cell line (MCF-7) was used as a model of cells. Metallic nanoparticles were characterized using UV-visible spectra and transmission electron microscopy (TEM). Cytotoxic effects of metallic nanoparticles on MCF-7 cells were followed by colorimetric SRB cell viability assays, microscopy, and cellular uptake. Nature of cell death was further investigated by DNA analysis and flow cytometry. Results: Treatment of MCF-7 with different concentrations of 5-10nm diameter of AgNPs inhibited cell viability in a dose-dependent manner, with IC50 value of $6.28{\mu}M$, whereas treatment of MCF-7 with different concentrations of 13-15nm diameter of AuNPs inhibited cell viability in a dose-dependent manner, with IC50 value of $14.48{\mu}M$. Treatment of cells with a IC50 concentration of AgNPs generated progressive accumulation of cells in the S phase of the cell cycle and prevented entry into the M phase. The treatment of cells with IC50 concentrations of AuNPs similarly generated progressive accumulation of cells in sub-G1 and S phase, and inhibited the entrance of cells into the M phase of the cell cycle. DNA fragmentation, as demonstrated by electrophoresis, indicated induction of apoptosis. Conclusions: Our engineered silver nanoparticles effectively inhibit the proliferation of human breast carcinoma cell line MCF-7 in vitro at high concentration ($1000{\mu}M$) through apoptotic mechanisms, and may be a beneficial agent against human carcinoma but further detailed study is still needed.

• Pediatric Infection and Vaccine
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• v.22 no.2
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• pp.97-105
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• 2015

Purpose: Serotyping pneumococcal isolates is important to monitor efficacy of pneumococcal vaccines. Because of difficulties of typing pnueumocci, a multiplex bead-based (multibead) serotyping assay was recently introduced. The aim of this study is to establish a new multibead serotyping assay and to apply this method to analyze clinical isolates of pneumococci in Korea. Methods: To establish the multibead serotyping assay, six key reagents were transferred from University of Alabama at Birmingham (UAB) to Ewha Center for Vaccine Evaluation and Study (ECVES): bead set coated with polysaccharide and monoclonal antibody pool were used in one multiplex inhibition-type immunoassay and 2 bead sets coated DNA probe and 2 primer pools were used in two multiplex PCR-based assays. After multibead serotyping assay was set up, 75 test samples of pneumococci were analyzed whether ECVES is able to identify serotype correctly. After confirming the performance, serotyping assay was applied to identify serotypes of 528 clinical isolates of pneumococci collected from 3 different hospitals. Results: After establishment of the multibead pneumococcal serotyping assay system at ECVES, 75 test samples were analyzed. There was no discrepancy of serotypes of 75 test samples between the results assigned at UAB and those at ECVES. The serotypes of 528 pneumococci isolated from patients or healthy subjects were determined in 94.3% of isolates (498/528). Conclusions: The multibead pneumococcal serotyping assay can be successfully established in Korea. With this method, surveillance of serotypes of pneumococci isolated from patients as well as healthy subjects could be studied.

• Parasites, Hosts and Diseases
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• v.52 no.1
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• pp.93-97
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• 2014

Subolesin (4D8), the ortholog of insect akirins, is a highly conserved protective antigen and thus has the potential for development of a broad-spectrum vaccine against ticks and mosquitoes. To date, no protective antigens have been characterized nor tested as candidate vaccines against Dermacentor silvarum bites and transmission of associated pathogens. In this study, we cloned the open reading frame (ORF) of D. silvarum 4D8 cDNA (Ds4D8), which consisted of 498 bp encoding 165 amino acid residues. The results of sequence alignments and phylogenetic analysis demonstrated that D. silvarum 4D8 (Ds4D8) is highly conserved showing more than 81% identity of amino acid sequences with those of other hard ticks. Additionally, Ds4D8 containing restriction sites was ligated into the pET-32(a+) expression vector and the recombinant plasmid was transformed into Escherichia coli rosetta. The recombinant Ds4D8 (rDs4D8) was induced by isopropyl ${\beta}$-D-thiogalactopyranoside (IPTG) and purified using Ni affinity chromatography. The SDS-PAGE results showed that the molecular weight of rDs4D8 was 40 kDa, which was consistent with the expected molecular mass considering 22 kDa histidine-tagged thioredoxin (TRX) protein from the expression vector. Western blot results showed that rabbit anti-D. silvarum serum recognized the expressed rDs4D8, suggesting an immune response against rDs4D8. These results provided the basis for developing a candidate vaccine against D. silvarum ticks and transmission of associated pathogens.