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The Koi fish (Cyprinus carpio var. koi) is an ornamental fish with a high selling value because of its attractive colors, color patterns, body shape, and swimming motion. Koi fish is extensively traded in the international fish market because of their popularity among hobbyists from numerous countries worldwide. This review discusses various aspects of Koi fish cultivation, including genetic involvement, selective breeding strategies, and management systems. By examining crucial factors such as water parameters, technological innovations, and evolving cultivation methods, this review explored their influence on the quality of Koi fish. Breakthrough technologies, such as ornamental fish warehousing and recirculation aquaculture systems, enhance breeding efficiency and profitability. Molecular sexing, feed optimization, and color enhancement strategies are central to pursuing superior Koi fish. Reproduction management, disease prevention, and risk reduction during transport underscore ongoing efforts to ensure their survival. Despite notable progress, several challenges remain, including limited genetic studies, gaps in disease research, and unexplored herbal alternatives. The active involvement of hobbyists and breeders in research initiatives is a pivotal force in unlocking the untapped potential. The holistic approaches to enhance production efficiency and improve care standards require further exploration, paving the way for a sustainable future in the evolving management of Koi fish cultivation.

Protecting the skin from the effects of UVB radiation using natural products is crucial in the cosmeceutical industry. This study aims to investigate the protective effects of Bangle (Zingiber cassumunar Roxb.) against UVB‐induced skin damage in Wistar albino rats. The rhizomes were macerated using 70% ethanol v/v, followed by n‐hexane to obtain n‐hexane soluble and n‐hexane insoluble fraction. The antioxidant properties of the ethanol extracts and n‐hexane soluble fraction were evaluated using a 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay. The study also examined the antiphotoaging properties through reactive oxygen species (ROS) scavenging assay, matrix metalloproteinase‐1 (MMP‐1) expression, and tyrosinase expression against UVB radiation in Wistar albino rats. The results demonstrated that the Z. cassumunar extract and fraction effectively converted DPPH radicals into a more stable compound. Analysis revealed the presence of Benzene, 4‐(1Z)‐1,3‐butadien‐1‐yl‐1,2‐dimethoxy‐ and (E)‐4‐(3,4‐Dimethoxyphenyl) but‐3‐en‐1‐ol as the primary compounds in both the extract and fraction, suggesting their contribution to the observed activity. Furthermore, Z. cassumunar compounds could reduce UVB‐induced ROS production and may protect against skin photoaging by changing the expression of MMP‐1 and tyrosinase levels in Wistar albino rats. These findings suggest that Z. cassumunar holds promise for preventing skin aging.

Gyrinops versteegii (Gilg.) Domke is one of the Thymelaeaceous family species with high economic value. The leaf synthesizes secondary metabolites (SMs) supporting herbal medicine, including a class of SMs that serve as sunscreen. However, the SMs are not equally distributed in the plant, depending on the organs, tissues or cell type. This research aimed to determine the spatial distribution of secondary metabolites in the leaves, especially in the epidermis and mesophyll tissues. The epidermis tissues were separated from the mesophyll using the carborundum abrasion (CA) technique, followed by the extraction of SMs using three different polarity solvents, namely methanol, hexane and chloroform. The photoprotective potential was evaluated using the SPF value with the spectrophotometry approach. Subsequently, the phytochemical analysis was done for the total tannin, flavonoid, and phenols content. The results showed that the highest SPF value was observed in the methanol epidermis extract with SPF value of 8.61, followed by methanol mesophyll extract (4.52), chloroform epidermis extract (3.97), chloroform mesophyll extract (2.52) and hexane epidermis extract (0.60) and hexane mesophyll extract (0.16). Total phenolic content (flavonoid, tannin and phenol) in epidermis extract with methanol solvent was higher (12.347 ± 0.652 mg GAE/g DW), followed by extract with chloroform and hexane solvents. The total phenolic content (flavonoid, tannin and phenol) of mesophyll extract with methanol solvent showed the highest content, followed by chloroform mesophyll and hexane extracts. The results of the histochemical analysis showed that photoprotective compounds accumulate in the epidermis and mesophyll tissue of the leaf of G. versteegii.

BACKGROUND: Type 2 diabetes mellitus affects glucose metabolism resulting in hyperglycemia. The bark of Cinnamomum burmannii (CB) and the leaves of Aquilaria malaccensis (AM) are believed to be effective for diabetes treatment. This study evaluated the synergistic effect of CB and AM, the extracts' phytochemical profiles, and the interaction between CB and AM metabolites with a-glucosidase through molecular docking. METHODS: The dried material was macerated with ethanol and then tested for a-amylase and a-glucosidase inhibitory activities and glucose diffusion inhibition in varied combination proportions. The extract fingerprinting was performed using a UV-Vis spectrophotometer followed by thin layer chromatography to determine the class of secondary metabolite in the extracts. Human maltase-glucoamylase (MGAM) receptor, ligands from acarbose and selected metabolites of CB and AM were studied in silico using UCSF Chimera, AutoDockTools, Autodock Vina Wizard (PyRx), and Biovia Discovery Studio software. RESULTS: The best ratio of CB:AM for a-amylase and a-glucosidase inhibition was 0.75:0.25 mg/mL, with inhibitory activities of 86.36 and 96.38 %, respectively. The best glucose diffusion inhibition was achieved at a ratio of 0.5:0.5 mg/mL CB and AM. The b-caryophyllene of CB and palustrol of AM had a significantly higher binding affinity of -10.7 kcal/mol and -10.2 kcal/mol, respectively than acarbose, which had a binding affinity of -8.1 kcal/mol. CONCLUSION: A ratio of CB to AM suppresses the activity of diabetes-related enzymes more efficiently. The in silico study suggested that the presence of b-caryophyllene in CB and palustrol in AM supported the synergistic activity. KEYWORDS: Aquilaria malaccensis, Cinnamomum burmannii, diabetes mellitus, a-amylase inhibition, a-glucose inhibition, glucose inhibition

Abstract Background ASEAN (Association of Southeast Asian Nations) is composed of ten Southeast Asian countries bound by socio-cultural ties that promote regional peace and stability. South Asia, located in the southern subregion of Asia, includes nine countries sharing similarities in geographical and ethno-cultural factors. Chikungunya is one of the most significant problems in Southeast and South Asian countries. Much of the current chikungunya epidemic in Southeast Asia is caused by the emergence of a virus strain that originated in Africa and spread to Southeast Asia. Meanwhile, in South Asia, three confirmed lineages are in circulation. Given the positive correlation between research activity and the improvement of the clinical framework of biomedical research, this article aimed to examine the growth of chikungunya virus-related research in ASEAN and South Asian countries. Methods The Scopus database was used for this bibliometric analysis. The retrieved publications were subjected to a number of analyses, including those for the most prolific countries, journals, authors, institutions, and articles. Co-occurrence mapping of terms and keywords was used to determine the current state, emerging topics, and future prospects of chikungunya virus-related research. Bibliometrix and VOSviewer were used to analyze the data and visualize the collaboration network mapping. Results The Scopus search engine identified 1280 chikungunya-related documents published by ASEAN and South Asian countries between 1967 and 2022. According to our findings, India was the most productive country in South Asia, and Thailand was the most productive country in Southeast Asia. In the early stages of the study, researchers investigated the vectors and outbreaks of the chikungunya virus. In recent years, the development of antivirus agents has emerged as a prominent topic. Conclusions Our study is the first to present the growth of chikungunya virus-related research in ASEAN and South Asian countries from 1967 to 2022. In this study, the evaluation of the comprehensive profile of research on chikungunya can serve as a guide for future studies. In addition, a bibliometric analysis may serve as a resource for healthcare policymakers.

Background and Aim: Guinea pigs (GPs) (Cavia porcellus) are not only kept as pets but also widely used in biological and biomedical research. At present, GPs are also used as a species for animal-assisted therapy (AAT). Consequently, assessing their health status is vital to determining their quality of life, usability for research, and prevention of spread of potential zoonotic diseases to patients using them for AAT. GPs are mainly sourced from animal markets supplied by traditional farms, where environmental factors and sanitation are not properly controlled. This study aimed to compare health status between GPs raised in uncontrolled (conventional farm) and controlled (animal facility) environments. Materials and Methods: Sample animals were obtained from a local animal market and transported to an animal facility. After 1 week of acclimatization, the health status of the animals, including general health condition, body weight, body temperature, complete blood count, liver function (alanine aminotransferase and bilirubin), renal function (blood urea nitrogen and creatinine), and presence of ectoparasites and endoparasites, was assessed. Then, the animals were maintained in the animal facility following the standard procedure for laboratory animals. After 2 months, the animals' health status was re-examined, assessing the same parameters. Results: Based on the evaluated parameters, GPs raised in an uncontrolled environment were found to have poorer health status than those raised in a controlled environment. There were significant differences in almost all parameters between GPs raised in controlled and uncontrolled environments. We found that the populations of two ectoparasites, Gyropus ovalis and Gliricola porcelli, and one endoparasite, Eimeria caviae>, decreased significantly following the movement of the animals from an uncontrolled to a controlled environment. Conclusion: GPs raised in an uncontrolled environment have poor health status. However, a controlled environment with better care management can improve the health status of GPs.

BACKGROUND: Aquilaria malaccensis has been consumed as herbal medicine, and in vitro study showed that the leaf extract possesses high antioxidant activities. A brief preliminary study indicated that A. malaccensis showed a promising immunomodulatory activity when evaluated using latex beads. This current study aimed to evaluate the immunomodulatory activity of A. malaccensis leaf extract on the macrophage, which was challenged with pathogenic bacteria Staphylococcus aureus. METHODS: Bioactivity was determined by evaluating the phagocytic capacity of macrophages isolated from Mus musculus against S. aureus. First, the cytotoxicity of extracts on macrophages was evaluated using MTT assays, and the IC50 value was used to determine the dose of immunomodulatory assays. The highest toxicity was observed on chloroform extract with an IC50 value of 111.4 µg/mL. Therefore, the treatment was 100 and 50 µg/mL. Two parameters, including the phagocytic activity and phagocytic capacity of macrophages infected with S. aureus, were used to evaluate immunomodulatory activity. The analysis of variance was done at p

The aim of this work was the in vitro expression of the recombinant fusion (F) protein of Newcastle disease virus (NDV). The pBT7-N-His-Fusion-NDV expression plasmid which carries the recombinant F protein encoding gene from local Indonesian isolates, was prepared and transformed into E. coli BL21 (DE3). To detect bacterial colonies carrying the recombinant plasmid, a restriction endonuclease analysis was performed using the EcoRI restriction endonuclease. These results showed that the pBT-N-His-Fusion-NDV plasmid was successfully isolated with a size of 4.601 bp, and three recombinant plasmids carrying the gene coding for the recombinant F protein of NDV were obtained. Selected recombinant plasmids were then in vitro by using a cell-free protein expression system followed by visualization of the recombinant F protein on a 12% SDS-PAGE gel both by Coomassie Brilliant Blue staining and Western blotting. Recombinant F protein was successfully in vitro expressed by using a cell-free protein expression system as indicated by a specific single protein band with a molecular mass of 25.6 kDa.

Objectives: Monitoring the spread of the G614 in specific locations is critical as this variant is highly transmissible and can trigger the emergence of other mutations. Therefore, a rapid and accurate method that can reliably detect the D614G mutation will be beneficial. This study aims to analyze the potential use of the two-step Reverse Transcriptase quantitative polymerase chain reaction - high resolution melting analysis (RT-qPCR-HRM) to detect a specific mutation in the SARS-CoV-2 genome. Methods: Six SARS-CoV-2 RNA samples were synthesized into cDNA and analyzed with the qPCR-HRM method in order to detect the D614G mutation in Spike protein of SARS-CoV-2. The primers are designed to target the specific Spike region containing the D614G mutation. The qPCR-HRM analysis was conducted simultaneously, and the identification of the SARS-CoV-2 variant was confirmed by conventional PCR and Sanger sequencing methods. Results: The results showed that the melting temperature (Tm) of the D614 variant was 79.39 ± 0.03 °C, which was slightly lower than the Tm of the G614 variant (79.62 ± 0.015 °C). The results of the HRM analysis, visualized by the normalized melting curve and the difference curve were able to discriminate the D614 and G614 variant samples. All samples were identified as G614 variants by qPCR-HRM assay, which was subsequently confirmed by Sanger sequencing. Conclusions: This study demonstrated a sensitive method that can identify the D614G mutation by a simple two-step RT-qPCR-HRM assay procedure analysis, which can be useful for active surveillance of the transmission of a specific mutation.

Cashew fruit peel is a waste produced from the cashew nut industry, and it has not been utilized optimally yet. Cashew peel extract has the potential to be used as a contraceptive agent, which capable of reducing reproductive capacity. However, its side effects on other tissue and organ such as liver not clearly studied yet. This study aims to determine the effect of ethanolic extracts of cashew peel on the histological structure of the white rat liver. In this study, 21 female white rats were used and be grouped for control (6 mice) which were treated with CMCMa 0.5% and 15 mice were treated with peel extract of 500 mg/kg body every day for one month. Liver for examination was collected sequentially at 3rd, 5th, 8th, 11th, and 14th of the estrous cycle. The liver was processed for histological observation and stained with Hematoxylin Eosin and Mallory Acid Fuchsin staining solution. The liver hepatocyte was observed for it abnormality and be scored to calculate the number of cell damage or abnormality. The result showed that peel extract-treated mouse liver was similar to control ones; we did not witness any evidence of fibrosis, pyknosis and cellular necrosis on either control or treated mouse. Statistical analysis by SPSS showed that the p-value between the control and treatment groups was 0.078 (> 0.05) so there was no significant difference between control and treatment. It could be concluded that ethanolic extracts of cashew nuts peel with a concentration of 500 mg/kg body weight caused no effect on the mouse liver histological structure. application with reduced-dosages of NPK fertilizers were arranged in a random block design with three replicates. The results show that large quantities of silica bodies attached to the surface of EFB fibers and amounting to 0.44% soluble Si. The FFB data indicated that the application of 75% NPK + 500 kg composted EFB + 2 L BioSilAc/ha/year on a five-year-old plant resulted in higher yield than that obtained from 100% standard dosage of NPK. The study also revealed that the application of EFB compost reduced 50% of BioSilAc dosage.

Tithonia diversifolia is a plant that has the potential to reduce blood glucose levels that is crucial in Diabetes Mellitus therapy. This study aims to determine the active compounds contained in the extract of T. diversifolia leaves decoction, the effect of decoction extract on the reduction of blood glucose, and the most effective decoction extract to reduce blood glucose levels as well. The study used a Completely Randomized Design (CRD) with the research treatment groups, namely: Kn = normal control (normal mice and not given leaves decoction extract), Ka = STZ control (DM mice, which are normal mice induced by 65mg/Kg of STZ), Kp = treatment control (normal mice given leaf decoction extract) , P1 = DM + mice were given young leaf decoction extract, P2 = DM + mice were given a mixture of young leaf and adult leaf decoction extract, P3 = DM + mice were given adult leaf decoction extract. The leaves used for decoction were 1-6 leaves from the shoots. The analysis of T. diversifolia leaves decoction compounds is using the visible spectrophotometers for tannins, phenols and flavonoids and GC-MS for terpenoid analysis. The result found that Tithonia diversifolia leaves decoction extracts contain tannins, flavonoids and phenols, while terpenoids are not detected. The leaves decoction extracts of T. diversifolia influenced the decreases in blood glucose levels of DM mice, especially the decoction extract of adult leaves which reduced blood glucose levels to 71.16%.

Dengue fever is caused by the mosquito-borne virus termed (DENV). However, DENV-2 has been identified as the most prevalent amongst the Indonesian pediatric urban population, in contrast with the other four serotypes. Therefore, it is important to reduce severe infection risk by adopting preventive measures, including through vaccine development. The aim of this study, therefore is to use various in silico tools in the design of epitope-based peptide vaccines (T-cell and B-cell types), based on the DENV-2 envelope glycoprotein sequences available. Therefore, in silico methods were adopted in the analysis of the retrieved protein sequences. This technique was required to determine the most immunogenic protein, and is achieved through conservancy analysis, epitope identification, molecular simulation, and allergenicity assessment. Furthermore, B4XPM1, and KAWLVHRQW were identified from positions 204-212, while the 77 to 85 peptide region was considered the most potent T-cell and B-cell epitopes. The interaction between KAWLVHRQW and HLA-C*12:03 occurs with maximum population coverage, alongside high conservancy (96.98%) and binding affinity. These results indicated a potential for the designed epitopes to demonstrate high immunity against DENV-2.

Aim: This study aimed to analyze the neuroprotective effect of Ocimum sanctum Linn. ethanolic extract (OSE) on human embryonic kidney-293 (HEK-293) cells as the in vitro model of neurodegenerative diseases. Materials and Methods: In this research, HEK-293 cells divided into five groups consisting of normal and healthy cells (NT), cells treated with Camptothecin 500 μM as the negative control, cells treated with trimethyltin 10 μM (TMT), cells treated with OSE 75 μg/ml, and cells pre-treated with OSE 75 μg/ml then induced by TMT 10 μM (OSE+TMT). MTT assay and phase contrast microscopy were applied to observe the cell viability quantitatively and morphological after Ocimum sanctum Linn extract treatment. Finally, the reverse transcription polymerase chain reaction was employed to study the expression of choline acetyltransferase (ChAT). Results: The MTT assay and phase contrast microscopy showed that OSE pre-treatment significantly increased the viability of TMT-induced apoptotic cells and maintained cell viability of the normal HEK-293 cells. Expression of ChAT markedly reduced on TMT treatment group, but OSE administration stabilized ChAT expression in TMT-induced HEK-293 cells. Conclusion: This present study proved that OSE administration has neuroprotective effect by increased HEK-293 cells viability and maintain ChAT expression.

Objective: To examine the anti-angiogenic potential of Artocarpus heterophyllus (A. heterophyllus) seed extract in chicken chorioallantoic membrane (CAM). Methods: This study used chicken CAM ex ovo culture to examine the potential anti-angiogenic activity of A. heterophyllus seed methanolic extract. Basic fibroblast growth factor was used to induce the ectopic formation of blood vessels on CAM treated with extract. Blood vessel number was assessed by macroscopic and microscopic observation, and compared and analyzed for all treatments and controls. Results: Macroscopic observation revealed that a dose of 35 μg/mL of methanolic extract of A. heterophyllus seeds could inhibit basic fibroblast growth factor-induced angiogenesis by 61% in chicken CAM ex ovo culture. This concurred with microscopic observations on the histological structure of blood vessels, which indicated that extract treatment repressed the formation of new blood vessels. Conclusions: This is the first study to report the anti-angiogenic effect of methanolic extract derived from A. heterophyllus seeds and its potential as a candidate for future anticancer therapy.

Streptomyces are a group of Gram-positive bacteria belonging to the Actinobacteria class, which are among the most important bacteria for producing secondary bioactive metabolites such as antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. Recently, we have isolated the Streptomyces sp. GMR22 from Cajuput rhizospheric soil at Wanagama Forest, Indonesia. GMR22 produced secondary metabolite that inhibited Candida albicans with IC50 of 62,5 μg/mL. The objective of this work was to reveal the novel secondary metabolites from GMR22 by genome mining approach. The antiSMASH 3.0 was used to predict gene clusters that encode the biosynthetic pathways of secondary metabolites in the genome of GMR22, and their core chemical structures. The pylogenomic analysis showed that GMR22 was closely related to Streptomyces bingchenggensis BCW1, as well as to the large genome size (9.5-12.7Mbp) groups of Streptomyces. AntiSMASH 3.0 analysis revealed that the genome of Streptomyces sp. GMR22 harbored at least 63 gene clusters that encode the biosynthetic pathways of secondary metabolites. It was the highest number of gene clusters had been observed among the members of Streptomyces groups, with PKS was predicted as the major groups of the identified gene cluster products. The results suggested that GMR22 could be a strong potential candidate of secondary bioactive metabolites source.

Aim: Avian encephalomyelitis (AE) is a viral disease which can infect various types of poultry, especially chicken. In Indonesia, the incidence of AE infection in chicken has been reported since 2009, the AE incidence tends to increase from year to year. The objective of this study was to analyze viral protein 2 (VP-2) encoding gene of AE virus (AEV) from various species of birds in field specimen by reverse transcription polymerase chain reaction (RT-PCR) amplification using specific nucleotides primer for confirmation of AE diagnosis. Materials and Methods: A total of 13 AEV samples are isolated from various species of poultry which are serologically diagnosed infected by AEV from some areas in central Java, Indonesia. Research stage consists of virus samples collection from field specimens, extraction of AEV RNA, amplification of VP-2 protein encoding gene by RT-PCR, separation of RT-PCR product by agarose gel electrophoresis, DNA sequencing and data analysis. Results: Amplification products of the VP-2 encoding gene of AEV by RT-PCR methods of various types of poultry from field specimens showed a positive results on sample code 499/4/12 which generated DNA fragment in the size of 619 bp. Sensitivity test of RT-PCR amplification showed that the minimum concentration of RNA template is 127.75 ng/μl. The multiple alignments of DNA sequencing product indicated that positive sample with code 499/4/12 has 92% nucleotide homology compared with AEV with accession number AV1775/07 and 85% nucleotide homology with accession number ZCHP2/0912695 from Genbank database. Analysis of VP-2 gene sequence showed that it found 46 nucleotides difference between isolate 499/4/12 compared with accession number AV1775/07 and 93 nucleotides different with accession number ZCHP2/0912695. Conclusions: Analyses of the VP-2 encoding gene of AEV with RT-PCR method from 13 samples from field specimen generated the DNA fragment in the size of 619 bp from one sample with sample code 499/4/12. The sensitivity rate of RT-PCR is to amplify the VP-2 gene of AEV until 127.75 ng/μl of RNA template. Compared to Genbank databases, isolate 499/4/12 has 85% and 92% nucleotide homology.

White spot syndrome virus (WSSV) is a devastating viral disease in shrimp aquaculture. Infection of WSSV in penaeid shrimps affects immune defense and changes gene expression. PmVRP15 has been reported as a part of the WSSV propagation pathway that is highly up-regulated in hemocytes at the acute phase of WSSV infection. This study analyzed the expression of PmVRP15 in local populations of giant tiger shrimp (Penaeus monodon) to be associated with susceptibility to WSSV. Tested populations consisted of an inbreeding population (G8) and outbreeding population (G8iA) from Jepara, Indonesia. Susceptibility was determined by cumulative mortality, median lethal time (LT50), and severity of infection at time of death. Though all populations were susceptible to WSSV, the frst mortality in G8 occurred at 18 hours post-infection (hpi) with mild infection, while frst mortality of G8iA occurred at 30 hpi with severe infection. The LT50 of G8 was signifcantly lower than that of G8iA, indicating that G8iA was less susceptible to WSSV than G8. Relative PmVRP15 transcripts of G8iA were insignifcantly down-regulated, whereas relative PmVRP15 transcripts of G8were insignifcantly upregulated. Although it’s still not conclusive, the results of this study suggest that PmVRP15 has weak potentialas a WSSV susceptibility marker in G8 and G8iA broodstock selection.

Marine actinomycetes is a group of bacteria that is highly potential in producing novel bioactive compound. It has unique characteristics and is different from other terrestrial ones. Extreme environmental condition is suspected to lead marine actinomycetes produce different types of bioactive compound found previously. The aim of this study was to explore the presence and diversity of NRPS genes in 14 anticancer-producing actinomycetes isolated from marine sediment in Indonesia. PCR amplification and restriction fragment analysis of NRPS genes with HaeIII from 14 marine actinomycetes were done to assess the diversity of NRPS genes. Genome mining of one species of marine actinomycetes (strain GMY01) also was employed towards this goal. The result showed that NRPS gene sequence diversity in 14 marine actinomycetes could be divided into 4 groups based on NRPS gene restriction patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that all isolates belong to genus of Streptomyces. Genome mining result showed that strain GMY01 harboring 10 different NRPS gene clusters that encode secondary metabolites, as pure NRPS or hybrid between NRPS and other compounds. These results indicated that marine actinomycetes having a high potential to be developed as source of anticancer drugs development.

Latar belakang: Survei virologi pada nyamuk vektor dapat digunakan sebagai Sistem Kewaspadaan Dini untuk mencegah penularan Demam dengue di suatu daerah. Pemeriksaan laboratoris untuk deteksi virus Dengue pada nyamuk seperti isolasi virus, Polymerase Chain Reaction (PCR) dan Direct Fluorescent-Antibody (DFA) memerlukan keahlian yang tinggi, peralatan yang mahal dan waktu yang lama. Suatu metode berdasarkan imunositokimia menggunakan antibody monoclonal DSSE10 memiliki beberapa kelebihan. Tujuan penelitian ini untuk mengevaluasi sensitifitas dan spesifitas pemeriksaan imunositokimia dibandingkan metode Reverse Transcription-Polymerase Chain Reaction (RT-PCR) untuk mendeteksi infeksi Virus Dengue 3. Metode: Penelitian eksperimental dilakukan di laboratorium Parasitologi Fakultas Kedokteran Universitas Gajah Mada (UGM) pada bulan Mei 2009-Oktober 2010. Sebanyak 22 Ae. aegypti yang diinfeksi virus Dengue 3 digunakan sebagai kelompok infeksius dan 35 nyamuk yang tidak diinfeksi sebagai kelompok non infeksius. Pemeriksaan imunositokimia Streptavidin Biotin Peroxidase Complex (SBPC) menggunakan antibodi monoklonal DSSE10 dilakukan pada sediaan head squash Ae .aegypti untuk mendeteksi antigen virus Dengue 3. Pemeriksaan RT-PCR sebagai baku emas diaplikasikan pada toraks nyamuk.Hasil: Nilai Kappa menunjukkan kesepakatan yang baik antara dua orang pemeriksa (0,63). Imunositokimia mendeteksi antigen virus Dengue-3 dengan sensitivitas yang sama dengan RT-PCR (sensitivitas 100%). Namun spesifisitas IC lebih rendah dibanding RT-PCR (spesifisitas 91%) karena beberapa hasil positif palsu muncul pada pemeriksaan ini. Kesimpulan: Metode IC memiliki nilai sensitivitas dan spesifisitas yang tinggi dibandingkan dengan metode RT-PCR. Metode IC ini dapat digunakan untuk surveilans virus Dengue pada nyamuk vektor. (Health Science Indones 2011;2:87-91).AbstractBackground: Virological surveillance provides an early warning sign for the risk of transmission in an area. Laboratory tests for dengue virus infection on mosquitoes include isolation of the virus, Polymerase Chain Reaction (PCR) and Direct Fluorescent-Antibody (DFA) requires a high level of technical skill, expensive equipment, and time-consuming. A method based on immunocytochemical (IC) using monoclonal antibody DSSE10 has several advantages. This study aimed to evaluate sensitivity and specificity IC assay compared with Reverse Transcription-Polymerase Chain Reaction (RT-PCR) as gold standard to detect Dengue Virus (DENV)-3 infections in mosquito Aedes aegypti.Methods: An experimental study was conducted in laboratory of Medical Parasitology, Faculty of Medicine, Universitas Gadjah Mada (UGM) in May 2009 until October 2010. A total of 22 artificially-infected adult Ae. aegypti mosquitoes of DENV 3 were used as infectious samples and 35 non-infected adult Ae. aegypti mosquitoes were used as normal ones. The IC Streptavidin Biotin Peroxidase Complex (SBPC) assay using monoclonal antibody DSSE10 was applied in mosquito head squash to detect Dengue virus antigen. RT-PCR as a gold standard was applied in mosquito thorax.Results: The kappa value showed a good agreement between two observers (kappa value 0.63). IC could detect dengue virus antigen as sensitive as RT-PCR (sensitivity 100%). But IC was less specific than RT-PCR (specificity 91%) because some false positive results were found in this method.Conclusion: The IC method has a high sensitivity and high specificity compared with RT-PCR. This IC method may be useful for virological surveillance of dengue infected Aedes mosquitoes. (Health Science Indones 2011;2:87-91).

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of manyregulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants.In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cell

Hepatitis B virus (HBV) can be classified into 8 genotypes, genotype A to H. Genotype of HBV is important for clinical and etiological investigations. Research for HBV genotyping, HBV transmission study using nested PCR and HBV genotyping based on RFLP using restriction enzymes have been reported. However, both of those methods have been not applied for HBV genotyping study among hepatitis B patients in endemic area, like Indonesia yet. Molecular genotyping of HBV will describe epidemiology, pathogenesis and clinical implication of HBV. Combination of nested PCR and RFLP (nested PCR-RFLP) method to determine HBV genotype in Indonesia is still less information. The objectives of study were to develop a system for HBV genotyping by nested PCR combined with RFLP (nested PCR-RFLP) method based on nucleotide sequence of surface protein encodinggene (S gene) in HBV genome and to confirm HBV genotypes which predominantly found among hepatitis B patients in Daerah Istimewa Yogyakarta Province and surrounding area. Total of 149 sera from chronic hepatitis B patients from Daerah Istimewa Yogyakarta and surrounding areas were collected for in this work. Viral DNA were extracted from sera of hepatitis B patients and used as template for first round nested PCR amplification using outer primers set. Amplicons of first round PCR were used as template for second round amplification using inner primers set. Then, amplicons of second round nested PCR were restriction digested by Sty I and Bsr I enzymes. For HBV genotyping then the restriction products were analyzed by RFLP based on restriction pattern. Results showed that the first round nested PCR amplification generated DNA fragments of whole S gene in length 1.233 bp, and in second round nested PCR amplification using inner primer set generated DNA fragments 585 bp in length. Genotype analysis for all samples using nested PCR-RFLP methods by restriction digested of Sty I and Bsr I enzymes found only 2 HBV genotypes among hepatitis B patients, namely genotype B and C. Quantificationdata showed that most of hepatitis B patients found infected by HBV genotype B (92,8%), genotype C (3,6%) and unidentified genotype (3,6%). Nested PCR-RFLP methods for HBV genotyping is simple and inexpensive for clinical diagnostic in large scale.

protein is also including in the HBV genome targeting into the nucleus through modulating carboxyl residues byphosphorylation. Nuclear localication Signal (NLS) in HBV core protein is inside the virion structure and it must beunmasked in order to function, perhaps by phosphorylation. Phosphorylation of of HBV core protein in turn couldbegin to alter capsid conformation. Staurosporine is a natural product originally isolated from bacteriumStreptomyces staurosporeus. Staurosporine was discovered to have biological activities ranging from anti-fungal toanti-hypertensive. The interest in these activities resulted in a large investigative effort in chemistry and biology andthe discovery of the potential for anti-cancer treatment. The main biological activity of Staurosporine is the inhibitionof protein kinases through the prevention of ATP binding to the kinase. In the present study, we have studied theintracellular localization of EGFP-Core fusion protein with triple HBV core and SV-40 nuclear localization signal atits carboxyl terminal in presence and absence of Staurosporine. We also to study the effect of Staurosporine treatmenton the intracellular localization of EGFP-Core fusion protein in the hepatocyte cells line of HepG2 cell. Resultsshowed that effect of Staurosporine is prevent the nuclear localization of EGFP-Core fusion protein into nucleusthrough an inhibition of the phosphorylation of core protein. Stauroporine also prevents cell division so that passivetrapping of core protein is inhibited.

Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3). This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM) with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4. This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

Bayer 41-4109 is heteroarylpyrimidine (HAP) which has been identified as potent of HBV capsid assemblyinhibitor. The present study was to study effect of Bayer 41-4109 treatment on the intracellular localization ofEGFP-Core fusion proteins into HepG2 cells. Three recombinant plasmids of pEGFP-Core with single, double andtriple NLS of HBV core (EGFP-Core 1C, 2C and 3C ) and two recombinant plasmids with single and triple NLS ofSV-40 (EGFP-Core 1 and 3 SV-40) were used in this work. After transient transfected into HepG2 cells and treatedwith Bayer 41-4109, the intracellular localization of expressed fusion proteins from all plasmid constructions weredetermined and quantified under confocal laser microscope. Results shown that Bayer 41-4109 treatment in HepG2cells inhibited the nuclear localization of EGFP-Core with single of triple HBV core NLS. As well as the constructionsof expressed fusion protein with single and triple SV-40 NLS (EGFP-Core 1 and 3 SV-40 NLS) showeddecreasing the nuclear localization after treated with Bayer 41-4109, even not as strong as EGFP-Core 1C and 3CNLS. Bayer 41-4109 has been identified as a potent inhibitors of HBV replication which has multiple effects on HBVcapsid assembly. It may inhibit virus replication by inducing assembly inappropriately and by misdirectingassembly decreasing the stability of normal capsids.Keywords: HBV capsid, Bayer 41-4109, EGFP-Core fusion protein, HepG2 cell

world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detectingdengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primersthat conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirusand there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp,the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virususing multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus.Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4,whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assaycan be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiologyand also evolutionary studies.Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR

Recently several methods for confirming Dengue Virus have been developed involve virus isolation, detection of virus antigen, and nucleic acid using PCR. It has been reported that rapid detection method for confirming DHF by Multiplex RT-PCR had been successfully developed. It was more effective than the other methods with a high sensitivity and specivicity were 100% at the early phase (day 1-3). This study was designed to develop rapid detection and serotyping methods for Dengue Virus using RT-PCR 2 primers (Dcon and preM) with annealing temperature was 57oC. The whole blood samples were collected from suspected dengue fever patients that had been confirmed with NS1 kit from R.S. Persahabatan DKI Jakarta and R.S. Prof. Dr. Sardjito DI Yogyakarta during Februari-August 2009. The PCR products showed that in 12 samples, 100 % were postitive with different pattern among the serotypes especially for DEN1 and DEN2, but not for DEN3 and Den4. This method was also able to confirm the double infection DEN2-DEN3, but not for the other ones because of the unspecific pattern. From the results, it indicated that the 2 primers can be a promising early detection and serotyping method of Dengue Virus which infected the DHF patients. Key words: Dengue Virus, DHF, early detection, serotyping, RT-PCR 2 primers.

Leptomycin B (LMB) was originally discovered as a potent anti-fungal antibiotic from Streptomyces species. The cellular target of LMB has been identified as the nuclear export receptor CRM-1 or exportin-1, which is involved in nuclear trafficking of cellular RNAs or proteins containing the nuclear export sequence (NES). CRM-1 is the main mediator of nuclear export in many cell types including hepatocyte cell lines. The ability of LMB to inhibit nuclear export has made it a useful tool in the study of the intracellular localization of many regulatory proteins. In this study, we evaluated the effect of nuclear export inhibitor LMB treatment on the intracellular localization of HBV core protein into the hepatocyte cell lines, Huh-7 and HepG2 cells. We also reported the quantification of the distribution of EGFP-Core fusion protein with redundant core NLS as well as SV-40 NLS into cell compartments. Results shown that in Huh-7 cells treatment of LMB caused retention of EGFP-Core fusion protein into the nucleus, so increased the nuclear localization of EGFP-Core and all variants. In HepG2 cells, although not significantly, treatment of LMB increased a number of nuclear localization in all EGFP-Core constructions, even the nuclear localization in HepG2 cells is not so high as in Huh-7 cells. Keywords: Leptomycin B, HBV, core protein, intracellular localization, NLS, Huh-7, HepG2 cell

Hepatitis B virus (HBV) can be classified into 8 genotypes, genotype A to H. Genotype of HBV is important for clinical and etiological investigations. Research for HBV genotyping, HBV transmission study using nested PCR and HBV genotyping based on RFLP using restriction enzymes have been reported. However, both of those methods have been not applied for HBV genotyping study among hepatitis B patients in endemic area, like Indonesia yet. Molecular genotyping of HBV will describe epidemiology, pathogenesis and clinical implication of HBV. Combination of nested PCR and RFLP (nested PCR-RFLP) method to determine HBV genotype in Indonesia is still less information. The objectives of study were to develop a system for HBV genotyping by nested PCR combined with RFLP (nested PCR-RFLP) method based on nucleotide sequence of surface protein encodinggene (S gene) in HBV genome and to confirm HBV genotypes which predominantly found among hepatitis B patients in Daerah Istimewa Yogyakarta Province and surrounding area. Total of 149 sera from chronic hepatitis B patients from Daerah Istimewa Yogyakarta and surrounding areas were collected for in this work. Viral DNA were extracted from sera of hepatitis B patients and used as template for first round nested PCR amplification using outer primers set. Amplicons of first round PCR were used as template for second round amplification using inner primers set. Then, amplicons of second round nested PCR were restriction digested by Sty I and Bsr I enzymes. For HBV genotyping then the restriction products were analyzed by RFLP based on restriction pattern. Results showed that the first round nested PCR amplification generated DNA fragments of whole S gene in length 1.233 bp, and in second round nested PCR amplification using inner primer set generated DNA fragments 585 bp in length. Genotype analysis for all samples using nested PCR-RFLP methods by restriction digested of Sty I and Bsr I enzymes found only 2 HBV genotypes among hepatitis B patients, namely genotype B and C. Quantificationdata showed that most of hepatitis B patients found infected by HBV genotype B (92,8%), genotype C (3,6%) and unidentified genotype (3,6%). Nested PCR-RFLP methods for HBV genotyping is simple and inexpensive for clinical diagnostic in large scale.

Bayer 41-4109 is heteroarylpyrimidine (HAP) which has been identified as potent of HBV capsid assembly inhibitor. The present study was to study effect of Bayer 41-4109 treatment on the intracellular localization of EGFP-Core fusion proteins into HepG2 cells. Three recombinant plasmids of pEGFP-Core with single, double and triple NLS of HBV core (EGFP-Core 1C, 2C and 3C ) and two recombinant plasmids with single and triple NLS of SV-40 (EGFP-Core 1 and 3 SV-40) were used in this work. After transient transfected into HepG2 cells and treated with Bayer 41-4109, the intracellular localization of expressed fusion proteins from all plasmid constructions were determined and quantified under confocal laser microscope. Results shown that Bayer 41-4109 treatment in HepG2 cells inhibited the nuclear localization of EGFP-Core with single of triple HBV core NLS. As well as the constructions of expressed fusion protein with single and triple SV-40 NLS (EGFP-Core 1 and 3 SV-40 NLS) showed decreasing the nuclear localization after treated with Bayer 41-4109, even not as strong as EGFP-Core 1C and 3C NLS. Bayer 41-4109 has been identified as a potent inhibitors of HBV replication which has multiple effects on HBV capsid assembly. It may inhibit virus replication by inducing assembly inappropriately and by misdirecting assembly decreasing the stability of normal capsids. Keywords: HBV capsid, Bayer 41-4109, EGFP-Core fusion protein, HepG2 cell

protein is also including in the HBV genome targeting into the nucleus through modulating carboxyl residues by phosphorylation. Nuclear localication Signal (NLS) in HBV core protein is inside the virion structure and it must be unmasked in order to function, perhaps by phosphorylation. Phosphorylation of of HBV core protein in turn could begin to alter capsid conformation. Staurosporine is a natural product originally isolated from bacterium Streptomyces staurosporeus. Staurosporine was discovered to have biological activities ranging from anti-fungal to anti-hypertensive. The interest in these activities resulted in a large investigative effort in chemistry and biology and the discovery of the potential for anti-cancer treatment. The main biological activity of Staurosporine is the inhibition of protein kinases through the prevention of ATP binding to the kinase. In the present study, we have studied the intracellular localization of EGFP-Core fusion protein with triple HBV core and SV-40 nuclear localization signal at its carboxyl terminal in presence and absence of Staurosporine. We also to study the effect of Staurosporine treatment on the intracellular localization of EGFP-Core fusion protein in the hepatocyte cells line of HepG2 cell. Results showed that effect of Staurosporine is prevent the nuclear localization of EGFP-Core fusion protein into nucleus through an inhibition of the phosphorylation of core protein. Stauroporine also prevents cell division so that passive trapping of core protein is inhibited.

world. We have evaluated the combination of one-step RT-PCR and multiplex nested PCR assays for detecting dengue viruses from clinical samples. Twelve patients were screened for the dengue virus, using a pair of primers that conserve for several Flavivirus. The results showed that in 12 suspect patients, 100% were positive for Flavivirus and there are some genotypic variation among them, that indicated by several RT-PCR products higher than 511 bp, the expected product for RT-PCR. Further assay was performed to clarify the presence and serotypes of dengue virus using multiplex nested PCR. Serotyping results indicated that 83,3% of samples can be confirmed for dengue virus. Among the dengue virus positive 16,7 % are dengue-2, 16.7 % are dengue-3, and the most common 50% are dengue-4, whereas dengue-1 were not found among the patients. The combination of RT-PCR and multiplex nested PCR assay can be used for rapid analysis dengue samples in early phase which is potentially useful for clinical, epidemiology and also evolutionary studies. Key words: Flavivirus, dengue virus, serotype, RT-PCR, multiplex nested PCR