In Silico Analysis of Actin Gene as a Candidate for DNA Non-Halal Detection Base on Real-Time PCR

Actin genes are genes that are common in organisms, and their expression is constitutive. These genes are used for gene normalization and internal control of DNA extraction, but the actin gene is not widely used for halal certification tests. Bioinformatic studies help to analyze the experiment through in silico more deeply before the experiment is carried out in laboratory, making it more efficient and time effective. uMelt is an analysis to predict the melting curve of target amplification in real-time PCR. Real-time PCR has been widely used for screening and detection of pork content in a product. This research aimed to explore actin gene as a candidate for testing pork using qPCR. The study was carried out in two main stages, namely alignment of the DNA sequence and analysis of the melting curve using the uMelt approach. The results showed a set of actin genes containing conserved regions that can be used as degenerate primers with different family-type coverages. Melting curve prediction with uMelt shows differences in tm peaks so as the types of samples can be easily identified. The use of bioinformatic applications such as uMelt helps in the simulation of predicting the melting curve to increase the precision of the analysis

Deteksi dan Kuantifikasi Cemaran Babi pada Sampel Olahan Daging Menggunakan Real-time PCR

 AbstrakMetode pengujian cemaran babi menjadi faktor penting dalam sertifikasi produk halal. Metode yang cepat dan robust diperlukan untuk deteksi dan kuantifikasi cemaran babi. Metode Real-time PCR atau dikenal dengan istilah quantitative PCR (qPCR) merupakan metode alternatif untuk deteksi dan kuantifikasi cemaran babi berdasarkan residu keberadaan DNAnya pada sampel olahan pangan. Metode ekstraksi DNA dan kit amplifikasi yang tahan terhadap inhibitor menjadi kunci keberhasilan penggunaan qPCR untuk pendeteksian dan kuantifikasi cemaran babi. Pendeteksian cemaran DNA dengan probe qPCR digunakan karena mempunyai kelebihan tahan terhadap inhibitor, cepat, spesifik, dan multipel target. Penelitian ini bertujuan untuk mendeteksi dan menguantifikasi cemaran DNA babi menggunakan metode ekstraksi DNA secara cepat dan qPCR. Tahapan penelitian ini adalah ekstraksi DNA, amplifikasi, deteksi, dan kuantifikasi DNA babi. Sampel berasal dari produk olahan pangan, seperti bakso, sosis, daging burger, siomay, kuah daging, dan daging isi roti. Hasil penelitian menunjukkan bahwa terdapat cemaran babi pada sampel bakso, daging burger, dan kuah bakso. Hasil yang didapatkan menunjukkan bakso memiliki persentase kontaminasi sejumlah 25%, sedangkan kuah daging sejumlah 12,5%. Hasil penelitian ini dapat direkomendasikan untuk laboratorium penguji makanan sebagai metode deteksi cemaran babi dalam produk pangan secara cepat dan akurat.AbstractPork contamination testing method is an important factor in halal product certification. A fast and robust method is needed for the detection and quantification of pig contamination. Real-time PCR method or commonly known as quantitative PCR (qPCR) is an alternative method for the detection and quantification of pork contamination based on the pig’s DNA residual presence in processed food samples. DNA extraction method and inhibitor-resistant amplification kit are the keys of successful qPCR implementation for the detection and quantification of pig contamination. Detection of DNA contamination with qPCR probe is used because it has some advantages, such as resistant to inhibitors, fast, specific, and multiple targets. This research aimed to detect and quantify pig’s DNA contamination using rapid DNA extraction method and qPCR. The stages of this research were pig’s DNA extraction, amplification, detection, and quantification. The samples taken from processed food products, such as meatballs, sausage, burgers’ meat, dumplings, meat broth, and meat filled in the bread. The results showed that there was pork contamination in the samples of meatballs, burgers’ meat, and meat broth. The results showed that the meatballs had a contamination percentage of 25%, while the meat broth had a contamination percentage of 12.5%. The results of this study can be a recommendation for food testing laboratories as a method of detecting the pork contamination in food products quickly and accurately

NS1’ Protein Expression in the JaOArS982 Strain of Japanese Encephalitis Virus Does Not Enhance Virulence in Mice

Using a mouse model, we previously demonstrated that subcutaneous infection with the JaTH160 strain of Japanese encephalitis virus (JEV) causes significantly higher virulence and stronger virus propagation in the brain compared with that of the JaOArS982 strain. We also showed that the JaTH160 strain, but not JaOArS982, expresses the NS1’ protein and that NS1’ enhances JEV production in avian cells and embryonated chicken eggs. In this study, we examined whether NS1’ expression affects virulence in mice infected with the JaOArS982 and JaTH160 strains using the corresponding recombinant viruses S982-IC and JaTH-IC. Expression of the NS1’ protein in S982-IC diminished the mortality in mice, whereas S982-IC viruses without NS1’ caused 40?60% mortality. However, the viral loads in the brains of these mice were not significantly different despite the dvariation in NS1’ expression. JaTH-IC viruses depleted of the NS1’ protein exhibited high mortality levels, similar to those of the virus expressing NS1’. Previous studies showed that the NS1’ protein plays a role in the enhanced virulence of the JEV SA14 strain in mice. However, our current data suggest that NS1’ protein expression in S982-IC reduces, rather than enhances, the mortality in mice. Thus, the effect of NS1’ on pathogenicity in vivo may vary among virus strains. Our data also suggest that the reduced mortality resulting from NS1’ expression in S982-IC is not simply due to viral replication in the brains. Further investigation is needed to uncover the mechanism by which NS1’ affects pathogenicity in JEV-infected animals

Identification of novel antiviral of fungus-derived brefeldin A against dengue viruses

Microbial natural products possess a wide range of biological and biochemical potential. Among them, fungal secondary metabolites are one of the most important sources for discovering new drugs or lead compounds. In the present study, we explored substances produced by the strain Penicillium sp. FKI-7127 for its antiviral activity. We identified brefeldin A as a novel antiviral agent against dengue viruses. The inhibitory effect of brefeldin A was confirmed by virus titer and immunofluorescence assay. Brefeldin A inhibited dengue viruses regardless of serotypes and other related viruses including Zika virus and Japanese encephalitis virus. Time-of-addition study showed that brefeldin A exerts its antiviral effect at an early stage of the dengue virus (DENV) life cycle. These studies demonstrate that (i) brefeldin A could be used as a lead compound for drug development of anti-DENV and other related viruses and (ii) fungal metabolites are a potential and valuable source for dengue virus drug discovery

Incubation of Denatured Samples Increased Reproducibility and Quality of Proteomic Profile of SELDI-TOF MS

Protein profiling with high-throughput proteomic technology, SELDI-TOF, is a new potential tool for diagnosis of human diseases. This advanced technique has increasingly been used for the detection of disease biomarker. However, analytical reproducibility is a significant challenge in SELDI-TOF profiling in order to have confidence in the results.  Here, we showed a simple step to improve its analytical performance. Incubation of denaturated samples overnight at 4oC increased significantly reproducibility and quality of proteomic profile of SELDI-TOF MS for IMAC30-Cu ProteinChip. This strategy could be concerned and apply to address reproducibility issue in this system.&nbsp

Inhibitory effect of the green tea molecule EGCG against dengue virusinfection

AbstractDengue virus (DENV) infection is a major public health problem worldwide; however, specific antiviral drugs against it arenot available. Hence, identifying effective antiviral agents for the prevention of DENV infection is important. In this study,we showed that the reportedly highly biologically active green-tea component epigallocatechin gallate (EGCG) inhibiteddengue virus infection regardless of infecting serotype, but no or minimal inhibition was observed with other flaviviruses,including Japanese encephalitis virus, yellow fever virus, and Zika virus. EGCG exerted its antiviral effect mainly at theearly stage of infection, probably by interacting directly with virions to prevent virus infection. Our results suggest thatEGCG specifically targets DENV and might be used as a lead structure to develop an antiviral drug for use against the virus

Incubation of Denatured Samples Increased Reproducibility and Quality of Proteomic Profile of SELDI-TOF MS

Protein profiling with high-throughput proteomic technology, SELDI-TOF, is a new potential tool for diagnosis of human diseases. This advanced technique has increasingly been used for the detection of disease biomarker. However, analytical reproducibility is a significant challenge in SELDI-TOF profiling in order to have confidence in the results. Here, we showed a simple step to improve its analytical performance. Incubation of denaturated samples overnight at 4o C increased significantly reproducibility and quality of proteomic profile of SELDI-TOF MS for IMAC30-Cu ProteinChip. This strategy could be concerned and apply to address reproducibility issue in this system

Dengue Virus neither Directly Mediates Hyperpermeability nor Enhances Tumor Necrosis Factor-α-Induced Permeability In Vitro

The mechanisms of endothelial barrier dysfunction in dengue disease remain poorly understood. Endothelial cell (EC) death due to virus infection or in combination with an infection-induced cytokine storm is deemed as one of the major causes of plasma leakage. Using an in vitro model of human endothelia and several dengue virus (DENV) strains (including a clinical isolate), the direct consequence of infection on endothelial permeability was investigated throughout the course of the infection. All employed DENV-2 strains were able to infect and replicate in ECs. Rather than increase endothelial permeability, DENV infection alone enhanced cell barrier integrity up to 7 days postinfection. Improved cell barrier function was mediated by type I interferon activation at the early phase of infection and by the survival advantage of the infected cells at the late phase of infection. Consistent with this phenomenon, DENV infection did not augment tumor necrosis factor-α-induced permeability. Our results prove that DENV infection does not directly account for vascular permeability; DENV neither induces hyperpermeability nor exacerbates the permeabilizing effect of cytokines. The contributory role of other factors on plasma leakage during dengue disease warrants further investigation

デングウイルスの感染はそれのみで血管の透過性を亢進する作用はなくTNF-αの透過性亢進作用を増強することもない

The mechanisms of endothelial barrier dysfunction in dengue disease remain poorly understood. Endothelial cell (EC) death due to virus infection or in combination with an infection-induced cytokine storm is deemed as one of the major causes of plasma leakage. Using an in vitro model of human endothelia and several dengue virus (DENV) strains (including a clinical isolate), the direct consequence of infection on endothelial permeability was investigated throughout the course of the infection. All employed DENV-2 strains were able to infect and replicate in ECs. Rather than increase endothelial permeability, DENV infection alone enhanced cell barrier integrity up to 7 days postinfection. Improved cell barrier function was mediated by type I interferon activation at the early phase of infection and by the survival advantage of the infected cells at the late phase of infection. Consistent with this phenomenon, DENV infection did not augment tumor necrosis factor-α-induced permeability. Our results prove that DENV infection does not directly account for vascular permeability; DENV neither induces hyperpermeability nor exacerbates the permeabilizing effect of cytokines. The contributory role of other factors on plasma leakage during dengue disease warrants further investigation.長崎大学学位論文 学位記番号:博(医歯薬)甲第635号 学位授与年月日:平成25年10月2日Author: Muhareva Raekiansyah, Lyre Anni Espada-Murao, Kenta Okamoto, Toru Kubo, Kouichi MoritaCitation: Japanese Journal of Infectious Diseases, 67(2), pp.86-94; 2014Nagasaki University (長崎大学)課程博