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"ABSTRAK Keloid muncul akibat proses fibrogenesis berlebih pada proses penyembuhan luka yang ditandai dengan meningkatnya proliferasi sel fibroblas. Sitoglobin (CYGB) merupakan salah satu anggota keluarga protein globin yang berperan penting dalam transpor oksigen di dalam sel. Dalam memenuhi kebutuhan ATPnya untuk proliferasi, fibroblas keloid memerlukan oksigen dalam jumlah yang cukup, sehingga diperlukan CYGB untuk mensuplai oksigen. Untuk membuktikan bahwa CYGB diperlukan oleh fibroblas keloid dilakukan penghambatan ekspresi CYGB menggunakan molekul siRNA. Sampel berasal dari kultur fibroblas keloid dari penelitian sebelumnya yang disimpan beku pada suhu -80ºC. Untuk penelitian ini setelah proses thawing, dilakukan kultur fibroblas dengan menggunakan medium DMEM low glucose. Sampel dibagi menjadi 3 kelompok perlakuan, kontrol; transfeksi dengan siRNA (+) CYGB; trasfeksi dengan siRNA (-) CYGB. Analisis hambatan ekspresi mRNA CYGB dilakukan dengan qRT-PCR dan analisis hambatan protein CYGB dilakukan dengan ELISA. Hasil penelitian menunjukkan terdapat penurunan ekspresi mRNA CYGB pada fibroblas yang ditransfeksi siRNA (+) CYGB dibandingkan dengan kontrol dan siRNA (-) CYGB. Kadar protein CYGB pada trasfeksi siRNA (+) CYGB juga menunjukan penurunan dibandingkan dengan kelompok kontrol dan siRNA (-) CYGB. Disimpulkan bahwa hambatan ekspresi CYGB menggunakan siRNA terbukti menurunkan ekspresi CYGB pada kultur fibroblas keloid.

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ABSTRACT Keloid arises from excessive fibrogenesis in wound healing process which is characterized by increased fibroblast cell proliferation. Cytoglobin (CYGB) is a member of the globin family proteins that play an important role in oxygen transport in cells. To meet its ATP requirements for proliferation, keloid fibroblasts require adequate amounts of oxygen, hence CYGB is needed for oxygen supply. To prove that CYGB is needed by keloid fibroblasts, Cygb expression is inhibited using siRNA molecules. The samples were keloid fibroblasts from previous studies which were stored frozen at -80ºC. After thawing process, fibroblasts were cultured using DMEM low glucose medium. The sample was divided into 3 treatment groups, control group; transfection with CYGB siRNA (+); tranfection with CYGB siRNA (-). Inhibition of mRNA CYGB expression was carried out with qRT-PCR and CYGB protein analysis was carried out by ELISA. The results showed a decrease in CYGB mRNA expression in CYGB siRNA (+) keloid fibroblasts compared to CYGB control and siRNA (-). CYGB protein levels in CYGB siRNA (+) transfection also showed a decrease compared to CYGB control and siRNA (-) group. It was concluded that inhibition of CYGB expression using siRNA was proven to reduce CYGB expression in cultures of keloid fibroblasts.

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"Some diseases, such as cancer, hereditary and genetic diseases, as well as viral infectious diseases, have been treated unsatisfied by the conventional therapy so far, and even more, by the gene therapy. Together sixth the pharmaceutical industries, researchers put their best effort to hunt some molecules that can be mow favorable for such kind of therapy. After a pivotal study reported in May, 2001, it is certain that Ribonucleic acid (RNA) could effectively silence gene expression in mammalian cell line, SQ it was then proposed in 2004 the term RNA therapeutics. Antisense RNA therapy which came into the atage earlier seemed to be the one that can answer all the problems in knocking out the unwanted messenger in gate expression. RNA interference (RNAi) concept, which came later us around 2QOQ, began to look like a possible contender. It was reported some studies that RNAi seem to have some more advantages over hath stronger gene-silencing effects and greater ease of use. However, the main obstacle of all kind of gene therapy is, undoubtedly, on the delivery of this molecule to enter the target eel!, and mostly to where it is needed most inside the body. Some studies on genetic Material delivery system have been reported, and their progress has been discussed."

"Latar Belakang: Penyakit avian influenza subtipe H5N1 Asian lineage yang mulai mewabah di kawasan Asia, Afrika dan Eropa sejak tahun 1997 selain menimbulkan kerugian ekonomi yang sangat signifikan juga mengancam aspek kesehatan manusia dimana sejumlah korban meninggal dunia karena infeksi virus yang bersifat zoonosis. Penanganan penyakit dilakukan dengan antiviral yang berbasis neuraminidase inhibitor. Permasalahan timbul sebagai akibat mutasi beberapa strain virus menjadi resisten terhadap antiviral yang ada. Penelitian ini bertujuan untuk melakukan desain antiviral alternatif berbasis siRNA terhadap gen nucleoprotein yang lebih sesuai terhadap virus avian influenza subtipe H5N1 yang bersirkulasi di Indonesia. Metode: Desain siRNA dilakukan secara in silico dengan program siDirect 2.0 berdasarkan 210 sekuen gen nucleoprotein virus H5N1 yang bersirkulasi di Indonesia. Dua kandidat siRNA-NP672 dan siRNA-NP1433 dipilih berdasarkan kajian bioinformatik. Selanjutnya, kedua kandidat siRNA-NP tersebut ditantang secara in vitro pada sel Mabin-Darby canine kidney (MDCK) terhadap virus H5N1 asal Indonesia clade 2.1.3 dan 2.3.2 dengan menggunakan siRNA-NP1496 sebagai pembanding. Paramater yang diamati adalah produksi virus dan ekspresi gen virus. Terakhir, analisa mutasi gen nucleoprotein virus H5N1 dilakukan untuk melihat paparan siRNA-NP secara berulang kali. Hasil: Kandidat siRNA-NP672 memberikan efek penurunan infeksi virus H5N1 yang lebih baik dalam menurunkan tingkat infeksi virus HPAI subtipe H5N1 baik clade 2.1.3 dan 2.3.2 secara in vitro pada sel MDCK yang dicerminkan dengan titer produksi virus dibandingkan dua desain lainnya yaitu siRNA-NP1433 dan siRNA-NP1496. Pemberian siRNA-NP672 juga memberikan efek peredaman yang lebih tinggi dan konsisten terhadap ekspresi gen-gen virus, antara lain nucleoprotein, polymerase acidic, hemagglutinin, neuraminidase, Matrix, dan non-structural. Hasil kajian bioinformatik terhadap struktur sekunder dan tersier RNA gen nucleoprotein menunjukkan bahwa target siRNA-NP672 lebih berinteraksi karena memiliki bagian bebas (loop) yang lebih banyak dibandingkan dua kandidat siRNA-NP lainnya. Selanjutnya, paparan siRNA-NP tidak memicu terjadinya mutasi gen target pada virus H5N1 baik clade 2.1.3 dan clade 2.3.2 setelah 3 kali paparan. Kesimpulan: Desain siRNA-NP672 menunjukkan prospek yang lebih baik dalam menurunkan tingkat infeksi virus avian influenza subtipe H5N1 baik clade 2.1.3 dan clade 2.3.2.

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Introduction: Avian influenza disease outbreak of subtype H5N1 Asian lineage that has spread in Asia, Africa, and European continental since 1997 caused massive economic drawbacks as well as a zoonotic threat where numerous deaths related to viral infection. The treatment of viral infection has been done with antiviral based on neuraminidase inhibitors. However, mutation of numerous virus strains has been confirmed that may lead to resistance against current antivirals. This study's objective was to design an alternative antiviral based on siRNA targeting nucleoprotein gene that is more suitable for the avian influenza viruses subtype H5N1 circulating in Indonesia. Methods: The siRNA design was accomplished in silico using the siDirect 2.0 program based on 210 nucleoprotein gene sequences of H5N1 viruses circulating in Indonesia. Two siRNA candidates (siRNA-NP672 and siRNA-NP1433) were chosen based on bioinformatic analyses. Subsequently, these siRNA-NP candidates were challenged in vitro in Mabin-Darby canine kidney cell culture against the Indonesian H5N1 both clade 2.1.3 and clade 2.3.2 using siRNA-NP1496 as a comparison. The parameters analyzed within the study are including virus production and viral gene expression level. Finally, mutation analysis was performed to evaluate the effect of three serial siRNA-NP exposures to the target gene of the H5N1 viruses. Results: The siRNA-NP672 provides a better reduction of the H5N1 viral infection, especially on viral production titer for both clade 2.1.3 and clade 2.3.2 compared to the two other siRNA candidates, including siRNA-NP1433 and siRNA-NP1496. The siRNANP672 also provides a better and more consistent reduction of viral gene expression levels, including nucleoprotein, polymerase acidic, hemagglutinin, neuraminidase, Matrix, dan non-structural. This finding was confirmed by bioinformatic analyses of the siRNA-NP672 biding site in the secondary and tertiary structure of the nucleoprotein gene which has more free parts (loop) compared to the two other siRNA-NP candidates. Subsequently, three serial exposures of siRNA-NP do not induce any mutation on the target site of the nucleoprotein gene of the H5N1 virus both clade 2.1.3 and 2.3.2. Conclusion: The design of siRNA-NP672 provides a better prospect to reduce the Indonesian avian influenza virus subtype H5N1 infection for both clade 2.1.3 and 2.3.2."

"Latar belakang: Pada penelitian sebelumnya, kami menemukan ekspresi sitoglobin Cygb pada keloid meningkat dibandingkan kulit normal, yang disertai dengan tingkat proliferasi sel fibroblas yang tinggi. Sitoglobin dilaporkan memiliki peran sebagai penangkal ROS yang dibutuhkan pada proses proliferasi sel. Di sisi lain, beberapa penelitian telah melaporkan ambiguitas dari peran Cygb, baik sebagai tumor supresor maupun onkogen. Oleh karena itu, penelitian ini bertujuan untuk mengetahui efek hambatan ekspresi gen Cygb terhadap proliferasi dan kadar ROS sel fibroblas keloid menggunakan small interfering RNA siRNA .Metode: Kami mengukur ekspresi mRNA dan tingkat protein Cygb menggunakan qRT-PCR dan ELISA, proliferasi sel menggunakan metode MTS, dan tingkat ROS menggunakan uji DCFHDA pada 3 kelompok yaitu kontrol, siRNA Cygb dan siRNA negatif. Hasil dari ketiga kelompok tersebut dibandingkan secara statistik. Kami juga menganalisis korelasi antara masing-masing variabel.Hasil: Tingkat ekspresi Cygb pada kelompok siRNA Cygb menurun dibandingkan dengan kelompok kontrol dan siRNA negatif. Sedangkan proliferasi sel dan tingkat ROS intraseluler meningkat sedikit namun signifikan pada kelompok siRNA Cygb dibandingkan dengan kelompok kontrol dan siRNA negatif. Tidak terdapat korelasi antara ekspresi Cygb dengan proliferasi sel, namun terdapat korelasi antara ekspresi Cygb dengan tingkat ROS, dan tingkat ROS dengan proliferasi sel.Kesimpulan: Pada sel fibroblas keloid, hambatan ekpresi gen Cygb menyebabkan peningkatan proliferasi sel dan kadar ROS intraseluler.

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Background In our previous work, we found the expression of Cytoglobin Cygb in keloid were significantly higher than those in normal skin, which accompanied by a high rate of fibroblasts cells proliferation. Cytoglobin is reported to have a role as ROS scavenger, which is required in cell proliferation. On the other hand, some studies have reported ambiguity role of Cygb, either as a tumor suppressor or oncogene. Therefore, we plan to elucidate the role of Cygb in the regulation of ROS and the proliferation of keloid fibroblast using small interfering RNA siRNA .Methods We measured mRNA expression and Cygb protein level using qRT PCR and ELISA, cell proliferation using MTS method, and ROS level using DCFHDA assay on 3 groups control group, siRNA Cygb group and siRNA negative group. The results of the three groups were compared statistically. We also analyzed the correlation between each variable.Results The expression level of Cygb on siRNA Cygb group were decreased compared to the control and siRNA negative group. Whereas the cells proliferation and intracellular ROS levels were increased slightly but significant in siRNA Cygb compared to control and siRNA negative group. There is no correlation between Cygb expression with cell proliferation, but there is a correlation between Cygb expression with ROS level, and ROS level with cell proliferation.Conclusion In keloid fibroblast cells, inhibition of Cygb gene expression leads to increased cell proliferation and intracellular ROS levels."

"Resistensi terhadap antibiotik di bidang kesehatan dapat dialami juga oleh antimikroba yang digunakan di bidang pangan. Selama ini di bidang pangan digunakan bakteriosin sebagai antimikroba. Bakteri penghasil bakteriosin terlindungi dari bakteriosin yang dihasilkannya karena memiliki bacteriocin immunity protein (bip). Gen bakteriosin disandikan bersama dengan gen imunitasnya dalam kondisi yang disebut sebagai quorum sensing. Pada penelitian sebelumnya, telah dilakukan konfirmasi uji aktivitas bacteriocin like inhibitory substance (BLIS) Weissella confusa MBF 8-1 terhadap beberapa bakteri patogen. Tujuan penelitian ini adalah mempelajari metode gene silencing dengan menggunakan model aktivitas BLIS dan bip.Pada penelitian ini dirancang sekuens siRNA bip dengan menggunakan informasi data sekuens dari basis data dan dari data whole genome sequence galur model Weissella confusa MBF8-1. Untuk membuktikan aktivitas gene silencing dari siRNA sintetik tersebut secara in vivo terhadap inang MBF 8-1 maka dilakukan esei zona hambat. Hasil rancangan siRNA yang diperoleh hanya menarget pada gene silencing di MBF 8-1. Berdasarkan analisis algoritma dan BLAST berhasil diperoleh rancangan siRNA kandidat utama, yaitu bip-a MBF 8-1_1 yang secara in vivo menunjukkan aktivitas gene silencing yang poten terhadap inangnya.

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Resistance to antibiotics in the health sector can be experienced by antimicrobial in the food industry. During this period, food industry has used bacteriocin as an antimicrobial. Bacteria is protected from its bacteriocin because it has bacteriocin immunity protein (bip). Bacteriocin gene is encoded with its immunity protein gene on a condition which was called as quorum sensing. In previous study, Weissella confusa MBF 8-1 possess Bacteriocin Like Inhibitory Substance (BLIS) activity against several pathogen bacteria. This study aimed to study gene silencing method using BLIS and bip activity as model.On this study siRNA bip sequence was designed using information from sequence database and model Weissella confusa MBF 8-1 whole genome sequence database. Disc dilution method was done to prove gene silencing activity of synthetic siRNA against its own host MBF 8-1. Result revealed that siRNA design is aimed as a gene silencing agent against MBF 8-1 alone. Based on algorithm analysis and BLAST, top rank bip-a MBF 8-1_1 siRNA design is potent and has proved its gene silencing activity against its own host."