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"Cathepsin dan calpain adalah anggota sistein protease yang merupakan enzim proteolitik yaitu enzim yang mengkatalisis pemecahan protein melalui hidrolisis ikatan peptida. Hidrolisis ikatan peptida pada ribuan protein baik di dalam maupun di luar sel berfungsi untuk mengontrol dinamika turn-over protein. Cathepsin merupakan enzim keluarga papain yang terletak di lisosom. Enzim tersebut memiliki 11 anggota yang berperan dalam pencernaan, presentasi antigen serta remodeling matriks. Calpain adalah kelompok sistein protease sitoplasmik yang aktivitasnya tergantung kalsium. Calpain mempunyai implikasi dalam proteolisis sejumlah protein intraseluler yang berhubungan dengan peningkatan kalsium intraseluler. Aktivasi protease tersebut berhubungan dengan pengikatan membran yang diikuti autolisis. Protein regulator seperti protein kinase C, actin-binding protein dan integrin sitoplasmik mengalami pemotongan oleh calpain, maka calpain dianggap berperan dalam proses signaling seluler.

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Cathepsin and calpain are cystein protease family as proteolytic enzyme that catalize breakdown of protein through hydrolysis of peptide bonds. Hydrolysis of peptide bond in many thousand proteins both intracellular and extracellular regulates the protein turnover. Cathepsin is papain family enzyme which is located in lysosome. This enzyme has 11 members that involve in digestive, antigen presenting and matrix remodeling, whereas calpain is cytoplasmic cystein protease and its activity is calcium dependent. Calpain has a role in proteolytic of intracellular protein related with calcium enhancement. Activation of that protease seems to correlate with membrane binding following autolysis. Some of regulator protein such as C kinase protein, actin- binding protein and cytoplasmic integrin are cut by calpain, therefore calpain has a role in cellular signaling process."

"PendahuluanPeriodontitis adalah penyakit pada jaringan penyangga gigi yang dikategorikan sebagai inflamasi tidak menular dan berkaitan dengan keadaan disbiosis biofilm. Penyakit tersebut memengaruhi seluruh jaringan periodontal dan dapat menyebabkan destruksi progresif pada tulang alveolar. Periodontitis dapat dipicu oleh bakteri seperti Aggregatibacter actinomycetemcomitans yang selanjutnya mempengaruhi osteoklastogenesis dan terjadinya kerusakan jaringan. Internalisasi dan proliferasi bakteri A. actinomycetemcomitans di dalam sel osteoklas dapat meningkatkan faktor virulensi seperti lipoposakarida (LPS) yang berperan dalam diferensiasi osteoklas yang ditandai dengan gen penanda, salah satunya Cathepsin K (CTSK). Penelitian ini bertujuan untuk menganalisis diferensiasi osteoklas melalui analisis ekspresi gen penanda diferensiasi osteoklas yaitu CTSK.MetodeOsteoklas (OC) diperoleh dari kultur primer bone marrow macrophage (BMM), yang dipaparkan dengan nuclear factor kappa-B ligand (RANKL) selama 3 hari. Kemudian diinfeksikan dengan bakteri A. actinomycetemcomitans (ATCC 29522) dengan perbandingan MOI 1:1 dan 1:5. Selanjutnya dievaluasi 1,5 jam dan 18 jam pasca infeksi (hpi) menggunakan RT-qPCR dengan teknik Livak (2-∆∆Ct).HasilEkspresi relatif gen CTSK pada BMM dengan perlakuan MOI 1:1 pada 1,5 hpi (2-∆∆Ct = 1,00) dan 18 hpi (2-∆∆Ct = 0,99), serta perlakuan MOI 1:5 pada 1,5 hpi (2-∆∆Ct = 1,00) dan 18 hpi (2-∆∆Ct = 1,04) cenderung tidak menunjukan adanya perubahan. Sedangkan pada OC dengan MOI 1:1 pada 1,5 hpi (2-∆∆Ct = 1,00) dan 18 hpi (2-∆∆Ct = 1,64), serta perlakuan MOI 1:5 pada 1,5 hpi (2-∆∆Ct = 1,00) dan 18 hpi (2-∆∆Ct = 3,27) cenderung menunjukan adanya peningkatan pada 18 hpi. Perbandingan kelompok OC 18 hpi pada perlakuan MOI 1:5 (2-∆∆Ct = 4,26) menunjukkan peningkatan ekspresi gen CTSK sekitar 4 kali dibanding perlakuan MOI 1:1 (2-∆∆Ct = 1,00).KesimpulanPeningkatan ekspresi gen CTSK pada osteoklas berkorelasi positif dengan jumlah bakteri yang menginfeksi dan waktu paparan bakteri dengan sel osteoklas. Peningkatan ekspresi CTSK tersebut diduga berhubungan dengan terjadinya internalisasi bakteri kedalam sel osteoklas.

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Introduction

Periodontitis is a disease affecting the supportive tissues of the teeth, categorized as a non-communicable inflammation associated with dysbiosis of the biofilm. This disease impacts the entire periodontal tissue and can cause progressive destruction of alveolar bone. Periodontitis can be triggered by bacteria such as Aggregatibacter actinomycetemcomitans, subsequently affecting osteoclastogenesis and tissue damage. The internalization and proliferation of A. actinomycetemcomitans bacteria inside osteoclast cells can directly increase virulence factors such as lipopolysaccharide (LPS), playing a role in osteoclast differentiation marked by genes, including Cathepsin K (CTSK). This study aims to analyze osteoclast differentiation through the analysis of the marker gene expression for osteoclast differentiation, namely CTSK.

Methods

Osteoclasts (OC) were obtained from primary cultures of bone marrow macrophages (BMM), exposed to nuclear factor kappa-B ligand (RANKL) for 3 days. They were then infected with A. actinomycetemcomitans bacteria (ATCC 29522) with a ratio of MOI 1:1 and 1:5. Subsequently, they were evaluated at 1.5 hours and 18 hours post-infection (hpi) using RT-qPCR with the Livak method (2-∆∆Ct).

Results

The relative expression of CTSK gene in BMM cells with MOI 1:1 treatment at 1.5 hpi (2-∆∆Ct = 1.00) and 18 hpi (2-∆∆Ct = 0.99), as well as MOI 1:5 treatment at 1.5 hpi (2-∆∆Ct = 1.00) and 18 hpi (2-∆∆Ct = 1.04), tended to show no significant changes. In OC with MOI 1:1 treatment at 1.5 hpi (2-∆∆Ct = 1.00) and 18 hpi (2-∆∆Ct = 1.64), as well as MOI 1:5 treatment at 1.5 hpi (2-∆∆Ct = 1.00) and 18 hpi (2-∆∆Ct = 3.27), there was a tendency to increase at 18 hpi. The comparison of OC groups at 18 hpi with MOI 1:5 (2-∆∆Ct = 4.26) showed a fourfold increase in CTSK gene expression compared to MOI 1:1 treatment (2-∆∆Ct = 1.00).

Conclusion

The increased expression of the CTSK gene in osteoclasts positively correlates with the number of infecting bacteria and the duration of bacterial exposure to osteoclast cells. This heightened CTSK expression is presumed to be associated with the internalization of bacteria into osteoclast cells."

"Pendahuluan: Aktivitas proliferasi, sintesis kolagen, dan hidrasi kulit akan menurun seiring proses penuaan kulit, sehingga kulit menua menjadi kusam, kendur, dan kering. Aquaporin-3 (AQP3) adalah protein kunci yang berperan pada proliferasi dan hidrasi keratinosit, sekarang menjadi target inovasi pengembangan kosmetika pelembab anti penuaan kulit. Penelitian ini bertujuan menganalisis kombinasi dua bahan alam yaitu ekstrak etanol Centella asiatica dan nanopartikel kitosan (EECA+NPK) terhadap proliferasi sel fibroblast dan keratinosit, sintesis kolagen I dan III serta ekspresi protein aquaporin-3 (AQP3) secara in vitro.Metode: Dilakukan uji proliferasi sel fibroblas dan keratinosit yang dianalisis dengan uji Microculture Tetrazolium (MTT), analisis sintesis kolagen I dan III menggunakan Enzyme Linked Immunosorbent Assay (ELISA) kit (COL1A1 dan COL3A1) setelah dipajankan dengan ekstrak etanol Centella asiaticadalam nanopartikel kitosan (EECA + NPK) pada beberapa konsentrasi selama 24, 48, dan 72 jam dibandingkan dengan asam retinoat, selanjutnya ekspresi aquaporin-3 (AQP3) dianalisis dengan teknik Imunositokimia menggunakan antibodi anti-aquaporin3 ab125219, kemudian dianalisis secara kuantitatif menggunakan ImageJ software.Hasil: EECA + NPK dapat meningkatkan proliferasi fibroblas 1.6 kali lipat dibandingkan kontrol. Optimal pada konsentrasi 6.25 mg/mL dan proliferasi sel keratinosit optimal pada konsentrasi 3.125 mg/mL, secara statistic tidak berbeda bermakna dengan AR. (EECA + NPK) dapat meningkatkan sintesis kolagen I setelah pajanan 72 jam dan kolagen III setelah pajanan 48 jam, secara statistic tidak berbeda bermakna dengan AR. Uji imunositokimia dengan antibodianti-aquaporin3 ab125219 (EECA + NPK) dapat meningkatkan ekspresi aquaporin 3 (AQP 3) pada sel fibroblas optimal pada konsentrasi 12.5 mg/mL dan sel keratinosit pada konsentrasi3.125 mg/mL setelah pajanan selama 24 jam, secara statistik berbeda dengan AR.Kesimpulan: Ekstrak etanol Centella asiatica dalam nanopartikel kitosan (EECA + NPK) dapat meningkatkan proliferasi fibroblas dan keratinosit, meningkatkan sintesis kolagen I dan III, serta ekspresi protein AQP3 pada sel fibroblas dan sel keratinosit.

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Introduction: Proliferation activity, collagen synthesis, and hydration of the skin will decrease with the process of aging, therefore, skin looks dull, sagging, and dry. Aquaporin-3 (AQP3) is a key protein that plays a role in keratinocyte proliferation and hydration, recently becomes the target of innovation development of anti-aging cosmetic moisturizer. This research aims to analyze a combination of two natural ingredients, Centella asiaticaethanolic extractand chitosan nanoparticles (EECA + NPK) to increased proliferation fibroblast cell and keratinocyte, synthesis type I and III collagen, and the expression of AQP3 in vitro.Methods: Microculture Tetrazolium Test was conducted to analyze the proliferation of fibroblast and keratinocyte. The synthesis of type I and III collagen in fibroblast were analyzed using Ezyme Linked Immunosorbent Assay (ELISA) kit (COL1A1 and COL3A1) after the exposure of CAEE + CNP in several concentrations for 24, 48, 72 hours and compared to Retinoic acid (RA). The expression of AQP 3 on fibroblast and keratinocyte was analyzed using antibody anti-aquaporin 3 ab125219 immunocytochemistry technique, then quantitively analyzed using Image-J software.Results: CAEE+ CNP increased the proliferation of fibroblas optimal result at 6.25mg/mL concentration and the proliferation of keratinocyte increased 1.5 time than control, optimal result at 3.125 mg/mL concentration, statistically were not significant different with RA.CAEE + CNP increased the synthesis collagen type I optimal after incubated for 72hours and the synthesis collagen type III optimal 48 hours, statistically were not significant different with RA.Using antibody anti-aquaporin 3 ab125219 immunocytochemistry examination indicated CAEE+ CNP increased the expression of AQP 3 on fibroblast and keratinocyte, optimal results at 12.5 mg/mL and 3.125 mg/mL respectively after 24 hours exposure.Conclusion: CAEE + CNP increased the proliferation of fibroblast and keratinocyte, synthesis of type I and III collagen, and the expression of AQP3 in fibroblast and keratinocyte;Introduction: Proliferation activity, collagen synthesis, and hydration of the skin will decrease with the process of aging, therefore, skin looks dull, sagging, and dry. Aquaporin-3 (AQP3) is a key protein that plays a role in keratinocyte proliferation and hydration, recently becomes the target of innovation development of anti-aging cosmetic moisturizer. This research aims to analyze a combination of two natural ingredients, Centella asiaticaethanolic extractand chitosan nanoparticles (EECA + NPK) to increased proliferation fibroblast cell and keratinocyte, synthesis type I and III collagen, and the expression of AQP3 in vitro."

"ABSTRAKSalah satu tanaman obat Indonesia yang berkhasiat sebagai antiinflamasi adalah mengkudu. Tujuan: Mengevaluasi perubahan protein total dan profil protein selHaCaT terinflamasi setelah dipapar ekstrak etanol buah mengkudu. Metode: Sel HaCaT terinflamasi LPS dipaparkan ekstrak etanol buah mengkudu 1%, 10%, dan 40%. Medium kultur dipisahkan dan protein sel diekstraksi menggunakan reagen Trizol. Protein sel dan protein dalam medium kultur diuji Bradford dan profil protein dievaluasi menggunakan metode SDS PAGE. Hasil dan Kesimpulan: Terdapat perubahan konsentrasi protein total dan jumlah pita protein medium dan sel pada semua kelompok perlakuan dan kelompok sel terinflamasi dibanding dengankelompok kontrol.

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ABSTRACTOne of Indonesian herb plant that reported has antiinflammations properties is noni.Objectives: To evaluate the changes of total protein and protein profile in inflamed HaCaT cell after ethanol extract of noni fruit’s exposure. Methods: Inflammed HaCaT cell culture are added by ethanol extract of noni fruits 1%, 10%, and 40%. The culture mediums are separated from the cell while the cell is extracted using Trizol reagent. Then, followed by Bradford assay and analized using SDS PAGE.Result and Conclussion: There are changes on the consentration of total proteins and the protein profile in the medium and cell of inflammed and treatment group compared to the control group."

"Plasmid penyandi protein fusi hemaglutinin dengan VP22, pcDNA3.1HAΔTMVP22, merupakan kandidat vaksin DNA yang diharapkan dapat menjadi salah satu alternatif vaksin pencegahan flu burung. Kemampuan kandidat vaksin untuk mengekspresikan antigen yang diharapkan perlu untuk dibuktikan sebelum diujikan pada hewan coba mencit. Ekspresi antigen tersebut diuji dengan cara mentransformasikan kandidat vaksin pada sel kultur mamalia. Penelitian ini bertujuan untuk mengetahui sistem ekspresi yang terdapat dalam kandidat vaksin pcDNA3.1HAΔTMVP22 dapat berfungsi seperti yang diharapkan. Dalam penelitian ini dilakukan transfeksi pcDNA3.1HAΔTMVP22 pada sel ovarium hamster cina (CHO K1) dan analisis ekspresi plasmid penyandi protein fusi hemaglutinin dengan VP22 dilakukan dengan uji imunostaining dan western blot. Setelah diinkubasi selama dua hari dilakukan imunostaining untuk mengetahui ekspresi protein hemaglutinin dan western blot terhadap supernatan kultur untuk mengetahui protein rekombinan yang dilepaskan di supernatan. Pengamatan imunostaining dengan mikroskop konvokal, menunjukkan bahwa protein rekombinan hemaglutinin-VP22 diekspresikan oleh sel CHO K1. Berdasarkan hasil uji western blot, keberadaan protein rekombinan di supernatan kultur belum dapat terdeteksi, karena antibodi yang digunakan dalam uji ini mengenali serum yang merupakan salah satu komponen media penumbuhan sel CHO K1.

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Plasmid encoding the fusion protein hemagglutinin with VP22, pcDNA3.1HAΔTMVP22, is one of DNA vaccine candidates which expects to be an alternative vaccine avian influenza prevention. The ability of candidate vaccines to express antigens is important to prove before tested in experimental animals mice. The antigen expression was tested by transforming vaccine candidate into mammalian cultured cells. The aims of this study is to determine the capability of vaccine candidate pcDNA3.1HAΔTMVP22 to express antigen once transformed into mammalian cell line. In this research, pcDNA3.1HAΔTMVP22 was transfected into Chinese Hamster Ovary (CHO K1) cells. Protein expression was measured by imunostaining and western blot assay. Immunostaining was performed to determine the expression of hemagglutinin protein inside CHO KI cells; the imunostained cells were observed using confocal microscope. The result showed if fusion protein hemagglutinin-VP22 can be produce in CHO K1 cells and can be recognized by rabbit H5N1 polyclonal antibody. Western blot was performed to determine the presence of recombinant protein in supernatant culuture. By using western blot assay performed in this study the presence of recombinant protein in the culture supernatant can not be detected. Protein band found in western blot assay gave unspesific is suspected as serum which is one component of CHO K1 cell growth media."

"ABSTRAKSuksesnya kehamilan sangat tergantung pada ketepatan dan sistematika proses implatasi dan plasentasi. Implantasi embrio membutuhkan sinkronisasi pada perkembangan trofoblas dan lapisan rahim. Ketidakmampuan embrio untuk menempel dan berimplantasi dengan baik adalah alasan utama kegagalan fertilisasi in vitro (IVF) atau Assisted Reproductive Technology (ART), serta infertilitas wanita, pre-eklampsia, intrauterine growth restriction (IUGR) atau lahir mati. Eksplan dapat menggambarkan proses in vivo secara mirip walaupun tidak dapat digunakan untuk meneliti tahap sel dan fungsional individu. Pada studi ini, kami ingin mengidentifikasi model sel untuk invasi trofoblas manusia. Kami melakukan pencarian literatur melalui PubMed search dan koleksi pribadi untuk menemukan berbagai jenis sel tropoblastic untuk membandingkan karakteristik utama dan umur untuk implantasi manusia dan plasentasi. Sebanyak 5 artikel dari 108 terpenuhi kriteria seleksi ini dipilih dari Pubmed dan 8 artikel tambahan dari koleksi pribadi kami.. Karena model eksplan sulit digunakan untuk mempelajari fungsi sel individu, kami menyimpulkan bahwa penggunaan HTR-8/ SVneo akan lebih unggul dari EVTs, karena EVTs bukan hanya sel invasif, tetapi abadi, murni dan mudah tersedia. Disarankan untuk menggunakan EVTs saat studi regulasi trofoblas dengan TGF. Kami berharap dalam jangka panjang, ulasan ini dapat membantu untuk meningkatkan angka kesuksesan kehamilan di Asia dan Indonesia.

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ABSTRACTA sucessful pregnancy highly determined by proper and systematic process of implatation and placentation. Embryo implantation requires careful synchrony of development between the trophoblast and the lining of uterine. Inability of the embryo to attached and implant properly is a major reason for in vitro fertilization (IVF) or Assisted Reproductive Technology (ART) failure and female infertility, pre-eclampsia, intrauterine growth restriction (IUGR) or stillbirth. Explants would closely mimic in vivo, but it cannot be use to study at individual cellular and functional level. In this study, we want to identify the cell model for human trophoblastic invasion. A literatures review using pubmed search engine along with our personal collection on various tropoblastic cell types to compare the main characteristics and lifespan of trophoblastic cell models for human implantation and placentation have been done. 5 out of 108 in total number articles were scutinized and selected from Pubmed along with 8 more articles from personal collection. Since explant model will be difficult to study the individual cell function, we concluded using HTR-8/SVneo will be more superior than primary EVTs, because they are not only an invasive cell line, but they are immortalized, pure and are easily available. It is recommended to use EVTs when study on the regulation of trophoblastic with TGF. We hope in the long term, this review can help to improved successful pregnancy rates in Asia and Indonesia."

"Latar Belakang: Penyakit arteri perifer (PAP) merupakan penyakit yang sering dijumpai serta memiliki morbiditas dan mortalitas yang bermakna. Iskemia tungkai akut merupakan salah satu PAP yang menyebabkan stres retikulum endoplasma dan kematian sel melalui apoptosis atau autofagia. Aktivasi Caspase secara berurutan memainkan peran penting dalam suatu fase apoptosis sei. Sementara itu, Beclin-l memiliki peran utama dalam autofagia. Endotelin-1 (ET -1) sebagai faktor transkripsi Monocyte Chemoattractant Protein-1 (MCP-1) telah diteliti dengan baik dalam perkembangan aterosklerosis tetapi tidak pada iskemia tungkai. Tujuan penelitian ini adalah untuk mengetahui peran ET-1 dalam patogenesis iskemia tungkai akut melaIui MCPIP, Beclin-1, dan Caspase-3. Bahan dan Metode: Tiga belas kelinci umur 5 bulan galur Selandia Bam White (NZW) dipilih untuk penelitian ini. Arteri femoralis kanan semua kelinci kemudian diikat dan dilakukan reseksi pembedahan sampai alirannya hilang, dikonfirmasi oIeh Doppler Laser Fluximetry. Semua kelinci kemudian secara acak dialokasikan untuk kelompok perlakuan dengan pemberian intravena ECE-1 inhibitor (CGS26303) 5 mglkg berat badanlhari selama 26 hari dan kelompok kontrol. Jaringan diambiI dari daerah otot iskemik dan dilakukan pemeriksaan untuk ekspresi protein MCPIP, Beclin-1 untuk biomarker autofagia serta Caspase-3 untuk biomarker apoptosis menggunakan imunohistokimia. Ekspresi gen diperiksa menggunakan real time polymerase chain reaction (RT-PCR) dan dinyatakan sebagai ekspresi gen relatif. Hasil: Selama periode follow-up, 2 kelinci mati karena infeksi. Oleh karena itu, tersisa 11 kelinci yang menjadi subjek penelitian ini. Empat kelinci dialokasikan untuk kelompok kontrol sementara 7 kelinci diberikan ECE-I sebagai kelompok perlakuan. Semua kelompok perlakuan positif memiliki antibodi terhadap MCPIP, Beclin-1, dan Caspase-3 tetapi tidak untuk kelompok kontrol (p = 0,003). Meskipun demikian, ekspresi gen relatif dari MCP1P, Beclin-1 dan Caspase-3 ditemukan bervariasi antara 2 kelompok dan tidak berbeda bermakna seeara statistik. Simpulan: Pada iskemia tungkai akut, ET-1 diduga memiliki peran penting dalam mengatur MCP1P, Beelin-1, dan Caspase-3. Jalur tersebut merupakan suatu alternatif baru mekanisme kematian sei. Intervensi melalui inhibisi ET -1 diduga dapat menunda proses kematian sel.

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Background: Peripheral arterial disease (PAD) is common and causes significant morbidity and mortality. Acute limb ischemia is one of the PAD that will develops endoplasmic reticulum stress and subsequently cell death through apoptosis or autophagy. Sequential activation of Caspases plays a major role in the execution phase of cell apoptosis while Beclin-I has a central role in autophagy. Endothelin-I (ET -1) as atranscription factor of Monocyte Chemoattractant Protein-I (MCP-1) has been well studied in the progression of atherosclerosis but not in limb ischemia. The aim of the study is to investigate the role of ET -1 in the pathogenesis of acute limb ischemia through MCPIP, Beclin-1 and Caspase-3 . Materials and Methods: Thirteen of 5-month-old New Zealand White (NZW) rabbits are chosen for this study. The right femoral artery of all rabbits are ligated and resected surgically until its flow disappear and confirmed by laser Doppler Fluximetry. All are then randomly. allocated for intravenous administration of ECE-I inhibitor (CGS26303) 5 mglkg body weight/day for 26 days (n=7) and control group (n=6). The tissue taken from ischemic muscle area was examined for protein expression of MCPIP, Beclin-1 for autophagy biomarker as well as Caspase-3 for apoptotic biomarker using immunohistochemistry technique. Gene expressions are examined using real time Polymerase Chain Reaction (PCR) and expressed as relative gene expression. Result: During period of follow-up, 2 rabbits died because of infection. Therefore there were still remain II rabits for subject of this study. Four were allocated for non-intervention as control group while 7 were for ECE-1 administration as intervention group. All of the intervention group has positive on antibody for MCPIP, Beclin-I and Caspase-3 but not for the control group (p = 0.003). However, the relative gene expression on MCPIP, Becline-1 and Caspase-3 varied between 2 groups and were not statistically different. Conclusion: In acute limb ischemia, ET-1 maya play major role in regulating MCPIP, Beclin-l and Caspase-3. This pathway may be proposed as a new alternative mechanism of cell death in this situation. Intervention of ET-I may delay the process of cell death."

"Tiamin berfungsi sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme karbohidrat. Mengingat pentingnya peran tiamin, maka dilakukan pengembangan teknik pengukuran tiamin yang analog dengan enzyme linked immunosorbent assay ( ELISA), dimana antibodi diganti dengan protein pengikat spesifik yaitu protein ikat tiamin kacang hijau (PITKH). Teknik pengukuran ini dilakukan secara kompetitif, kompetitor akan dikompetisikan dengan tiamin bebas yang akan diukur. Kompetitor tersebut berupa konjugat antara tiamin-biotin. Tiamin murni diikatkan dengan biotin menggunakan senyawa perangkai yaitu glutaraldehid. Pada analisis LC-MS ditemukan 3 puncak, . Puncak ke 3 merupakan konjugat tiamin-biotin. Dibuat kurva standar dan diperoleh persamaan garis lurus dengan nilai R2= 0,9986. Uji validasi menggunakan konjugat tiamin-biotin menunjukan nilai coefficient of variation (CV) = 3,81%, nilai ini lebih kecil dari CV Horwitz = 8,12%, akurasi dengan nilai Recovery (R) =94 %-98%. Hasil ini menunjukan syarat pengukuran dengan teknik ELPLA sudah terpenuhi, dengan presisi dan akurasi yang baik. Aplikasi pengukuran kadar tiamin pada serum normal sebanyak 23 sampel didapatkan kadar tiamin berkisar 2,62-9,76 μg/ml. Dengan demikian, teknik ELPLA dengan konjugat tiamin-biotin sebagai kompetitor dapat digunakan pada pengukuran kadar tiamin dalam serum

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Thiamine has a coenzyme function in several enzymes involved in carbohydrate metabolism. Considering the important role of thiamine, a thiamine measurement technique analogous to the enzyme linked immunosorbent assay (ELISA) was developed, that the antibody was replaced by a specific binding protein named mung bean thiamine binding protein (MBTBP). The measurement technique was carried out competitively in which competitors would be competed with free thiamine to be measured. The competitor is a thiamine-biotin bond. Pure thiamine was bound to biotin using a coupling compound called glutaraldehyde. In the LC-MS analysis we found 3 peaks. The third peak was the thiamine-biotin conjugate. A standard curve was made and the value of its straight line equation was obtained R2= 0,9986. The validation test using thiamine-biotin conjugate showed coefficient of variation (CV) value = 3,81% which was smaller than Horwitz CV = 8,12%, with the accuracy of the Recovery (R) value = 94% – 98%. These results indicated that the measurement requirements for the ELPLA technique had been met with good precision and accuracy. The application of the serum measurements to 23 samples showed thiamine levels ranging from 2,62- 9,76 μg/ml. Thus, the ELPLA technique with thiamine-biotin conjugate as a competitor could be used in the measurement of serum thiamine levels"

"Tiamin berfungsi sebagai koenzim untuk beberapa enzim yang terlibat dalam metabolisme karbohidrat. Mengingat pentingnya peran tiamin, maka dilakukan pengembangan teknik pengukuran tiamin yang analog dengan enzyme linked immunosorbent assay (ELISA), dimana antibodi diganti dengan protein pengikat spesifik yaitu protein ikat tiamin kacang hijau (PITKH). Teknik pengukuran ini dilakukan secara kompetitif, kompetitor akan dikompetisikan dengan tiamin bebas yang akan diukur. Kompetitor tersebut berupa konjugat antara tiamin-biotin. Tiamin murni diikatkan dengan biotin menggunakan senyawa perangkai yaitu glutaraldehid. Pada analisis LC-MS ditemukan 3 puncak, . Puncak ke 3 merupakan konjugat tiamin-biotin. Dibuat kurva standar dan diperoleh persamaan garis lurus dengan nilai R2= 0,9986. Uji validasi menggunakan konjugat tiamin-biotin menunjukan nilai coefficient of variation (CV) = 3,81%, nilai ini lebih kecil dari CV Horwitz = 8,12%, akurasi dengan nilai Recovery (R) =94 %-98%. Hasil ini menunjukan syarat pengukuran dengan teknik ELPLA sudah terpenuhi, dengan presisi dan akurasi yang baik. Aplikasi pengukuran kadar tiamin pada serum normal sebanyak 23 sampel didapatkan kadar tiamin berkisar 2,62-9,76 µg/ml. Dengan demikian, teknik ELPLA dengan konjugat tiamin-biotin sebagai kompetitor dapat digunakan pada pengukuran kadar tiamin dalam serum.

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Thiamine has a coenzyme function in several enzymes involved in carbohydrate metabolism. Considering the important role of thiamine, a thiamine measurement technique analogous to the enzyme linked immunosorbent assay (ELISA) was developed, that the antibody was replaced by a specific binding protein named mung bean thiamine binding protein (MBTBP). The measurement technique was carried out competitively in which competitors would be competed with free thiamine to be measured. The competitor is a thiamine-biotin bond. Pure thiamine was bound to biotin using a coupling compound called glutaraldehyde. In the LC-MS analysis we found 3 peaks. The third peak was the thiamine-biotin conjugate. A standard curve was made and the value of its straight line equation was obtained R2= 0,9986. The validation test using thiamine-biotin conjugate showed coefficient of variation (CV) value = 3,81% which was smaller than Horwitz CV = 8,12%, with the accuracy of the Recovery (R) value = 94% – 98%. These results indicated that the measurement requirements for the ELPLA technique had been met with good precision and accuracy. The application of the serum measurements to 23 samples showed thiamine levels ranging from 2,62- 9,76 µg/ml. Thus, the ELPLA technique with thiamine-biotin conjugate as a competitor could be used in the measurement of serum thiamine levels.

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"Demam dengue (DD) merupakan penyakit infeksi virus dengue (DENV) dengan vektor Aedes aegypti dan Aedes albopictus. Secara global, terjadi peningkatan kasus DD sebanyak enam kali lipat dari tahun 2010 hingga 2016 namun sampai saat ini regimen terapi DD adalah terapi suportif yaitu terapi cairan dan simptomatik. Ekstrak Curcuma longa telah diteliti memiliki potensi sebagai antiviral untuk DENV. Namun, mekanisme penghambatannya masih belum diketahui sehingga penelitian ini dilakukan untuk mengetahui efek ekstrak Curcuma longa terhadap penghambatan reseptor dan penempelan virus dengue serotipe 2 (DENV-2) secara in vitro dan ikatan curcumin dengan protein E secara in silico.Penelitian ini merupakan studi eksperimental untuk menganalisa mekanisme kerja dari ekstrak Curcuma longa sebagai antivirus terhadap DENV-2 menggunakan sel Vero sebagai sel uji dan in silico untuk mengetahui ikatan energi curcumin dengan protein E DENV. Focus assay dan MTT Assay digunakan untuk menilai penghambatan reseptor dan protein virus, serta viabilitas sel secara berturut-turut. Konsentrasi ekstrak Curcuma longa yang digunakan yaitu dua kali IC50 (17,91 μg/mL). DMSO digunakan sebagai kontrol.Persentase hambat pada reseptor sel dan protein virus masing-masing adalah 98,67 ± 1,33% dan 2,29 ± 1,19%. Persentase viabilitas sel pasca pemberian ekstrak Curcuma longa adalah 97,07 ± 0,50%. Energi ikatan pada konformasi terbaik curcumin dengan protein E bernilai -2,71 kkal/mol dengan konstanta inhibisi 10,34 mM. Ekstrak Curcuma longa memiliki efek penghambatan reseptor sel lebih baik daripada protein E virus serta memiliki ikatan yang relatif baik dengan protein E.

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Dengue fever (DF) is an disease caused by dengue virus infection (DENV). From 2010 to 2016, there has been a sixfold increase of DF cases globally. However, therapy for DF currently only consist of supportive treatments. Curcuma longa (turmeric) extract has been studied and its potential antiviral activity against dengue serotype 2 virus was found but inhibitory mechanism is still unknown. This research aims to find the inhibitory effect of turmeric extract against cell receptor and attachment protein of DENV-2 in vitro and binding energy between curcumin and dengue protein E in silico.

Experimental, in vitro study was done to analyze inhibitory mechanism of turmeric extract as antivirus to DENV-2 using Vero cell as test cell. In silico calculation of binding energy between curcumin and DENV protein E was also done using a docking software. Focus assay and MTT assay were used to evaluate receptor and viral attachment protein inhibition as well as cell viability, respectively. Turmeric extract concentration used was twice of IC50 (17,91 μg/mL) . DMSO was used as control.

Inhibition percentage on cell receptor and viral attachment protein yielded 98,67±1,33% and 2,29±1,19% respectively. Viability percentage of the cells after treatment with turmeric extract is 97,07 ± 0,50%. Binding energy at the best conformation between curcumin and viral protein E is -2.71 kcal/mol with inhibition constant of 10,34 mM. Turmeric extract has a higher inhibition effect against cell receptor compared to viral attachment protein and has a relatively strong bond with protein E."