P0977LOW EXPRESSION OF AUTOPHAGY-RELATED PROTEIN 5 (ATG5) LEADS TO SUPPRESSION OF AUTOPHAGY IN PATIENTS WITH DIABETIC NEPROPATHY AND RETINOPATHY

Background and Aims Autophagy is a catabolic mechanism that involves lysosomal-dependent degradation of unnecessary or dysfunctional intracellular components. Autophagy plays role in many biological processes, including diabetic nephropathy (DN) and diabetic retinopathy (DR). Autophagy-related gene 5 (ATG5) is one of the most important participants in the autophagy mechanism. Deficiencies in ATG5 protein levels are associated with several diseases by influencing the level of autophagy pathway. Our study's aim was to investigate if aberrant expression of ATG5 protein or Atg5 gene is associated with DN or DR. Method The study included 120 human participants in 4 groups – Healthy, diabetic (DM), DN and DR; and 10 mice in 2 groups – healthy and DN. Western blot analyses of ATG5 and its downstream collaborator LC3B were performed on human white blood cell (WBC) lysates and murine renal lysates. Immunohistochemical analysis was performed on mice renal tissues. Quantitative real-time PCR analysis of ATG5 was performed on total mRNA isolated from human WBC. Results Conclusion

Protocol for Identifying Natural Agents That Selectively Affect Adhesion, Thickness, Architecture, Cellular Phenotypes, Extracellular Matrix, and Human White Blood Cell Impenetrability of Candida albicans Biofilms

ABSTRACT In the screening of natural plant extracts for antifungal activity, assessment of their effects on the growth of cells in suspension or in the wells of microtiter plates is expedient. However, microorganisms, including Candida albicans, grow in nature as biofilms, which are organized cellular communities with a complex architecture capable of conditioning their microenvironment, communicating, and excluding low- and high-molecular-weight molecules and white blood cells. Here, a confocal laser scanning microscopy (CLSM) protocol for testing the effects of large numbers of agents on biofilm development is described. The protocol assessed nine parameters from a single z-stack series of CLSM scans for each individual biofilm analyzed. The parameters included adhesion, thickness, formation of a basal yeast cell polylayer, hypha formation, the vertical orientation of hyphae, the hyphal bend point, pseudohypha formation, calcofluor white staining of the extracellular matrix (ECM), and human white blood cell impenetrability. The protocol was applied first to five plant extracts and derivative compounds and then to a collection of 88 previously untested plant extracts. They were found to cause a variety of phenotypic profiles, as was the case for 64 of the 88 extracts (73%). Half of the 46 extracts that did not affect biofilm thickness affected other biofilm parameters. Correlations between specific effects were revealed. The protocol will be useful not only in the screening of chemical libraries but also in the analysis of compounds with known effects and mutations.

Determining the Genetic Similarities and Variability of Javanese and Arab Ethnic Families with DNA Fingerprint in Malang East Java Indonesia

PENENTUAN SIMILARITAS DAN VARIABILITAS GENETIK PADA KELUARGA ETNIS JAWA DAN ARAB DENGAN DNA FINGERPRINT DI MALANG, JAWA TIMUR, INDONESIA ABSTRAKLebih dari sepertiga genom manusia terdiri dari urutan daerah berulang (Repeat area) yang terdiri dari Minisatellite atau Variant Number Of Tandem Repeats (VNTR) dan Microsatellite atau Short Tandem Repeat (STR). STR sebagai daerah berulang dengan rentang alel yang pendek sering digunakan untuk tes paternitas, penelitian penyakit genetik dalam bidang kesehatan, arkeologi molekular, maupun kasus kriminalitas dalam bidang forensic. Tujuan dari penelitian ini adalah untuk mengidentifikasi DNA Fingerprint pada etnis Jawa – Arab dengan menentukan similaritas dan variabilitas genetiknya. Bahan dan metode yang digunakan untuk mengerjakan adalah menggunakan sel darah putih manusia yang berasal dari tiga generasi dalam tiga keluarga yang terdiri dari : (1) Nenek – Ibu, Ayah – anak perempuan, (2) Kakek – Ibu, Ayah – Anak perempuan, (3) Kakek, Nenek – Ibu, Ayah – Anak laki-laki. Isolasi DNA pada tiap sampel diperoleh dengan salting out, selanjutnya Amplifikasi PCR dengan menggunakan 13 CODIS yang meliputi TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, VWA, D13S317, D16S539, D18S51, D21S11 dan amelogenin yang dapat dilihat melalui hasil elektroforesis gel poliakrilamid 8% dengan Chemidoc Gel Imaging. Analisis profil pita pada tiap individu untuk menentukan similaritas dan variabilitas genetik serta pola alel dengan menggunakan software Quantity One. Variasi pola pita DNA dianalisis dengan menggunakan program software GENEPOP package versi 4.2 yang akan didapat frekuensi alel, heterozigositas, dan migrasi alel. Berdasarkan identifikasi yang dilakukan diperoleh bahwa nilai heterozigositas pada populasi III (93.8461%) memiliki nilai heterozigositas lebih tinggi dibandingkan dengan populasi I (88.4615%) dan II (76.9230%) dan telah terjadi migrasi alel 0.341373%. Adanya persentase migrasi alel tersebut meskipun kecil menunjukkan telah terjadi Breeding diantara populasi Jawa dengan populasi Arab sehingga meningkatkan rata-rata nilai heterozigositas pada tiap populasi. Pola alel heterozigot dengan berdasarkan nilai heterozigositas, jumlah alel pada D21S11, VWA dan THO1 dapat direkomendasikan sebagai penanda molekular untuk identifikasi variasi genetik.Kata kunci: Etnis Jawa–Arab, DNA Fingerprint, 13 CODIS DETERMINING THE GENETIC SIMILARITIES AND VARIABILITY OF JAVANESE AND ARAB ETHNIC FAMILIES WITH DNA FINGERPRINT IN MALANG EAST JAVA INDONESIAABSTRACTMore than one-third of human genome consists of repetitive sequence region (Repeat Area) which consist of Minisatellite or Variant Number Of Tandem Repeats (VNTR) and Microsatellite or Short Tandem Repeat (STR). Based on its short allele range STR can be used for the paternity testing study of genetics disease, molecular archeology, as well as in forensic crime cases. The aim of this study is to identify Javanese – Arab Ethnic DNA fingerprint in determining the similarities and genetic variability. Materials and methods to accomplish this, we used human white blood cell from three generations of three family consists of: (1) grandmother-mother, father-daughter, (2) grandfather-mother, father-daughter, (3) grandfather, grandmother–mother, father-son. DNA blood samples were Isolated by salting out, furthermore PCR amplification used by applying 13 CODIS which consists of TPOX, D3S1358, FGA, D5S818, CSF1PO, D7S820, D8S1179, TH01, VWA, D13S317, D16S539, D18S51, D21S11 and amelogenin, and then it was visualized by 8% polyacrylamid gel. The Fingerprint profile was visualized by 8% polyacrylamide gel and took the picture by ChemiDoc gel Imaging and measure the intensity band pattern by Quantity One software. Variations in the pattern of DNA bands were analyzed using the program GENEPOP software package version 4.2 that will be obtained allele frequencies, heterozygosity, and allele migration. Based on identification, this result showed analysis heterozygosity values, population III (93.8461%) have higher heterozygosity values compared with the population I (88.4615%) and II (76.9230%) and migration of alleles 0.341373%. The percentage of the migration though minor allele had occurred Breeding populations between Java to the Patterns of heterozygous alleles with values based on heterozygosity, number of alleles at D21S11,VWA and THO1 can be recommended as a molecular marker for the identification of genetic variation.Keywords: Javanese – Arab Ethnics, DNA fingerprint, 13 CODIS