Various gene modification techniques to discover molecular targets for nonhormonal male contraceptives: A review

The identification and characterization of relevant targets are necessary for developing nonhormonal male contraceptives. The molecules must demonstrate that they are necessary for reproduction. As a result, a sophisticated technique is required to identify the molecular targets for nonhormonal male contraceptives. Genetic modification (GM) techniques are one method that can be applied. This technique has been widely used to study gene function that effected male fertility and has resulted in the discovery of numerous nonhormonal male contraceptive target molecules. We examined GM techniques and approaches used to investigate genes involved in male fertility as potential targets for nonhormonal contraceptives. The discovery of nonhormonal contraceptive candidate molecules was increased by using GM techniques, especially the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 method. The discovery of candidate nonhormonal contraceptive molecules can be a wide-open research for the development of nonhormonal male contraceptives. Therefore, we are believing that one day nonhormonal male contraceptives will be released

The effect of ethanolic leaves extract of soursop (Annona muricata L.) on human colorectal cancer cell line: cell viability and in silico study to cyclin D1 protein

Latar Belakang: Kanker kolorektal merupakan transformasi patologis dari epitel kolon dan rektum normal menjadi massa jaringan abnormal, perubahan ini terjadi karena ekspresi berlebih dari protein cyclin D1 yang menginduksi proliferasi sel kolorektal secara berlebihan. Pengobatan dan pencegahan kanker kolorektal dapat dilakukan secara alami dengan mengonsumsi ekstrak daun Annona muricata L. (sirsak). Sirsak dikenal karena banyak komponen fitokimia yang berfungsi sebagai anti kanker. Metode: Penelitian ini menggunakan sel kanker kolorektal HT-29 yang diberi ekstrak etanol daun sirsak dan 5 Fluorourasil (5-FU). Tujuannya untuk menemukan konsentrasi sitotoksisitas yang dapat menghambat 50% populasi sel HT-29 (CC50) dan konsentrasi yang didapat sebelumnya akan diuji dengan metode uji MTT. Analisis docking molekuler dilakukan antara molekul-molekul dari ekstrak etanol daun sirsak terhadap protein Cyclin D1 menggunakan perangkat lunak molecular operating environment (MOE) 2013.08. Hasil: CC50 ekstrak etanol daun sirsak adalah 278 μg / mL dan 5-FU adalah 88 μg / mL. Persentase terendah sel HT-29 yang layak adalah 2 x CC50 setelah perlakuan ekstrak etanol daun sirsak (40,4 ± 1,3%) dibandingkan dengan 5-FU (52,8 ± 4,3%), kontrol pelarut ( 97,2 ± 1,4%), dan kontrol sel (100%). Analisis docking molekuler untuk protein cyclin D1 diperoleh asam N-hexadecanoic dan molekul phytol sebagai kandidat yang baik untuk menghambat protein cyclin D1. Kesimpulan: Ekstrak etanol daun sirsak dapat menurunkan viabilitas sel kultur kanker kolon HT-29 dan berdasarkan analisis molekular docking dilihat dari energi bebas gibbs (ΔG) dan afinitas tertinggi (pKi) diperoleh N-hexadecanoic dan molekul phytol sebagai penghambat protein cyclin D1. (Health Science Journal of Indonesia 2019;10(2):96-102) Kata Kunci: Kanker kolorektal HT-29, ekstrak etanol daun sirsak, viabilitas sel, molecular docking, cyclin D1   Abstract Introduction: Colorectal cancer is a pathological transformation of normal colon and rectum epithelial that becomes an abnormal tissue mass, due to the overexpression of cyclin D1 protein that inducing excessive proliferation of colorectal cell. The treatment and prevention of colorectal cancer could be done naturally by consuming leaves extract of Annona muricata L. (soursop). Soursop is known for many phytochemical components that serve as an anti-cancer. Methods: This study was used HT-29 colorectal cancer cell that treated with ethanolic leaves extract of soursop and 5-Fluorourasil (5-FU) to find the cytotoxicity concentration that can inhibit 50% of HT-29 cell population (CC50) and the next concentrations of them were treated for next treatment with MTT assay. Molecular docking analysis of the compounds of ethanolic leaves extract of soursop to cyclin D1 protein used molecular operating environment (MOE) 2013.08 software. Results: CC50 of ethanolic leaves extracts of soursop was 278 μg/mL dan 5-FU was 88 μg/mL. The lowest percentage of viable HT-29 cell was 2 x CC50 after ethanolic leaves extract of soursop treatment (40,4±1,3%) was compared to 5-FU (52,8±4,3%), solvent control (97,2±1,4%), and cells control (100%). Analysis of molecular docking to cyclin D1 protein was obtained N-hexadecanoic acid and phytol molecules as good candidates to inhibit cyclin D1 protein. Conclusions: The ethanolic leaves extract of soursop could be a good alternative treatment for colorectal cancer and its compounds had ability to inhibit cyclin D1 protein (the highest gibbs free energy (ΔG) and affinity (pKi)). (Health Science Journal of Indonesia 2019;10(2):96-102) Keywords: Colorectal cancer, ethanolic leaves extract of soursop, cell viability, molecular docking, cyclin D

Progesterone decrease plasma membrane in human sperm with subnormal hypoosmotic swelling test scores

Background Progesterone (P4) is known as a female hormone affecting oocyte maturation and developing uterine wall. A proteomic study identified several receptors including P4 receptors on human sperm. The role of P4 in human sperm cells remains unknown as to whether P4 has non-genomic effects on human sperm. The present study aims to determine the effect of progesterone (P4) on the hyperactivated motility and membrane integrity of human sperm cells. Methods Semen from normal individuals was obtained from donors. The semen was washed by gradient density centrifugation. P4 was added to each semen sample to final concentrations of 0 (control), 250, 500, 750 and 1000 ng/mL. After the sample treatment was completed, the sperm membrane integrity was assessed with the hypoosmotic swelling test (sodium citrate dihydrate and D-fructose) and the hyperactivated sperm motility parameter was determined with the Computer Assisted Sperm Analyzer [CASA] (Hamilton Thorne, IVOS II, USA). The percentage was then compared between the treatment groups and the control group. The percentage differences were analyzed with the Sigmastat version 2.0 statistical program. Results Administration of P4 increased sperm hyperactivated motility when compared with the control group at a concentration of 500 ng/mL, but the increase was statistically not signicant (p>0.05). In contrast, P4 decreased sperm membrane integrity significantly (p=0.042). And the mean of plasma membrane integrity in all groups was subnormal hypoosmotic swelling test score. Conclusion Progesterone administration tends to increase sperm hyperactivated motility. The integrity of plasma sperm membrane was affected by progesterone

In silico docking analysis of beta-defensin 20 against cation channel sperm-associated protein 1–4 to predict its role in the sperm maturation

Beta-defensin 20 (DEFB20) is widely expressed in the epididymis with gene features involved in epididymal sperm maturation. However, the action mechanism and function of DEFB20 in sperm maturation are still unclear. One of the important roles of beta-defensin is the ion channel activity. The cation channel sperm-associated protein (CatSper) alpha is an ion channel protein found on the sperm surface. This study aimed to investigate the interaction between DEFB20 and CatSper1–4 protein in relation to the sperm maturation process. Protein sequences were obtained from the National Center for Biotechnology Information (NCBI). Protein modeling and validation were carried out by using the Robetta modeling server and the Ramachandran plot method. Rosetta web server was used for the docking analysis. The results revealed a natural interaction between DEFB20 and CatSper1–4. The interaction occurred at the cation channel (close to the casein kinase II), ion transport protein, and kinase c phosphorylation of the CatSper1–4 active site. The DEFB20 region interacting with CatSper2–4 was the beta-defensin domain, while with CatSper1 was the non-beta-defensin domain. Based on the analysis, DEFB20 may interact with CatSper α subunits, particularly CatsSper1, to affect ion channel activity during sperm maturation

Genotype distribution of methylenetetrahydrofolate reductase A1298C and C677T gene in Indonesian infertile men

Background: Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme of folate and methionin metabolism, making it crucial for DNA synthesis and methylation. Variants of MTHFR C677T and A1298C gene result in reduced plasma folate levels and increase the susceptibility to spermatogenic arrest. This research aims to analyses MTHFR C677T and A1298C gene polymorphism in Indonesian infertile men with azoospermia and oligozoospermia.Methods: This cross sectional study takes 3 mL of blood from 150 infertile men with oligozoospermia and azoospermia. MTHFR gene is analyzed using polymerase chain reaction technique (PCR) with specific primers. PCR-RFLP analysis of the MTHFR gene using restriction enzymes MboII and HinfI determines allotypes, both of SNP A1298C and C677T in oligozoospermia and azoospermia in Indonesian population.Results: The results show that the distribution of allotypes of MTHFR gene SNP A1298C and A677T is not significantly different (p&gt;0.05) between patient groups with oligozoospermia and azoospermia.Conclusion: MTHFR gene polymorphisms, both of SNP A1298C and C677T are not associated with male infertility in Indonesian men including patients with severe oligozoospermia and azoospermia. (Med J Indones 2012;21:23-7)Keywords: DNA methylation, MTHFR, spermatogenic arrest</p

Gene Expression of Sperm Associated Antigen 8 and Ran-Binding Protein 9 on Azoospermic Male : Its Association with Spermatogenic Arrest

Proteins that play an  important role in the transcription process during spermatogenesis are CREMs that bind to their ACT activators that are suspected to be regulated by SPAG8 and RANBP9. Until now the role of both genes in the spermatogenic arrest process is not known. This study aims to determine the relative expression of Spag8 and RanBP9 on spermatogenic arrest and to analyze the correlation of expression of both genes. This study is a cross sectional study using a sample of testicular biopsy with Johnsen 2 to 8 score. Relative expression analysis of Spag8 and RanBP9 using qRT-PCR technique with Livak calculation. The data obtained were analyzed statistically using ANOVA one way test for Spag8 and Kruskal Wallis test for RanBP9 with significance value p &lt;0,05. The results of this study show that the relative expression of Spag8 and RanBP9 is highest on Johnsen 3 scores and is statistically significantly different (p &lt;0.05). There is a positive correlation with a very strong correlation strength between SPAG8 and RANBP9 expressions. Based on the results of this study shows that both of these genes are candidates for spermatogenic arrest

Genetic Variation at 5'-UTR of CD40 Gene and Patients Characteristics: Associated with Relapse in Graves' Disease

More than 50% patients graves' disease (GD) can relapse after remission period. This can be influenced by treatment, gender, age of diagnosis and genetic. One of the genes that regulate immune response is CD40 gene, which is found on the surface of Lymphocyte B. The aim of this research was to determine genetic variation at 5'-UTR of CD40 gene and the role of age and gender that influence the risk for relapse. This study was conducted in Dr Cipto Mangunkusumo Hospital from August to December 2014. This study was a case-control study comparing 30 relapse patients and 30 non-relapse patients after treatment with anti-thyroid drug was terminated. Genetic variation was analyzed with PCR-RFLP and clinical characteristic by medical record. In this research, we found that both genotype and alleles 5'-UTR of CD40 gene variation have no association with risk for relapse (p&gt;0.05), but age of diagnosis was considered significant with risk for relapse (p=0.001). In conclusion, genetic variation in 5'-UTR CD40 gene is not risk for relapse but age of diagnosis may be a risk for relapse in GD patients

EFFECT OF HUMAN ADIPOSE-DERIVED STEM CELL IN COLLAGEN GEL ON RELATIVE EXPRESSION LEVEL OF VASCULAR ENDOTHELIAL GROWTH FACTOR-A OF DEEP DERMAL BURN HEALING

Objectives: The objectives of this research was to measure the relative expression levels of vascular endothelial growth Factor-A (VEGF-A) in the deep dermal burns treated with human adipose-derived stem cell (hADSC) in collagen gel in each observational day (days 7, 14, 21, and 28).Methods: This study used 20 male Sprague Dawley rats, divided into four groups of observation days. Each rat received three burn injuries and then given different treatments (hADSC in collagen gel, collagen gel, and control). Deep dermal burn injury on the dorsal was made by placing a metal plate with 250°C for 15 s. Relative expression level of VEGF-A measurement with quantitative reverse transcription polymerase chain reaction.Results: On the 7th day, the relative expression level of VEGF-A in the wound treated with hADSC in collagen gel was significantly different from the scaffold collagen and control group (p&lt;0.05), whereas the control and scaffold collagen group was not significantly different. The relative expression level of VEFG-A in wound treated with hADSC in collagen gel, collagen gel only, and control was (mean±standard error of the mean) 17.93±4.37, 7.54±2.63, and 5.44±1.59, respectively. On the next observation days, the result showed that the relative expression level of VEGF-A was not significantly different between the three treatments. The relative expression level of VEGF-A has decreases from day 7 to 28 days. The decrease of VEGF-A relative expression level hADSC in collagen gel group was significantly different on the 7th day to the 21st and 28th days (p&lt;0.05).Conclusion: The provision of hADSC in scaffold collagen increases the relative expression of VEGF-A early in the wound healing process compared to the without a hADSC group

HUBUNGAN KADAR SERUM AMH DENGAN JUMLAH MUTASI PADA GEN PROMOTER AMH (ANTI-MULLERIAN HORMONE) PADA PASIEN SOPK (SINDROM OVARIUM POLIKISTIK)

Anti-Mullerian Hormone (AMH) adalah anggota dari kelompok Transforming Growth Factor-β yang berperan penting dalam regulasi folikulogenesis reproduksi wanita. Peningkatan kadar AMH 2-3 kali lipat ditemukan pada pasien SOPK (Sindrom Ovarium Polikistik) dibandingkan dengan wanita dengan ovulasi normal. Tujuan dari penelitian ini adalah untuk mengetahui hubungan kadar serum AMH dengan jumlah mutasi pada gen promoter AMH (Anti-Mullerian Hormone) pada pasien SOPK (Sindrom Ovarium Polikistik). Besar sampel adalah 114 pasien yang terdiri dari 60 pasien SOPK dan 54 pasien bukan SOPK sebagai kontrol. Kadar AMH didapatkan dari rekam medis pasien di Klinik Yasmin IVF RSCM Kencana Hospital, Jakarta. Analisis molekuler dan genotipe dilakukan dengan PCR dan sekuensing dilanjutkan dengan analisis bioinformatika. Terdapat 60 mutasi titik pada varian promotor gen AMH. Jenis mutasi varian tertinggi yang ditemukan adalah -674 G/A (100%), diikuti oleh -245 C/CT (88,2%), dan -444 A/G (17,9%) di seluruh sampel. Berdasarkan hasil uji Wilcoxon Signed Rank test, jumlah mutasi pada kelompok SOPK berpengaruh nyata terhadap AMH serum (p0,05). Jumlah mutasi pada promotor gen mempengaruhi kadar serum AMH pada PCOS