Pub Date : 2022-06-29DOI: 10.22146/ijbiotech.65822
Sotharith Phon, A. Ningrum, L. D. Witasari
Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.41 U/mg (Vmax) and 0.25 mg/mL (KM). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.
{"title":"Purification and characterization of thermostable serine alkaline protease from Geobacillus sp. DS3 isolated from Sikidang crater, Dieng plateau, Central Java, Indonesia","authors":"Sotharith Phon, A. Ningrum, L. D. Witasari","doi":"10.22146/ijbiotech.65822","DOIUrl":"https://doi.org/10.22146/ijbiotech.65822","url":null,"abstract":"Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.41 U/mg (Vmax) and 0.25 mg/mL (KM). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43328740","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-06-29DOI: 10.22146/ijbiotech.70445
Nurmi Nadhira, A. Wafa, Wahyu Indra Duwi Fanata, H. S. Addy
Rice (Oryza sativa L.) production is limited by bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo). For decades, researchers have attempted to control this disease by growing plants with blight‐resistant Xa genes. Genetic resources often vary between rice varieties, and there is little information about the genetic resources of the pigmented rice varieties widely grown in Indonesia and their resistance genes against Xoo. The purpose of this study was to determine the expression of Xa genes in pigmented rice such as Inpari 24 and Cempo Merah (red‐pigmented) along with Hitam Bantul (black‐pigmented) and white rice varieties IR64 and Ciherang, and to evaluate their resistance to BLB. All varieties carried the Xa4, Xa10 and xa13 genes but varied in the Xa1, Xa7 and Xa21 genes. The rice varieties expressed some of these genes only after inoculation with Xoo. Disease assessment categorised the three different pigmented rice varieties as resistant (Ciherang, Cempo Merah and Hitam Bantul), while IR64 (white) and Inpari 24 (red) were moderately resistant. There was no specific pattern of Xa genes possession, quality of expression or resistance level to X. oryzae pv. oryzae. Therefore, when breeding plants, the selection of parental variety must be considered in terms of the possession and expression of Xa genes such as Xa10 as a molecular marker for resistance.
{"title":"Resistance gene expression in selected Indonesian pigmented rice varieties against infection by Xanthomonas oryzae pv. oryzae","authors":"Nurmi Nadhira, A. Wafa, Wahyu Indra Duwi Fanata, H. S. Addy","doi":"10.22146/ijbiotech.70445","DOIUrl":"https://doi.org/10.22146/ijbiotech.70445","url":null,"abstract":"Rice (Oryza sativa L.) production is limited by bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv. oryzae (Xoo). For decades, researchers have attempted to control this disease by growing plants with blight‐resistant Xa genes. Genetic resources often vary between rice varieties, and there is little information about the genetic resources of the pigmented rice varieties widely grown in Indonesia and their resistance genes against Xoo. The purpose of this study was to determine the expression of Xa genes in pigmented rice such as Inpari 24 and Cempo Merah (red‐pigmented) along with Hitam Bantul (black‐pigmented) and white rice varieties IR64 and Ciherang, and to evaluate their resistance to BLB. All varieties carried the Xa4, Xa10 and xa13 genes but varied in the Xa1, Xa7 and Xa21 genes. The rice varieties expressed some of these genes only after inoculation with Xoo. Disease assessment categorised the three different pigmented rice varieties as resistant (Ciherang, Cempo Merah and Hitam Bantul), while IR64 (white) and Inpari 24 (red) were moderately resistant. There was no specific pattern of Xa genes possession, quality of expression or resistance level to X. oryzae pv. oryzae. Therefore, when breeding plants, the selection of parental variety must be considered in terms of the possession and expression of Xa genes such as Xa10 as a molecular marker for resistance.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49404094","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-29DOI: 10.22146/ijbiotech.65728
Yustina Carolina Febrianti Salsinha, Alfino Sebastian, E. Sutiyanti, Y. A. Purwestri, D. Indradewa, D. Rachmawati
Response by plants to drought occurs through a series of mechanisms that involve transcription regulation. This research was conducted to study transcription factors (TF) and physiological changes in the drought response of local rice cultivars from East Nusa Tenggara (Nusa Tenggara Timur, NTT) during drought stress. Using three NTT local rice cultivars (Boawae Seratus Malam (BSM), Gogo Jak (GJ), and Kisol Manggarai (KM)) and the fraction of transpirable soil water (FTSW) method with two treatment levels, FTSW 1 (control) and FTSW 0.2 (severe stress), we analyzed the TF expression of OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6. Based on the result, the highest level of TF expression occurred in the BSM, followed by the GJ and KM cultivars. Analysis of physiological characteristics showed an association between TF expression levels and physiological response, with the BSM cultivar showing high pigment levels, high proline content, and lower H2O2 levels. A linkage was also found in relation to water conservation, as indicated by the higher relative water content and cell membrane stability index in the BSM cultivar in contrast to lower electronic leakage and malondialdehyde percentage when exposed to drought. Based on the results, it can be concluded that the BSM cultivar can be considered as a drought‐tolerant local cultivar according to morpho‐physiological analysis. In this study, all NTT local rice cultivars showed a subtle upregulation of stress‐responsive transcription factors OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6 as responses to drought stress.
植物对干旱的反应是通过一系列涉及转录调控的机制发生的。本研究旨在研究东努沙登加拉(Nusa Tenggara Timur, NTT)水稻品种干旱胁迫下转录因子(TF)及其生理响应的变化。以3个NTT地方水稻品种Boawae Seratus Malam (BSM)、Gogo Jak (GJ)和Kisol Manggarai (KM)为研究对象，采用土壤水分蒸发量(FTSW)法，在FTSW 1(对照)和FTSW 0.2(重度胁迫)两个处理水平下，分析了OsDREB1A、OsDREB2A、OsWRKY45和OsNAC6的TF表达。结果表明，TF在BSM中表达量最高，其次是GJ和KM品种。生理特性分析表明，TF表达水平与生理反应相关，BSM品种表现出高色素含量、高脯氨酸含量和低H2O2水平。在水分保护方面也发现了联系，这表明BSM品种的相对含水量和细胞膜稳定性指数较高，而暴露于干旱时的电子渗漏和丙二醛百分比较低。结果表明，从形态生理分析来看，BSM品种可视为耐旱品种。在本研究中，所有NTT地区水稻品种在干旱胁迫下均表现出胁迫响应转录因子OsDREB1A、OsDREB2A、OsWRKY45和OsNAC6的轻微上调。
{"title":"The relationship between morpho‐physiological changes and expression of transcription factors in NTT local rice cultivars as a response to drought stress","authors":"Yustina Carolina Febrianti Salsinha, Alfino Sebastian, E. Sutiyanti, Y. A. Purwestri, D. Indradewa, D. Rachmawati","doi":"10.22146/ijbiotech.65728","DOIUrl":"https://doi.org/10.22146/ijbiotech.65728","url":null,"abstract":"Response by plants to drought occurs through a series of mechanisms that involve transcription regulation. This research was conducted to study transcription factors (TF) and physiological changes in the drought response of local rice cultivars from East Nusa Tenggara (Nusa Tenggara Timur, NTT) during drought stress. Using three NTT local rice cultivars (Boawae Seratus Malam (BSM), Gogo Jak (GJ), and Kisol Manggarai (KM)) and the fraction of transpirable soil water (FTSW) method with two treatment levels, FTSW 1 (control) and FTSW 0.2 (severe stress), we analyzed the TF expression of OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6. Based on the result, the highest level of TF expression occurred in the BSM, followed by the GJ and KM cultivars. Analysis of physiological characteristics showed an association between TF expression levels and physiological response, with the BSM cultivar showing high pigment levels, high proline content, and lower H2O2 levels. A linkage was also found in relation to water conservation, as indicated by the higher relative water content and cell membrane stability index in the BSM cultivar in contrast to lower electronic leakage and malondialdehyde percentage when exposed to drought. Based on the results, it can be concluded that the BSM cultivar can be considered as a drought‐tolerant local cultivar according to morpho‐physiological analysis. In this study, all NTT local rice cultivars showed a subtle upregulation of stress‐responsive transcription factors OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6 as responses to drought stress.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43164502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-29DOI: 10.22146/ijbiotech.67506
Ayu Dewi Ni Nyoman, Ni Made Pramita Widya Suksmarini, Anak Agung Ngurah Satya Pranata, A. Y. Rompis, I. W. J. Sumadi
Mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog gene) and BRAF (v‐Raf murine sarcoma viral oncogene homolog B1) gene play a significant role in primary resistance to colorectal cancer therapy. Around 85‐90% of KRAS mutations in colorectal cancer occur in exon 2 (codon 12 and 13), whereas approximately 96% of BRAF mutations occur in exon 15 codon 600 (V600E). This study aimed to determine the prevalence and mutation characteristics of the KRAS and BRAF genes in colorectal cancer patients in Bali. The DNA was isolated from 44 formalin‐fixed paraffin‐embedded colorectal cancer samples which were stored in the Department of Pathology, Sanglah General Hospital in 2017. Detection of mutation was carried out by polymerase chain reaction (PCR) and direct sequencing. Out of 44 samples, only 27 were successfully amplified and sequenced. Our findings showed six samples (22.2%) with mutated KRAS at codons 12 and 13 (including two samples with G12D, one sample with G12V, and three samples with G13D). Interestingly, we found three samples (11.1%) of BRAF mutation, including two samples with V600E mutation and one with V600L mutation. Taken together, our results showed that KRAS and BRAF mutations were identified and occurred exclusively. Further studies are essential to identify the correlation of these mutations with colorectal cancer prognosis and response to chemotherapy
KRAS (Kirsten大鼠肉瘤病毒癌基因同源基因)和BRAF (v - Raf小鼠肉瘤病毒癌基因同源基因B1)基因的突变在结直肠癌治疗的原发性耐药中起重要作用。结直肠癌中约85% - 90%的KRAS突变发生在外显子2(密码子12和13)，而约96%的BRAF突变发生在外显子15密码子600 (V600E)。本研究旨在确定巴厘岛结直肠癌患者KRAS和BRAF基因的患病率和突变特征。该DNA是从2017年存放在Sanglah总医院病理科的44份福尔马林固定石蜡包埋结直肠癌样本中分离出来的。采用聚合酶链反应(PCR)和直接测序法检测突变。在44个样本中，只有27个样本被成功扩增和测序。结果显示，6个样本(22.2%)在12和13密码子处发生KRAS突变(其中2个样本为G12D, 1个样本为G12V, 3个样本为G13D)。有趣的是，我们发现了3个BRAF突变样本(11.1%)，其中V600E突变样本2个，V600L突变样本1个。综上所述，我们的结果表明KRAS和BRAF突变是确定的，并且是排他发生的。需要进一步的研究来确定这些突变与结直肠癌预后和化疗反应的相关性
{"title":"The prevalence of KRAS and BRAF mutation in colorectal cancer patients in Bali","authors":"Ayu Dewi Ni Nyoman, Ni Made Pramita Widya Suksmarini, Anak Agung Ngurah Satya Pranata, A. Y. Rompis, I. W. J. Sumadi","doi":"10.22146/ijbiotech.67506","DOIUrl":"https://doi.org/10.22146/ijbiotech.67506","url":null,"abstract":"Mutations in the KRAS (Kirsten rat sarcoma viral oncogene homolog gene) and BRAF (v‐Raf murine sarcoma viral oncogene homolog B1) gene play a significant role in primary resistance to colorectal cancer therapy. Around 85‐90% of KRAS mutations in colorectal cancer occur in exon 2 (codon 12 and 13), whereas approximately 96% of BRAF mutations occur in exon 15 codon 600 (V600E). This study aimed to determine the prevalence and mutation characteristics of the KRAS and BRAF genes in colorectal cancer patients in Bali. The DNA was isolated from 44 formalin‐fixed paraffin‐embedded colorectal cancer samples which were stored in the Department of Pathology, Sanglah General Hospital in 2017. Detection of mutation was carried out by polymerase chain reaction (PCR) and direct sequencing. Out of 44 samples, only 27 were successfully amplified and sequenced. Our findings showed six samples (22.2%) with mutated KRAS at codons 12 and 13 (including two samples with G12D, one sample with G12V, and three samples with G13D). Interestingly, we found three samples (11.1%) of BRAF mutation, including two samples with V600E mutation and one with V600L mutation. Taken together, our results showed that KRAS and BRAF mutations were identified and occurred exclusively. Further studies are essential to identify the correlation of these mutations with colorectal cancer prognosis and response to chemotherapy","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43598035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-29DOI: 10.22146/ijbiotech.64933
A. L. Dewi, D. K. Paramita, J. Fachiroh
Rs1948 A>G is a single nucleotide variation (SNV) in the 3’‐UTR of CHRNB4. Genotyping the synonymous CHRNB4 rs1948 may be useful in identifying a lung cancer susceptibility gene. The study aimed to develop a simple and easy tetra‐primer amplification refractory mutation system (ARMS PCR) for CHRNB4 rs1948. The following steps were taken to optimize tetra‐primer ARMS PCR: 1) determining the gene sequence and position of a single mutation; 2) developing outer and inner primers; 3) amplification of target gene fragments via PCR using an outer primer; 4) genotyping PCR product using Sanger sequencing; 5) determining the optimal annealing temperature and PCR cycle; 6) determining optimal outer and inner primer ratio; and 7) testing the reproducibility of the PCR program and final validation with Sanger sequencing. Genotype (PCR result) was visualized with 3% agarose gel electrophoresis. Optimum condition was determined as annealing temperature of 64.8 ºC and 35 cycles, outer and inner primer ratio of 1:6, and DNA volume of 3 µL. Sanger sequencing confirmed the results of the tetra‐primer ARMS PCR and it was shown that ARMS PCR was able to identify three different variants of CHRNB4 rs1948.
{"title":"Tetra‐primer amplification refractory mutation system (ARMS) PCR used to detect 3’UTR rs1948 mutation in CHRNB4","authors":"A. L. Dewi, D. K. Paramita, J. Fachiroh","doi":"10.22146/ijbiotech.64933","DOIUrl":"https://doi.org/10.22146/ijbiotech.64933","url":null,"abstract":"Rs1948 A>G is a single nucleotide variation (SNV) in the 3’‐UTR of CHRNB4. Genotyping the synonymous CHRNB4 rs1948 may be useful in identifying a lung cancer susceptibility gene. The study aimed to develop a simple and easy tetra‐primer amplification refractory mutation system (ARMS PCR) for CHRNB4 rs1948. The following steps were taken to optimize tetra‐primer ARMS PCR: 1) determining the gene sequence and position of a single mutation; 2) developing outer and inner primers; 3) amplification of target gene fragments via PCR using an outer primer; 4) genotyping PCR product using Sanger sequencing; 5) determining the optimal annealing temperature and PCR cycle; 6) determining optimal outer and inner primer ratio; and 7) testing the reproducibility of the PCR program and final validation with Sanger sequencing. Genotype (PCR result) was visualized with 3% agarose gel electrophoresis. Optimum condition was determined as annealing temperature of 64.8 ºC and 35 cycles, outer and inner primer ratio of 1:6, and DNA volume of 3 µL. Sanger sequencing confirmed the results of the tetra‐primer ARMS PCR and it was shown that ARMS PCR was able to identify three different variants of CHRNB4 rs1948.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45880666","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-29DOI: 10.22146/ijbiotech.66854
Tsania Taskia Nabila, A. Wasiati, A. Jati, Annisa Khumaira
Molecular detection needs to be augmented for COVID‐19 detection in Indonesia using the PCR method with primer‐based gene analysis. This is necessary because the RNA of the SARS‐CoV‐2 virus, the causative infectious agent of the pandemic, has been mutated. Therefore, this study aimed to develop a primer design for determining SARS‐CoV‐2 clades in Indonesia using phylogenomic analysis. Data were obtained from 38 GISAID (Global Initiative on Sharing All Influenza Data) viruses and the relationships were analyzed using maximum likelihood (ML) phylogenomic analysis with a substitution model of generalized time‐reversible (GTR) to construct the tree topology. The results showed that the five types of SARS‐CoVs‐2 clades in Indonesia were L, G, GH, GR, and O. It also indicated that the GH region had the highest rate of clade at 50%, with the S clade affecting its formation. Furthermore, the genome sequences of the GH type used to design its primer were based on three genes, namely RdRp, S, and N. The RdRp and N genes were found to be conserved and hardy mutants, while the S gene occurred repeatedly. Several previous studies have stated that the designed primers produced missense mutations compared to another in silico. Therefore, three sets of primers were achieved from the GC contents and clamps, Tm range, and structural secondary indicator standards.
{"title":"The design of Indonesian SARS-CoV-2 primers based on phylogenomic analysis of their clades","authors":"Tsania Taskia Nabila, A. Wasiati, A. Jati, Annisa Khumaira","doi":"10.22146/ijbiotech.66854","DOIUrl":"https://doi.org/10.22146/ijbiotech.66854","url":null,"abstract":"Molecular detection needs to be augmented for COVID‐19 detection in Indonesia using the PCR method with primer‐based gene analysis. This is necessary because the RNA of the SARS‐CoV‐2 virus, the causative infectious agent of the pandemic, has been mutated. Therefore, this study aimed to develop a primer design for determining SARS‐CoV‐2 clades in Indonesia using phylogenomic analysis. Data were obtained from 38 GISAID (Global Initiative on Sharing All Influenza Data) viruses and the relationships were analyzed using maximum likelihood (ML) phylogenomic analysis with a substitution model of generalized time‐reversible (GTR) to construct the tree topology. The results showed that the five types of SARS‐CoVs‐2 clades in Indonesia were L, G, GH, GR, and O. It also indicated that the GH region had the highest rate of clade at 50%, with the S clade affecting its formation. Furthermore, the genome sequences of the GH type used to design its primer were based on three genes, namely RdRp, S, and N. The RdRp and N genes were found to be conserved and hardy mutants, while the S gene occurred repeatedly. Several previous studies have stated that the designed primers produced missense mutations compared to another in silico. Therefore, three sets of primers were achieved from the GC contents and clamps, Tm range, and structural secondary indicator standards.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44354575","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-29DOI: 10.22146/ijbiotech.69337
S. Afidah, Inyana Dwi Agustien, P. Dewanti, B. Sugiharto
Sucrose‐phosphate synthase (SPS) is a key enzyme catalyzing the formation of sucrose‐6‐phosphate through the transfer of uridine‐diphosphate glucose (UDP‐G) as a donor to fructose‐6‐phosphate (F6P) as an acceptor. Plant SPS consists of three main domains: N‐terminal, glycosyltransferase, and C‐terminal domains. Among these, the N‐terminal domain is involved in regulating the allosteric activator glucose‐6‐phosphate (G6P). This study was directed toward determining the regulation and characterization of N‐terminal truncated SPS in transgenic tomato. In this study, the N‐terminal truncated mutant of sugarcane SPS (ΔN‐SoSPS1) and full‐length sugarcane SPS (FL‐SoSPS1) were expressed into tomato plants to verify the functional role and importance of the N‐terminal domain in plant SPS. Overexpression of ΔN‐SoSPS1 led to an up to 3‐fold increase in the specific activity of SPS compared to non‐transformant plants (WT), while the specific activity of ΔN‐SoSPS1 was higher than FL‐SoSPS1 in transgenic tomato plants. Unlike WT and FL‐SoSPS1, the ΔN‐SoSPS1 mutant was not allosterically regulated by G6P. These results indicated that deletion of the N‐terminal domain promotes the loss of allosteric activation by G6P and increases binding affinity between enzyme and substrate.
{"title":"Increased activity of sugarcane sucrose‐phosphate synthase in transgenic tomato in response to N‐terminal truncation","authors":"S. Afidah, Inyana Dwi Agustien, P. Dewanti, B. Sugiharto","doi":"10.22146/ijbiotech.69337","DOIUrl":"https://doi.org/10.22146/ijbiotech.69337","url":null,"abstract":"Sucrose‐phosphate synthase (SPS) is a key enzyme catalyzing the formation of sucrose‐6‐phosphate through the transfer of uridine‐diphosphate glucose (UDP‐G) as a donor to fructose‐6‐phosphate (F6P) as an acceptor. Plant SPS consists of three main domains: N‐terminal, glycosyltransferase, and C‐terminal domains. Among these, the N‐terminal domain is involved in regulating the allosteric activator glucose‐6‐phosphate (G6P). This study was directed toward determining the regulation and characterization of N‐terminal truncated SPS in transgenic tomato. In this study, the N‐terminal truncated mutant of sugarcane SPS (ΔN‐SoSPS1) and full‐length sugarcane SPS (FL‐SoSPS1) were expressed into tomato plants to verify the functional role and importance of the N‐terminal domain in plant SPS. Overexpression of ΔN‐SoSPS1 led to an up to 3‐fold increase in the specific activity of SPS compared to non‐transformant plants (WT), while the specific activity of ΔN‐SoSPS1 was higher than FL‐SoSPS1 in transgenic tomato plants. Unlike WT and FL‐SoSPS1, the ΔN‐SoSPS1 mutant was not allosterically regulated by G6P. These results indicated that deletion of the N‐terminal domain promotes the loss of allosteric activation by G6P and increases binding affinity between enzyme and substrate.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47322709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-03-29DOI: 10.22146/ijbiotech.64021
Pangesti Drawina, A. Putra, T. Nasihun, Yan Wisnu Prajoko, Bayu Tirta Dirja, N. Amalina
The healing process of a full‐thickness wound involves a complex cascade of cellular responses to reverse skin integrity formation. These processes require growth factors, particularly platelet‐derived growth factor (PDGF). Conversely, hypoxic mesenchymal stem‐cell‐conditioned medium (HMSC‐CM)‐contained growth factors notably contribute to acceleration of wound healing. This study aims to investigate the role of HMSC‐CM in controlling the serial levels of PDGF associated with accelerated wound closure in full‐thickness wounds. Twenty male Wistar rats with full‐thickness wounds were developed as animal models. The animals were randomly assigned to four groups, comprising two treatment groups (treated using HMSC‐CM at a high dose as P1 and at a low dose as P2), a control group (administration of base gel), and sham group (healthy group). PDGF levels were examined using an enzyme‐linked immunosorbent assay. Using ImageJ software, wound closure percentages were determined photographically. The study showed that there was a significant increase in PDGF levels on days 3 and 6 after HMSC‐CM treatment, followed by a decrease in PDGF levels on day 9. In line with these findings, wound closure percentage also increased significantly on days 6 and 9. In the rat model, HMSC‐CM administration may promote acceleration of wound closure by increasing serial PDGF levels in the full‐thickness wound.
{"title":"Increased serial levels of platelet‐derived growth factor using hypoxic mesenchymal stem cell‐conditioned medium to promote closure acceler‐ ation in a full‐thickness wound","authors":"Pangesti Drawina, A. Putra, T. Nasihun, Yan Wisnu Prajoko, Bayu Tirta Dirja, N. Amalina","doi":"10.22146/ijbiotech.64021","DOIUrl":"https://doi.org/10.22146/ijbiotech.64021","url":null,"abstract":"The healing process of a full‐thickness wound involves a complex cascade of cellular responses to reverse skin integrity formation. These processes require growth factors, particularly platelet‐derived growth factor (PDGF). Conversely, hypoxic mesenchymal stem‐cell‐conditioned medium (HMSC‐CM)‐contained growth factors notably contribute to acceleration of wound healing. This study aims to investigate the role of HMSC‐CM in controlling the serial levels of PDGF associated with accelerated wound closure in full‐thickness wounds. Twenty male Wistar rats with full‐thickness wounds were developed as animal models. The animals were randomly assigned to four groups, comprising two treatment groups (treated using HMSC‐CM at a high dose as P1 and at a low dose as P2), a control group (administration of base gel), and sham group (healthy group). PDGF levels were examined using an enzyme‐linked immunosorbent assay. Using ImageJ software, wound closure percentages were determined photographically. The study showed that there was a significant increase in PDGF levels on days 3 and 6 after HMSC‐CM treatment, followed by a decrease in PDGF levels on day 9. In line with these findings, wound closure percentage also increased significantly on days 6 and 9. In the rat model, HMSC‐CM administration may promote acceleration of wound closure by increasing serial PDGF levels in the full‐thickness wound.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45667960","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2022-02-11DOI: 10.21203/rs.3.rs-1319627/v1
Elnora Listianto Lie, T. Hermawan, Kholis Abdurachmin Audah
Background: SARS-CoV-2 virus is the cause of the global pandemic since the end of the year 2019. Since then, the virus has had mutations that cause different types of variants with various effects on those infected. This has complicated human intervention for prevention. Indonesia is one of the countries which was heavily affected by the pandemic specifically from May to August 2021, and it is a country that has recorded many distinct isolates.Methods: GISAID database was used to obtain the Indonesian isolates while NCBI BLAST was utilized for comparison of all variants, and MAFFT version 7 for multi comparison.Results: There were 9,488 isolates in Indonesia as of November 2021 where most include the Delta variant. Most of the isolates have mutations common to the ones from other countries. Although there are some atypical ones such as the mutation V1264L in the Delta variant that was suspected play a role in worsening the pandemic.Conclusions: The Delta variant had the most mutations in the Spike protein, when compared to the Alpha and Beta variants, giving its important roles in infectivity and vigorous entry into cells, explaining why in the period of May to November 2021 in Indonesia, there was a rocket of cases for the Delta variant unlike the other variants
背景:自2019年底以来，SARS-CoV-2病毒是全球大流行的原因。从那以后，这种病毒发生了突变，导致不同类型的变异，对感染者产生了不同的影响。这使得人为干预预防变得复杂。特别是在2021年5月至8月期间，印度尼西亚是受该大流行严重影响的国家之一，该国记录了许多不同的分离病例。方法:采用GISAID数据库获取印尼分离株，采用NCBI BLAST对各变异进行比较，采用MAFFT version 7进行多株比较。结果:截至2021年11月，印度尼西亚有9488株分离株，其中大多数包括Delta变体。大多数分离株具有与来自其他国家的菌株相同的突变。尽管有一些非典型的变异，如Delta变异中的V1264L突变，被怀疑在大流行恶化中发挥了作用。结论:与α和β变异体相比，Delta变异体的Spike蛋白突变最多，这说明它在传染性和快速进入细胞方面发挥了重要作用，这解释了为什么在2021年5月至11月期间，Delta变异体与其他变异体不同，在印度尼西亚出现了大量病例
{"title":"Whole Genome Sequences Analyses of Indonesian Isolates SARS-CoV-2 Variants and their Clinical Manifestations ","authors":"Elnora Listianto Lie, T. Hermawan, Kholis Abdurachmin Audah","doi":"10.21203/rs.3.rs-1319627/v1","DOIUrl":"https://doi.org/10.21203/rs.3.rs-1319627/v1","url":null,"abstract":"\u0000 Background: SARS-CoV-2 virus is the cause of the global pandemic since the end of the year 2019. Since then, the virus has had mutations that cause different types of variants with various effects on those infected. This has complicated human intervention for prevention. Indonesia is one of the countries which was heavily affected by the pandemic specifically from May to August 2021, and it is a country that has recorded many distinct isolates.Methods: GISAID database was used to obtain the Indonesian isolates while NCBI BLAST was utilized for comparison of all variants, and MAFFT version 7 for multi comparison.Results: There were 9,488 isolates in Indonesia as of November 2021 where most include the Delta variant. Most of the isolates have mutations common to the ones from other countries. Although there are some atypical ones such as the mutation V1264L in the Delta variant that was suspected play a role in worsening the pandemic.Conclusions: The Delta variant had the most mutations in the Spike protein, when compared to the Alpha and Beta variants, giving its important roles in infectivity and vigorous entry into cells, explaining why in the period of May to November 2021 in Indonesia, there was a rocket of cases for the Delta variant unlike the other variants","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2022-02-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48126468","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2021-12-31DOI: 10.22146/ijbiotech.63023
M. R. Diansyah, Annisa Annisa, W. Kusuma
Parkinson’s disease is the second‐most‐common neurodegenerative disorder and can reduce patients’ quality of life. The disease is caused by abnormalities in dopaminergic neurons, such as reactive oxygen species (ROS) imbalance leading to programmed cell death, protein misfolding, and vesicle trafficking. Protein‐protein interaction (PPI) analysis has been demonstrated to understand better candidate proteins that might contribute to multifactorial neurodegenerative diseases, particularly in Parkinson’s disease. PPI analysis can be obtained from experiments and computational predictions. However, experiment data is often limited in interactome coverage. Therefore, additional computational prediction methods are required to provide more comprehensive PPI information. PPI can be represented as protein‐protein networks and analyzed based on centrality measures. The previous study has shown that top‐k skyline query, a method using dominance rule‐based centrality measures, reveals important protein candidates in Parkinson’s diseases. This study applied the top‐k skyline query to PPIs containing experiment and prediction data to find important proteins in Parkinson’s disease. The result shows that alpha‐synuclein (SNCA) is the most important protein and is expected to be a potential biomarker candidate for Parkinson’s disease.
{"title":"Analysis using top‐k skyline query of protein‐protein interaction reveals alpha‐synuclein as the most important protein in Parkinson’s disease","authors":"M. R. Diansyah, Annisa Annisa, W. Kusuma","doi":"10.22146/ijbiotech.63023","DOIUrl":"https://doi.org/10.22146/ijbiotech.63023","url":null,"abstract":"Parkinson’s disease is the second‐most‐common neurodegenerative disorder and can reduce patients’ quality of life. The disease is caused by abnormalities in dopaminergic neurons, such as reactive oxygen species (ROS) imbalance leading to programmed cell death, protein misfolding, and vesicle trafficking. Protein‐protein interaction (PPI) analysis has been demonstrated to understand better candidate proteins that might contribute to multifactorial neurodegenerative diseases, particularly in Parkinson’s disease. PPI analysis can be obtained from experiments and computational predictions. However, experiment data is often limited in interactome coverage. Therefore, additional computational prediction methods are required to provide more comprehensive PPI information. PPI can be represented as protein‐protein networks and analyzed based on centrality measures. The previous study has shown that top‐k skyline query, a method using dominance rule‐based centrality measures, reveals important protein candidates in Parkinson’s diseases. This study applied the top‐k skyline query to PPIs containing experiment and prediction data to find important proteins in Parkinson’s disease. The result shows that alpha‐synuclein (SNCA) is the most important protein and is expected to be a potential biomarker candidate for Parkinson’s disease.","PeriodicalId":13452,"journal":{"name":"Indonesian Journal of Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2021-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43716765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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