Indonesian Journal of Biotechnology
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Analysis of ethylene biosynthesis gene expression profile during titanium dioxide (TiO2) treatment to develop a new banana postharvest technology
No full textBanana is an important crop that demands proper methods in postharvest handling. As a climacteric fruit, thebanana fruit ripening process is affected by ethylene. Several methods have been developed to extend the shelf life of a banana, such as using ethylene scrubbers. In this study, ttanium dioxide (TiO2), a photocatalyst, was used as an alternatve method to delay the fruit ripening process. The effect of TiO2 on the ripening‐related gene MaACS1 was investgated. Banana fruits were placed in a TiO2‐coated glass chamber and observed for ten days. Fruit ripening in the treated chamber was delayed for eight days compared to the control. Total RNA was extracted from control and TiO2‐treated fruit pulp and synthesized into cDNA. Reverse transcripton PCR was performed to investgate the gene expression, which showed that MaACS1 expression was relatvely lower than treated control. The fnding of these studies suggested that the TiO2 chamber has the potental to extend the shelf life of banana by delaying its ripening process and decreasing the expression of MaACS1. To the best of our knowledge, no previous study has investgated the effect of TiO2 on the expression of genes related to banana fruit ripening
Isolation, identification, and detection of ACC deaminase gene-encoding rhizobacteria from rhizosphere of stressed pineapple
No full textACC deaminase is a microbial cytoplasmic enzyme that cleaves ACC, a precursor of ethylene, in the stressed plant. The aims of this study were to isolate, identify, and detect the presence of ACC deaminase gene-encoding rhizobacteria from the rhizospheric soil of pineapple plants that have been exposed to abiotic and biotic stress, specifically herbicide, flooding, and Phytophthora spp. stress. A total of 49 rhizobacterial isolates were obtained, seven of which were observed for their growth on DF medium containing 3 mM L-1 ACC. The four best-growing isolates were selected for genomic DNA extraction. They were molecularly identified as Stenotrophomonas maltophilia (3), Burkholderia territorii (2A), Pseudomonas oryzihabitans (5B), and Bacillus tropicus (1E). A set of primers, 105F-acdS 5’-TGCCAAGCGTGAAGACTGC-3’ and 244R-acdS 5’-GGGTCTGGTTCGACTGGAT-3’, were constructed to amplify the ACC deaminase gene (acdS). Based on melt peak curve analysis, four products appeared to show a specific single peak at 86, 89, 87, and 89.5°C, indicating a single product was produced. In addition, a Blast search showed that these four products met the ACC deaminase feature and their acdS sequences were clustered into an ancestral group compared with the bacterial strains deposited in GenBank. These results suggest that ACC deaminase gene-encoding rhizobacteria from a pineapple plantation of tropical origin may affect the acdS sequences and may contribute to the host plant’s stress tolerance
Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production
No full textRecombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained
Inverse correlation of kidney interstitial cells expansion with hemoglobin level and erythropoietin expression in single and repeated kidney ischemic/reperfusion injury in mice
No full textIschemic/reperfusion injury (IRI) causes acute kidney injury that may lead to chronic kidney disease. We investigated the correlation between kidney interstitial cells expansion, hemoglobin level, and erythropoietin expression as the chronic effects of single and repeated kidney IRI in mice. We created an IRI model using male Swiss mice by clamping the bilateral renal pedicles. Subjects were divided into four groups that contained six mice each: control/sham operation, single acute IRI, single chronic IRI, and repeated IRI. Our results showed that the single chronic and repeated IRI groups significantly increased the tubular injury score, decreased the hemoglobin level, and increased erythropoietin expression compared with the control. Lower hemoglobin levels in all of the groups compared with the control was associated with erythropoietin resistance. In single chronic and repeated kidney IRI, there were decreased creatinine levels compared with the control. The decreased creatinine levels from the single acute IRI group to the single chronic IRI group, suggesting a repair phase of IRI starting on day 7 occurred in the single chronic IRI group. A macrophage marker, CD68, and an inflammatory mediator marker, MCP-1, significantly increased in all IR groups, indicating inflammation occurred due to IRI. In conclusion, chronic and repeated kidney IRI induced interstitial cells expansion and inflammation associated with anemia
The establishment of PCR amplification, cloning, and sequencing of bovine herpesvirus 1 (BHV-1) glycoprotein D gene isolated in Indonesia
No full textConsidering the increasing incidence of infectious bovine rhinotracheitis (IBR) in Indonesia, it was necessary to conduct a more in-depth study of bovine herpesvirus-1 (BHV-1) as the causative agent of IBR disease. Previous research reports indicate that the BHV-1 subtypes found in Indonesia are subtype 1.1. Currently, IBR field case detection in Indonesia still uses the serological method (ELISA), which has the potential to give false positive results and cannot explain the virus subtype. Other detection methods, such as viral isolation, take longer and require adequate resources. This study aimed to determine the BHV-1 subtypes of Indonesian isolates using molecular techniques. Nested PCR using two pairs of primers was successfully used to amplify the glycoprotein D (gD) gene. The gD gene fragment was cloned into the pGEM-T plasmid. Analysis of the gD gene sequence was subsequently carried out to determine the BHV-1 character of the Indonesian isolates. The results indicated that the isolates were different from the previous isolates, and had similarities (100%) with subtype 1.2 strain SP1777 and SM023
The characterization of bacteriocins produced by Lactobacillus plantarum strains isolated from traditional fermented foods in Indonesia and the detection of its plantaricin-encoding genes
No full textLactobacillus plantarum is widely found in either anaerobic plant matter or fermented foods, and it has been recognized as producing antimicrobial bacteriocins. This study aimed to characterize the antimicrobial bacteriocins of L. plantarum and detect its genes that encode plantaricins. Samples were isolated from traditional fermented foods from Indonesia. Antimicrobial activity was evaluated using the agar diffusion assay procedure. The titration method applied the maximum amounts of lactic acid at 1054 mg/mL and hydrogen peroxide at 3.85 mg/mL. Based on the results, the supernatant of the L. plantarum strains appeared to have a broad spectrum of antimicrobial activity against pathogens, which would be active at pH 2.0–12.0 and stable temperature. In addition, almost all of the L. plantarum strains contained plantaricin-encoding genes (e.g. plnA, plnF,plnJK, and plnW), which were grouped into one cluster as indicated by phylogenetic analysis. Therefore, this study discovered clear evidence of the potential of some L. plantarum strains to act as antimicrobial agents
Molecular characterization of ageratum enation virus and beta satellite associated with leaf curl disease of fenugreek in India
No full textCisplatn is one of the chemotherapy for the treatment of triple‐negatve breast cancer (TNBC), but its effectveness is limited because of the phenomenon of chemoresistance. miR‐638 was shown to regulate chemoresistance; however, it has never been validated in the cisplatn‐resistant tumor from patents. This present study aimed to identfy the key gene regulatory networks of miR‐638 and evaluate the potental role of the miR‐638 and its targets as potental prognosis biomarkers for cisplatn‐resistance triple‐negatve breast cancer patents. The miR‐638 target was obtained from the miRecords database while the mRNA of chemoresistance biomarker candidate was obtained from the GSE18864 of GEO database, which is mRNA of cisplatn‐resistance TNBC patents. CCND1 and FZD7 are potental candidates for cisplatn chemoresistance biomarkers in patents with TNBC. Moreover, a Kaplan‐Meier survival plot showed that breast cancer patents with low mRNA levels of FZD7 had signifcantly worse overall survival than those in higher mRNA expression group. Taken together, miR‐638 plays a role in cisplatn resistance mechanism through a mechanism involving its target gene CCND1 and FZD7. Overall, miR‐638, CCND1, and FZD7 are candidates for cisplatn biomarker resistance in TNBC
The expression of growth factor signaling genes in co-culture IVM
No full textThe objective of this study was to determine the expression of growth factor signaling genes in human adiposederived stem cells (ASCs), porcine oocytes, and cumulus during in vitro maturation (IVM). The human ASCs (from 2 young and 2 old donors) were used for the co-culture IVM system. The maturation rate was examined based on polar body extrusion. The expression of the growth factor signaling genes from ASCs, oocytes, and cumulus were measured using qPCR. All data were analyzed using ANOVA followed by Tukey’s test. The expression of the h-IGF1 signaling genes from human ASCs cells showed similar values in all groups and the h-FGF2 expressions were higher in the young donors than the old ones. The p-FGF2, p-FGFR2, and p-TGFβ1 expressions in the oocytes as well as p-IGFR in the cumulus that were co-cultured from the young donors showed higher values than the old and control groups. The apoptotic ratio (p-BAX/p-BCL2) from the oocytes and cumulus in both co-culture groups also showed lower levels than the control (P<0.05). Oocyte maturation rates were significantly increased in all co-cultured groups (Y1 (85.9 ± 2.2%), Y2 (91.2 ± 1.1%), O1 (86.3 ± 1.5%), and O2 (86.5 ± 2.3%)) compared with the control (76.7 ± 1.1%; P<0.05). Although the expression of growth factor signaling genes was varied, young donors’ ASCs might support in vitro maturation beħer than those from old donors
Allelic diversity of butyrophilin (BTN1A1) gene in Indian bovines
No full textIndian milch bovines comprises of 58.56% of total livestock population (512.05 million) in the country and primarily includes native and crossbred cattle (37.28%) and water buffaloes (21.28%). Milk and milk products are essential food items of Indian diet especially in children, old and senile. Milk fat is an important constituent of milk and has an economic value and its percentage in milk varies betweem species and breeds within species. Butyrophilin (BTN1A1) a membrane protein regulates secretion of lipids and size of a fat globule in milk. Present study was conducted in 538 bovines of 11 breeds/populations adapted to different parts of India, with an aim to screen and determine the major allele of BTN1A1 gene using PCR-RFLP based test. Results indicate that exon 8 of BTN1A1 gene is polymorphic in Tharparkar, Sahiwal, Jhari and Belahi populations of native cattle and Holstein Friesian and Jersey crossbreds where as the same exon was monomorphic in Murrah, Chilika, Gojri, Chhattisgarhi and Bargur populations of water buffalo. We conclude that variations in BTN1A1 gene can serve as an excellent genetic marker while selecting cows for higher milk fat and can be applied while formulating their breeding plans
Evaluation of potential gene expression as early markers of insulin resistance and non-alcoholic fatty liver disease in the Indonesian population
No full textEarly detection of insulin resistance (IR) or non-alcoholic fatty liver disease (NAFLD) is crucial to preventing future risks of developing chronic diseases. The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR), Liver Fat Score (LFS), and Fatty Liver Index (FLI) are generally employed to measure severity stages of IR and NAFLD. The study of gene expressions could explain the molecular mechanisms that occur early on in IR and NAFLD; thus providing potential early markers for both diseases. This study was conducted to evaluate the gene expressions that could potentially be early markers of IR and NAFLD. All participants (n = 21) had normal blood glucose and were categorized as without hepatosteatosis (n = 10), at higher risk of hepatosteatosis (n = 6), and hepatosteatosis (n = 5). Gene expression analysis was performed using the 2-∆∆CT relative quantification method. There were significant differences in galnt2 (p < 0.002) and sirt1 (p < 0.010) expression between the first and the third tertiles of HOMA-IR; and in ptpn1 (p < 0.012) expression between the first and the second tertiles of LFS. In conclusion, the expressions of galnt2 and sirt1 could be used as early markers of IR, while the expression of ptpn1 could be employed as an early marker of NAFLD