Indonesian Journal of Biotechnology
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Development of nanocomplex mimic‐hsa‐miR‐143‐3p loaded exosome (exo‐miR) to inhibit viability, migration and proliferation of triple‐negative breast cancer
No full textBreast cancer represents the highest number of cancer cases in Indonesia, with triple‐negative breast cancer (TNBC) being a common subtype (10–15%). MicroRNAs play a role in cancer epigenetics and contributing as core factors to the disease. The expression of miR‐143‐3p have been found to be lower in breast cancer samples from Yogyakarta and Central Java. It is known that miR‐143‐3p functions as a tumor suppressor in breast cancer, and its overexpression corresponds with an increased survival rate. The structure of miRNA is quickly degraded, an enhanced delivery system for miRNA is required. Exosomes are indeed emerging as natural delivery agent. A new approach represents that exosomes will be transfected with mimic‐hsa‐miR‐143‐3p yield an exo‐miR. The research aimed to examine how exo‐miR affects viability, migration, and proliferation using 4T1 cell line. The Exo‐Fect‐based method was used to transfect mimic‐hsa‐miR‐143‐3p into exosomes. The MTT assay, wound healing assay, and colony formation assay were used as functional assay. The MTT assay revealed that 7.5 µL/ 250,000 particles exo‐miR obtained a lower percentage of cell viability (58%) than the control (99.7%). The wound healing assay showed that transfection of 37.5 µL/ 1,250,000 particles exo‐miR was able to suppress migration by the percentage of wound closure (67%) compared to the control (100%). Exo‐miR also had a significant (p < 0.001) effect on colony‐forming abilities, as shown by fewer colonies (32) compared to the control (132). This findings demonstrated that exo‐miR represents a promising targeted approach in cancer therapy
Expression and purification of recombinant human granulocyte colony‐stimulating factor (rG‐CSF) from Pichia pastoris
No full textRecent advances in biotechnology have sparked global interest in developing biosimilar drugs, particularly those containing physiologically active proteins, such as growth factors and cytokines. The methylotrophic yeast Pichia pastoris can produce and secrete fully active heterologous proteins with strong secretory capacity and low levels of native proteins and has the ability to achieve high cell densities. In this study, a yeast‐based system was used to express and purify recombinant human granulocyte colony‐stimulating factor (rG‐CSF). Cultures were induced every 12 h for 48 h to express rG‐CSF, and parameters such as cell density, media pH, and cell dry weight were observed. Cell density increased along with the corresponding secretion of rG‐CSF during the induction period, as determined by Western blot assay, while the pH of the media remained stable. Ammonium sulfate at different saturation levels was used to precipitate the recombinant protein, with the highest total protein content determined spectrophotometrically at 29.6 µg/mL. Ni‐NTA resin with affinity column purification was used to purify the recombinant protein. The purified protein showed rG‐CSF with a molecular weight of approximately 18 kDa based on SDS‐PAGE analysis and immuno slot blot assay detected in purple. Overall, the study results indicated that the production and purification of rG‐CSF was successful, although optimization was required. The long‐term goal of this research is to discover alternative methods and sources for producing biosimilars of the therapeutic protein rG‐CSF, which can be utilized in the pharmaceutical industry to support health programs, particularly cancer treatment
Detection and quantification of pork and rat DNA in processed meats using multiplex quantitative Real‐Time PCR (m‐qPCR)
No full textIn addition to the issue of pork contamination, processed meats frequently contain traces of rat meat. Therefore, detection and quantification of the pork and rat DNA in cases of meat and processed meat adulteration are necessary. In the current study, two gene targets of the cytochrome b for pigs and the Mt‐atp6 of Rattus norvegicus for rats were used in the absolute multiplex quantitative real‐time PCR (m‐qPCR). The sample DNA was amplified with a standard as positive control in the various concentration of 1000 pg, 100 pg, 10 pg, 0.1 pg, 0.01 pg, and 0.001 pg. There were 25 processed meat samples and 5 fresh meat samples identified in this study. Among the total of 30 samples assessed, 6 samples were successfully detected and quantified their pork and rat DNA contamination. One sample was contaminated with pork DNA with a concentration of 2.451×10‐4 pg (“Meatball 3). Five samples were contaminated with rat DNA with a concentration of 3.603×10‐11 pg (“Sempol 3”), 2.196×10‐10pg (“Meatball 6”), 4.908×10‐11 pg (“Siomay 3”), 1.489×10‐10 pg (“Grinding 2”), and 3.564×10‐10 pg (“Grinding 4”). In this study, we have discovered that the contamination of pork and rat were detected in the samples. It suggested that this method is applicable for detecting the contaminant in processed meat sample
Genes expression analysis of EgUnk1, EgZFP2, and EgIPK2b in oil palm using Ct value correction and two relative quantification approaches
No full textThe determination of transcript accumulation values significantly affects gene expression in oil palm. Various genes are involved in pathogen infection, including probable 2‐oxoglutarate‐dependent dioxygenase At5g05600 (EgUnk3), zinc finger protein 2‐like (EgZFP2), and inositol polyphosphate multikinase beta‐like (EgIPK2b). Gene expression is typically measured using relative quantitative methods to calculate differences in quantitative values in the expression levels of targeted genes compared to a reference gene. However, the effectiveness of these methods in assessing the expression of EgUnk3, EgZFP2, and EgIPK2b, which are involved in Ganoderma boninense infection in oil palm seedlings, requires evaluation. This study aimed to establish an effective and straightforward method for analyzing the expression of EgUnk1, EgZFP2, and EgIPK2b genes in oil palm seedlings infected with G. boninense, utilizing Ct value correction through regression coefficients on the 2‐ΔΔCt and E‐ΔΔCt approaches. A correlation regression revealed values of 0.28, ‐0.32, and 0.29 for delta Ct of EgUnk1, EgZFP2, and EgIPK2b, respectively. However, a negative correlation in the Ct mean was corrected by linear regression for the targeted genes: ‐0.55, ‐0.81, and ‐0.29 for EgUnk1, EgZFP2, and EgIPK2b, respectively. The amplification factor (E) and efficiency value (R) using the EgActin gene were 1.95 and 94.92%, respectively. Normalization of log10 on the fold change value 2‐ΔΔCt and 1.95‐ΔΔCt approaches using the regression coefficient yielded consistent results for the EgUnk1, EgZFP2, and EgIPK2b genes. Overall, EgUnk3 and EgIPK2b genes exhibited downregulated expression in susceptible oil palm seedlings (‐0.60 for 2(‐ΔΔCt) and ‐0.58 for 1.95(‐ΔΔCt)), whereas EgIPK2b gene showed up‐regulated and the highest value in inoculated resistant seedlings (1.39 for 2(‐ΔΔCt) and 1.34 for 1.95(‐ΔΔCt)). Basal stem rot disease (BSR) in oil palm decreased EgUnk1 and EgIPK2b expression in susceptible seedlings but increased EgZFP2 gene expression in resistant ones. The results of this research provide valuable corrections to Ct values obtained directly from RT‐qPCR machines using simple linear regression. Consequently, the Ct values of target genes and reference genes exhibit smaller bias values, rendering gene expression levels more reliable
Improving transient gene expression and agroinfiltration‐based transformation effectiveness in Indonesian orchid Phalaenopsis amabilis (L.) Blume
No full textTransient gene expression is an approach used to study transient genes across various species, with infiltration by Agrobacterium tumefaciens (agroinfiltration) being a commonly used method. Agroinfiltration offers a simple and effective means of delivering transgenes into the plant genome. An alternative method for enhancing the quality and productivity of orchids as ornamental plants is genetic modification through agroinfiltration. Although Agrobacterium‐mediated genetic transformation by immersion has been used on the Phalaenopsis amabilis (L.) Blume species of orchid, transformation efficiency using the immersion technique remains relatively low and the method itself is challenging due to its requirement for aseptic handling. The application of agroinfiltration in P. amabilis has not previously been reported. This study investigates the impact of the injection site, acetosyringone concentration, bacterial density (OD600), and injection volume to determine the optimum conditions for agroinfiltration on P. amabilis. The results demonstrated that injection site had a noticeably distinct impact on transformation effectiveness, with the abaxial position of the leaf being the optimal site for Agrobacterium culture suspension injection. While adjustments in acetosyringone concentration, bacterial density (OD600), and injection volume did not significantly affect transformation efficiency, they did influence the peak time of GFP fluorescence. Acetosyringone at a concentration of 200 µM, an OD600 of 1.0 for Agrobacterium culture, and an injection volume of 500 µL effectively accelerated GFP expression duration
Expression profiles of XIK1 and OsSWEET14 genes in parental and back‐ crossing rice lines after Xanthomonas oryzae pv. oryzae infection
No full textOryza sativa L. ssp. indica (RD47 cultivar) is a major commercial rice variety known for its highly stable yields. However, it is highly susceptible to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). While previous research has focused on improving rice cultivars through breeding programs, no reports involved the interaction between Xoo infection and gene expression. This study aimed to analyze the relationship between bacterial blight disease and gene expression, focusing on two resistance genes (Xa21 and XIK1) and one susceptible gene (OsSWEET14). Gene expression analysis revealed that the Xa21 gene conferred effective resistance against bacterial blight Xoo16PK002 infection, providing high and moderate resistance to bacterial blight symptoms in two rice varieties carrying the Xa21 gene, IRBB21 and the near–isogenic RD47–Xa21 BC4F4, respectively. Additionally, the Xa21 gene directly induced XIK1 expression in both resistance rice cultivars. Moreover, one susceptible gene, OsSWEET14, was consistently up–regulated in only the bacterial blight–susceptible indica rice cultivar RD47. Therefore, the up–regulation of resistance genes and the suppression of susceptible genes contributed to the improvement of bacterial blight disease in the RD47 cultivar. Xa21 emerged as a criti‐ cally important gene in directly inducing mechanisms against Xoo, thereby promoting the reduction of bacterial blight disease
Identification of medium‐grain rice based on GS3, a gene linked to rice grain size
No full textPrevious studies have used molecular markers associated with the GS3 gene to differentiate between short and long rice. However, there are three classifications of grain size: long, short, and medium. The identification of medium‐grain rice using these markers linked to the GS3 gene is yet to be confirmed. Hence, this study aimed to identify medium‐grain rice through phenotyping and genotyping. Grain characteristics including grain length (GL), grain width (GW), and the length‐to‐width ratio (GL/GW) were measured using SmartGrain software. The genotype was then amplified with the GS3 gene‐linked DRR‐GL (double round‐robin for grain length) molecular marker. The results revealed that medium‐grain rice, as identified by the DRR‐GL marker, exhibited DNA bands at the position of 150 bp. These bands differed from those observed in long‐grain rice, but they were consistent with those found in short‐grain rice. The genotypic results further indicated that PCR products obtained with the DRR‐GL marker in medium‐grain rice accounted for 86.8% of the phenotypic variation in grain size. This study provides fundamental genetic insights into the identification of medium‐grain rice and contributes to optimizing effects on rice breeding related to grain size
Protective effects of Zingiber cassumunar Roxb. extract against UVB‐induced oxidative stress in Wistar albino rats (Rattus novergicus Berkenhout, 1769)
No full textProtecting the skin from the effects of UVB radiation using natural products is crucial in the cosmeceutical industry. This study aims to investigate the protective effects of Bangle (Zingiber cassumunar Roxb.) against UVB‐induced skin damage in Wistar albino rats. The rhizomes were macerated using 70% ethanol v/v, followed by n‐hexane to obtain n‐hexane soluble and n‐hexane insoluble fraction. The antioxidant properties of the ethanol extracts and n‐hexane soluble fraction were evaluated using a 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay. The study also examined the antiphotoaging properties through reactive oxygen species (ROS) scavenging assay, matrix metalloproteinase‐1 (MMP‐1) expression, and tyrosinase expression against UVB radiation in Wistar albino rats. The results demonstrated that the Z. cassumunar extract and fraction effectively converted DPPH radicals into a more stable compound. Analysis revealed the presence of Benzene, 4‐(1Z)‐1,3‐butadien‐1‐yl‐1,2‐dimethoxy‐ and (E)‐4‐(3,4‐Dimethoxyphenyl) but‐3‐en‐1‐ol as the primary compounds in both the extract and fraction, suggesting their contribution to the observed activity. Furthermore, Z. cassumunar compounds could reduce UVB‐induced ROS production and may protect against skin photoaging by changing the expression of MMP‐1 and tyrosinase levels in Wistar albino rats. These findings suggest that Z. cassumunar holds promise for preventing skin aging
Overexpression and purification of YidRv gene from the hypervirulent Klebsiella pneumoniae, and the ability of the gene product in inducing a humoral response
No full textThe yidRv gene, isolated from the hypervirulent Klebsiella pneumoniae (hvKP), is a novel gene with an unknown function; however, it has exhibited high homology to the yidR, a gene recognized as potential vaccine candidate. The aim of this study was to clone the yidRv gene from the Indonesian hvKP and to investigate its overexpression in Escherichia coli. In the experiment, yidRv was cloned into pET21 to construct pYik23. Recombinant protein YidRv was produced by growing E. coli BL21 (DE3)/pYik23 in LB medium with ampicillin at 29 °C, inducing protein synthesis with 0.5 mM IPTG for 20 hours. Purification was performed using Ni‐NTA resin, and the purified protein (50 µg) was administered to BALB/c mice to test for the production of IgG, IgM and IgA on 2 days before and day 19th and 37th after the first vaccination. The results show a significant induction of IgG and IgM, but not of IgA antibodies. In conclusion, the yidRv gene was overexpressed in E. coli BL21 (DE3) at high levels in soluble form, with the recombinant protein able to be purified to 90% homogeneity. The recombinant YidRv demonstrated the ability to stimulate the generation of both IgM and IgG antibodies
Antimicrobial compounds from intracellular and extracellular secondary metabolites of Actinobacteria InaCC A759
No full textThe World Health Organization (WHO) has determined a list of pathogens that require the development of new antimicrobials due to resistance problems; these include Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus. In addition, Mycobacterium smegmatis has been used for antimycobacterial discovery to address the increasing burden of tuberculosis. In this study, optimization of antimicrobial activity, secondary metabolite profiling, and strain identification was conducted on Actinobacteria InaCC A759. Intracellular and extracellular extracts of Actinobacteria InaCC A759 were found to have different antimicrobial activities. The minimum inhibitory concentration (MIC) values of the extract to inhibit the growth of M. smegmatis, E. coli, and P. aeruginosa were 50, 25, and 100 µg/mL (intracellular), and 25, 25, and 100 µg/mL (extracellular), respectively. However, neither extract was able to inhibit the growth of S. aureus. Metabolite profiling using High resolution‐mass spectrometry (HR‐MS) resulted in differences in the major compound between the two extracts of Actinobacteria InaCC A759, namely n‐acetyltyramine (C10H13NO2/179.0945) (24.24%) (intracellular) and palmitic acid (C16H32O2/273.27034) (86.92%) (extracellular). Based on molecular analysis of the 16S rRNA gene, Actinobacteria InaCC A759 is identical to the Streptomyces olivaceus strain FoRh46. The antimicrobial activity and secondary metabolites profiles of Streptomyces olivaceus InaCC A759 have not been previously reported