Displaying publications 21 - 40 of 104 in total

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  1. Production and characterization of graphene from carbonaceous rice straw by cost-effect extraction
    Uda MNA, Gopinath SCB, Hashim U, Halim NH, Parmin NA, Uda MNA, et al.
    3 Biotech, 2021 May;11(5):205.
    PMID: 33868892 DOI: 10.1007/s13205-021-02740-9
    This paper describes the synthesis of graphene-based activated carbon from carbonaceous rice straw fly ash in an electrical furnace and the subsequent potassium hydroxide extraction. The produced graphene has a proper morphological structure; flakes and a rough surface can be observed. The average size of the graphene was defined as up to 2000 nm and clarification was provided by high-resolution microscopes (FESEM and FETEM). Crystallinity was confirmed by surface area electron diffraction. The chemical bonding from the graphene was clearly observed, with -C=C- and O-H stretching at peaks of 1644 cm-1 and 3435 cm-1, respectively. Impurities in the graphene were found using X-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy. The measured size, according to zeta-potential analysis, was 8722.2 ± 25 nm, and the average polydispersity index was 0.576. The stability of the mass reduction was analyzed by a thermogravimetric at 100 °C, with a final reduction of ~ 11%.
  2. Microwave-assisted solvent-free extraction of essential oil from Coleus aromaticus: anti-phytopathogenic potential for fruit post-harvesting
    Gunny AAN, Fang LP, Misnan NM, Gopinath SCB, Salleh NHM, Hashim RHR, et al.
    3 Biotech, 2021 Apr;11(4):166.
    PMID: 33816043 DOI: 10.1007/s13205-021-02701-2
    This work evaluates the fungicidal effect of essential oil from Coleus aromaticus (C. aromaticus) by solvent-free microwave-assisted extraction with a yield of 0.54%. Fourier-transform infrared spectroscopy was utilised to identify the functional groups, which were O-H, C-O, C-H, and C=C. Gas chromatography-mass spectrometry analysis was performed to determine the primary essential oil components, namely, thymol (92.62%), thymoquinone (2.64%), creosol (1.77%), linalool (1.68%), p-Cymene-2,5-diol (0.73%), and p-Cymene (0.56%). The inhibitory effect of essential oil extracted from C. aromaticus against the isolated fungi, Aspergillus niger from mango, was investigated. The mycelial growth inhibition of the extracted essential oil by the poisoned food test and disc diffusion assay showed the reduction at 79.63 ± 1.7 and 70.45 ± 6.54%, respectively. In vivo experiment was conducted with artificially wounded and unwounded mangoes, applying the extracted essential oil to the wounded mangoes inoculated with A. niger that could decrease the disease incidence from 100 to 58.33 ± 14.43%. Meanwhile, the treatment of the extracted essential oil did not affect the quality of the mango and it also shows improvement in weight loss reduction of the mango in comparison to the chemical fungicide and untreated mango. Hence, essential oil from C. aromaticus by solvent-free microwave-assisted extraction could be used as an effective control for the fruit spoilage and potential source of fruit preservative.
  3. Fulltext Hepatoprotective effect of bone marrow-derived mesenchymal stromal cells in CCl4-induced liver cirrhosis
    Aithal AP, Bairy LK, Seetharam RN, Kumar N
    3 Biotech, 2021 Feb;11(2):107.
    PMID: 33564610 DOI: 10.1007/s13205-021-02640-y
    Bone marrow mesenchymal stromal cells (BM-MSCs) are multipotent stem cells which are ideal candidates for use in regenerative medicine. The objectives of this study were to evaluate the hepatoprotective effect of BM-MSC and its combination treatment with silymarin in carbon tetrachloride (CCl4)-induced liver cirrhosis animal model and to investigate whether tail vein or portal vein infusion was the ideal route for BM-MSC transplantation. 36 female Wistar rats were randomly divided into six groups (n = 6): Group 1 (normal control), Group 2 (received only CCl4, disease model), Group 3 (CCl4 + BM-MSCs through tail vein), Group 4 (CCl4 + BM-MSCs through portal vein), Group 5 (CCl4 + silymarin), Group 6 (CCl4 + BM-MSCs + silymarin). On the 21st day after treatment, blood samples were collected for biochemical estimations. After the experiment, the rats were sacrificed. Liver was dissected out and processed for histopathology and scanning electron microscopy studies. Liver enzyme and marker analysis, histopathological studies indicated that the combination of BM-MSCs and silymarin was effective in treating liver cirrhosis. Transplanted BM-MSCs in combination with silymarin ameliorated the liver tissue damage through their immunoregulatory activities. Among the two routes, the intravenous administration of cells through the tail vein was found to be more effective and safe.
  4. Fulltext Bacterial and eukaryotic microbial communities in urban water systems profiled via Illumina MiSeq platform
    Ting ASY, Zoqratt MZHM, Tan HS, Hermawan AA, Talei A, Khu ST
    3 Biotech, 2021 Feb;11(2):40.
    PMID: 33479595 DOI: 10.1007/s13205-020-02617-3
    Microbial communities from a lake and river flowing through a highly dense urbanized township in Malaysia were profiled by sequencing amplicons of the 16S V3-V4 and 18S V9 hypervariable rRNA gene regions via Illumina MiSeq. Results revealed that Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the dominant prokaryotic phyla; whereas, eukaryotic communities were predominantly of the SAR clade and Opisthokonta. The abundance of Pseudomonas and Flavobacterium in all sites suggested the possible presence of pathogens in the urban water systems, supported by the most probable number (MPN) values of more than 1600 per 100 mL. Urbanization could have impacted the microbial communities as transient communities (clinical, water-borne and opportunistic pathogens) coexisted with common indigenous aquatic communities (Cyanobacteria). It was concluded that in urban water systems, microbial communities vary in their abundance of microbial phyla detected along the water systems. The influences of urban land use and anthropogenic activities influenced the physicochemical properties and the microbial dynamics in the water systems.

    Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-020-02617-3.

  5. Fulltext The effects of amino acids and fatty acids on the disease resistance of Epinephelus fuscoguttatus in response to Vibrio vulnificus infection
    Nurdalila AA, Natnan ME, Baharum SN
    3 Biotech, 2020 Dec;10(12):544.
    PMID: 33240745 DOI: 10.1007/s13205-020-02543-4
    Mass mortality resulting from bacterial infection poses a major problem in the grouper aquaculture industry. The purpose of this study was to profile the metabolites released in challenged fish and to reconstruct the metabolic pathways of brown marble grouper (Epinephelus fuscoguttatus) in response to Vibrio vulnificus infection. Metabolite profiles from control and challenged treatment groups after feeding were determined using gas chromatography-mass spectrometry (GC-MS). Forty metabolites were identified from the GC-MS analysis. These metabolites comprised of amino acids, fatty acids, organic acids and carbohydrates. The profiles showed the highest percent area (33.1%) for leucine from the amino acid class in infected fish compared to the control treatment group (12.3%). Regarding the fatty acid class, a higher percent area of the metabolite 8,11-eicosadienoic acid (27.04%) was observed in fish infected with V. vulnificus than in the control treatment group (22.5%). Meanwhile, in the carbohydrate class, glucose (47.0%) was the metabolite in the carbohydrate class present at highest percentage in the control treatment group compared to infected fish (30.0%). Our findings highlight the importance of a metabolic analysis for understanding the changes of metabolites in E. fuscoguttatus in response to bacterial infections.
  6. Fulltext A rapid RNA extraction method from oil palm tissues suitable for reverse transcription quantitative real-time PCR (RT-qPCR)
    Badai SS, Rasid OA, Parveez GKA, Masani MYA
    3 Biotech, 2020 Dec;10(12):530.
    PMID: 33214977 DOI: 10.1007/s13205-020-02514-9
    Cetyltrimethylammonium bromide (CTAB) is the preferred detergent in RNA extraction of oil palm tissues. However, the CTAB-based protocol is time-consuming. In this study, a combination of the CTAB-based method and silica-based purification reduced the extraction time from two days to five hours. Quality of total RNA from 27 different tissues of oil palm was shown to have an RNA integrity number (RIN) value of more than seven. The extracted RNA was evaluated by RT-qPCR using three reference oil palm genes (GRAS, CYP2, and SLU7) and three putative mesocarp-specific transcripts annotated as WRKY DNA-binding protein 70 (WRKY-70), metallothionein (MT) and pentatricopeptide repeat (PPR) genes. Tissue-specific expression profiling across complete developmental stages of mesocarp and vegetative tissues was determined in this study. Overall, the RNA extraction protocol described here is rapid, simple and yields good quality RNAs from oil palm tissues.
  7. Fulltext Anti-snake venom and methanolic extract of Andrographis paniculata: a multipronged strategy to neutralize Naja naja venom acetylcholinesterase and hyaluronidase
    Nayak AG, Kumar N, Shenoy S, Roche M
    3 Biotech, 2020 Nov;10(11):476.
    PMID: 33083200 DOI: 10.1007/s13205-020-02462-4
    The study investigates the ability of methanolic extract of Andrographis paniculata (MAP) to supplement polyvalent anti-snake venom (ASV) in inhibiting neurotoxic enzyme acetylcholinesterase (AChE) and 'spreading factor' hyaluronidase from Naja naja (N.N) venom. AChE and hyaluronidase activity were measured in 100 or 200 µg of crude venom, respectively, and designated as 'control'. In Test Group I, enzyme assays were performed immediately after the addition of ASV/MAP/ASV + MAP to the venom. Inhibition of AChE by ASV (100-367 µg) was 12-17%, and of hyaluronidase (22-660 µg) was 33-41%. Under the same conditions, MAP (100-400 µg) inhibited AChE and hyaluronidase to the extent of 17-33% and 17-52%, respectively. When ASV (220 µg) and MAP (100-200 µg) were added together, AChE and hyaluronidase were inhibited to a greater extent from 39-63 to 36-44%, than when either of them was used alone. In Test Group 2, the venom was incubated with ASV/MAP/ASV + MAP for 10-30 min at 37 °C prior to the assay which enhanced AChE inhibition by 6%, 82% and 18% respectively, when compared to Test Group I. Though there was no change in inhibition of hyaluronidase in the presence of ASV, MAP could further increase the extent of inhibition by 27% and ASV + MAP upto 4%. In Test Group III, venom and substrate were incubated for 90 min and hyaluronidase activity was measured after the addition of inhibitors. Here, ASV + MAP caused increased inhibition by 69% compared to ASV alone. The study confirms the ability of phytochemicals in MAP to contribute to a multipronged strategy by supplementing, thereby augmenting the efficacy of ASV.
  8. Fulltext Cytotoxicity and insulin resistance reversal ability of biofunctional phytosynthesized MgO nanoparticles
    Jeevanandam J, Chan YS, Danquah MK
    3 Biotech, 2020 Nov;10(11):489.
    PMID: 33123456 DOI: 10.1007/s13205-020-02480-2
    The present study investigates the cytotoxicity of hexagonal MgO nanoparticles synthesized via Amaranthus tricolor leaf extract and spherical MgO nanoparticles synthesized via Amaranthus blitum and Andrographis paniculata leaf extracts. In vitro cytotoxicity analysis showed that the hexagonal MgO nanoparticles synthesized from A. tricolor extract demonstrated the least toxicity to both diabetic and non-diabetic cells at 600 μl/ml dosage. The viability of the diabetic cells (3T3-L1) after incubation with varying dosages of MgO nanoparticles was observed to be 55.3%. The viability of normal VERO cells was 86.6% and this stabilized to about 75% even after exposure to MgO nanoparticles dosage of up to 1000 μl/ml. Colorimetric glucose assay revealed that the A. tricolor extract synthesized MgO nanoparticles resulted in ~ 28% insulin resistance reversal. A reduction in the expression of GLUT4 protein at 54 KDa after MgO nanopaSrticles incubation with diabetic cells was observed via western blot analysis to confirm insulin reversal ability. Fluorescence microscopic analysis with propidium iodide and acridine orange dyes showed the release of reactive oxygen species as a possible mechanism of the cytotoxic effect of MgO nanoparticles. It was inferred that the synergistic effect of the phytochemicals and MgO nanoparticles played a significant role in delivering enhanced insulin resistance reversal capability in adipose cells.
  9. Fulltext Validation of target proteins of down-regulated miR-221-5p in HeLa cells treated with Polyalthia longifolia leaf extract using label-free quantitative proteomics approaches
    Shanmugapriya, Othman N, Sasidharan S
    3 Biotech, 2020 Sep;10(9):399.
    PMID: 32850286 DOI: 10.1007/s13205-020-02396-x
    The current study was conducted to validate the target proteins of down-regulated miR-221-5p in HeLa cells treated with P. longifolia leaf extract. The validation was done by label-free quantitative proteomics approaches, Gene Ontology (GO) and protein-protein interaction analyses after the cells transfected with miRNA mimics or miRNA inhibitor. The LC-ESI-MS/MS identified a total of 1061, 668, 564 and 940 proteins from untransfected and untreated HeLa cells, untransfected P. longifolia leaf extract-treated HeLa cells, miR-221-5p mimic-transfected P. longifolia leaf extract-treated HeLa cells and anti-miR-221-5p-transfected P. longifolia leaf extract-treated HeLa cells, respectively. The proteomic, GO and protein-protein interaction analyses showed that P. longifolia treatment regulated various protein expressions in HeLa cells, namely tropomyosin, PRKC apoptosis WT1 regulator protein (PAWR), alpha-enolase and beta-enolase, which induced apoptotic cell death after the down-regulation of miR-221-5p. Conclusively, this study showed P. longifolia leaf extract's vital contribution in regulating various protein expressions in HeLa cervical cancer cells to induce apoptotic cell death after downregulation miR-221-5p.
  10. Fulltext Anxiety determination by antibody-conjugated nanoparticles on an interdigitated electrode sensor
    Wang X, Gopinath SCB, Li J
    3 Biotech, 2020 Sep;10(9):377.
    PMID: 32802719 DOI: 10.1007/s13205-020-02370-7
    This work focused on the detection of cortisol on an interdigitated electrode sensor surface using an anti-cortisol antibody. To improve immobilization, antibodies were conjugated with silver nanoparticles and attached to the surface of the sensor. Cortisol interacted in a dose-dependent manner on the antibody-immobilized sensor surface, and current changes were observed. Linear regression analysis was performed by a 3σ calculation, and the limit of detection fell into the range of 0.01 and 0.1 ng/mL. The sensitivity of cortisol was calculated to be 0.01 ng/mL and the sensor discriminated against other hormones, namely norepinephrine and progesterone, with higher selectivity for cortisol. This result represented the selective detection of cortisol with high performance, which can help to determine anxiety disorders.
  11. Fulltext Enhancement of nitrification efficiency during sludge bulking by magnetic field under long sludge retention time
    Zaidi NS, Muda K, Sohaili J, Loan LW, Sillanpää M
    3 Biotech, 2020 Sep;10(9):408.
    PMID: 32904368 DOI: 10.1007/s13205-020-02398-9
    The aim of the present study is to investigate the potential of magnetic field application as an alternative approach for controlling sludge bulking due to long sludge retention time (SRT) while enhancing nitrification efficiency upon the occurrence. Two sequencing batch reactors, reactor A (SBRA, magnetic field intensity 88.0 mT) and reactor B (SBRB, control) were operated under long SRT to induce the growth of filamentous microorganisms. The effect of magnetic field on nitrification, viz. ammonia-nitrogen (NH4-N) and nitrite removal, as well as biomass properties were studied under the sludge bulking condition. Results indicated that nitrification efficiency of SBRA was consistently higher with 90% NH4-N removal and 74-81% nitrite removal, which could be credited to the enhanced biomass properties of activated sludge due to the induced magnetic field. Metabolism activity and biodegradability of aerobic bacteria were also enhanced through the application of magnetic field, even under long SRT condition. This was evidenced by the average oxygen uptake rate (OUR) in SBRA that was higher with 11.7 ± 1.2 mg/L·h compared to SBRB with 9.5 ± 0.4 mg/L·h. Occurrence of filamentous sludge bulking was likewise minimized.
  12. Fulltext Synthesis and characterization of reduced graphene oxide using the aqueous extract of Eclipta prostrata
    Chuah R, Gopinath SCB, Anbu P, Salimi MN, Yaakub ARW, Lakshmipriya T
    3 Biotech, 2020 Aug;10(8):364.
    PMID: 32832325 DOI: 10.1007/s13205-020-02365-4
    In this study, biological deoxygenation of graphene oxide (GO) using an Eclipta prostrata phytoextract was performed via the infusion method. The presence of oxide groups on the surface of graphene and removal of oxides groups by reduction were characterized through morphological and structural analyses. Field emission scanning electron microscopy images revealed that the synthesized GO and rGO were smooth and morphologically sound. Transmission electron microscopy images showed rGO developing lattice fringes with smooth edges and transparent sheets. Atomic force microscopy images showed an increase in the surface roughness of graphite oxide (14.29 nm) compared with that of graphite (1.784 nm) due to the presence of oxide groups after oxidation, and the restoration of surface roughness to 2.051 nm upon reduction. Energy dispersive X-ray analysis indicated a difference in the carbon/oxygen ratio between GO (1.90) and rGO (2.70). Fourier-transform infrared spectroscopy spectrum revealed peak stretches at 1029, 1388, 1578, and 1630 cm-1 for GO, and a decrease in the peak intensity after reduction that confirmed the removal of oxide groups. X-ray photoelectron microscopy also showed a decrease in the intensity of oxygen peak after reduction. In addition, thermogravimetric analysis suggested that rGO was less thermally stable than graphite, graphite oxide, and GO, with rGO decomposing after heating at temperatures ranging from room temperature to 600 °C.
  13. Fulltext Transcriptomic analysis of multi-drug resistant Escherichia coli K-12 strain in response to Lavandula angustifolia essential oil
    Lai PJ, Ng EV, Yang SK, Moo CL, Low WY, Yap PS, et al.
    3 Biotech, 2020 Jul;10(7):313.
    PMID: 32596098 DOI: 10.1007/s13205-020-02304-3
    To better understand the synergistic antibacterial activity between piperacillin and Lavandula angustifolia essential oil (LEO) against multidrug-resistant Escherichia coli, we performed microarray transcriptomic analysis of LEO when used alone and in combination with piperacillin against the non-treated control. In total, 90 genes were differentially expressed after the combination of LEO and piperacillin treatment. Among the up-regulated genes, nfsB, nemA, fruA, nfsB, nemA are known to control microbial metabolism and nitrotoluene degradation, which were observed only in the LEO-piperacillin combinatory treatment. Four candidate genes from the microarray result, srIA, srID, waaR and nfsB, were validated by qRT-PCR as these genes showed differential expression consistently in the two methods. Biochemical pathway analysis showed that there was upregulation of genes involved in several biological processes including fructose and mannose metabolism, phosphotransferase system (PTS), lipopolysaccharide biosynthesis and nitrotoluene degradation. Genes involved in microbial metabolism in diverse environments were found both up- and down-regulated in LEO-piperacillin combinatory treatment. Our study provides new information concerning the transcriptional changes that occur during the LEO and piperacillin interaction against the multidrug-resistant bacteria and contributes to unravel the mechanisms underlying this synergism.
  14. Fulltext Promising discovery of beneficial Escherichia coli in the human gut
    Nakkarach A, Foo HL, Song AA, Nitisinprasert S, Withayagiat U
    3 Biotech, 2020 Jul;10(7):296.
    PMID: 32550113 DOI: 10.1007/s13205-020-02289-z
    Ingested dietary fibres are hydrolysed by colon microbiota to produce energy-providing short-chain fatty acids (SCFA) that stimulate anti-inflammatory effects. SCFA-producing bacteria were screened from bacteria isolated from human faeces using bromothymol blue as an acid indicator and gas chromatography for SCFA profiling. The beneficial functions (antagonistic activity, haemolytic activities, antibiotic susceptibility, mucus adherent percentage and toxin gene detection) were evaluated for the top five SCFA-producing bacteria isolated from three healthy volunteers that identified as Escherichia coli strains. They produced acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic acids at average concentrations of 15.9, 1.8, 1.1, 1.9, 1.8, 2.7 and 3.4 mM, respectively. The SCFA production by E. coli strains was rapidly increased during the first 8 h of incubation and gradually decreased after 16 h of incubation. All E. coli strains showed acid and bile tolerance, resulting in a survival rate greater than 70% with no haemolytic activity, mucus adherence greater than 40% and susceptibility to conventional antibiotics. Hence, the selected E. coli strains exhibited promising probiotic properties with neither enterotoxin nor LPS producibility was detected. The present results confirm the existence of friendly and harmless E. coli strains in human microbiota as potential probiotics.
  15. Fulltext MicroRNA-155 complementation on a chemically functionalized dual electrode surface for determining breast cancer
    Gopinath SCB, Perumal V, Xuan S
    3 Biotech, 2020 Jun;10(6):270.
    PMID: 32523864 DOI: 10.1007/s13205-020-02261-x
    This study correlated and quantified the expression of microRNA-155 with breast cancer to determine breast cancer progression. The target microRNA-155 sequence was identified by complementation on a capture-probe sequence-immobilized interdigitated dual electrode surface. The sensitivity was found to be 1 fM, and the limit of detection fell between 1 and 10 fM. The specific sequence selectivity with single mismatches, triple mismatches, and noncomplementary bases failed to complement the capture-probe sequence. The obtained results demonstrate the selective determination of the microRNA-155 sequence and can help to diagnose breast cancer.
  16. Fulltext Targeted DNA complementation on a 1,1'-carbonyldiimidazole-functionalized surface for identifying Mycobacterium tuberculosis
    Xu S, Xue Y, Guo F, Xu M, Gopinath SCB, Mao X
    3 Biotech, 2020 May;10(5):227.
    PMID: 32373419 DOI: 10.1007/s13205-020-02216-2
    Herein, a rapid and sensitive current-volt measurement was developed for identifying the IS6110 DNA sequence to diagnose Mycobacterium tuberculosis (TB). An aminated capture probe was immobilized on a 1,1'-carbonyldiimidazole-functionalized interdigitated electrode (IDE) silica substrate, and the target sequence was detected by complementation. It was found that all tested concentrations displayed a higher response in current changes than the control, and the limit of detection was 10 fM. The sensitivity ranged from 1 to 10 fM. The control sequences with single-, triple-mismatch and noncomplementary sequences showed great discrimination. This rapid and easy DNA detection method helps to identify M. tuberculosis for early-stage diagnosis of TB.
  17. Fulltext Functional analysis of down-regulated miRNA-221-5p in HeLa cell treated with polyphenol-rich Polyalthia longifolia as regulators of apoptotic HeLa cell death
    Shanmugapriya, Sasidharan S
    3 Biotech, 2020 May;10(5):206.
    PMID: 32346497 DOI: 10.1007/s13205-020-02193-6
    MicroRNAs are endogenous small non-coding-RNAs that control gene expression and cancer development. Previous studies reported that Polyalthia longifolia treatment induced apoptotic cell death in HeLa cells by down-regulation of miR-221-5p. Hence, the current study was conducted to validate the down-regulated miR-221-5p in HeLa cells. Functional analysis of miR-221-5p was conducted through the gain-of-function, and loss-of-function approach and the miRNA expression was quantified by a real-time polymerase chain reaction. The P. longifolia treatment significantly (p 
  18. Fulltext Mycorrhiza: a natural resource assists plant growth under varied soil conditions
    Huey CJ, Gopinath SCB, Uda MNA, Zulhaimi HI, Jaafar MN, Kasim FH, et al.
    3 Biotech, 2020 May;10(5):204.
    PMID: 32337150 DOI: 10.1007/s13205-020-02188-3
    In this overview, the authors have discussed the potential advantages of the association between mycorrhizae and plants, their mutual accelerated growth under favorable conditions and their role in nutrient supply. In addition, methods for isolating mycorrhizae are described and spore morphologies and their adaptation to various conditions are outlined. Further, the significant participation of controlled greenhouses and other supported physiological environments in propagating mycorrhizae is detailed. The reviewed information supports the lack of host- and niche-specificity by arbuscular mycorrhizae, indicating that these fungi are suitable for use in a wide range of ecological conditions and with propagules for direct reintroduction. Regarding their prospective uses, the extensive growth of endomycorrhizal fungi suggests it is suited for poor-quality and low-fertility soils.
  19. Isolation and functional characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from glyphosate-tolerant Pseudomonas nitroreducens strains FY43 and FY47
    Tan XL, Othman RY, Teo CH
    3 Biotech, 2020 Apr;10(4):183.
    PMID: 32257739 DOI: 10.1007/s13205-020-02176-7
    5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of EPSPS gene for high level of glyphosate tolerance is important to generate glyphosate-tolerant crops. In this study, we report the isolation and characterization of EPSPS genes of glyphosate-tolerant Pseudomonas nitroreducens strains FY43 and FY47. Both P. nitroreducens strains FY43 and FY47, which showed glyphosate tolerance up to 8.768% (518.4 mM, 32 × higher than field application), were isolated from soil samples collected from oil palm plantation with a long history of glyphosate application. The glyphosate tolerance property of EPSPS genes of strains FY43 and FY47 was functionally characterized by expressing the genes in Escherichia coli strain BL21(DE3). Error-prone PCR was performed to mutagenize native EPSPS gene of strains FY43 and FY47. Ten mutagenized EPSPS with amino acid changes (R21C, N265S, A329T, P71L, T258A, L184F, G292C, G292S, L35F and A242V) were generated through error-prone PCR. Both native and mutated EPSPS genes of strains FY43 and FY47 were introduced into Escherichia coli strain BL21(DE3) and transformants were selected on basal salt medium supplemented with 8.768% (518.4 mM) glyphosate. Mutants with mutations (R21C, N265S, A329T, P71L, T258A, L35F, A242V, L184F and G292C) showed sensitivity to 8.768% glyphosate, whereas glyphosate tolerance for mutant with G292S mutation was not affected by the mutation.
  20. Box-Behnken supported development and validation of robust HPTLC method: an application in estimation of punarnavine in leaf, stem, and their callus of Boerhavia diffusa Linn
    Ahmad W, Husain I, Ahmad N, Amir M, Sarafroz M, Ansari MA, et al.
    3 Biotech, 2020 Apr;10(4):165.
    PMID: 32206499 DOI: 10.1007/s13205-020-2154-1
    Boerhavia diffusa (BD) Linn. (Nyctaginaceae) is one of the most commonly used herbs in the Indian traditional system of medicine for the urinary disorders. The aim of the current investigation was to carry out initiation, development, and maintenance of BD callus cultures and quantitative estimation of punarnavine in plant and callus extracts. Leaves and stem of BD were used as explant for the tissue culture studies using Murashige and Skoog (MS) basal medium. MS Media comprising 2,4-Dichlorophenoxy acetic acid (2,4-D) (1 ppm) and 2,4-D (1 ppm) + Indole-3-acetic acid (IAA) (1.0 ppm) were found to yield friable callus from leaf explant; similarly, 2,4-D (0.3 ppm) + IAA (0.75 ppm) + Kinetin (0.3 ppm) and 2,4-D (0.5 ppm) + Naphthalene acetic acid (NAA) (1.5 ppm) + Kinetin (0.3 ppm) were found to yield friable callus from the stem explant. High-performance thin-layer chromatography method was been developed for the quantitative estimation of punarnavine (Rf = 0.73) using mobile phase containing toluene: ethyl acetate: formic acid in the ratio (7.0:2.5:0.7, v/v/v) at 262 nm. The validated method was found linear (r2 = 0.9971) in a wide range (100-1000 ng spot-1), precise, accurate, and robust. The values of limit of detection, LOD = 30.3 ng spot-1, and limit of quantification, LOQ = 100.0 ng spot-1. The robustness of the method was proved by applying the Box-Behnken design (BBD). The developed method found appropriate for the quality control of medicinal plants containing punarnavine as a constituent.
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