GDSL esterase is designated as a member of Family II of lipolytic enzymes known to catalyse the synthesis and hydrolysis of ester bonds. The enzyme possesses a highly conserved motif Ser-Gly-Asn-His in the four conserved blocks I, II, III and V respectively. The enzyme characteristics, such as region-, chemo-, and enantioselectivity, help in resolving the racemic mixture of single-isomer chiral drugs. Recently, crystal structure of GDSL esterase from Photobacterium J15 has been reported (PDB ID: 5XTU) but not in complex with substrate. Therefore, GDSL in complex with substrate could provide insights into the binding mode of substrate towards inactive form of GDSL esterase (S12A) and identify the hot spot residues for the designing of a better binding pocket. Insight into molecular mechanisms is limited due to the lack of crystal structure of GDSL esterase-substrate complex. In this paper, the crystallization of mutant GDSL esterase (S12A) (PDB ID: 8HWO) and its complex with butyric acid (PDB ID: 8HWP) are reported. The optimized structure would be vital in determining hot spot residue for GDSL esterase. This preliminary study provides an understanding of the interactions between enzymes and hydrolysed p-nitro-phenyl butyrate. The information could guide in the rational design of GDSL esterase in overcoming the medical limitations associated with racemic mixture.
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.
Sanitary landfilling is the most common way to dispose solid urban waste; however, improper landfill management may pose serious environmental threats through discharge of high strength polluted wastewater also known as leachate. The treatment of landfill leachate to fully reduce the negative impact on the environment, is nowadays a challenge. In this study, an aerobic sequencing batch reactor (ASBR) was proposed for the treatment of locally obtained real landfill leachate with initial ammoniacal nitrogen and chemical oxygen demand (COD) concentration of 1800 and 3200 mg/L, respectively. ASBR could remove 65 % of ammoniacal nitrogen and 30 % of COD during seven days of treatment time. Thereafter, an effective adsorbent, i.e., zeolite was used as a secondary treatment step for polishing the ammoniacal nitrogen and COD content that is present in leachate. The results obtained are promising where the adsorption of leachate by zeolite further enhanced the removal of ammoniacal nitrogen and COD up to 96 and 43 %, respectively. Furthermore, this combined biological-physical treatment system was able to remove heavy metals, i.e. aluminium, vanadium, chromium, magnesium, cuprum and plumbum significantly. These results demonstrate that combined ASBR and zeolite adsorption is a feasible technique for the treatment of landfill leachate, even considering this effluent's high resistance to treatment.
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.
Enzymatic catalysis is considered to be among the most environmental friendly processes for the synthesis of fine chemicals. In this study, lipase from Thermomyces lanuginosus (Lecitase Ultra™) was used to catalyze the synthesis of flavor esters, i.e., methyl butanoate and methyl benzoate by esterification of the acids with methanol in a microfluidic system. Maximum reaction rates of 195 and 115 mM min(-1) corresponding to catalytic efficiencies (k cat/K M) of 0.30 and 0.24 min(-1) mM(-1) as well as yield conversion of 54 and 41 % were observed in methyl butanoate and methyl benzoate synthesis, respectively. Catalytic turnover (k cat) was higher for methyl butanoate synthesis. Rate of synthesis and yield decreased with increasing flow rates. For both esters, increase in microfluidic flow rate resulted in increased advective transport over molecular diffusion and reaction rate, thus lower conversion. In microfluidic synthesis using T. lanuginosus lipase, the following reaction conditions were 40 °C, flow rate 0.1 mL min(-1), and 123 U g(-1) enzyme loading found to be the optimum operating limits. The work demonstrated the application of enzyme(s) in a microreactor system for the synthesis of industrially important esters.
Water-immiscible substrate, diesel, was supplied as the main substrate in the fermentation of Pseudomonas aeruginosa USM-AR2 producing rhamnolipid biosurfactant, in a stirred tank bioreactor. In addition to the typical gas-aqueous system, this system includes gas-hydrocarbon-aqueous phases and the presence of surfactant (rhamnolipid) in the fermentation broth. The effect of diesel dispersion on volumetric oxygen transfer coefficient, k L a, and thus oxygen transfer, was evaluated at different agitations of 400, 500 and 600 rpm. The oxygen transfer in this oil-water-surfactant system was shown to be affected by different oil dispersion at those agitation rates. The highest diesel dispersion was obtained at 500 rpm or impeller tip speed of 1.31 m/s, compared to 400 and 600 rpm, which led to the highest k L a, growth and rhamnolipid production by P. aeruginosa USM-AR2. This showed the highest substrate mixing and homogenization at this agitation speed that led to the efficient substrate utilization by the cells. The oxygen uptake rate of P. aeruginosa USM-AR2 was 5.55 mmol/L/h, which showed that even the lowest k L a (48.21 h-1) and hence OTR (57.71 mmol/L/h) obtained at 400 rpm was sufficient to fulfill the oxygen demand of the cells. The effect of rhamnolipid concentration on k L a showed that k L a increased as rhamnolipid concentration increased to 0.6 g/L before reaching a plateau. This trend was similar for all agitation rates of 400, 500 and 600 rpm, which might be due to the increase in the resistance to oxygen transfer (k L decrease) and the increase in the specific interfacial area (a).
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.
Cyanobacteria bioactive compounds are chemical treasure troves for product discovery and development. The wound healing effects and antioxidant capacities of water extracts from Nostoc NIES-2111_MUM004 were evaluated via in vitro wound scratch assay and three antioxidant assays respectively. Results showed that the water extracts were protein-rich and exhibited good antioxidant properties in ABTS radical scavenging (11.27 ± 0.205 mg TAE g-1 extract), Ferric reducing antioxidant power (1652.71 ± 110.71 mg TAE g-1 extract) and β-carotene bleaching assay (354.90 ± 31.80 mg TAE g-1 extract). Also, extracts were non-cytotoxic in concentrations up to 250 µg/mL as reflected in cytotoxicity assay. Importantly, water extracts showed considerable proliferation and migration activity at 125 µg/mL with wound closure rate as high as 42.67%. Statistical correlation revealed no significant relationship (p > 0.05) between protein fraction and the wound healing properties, confirming that phycobiliproteins were not solely responsible for wound healing activities. Subsequent Q-TOF-LCMS analysis identified six protein families involved in enhancing the proliferation and migration of epithelial cells. These findings are antecedent in the uncovering of continuous supplies of bioactive compounds from new and sustainable sources. Ultimately, enriching the microalgae menu for applications in pharmaceutical, nutraceutical and cosmeceuticals.
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.
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.
Uncontrolled disposal of feathers from the poultry industry and slaughterhouses is environmentally undesirable. The feathers are composed of approximately 90% of keratin which is an important ingredient of cosmetics, shampoos and hair treatment creams. This study aimed to determine the optimum conditions for the extraction of keratin from chicken feathers. The extraction of keratin using various reducing agents was studied using statistical experimental design. In the extraction process, pH, temperature, ratio of reducing agents, mass of chicken feathers and incubation time were analyzed. The keratin in the total extracted protein was purified by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and further characterized using amino acids profile analysis. The surface morphology and chemical composition were studied by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analysis. Sodium sulfide (Na2S) yielded 84.5% of keratin as compared to sodium hydroxide (43.8), urea mixture (50.6), mixture of sodium dodecyl sulfate (SDS) and sodium bisulfite (18.3) and a mixture of Na2S and sodium hydroxide (41.5%) under optimized conditions. The optimum yield of keratin was achieved at 80.9 °C in 9.5 h with 0.05 M sodium sulfide using response surface methodology (RSM). Among the five parameters screened, pH was found not to be significant because the p value was greater than 0.05.
In current practice, oil palm frond leaflets and stems are re-used for soil nutrient recycling, while the petioles are typically burned. Frond petioles have high commercialization value, attributed to high lignocellulose fiber content and abundant of juice containing free reducing sugars. Pressed petiole fiber is the subject of interest in this study for the production of lignocellulolytic enzyme. The initial characterization showed the combination of 0.125 mm frond particle size and 60% moisture content provided a surface area of 42.3 m2/g, porosity of 12.8%, and density of 1.2 g/cm3, which facilitated fungal solid-state fermentation. Among the several species of Aspergillus and Trichoderma tested, Aspergillus awamori MMS4 yielded the highest xylanase (109 IU/g) and cellulase (12 IU/g), while Trichoderma virens UKM1 yielded the highest lignin peroxidase (222 IU/g). Crude enzyme cocktail also contained various sugar residues, mainly glucose and xylose (0.1-0.4 g/L), from the hydrolysis of cellulose and hemicellulose. FT-IR analysis of the fermented petioles observed reduction in cellulose crystallinity (I900/1098), cellulose-lignin (I900/1511), and lignin-hemicellulose (I1511/1738) linkages. The study demonstrated successful bioconversion of chemically untreated frond petioles into lignin peroxidase and xylanase-rich enzyme cocktail under SSF condition.
In this study, RNA sequencing of several Hevea brasiliensis clones grown in Malaysia with different annual rubber production yields and disease resistance was performed on the Illumina platform. A total of 29,862,548 reads were generated, resulting in 101,269 assembled transcripts that were used as the reference transcripts. A similarity search against the non-redundant (nr) protein databases presented 83,771 (83%) positive BLASTx hits. The transcriptome was annotated using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the Pfam database. A search for putative molecular markers was performed to identify single-nucleotide polymorphisms (SNPs). Overall, 3,210,629 SNPs were detected and a total of 1314 SNPs associated with the genes involved in MVA and MEP pathways were identified. A total of 176 SNP primer pairs were designed from sequences that were related to the MVA and MEP pathways. The transcriptome of RRIM 3001 and RRIM 712 were subjected to pairwise comparison and the results revealed that there were 1262 significantly differentially expressed genes unique to RRIM 3001, 1499 significantly differentially expressed genes unique to RRIM 712 and several genes related to the MVA and MEP pathways such as AACT, HMGS, PMK, MVD, DXS and HDS were included. The results will facilitate the characterization of H. brasiliensis transcriptomes and the development of a new set of molecular markers in the form of SNPs from transcriptome assembly for the genotype identification of various rubber varieties with superior traits in Malaysia.
In the present study, Karanjin and Pongapin, two important furanoflavone, constituents of Pongamia pinnata were studied in the management of Psoriasis. Presently, we have experimentally studied the free radical quenching property of Karanjin and Pongapin. A modified method was used to estimate the scavenging effect of the Karanjin (the highest activity of 95.60%) and Pongapin (68.05%) compared to the ascorbic acid as standard (11.60%) against nitric oxide. Furthermore, Molecular docking studies were performed using CLC drug discovery workbench software version 3.0 of the studied flavones (Karanjin and Pongapin) with the receptors responsible for psoriasis (viz. IL-17A, IL-17F, IL-23, RORγt, and TLR-7). Docking scores of Karanjin and Pongapin with different studied receptors were found to be comparable to that of Methotrexate, a known drug for treating Psoriasis. Docking results suggest that Karanjin and Pongapin might also help in controlling the disease. Overall, our results indicate that flavones (Karanjin and Pongapin) could be a natural and better alternative in curing psoriasis without any side effects.
Long noncoding RNAs (lncRNAs) are implicated in various biological processes, such as cell proliferation, differentiation, apoptosis, migration, and invasion. They are also key players in various biological pathways. LncRNA was considered as 'translational noise' before 1980s. It has been reported that lncRNAs are aberrantly expressed in different cancers, either as oncogene or tumor suppressor gene. Therefore, more and more lncRNAs are recognized as potential diagnostic biomarkers and/or therapeutic targets. As competitive endogenous RNA, lncRNAs can interact with microRNA to alter the expression of target genes, which may have extensive clinical implications in cancers, including diagnosis, treatment, prognosis, and chemoresistance. This review comprehensively summarizes the functions and clinical relevance of lncRNAs in digestive system cancers, especially as a potential tool to overcome chemoresistance.
Cortisol is a stress hormone released from the adrenal glands and is responsible for both hyperglycemia and hypertension during pregnancy. These factors make it mandatory to detect the levels of cortisol during pregnancy to identify and treat hypoglycemia and hypertension. In this study, cortisol levels were quantified with an aptamer-conjugated gold nanorod using an electrochemical interdigitated electrode sensor. The surface uniformity was analyzed by high-power microscopy and 3D-nanoprofiler imaging. The detection limit was determined to be 0.01 ng/mL, and a linear regression indicated that the sensitivity range was in the range of 0.01-0.1 ng/mL, based on a 3σ calculation. Moreover, the specificity of the aptamer was determined by a binding analysis against norepinephrine and progesterone, and it was clearly found that the aptamer specifically recognizes only cortisol. Further, the presence of cortisol was detected in the serum in a dose-dependent manner. This method is useful to detect and correlate multiple pregnancy-related diseases by quantifying the levels of cortisol.
In this study, an alpha-amylase enzyme from a locally isolated Aspergillus flavus NSH9 was purified and characterized. The extracellular α-amylase was purified by ammonium sulfate precipitation and anion-exchange chromatography at a final yield of 2.55-fold and recovery of 11.73%. The molecular mass of the purified α-amylase was estimated to be 54 kDa using SDS-PAGE and the enzyme exhibited optimal catalytic activity at pH 5.0 and temperature of 50 °C. The enzyme was also thermally stable at 50 °C, with 87% residual activity after 60 min. As a metalloenzymes containing calcium, the purified α-amylase showed significantly increased enzyme activity in the presence of Ca2+ ions. Further gene isolation and characterization shows that the α-amylase gene of A. flavus NSH9 contained eight introns and an open reading frame that encodes for 499 amino acids with the first 21 amino acids presumed to be a signal peptide. Analysis of the deduced peptide sequence showed the presence of three conserved catalytic residues of α-amylase, two Ca2+-binding sites, seven conserved peptide sequences, and several other properties that indicates the protein belongs to glycosyl hydrolase family 13 capable of acting on α-1,4-bonds only. Based on sequence similarity, the deduced peptide sequence of A. flavus NSH9 α-amylase was also found to carry two potential surface/secondary-binding site (SBS) residues (Trp 237 and Tyr 409) that might be playing crucial roles in both the enzyme activity and also the binding of starch granules.
Pseudogenes in the Escherichia coli genome are assumed to be non-functional. In this study, Keio collection BW25113∆yqiG and YqiG-producing strain (BW25113/pCA24N-YqiG) were used to evaluate the importance of pseudogene yqiG in hydrogen metabolism. Our results show pseudogene protein YqiG was identified as an essential protein in the production of biohydrogen from glucose. The mutant yqiG decreased biohydrogen production from 37 µmol mg-1 protein to 6 µmol mg-1 protein compared to the wild-type strain, and glucose consumption was reduced by 80%. Through transcriptional analysis, we found that the yqiG mutation represses pflB transcription tenfold; pflB encodes pyruvate-formate lyase, one of the key enzymes in the anaerobic metabolism of E. coli. Moreover, production of YqiG stimulated glycolysis and increased biohydrogen productivity 1.5-fold compared to that of the wild-type strain. Thus, YqiG is important for the central glycolysis reaction and is able to influence hydrogen metabolism activity in E. coli.
Proteomic analysis was conducted to identify the rice root proteins induced by exogenous proline and their involvement in root growth. Proteins were extracted from the root tissues grown under two conditions, T1 (control) and T2 (10 mM proline), and profiled by two-dimensional polyacrylamide gel electrophoresis. Seventeen of 30 differentially expressed proteins were identified by mass spectrometry. Proline-treated rice roots showed up-regulation and down-regulation of nine and eight proteins, respectively, when compared to those in the control. Among the differentially expressed proteins, the down-regulation of glutathione reductase and peroxidase could be involved in the regulation of cellular hydrogen peroxide and reactive oxygen species levels that modulate the root cell wall structure. Differentially expressed proteins identified as pathogenesis-related proteins might be related to stress adaptive mechanisms in response to exogenous proline treatment. In addition, differentially expressed protein identified as the fructose-bisphosphate aldolases and cytochrome c oxidase might be associated with energy metabolism, which is needed during root developmental process. This is the first attempt to study the changes in rice root proteome treated with proline. The acquired information could open new avenues for further functional studies on the involvement of proline in modulating root development and its relation to stress adaptation of plants.
Banana peel (BP) is a major waste produced by fruit processing industries. Pre-treatment of BP at different temperatures led to 40% reduction in saponin at 100 °C (from 9.5 to 5.7 mg/g). Sequential mixed culture of Phanerochaete chrysosporium (P. chrysosporium) and Candida utilis (C. utilis) gave highest protein enrichment (88.93 mg/g). There is 26% increase in protein synthesis (from 88.93 to 111.78 mg/g) after media screening. Inclusion of KH2PO4, FeSO4·7H2O, wheat flour and sucrose in the media contributed positively to protein synthesis, while elevated concentration of urea, peptone, K2HPO4, KCl, NH4H2PO4, and MgSO4.7H2O are required to reach optimum protein synthesis. Total soluble sugar (TSS), total reducing sugar (TRS) and total carbohydrate (CHO) consumption varied with respect to protein synthesis in all experimental runs. Optimum protein synthesis required 6 days and inclusion of 5% sucrose, 0.6% NH4H2PO4, 0.4% KCl, and 0.5% MgSO4·7H2O as concentration media constituents to reach 140.95 mg/g protein synthesis equivalent to 300% increase over the raw banana peel protein content (35.0 mg/g).