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.
The ability of gellan gum-immobilised cells of the heavy metal-tolerant bacterium Alcaligenes sp. AQ05-001 to utilise both heavy metal-free and heavy metal-polluted feathers (HMPFs) as substrates to produce keratinase enzyme was studied. Optimisation of the media pH, incubation temperature and immobilisation parameters (bead size, bead number, gellan gum concentration) was determined for the best possible production of keratinase using the one-factor-at-a-time technique. The results showed that the immobilised cells could tolerate a broader range of heavy metal concentrations and produced higher keratinase activity at a gellan gum concentration of 0.8% (w/v), a bead size of 3 mm, bead number of 250, pH of 8 and temperature of 30 °C. The entrapped bacterium was used repeatedly for ten cycles to produce keratinase using feathers polluted with 25 ppm of Co, Cu and Ag as substrates without the need for desorption. However, its inability to tolerate/utilise feathers polluted with Hg, Pb, and Zn above 5 ppm, and Ag and Cd above 10 ppm resulted in a considerable decrease in keratinase production. Furthermore, the immobilised cells could retain approximately 95% of their keratinase production capacity when 5 ppm of Co, Cu, and Ag, and 10 ppm of As and Cd were used to pollute feathers. When the feathers containing a mixture of Ag, Co, and Cu at 25 ppm each and Hg, Ni, Pb, and Zn at 5 ppm each were used as substrates, the immobilised cells maintained their operational stability and biological activity (keratinase production) at the end of 3rd and 4th cycles, respectively. The study indicates that HMPF can be effectively utilised as a substrate by the immobilised-cell system of Alcaligenes sp. AQ05-001 for the semi-continuous production of keratinase enzyme.
In this study, Gelidium elegans is investigated for ethanol production. A combination of factors including different temperatures, acid concentration and incubation time was evaluated to determine the suitable saccharification conditions. The combination of 2.5% (w/v) H2SO4 at 120 °C for 40 min was selected for hydrolysis of the seaweed biomass, followed by purification, and fermentation to yield ethanol. The galactose and glucose were dominant reducing sugars in the G. elegans hydrolysate and under optimum condition of dilute acid hydrolysis, 39.42% of reducing sugars was produced and fermentation resulted in ethanol concentration of 13.27 ± 0.47 g/L. A modified method was evaluated for sample preparation for gas chromatography (GC) analysis of the ethanol content. A solvent mixture of acetonitrile and iso-butanol precipitated dissolved organic residues and reduced water content in GC samples at least by 90%. Results showed that this method could be successfully used for bioethanol production from seaweed.
Initially, a new moderate halophilic strain was locally isolated from seawater. The partial 16S rRNA sequence analysis positioned the organism in Marinobacter genus and was named 'Marinobacter litoralis SW-45'. This study further demonstrates successful utilization of the halophilic M. litoralis SW-45 lipase (MLL) for butyl ester synthesis from crude palm fruit oil (CPO) and kernel oil (CPKO) in heptane and solvent-free system, respectively, using hydroesterification. Hydrolysis and esterification of enzymatic [Thermomyces lanuginosus lipase (TLL)] hydrolysis of CPO and CPKO to free fatty acids (FFA) followed by MLL-catalytic esterification of the concentrated FFAs with butanol (acyl acceptor) to synthesize butyl esters were performed. A one-factor-at-a-time technique (OFAT) was used to study the influence of physicochemical factors on the esterification reaction. Under optimal esterification conditions of 40 and 45 °C, 150 and 230 rpm, 50% (v/v) biocatalyst concentration, 1:1 and 5:1 butanol:FFA, 9% and 15% (w/v) NaCl, 60 and 15 min reaction time for CPO- and CPKO-derived FFA esterification system, maximum ester conversion of 62.2% and 69.1%, respectively, was attained. Gas chromatography (GC) analysis confirmed the products formed as butyl esters. These results showed halophilic lipase has promising potential to be used for biosynthesis of butyl esters in oleochemical industry.
This investigation demonstrates an efficient method of propagation, short-term conservation, and germplasm exchange for Plectranthus amboinicus (Lour.) Spreng. encapsulated propagules. In vitro-derived shoot apices (shoot tips and nodal segments) which showed 100% survival on MS medium supplemented with 0.4 mg/L 6-benzylaminopurine were selected for encapsulation studies. Shoot apices measuring about 3-5 mm in size showed the ability to break the beads and exhibited 100% survival and regrowth. The combination of 3% (w/v) sodium alginate and 100 mM CaCl2 was found to be ideal for forming uniformally spherical beads, and successive preservation of encapsulated shoot apices into plantlets. The encapsulated shoot tips were relatively more effective than the nodal segments in terms of shoot growth and multiplication. Encapsulated shoot tips retained the ability to regrow (63.3%) for up to 40 days when maintained at 4 °C. Encapsulated shoot tips effectively converted into plantlets on agar medium (78%) and peat moss (58%) under in vitro conditions. Encapsulated shoot tips on agar medium showed a higher shoot regeneration (9.91 ± 0.15 shoots per explant) ability than the peat moss (5.71 ± 0.34 shoots per explant), while the highest rooting (12.16 ± 0.23 roots per explant) was observed on peat moss. Thus, calcium alginate encapsulation holds latent qualities that could be explored to develop a future alternative method of propagation, short-term storage and germplasm distribution for elite genotypes of Plectranthus sp.
An ecologically important tropical freshwater microalga, Scenedesmus quadricauda, was exposed to Ni toxicity under two temperature regimes, 25 and 35 °C to investigate the interactive effects of warming and different Ni concentrations (0.1, 1.0 and 10.0 ppm). The stress responses were assessed from the growth, photosynthesis, reactive oxygen species (ROS) generation and metabolomics aspects to understand the effects at both the physiological and biochemical levels. The results showed that the cell densities of the cultures were higher at 35 °C compared to 25 °C, but decreased with increasing Ni concentrations at 35 °C. In terms of photosynthetic efficiency, the maximum quantum yield of photosystem II (Fv/Fm) of S. quadricauda remained consistent across different conditions. Nickel concentration at 10.0 ppm affected the maximum rate of relative electron transport (rETRm) and saturation irradiance for electron transport (Ek) in photosynthesis. At 25 °C, the increase of non-photochemical quenching (NPQ) values in cells exposed to 10.0 ppm Ni might indicate the onset of thermal dissipation process as a self-protection mechanism against Ni toxicity. The combination of warming and Ni toxicity induced a strong oxidative stress response in the cells. The ROS level increased significantly by 40% after exposure to 10.0 ppm of Ni at 35 °C. The amount of Ni accumulated in the biomass was higher at 25 °C compared to 35 °C. Based on the metabolic profile, temperature contributed the most significant differentiation among the samples compared to Ni treatment and the interaction between the two factors. Amino acids, sugars and organic acids were significantly regulated by the combined factors to restore homeostasis. The most affected pathways include sulphur, amino acids, and nitrogen metabolisms. Overall, the results suggest that the inhibitory effect of Ni was lower at 35 °C compared to 25 °C probably due to lower metal uptake and primary metabolism restructuring. The ability of S. quadricauda to accumulate substantial amount of Ni and thrive at 35 °C suggests the potential use of this strain for phycoremediation and outdoor wastewater treatment.
Empty fruit bunch (EFB) and palm oil mill effluent (POME) are the major wastes generated by the oil palm industry in Malaysia. The practice of EFB and POME digester sludge co-composting has shown positive results, both in mitigating otherwise environmentally damaging waste streams and producing a useful product (compost) from these streams. In this study, the bacterial ecosystems of 12-week-old EFB-POME co-compost and POME biogas sludge from Felda Maokil, Johor were analysed using 16S metagenome sequencing. Over ten phyla were detected, with Chloroflexi being the predominant phylum, representing approximately 53% of compost and 23% of the POME microbiome reads. The main bacterial lineage found in the compost and POME was Anaerolinaceae (Chloroflexi) with 30% and 18% of the total gene fragments, respectively. The significant differences between compost and POME communities were abundances of Syntrophobacter, Sulfuricurvum and Coprococcus. No methanogens were identified due to the bias in general 16S primers to eubacteria. The preponderance of anaerobic species in the compost and high abundance of secondary metabolite fermenting bacteria is due to an extended composting time, with anaerobic collapse of the pile due to the tropical heat. Predictive functional profiles of the metagenomes using 16S rRNA marker genes suggest that the presence of enzymes involved in degradation of polysaccharides such as glucoamylase, endoglucanase and arabinofuranosidase, all of which were strongly active in POME. Eubacterial species associated with cellulytic methanogenesis were present in both samples.
Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9 µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3 M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45 min at 65 °C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.
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.
Tree species in the Aquilarieae tribe of the Thymelaeaceae family produce agarwood, a natural product highly valued for its fragrance, but the species are under threat due to indiscriminate harvesting. For conservation of these species, molecular techniques such as DNA profiling have been used. In this study, we assessed cross-amplification of microsatellite markers, initially developed for three Aquilaria species (A.crassna, A.malaccensis, and A.sinensis), on ten other agarwood-producing species, including members of Aquilaria (A.beccariana, A.hirta, A.microcarpa, A.rostrata, A.rugosa, A.subintegra, and A.yunnanensis) and Gyrinops (G.caudata, G.versteegii, and G.walla), both from the Aquilarieae tribe. Primers for 18 out of the 30 microsatellite markers successfully amplified bands of expected sizes in 1 sample each of at least 10 species. These were further used to genotype 74 individuals representing all the 13 studied species, yielding 13 cross-amplifiable markers, of which only 1 being polymorphic across all species. At each locus, the number of alleles ranged from 7 to 23, indicating a rather high variability. Four markers had relatively high species discrimination power. Our results demonstrated that genetic fingerprinting can be an effective tool in helping to manage agarwood genetic resources by potentially supporting the chain-of-custody of agarwood and its products in the market.
Chitinase is an enzyme that catalyzes the degradation of chitin, commonly induced upon the attack of pathogens and other stresses. A cDNA (MsChi1) was isolated from Metroxylon sagu and expressed predominantly in the inflorescence tissue of M. sagu, suggesting its role in developmental processes. The chitinase cDNA was detected and isolated via differential display and rapid amplification of cDNA ends (RACE). Primers specific to M. saguchitinase were used as probes to amplify the 3'-end and 5'-end regions of chitinase cDNA. Transcript analysis showed that chitinase is expressed in inflorescence and meristem tissues but was not detected in the leaf tissue. Sequence analysis of amplified cDNA fragments of 3'-end and 5'-end regions indicated that the chitinase cDNA was successfully amplified. The M. saguchitinase cDNA isolated was approximately 1,143 bp long and corresponds to 312 predicted amino acids. Alignments of nucleotide and amino acid have grouped this chitinase to family 19 class I chitinase.
The present study is aimed to evaluate the effects of sub-acute toxicity testing of copper sulphate (CuSO4), on behavioural, histological and biochemical changes of the Oreochromis mossambicus (black tilapia) blood tissues. The effects were assessed according to the previous results on sub-acute toxicity test after exposing fish to several concentrations (0.0, 2.5, 5.0, and 10.0 mg/L). The observations of scanning electron microscope, and transmission electron microscope studies revealed severe histopathological changes on the surface and the cellular changes in blood tissues, respectively. The morphological alterations in blood involved irregular structure of red blood cell and blood clot formation. CuSO4 affected the biochemical alteration of the blood cholinesterase also known as serum cholinesterase (ChE). Blood ChE inhibited up to 80% of activity when exposed to 10.0 mg/L CuSO4. The findings from this study can further improve the quality standards of aquaculture industry and the fundamental basis in selecting suitable strains among freshwater fish species to be used as bioindicator.
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.
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.
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.
Increasing evidence suggests a sizable involvement of hemotoxins in the morbidity associated with envenomation by the Indian spectacled cobra, Naja naja (N.N). This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in reversing the hemostatic abnormalities, viz. activated partial thromboplastin time(aPTT), prothrombin time(PT) and thrombin time(TT) in citrated plasma. These parameters were assessed in 2 groups of experiments. Group 1: Without the prior incubation of plasma with venom and Group 2: With prior incubation of plasma with venom for 90 min at 37°C. Venom caused significant (p
Over recent years, keratin has gained great popularity due to its exceptional biocompatible and biodegradable nature. It has shown promising results in various industries like poultry, textile, agriculture, cosmetics, and pharmaceutical. Keratin is a multipurpose biopolymer that has been used in the production of fibrous composites, and with necessary modifications, it can be developed into gels, films, nanoparticles, and microparticles. Its stability against enzymatic degradation and unique biocompatibility has found their way into biomedical applications and regenerative medicine. This review discusses the structure of keratin, its classification and its properties. It also covers various methods by which keratin is extracted like chemical hydrolysis, enzymatic and microbial treatment, dissolution in ionic liquids, microwave irradiation, steam explosion technique, and thermal hydrolysis or superheated process. Special emphasis is placed on its utilisation in the form of hydrogels, films, fibres, sponges, and scaffolds in various biotechnological and industrial sectors. The present review can be noteworthy for the researchers working on natural protein and related usage.
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%.
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.
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.