Withaferin A (WA), a bioactive constituent derived from Withania somnifera plant, has been shown to exhibit many qualifying properties in attenuating several metabolic diseases. The current investigation sought to elucidate the protective mechanisms of WA (1.25 mg/kg/day) on pre-existing obese mice mediated by high-fat diet (HFD) for 12 weeks. Following dietary administration of WA, significant metabolic improvements in hepatic insulin sensitivity, adipocytokines with enhanced glucose tolerance were observed. The hepatic oxidative functions of obese mice treated with WA were improved via augmented antioxidant enzyme activities. The levels of serum pro-inflammatory cytokines and hepatic mRNA expressions of toll-like receptor (TLR4), nuclear factor κB (NF-κB), tumor necrosis factor-α (TNF-α), chemokine (C-C motif) ligand-receptor, and cyclooxygenase 2 (COX2) in HFD-induced obese mice were reduced. Mechanistically, WA increased hepatic mRNA expression of peroxisome proliferator-activated receptors (PPARs), cluster of differentiation 36 (CD36), fatty acid synthase (FAS), carnitine palmitoyltransferase 1 (CPT1), glucokinase (GCK), phosphofructokinase (PFK), and phosphoenolpyruvate carboxykinase (PCK1) that were associated with enhanced lipid and glucose metabolism. Taken together, these results indicate that WA exhibits protective effects against HFD-induced obesity through attenuation of hepatic inflammation, oxidative stress, and insulin resistance in mice.
Brewer's spent grain (BSG) is a major by-product in the beer-brewing process which contributes to 85% of the entire generated by-product in the brewing process. BSG is rich in proteins, and most of the malt proteins (74-78%) remain insoluble in BSG after the mashing process. Solid-state fermentation (SSF) is a promising bioprocess that enables microorganisms to survive in environments with minimal water and has shown to enhance the nutritional composition of BSG. In this review, the potential application of protein, amino acids (proline, threonine, and serine), phenolic contents, and soluble sugars (glucose, fructose, xylose, arabinose, and cellobiose) extracted from BSG by various microorganisms using SSF is explored. Incorporation of BSG into animal feed, human diets, and as a substrate for microorganisms are the prospects that could be implemented in the industrial scale. This review also discussed various advances to improve the fermentation yield such as symbiotic fermentation, the addition of nitrogen supplements, and an optimal mixture of the agro-industrial waste substrate. Future perspectives on SSF are also addressed to provide important ideas for immediate and future studies. However, challenges include optimizing SSF conditions and design of bioreactors, and operational costs must be addressed in the future to overcome current obstacles. Overall, this mini review highlights the potential benefits of BSG utilization and SSF in a sustainable way.
Replication-competent oncolytic adenovirus (TOA2) gene therapy is a recently introduced anti-tumor treatment regimen with superior results. The biodistribution studies of virus vector-based medicine seem more cautious and have been given much attention recently in terms of its quality and safety in preclinical trials. The current study determined the biodistribution and safety of a replication-competent adenovirus in different organs to predict its toxicity threshold. The present study has used TOA2, while biodistribution analysis was performed in human lung carcinoma A549-induced tumor-bearing nude mice model. Intratumoral injection was applied onto tumor-bearing mice with the adenovirus (3×1010 VP per mouse). Mice were sacrificed at the end of the experiment and the organs were dissected. Biodistribution analysis was done with complete hexon gene detection in each organ using quantitative real-time polymerase chain reaction (qRT-PCR). The biodistribution and concentration profiles showed that the TOA2 is well distributed in the entire tumor tissue. After dose 3 at day 11, the concentration of the virus has increased in the tumor tissue from 2240.54 (± 01.69) copies/100 ng genome to 13,120.28 (± 88.21) copies/100 ng genome on the 18th day, which eventually approached 336.45 (± 23.41) copies/100ng genome on the day 36. On the contrary, the concentration of the same decreased in the order of the liver, kidney, spleen, lung, and heart over time but no distributional traces in gonads. But the concentration found decreased dramatically in blood and other organs, while at the end of the experiment no detectable distribution was seen besides tumor tissue. The study confirms that adenovirus-based tumor therapy using conditionally replicating competent oncolytic TOA2 exhibited great efficiency with no toxicity at all.
The present studies are to evaluate the ability of PB to induce weight loss and urine metabolite profile of Piper betle L. (PB) leaf extracts using metabolomics approach. Dried PB leaves were extracted with ethanol 70% and the studies were performed in different groups of rats fed with high fat (HFD) and normal diet (ND). Then, fed with the PB extract with 100, 300, and 500 mg/kg and two negative control groups given water (WTR). The body weights were monitored and evaluated. Urine was collected and 1H NMR-based metabolomics approach was used to detect the metabolite changes. Results showed that PB-treated group demonstrated inhibition of body weight gain. The trajectory of urine metabolites showed that PB-treated group gave the different distribution from week 12 to 16 compared with the control groups. In 1H NMR metabolomic approach analysis, the urine metabolites gave the best separation in principle component 1 and 3, with 40.0% and 9.56% of the total variation. Shared and unique structures (SUS) plot model showed that higher concentration PB-treated group was characterized by high level of indole-3-acetate, aspartate, methanol, histidine, and creatine, thus caused an increased the metabolic function and maintaining the body weight of the animals treated.
Patients are turning into herbs for the management of diabetes, which cause increasing in the demand of plant-based alternative medicines. Ficus deltoidea or locally known as "Mas Cotek" in Malaysia is a famous herbal plant. However, many varieties of F. deltoidea existed with varied antidiabetic activities inspire us to evaluate in vivo antidiabetic activity of the most available varieties of F. deltoidea. Therefore, antihyperglycemic effect of different varieties of F. deltoidea at dose 250 mg/kg was evaluated on streptozotocin-nicotinamide-induced diabetic rats and further assessed their urinary metabolites using proton nuclear magnetic resonance (1H-NMR). The hyperglycemic blood level improved towards normoglycemic state after 30 days of treatment with standardized extracts of F. deltoidea var. trengganuensis, var. kunstleri, and var. intermedia. The extracts also significantly managed the biochemical parameters in diabetic rats. Metabolomics results showed these varieties were able to manage the altered metabolites of diabetic rats by shifting some of the metabolites back to their normal state. This knowledge might be very important in suggesting the use of these herbs in long-term treatment for diabetes. The most potential variety can be recommended, which may be useful for further pharmacological studies and herbal authentication processes.
The sustainability of nitrile glove production process is essential both in the financial and energy perspective. Nitrile glove has the lowest material cost with positive mechanical and chemical performance quality for the disposable glove market. Nitrile glove also holds a major market in disposable gloves sector, and nitrile rubber compounds may contribute to the huge reduction of the capital cost for a pair of surgical gloves due to the inexpensive raw material compares with other synthetic polyisoprene or neoprene. Hence, blending of bio-additive into the nitrile latex might support the 3 pillars of sustainability for environmental, societal, and financial sector. Bio-additives helps increase the degradation rate of gloves under natural conditions. Bio-based substances could be derived from food waste, natural plants, and aquatic plants like micro- and macro algae. Furthermore, antimicrobial agent (e.g. brilliant green and cyclohexadiene) is the trend in surgical glove for coated as protecting layer, due to the capability to remove pathogens or bacterial on the surgeon hands during operation period. Besides, the section in energy recovery is a proposing gateway for reducing the financial cost and makes the process sustainable.
Oil sands tailings, a slurry of alkaline water, silt, clay, unrecovered bitumen, and residual hydrocarbons generated during bitumen extraction, are contained in ponds. Indigenous microbes metabolize hydrocarbons and emit greenhouse gases from the tailings. Metabolism of hydrocarbons in tailings ponds of two operators, namely, Canadian Natural Upgrading Limited (CNUL) and Canadian Natural Resources Limited (CNRL), has not been comprehensively investigated. Previous reports have revealed sequential and preferential hydrocarbon degradation of alkanes in primary cultures established from CNUL and CNRL tailings amended separately with mixtures of hydrocarbons (n-alkanes, iso-alkanes, paraffinic solvent, or naphtha). In this study, activation pathway of hydrocarbon biodegradation in these primary cultures was investigated. The functional gene analysis revealed that fumarate addition was potentially the primary activation pathway of alkanes in all cultures. However, the metabolite analysis only detected transient succinylated 2-methylpentane and 2-methylbutane metabolites during initial methanogenic biodegradation of iso-alkanes and paraffinic solvent in all CNUL and CNRL cultures amended with iso-alkanes and paraffinic solvent. Under sulfidogenic conditions (prepared only with CNUL tailings amended with iso-alkanes), succinylated 2-methylpentane persisted throughout incubation period of ~ 1100 days, implying dead-end nature of the metabolite. Though no metabolite was detected in n-alkanes- and naphtha-amended cultures during incubation, assA/masD genes related to Peptococcaceae were amplified in all CNUL and CNRL primary cultures. The findings of this present study suggest that microbial communities in different tailings ponds can biodegrade hydrocarbons through fumarate addition as activation pathway under methanogenic and sulfidogenic conditions.
Free laccase and fungal biomass from white-rot fungi were compared in the thermokinetics study of the laccase-catalyzed decolorization of an azo dye, i.e., Trypan Blue. The decolorization in both systems followed a first-order kinetics. The apparent first-order rate constant, k1', value increases with temperature. Apparent activation energy of decolorization was similar for both systems at ∼ 22 kJ mol(-1), while energy for laccase inactivation was 18 kJ mol(-1). Although both systems were endothermic, fungal biomass showed higher enthalpy, entropy, and Gibbs free energy changes for the decolorization compared to free laccase. On the other hand, free laccase showed reaction spontaneity over a wider range of temperature (ΔT = 40 K) as opposed to fungal biomass (ΔT = 15 K). Comparison of entropy change (ΔS) values indicated metabolism of the dye by the biomass.
Human adipose-derived stem cells (ASCs) have generated a great deal of excitement in regenerative medicine. However, their safety and efficacy issue remain a major concern especially after long-term in vitro expansion. The aim of this study was to investigate the fundamental changes of ASCs in long-term culture by studying the morphological feature, growth kinetic, surface marker expressions, expression level of the senescence-associated genes, cell cycle distribution and ß-galactosidase activity. Human ASCs were harvested from lipoaspirate obtained from 6 patients. All the parameters mentioned above were measured at P5, P10, P15 and P20. Data were subjected to one-way analysis of variance with a Tukey post hoc test to determine significance difference (P < 0.05). The data showed that growth of ASCs reduced in long-term culture and the ß-galactosidase activity was significantly increased at later passage (P20). The morphology of ASCs in long-term culture showed the manifestation of senescent feature at P15 and P20. Significant alteration in the senescence-associated genes expression levels was observed in MMP1, p21, Rb and Cyclin D1 at P15 and P20. Significant increase in CD45 and HLA DR DQ DP surface marker was observed at P20. While cell cycle analysis showed significant decrease in percentage of ASCs at S and G2/M phase at later passage (P15). Our data showed ASCs cultured beyond P10 favours the senescence pathway and its clinical usage in cell-based therapy may be limited.
Fish protein hydrolysate (FPH) has shown immense potential as a dietary protein supplement and immunostimulant in aquaculture, especially in Nile tilapia production. Four isoproteic diets (30% crude protein) were prepared by including FPH at varying percentages (0%, 0.5%, 1%, and 2%). Nile tilapia fed with FPH diets for 90 days, and their growth performance, feed utilization, blood biochemistry, liver and gut morphology, and resistance against Streptococcus iniae were investigated. The findings revealed that diets physical attributes such as pellet durability index and water stability were remarkably (p
Enzymatic halogenation captures scientific interest considering its feasibility in modifying compounds for chemical diversity. Currently, majority of flavin-dependent halogenases (F-Hals) were reported from bacterial origin, and as far as we know, none from lichenized fungi. Fungi are well-known producers of halogenated compounds, so using available transcriptomic dataset of Dirinaria sp., we mined for putative gene encoding for F-Hal. Phylogenetic-based classification of the F-Hal family suggested a non-tryptophan F-Hals, similar to other fungal F-Hals, which mainly act on aromatic compounds. However, after the putative halogenase gene from Dirinaria sp., dnhal was codon-optimized, cloned, and expressed in Pichia pastoris, the ~63 kDa purified enzyme showed biocatalytic activity towards tryptophan and an aromatic compound methyl haematommate, which gave the tell-tale isotopic pattern of a chlorinated product at m/z 239.0565 and 241.0552; and m/z 243.0074 and 245.0025, respectively. This study is the start of understanding the complexities of lichenized fungal F-hals and its ability to halogenate tryptophan and other aromatic. compounds which can be used as green alternatives for biocatalysis of halogenated compounds.
Enteroendocrine cells are the largest population of hormone-producing cells in the body and play important roles in many aspects of body functions. The enteroendocrine cell population is divided into different subpopulations that secrete different hormones and peptides. Characterization of each subpopulation is particularly useful for analyzing the cellular mechanisms responsible for specific cell types. Therefore, the necessity of a pure cell line for a specific study purpose was the important motivation for the separation of cell lines for each subpopulation of enteroendocrine cells. The present research introduces a method for the isolation of L-cells, one of the important subpopulations of enteroendocrine cells. The antibiotic selection method was conducted in order to isolate the L-cells from a heterogonous population of intestinal cell line. In this method, a neomycin resistance gene (as selected marker) was expressed under the control of a specific promoter of L-cells. After transfection of manipulated plasmid, only the cells which determine the specific promoter and express neomycin resistance protein would be able to survive under Geneticin antibiotic treatment condition. In order to confirm that the isolated cells were L-cells, reverse transcriptase polymerase chain reaction (PCR) and quantitative PCR assays were performed. Based on the results, the isolated cells were pure L-cells that could be able to express specific mRNA of L-cells efficiently. This technique provides a unique method for the isolation and purification of any cell line. The purified isolated L-cells by this method can be used for future studies and for analyzing cellular mechanisms that involve L-cells' functions.
Synthesis and anticancer studies of three symmetrically and non-symmetrically substituted silver(I)-N-Heterocyclic carbene complexes of type [(NHC)2-Ag]PF6 (7-9) and their respective (ligands) benzimidazolium salts (4-6) are described herein. Compound 5 and Ag-NHC-complex 7 were characterized by the single crystal X-ray diffraction technique. Structural studies for 7 showed that the silver(I) center has linear C-Ag-C coordination geometry (180.00(10)o). Other azolium and Ag-NHC analogues were confirmed by H1 and C13-NMR spectroscopy. The synthesized analogues were biologically characterized for in vitro anticancer activity against three cancer cell lines including human colorectal cancer (HCT 116), breast cancer (MCF-7), and erythromyeloblastoid leukemia (K-562) cell lines and in terms of in vivo acute oral toxicity (IAOT) in view of agility and body weight of female rats. In vitro anticancer activity showed the values of IC50 in range 0.31-17.9 μM in case of K-562 and HCT-116 cancer cell lines and 15.1-35.2 μM in case of MCF-7 while taking commercially known anticancer agents 5-fluorouracil, tamoxifen, and betulinic acid which have IC50 values 5.2, 5.5, and 17.0 μM, respectively. In vivo study revealed vigor and agility of all test animals which explores the biocompatibility and non-toxicity of the test analogues.
The current study reports the synthesis of sustainable nano-hydroxyapatite (nHAp) using a wet chemical precipitation approach. The materials used in the green synthesis of nHAp were obtained from environmental biowastes such as HAp from eggshells and pectin from banana peels. The physicochemical characterization of obtained nHAp was carried out using different techniques. For instance, X-ray diffractometer (XRD) and FTIR spectroscopy were used to study the crystallinity and synthesis of nHAp respectively. In addition, the morphology and elemental composition of nHAP were studied using FESEM equipped with EDX. HRTEM showed the internal structure of nHAP and calculated its grain size which was 64 nm. Furthermore, the prepared nHAp was explored for its antibacterial and antibiofilm activity which has received less attention previously. The obtained results showed the potential of pectin-bound nHAp as an antibacterial agent for various biomedical and healthcare applications.
Garcinia mangostana pericarp is a good source of natural antioxidants with numerous functional properties. The conventional approaches for the recovery of antioxidants from Garcinia mangostana pericarp require long processing time and high temperature, which may cause degradation or loss of bioactivity of antioxidants, and often result in low recovery efficiency. In this study, the extraction of antioxidants from Garcinia mangostana pericarp was investigated using a polyethylene glycol (PEG)/citrate aqueous biphasic system (ABS) with the addition of surfactants. The optimum condition for the recovery of antioxidants was achieved in PEG 1000/citrate ABS of pH 8 with tie-line length (TLL) of 48.3% (w/w), volume ratio (VR) of 1.6, 0.2% (w/w) sample loading and addition of 1.0% (w/w) Tween 85. The antioxidants were recovered in the PEG-rich top phase with a high K value of 18.23 ± 0.33 and a recovery yield of 92.01% ± 0.09. The findings suggested that the addition of surfactants to polymer/salt ABS can enhance the recovery of antioxidants from Garcinia mangostana pericarps by conserving the antioxidative properties.
In this study, the ability of Cupriavidus sp. USMAA2-4 to synthesize polyhydroxyalkanoates (PHA) containing 4-hydroxyvalerate monomer (4HV) was studied through one-stage cultivation using γ-valerolactone as the carbon precursor. The presence of 4HV monomer unit in the polymer was detected through gas chromatography analysis, proving the capability of this wild strain bacterium to produce poly(3-hydrxybutyrate-co-3-hydroxyvalerate-co-4-hydroxyvalerate) [P(3HB-co-3HV-co-4HV)] terpolymer. Existence of a 4HV monomer unit in the PHA produced was further confirmed through (13)C and (1)H NMR analysis. P(3HB-co-88 % 3HV-co-1 % 4HV) terpolymer with the highest PHA content of 63 wt% was obtained through combination of 0.14 wt% C of γ-valerolactone with 0.42 wt% C of oleic acid. Various compositions of P(3HB-co-3HV-co-4HV) terpolymer with 3HV and 4HV compositions ranging from 11 to 94 mol% and from 1 to 4 mol%, respectively, were acquired by manipulating γ-valerolactone and oleic acid concentrations. The molecular weight and the thermal and mechanical properties of four different compositions of terpolymers-P(3HB-co-91 % 3HV-co-1 % 4HV), P(3HB-co-55 % 3HV-co-2 % 4HV), P(3HB-co-27 % 3HV-co-2 % 4HV), and P(3HB-co-9 % 3HV-co-1 % 4HV)-were characterized. Among these terpolymers, P(3HB-co-27 % 3HV-co-2 % 4HV) terpolymer with a molecular weight of 5.7 (10(5) Da) exhibited the highest elongation to break (264 %). The monomer unit compositional distributions of these terpolymers were investigated through acetone-water fractionation analysis. The results suggested that these produced terpolymers had broad 3HV compositional distribution and narrow 4HV compositional distribution.
Tuberculosis (TB), an epidemic disease, affects the world with death rate of two million people every year. The bacterium Mycobacterium tuberculosis was found to be a more potent and disease-prolonged bacterium among the world due to multi-drug resistance. Emergence of new drug targets is needed to overcome the bacterial resistance that leads to control epidemic tuberculosis. The pathway thiamine biosynthesis was targeting M. tuberculosis due to its role in intracellular growth of the bacterium. The screening of enzymes involved in thiamin biosynthesis showed novel target thiazole synthase (ThiG) involved in catalysis of rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine. We carried out homology modeling for ThiG to understand the structure-function relationship, and the model was refined with MD simulations. The results showed that the model predicted with (α + β)8-fold of synthase family proteins. Molecular docking of ThiG model with substrate DXP showed binding mode and key residues ARG46, ASN69, THR41, and LYS96 involved in the catalysis. First-line anti-tuberculosis drugs were docked with ThiG to identify the inhibition. The report showed the anti-tuberculosis drugs interact well with ThiG which may lead to block thiamin biosynthesis pathway.
Solid-state fermentation (SSF) was employed to enhance the nutritive values of palm kernel cake (PKC) for poultry feeding. Aspergillus flavus was isolated from local PKC and utilized to increase the mannose content of PKC via the degradation of β-mannan in PKC; evaluation was done for batch SSF in Erlenmeyer flasks and in a novel laterally aerated moving bed (LAMB) bioreactor. The optimum condition for batch SSF in flasks was 110% initial moisture content, initial pH 6.0, 30 °C, 855 μm particle size, and 120 h of fermentation, yielding 90.91 mg mannose g⁻¹ dry PKC (5.9-fold increase). Batch SSF in the LAMB at the optimum condition yielded 79.61 mg mannose g⁻¹ dry PKC (5.5-fold increase) within just 96 h due to better heat and mass transfer when humidified air flowed radially across the PKC bed. In spite of a compromise of 12% reduction in mannose content when compared with the flasks, the LAMB facilitated good heat and mass transfer, and improved the mannose content of PKC in a shorter fermentation period. These attributes are useful for batch production of fermented PKC feed in an industrial scale.
Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
E6 and E7 human papillomavirus (HPV) oncoproteins play a significant role in the malignant transformation of infected cervical cancer cells via suppression of tumour suppressor pathways by targeting p53 and pRb, respectively. This study aimed to investigate the anticancer effects of Oroxylum indicum (OI) leaves' methanol extract on SiHa cervical cancer cells. Expression of apoptosis-related proteins (Bcl-2, caspase (cas)-3, and cas-9), viral oncoproteins (E6 and E7), and tumour suppressor proteins (p53 and pRb) were evaluated using western blot analysis before and after E6/E7 small interfering RNAs (siRNAs) transfection. In addition, the E6/E7 mRNA expression levels were assessed with real-time (RT)-PCR. The present study showed that the OI extract effectively hindered the proliferation of SiHa cells and instigated increments of cas-3 and cas-9 expressions but decreased the Bcl-2 expressions. The OI extract inhibited E6/E7 viral oncoproteins, leading to upregulation of p53 and pRb tumour suppressor genes in SiHa cells. Additionally, combinatorial treatment of OI extract and gossypin flavonoid induced restorations of p53 and pRb. Treatment with OI extract in siRNA-transfected cells also further suppressed E6/E7 expression levels and further upregulations of p53 and pRb proteins. In conclusion, OI extract treatment on siRNAs-transfected SiHa cells can additively and effectively block E6- and E7-dependent p53 and pRb degradations. All these data suggest that OI could be explored for its chemotherapeutic potential in cervical cancer cells with HPV-integrated genomes.