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Background: Recently, the microwave has shown tremendous research interest and potential for the extraction of phytoconstituents from plant resources as there is a need for the advancement of performance in the extraction method. Extraction is the first basic step in the preparation of crude extract followed by isolation, and characterization of isolated components related to current analytical chemistry. The current use of microwaves implements green technology and satisfies the demand for getting the maximum yield of the target compound. Method: Research articles related to the current research topics on Microwave-Assisted Extraction (MAE) were collected and reviewed. Results: MAE promises to be a novel extraction technique for the extraction of phytoconstituents from natural sources than other conventional extraction techniques. Conclusion: Microwave-assisted extraction is proved to be advantageous as this method permits specificity, higher yield, selectivity, and extraction efficiency over traditional methods such as Soxhlet extraction, maceration and others along with reduced time and solvent consumption, less environmental pollution, and reduced degradation of thermolabile constituents.

Amyloid [Formula: see text] (A[Formula: see text]) peptide monomers polymerize to form insoluble amyloid fibril aggregates and accumulate as senile plaques which eventually leads to cognitive impairment. Modulating abnormal amyloid aggregation can be considered a therapeutic target for Alzheimer’s disease. Recent studies support that Curcumin interferes with larger protein aggregate formation by destabilizing the salt bridge (Asp 23-Lys 28) of A[Formula: see text] protein. The chemical library of curcumin derivative with pyrazole, isoxazole, and isothiazole showed considerable binding affinity comparable to that of curcumin. In silico docking studies of the library of the compound, revealed strong binding affinity with A[Formula: see text] protein and [Formula: see text]-secretase enzyme (BACE1). De novo ligand design coupled with manual pharmacophore mapping of our best-fitting lead revealed another ligand having a potential binding affinity with both A[Formula: see text] protein and BACE-1. Both the compounds passed Lipinski’s Rule of Five, in silico toxicity testing by admetSAR, and pharmacophore overlaps with Verubecestat, a compound under clinical trial against Alzheimer’s disease. MD dynamic simulation study revealed the stability of protein after it binds to our ligand. Secondary structure determination was also done to observe the changes in [Formula: see text] and [Formula: see text] sheets of the protein with and without ligand binding. Ligand-based drug design was also carried out via pharmacophore mapping and searching the molecules via zinc database.

Aims: Design isoxazole bearing leads as dual inhibitors against Amyloid β and BACE-1 loop in protein fibrillation. Background: Protein fibrillation is one of the key reasons for several diseases, namely Alzheimer’s, Parkinson’s, and many others. One of the key strategies of preventing protein fibrillation is destabilizing the protein fibrils themselves or inhibiting the amyloid fibril-forming pathway in the initial stage. Introduction: Attempts have been taken to design newer leads to inhibit protein fibrillation by targeting the β-amyloidogenesis pathway in the brain. To exploit interfenestration between Amyloid β -42 protein and BACE-1 (β-site amyloid precursor protein cleaving enzyme) for amyloidogenesis, studies are undertaken to design dual inhibitors against the same. Method: In vitro binding interactions were found using docking, de novo ligand design, and MD simulation study. Results: Three compounds bearing an isoxazole heterocyclic nucleus were designed which could successfully bind to the hydrophobic raft and salt bridge residues Asp 23-Lys-26 of Amyloid β, destabilizing the growing fibril. Additionally, one of our candidate compounds exhibited force of interaction with Thr232 at the S3 pocket of BACE-1, interacted with key residue Asp228, Tyr71, and Thr72 of the β-hairpin flap and hydrogen bonding with Gly11 at loop 10s. Conclusion: Protein flexibility dynamics of the Aβ-42 protein revealed that there is a considerable conformational change of the same with or without ligand binding. The lower RMSF of the bound region and reprogramming residual contacts within the Aβ-42 protein suggested successful binding of the ligand with the protein, lowering the access for further β-β dimerization.

The isolated local recurrence in a patient previously treated for early-stage invasive breast cancer presents a unique challenge to the oncologist. The main objectives of this study were to identify breast carcinoma patients who are prone to develop early recurrence, to study the feasibility of resection in case of locally recurrent breast carcinoma and determine the incidence of distant metastasis associated with locally recurrent breast carcinoma. The parameters namely age, previous clinical TNM staging, previous Histological grade, lymphovascular invasion, previous types of surgery, previous immunohistochemistry and previous chemoradiotherapy had shown very strong positive significance. There was significant association between previous findings of IHC and present findings of IHC (p=0.0087) but there was no significant association between clinical stage and resectability (p=0.95), between status of LVI and resectability (p=0.71) and between Histopathological Grade and resectability (p=0.43). Local recurrences after primary resection are seen to be mostly resectable with negative margin; however, whether it results in survival benefit can be studied in long term study.

Purpose: The sole purpose of this study is to improve the solubility and dissolution of telmisartan by cocrystallization technique and apply a computational simulation approach to assess the nature of chemical interactions between telmisartan and coformer as well as the solvent contribution to the molecules for furnishing cocrystallization. Methods: The effects of various concentrations of coformer i.e. oxalic acid on physicochemical parameters and drug release were investigated. Results: Solubility studies suggested that cocrystallization technique with oxalic acid helps to increase the solubility of pure telmisartan of about 7 folds and drug release study revealed that telmisartan-oxalic acid cocrystals showed greater dissolution as compared to pure telmisartan. SEM study suggested that prepared telmisartan cocrystals showed rhomboid-shaped crystals with sharp edges and smooth surface. FTIR study revealed that shifting in the vibrational frequencies of C=O group of telmisartan in telmisartan- oxalic acid cocrystal indicates the formation of supra molecular hetero synthon of the cocrystal. DSC and XRD studies confirmed the formation of telmisartan-oxalic acid cocrystals. Computational simulation approach revealed that telmisartan and oxalic acid can interact with each other in the presence of methanol and water where oxalic acid can form interactions principally with the others. The interactions, thereof, may form several associations or bondings in between the drug and carrier modifying the planarity, bond energy, bond angles of both which subsequently lead to cocrystallization. Conclusion: So, the present research concluded that prepared telmisartan-oxalic acid cocrystal is a successful application of crystal engineering approach to improve the physicochemical properties as well as to enhance the solubility and dissolution of telmisartan.

Mangiferin (MGF), from Mangifera indica is well reported for its hypoglycemic activity and hypolipidemic activity. However, MGF suffers therapeutic limitation due to poor solubility causing disparaging bioavailability. Herein to address this problem, we have incorporated MGF in alginate grafted N-succinylated chitosan (NSC) nanomatrix. Characterization by molecular docking, FT-IR and 2D-NMR (COSY) has revealed that MGF could reinforce interaction with NSC. The OH and CH2OH groups of MGF may set interactions with pyranosic OH, CH2OH, NH2 (or NH-succinyl and COOH-succinyl) of NSC. The NSC-MGF nanoconjugate revealed a spherical particle geometry of 100 ∼ 200 nm size. The encapsulated MGF showed 100% release in vitro. In vivo, NSC-MGF nanoconjugate revealed blood glucose lowering from 300 mg/dL to ∼ 90 mg/dL as well as ∼ 37% lowering of total plasma cholesterol. This is well comparative to the earlier reports which acknowledged only 1 ∼ 36% lowering of plasma cholesterol with MGF. Furthermore, NSC-MGF lowered serum trigyceride to ∼ 61%, while in earlier studies, only 10 ∼ 40% serum triglycerides reduction was found with solitary MGF.

Elevated serum interleukins (IL-6 IL-1beta) over baseline concentration help in blastocyst adhesion to the uterine endometrium in the early phase of pregnancy. A nano PLA (Piperolactam A)-HPBCD (2-hydroxy-propyl-beta-cyclodextrin) inclusion complex was developed as an interleukin down-regulator that exhibited 100% anti-implantation activity in rodents at a dose as low as 2.5-5.0mg/kg. On metabolomics study among major glyco-lipo-protein metabolites only serum low-density lipoprotein (LDL) or very low-density lipoprotein (VLDL) levels revealed alteration by the formulation. Administration of PLA-HPBCD did not cause changes in serum estrogen and progesterone levels. However IL-6 and IL-1beta failed to increase post PLA-HPBCD administration hence is assumed to be the mode of the drugs abortifacient action. In addition absence of signs of either acute or chronic toxicity suggests the formulation considerably non-toxic. Therefore the nano-PLA conjugate promises as a non-steroidal contraceptive lead apart from ormeloxifene the only non-steroidal anti-fertility agent currently available globally. Copyright (c) 2018. Published by Elsevier Inc.

Protein and protein-polysaccharide cages (PPCs) have emerged as a promising tool for drug delivery in the last few years. The primary criterion of selecting protein as a carrier is its biocompatibility, which ensures safe delivery of its cargo without eliciting adverse immunogenic response. Second, due to its properties such as unique sequences, secondary and tertiary structures, folding, clustering, domain-to-domain interactions, entangling between its chains with ionic, covalent, van der Waals, or hydrogen bonding interactions, and casting bridges between its multimers, protein leverages a unique display of cage-like structures that could be used as host to accommodate guest drug molecules with improved stability. Also, PPCs have evolved as unique nanohybrids that allow novel interactions with guest molecules and wrap them with firm interactions vis-a-vis binding forces. When attributed to such kind of payloads, protein cages (PCs) and PPC could be exploited to deliver small to macromolecular drug molecules, control their release inside body, and deliver them in either stimuli-responsive or target-specific manner. These cages may further be engineered by metal-guided intra or intermeric cross-linking, metal lacing, chemical-guided disulfide or amide bridge formation, ligand-guided epitope appending, or aptamer-tagged target furnishing. Such kind of engineering could be employed to shape the PC for improving cage stability, cavity architecture, target specificity, cellular uptake, and release property. In this chapter, we have presented all traditional PCs, their architectures, release properties, engineering approaches, and their biomedical applications. In addition, comparatively newer engineering approaches such as posttranslational modification of proteins to tune their in vivo properties have also been discussed. The perturbations of PPC in drug delivery and therapeutic applications have also been reviewed. It is noteworthy that various engineering approaches such as electrostatic complexation, chemical coupling, and electrospinning have been exploited in contemporary ages to engineer PPCs for improving their intracellular uptakes, loading capacity, in vivo drug release as well as stability. A succinct and magnanimous highlight of all these manipulations has been a major chord in this chapter, especially focusing their applications in recent days of biomedical world.

Natural products have widely been used in applications ranging from antibacterial, antiviral, antifungal, and various other medicinal applications. The use of these natural products was recognized way before the establishment of basic chemistry behind the disease and the chemistry of plant metabo-lites. After the establishment of plant chemistry, various new horizons evolved, and the application of natural products breached the orthodox limitations. In one such interdisciplinary area, the use of plant materials in the synthesis of nanoparticles (NPs) has exponentially emerged. This advancement has offered various environment-friendly methods where hazardous chemicals are completely replaced by natural products in the sophisticated and hectic synthesis processes. This review is an attempt to under-stand the mechanism of metal nanoparticlessynthesis using plant materials. It includes details on the role of the plant’s secondary metabolites in the synthesis of nanoparticles including the mechanism of action. In addition, the use of these nanomaterials has widely been discussed along with the possible mechanism behind their antimicrobial and catalytic action.

Previously, we published cloning, overexpression, characterization and subsequent exploitation of a carbonyl reductase (cr) gene, belonging to general family aldo-keto reductase from Candida glabrata CBS138 to convert keto ester (COBE) to a chiral alcohol (ethyl-4-chloro-3-hydroxybutanoate or CHBE). Exploiting global transcription factor CRP, rDNA and transporter engineering, we have improved batch production of CHBE by trinomial bioengineering. Herein, we present the exploration of cr gene in Candida glabrata CBS138 through genome mining approach, in silico validation of its activity and selection of its biocatalytic phase. For exploration of the gene under investigation, 3 template genes were chosen namely Saccharomyces cerevisae YDR541c, YGL157w and YOL151w. The CR showed significant homology match, overlapping of substrate binding site and NADPH binding site with the template proteins. The binding affinity of COBE toward CR (−4.6 Kcal/ mol) was found higher than that of the template proteins (−3.5 to −4.5 Kcal/ mol). Biphasic biocatalysis with cofactor regeneration improved product titer 4∼5 times better than monophasic biotransformation. Currently we are working on DNA Shuffling as a next level of strain engineering and we demonstrate this approach herein as a future strategy of biochemical engineering.

Objectives Introduction of multiple molecules in a single inclusion complex, albeit cheaper, lacks conclusive attempts in earlier drug delivery reports. This manuscript emphasizes simultaneous incorporation of two anticancer drugs, gefitinib (G) and simvastatin (S), in a single molecule of β-cyclodextrin for the first time to achieve effective drug delivery. Methods The inclusion complex (GSBCD) was prepared by cosolvent evaporation technique using β-cyclodextrin (BCD) as carrier. Characterization of GSBDC was performed by Fourier transform infrared spectroscopy, COSY, differential scanning calorimetry, X-ray diffraction and dynamic light scattering analyses, which were ascribed to the complex formation inside BCD cavity, micronization of drugs and conversion to amorphous state. Key findings The complex revealed entrapment of G and S in 3 ± 0.48: 2 ± 0.19 molar ratio and showed more than 3.5 and 10 fold increase in drug release in in vitro and in vivo, respectively. Docking and COSY studies revealed molecular alignment into BCD central cavity that been achieved via hydrogen bonding between certain groups of the ligands (G and S) and the polar heads of BCD. Partial incorporation of the molecular backbone inside inclusion complex suggests superficial contact with the solvent indicating slow steady release kinetics. Conclusions This approach of forming inclusion complex with multiple molecules within a single cavity can be a landmark for further studies in drug delivery.

Strain engineering tools are usually adopted to improve microorganism tolerance towards stressful environments in bioindustries. However, classical strain engineering techniques of using UV/chemical mutagens are often both labor- and time- intensive. In recent years, transcriptional engineering approach has started to attract attention in strain engineering. The better efficiency of this method has made it more preferable over the classical methods. Here in this thesis, I would adopt error-prone PCR technique to engineer global transcription factor cAMP receptor protein (CRP), which can regulate more than 400 genes in Escherichia coli, to enhance its tolerance against organic solvents, oxidative stress, and low pH. E. coli DH5α-∆crp strain was transformed with the plasmid-containing crp mutants, which were generated from error-prone PCR, followed by a selection under various stresses. All selected mutants exhibited much better tolerance than the wild type (WT) against respective stress. For example, the best toluene tolerant mutant showed growth even in 0.23% (v/v) toluene 0.51 h-1 whereas the growth of WT was completely inhibited. The best oxidative stress mutant demonstrated viability in 12 mM H2O2, while WT growth was halted at 6 mM H2O2. The best mutant identified under various stresses were then subjected to various characterizations, including cross-tolerance check, DNA microarray analysis, quantitative real time PCR and enzyme assay. For instance, toluene-tolerant mutant was also able to have improved growth against n-hexane, cyclohexane and p-xylene while oxidative-stress-tolerant mutant also exhibits thermotolerance. DNA microarray analysis and qRT-PCR results have demonstrated that the modifications to CRP would not only bring differential expression in CRP-regulated genes but also those non-CRP-regulated genes. In conclusion, random mutagenesis of CRP can provide an efficient alternative for E. coli strain engineering. DOCTOR OF PHILOSOPHY (SCBE)

Graphene Oxide (GO) has been extensively studied in the field of biomedical sciences as one of the most promising biomaterials due to its exceptional physiochemical properties. Experts have long favored anticancer drug cocktails over single drugs, given that the former may provide a more balanced molecular basis for novel chemotherapeutic strategies. Here, we investigated a combinatorial anticancer drug treatment involving the well-proven anticancer drugs quercetin and gefitinib and compared it with gefitinib and quercetin loaded separately onto polyvinylpyrrolidone (PVP)-functionalized graphene oxide (GO-PVP). The loading and cancer cell cytotoxicity of the individual drug systems and their combined loading onto GO-PVP nanovehicles were investigated in PA-1 ovarian cancer cells and compared to their effects on IOSE-364 ovarian epithelial cells. In this report, the combined drug system loaded on the GO-PVP nanovehicle was found to be significantly more toxic than the individual drug loaded systems, as well as the free drugs, toward PA-1 cells compared to the toxicity toward IOSE-364 cells. The combined drug system loaded on the GO-PVP nanovehicle is likely to be more successful than individual drug therapy, given the stronger impact of the combinatorial approach and the efficiency of chemotherapeutic delivery.

Nateglinide (NAT) and Pioglitazone (PIO) are an antidiabetic drugs combination and currently under clinical trial in countries like Japan. In this study, an alternative, a simple, sensitive high-performance liquid chromatography method has been developed (limit of detection: 15 ng/mL and limit of quantification: 50 ng/mL) for simultaneous estimation of this drug combination in rat plasma. Most remarkably, bioavailability of NAT has been increased markedly on coadministration with PIO, than when it was administered alone. Thus, PIO is assumed to retard the catabolism of NAT by inhibiting metabolic liver-microsomal enzyme, especially CYP2C9. Using a Waters Nova-Pak C 18 column (150 × 3.9 mm, 4 μm) and a mobile phase of acetonitrile: 10 mM KH2PO4 (60: 40, V/V (volume by volume)) pH 3.5, the analysis was performed at 210 nm with a flow rate of 1.5 mL/min. In silico docking via molecular dynamics simulation revealed that NAT-CYP2C9 binding affinity may be reduced after PIO attachment, presumably due to the binding site overlapping of the two drugs. Thus, it has been proposed that NAT and PIO may be an efficient synergistic fixed dose combination against diabetes mellitus, and the above method can foster a simple but highly sensitive bioanalytical estimation for routine analysis.

Knowledge of usage of traditional medicinal plant databases has become a paradigm of immense importance due to intense utilization of natural products across the globe. Digitization of natural products has crept in via four major approaches: data preservation in various web databases, virtual screening of different drug target networks, a bioinformatics-guided approach for lead optimization, and in silico approaches such as docking or molecular modeling for drug discovery. Several universal natural product databases have been created such as UNPD, CMKb, ebDB, ZINC, TCM, UNIIQUIM, NuBBE, pANAPL, InvertNet, CamMED NP, and DIVERSet where information on more than 19 million compounds has been stored. In addition, different digitization tools have been created for various purposes such as identification tools (EDIT's cybertaxonomy platform, Electronic Field Guide, Medical Fungi Identification Website, Free Delta, and Meka), digitization tools (Bauble, Bibmaster, Biota, and Biotica), and biodiversity together with ecological modeling tools (ADE4, APE, DIVA-GIS, GARP, LAMARC, Molphy, and others).

Proteins, particularly enzymes, are often used in bioreactors to catalyze biosynthetic reactions. However, one of the major challenges in applying proteins in bioreactors is enzyme stability and recovery. This review is an enlightening discussion on recent trends and advancements used to alleviate these limitations completely or to minimize them. Two major strategies, polymer-based enzyme immobilization and polymeric enzyme nanoreactors have been discussed systematically. Further, this review puts light on various methods such as smart polymer-based supports which have been exploited for their ability to regulate ligand-protein interactions. In addition, chemical moieties like protein-based microcrystals, cross-linked enzyme aggregates, and polymer-based nanoreactors have also been discussed in a comprehensive way focusing more on their unique applicability and target-specific actions. Polymer-based nanoreactors are described in detail in vivo together with enhancement of the enzyme stability and controll...

A simple, sensitive, selective and reproducible method based on a reversed-phase chromatography was developed for the determination of Praziquantel in rat plasma using internal standard as Diazepam. Praziquantel & Diazepam was separated on a C18 column Enable (250 mm × 4.6 mm, 5 µm particle size: Spinco Biotech Pvt Ltd), with retention times of 6.4 & 8.5 min. Ultraviolet detection was set at 225 nm. The mobile phase consisted of acetonitrile and water (60:40, v/v), running through the column at a flow rate of 1.0 mL min-1. The chromatographic analysis was operated at an ambient temperature. Sample preparation (200 µl plasma) was done by protein precipitation using perchloric acid. Calibration curve in plasma was plotted at different concentration level 5, 50, 100, 500, 1000, 2000, 3000 & 5000 ng mL-1 were found to be linear with correlation coefficients (r) is 0.9989. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was within 15% (relative standard deviation: R.S.D. should be less than 15 according to CDER guidance for Bio-analytical Method Validation). Good accuracy was observed for both the intra-day or inter-day assays, as indicated by the minimal deviation of mean values. Limit of quantitation (LOQ) was accepted as 5 ng mL-1 using 200 µl samples. The mean recovery was found to be greater than 90% for praziquantel. The method appears to be robust and has been applied for pharmacokinetic study of praziquantel, in three different groups of rats following a single oral dose of 40 mg kg-1 body weight of praziquantel.

A carbonyl reductase ( cr ) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363 mM with highest turnover number (112.77 ± 3.95 s −1 ). Solitary recombineering of such catalytic cell reproduced CHBE 161.04 g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp ( crp *, F136I) into this heterologous E. coli host yielded CHBE 477.54 g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88 g/L/gDCW. The CHBE production has thus been upscaled to 8–12 times than those reported so far. qRT-PCR studies revealed that both membrane efflux channels such as acrAB as well as ROS scavenger genes such as ahpCF have been activated by engineering crp . Moreover, membrane protecting genes such as manXYZ together with solvent extrusion associated genes such as glpC have been upregulated inside mutant host. Although numerous proteins have been investigated to convert COBE to CHBE; this is the first approach to use engineering triad involving crp engineering, recombinant DNA engineering and transporter engineering together for improving cell performance during two-phase biocatalysis.

Leishmaniasis is an epidemic in various countries, and the parasite Leishmania donovani is developing resistance against available drugs. In the present study the antileishmanial action of piperolactam A (PL), isolated after bioactivity guided fractionation from root extracts of Piper betle was accentuated in detail. Activity potentiation was achieved via cyclodextrin complexation. Crude hydro-ethanolic extract (PB) and three fractions obtained from PB and fabricated PL-hydroxypropyl-β-cyclodextrin (HPBCD) nanoparticles were evaluated for antileishmanial activity. Tests were performed against L . donovani wild-type, sodium stibogluconate, paromomycin and field isolated (GE1) resistant strains in axenic amastigote and amastigote in macrophage models. PL-HPBCD complex was characterized and FITC loaded HPBCD nanoparticles were assessed for macrophage internalization in confocal microscopic studies. Isolated and purified PL from most potent, alkaloid rich ethyl acetate fraction of PB showed high level of antileishmanial activities in wild-type (IC 50 = 36 μM), sodium stibogluconate resistant (IC 50 = 103 μM), paromomycin resistant (IC 50 = 91 μM) and field isolated resistant (IC 50 = 72 μM) strains together with cytotoxicity (CC 50 = 900 μM) in mouse peritoneal macrophage cells. Inclusion of PL in HPBCD nanoparticles resulted in 10-fold and 4–10-fold increase in selectivity indexes (CC 50 /IC 50 ) for wild-type and drug resistant strains, respectively. Drug-carrier interactions were clearly visualized in FT-IR studies. Complete incorporation of PL in HPBCD cavity was ascertained in DSC and XRD analyses. 180 nm size stable nanospheres showed macrophage internalization within 1 h of incubation. Piperolactam A (PL), a representative of the inchoate skeleton of aristolactam chassis might be the source of safe and affordable antileishmanial agents for the cure of deadly Leishmania infections.

Fertility control is a burning problem all over the world to regulate population overflow and maintain ecological balance. This study is an in-silico approach to explore a non-steroidal lead as contraceptive agent in order to avoid several contraindications generated by steroidal analogues. Piperolactam A, an aristolactam isolated from Piper betle Linn. showed binding affinity towards estrogen and progesterone receptor as -8.9 and -9.0Kcal/mol (inhibition constant Ki=0.294μM and 0.249μM) respectively which is even larger than that of reported antagonists such as Rohitukine and OrgC (binding affinity -8.7 and -8.4Kcal/mol; Ki 0.443μM and 0.685μM respectively). The binding site exploration displayed more hydrogen bonding of Piperolactam A (His 524, Leu 346, Thr 347) than Rohitukine and OrgC (Leu 718) with associated receptors which was further confirmed by molecular dynamics simulations. The drug-likeliness of the compound has been proved from its tally with Lipinsky's Rule of Five and lowered toxicity such as cardiac toxicity, liver toxicity, mutagenicity and ecological toxicity. Endocrine disruptome and later docking guided molecular simulations revealed that Piperolactam A has weaker binding affinity and/or lower probability of binding with nuclear receptors especially hERG and cytochrome P450. The high Caco-2 permeability suggested more bioavailability hence more therapeutic efficacy of the drug.

Bioprocesses such as production of organic acids or acid hydrolysis of bioresources during biofuel production often suffer limitations due to microbial sensitivity under acidic conditions. Approaches for improving the acid tolerance of these microbes have mainly focused on using metabolic engineering tools. Here, we tried to improve strain acidic tolerance from its transcription level, i.e . we adopted error-prone PCR method to engineer global regulator cAMP receptor protein (CRP) of Escherichia coli to improve its performance at low pH. The best mutant AcM1 was identified from random mutagenesis libraries based on its growth performance. AcM1 almost doubled (0.113 h −1 ) the growth rate of the control (0.062 h −1 ) at pH 4.24. It also demonstrated better thermotolerance than the control at 48 °C, whose growth was completely inhibited at this temperature. Quantitative real time reverse transcription PCR results revealed a stress response overlap among low pH stress-, oxidative stress- and osmotic stress-related genes. The chief enzyme responsible for cell acid tolerance, glutamate decarboxylase, demonstrated over twofold activity in AcM1 compared to the control. Differential binding properties of AcM1 mutant CRP with Class-I, II, and III CRP-dependent promoters suggested that modifications to native CRP may lead to transcription profile changes. Hence, we believe that transcriptional engineering of global regulator CRP can provide a new strain engineering alternative for E. coli .

Organic solvents are often applied in biphasic biocatalysis or involved in bioremediation, but native microorganisms usually have low tolerance toward these organic solvents and cannot survive in the stressful environment. Here, we aimed to improve the organic solvent tolerance of Escherichia coli by engineering its global transcription factor cAMP receptor protein (CRP). Toluene was chosen as model organic solvent. The mutated crp genes were generated via error-prone PCR and three toluene-tolerant mutants M1–M3 were isolated from random mutagenesis libraries by enrichment selection. All mutants exhibited much better growth than the wild type upon exposure to 0.2–0.23% (v/v) toluene, with M2 the best. When toluene concentration was at 0.2%, M1–M3 shared similar growth rate at 0.68 h −1 despite the elongated 20-h lag phase, with WT exhibiting null growth. In 0.23% toluene, the growth rate of M2 was found to be 0.51 h −1 while the growth of WT was completely inhibited. M2 also demonstrated excellent growth in other organic solvents such as n -hexane, cyclohexane and p -xylene as compared to the wild type. Our quantitative real-time reverse transcription PCR analysis with organic-solvent stress associated genes indicated that amino acid mutations in CRP would lead to changes in the expression level of genes that are not regulated by CRP. The field emission scanning electron microscopy images revealed that both wild type and M2, though combating toluene stress, did not have any significant changes in their cellular size and shape.