Mutational analysis of flavonol synthase of M. pinnata towards enhancement of binding affinity: a computational approach.

Millettia pinnata is an important medicinal plant that has been used as a treatment of various diseases due to presence of wide range of pharmacological properties. The plant contains quercetin, kaempferol, karanjin, pongaglabrone, kanjone, kanugin, gammatin, pongaglabol, and other bioflavonoids. Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, and antidiabetic activities etc. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols, i.e. biosynthesis of kaempferol from dihydrokaempferol. The current work examined the binding affinity-based approach to improve the enzyme catalytic activity using computational methods. Sequential site-directed mutagenesis was used to create four mutants with the goal to increase hydrogen bonds and further improving the ligand (dihydrokaempferol) binding efficiency. Simulations were done to monitor the stability of the mutants followed by molecular docking to confirm interactions with ligand. For structure validation, various dynamic analysis like RMSD, RMSF, ROG, SASA, H-bond, PCA, DCCM, and FEL were performed, which predicts the stability of wild-type (WT) proteins and mutants. The Mutant_2 and Mutant_3 showed maximum H-bonding and better stability than other mutants and WT that proved higher affinity suggesting improved catalysis. Mutant_2 and Mutant_3 exhibited binding affinities of -7.6 and -8.2 kcal/mol, respectively for the ligand. The outcome of present study will provide significant improvement in synthesis of kaempferol and other plant-based flavonoids.Communicated by Ramaswamy H. Sarma.

The molecular docking and molecular dynamics study of flavonol synthase and flavonoid 3'-monooxygenase enzymes involved for the enrichment of kaempferol.

Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, antidiabetic activities etc. It has been reported in many vegetables, fruits, herbs and medicinal plants. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols. Whereas flavonoid 3'-monooxygenase (F3'H, EC 1.14.14.82) catalyses the hydroxylation of dihydroflavonol, and flavonol. FLS is involved in the synthesis of the kaempferol whereas F3'H causes degradation of kaempferol. The present study aimed to analyse the binding affinity, stability and activating activity of enzyme FLS as well as inhibitory activity of enzyme F3'H involved in the enrichment of the kaempferol using the in-silico approaches. Computational study for physico-chemical properties, conserved domain identification, 3-D structure prediction and its validation, conservation analysis, molecular docking followed by molecular dynamics analysis of FLS and F3'H, protein-activator (FLS-LIG Complex) and protein-inhibitor (F3'H-LIG Complex) complexes have been performed. Other structural analyses like root mean square fluctuation (RMSF), root mean square deviation (RMSD), surface area solvent accessibility (SASA), radius of gyration (Rg), hydrogen bond analysis, principal component analysis (PCA), Poisson-Boltzmann analysis (MM_PBSA) and the dynamic cross correlation map (DCCM) analysis to explore the structural, functional and thermodynamic stability of the proteins and the complexes were also studied. The molecular docking result showed that FLS binds strongly with the activator ascorbate (CID _54670067) while F3'H binds with the inhibitor ketoconazole (CID_456201). The most powerful inhibitor (ketoconazole for F3'H) and activator (ascorbate for FLS) is determined by computing the thermodynamic binding free energy through MM_PBSA analysis. The current work provides wide-ranging structural and functional information about FLS and F3'H enzymes showing detailed molecular mechanism of kaempferol biosynthesis and its degradation and hence kaempferol enrichment. Finding of the present work opens up new possibilities for future research towards enrichment of kaempferol by using activator (ascorbate) for FLS and inhibitor (ketoconazole) for F3'H as well as for its large-scale production using in vitro approaches.Communicated by Ramaswamy H. Sarma.

Adaptive resilience of roadside trees to vehicular emissions via leaf enzymatic, physiological, and anatomical trait modulations.

Unplanned urbanization and heavy automobile use by the rapidly growing population contribute to a variety of environmental issues. Roadside plants can mitigate air pollution by modifying their enzymatic activity, physiological and anatomical traits. Plant enzymes, physiological and anatomical traits play an important role in adaptation and mitigation mechanisms against vehicular emissions. There is a significant gap in understanding of how plant enzymes and anatomical traits respond or how they participate in modulating the effect of vehicular emissions/air pollution. Modulation of leaf anatomical traits is also useful in regulating plant physiological behavior. Hence, the present study was conducted to evaluate the effects of vehicular pollution on the enzymatic activity, physiological, and anatomical traits of plant species that grow in forests (S1) and alongside roads (S2-1 km away from the S1 site) during different seasons. The present study examines four commonly found roadside tree species i.e. Grevillea robusta, Cassia fistula, Quercus leucotrichophora and Cornus oblonga. The study found that the activities of catalase and phenylalanine ammonium enzymes were higher in G. robusta species of roadside than control site (S1). Non-enzymatic antioxidants such as flavonoid and phenol were also found in higher concentrations in roadside tree species during the summer season. However, the measured values of physiological traits were higher in Q. leucotrichophora tree species of S1 during the summer season. When compared to the other species along the roadside, Q. leucotrichophora had the highest number of stomata and epidermal cells during the summer season. Hence, we found that tree species grown along the roadside adapted towards vehicular emissions by modulating their enzymatic, physiological, and anatomical traits to mitigate the effect of air pollution.

Removal of inorganic toxic contaminants from wastewater using sustainable biomass: A review.

Lignocellulosic biomass is most abundant, ecofriendly and sustainable material on this green planet which has received great attention due to exhaustion of petroleum reserves and various environmental complications. Due to its abundance and sustainability, it has been opted in number of advanced applications i.e. synthesis of green chemicals, biofuels, paper, packaging, biocomposite and for discharge of toxic contaminants from wastewaters. Utilization of sustainable biomass for removal of toxic pollutants from wastewater is robust technique due to its low-cost and easy availability. In this review, we have summarized removal of inorganic pollutants by sustainable lignocellulosic biomass in their natural as well as in chemically functionalized form. Various techniques for modification of sustainable biomass have been discussed and it was found that modified biomass showed better biosorption ability as compared to natural biomass. We conclude that modified biomass biosorbents are useful for removal of toxic inorganic pollutants to deficient levels. Several modification strategies can improve the qualities of biosorbent, however grafting is the most successful among them, as demonstrated in this work. The numerous grafting methods using a free radical grafting process are also summarized in this review article. This review also gathers studies comparing sorption capabilities with and without modification using modified and unmodified biosorbents. Chemically modified cellulosic biomass is favoured over untreated biomass because it has a higher adsorption efficiency, which is favoured by a large number of reactive binding sites, improved ion-exchange characteristics, and more functional groups available after modification.

Cellulose-g-poly-(acrylamide-co-acrylic acid) polymeric bioadsorbent for the removal of toxic inorganic pollutants from wastewaters.

Cellulose biopolymer was functionalized by free graft copolymerization and used as a new adsorbent to eliminate toxic inorganic pollutants from wastewater. Functional graft copolymers from cellulose were characterized by FT-IR spectroscopy, XRD, TGA, and SEM-EDX techniques.Cellulose-g-poly-(acrylamide-co-acrylic acid) polymer adsorbent showed high adsorption capacities for the Cd2+, Cu2+, Pb2+ and Zn2+ toxic metal ions, which were evidenced by the comparison with unmodified cellulose-richsamples. Cellulose-g-poly-(acrylamide-co-acrylic acid) polymer adsorbent was optimized with various adsorption parameters such as the effect of pH, contacttime, temperature, and metallic ions concentration. The maximum monolayer capacity qm calculated for Cd2+, Cu2+, Pb2+, Zn2+ were 101.73, 61.84, 209.64, and 55.04 mgg-1 respectively. Thus, these results proved that cellulose-g-poly-(acrylamide-co-acrylic acid) graft copolymer adsorbent from cellulose-rich biomass could potentially be used for the removal of pollutants from wastewater.

Clinical Course of Nonfunctional Pituitary Microadenoma in Children: A Single-Center Experience.

CONTEXT: Pituitary lesions consistent with microadenomas are increasingly discovered by MRI. Sparse data are available on the long-term clinical and imaging course of such lesions in children. OBJECTIVE: The aim of this study was to define the clinical and imaging course of pituitary lesions representing or possibly representing nonfunctioning microadenomas in children to guide clinical management. DESIGN: Retrospective observational study. METHODS: The clinical data warehouse at a tertiary care academic children's hospital was queried with the terms "pituitary" AND "microadenoma" and "pituitary" AND "incidentaloma." The electronic health records of the identified subjects were reviewed to extract data on the clinical and imaging course. RESULTS: A total of 78 children had nonfunctioning pituitary lesions incidentally discovered during clinical care, of which 44 (56%) were reported as presumed or possible microadenomas. In the children with microadenoma (median age 15 years, interquartile range 2), a majority (70%) underwent imaging for nonendocrine symptoms, the most common being headache (n = 16, 36%). No significant increase in the size of the microadenoma or cysts or worsening of pituitary function was seen over the average clinical follow-up of 4.5 ± 2.6 years. Four cases of drug-induced hyperprolactinemia resolved with discontinuation of the offending medication. CONCLUSIONS: Asymptomatic pituitary lesions representing cysts, microadenomas, or possible microadenomas follow a benign course in children. In the absence of new endocrine or visual symptoms, repeat MRI may not be needed, and if performed, should be done in no less than a year. When possible, it is prudent to discontinue hyperprolactinemia-inducing medications before imaging.

Assessment of the role of early dynamic PET/CT with 18F-fluorocholine in detection of parathyroid lesions in patients with primary hyperparathyroidism.

OBJECTIVE: The aim of the present study was to assess the utility of early dynamic PET/computed tomography with fluorine-18-fluorocholine (F-FCH) in detecting parathyroid lesions and in differentiating parathyroid lesions from cervical lymph nodes (LNs). PATIENTS AND METHODS: A prospective study was conducted on 14 patients with clinical and biochemical evidence of primary hyperparathyroidism by having a positive Tc-sestaMIBI scan. Patients underwent early dynamic F-FCH PET/computed tomography scan, after the administration of 5-8 mCi (185-296 MBq) at 1 min per frame for 15 min. Delayed static images of 2-3 min per bed position were taken between 45 and 60 min. 3D-VOI's were plotted on parathyroid adenoma, cervical LN and thyroid. Dynamic and static images were interpreted by two expert nuclear medicine physicians independently and the following parameters were calculated for parathyroid adenoma and cervical LN: maximum standardized uptake value (SUVmax), time activity curve for SUVmax, t-peak. Adenoma to thyroid ratio (A/T) and cervical LN to thyroid ratio were calculated for each dynamic and static image. RESULTS: Fourteen (eight females and six males) patients were included in the study. All patients showed a higher SUVmax in the adenoma and the cervical LN in the early dynamic images as compared with delayed static images. A/T ratio obtained in the dynamic and static images were compared and found to have insignificant difference (P=0.2255). The difference between mean A/T and LN to thyroid ratio was found to be significant (P=0.0117) during the dynamic study. CONCLUSION: A possible explanation of higher SUVmax in the dynamic images in adenomas may be due to the increased vascularity/early F-FCH uptake. Results indicate early dynamic imaging could suffice, without the need for a delayed image after 45 min, and this technique could adequately differentiate a parathyroid adenoma from a cervical LN.

Comparability of Early Dynamic and Conventional Static Imaging With 18F-Fluorocholine PET/CT in a Patient With Primary Hyperparathyroidism.

Recent studies have established the superiority of FCH PET/CT over MIBI scan in accurate preoperative localization of parathyroid adenomas. In this patient, we compared both early dynamic and conventional static imaging to see if early imaging could suffice. We describe a 15-year-old boy with primary hyperparathyroidism, in whom early dynamic FCH PET/CT was performed for 15 minutes after injection, followed by conventional static image at 60 minutes. Early dynamic images accurately localized the parathyroid adenoma. Also, higher SUVmax was observed on dynamic imaging when compared with conventional static images, but parathyroid-to-thyroid ratio was higher in the latter.

Molecular Modeling, Synthesis, and Anti-HIV Activity of Novel Isoindolinedione Analogues as Potent Non-nucleoside Reverse Transcriptase Inhibitors.

Different isoindolinedione derivatives bearing imine, amide, thioamide, and sulfonamide linkages have been designed in silico using discovery studio software (BIOVIA, San Diego, CA, USA), synthesized, and evaluated for their anti-HIV activity. SAR studies revealed that the linkages in these molecules did affect their anti-HIV activity and the molecules having sulfonamide linkages were the most potent HIV-RT inhibitors as the S=O bonds of the sulfonamide moiety interacted with Lys103 (NH or carbonyl or both) and Pro236; the NH part of the sulfonamide linkage formed bond with carbonyl of Lys101. blood-brain barrier (BBB) plots were also studied, and it was found that all the designed molecules have potential to cross BBB, a very vital criteria for anti-HIV drugs. In vitro screening was performed using HIV-1 strain IIIB in MT-4 cells using the MTT assay, and it was seen that some of these molecules were effective inhibitors of HIV-1 replication at nanomolar concentration with selectivity indices ranging from 33.75 to 73.33 under in vitro conditions. Some of these molecules have shown good anti-HIV activity at 3-4 nm concentrations. These derivatives have potential to be developed as lead molecules effective against HIV-1. Novel isoindolinedione derivatives as probable NNRTIs have been synthesized and characterized. Some of these molecules have shown good anti-HIV activity at 3-4 nm concentrations.

Anti-HIV drug development: structural features and limitations of present day drugs and future challenges in the successful HIV/AIDS treatment.

Acquired Immune Deficiency Syndrome (AIDS), an immuno-compromized condition, a sequel to untreated human immunodeficiency virus (HIV) infection, inviting several life-threatening diseases, has become one of the most fatal disorders in the recent past because of HIV strain variance due to mutations, passive latency and reservoirs helping in replenishing and reviving the HIV-1 proviral DNA. Scientific efforts have led to the discovery of several effective drugs against HIV and lowered the morbidity and mortality all over the world. However, despite availability of a good number of anti-HIV drugs, the problem, for the foreseeable reasons, stands out as the most chronic disease due to the less tolerability and low accessibility of drugs, life-long expensive treatment, and above all, the emergence of drug resistant viral strains. This review dwells upon HIV infection and its proliferation inside the host system, drug targets, different types of drugs, their structural features and mode of interaction with viral targets and drug regimens. It further focuses on topics of latest interest regarding drug development, fixed dose combinations (FDCs), the limitations of present day drugs with their structural features along with their pharmacodynamics, pharmacokinetics and pharmacogenomics and the challenges in finding a permanent cure for HIV/AIDS.

Curcumin bioconjugates: studies on structure-activity relationship and antibacterial properties against clinically isolated strains.

Curcumin bioconjugates, with folic acid, fatty acids and dipeptide, have shown much lower MIC than curcumin against clinically isolated Gram-positive, S.viridians, and Gram-negative bacterial strains, E. coli, P. mirabilis and K. pneumoniae. Polynomial regression analysis was performed to establish a correlation between lipophilicity (logP) and antibacterial activity (pMIC), which showed the efficacy of these molecules against the bacterial strains in the following order: E. coli > S viridans = K. pneumoniae > P. mirabilis. The regression coefficients (R(2) = 0.62 to 0.91) derived for each strain were correlated significantly and led to a conclusion that it was the amphiphilic nature that governed the antibacterial activity. Thus, the bioconjugate 2, having folic acid attached at active methylene site of curcumin with free phenolic hydroxyls, showed the best result.

Rhodium(II) acetate-catalyzed stereoselective synthesis, SAR and anti-HIV activity of novel oxindoles bearing cyclopropane ring.

Novel oxindole derivatives bearing substituted cyclopropane ring have been designed on the basis of docking studies with HIV-1 RT using the software DS 2.5 and synthesized as probable NNRTIs against HIV-1 using rhodium(II) acetate-catalyzed stereoselective cyclopropanation reaction. The cyclopropane isomer, having trans relationship with respect to carbonyl of lactam moiety and functional group on the cyclopropane ring, was the major product in all cases along with a small amount of cis and methylene products. The trans isomers interacted well with HIV-1 RT through H-bonding with amino acids, like Lys101, Lys103, His235, Tyr318, constituting the non-nucleoside inhibitor binding pocket (NNIBP) during docking experiments. However, the compounds showed very little activity when subjected to in vitro anti-HIV-1 screening using ß-galactosidase assay (TZM-bl cells) and GFP quantification (CEM-GFP cells). The very low level of in vitro HIV inhibition, in comparison to predicted EC(50) values on the basis of computational studies, during CEM-GFP screening using AZT as positive control indicated that probably the HIV RT is not the viral target and the molecules work through some different mechanism.

Synthesis, structure-activity relationship and antiviral activity of 3'-N,N-dimethylamino-2',3'-dideoxythymidine and its prodrugs.

A probable NRTI molecule, viz. 3'-N,N-dimethylamino-2',3'-dideoxythymidine (4) and its 5'-O-carboxyl ester prodrugs - 5'-(N-alpha-BOC-L-phenylalanyl)-3'-N,N-dimethylamino-2',3'-dideoxythymidine (5), 5'-L-phenylalanyl-3'-N,N-dimethylamino-2',3'-dideoxythymidine (6) and 5'-decanoyl-3'-N,N-dimethylamino-2',3'-dideoxythymidine (7) have been synthesized and screened against HIV, HSV-1 and 2, parainfluenza-3, vesicular stomatitis and several other viruses. The compound 6 showed good antiviral activity with EC(50) value 0.03 microM (SI=8) against VSV in Hela and HEL cell lines. However, the lead compound 4 and its derivatives 5, 6 and 7 showed no remarkable activity against HIV-1 and other viruses. Molecular docking studies with HIV-1 RT using DS 2.5 and pymol softwares have shown marked differences in the interaction patterns between the lead compound 4 and AZT.

Design and synthesis of novel oxindoles as potential non-nucleosidic reverse transcriptase inhibitors against HIV.

We describe here the synthesis of novel oxindoles by cycloproponation of its diazo derivatives. The synthesis involves the use of allyl methyl sulfide and allyl alcohol as olefins and rhodium acetate as catalyst. These oxindoles have been designed to act as non-nucleosidic reverse transcriptase inhibitors (NNRTIs) against HIV.