Wound Healing Activity of Topical Herbal Gels Containing Barringtonia acutangula Fruit Extract: In silico and In vivo Studies.

The current study describes the efficacy of B. acutangula fruit extract in wound healing via incorporation within topical gels. B. acutangula fruit extract was produced by solvent extraction method. The bioactive extract was incorporated within Carbopol 940-based topical gels, which were applied topically over the excision and incision wounds. The change in healing process was observed till 20 days. The percentages of closure of excision wound area were 92.89 % and 93.43 %, when treated with topical herbal gels containing B. acutangula fruit extract of 5 % and 10 %, respectively. The tensile strengths of incision area in rats treated with topical herbal gels containing 5 % and 10 % methanol extract of B. acutangula fruits were found to be 25±5.12 g and 30±4.10 g, respectively. The wound healing activity of topical herbal gels containing B. acutangula fruit extract in rats was found to be significant when compared with that of the reference standard and untreated groups. In addition, in silico studies suggested about good skin permeability and binding to the proteins responsible for delaying wound healing. It can be concluded that this topical herbal gels containing B. acutangula fruit extract could be used clinically for the treatment of wounds.

Islet Dimensions and Its Impact on the Cellular Composition and Insulin-Secreting Capacity: Insights Into the Role of Non-beta Cells.

Studies have underscored the significance of islet dimensions, encompassing i) the necessity for islets to maintain an optimal diameter to sustain functional activity; ii) larger islets exhibit an intermingled architecture of alpha and beta cells, enhancing functional activity through paracrine effects; iii) non-alpha/beta (NAB) cells play a significant role in regulating beta cells; and iv) there is a preferential loss of larger islets in cases of type 2 diabetes mellitus. To delve deeper into these aspects, the authors documented the cellular composition in islets of various dimensions and regions of the pancreas, along with their secreting capacity, using the expression of the myosin Va motor protein in nine non-diabetic adult human pancreases. The proportion of NAB cells was found to be higher in intermediate islets and significantly lower in smaller and larger islets. By comparing the differences in islet composition, where NAB cells increase from smaller to intermediate islets, leading to a decrease in the proportion of alpha and beta cells, and in larger islets, there is a higher proportion of beta and alpha cells similar to smaller islets, we propose the hypothesis that NAB cells proliferate as islets increase in size. Furthermore, in larger islets, these NAB cells convert into alpha and beta cells, resulting in the scattered, intermingled arrangement observed in larger islets. The higher intensity of myosin Va expression in the islets of the tail region, along with a similar proportion of NAB cells in intermediate islets of the tail region compared to larger islets, leads to decreased inhibitory stimuli to beta cells and an increased insulin-secreting capacity.

Biochemical and in silico analysis of the binding mode of erastin with tubulin.

Erastin (ERN) is a small molecule that induces different forms of cell death. For example, it has been reported to induce ferroptosis by disrupting tubulin subunits that maintain the voltage-dependent anion channels (VDACs) of mitochondria. Although its possible binding to tubulin has been suggested, the fine details of the interaction between ERN and tubulin are poorly understood. Using a combination of biochemical, cell-model and in silico approaches, we elucidate the interactions of ERN with tubulin and their biological manifestations. After confirming ERN's antiproliferative efficacy (IC50, 20 ± 3.2 M) and induction of cell death in the breast cancer cell line MDA-MB-231, the binding interactions of ERN with tubulin were examined. ERN bound to tubulin in a concentration-dependent manner, disorganizing the structural integrity of the protein, as substantiated via the tryptophan-quenching assay and the aniline-naphthalene sulfonate binding assay, respectively. In silico studies based on molecular docking revealed a docking score of -5.863 kcal/mol, suggesting strong binding interactions of ERN with tubulin. Additionally, molecular dynamics simulation and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analyses evinced the binding free energy (ΔGbinding) of -31.235 kcal/mol, substantiating strong binding affinity of ERN with tubulin. Ligplot analysis showed hydrogen bonding with specific amino acids (Asn A226, Thr A223, Gln B247 and Val B355). QikProp-based ADME (absorption, distribution, metabolism and excretion) assessment showed considerable therapeutic potential for ERN.Communicated by Ramaswamy H. Sarma.

Exploring the antifilarial potential of an important medicinal plant Typhonium trilobatum (L. Schoot): Isolation, characterization, and structural elucidation of bioactive compounds against Brugia malayi.

ETHNOPHARMACOLOGY RELEVANCE: The plant Typhonium trilobatum has been utilized in traditional medicine for the treatment of many ailments, including parasitic infections. Recent examinations indicate that the bioactive substances from this plant may have antiparasitic activities against Brugia malayi, which have not been determined. PURPOSE: The parasitic nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti causing lymphatic filariasis, remain a significant challenge to global public health. Given the ongoing nature of this enduring menace, the current research endeavours to examine the efficacy of an important medicinal plant, Typhonium trilobatum. METHODS: Different extracts of the T. trilobatum tubers were evaluated for their antiparasitic activity. The most prominent extract was subjected to Gas Chromatography Mass Spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC) followed by Column Chromatography for isolating bioactive molecules. The major compounds were isolated and characterized based on different spectroscopic techniques (FTIR, NMR and HRMS). Further, the antiparasitic activity of the isolated compounds was evaluated against B. malayi and compared with clinically used antifilarial drugs like Diethylcarbamazine and Ivermectin. RESULTS: The methanolic extract of the tuber exhibited significant antiparasitic activity compared to the other extracts. The bioactive molecules isolated from the crude extract were identified as Linoleic acid and Palmitic acid. Antiparasitic activity of both the compounds has been performed against B. malayi and compared with clinically used antifilarial drugs, Ivermectin and DEC. The IC50 value of Linoleic acid was found to be 6.09 ± 0.78 µg/ml after 24 h and 4.27 ± 0.63 µg/ml after 48 h, whereas for Palmitic acid the value was 12.35 ± 1.09 µg/ml after 24 h and 8.79 ± 0.94 µg/ml after 48 h. The IC50 values of both the molecules were found to be similar to the standard drug Ivermectin (IC50 value of 11.88 ± 1.07 µg/ml in 24 h and 2.74 ± 0.43 µg/ml in 48 h), and much better compared to the DEC (IC50 values of 194.2 ± 2.28 µg/ml in 24 h and 101.8 ± 2.06 µg/ml in 48 h). Furthermore, it has been observed that both the crude extracts and the isolated compounds do not exhibit any detrimental effects on the J774.A.1 macrophage cell line. CONCLUSION: The isolation and characterization of bioactive compounds present in the methanolic tuber extract of Typhonium trilobatum were explored. Moreover, the antimicrofilarial activity of the crude extracts and its two major compounds were determined using Brugia malayi microfilarial parasites without any significant side effects.

Probabilistic modelling is superior to deterministic approaches in the human health risk assessment: an example from a tribal stretch in central India.

This case drew national attention in 2018. About 100 people died and more than 300 hospitalized in a span of few years in a village of 1200 people in a tribal stretch in central India. Medical teams visiting the area reported severe renal failure and blamed the local eating and drinking habits as causative factors. This human health assessment based on geochemical investigations finds nitrate (NO3-) and fluoride (F-) pollution as well in village's groundwater. Both deterministic and probabilistic techniques are employed to decipher the contamination pathways and extent of contamination. Source apportionments of NO3- and F- and their relationship with other ions in groundwater are carried out through chemometric modelling. Latent factors controlling the hydrogeochemistry of groundwater too are explored. While hazard quotients ([Formula: see text]) of the chemical parameters ([Formula: see text] and [Formula: see text]) identify ingestion as the prominent pathway, the calculated risk certainty levels (RCL) of the hazard index (HI) values above unity are compared between the deterministic and probabilistic approaches. Deterministic model overestimates the HI values and magnify the contamination problems. Probabilistic model gives realistic results that stand at infants ([Formula: see text] = 34.03%, [Formula: see text] = 24.17%) > children ([Formula: see text] = 23.01%, [Formula: see text] = 10.56%) > teens ([Formula: see text] = 13.17%, [Formula: see text] = 2.00%) > adults ([Formula: see text] = 11.62%, [Formula: see text] = 1.25%). Geochemically, about 90% of the samples are controlled by rock-water interaction with Ca2+-Mg2+-HCO3- (~ 56%) as the dominant hydrochemical facies. Chemometric modelling confirms Ca2+, Mg2+, HCO3-, F-, and SO42- to originate from geogenic sources, Cl- and NO3- from anthropogenic inputs and Na+ and K+ from mixed factors. The area needs treated groundwater for human consumption.

Exploration of efficacy, cellular responses, and safety profile of novel 9-(3-Pyridyl) noscapine derivatives as promising anticancer candidates.

This study presented a novel derivative of the antitussive compound noscapine, named 9-3-Pyridyl noscapine (PYNos), to enhance its anticancer potential. Through in silico investigations, PYNos exhibited strong interactions with microtubules, inhibiting cancer cell proliferation both alone and in combination with docetaxel. Docking scores highlighted the affinity of PYNos -5.67 kcal/mol and docetaxel -4.94 kcal/mol to microtubules. When docked with tubulin-DOX co-complex, PYNos displayed a synergistic score of -8.99 kcal/mol. MTT assays on MCF-7 breast cancer cells showed PYNos IC50 values of 11.0 µM (48 h) and 8.4 µM (72 h), while docetaxel had three orders of magnitude lower IC50 values: 0.028 µM (48 h) and 0.015 µM (72 h). Combining PYNos (25 µM) and docetaxel (0.01 µM) reduced proliferation by 50% at both time points. Isobologram analysis confirmed strong antiproliferative synergy (sum FIC <1) at 48 and 72 h. Our comprehensive evaluation encompassing apoptosis and cell cycle arrest patterns further validated the synergistic advantages of this combination. In a xenograft mice model using MCF-7 cells, the PYNos-docetaxel co-treatment resulted in significant tumor regression, showcasing promising induction of apoptosis while mitigating docetaxel-associated toxicity. In summary, our findings underscore the substantial microtubule interactions facilitated by 9-3-Pyridyl noscapine, revealing its synergistic potential with docetaxel and establishing a solid foundation for advancing cancer therapeutic strategies.Communicated by Ramaswamy H. Sarma.

Rational design of novel microtubule targeting anticancer drugs N-imidazopyridine noscapinoids: Chemical synthesis and experimental evaluation based on in vitro using breast cancer cells and in vivo using xenograft mice model.

We present N-imidazopyridine-noscapinoids, a new class of noscapine derivatives that bind to tubulin and exhibit antiproliferative activity against triple positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells. The N-atom of the isoquinoline ring of noscapine scaffold was altered in silico by coupling the imidazo [(Ye et al., 1998; Ke et al., 2000) 1,21,2-a] pyridine pharmacophore to rationally develop a series of N-imidazopyridine-noscapinoids (7-11) with high tubulin binding affinity. The predicted ΔGbinding of the N-imidazopyridine-noscapinoids 7-11 varied from -27.45 to -36.15 kcal/mol, a much lower value than noscapine with ΔGbinding -22.49 kcal/mol. The cytotoxicity of N-imidazopyridine-noscapinoids was evaluated using hormone dependent MCF-7, triple negative MDA-MB-231 breast cancer cell lines and primary breast cancer cells. The cytotoxicity of these compounds (represented as IC50 concentration) ranges between 4.04 and 33.93 µM against breast cancer cells without affecting normal cells (IC50 value > 952 µM). All the compounds (7-11) perturbed the cell cycle progression at G2/M phase and triggered apoptosis. Among all the N-imidazopyridine-noscapinoids, N-5-Bromoimidazopyridine-noscapine (9) showed promising antiproliferative activity and was selected for detailed investigation. The onset of apoptosis treated with 9 using MDA-MB-231 revealed morphological changes like cellular shrinkage, chromatin condensation, membrane blebbing, and apoptotic bodies formation. Along with elevated reactive oxygen species (ROS), there was a loss of mitochondrial membrane potential, suggesting induction of apoptosis to cancer cells. Compound 9 was also found to significantly regress the implanted tumour in nude mice as xenografts of MCF-7 cells without any apparent side effects after drug administration. We conclude that N-imidazopyridine-noscapinoids possess excellent potential as a promising drug for treating breast cancers.

Variation in Yield, Chemical Composition and Biological Activities of Essential Oil of Three Curcuma Species: A Comparative Evaluation of Hydrodistillation and Solvent-Free Microwave Extraction Methods.

The essential oils of three medicinally important Curcuma species (Curcuma alismatifolia, Curcuma aromatica and Curcuma xanthorrhiza) were extracted using conventional hydro-distillation (HD) and solvent free microwave extraction (SFME) methods. The volatile compounds from the rhizome essential oils were subsequently analysed by GC-MS. The isolation of essential oils of each species was carried out following the six principles of green extraction and comparison was made between their chemical composition, antioxidant, anti-tyrosinase and anticancer activities. SFME was found to be more efficient than HD in terms of energy savings, extraction time, oil yield, water consumption and waste production. Though the major compounds of essential oils of both the species were qualitatively similar, there was a significant difference in terms of quantity. The essential oils extracted through HD and SFME methods were dominated by hydrocarbon and oxygenated compounds, respectively. The essential oils of all Curcuma species exhibited strong antioxidant activity, where SFME was significantly better than HD with lower IC50 values. The anti-tyrosinase and anticancer properties of SFME-extracted oils were relatively better than that of HD. Further, among the three Curcuma species, C. alismatifolia essential oil showed the highest rates of inhibition in DPPH and ABTS assay, significantly reduced the tyrosinase activity and exhibited significant selective cytotoxicity against MCF7 and PC3 cells. The current results suggested that the SFME method, being advanced, green and fast, could be a better alternative for production of essential oils with better antioxidant, anti-tyrosinase and anticancer activities for application in food, health and cosmetic industries.

Deciphering the antibiofilm potential of 2-Phenylethyl methyl ether (PEME), a bioactive compound of Kewda essential oil against Staphylococcus aureus.

Opportunistic pathogenic bacteria and their pathogenicity linked with biofilm infections become a severe issue as they resist the actions of multiple antimicrobial drugs. Naturally derived drugs having antibiofilm properties are more effective than chemically synthesized drugs. The plant derived essential oils are a rich source of phytoconstituents with widespread pharmacological values. In the present investigation, a major phytoconstituent, 2-Phenyl Ethyl Methyl Ether (PEME) of Kewda essential oil extracted from the flowers of Pandanus odorifer was explored for its prospective antimicrobial and anti-biofilm properties against ESKAPE pathogenic bacterial strains, Staphylococcus aureus and MTCC 740. The minimum inhibitory concentration (MIC) of PEME was found to be 50 mM against the tested bacterial strains. A gradual decrease in biofilm production was observed when PEME was treated with the sub-MIC concentration. The reduction in biofilm formation was noticeable from qualitative assay i.e., Congo Red Agar Assay (CRA) and further quantified by crystal violet staining assay. The decline in exopolysaccharides production was quantified, with the highest inhibition against MTCC 740 with a decrease of 71.76 ± 4.56% compared to untreated control. From the microscopic analysis (light and fluorescence microscopic method), PEME exhibited inhibitory effect on biofilm formation on the polystyrene surface. The in silico studies stated that PEME could invariably bind to biofilm associated target proteins. Further, transcriptomic data analysis suggested the role of PEME in the down-regulation of specific genes, agrA, sarA, norA and mepR, which are critically associated with bacterial virulence, biofilm dynamics and drug resistance patterns in S. aureus. Further, qRT-PCR analysis validated the role of PEME on biofilm inhibition by relative downregulation of agrA, sarA, norA and mepR genes. Further, advanced in silico methodologies could be employed in future investigations to validate its candidature as promising anti-biofilm agent.

Antiproliferative Noscapinoids Bearing an Amidothiadiazole Scaffold as Apoptosis Inducers: Design, Synthesis and Molecular Docking.

Noscapine an FDA-approved antitussive agent. With low cytotoxicity with higher concentrations, noscapine and its derivatives have been shown to have exceptional anticancer properties against a variety of cancer cell lines. In order to increase its potency, in this study, we synthesized a series of new amido-thiadiazol coupled noscapinoids and tested their cytotoxicity in vitro. All of the newly synthesised compounds demonstrated potent cytotoxic potential, with IC50 values ranging from 2.1 to 61.2 µM than the lead molecule, noscapine (IC50 value ranges from 31 to 65.5 µM) across all cell lines, without affecting normal cells (IC50 value is>300 µM). Molecular docking of all these molecules with tubulin (PDB ID: 6Y6D, resolution 2.20 Å) also revealed better binding affinity (docking score range from -5.418 to -9.679 kcal/mol) compared to noscapine (docking score is -5.304 kcal/mol). One of the most promising synthetic derivatives 6aa (IC50 value ranges from 2.5 to 7.3 µM) was found to bind tubulin with the highest binding affinity (ΔGbinding is -28.97 kcal/mol) and induced apoptosis in cancer cells more effectively.

Development of 9-(N-arylmethylamino) congeners of noscapine: the microtubule targeting drugs for the management of breast cancer.

Noscapine is a natural lead molecule with anticancer activity at a higher concentrations. So, there is an urge for the development of more potent derivatives of noscapine. In this study, we have approached for development of 9-N-arylmethylamino derivatives of noscapine that kills cancer cells without affecting the normal cells. They were designed by substituting N-aryl methyl pharmacophore at the C-9 position and screened out top-ranked three derivatives 13a-c using molecular docking. Further, their theoretical free energy of binding with tubulin was calculated followed by chemical synthesis and experimental validation. In vitro antiproliferative activity of noscapine and its 9-N-arylmethylamino derivatives (13a-c) was carried out using MCF-7 (a triple receptors positive) and MDA-MB-231 (a triple receptor negative) breast cancer cell lines. Further, cytotoxicity to normal cells was examined using human embryonic kidney cells (HEK cells). Inhibition to cell cycle progression and induction of apoptosis was monitored using FACS. The binding of noscapine and 13a-c with tubulin was examined using fluorescence quenching assay. The 9-N-arylmethylamino derivatives of noscapine (13a-c) were found to inhibit the proliferation of cancer cells at a much lower concentration (IC50 values range between 9.1 to 47.3 µM) compared to noscapine (IC50 value is 45.8-59.3 µM). Surprisingly, the proliferation of HEK cells was not inhibited even at a concentration of 100 µM (cytotoxicity is < 5%). These derivatives induced apoptosis by arresting cells at G2/M-phase and also bind to tubulin. The 9-(N-arylmethylamino) noscapinoids have the potential to be a novel therapeutic agent for the treatment of breast cancer. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03445-3.

Identification of potent EGFR-TKD inhibitors from NPACT database through combined computational approaches.

Cancer is the world's second leading cause of death, and there are no approved herbal therapies. The epidermal growth factor receptor tyrosine kinase (EGFR-TK) receptor is a transmembrane protein with eight domains that is found in almost every cancer type and plays an important role in abnormal cell cellular function and causes malignant outcomes. The current study aimed to virtually screen phytochemicals from the NPACT database against EGFR-TKD and also to identify potential inhibitors of this transmembrane protein among plant candidates for anticancer drug development. The docking scores of the chosen phytochemicals were compared with the control (erlotinib). Kurarinone, (2S)-2-methoxykurarnione, and Sophoraflavanone-G exhibited a stronger binding affinity of -18.102 kcal/mol, -14.243 kcal/mol, and -13.759 kcal/mol than erlotinib -12.783 kcal/mol. Moreover, several online search engines were used to predict ADME and toxicity. The drug-likeness of selected phytochemicals was higher than the reference (erlotinib). A 100 ns molecular dynamic (MD) simulation was also applied to the docked conformations to examine the stability and molecular mechanics of protein-ligand interactions. Furthermore, the calculated molecular mechanics Poisson Boltzmann surface area energy of (2S)-2-methoxykurarnione was found to be -129.555 ± 0.512 kJ/mol, which approximately corresponds to the free energy of the reference molecule -130.595 ± 0.908 kJ/mol. We identify phytoconstituents present in Sophora flavescens from the NPACT database, providing key insights into tyrosine kinase inhibition and may serve as better chemotherapeutic agents. Experimental validation is required to determine the anti-EGFR potency of the potent lead molecules discussed in this study.Communicated by Ramaswamy H. Sarma.

Substrate-directed Synthesis of Isocoumarin and 3-Ylidenephthalide Conjugated Noscapinoids and their Antiproliferative Activities.

A series of new noscapinoids designed; synthesized and assessed whether its 3-ylidenephthalide and isocoumarin conjugates improved cytotoxicity. Cu-catalysed Sonogashira coupling of N-propargyl noscapine with 2-bromobenzoic acids followed by in-situ substrate-directed 5-exo-dig or 6-endo-dig cyclization produced 3-ylidenephthalide 6 a-6 f and isocoumarin 7 a-7 h analogues in very good yields. In comparison to the lead drug, noscapine, all the newly synthesised derivatives exhibited strong cytotoxic potential in vitro with IC50 ranging from 5.4 µM to 39.5 µM across the evaluated panel of cancer cell lines, without harming normal cells (IC50 >300 µM).

Folate receptor-targeted PLGA-PEG nanoparticles for enhancing the activity of genistein in ovarian cancer.

Genistein (GEN), a natural isoflavone possesses a wide range of pharmacological properties and nutraceutical applications. GEN has been studied for its anticancer activity against different types of cancers, but its use in clinical practice is limited due to its low water solubility, rapid metabolism and excretion, lack of cancer cell targeting and poor bioavailability. In the present study, we investigated folate receptor-targeted and PEGylated poly(lactide-co-glycolide) nanoparticles (PLGA-PEG-FA NPs) containing GEN for targeted delivery to ovarian cancer cells. PLGA-PEG and PLGA-PEG-FA polymer conjugates were synthesized and characterized. Nano-precipitation method was employed for the fabrication of NPs of PLGA, PLGA-PEG and PLGA-PEG-FA containing GEN. GEN containing PLGA-PEG and PLGA-PEG-FA NPs prepared were small (104.17 ± 1.61 and 125.41 ± 3.11 nm, respectively) and exhibited sustained release of GEN for around six days. Folate-decorated PLGA-PEG NPs showed increased cellular uptake in comparison to non-targeted PLGA-PEG NPs. The GEN containing PLGA-PEG-FA NPs showed superior anticancer activity than non-targeted PLGA and PLGA-PEG NPs in folate receptor-overexpressing ovarian cancer cell line, SKOV-3. The IC50 of GEN, GEN encapsulated NPs of PLGA, PLGA-PEG and PLGA-PEG-FA were 51.48, 26.70, 23.43 and 11.98 µg/ml, respectively. Folate-targeted PLGA nanoparticles could be developed for potential target-specific delivery of GEN in the treatment of ovarian cancer.

Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs.

BACKGROUND: Kinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood. RESULTS: We purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family. CONCLUSIONS: Together, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

In vitro characterization and molecular dynamic simulation of shikonin as a tubulin-targeted anticancer agent.

Shikonin (SK), a naphthoquinone compound from the purple gromwell, Lithospermum erythrorhizon, possesses a considerable antiproliferative potential. By using a combination of biophysical techniques, cellular assays, immunofluorescence imaging, and molecular dynamic simulation, we identified a possible mechanism of action of SK. SK inhibited the viability of the triple negative breast cancer cells MDA-MB-231 (IC50 of 1 ± 0.1 µM), and its inhibitory effect was irreversible. It strongly suppressed the clonogenic and migratory potential of the cells. Although SK did not show any phase-specific inhibition of cell cycle progression, it induced apoptosis as confirmed by annexin-V-based flow cytometry and Western immunoblotting of PARP1. Probing further into its mechanism using a tryptophan-quenching assay, it was found that SK binds the microtubule-building protein tubulin with a dissociation constant (Kd) of 8 ± 2.7 µM, without grossly damaging the tertiary structure of the protein. The drug-bound tubulin could not assemble microtubules properly in vitro as confirmed by polymer mass analysis, turbidimetry analysis, and transmission electron microscopy, and in cells, as visualized by immunofluorescence imaging. In cells, SK also suppressed the dynamicity of microtubules as indicated by considerable acetylation of the cellular microtubules. The fine details of tubulin-SK interactions were then elucidated using molecular docking and molecular dynamic simulation. The free energy change of the interaction (ΔGbind,pred) was found to be -14.60 kcal/mol and the binding involved both the intermolecular van der Waals (ΔEvdw) and the electrostatic (ΔEele) interactions. Taken together, our data provide evidence for a possible mechanism of action of SK as a tubulin-targeted anticancer agent.

Biology and biotechnological aspect of sweet potato (Ipomoea batatas L.): a commercially important tuber crop.

MAIN CONCLUSION: This review highlights the economic importance of sweet potato and discusses new varieties, agronomic and cultivation practices, pest and disease control efforts, plant tissue culture protocols, and unexplored research areas involving this plant. Abstract Sweet potato is widely consumed in many countries around the world, including India, South Africa and China. Due to its valuable nutritional composition and highly beneficial bioactive compounds, sweet potato is considered a major tuber crop in India. Based on the volume of production, this plant ranks seventh in the world among all food crops. Sweet potato is considered a "Superfood" by the 'Centre for Science in the Public Interest' (CSPI), USA. This plant is mostly propagated through vegetative propagation using vine cuttings or tubers. However, this process is costly, labour-intensive, and comparatively slow. Conventional propagation methods are not able to supply sufficient disease-free planting materials to farmers to sustain steady tuber production. Therefore, there is an urgent need to use various biotechnological approaches, such as cell, tissue, and organ culture, for the large-scale production of healthy and disease-free planting material for commercial purposes throughout the year. In the last five decades, a number of tissue culture protocols have been developed for the production of in vitro plants through meristem culture, direct adventitious organogenesis, callus culture and somatic embryogenesis. Moreover, little research has been done on synthetic seed technology for the in vitro conservation and propagation of sweet potato. The current review comprehensively describes the biology, i.e., plant phenotypic description, vegetative growth, agronomy and cultivation, pests and diseases, varieties, and conventional methods of propagation, as well as biotechnological implementation, of this tuber crop. Furthermore, the explored and unexplored areas of research in sweet potato using biotechnological approaches have been reviewed.

In silico design of novel tubulin binding 9-arylimino derivatives of noscapine, their chemical synthesis and cellular activity as potent anticancer agents against breast cancer.

We present a series of 9-arylimino derivatives of noscapine (an antitussive plant alkaloid) that binds to tubulin and displaying anticancer activity against a panel of breast cancer cells. These compounds were rationally designed by coupling of Schiff base containing imine groups at position-9 of the isoquinoline ring of noscapine. Based on a combination of Glide docking and free energy of binding (FEB) calculation, we have screened a panel of three 9-compounds, 12-14 with improved binding affinity with tubulin compared to noscapine. The predicted FEB is -6.166 kcal/mol for 12, -6.411 kcal/mol for 13 and -7.512 kcal/mol for 14. In contrast, the predicted FRB of noscapine is -5.135 kcal/mol. These novel derivatives were strategically synthesized and validated their anticancer activity based on cellular studies using two human breast adenocarcinoma, MCF-7 and MDAMB-231, as well as with a panel of primary breast tumor cells isolated from patients. Interestingly, all these derivatives inhibited cellular proliferation in all the cancer cells that ranged between 3.6 and 26.4 µM, which is 11.02-2.03 fold lower than that of noscapine. Unlike previously reported derivatives of noscapine that arrest cells in the S-phase, these novel derivatives effectively inhibit proliferation of cancer cells, arrest the cell cycle in the G2/M-phase and induced apoptosis. Thus, we conclude that 9-arylimino derivatives of noscapine have great potential to be a novel therapeutic agent for breast cancers.Communicated by Ramaswamy H. Sarma.

Development of 1,3-diynyl derivatives of noscapine as potent tubulin binding anticancer agents for the management of breast cancer.

We developed 1,3-diynyl derivatives of noscapine (an opium alkaloid) through in silico combinatorial approach and screened out a panel of promising derivatives that bind tubulin and display anticancer activity. The selected derivatives such as 9-4-tBu-Ph-Diyne (20p), 9-3,4-Di-Cl-Diyne (20k) and 9-3,4-Di-F-Diyne (22s) noscapinoids revealed improved predicted binding energy of -6.676 kcal/mol for 20p, -7.294 kcal/mol for 20k and -7.750 kcal/mol for 20s respectively in comparison to noscapine (-5.246 kcal/mol). These 1,3-diynyl derivatives (20p, 29k and 20s) were strategically synthesized in high yields by regioselective modification of noscapine scaffold and HPLC purified (purity is >96%). The decrease in intrinsic fluorescence of purified tubulin to 8.39%, 17.39% and 25.47% by 20p, 20k and 20s respectively, compared to control suggests their binding capability to tubulin. Their cytotoxicity activity was validated based on cellular studies using two human breast adenocarcinoma (MCF-7 and MDA-MB-231), a panel of primary breast tumor cells and one normal human embryonic kidney cell (293 T). The 1,3-diynyl noscapinoids, 20p, 20k and 20s inhibited cellular proliferation in all the cancer cells that ranged between 6.2 and 38.9 µM, without affecting the normal healthy cells (cytotoxicity is <5% at 100 µM). Further, these novel derivatives arrest cell cycle in the G2/M-phase, followed by induction of apoptosis to cancer cells. Thus, we conclude that 1,3-diynyl-noscapinoids have great potential to be a novel therapeutic agent for breast cancers.Communicated by Ramaswamy H. Sarma.