Ethyl palmitate, an anti-chikungunya virus principle from Sauropus androgynus, a medicinal plant used to alleviate fever in ethnomedicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Sauropus androgynus is a medicinal shrub used for the treatment of fever in ethnomedical traditions in various Southeast Asian countries. AIM OF THE STUDY: This study was aimed to identify antiviral principles from S. androgynus against Chikungunya virus (CHIKV), a major mosquito-borne pathogen that re-emerged in the last decade, and to unravel their mechanism of action. MATERIALS AND METHODS: Hydroalcoholic extract of S. androgynus leaves was screened for anti-CHIKV activity using cytopathic effect (CPE) reduction assay. The extract was subjected to activity guided isolation and the resultant pure molecule was characterized by GC-MS, Co-GC and Co-HPTLC. The isolated molecule was further evaluated for its effect by plaque reduction assay, Western blot and immunofluorescence assays. In silico docking with CHIKV envelope proteins and molecular dynamics simulation (MD) analyses were used to elucidate its possible mechanism of action. RESULTS: S. androgynus hydroalcoholic extract showed promising anti-CHIKV activity and its active component, obtained by activity guided isolation, was identified as ethyl palmitate (EP), a fatty acid ester. At 1 µg/mL, EP led to 100% inhibition of CPE and a significant 3 log10 reduction in CHIKV replication in Vero cells at 48 h post-infection. EP was highly potent with an EC50 of 0.0019 µg/mL (0.0068 µM) and a very high selectivity index. EP treatment significantly reduced viral protein expression, and time of addition studies revealed that it acts at the stage of viral entry. A strong binding to the viral envelope protein E1 homotrimer during entry, thus preventing viral fusion, was identified as a possible mechanism by which EP imparts its antiviral effect. CONCLUSIONS: S. androgynus contains EP as a potent antiviral principle against CHIKV. This justifies the use of the plant against febrile infections, possibly caused by viruses, in various ethnomedical systems. Our results also prompt more studies on fatty acids and their derivatives against viral diseases.

Identification of two ISG15 homologues involved in host immune response against RGNNV in Asian seabass (Lates calcarifer).

Interferon Stimulated Gene (ISG)15 is a ubiquitin-like protein that is induced upon viral infections. Our study reports the identification of two homologues of ISG15 in the Asian seabass designated LcISG15A and LcISG15B. The cloned LcISG15A cDNA fragment contained a 474 bp ORF encoding a 157 amino acid protein whereas LcISG15B featured a 498 bp ORF encoding a slightly longer protein of 165 amino acids. Both proteins featured the two tandem ubiquitin-like domains and the C-terminal LRGG motif characteristic of ISG15. The LcISG15B protein has a 10-amino acid C-terminal extension after the LRGG motif. Molecular docking studies revealed that LcISG15A showed more conformational variability of the ubiquitin domains and catalytic function than LcISG15B. The Lates ISG15A and ISG15B genes, reside close in the genome, share the same basic structure with two exons and an intron, but only the second exon encoding the protein. These genes also featured the IFN-stimulatory response elements (ISRE) in the promoter region and ATTTA instability motif in the 3' UTR region. Leukocyte-rich organs such as the head kidney, heart, spleen, and gill showed higher levels of ISG15A and ISG15B basal expression. Poly (I:C) injection rapidly upregulated the transcription of both the ISG15 genes in these tissues in Lates. In-vivo viral infection by red-spotted grouper nervous necrosis virus also induced upregulation of ISG15 genes in the head kidney, spleen, heart and gill. These findings indicate that the two ISG15 homologues may play a crucial role in innate antiviral immunity and could be used to improve prophylactic strategies and develop species-specific immunological tools for Lates calcarifer.

Comparative proteomic analysis of saline tolerant, phosphate solubilizing endophytic Pantoea sp., and Pseudomonas sp. isolated from Eichhornia rhizosphere.

Soil salinization is a major stress affecting crop production on a global scale. Application of stress tolerant plant growth promoting rhizobacteria (PGPR) in saline soil can be an ideal practice for improving soil fertility. Rhizospheric microbiota of stress tolerant Eichhornia crassipes was screened for saline tolerant phosphate solubilizing bacteria, and the two isolates showing maximum solubilization index at 1 M NaCl were subjected to further analyses. The isolates were identified as Pantoea dispersa and Pseudomonas aeruginosa. Among the two isolates, P. dispersa PSB1 showed better phosphorus (P) solubilization potential under saline stress (335 ± 30 mg/L) than P. aeruginosa PSB5 (200 ± 24 mg/L). The mechanisms of P-solubilization, such as the production of organic acids and phosphatase were found to be influenced negatively by saline stress. The adaptive mechanisms of the isolates to overcome salt stress were analyzed by protein profiling which revealed salt stress induced modulations in protein expression involved in amino acid biosynthesis, carbon metabolisms, chemotaxis, and stress responses. Survival mechanisms such as protein RecA, LexA repressor and iron-sulfur cluster synthesis were upregulated in both the organisms under saline stress. P. dispersa PSB1 showed improved defense mechanisms such as the production of osmotolerants, redox enzymes, and quorum quenchers under saline stress, which may explain its better P solubilization potential than the P. aeruginosa PSB5. This study emphasizes the need for molecular approaches like proteome analysis of PGPR for identifying novel traits like stress tolerance and plant growth promotion before developing them as biofertilizers and biocontrol formulations.

Supramolecular Hydrogel Based Post-Surgical Implant System for Hydrophobic Drug Delivery Against Glioma Recurrence.

Purpose: The brain, protected by the cranium externally and the blood-brain barrier (BBB) internally, poses challenges in chemotherapy of aggressive brain tumors. Maximal tumor resection followed by radiation and chemotherapy is the standard treatment protocol; however, a substantial number of patients suffer from recurrence. Systemic circulation of drugs causes myelodysplasia and other side effects. To address these caveats, we report facile synthesis of a polyester-based supramolecular hydrogel as a brain biocompatible implant for in situ delivery of hydrophobic drugs. Methods: Polycaprolactone-diol (PCL) was linked to polyethyleneglycol-diacid (PEG) via an ester bond. In silico modeling indicated micelle-based aggregation of PCL-PEG co-polymer to form a supramolecular hydrogel. Brain biocompatibility was checked in Sprague Dawley rat brain cortex with MRI, motor function test, and histology. Model hydrophobic drugs carmustine and curcumin entrapment propelled glioma cells into apoptosis-based death evaluated by in vitro cytotoxicity assays and Western blot. In vivo post-surgical xenograft glioma model was developed in NOD-SCID mice and evaluated for efficacy to restrict aggressive regrowth of tumors. Results: 20% (w/v) PCL-PEG forms a soft hydrogel that can cover the uneven and large surface area of a tumor resection cavity and maintain brain density. The PCL-PEG hydrogel was biocompatible, and well-tolerated upon implantation in rat brain cortex, for a study period of 12 weeks. We report for the first time the combination of carmustine and curcumin entrapped as model hydrophobic drugs, increasing their bioavailability and yielding synergistic apoptotic effect on glioma cells. Further in vivo study indicated PCL-PEG hydrogel with a dual cargo of carmustine and curcumin restricted aggressive regrowth post-resection significantly compared with control and animals with intravenous drug treatment. Conclusion: PCL-PEG soft gel-based implant is malleable compared with rigid wafers used as implants, thus providing larger surface area contact. This stable, biocompatible, supramolecular gel without external crosslinking can find wide applications by interchanging formulation of various hydrophobic drugs to ensure and increase site-specific delivery, avoiding systemic circulation.

Acetylation of Isoniazid Is a Novel Mechanism of Isoniazid Resistance in Mycobacterium tuberculosis.

Isoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a prodrug which is activated by the intracellular KatG enzyme of Mycobacterium tuberculosis The activated drug hinders cell wall biosynthesis by inhibiting the InhA protein. INH-resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH-resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study, we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze the transfer of the acetyl group to INH from acetyl coenzyme A (acetyl-CoA). High-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. A drug susceptibility assay and confocal analysis showed that Mycobacterium smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Mutant proteins of Rv2170 failed to acetylate INH. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at MICs that inhibited wild-type strains. Besides, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity, although this needs to be tested in INH-resistant clinical strains.

DNA Binding and Molecular Dynamic Studies of Polycyclic Tetramate Macrolactams (PTM) with Potential Anticancer Activity Isolated from a Sponge-Associated Streptomyces zhaozhouensis subsp. mycale subsp. nov.

A sponge-associated actinomycete (strain MCCB267) was isolated from a marine sponge Mycale sp. collected in the Indian Ocean off the Southeast coast of India. Phylogenetic studies of this strain using 16S rRNA gene sequencing showed high sequence similarity to Streptomyces zhaozhouensis. However, strain MCCB267 showed distinct physiological and biochemical characteristic features and was thus designated as S. zhaozhouensis subsp. mycale. subsp. nov. A cytotoxicity-guided fractionation of the crude ethyl acetate extract of strain MCCB267 culture medium yielded four pure compounds belonging to the polycyclic tetramate macrolactam (PTM) family of natural products: ikarugamycin (IK) (1), clifednamide A (CF) (2), 30-oxo-28-N-methylikarugamycin (OI) (3), and 28-N-methylikarugamycin (MI) (4). The four compounds exhibited promising cytotoxic activity against NCI-H460 lung carcinoma cells in vitro, by inducing cell death via apoptosis. Flow cytometric analysis revealed that 1, 3, and 4 induced cell cycle arrest during G1 phase in the NCI-H460 cell line, whereas 2 induced cell arrest in the S phase. A concentration-dependent accumulation of cells in the sub-G1 phase was also detected upon treatment of the cancer cell line with compounds 1-4. The in vitro cytotoxicity studies were supported by molecular docking and molecular dynamic simulation analyses. An in silico study revealed that the PTMs can bind to the minor groove of DNA and subsequently induce the apoptotic stimuli leading to cell death. The characterization of the isolated actinomycete, the study of the mode of action of the four PTMs, 1-4, and the molecular docking and molecular dynamic simulations analyses are herein described.

In silico approaches illustrate the evolutionary pattern and protein-small molecule interactions of quinolone synthase from Aegle marmelos Correa.

Quinolone synthase from Aegle marmelos (AmQNS) is a Rutacean-specific plant type III polyketide synthase that synthesizes quinolone, acridone, and benzalacetone with therapeutic potential. Simple architecture and broad substrate affinity of AmQNS make it as one of the target enzymes to produce novel structural scaffolds. Another unique feature of AmQNS despite its high similarity to acridone forming type III polyketide synthase from Citrus microcarpa is the variation in the product formation. Hence, to explore the characteristic features of AmQNS, an in-depth sequence and structure-based bioinformatics analyses were performed. Our studies indicated that AmQNS and its nearest homologs have evolved by a series of gene duplication events and strong purifying selection pressure constrains them in the evolutionary process. Additionally, some amino acid alterations were identified in the functionally important region(s), which can contribute to the functional divergence of the enzyme. Prediction of favorable amino acid substitutions will be advantageous in the metabolic engineering of AmQNS for the production of novel compounds. Furthermore, comparative modeling and docking studies were utilized to investigate the structural behavior and small molecule interaction pattern of AmQNS. The observations and results reported here are crucial for advancing our understanding of AmQNS's phylogenetic position, selection pressure, evolvability, interaction pattern and thus providing the foundation for further studies on the structural and reaction mechanism.

Molecular simulation and in vitro evaluation of chitosan nanoparticles as drug delivery systems for the controlled release of anticancer drug cytarabine against solid tumours.

The present work is an attempt to integrate the molecular simulation studies with in vitro cytotoxicity of cytarabine-loaded chitosan nanoparticles and exploring the potential of this formulation as therapeutics for treating solid tumours. The molecular simulation was performed using GROMACS v5.4 in which, chitosan polymer (CHT; six molecules) was used to study the encapsulation and release of a single molecule of cytarabine. Root Mean Square Deviation (RMSD) of the Cα atom of cytarabine (CBR) molecule shows that CBR starts to diffuse out of the CHT polymer binding pocket around 10.2 ns, indicated by increased fluctuation of RMSD at pH 6.4, while the drug diffusion is delayed at pH 7.4 and starts diffusing around 17.5 ns. Cytarabine-loaded chitosan nanoparticles (CCNP), prepared by ionic gelation method were characterized for encapsulation efficiency, particle size and morphology, zeta potential, crystallinity and drug release profile at pH 6.4 and 7.4. CCNPs showed 64% encapsulation efficiency with an average diameter of 100 nm and zeta potential of + 53.9 mV. It was found that cytarabine existed in amorphous state in nanoformulation. In vitro release studies showed 70% cytarabine was released from the chitosan-based nanoformulation release at pH 6.4, which coincides with the pH of tumour microenvironment. Cytotoxicity against breast cancer cell line (MCF 7) was higher for nanoformulation compared to free cytarabine. Haemocompatibility studies showed that chitosan-based nanoformulation is safe, biocompatible and nonhaemolytic in nature; hence, can be used as a safe drug delivery system. Taken together, our study suggests that chitosan nanoformulation would be an effective strategy for the pH-dependent release of cytarabine against solid tumours and might impart better therapeutic efficiency.

Bacoside A Induces Tumor Cell Death in Human Glioblastoma Cell Lines through Catastrophic Macropinocytosis.

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumor with an extremely poor prognosis. Recent evidences have shown that the "biomechanical imbalances" induced in GBM patient-derived glioblastoma cells (GC) and in vivo via the administration of synthetic small molecules, may effectively inhibit disease progression and prolong survival of GBM animal models. This novel concept associated with de novo anti-GBM drug development has however suffered obstacles in adequate clinical utility due to the appearance of unrelated toxicity in the prolonged therapeutic windows. Here, we took a "drug repurposing approach" to trigger similar physico-chemical disturbances in the GBM tumor cells, wherein, the candidate therapeutic agent has been previously well established for its neuro-protective roles, safety, efficacy, prolonged tolerance and excellent brain bioavailability in human subjects and mouse models. In this study, we show that the extracts of an Indian traditional medicinal plant Bacopa monnieri (BM) and its bioactive component Bacoside A can generate dosage associated tumor specific disturbances in the hydrostatic pressure balance of the cell via a mechanism involving excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/CaMK2A) enzyme that is further involved in the release of calcium from the smooth endoplasmic reticular networks. High intracellular calcium stimulated massive macropinocytotic extracellular fluid intake causing cell hypertrophy in the initial stages, excessive macropinosome enlargement and fluid accumulation associated organellar congestion, cell swelling, cell rounding and membrane rupture of glioblastoma cells; with all these events culminating into a non-apoptotic, physical non-homeostasis associated glioblastoma tumor cell death. These results identify glioblastoma tumor cells to be a specific target of the tested herbal medicine and therefore can be exploited as a safe anti-GBM therapeutic.

Extracellular Proton Concentrations Impacts LN229 Glioblastoma Tumor Cell Fate via Differential Modulation of Surface Lipids.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer with marginal survival rates. GBM extracellular acidosis can profoundly impact its cell fate heterogeneities and progression. However, the molecules and mechanisms that enable GBM tumor cells acid adaptation and consequent cell fate competencies are weakly understood. Since extracellular proton concentrations (pHe) directly intercept the tumor cell plasma membrane, surface lipids must play a crucial role in pHe-dependent tumor cell fate dynamics. Hence, a more detailed insight into the finely tuned pH-dependent modulation of surface lipids is required to generate strategies that can inhibit or surpass tumor cell acid adaptation, thereby forcing the eradication of heterogeneous oncogenic niches, without affecting the normal cells. RESULTS: By using image-based single cell analysis and physicochemical techniques, we made a small-scale survey of the effects of pH ranges (physiological: pHe 7.4, low: 6.2, and very low: 3.4) on LN229 glioblastoma cell line surface remodeling and analyzed the consequent cell fate heterogeneities with relevant molecular targets and behavioral assays. Through this basic study, we uncovered that the extracellular proton concentration (1) modulates surface cholesterol-driven cell fate dynamics and (2) induces 'differential clustering' of surface resident GM3 glycosphingolipid which together coordinates the proliferation, migration, survival, and death reprogramming via distinct effects on the tumor cell biomechanical homeostasis. A novel synergy of anti-GM3 antibody and cyclophilin A inhibitor was found to mimic the very low pHe-mediated GM3 supraclustered conformation that elevated the surface rigidity and mechano-remodeled the tumor cell into a differentiated phenotype which eventually succumbed to the anoikis type of cell death, thereby eradicating the tumorigenic niches. CONCLUSION AND SIGNIFICANCE: This work presents an initial insight into the physicochemical capacities of extracellular protons in the generation of glioblastoma tumor cell heterogeneities and cell death via the crucial interplay of surface lipids and their conformational changes. Hence, monitoring of proton-cholesterol-GM3 correlations in vivo through diagnostic imaging and in vitro in clinical samples may assist better tumor staging and prognosis. The emerged insights have further led to the translation of a 'pH-dependent mechanisms of oncogenesis control' into the surface targeted anti-GBM therapeutics.

An additional record of Fejervarya manoharani Garg and Biju from the Western Ghats with a description of its complete mitochondrial genome.

An additional sighting of newly described frog species, Fejervarya manoharani Garg and Biju, outside of the type locality along with their morphological data is reported herewith. We are also providing the whole DNA sequence of the mitochondrial genome with its gene organization as additional data to distinguish this species from its congeners. The mitogenome of F. manoharani was 17,654 bp in length. It contains 38 genes including two rRNAs, 23 tRNAs, 13 protein-coding genes and a control region. Similar to other dicroglossid frogs, a tandem duplication of tRNAMet was found. The ND5 gene was located at the 3' end of the control region like in three other Fejervarya species for which mitogenomic data are available. A rearrangement of four tRNA genes, namely Leucine, Threonine, Proline, and Phenylalanine, between ND5 and 12S rRNA, differing from other Fejervarya species, was also observed.

Marine derived compounds as binders of the White spot syndrome virus VP28 envelope protein: In silico insights from molecular dynamics and binding free energy calculations.

White spot syndrome virus (WSSV) remains as one of the most dreadful pathogen of the shrimp aquaculture industry owing to its high virulence. The cumulative mortality reaches up to 100% within in 2-10days in a shrimp farm. Currently, no chemotherapeutics are available to control WSSV. The viral envelope protein, VP28, located on the surface of the virus particle acts as a vital virulence factor in the initial phases of inherent WSSV infection in shrimp. Hence, inhibition of envelope protein VP28 could be a novel way to deal with infection by inhibiting its interaction in the endocytic pathway. In this direction, a timely attempt was made to recognize a potential drug candidate of marine origin against WSSV using VP28 as a target by employing in silico docking and molecular dynamic simulations. A virtual library of 388 marine bioactive compounds was extracted from reports published in Marine Drugs. The top ranking compounds from docking studies were chosen from the flexible docking based on the binding affinities (ΔGb). In addition, the MD simulation and binding free energy analysis were implemented to validate and capture intermolecular interactions. The results suggested that the two compounds obtained a negative binding free energy with -40.453kJ/mol and -31.031kJ/mol for compounds with IDs 30797199 and 144162 respectively. The RMSD curve indicated that 30797199 moves into the hydrophobic core, while the position of 144162 atoms changes abruptly during simulation and is mostly stabilized by water bridges. The shift in RMSD values of VP28 corresponding to ligand RMSD gives an insight into the ligand induced conformational changes in the protein. This study is first of its kind to elucidate the explicit binding of chemical inhibitor to WSSV major structural protein VP28.

Moult-inhibiting fusion protein augments while polyclonal antisera attenuate moult stages and duration in Penaeus monodon.

Moulting in crustaceans is regulated by moult-inhibiting hormone (MIH) of the CHH family neuropeptides. The inhibitory functions of MIH have pivotal roles in growth and reproduction of Penaeus monodon. In this study, we report the expression of a thioredoxin-fused mature MIH I protein (mf-PmMIH I) of P. monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmMIH I). The mature MIH I gene of 231bp, that codes for 77 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The translation expression vector construct (mf-PmMIH I+pET32a+) upon induction produced 29.85kDa mature MIH I fusion protein (mf-PmMIH I). The purified fusion protein was used as exogenous MIH I and as antigen to raise polyclonal antisera. When fusion protein (mf-PmMIH I) was injected into D2 and D3 stages of juvenile shrimp, the moult cycle duration was extended significantly to 16.67±1.03 and 14.67±1.03days respectively compared to that of 11.67±1.03days in controls. Moult duration was further reduced to 8.33±0.82days when polyclonal antiserum (anti-mf-PmMIH I - 1:500 dilutions) was injected. Anti-mf-PmMIH I immunolocalized MIH I producing neurosecretory cells in the eyestalk of P. monodon. In short, the present manuscript reports an innovative means of moult regulation in P. monodon with thioredoxin fused MIH I and antisera developed.

Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor.

Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence ZoCDPK1 is operating through NAC TF mediated ABA-independent, cold non-responsive stress signaling pathway in ginger.

Resveratrol--a potential inhibitor of biofilm formation in Vibrio cholerae.

Resveratrol, a phytochemical commonly found in the skin of grapes and berries, was tested for its biofilm inhibitory activity against Vibrio cholerae. Biofilm inhibition was assessed using crystal violet assay. MTT assay was performed to check the viability of the treated bacterial cells and the biofilm architecture was analysed using confocal laser scanning microscopy. The possible target of the compound was determined by docking analysis. Results showed that subinhibitory concentrations of the compound could significantly inhibit biofilm formation in V. cholerae in a concentration-dependent manner. AphB was found to be the putative target of resveratrol using docking analysis. The results generated in this study proved that resveratrol is a potent biofilm inhibitor of V. cholerae and can be used as a novel therapeutic agent against cholera. To our knowledge, this is the first report of resveratrol showing antibiofilm activity against V. cholerae.

Dry powder cationic lipopolymeric nanomicelle inhalation for targeted delivery of antitubercular drug to alveolar macrophage.

Excipients having self-assembling properties are less explored in the field of dry powder inhalation (DPI) technology. An amphiphilic lipopolymer system was developed using stearic acid (SA) and branched polyethyleneimine (BPEI) (1800 Dalton), at different proportions by covalent conjugation. A molecular dynamic (MD) simulation tool was employed for predicting the carrier behavior in a polar in vivo condition. The structural characterization was carried out using nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared (FTIR) spectroscopy. The physical nature of the lipopolymer was analyzed by differential scanning calorimetry. Determination of zeta potential and diameter of the micelles showed existence of cationic particles in the nano size range when a lower number of primary amino groups of BPEI was grafted with SA. The rifampicin (RIF)-loaded lipopolymer was also formulated further into spray-dried microparticles. Powder X-ray diffraction (PXRD) studies revealed that the RIF API (active pharmaceutical ingredient) exists as molecular dispersion in spray-dried microparticles. Topological analysis of the spray-dried nanomicelle was carried out using scanning electron microscopy (SEM). A large population of the drug-carrying particles were found to be under the inhalable size range (fine particle fraction 67.88% ± 3%). In vitro drug release kinetics from spray-dried nanomicelles were carried out at lung fluid pH.

Expression and functional analysis of two osmotin (PR5) isoforms with differential antifungal activity from Piper colubrinum: prediction of structure-function relationship by bioinformatics approach.

Osmotin, a pathogenesis-related antifungal protein, is relevant in induced plant immunity and belongs to the thaumatin-like group of proteins (TLPs). This article describes comparative structural and functional analysis of the two osmotin isoforms cloned from Phytophthora-resistant wild Piper colubrinum. The two isoforms differ mainly by an internal deletion of 50 amino acid residues which separates them into two size categories (16.4 kDa-PcOSM1 and 21.5 kDa-PcOSM2) with pI values 5.6 and 8.3, respectively. Recombinant proteins were expressed in E. coli and antifungal activity assays of the purified proteins demonstrated significant inhibitory activity of the larger osmotin isoform (PcOSM2) on Phytophthora capsici and Fusarium oxysporum, and a markedly reduced antifungal potential of the smaller isoform (PcOSM1). Homology modelling of the proteins indicated structural alterations in their three-dimensional architecture. Tertiary structure of PcOSM2 conformed to the known structure of osmotin, with domain I comprising of 12 ß-sheets, an α-helical domain II and a domain III composed of 2 ß-sheets. PcOSM1 (smaller isoform) exhibited a distorted, indistinguishable domain III and loss of 4 ß-sheets in domain I. Interestingly, an interdomain acidic cleft between domains I and II, containing an optimally placed endoglucanase catalytic pair composed of Glu-Asp residues, which is characteristic of antifungal PR5 proteins, was present in both isoforms. It is well accepted that the presence of an acidic cleft correlates with antifungal activity due to the presence of endoglucanase catalytic property, and hence the present observation of significantly reduced antifungal capacity of PcOSM1 despite the presence of a strong acidic cleft, is suggestive of the possible roles played by other structural features like domain I or/and III, in deciding the antifungal potential of osmotin.

Evolutionary Implications and Physicochemical Analyses of Selected Proteins of Type III Polyketide Synthase Family.

Type III polyketide synthases have a substantial role in the biosynthesis of various polyketides in plants and microorganisms. Comparative proteomic analysis of type III polyketide synthases showed evolutionarily and structurally related positions in a compilation of amino acid sequences from different families. Bacterial and fungal type III polyketide synthase proteins showed <50% similarity but in higher plants, it exhibited >80% among chalcone synthases and >70% in the case of non-chalcone synthases. In a consensus phylogenetic tree based on 1000 replicates; bacterial, fungal and plant proteins were clustered in separate groups. Proteins from bryophytes and pteridophytes grouped immediately near to the fungal cluster, demonstrated how evolutionary lineage has occurred among type III polyketide synthase proteins. Upon physicochemical analysis, it was observed that the proteins localized in the cytoplasm and were hydrophobic in nature. Molecular structural analysis revealed comparatively stable structure comprising of alpha helices and random coils as major structural components. It was found that there was a decline in the structural stability with active site mutation as prophesied by the in silico mutation studies.

Kernel based machine learning algorithm for the efficient prediction of type III polyketide synthase family of proteins.

Type III Polyketide synthases (PKS) are family of proteins considered to have significant roles in the biosynthesis of various polyketides in plants, fungi and bacteria. As these proteins shows positive effects to human health, more researches are going on regarding this particular protein. Developing a tool to identify the probability of sequence being a type III polyketide synthase will minimize the time consumption and manpower efforts. In this approach, we have designed and implemented PKSIIIpred, a high performance prediction server for type III PKS where the classifier is Support Vector Machines (SVMs). Based on the limited training dataset, the tool efficiently predicts the type III PKS superfamily of proteins with high sensitivity and specificity. The PKSIIIpred is available at http://type3pks.in/prediction/. We expect that this tool may serve as a useful resource for type III PKS researchers. Currently work is being progressed for further betterment of prediction accuracy by including more sequence features in the training dataset.