In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGF Serine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties

Copyright © 2019 American Chemical Society. Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity

The transition between stochastic and deterministic behavior in an excitable gene circuit

We explore the connection between a stochastic simulation model and an ordinary differential equations (ODEs) model of the dynamics of an excitable gene circuit that exhibits noise-induced oscillations. Near a bifurcation point in the ODE model, the stochastic simulation model yields behavior dramatically different from that predicted by the ODE model. We analyze how that behavior depends on the gene copy number and find very slow convergence to the large number limit near the bifurcation point. The implications for understanding the dynamics of gene circuits and other birth-death dynamical systems with small numbers of constituents are discussed.Comment: PLoS ONE: Research Article, published 11 Apr 201

High-Throughput Screening to Identify Plant Derived Human LDH-A Inhibitors

Aims: Lactate dehydrogenase (LDH)-A is highly expressed in diverse human malignant tumors, parallel to aggressive metastatic disease, resistance to radiation /chemotherapy and clinically poor outcome. Although this enzyme constitutes a plausible target in treatment of advanced cancer, there are few known LDH-A inhibitors. Study Design: In this work, we utilized a high-throughput enzyme micro-array format to screen and evaluate > 900 commonly used medicinal plant extracts (0.00001-.5 mg/ml) for capacity to inhibit activity of recombinant full length human LDHA; EC .1.1.1.27. Methodology: The protein sequence of purified enzyme was confirmed using 1D gel electrophoresis- MALDI-TOF-MS/MS, enzyme activity was validated by oxidation of NADH (500μM) and kinetic inhibition established in the presence of a known inhibitor (Oxalic Acid). Results: Of the natural extracts tested, the lowest IC50s [<0.001 mg/ml] were obtained by: Chinese Gallnut (Melaphis chinensis gallnut), Bladderwrack (Fucus vesiculosus), Kelp (Laminaria Japonica) and Babul (Acacia Arabica). Forty-six additional herbs contained significant LDH-A inhibitory properties with IC50s [<0.07 mg/ml], some of which have common names of Arjun, Pipsissewa, Cinnamon, Pink Rose Buds/ Petals, Wintergreen, Cat’s Claw, Witch Hazel Root and Rhodiola Root. Conclusion: These findings reflect relative potency by rank of commonly used herbs and plants that contain human LDH-A inhibitory properties. Future research will be required to isolate chemical constituents within these plants responsible for LDH-A inhibition and investigate potential therapeutic application

Structural Study of Carbon Coated TiO2 Anatase Nanoparticles as High Performance Anode Materials for Na Ion Batteries

In this work, we study the electronic and atomic structural modifications occurring in TiO2 anatase nanoparticles as anode materials in Na ion batteries upon sodiation and desodiation. The structural investigation is performed over both long and short range order by combining a comprehensive extended X ray absorption fine structure EXAFS characterization with X ray diffraction XRD . The evolution of the electronic structure upon cycling is qualitatively investigated by X ray absorption near edge structure XANES analysis. The goal of this work is to correlate the outstanding electrochemical performance of carbon coated TiO2 anatase nanoparticles in sodium batteries with the electronic and structural modifications induced during the sodiation and desodiation processes upon cycling. This work also demonstrates for the first time a coherent explanation of the structural changes observed, where an electrochemically induced short range ordering is revealed upon cyclin

Bioactivity-Guided Isolation of Neuritogenic Factor from the Seeds of the Gac Plant (Momordica cochinchinensis)

Nerve growth factor (NGF) is an endogenously produced protein with the capacity to induce central nervous system (CNS) neuronal differentiation and repair. NGF signaling involves its binding to tropomyosin-related kinase (Trk) receptors, internalization, and initiation of phosphorylation cascades which cause microtubule reorganization and neuronal outgrowth. Because NGF cannot cross the blood-brain barrier, its therapeutic use is limited. Synthetic peptides that can act as NGF receptor agonists (NGF mimetics) are known to attenuate neurodegenerative pathologies in experimental models of Alzheimer’s disease and Parkinson’s disease; however, the existence of plant-based NGF mimetics is uncertain. For this reason, we recently completed a high throughput screening of over 1100 nutraceuticals (vitamins, herbal plant parts, polyphenolics, teas, fruits, and vegetables) to identify neuritogenic factor using a PC-12 cell model. Remarkably we found only one, commonly known as the seed of Gac plant (Momordica cochinchinensis) (MCS). In the current study, we further investigated this seed for its neuritogenic effect using bioactivity-guided chemical separations. The data show no biological neuritogenic activity in any chemical solvent fraction, where activity was exclusive to the crude protein. MSC crude proteins were then separated by 1D electrophoresis, where the active neuritogenic activity was confirmed to have a molecular mass of approximately 17 kDa. Subsequently, the 17kDa band was excised, digested, and run on a UPLC-MS/MS with a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer with data evaluated diverse tools such as X! Tandem, OMS, and K-score algorithms. Proteomic evaluation of the 17kDa band confirmed evidence for 11S globulin subunit beta, napin, oleosin, Momordica trypsin inhibitors (TI) MCoTI-I /II, and many isoforms of Two Inhibitor Peptide Topologies (TIPTOPs). While all peptides identified correspond to the genus/species, Momordica cochinchinensis and Cucumis Sativus, a significant limitation of the analysis is the nonexistence of full annotation for the Momordica cochinchinensis proteome. In conclusion, these findings demonstrate that there is a stable protein within MCS having a mass of 17kDa with the capacity to induce neurite outgrowth. Future work will be required to establish the therapeutic value of the MCS for the treatment of neurodegenerative diseases

Functional immune boosters; the herb or its dead microbiome? Antigenic TLR4 agonist MAMPs found in 65 medicinal roots and algae’s

Background: Humans have been consuming medicinal plants (as herbs/ spices) to combat illness for centuries while ascribing beneficial effects predominantly to the plant/phytochemical constituents, without recognizing the power of obligatory resident microorganism’ communities (MOCs) (live/dead bacteria, fungus, yeast, molds etc.) which remain after industrial microbial reduction methods. Very little is known about the taxonomic identity of residual antigenic microbial associated molecular patterns (MAMPs) debris in our botanical over the counter (OTC) products, which if present would be recognized as foreign (non-self) antigenic matter by host pattern recognition receptors (PRRs) provoking a host immune response; this the basis of vaccine adjuvants. As of today, only few research groups have removed the herbal MAMP biomass from herbs, all suggesting that immune activation may not be from the plant but rather its microbial biomass; a hypothesis we corroborate. Purpose: The purpose of this work was to conduct a high through put screening (HTPS) of over 2500 natural plants, OTC botanical supplements and phytochemicals to elucidate those with pro-inflammatory; toll like receptor 4 (TLR4) activating properties in macrophages. Study Design: The HTPS was conducted on RAW 264.7 cells vs. lipopolysaccharide (LPS) E. coli 0111:B4, testing iNOS / nitric oxide production (NO2–) as a perimeter endpoint. The data show not a single drug/chemical/ phytochemical and approximately 98 % of botanicals to be immune idle (not effective) with only 65 pro-inflammatory (hits) in a potency range of LPS. Method validation studies eliminated the possibility of false artifact or contamination, and results were cross verified through multiple vendors/ manufacturers/lot numbers by botanical species. Lead botanicals were evaluated for plant concentration of LPS, 1,3:1,6-β-glucan, 1,3:1,4-β-D-glucan and α-glucans; where the former paralleled strength in vitro. LPS was then removed from plants using high-capacity endotoxin poly lysine columns, where bioactivity of LPS null “plant” extracts were lost. The stability of E.Coli 0111:B4 in an acid stomach mimetic model was confirmed. Last, we conducted a reverse culture on aerobic plate counts (APCs) from select hits, with subsequent isolation of gram-negative bacteria (MacConkey agar). Cultures were 1) heat destroyed (retested/ confirming bioactivity) and 2) subject to taxonomical identification by genetic sequencing 18S, ITS1, 5.8 s, ITS2 28S, and 16S. Conclusion: The data show significant gram negative MAMP biomass dominance in A) roots (e.g. echinacea, yucca, burdock, stinging nettle, sarsaparilla, hydrangea, poke, madder, calamus, rhaponticum, pleurisy, aconite etc.) and B) oceanic plants / algae’s (e.g. bladderwrack, chlorella, spirulina, kelp, and “OTC Seamoss-blends“ (irish moss, bladderwrack, burdock root etc), as well as other random herbs (eg. corn silk, cleavers, watercress, cardamom seed, tribulus, duckweed, puffball, hordeum and pollen). The results show a dominance of gram negative microbes (e.g. Klebsilla aerogenes, Pantoae agglomerans, Cronobacter sakazakii), fungus (Glomeracaea, Ascomycota, Irpex lacteus, Aureobasidium pullulans, Fibroporia albicans, Chlorociboria clavula, Aspergillus_sp JUC-2), with black walnut hull, echinacea and burdock root also containing gram positive microbial strains (Fontibacillus, Paenibacillus, Enterococcus gallinarum, Bromate-reducing bacterium B6 and various strains of Clostridium). Conclusion: This work brings attention to the existence of a functional immune bioactive herbal microbiome, independent from the plant. There is need to further this avenue of research, which should be carried out with consideration as to both positive or negative consequences arising from daily consumption of botanicals highly laden with bioactive MAMPS