Schisandrin B Suppresses Colon Cancer Growth by Inducing Cell Cycle Arrest and Apoptosis: Molecular Mechanism and Therapeutic Potential.

Colon cancer is among the most lethal and prevalent malignant tumors in the world, and the lack of effective therapies highlights the need for novel therapeutic approaches. Schisandrin B (Sch B), a lignan extracted from the fruit ofSchisandra chinensis, has been reported for its anticancer properties. However, to date, no studies have been done to characterize the exact molecular mechanisms underlying the antitumorigenic effects of Sch B in colon cancer. This study aimed to explore the antitumorigenic effects of Sch B in colon cancer and to understand the underlying therapeutic mechanism. A comprehensive analysis of the molecular mechanism underlying the antitumorigenic effects of Sch B on human colon cancer cells was performed using a combination of Raman spectroscopy, RNA-seq, computational docking, and molecular biological experiments. The in vivo efficacy was evaluated by a mouse xenograft model. Sch B reduced cell proliferation and triggered apoptosis in human colon cancer cell lines. Raman spectroscopy, computational, RNA-seq, and molecular and cellular studies revealed that Sch B activated unfolded protein responses by interacting with CHOP and upregulating CHOP, which thereby induced apoptosis. CHOP knockdown alleviated the Sch B-induced reduction in cell viability and apoptosis. Sch B reduced colon tumor growth in vivo. Our findings demonstrated that Sch B induced apoptosis and inhibited cell proliferation and tumor growth in vitro and in vivo. These results provided an essential background for clinical trials examining the effects of Sch B in patients with colon cancer.

A Direct Analysis of ß-N-methylamino-l-alanine Enantiomers and Isomers and Its Application to Cyanobacteria and Marine Mollusks.

Of the wide variety of toxic compounds produced by cyanobacteria, the neurotoxic amino acid ß-N-methylamino-l-alanine (BMAA) has attracted attention as a result of its association with chronic human neurodegenerative diseases such as ALS and Alzheimer's. Consequently, specific detection methods are required to assess the presence of BMAA and its isomers in environmental and clinical materials, including cyanobacteria and mollusks. Although the separation of isomers such as ß-amino-N-methylalanine (BAMA), N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobutyric acid (DAB) from BMAA has been demonstrated during routine analysis, a further compounding factor is the potential presence of enantiomers for some of these isomers. Current analytical methods for BMAA mostly do not discriminate between enantiomers, and the chiral configuration of BMAA in cyanobacteria is still largely unexplored. To understand the potential for the occurrence of D-BMAA in cyanobacteria, a chiral UPLC-MS/MS method was developed to separate BMAA enantiomers and isomers and to determine the enantiomeric configuration of endogenous free BMAA in a marine Lyngbya mat and two mussel reference materials. After extraction, purification and derivatization with N-(4-nitrophenoxycarbonyl)-l-phenylalanine 2-methoxyethyl ester ((S)-NIFE), both L- and D-BMAA were identified as free amino acids in cyanobacterial materials, whereas only L-BMAA was identified in mussel tissues. The finding of D-BMAA in biological environmental materials raises questions concerning the source and role of BMAA enantiomers in neurological disease.

The Effects of Long-term, Low-dose ß-N-methylamino-L-alanine (BMAA) Exposures in Adult SODG93R Transgenic Zebrafish.

ß-N-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid produced by cyanobacteria, which has been implicated in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). It is postulated that chronic exposure to BMAA can lead to formation of protein aggregates, oxidative stress, and/or excitotoxicity, which are mechanisms involved in the etiology of ALS. While specific genetic mutations are identified in some instances of ALS, it is likely that a combination of genetic and environmental factors, such as exposure to the neurotoxin BMAA, contributes to disease. We used a transgenic zebrafish with an ALS-associated mutation, compared with wild-type fish to explore the potential neurotoxic effects of BMAA through chronic long-term exposures. While our results revealed low concentrations of BMAA in the brains of exposed fish, we found no evidence of decreased swim performance or behavioral differences that might be reflective of neurodegenerative disease. Further research is needed to determine if chronic BMAA exposure in adult zebrafish is a suitable model to study neurodegenerative disease initiation and/or progression.

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.

Understanding the Anion-Templated, OSDA-Free, Interzeolite Conversion Synthesis of High Silica Zeolite ZK-5.

High silica zeolite ZK-5 (framework Si/Al=4.8) has been prepared by interzeolite conversion from ultrastable zeolite Y via a co-templating route using alkali metal cations and nitrate anions but without organic structure directing agents. The mechanism, which involves zeolite framework - alkali metal cation - nitrate anion ordering, has been established by a combination of chemical and thermal analyses, Raman spectroscopy, computational modelling, and X-ray powder diffraction. Ammonium exchange gives ZK-5 with occluded ammonium nitrate and subsequent heating gives microporous zeolite ZK-5.

Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E.

Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.

Site-Specific Iron Substitution in STA-28, a Large Pore Aluminophosphate Zeotype Prepared by Using 1,10-Phenanthrolines as Framework-Bound Templates.

An AlPO4 zeotype has been prepared using the aromatic diamine 1,10-phenanthroline and some of its methylated analogues as templates. In each case the two template N atoms bind to a specific framework Al site to expand its coordination to the unusual octahedral AlO4 N2 environment. Furthermore, using this framework-bound template, Fe atoms can be included selectively at this site in the framework by direct synthesis, as confirmed by annular dark field scanning transmission electron microscopy and Rietveld refinement. Calcination removes the organic molecules to give large pore framework solids, with BET surface areas up to 540 m2 g-1 and two perpendicular sets of channels that intersect to give pore space connected by 12-ring openings along all crystallographic directions.

DIVERSet JAG Compounds Inhibit Topoisomerase II and Are Effective Against Adult and Pediatric High-Grade Gliomas.

High-grade gliomas (HGGs) are aggressive primary brain tumors with local invasive growth and poor clinical prognosis in both adult and pediatric patients. Clinical response is compounded by resistance to standard frontline antineoplastic agents, an absence of novel therapeutics, and poor in vitro models to evaluate these. We screened a range of recently identified anticancer compounds in conventional adult, pediatric, and new biopsy-derived HGG models. These in vitro lines showed a range of sensitivity to standard chemotherapeutics, with varying expression levels of the prognostic markers hypoxia-induced factor (HIF) 1α and p53. Our evaluation of lead DIVERSet library compounds identified that JAG-6A, a compound that was significantly more potent than temozolomide or etoposide, was effective against HGG models in two-dimensional and three-dimensional systems; mediated this response by the potent inhibition of topoisomerase Iiα; remained effective under normoxic and hypoxic conditions; and displayed limited toxicity to non-neoplastic astrocytes. These data suggest that JAG-6A could be an alternative topoisomerase IIα inhibitor and used for the treatment of HGG.

A Bifunctional MOF Catalyst Containing Metal-Phosphine and Lewis Acidic Active Sites.

Post-synthetic modification of the hafnium metal-organic framework MOF-808(Hf) to include triarylphosphine ligands is reported. Sulfonated phenylphosphines are incorporated without oxidation to give a "MOF ligand" that can complex late transition metals such as Ir and Rh to give a bifunctional catalyst containing both metal-phosphine complexes and the Lewis acidic framework hafnium metal sites. The metallated phosphine-bearing MOFs act as fully heterogeneous bifunctional catalysts for tandem reductive amination and hydroaminomethylation reactions.

Analysis of Neurotoxic Amino Acids from Marine Waters, Microbial Mats, and Seafood Destined for Human Consumption in the Arabian Gulf.

Human health risks associated with exposure to algal and cyanobacterial toxins (phycotoxins) have been largely concerned with aquatic habitats. People inhabiting desert environments may be exposed to phycotoxins present in terrestrial environments, where cyanobacterial crusts dominate. Seafood comprises a significant portion of the human diet in desert environments proximal to an ocean or sea. Consequently, in addition to terrestrial exposure to cyanotoxins, the potential exists that seafood may be an important exposure route for cyanotoxins in desert regions. Understanding the possible risk of exposure from seafood will help create cyanotoxin health guidelines for people living in environments that rely on seafood. Commonly-consumed local seafood products destined for human consumption were purchased from a fish market in Doha, Qatar. Organs were excised, extracted, and analyzed for the neurotoxic amino acid ß-N-methylamino-L-alanine (BMAA) and the isomers 2,4-diaminobutyric acid (DAB) and N-2(aminoethyl)glycine (AEG). The presence and concentration of neurotoxic amino acids were investigated in organisms from various trophic levels to examine the potential for biomagnification. Although BMAA and isomers were detected in marine microbial mats, as well as in marine plankton net trawls associated with diatoms and dinoflagellates, in seafood, only AEG and DAB were present at low concentrations in various trophic levels. The findings of this study suggest that exposure to neurotoxic amino acids through seafood in the Arabian Gulf may be minor, yet the presence of BMAA in phytoplankton confirms the need for further monitoring of marine waters and seafood to protect human health.

Base-Catalyzed Aryl-B(OH)2 Protodeboronation Revisited: From Concerted Proton Transfer to Liberation of a Transient Aryl Anion.

Pioneering studies by Kuivila, published more than 50 years ago, suggested ipso protonation of the boronate as the mechanism for base-catalyzed protodeboronation of arylboronic acids. However, the study was limited to UV spectrophotometric analysis under acidic conditions, and the aqueous association constants (Ka) were estimated. By means of NMR, stopped-flow IR, and quenched-flow techniques, the kinetics of base-catalyzed protodeboronation of 30 different arylboronic acids has now been determined at pH > 13 in aqueous dioxane at 70 °C. Included in the study are all 20 isomers of C6HnF(5-n)B(OH)2 with half-lives spanning 9 orders of magnitude: <3 ms to 6.5 months. In combination with pH-rate profiles, pKa and ΔS⧧ values, kinetic isotope effects (2H, 10B, 13C), linear free-energy relationships, and density functional theory calculations, we have identified a mechanistic regime involving unimolecular heterolysis of the boronate competing with concerted ipso protonation/C-B cleavage. The relative Lewis acidities of arylboronic acids do not correlate with their protodeboronation rates, especially when ortho substituents are present. Notably, 3,5-dinitrophenylboronic acid is orders of magnitude more stable than tetra- and pentafluorophenylboronic acids but has a similar pKa.

Inhibition of homologous phosphorolytic ribonucleases by citrate may represent an evolutionarily conserved communicative link between RNA degradation and central metabolism.

Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.

Phase I clinical trial of safety of L-serine for ALS patients.

We performed a randomized, double-blind phase I clinical trial for six months on the effects of oral L-serine in patients with ALS. The protocol called for enrollment of patients with a diagnosis of probable or definite ALS, age 18-85 years, disease duration of less than three years and forced vital capacity (FVC) ≥ 60%. Patients were randomly assigned to four different oral twice-daily dose regimens (0.5, 2.5, 7.5, or 15 g/dose). Blood, urine and CSF samples, ALS Functional Rating Scale-Revised (ALSFRS-R) scores and forced vital capacity (FVC) were obtained throughout the trial. Disease progression was compared with matched historical placebo controls from five previous ALS therapeutic trials. Of 20 patients enrolled, one withdrew before receiving study drug and two withdrew with gastro-intestinal problems. Three patients died during the trial. L-serine was generally well tolerated by the patients and L-serine did not appear to accelerate functional decline of patients as measured by slope of their ALSFRS-R scores. Based on this small study, L-serine appears to be generally safe for patients with ALS.

Protodeboronation of Heteroaromatic, Vinyl, and Cyclopropyl Boronic Acids: pH-Rate Profiles, Autocatalysis, and Disproportionation.

pH-rate profiles for aqueous-organic protodeboronation of 18 boronic acids, many widely viewed as unstable, have been studied by NMR and DFT. Rates were pH-dependent, and varied substantially between the boronic acids, with rate maxima that varied over 6 orders of magnitude. A mechanistic model containing five general pathways (k1-k5) has been developed, and together with input of [B]tot, KW, Ka, and KaH, the protodeboronation kinetics can be correlated as a function of pH (1-13) for all 18 species. Cyclopropyl and vinyl boronic acids undergo very slow protodeboronation, as do 3- and 4-pyridyl boronic acids (t0.5 > 1 week, pH 12, 70 °C). In contrast, 2-pyridyl and 5-thiazolyl boronic acids undergo rapid protodeboronation (t0.5 ≈ 25-50 s, pH 7, 70 °C), via fragmentation of zwitterionic intermediates. Lewis acid additives (e.g., Cu, Zn salts) can attenuate (2-pyridyl) or accelerate (5-thiazolyl and 5-pyrazolyl) fragmentation. Two additional processes compete when the boronic acid and the boronate are present in sufficient proportions (pH = pKa ± 1.6): (i) self-/autocatalysis and (ii) sequential disproportionations of boronic acid to borinic acid and borane.

A zeolite family with expanding structural complexity and embedded isoreticular structures.

The prediction and synthesis of new crystal structures enable the targeted preparation of materials with desired properties. Among porous solids, this has been achieved for metal-organic frameworks, but not for the more widely applicable zeolites, where new materials are usually discovered using exploratory synthesis. Although millions of hypothetical zeolite structures have been proposed, not enough is known about their synthesis mechanism to allow any given structure to be prepared. Here we present an approach that combines structure solution with structure prediction, and inspires the targeted synthesis of new super-complex zeolites. We used electron diffraction to identify a family of related structures and to discover the structural 'coding' within them. This allowed us to determine the complex, and previously unknown, structure of zeolite ZSM-25 (ref. 8), which has the largest unit-cell volume of all known zeolites (91,554 cubic ångströms) and demonstrates selective CO2 adsorption. By extending our method, we were able to predict other members of a family of increasingly complex, but structurally related, zeolites and to synthesize two more-complex zeolites in the family, PST-20 and PST-25, with much larger cell volumes (166,988 and 275,178 cubic ångströms, respectively) and similar selective adsorption properties. Members of this family have the same symmetry, but an expanding unit cell, and are related by hitherto unrecognized structural principles; we call these family members embedded isoreticular zeolite structures.

Grazing livestock are exposed to terrestrial cyanobacteria.

While toxins from aquatic cyanobacteria are a well-recognised cause of disease in birds and animals, exposure of grazing livestock to terrestrial cyanobacteria has not been described. This study identified terrestrial cyanobacteria, predominantly Phormidium spp., in the biofilm of plants from most livestock fields investigated. Lower numbers of other cyanobacteria, microalgae and fungi were present on many plants. Cyanobacterial 16S rDNA, predominantly from Phormidium spp., was detected in all samples tested, including 6 plant washings, 1 soil sample and ileal contents from 2 grazing horses. Further work was performed to test the hypothesis that ingestion of cyanotoxins contributes to the pathogenesis of some currently unexplained diseases of grazing horses, including equine grass sickness (EGS), equine motor neuron disease (EMND) and hepatopathy. Phormidium population density was significantly higher on EGS fields than on control fields. The cyanobacterial neurotoxic amino acid 2,4-diaminobutyric acid (DAB) was detected in plant washings from EGS fields, but worst case scenario estimations suggested the dose would be insufficient to cause disease. Neither DAB nor the cyanobacterial neurotoxins ß-N-methylamino-L-alanine and N-(2-aminoethyl) glycine were detected in neural tissue from 6 EGS horses, 2 EMND horses and 7 control horses. Phormidium was present in low numbers on plants where horses had unexplained hepatopathy. This study did not yield evidence linking known cyanotoxins with disease in grazing horses. However, further study is warranted to identify and quantify toxins produced by cyanobacteria on livestock fields, and determine whether, under appropriate conditions, known or unknown cyanotoxins contribute to currently unexplained diseases in grazing livestock.

An aluminophosphate molecular sieve with 36 crystallographically distinct tetrahedral sites.

The structure of the new medium-pore aluminophosphate molecular sieve PST-6 is determined by the combined use of rotation electron diffraction tomography, synchrotron X-ray powder diffraction, and computer modeling. PST-6 was prepared by calcination of another new aluminophosphate material with an unknown structure synthesized using diethylamine as a structure-directing agent, which is thought to contain bridging hydroxy groups. PST-6 has 36 crystallographically distinct tetrahedral sites in the asymmetric unit and is thus crystallographically the most complex zeolitic structure ever solved.

Controlled release of 5-fluorouracil from microporous zeolites.

Zeolite particles with different pore diameter and particle size were loaded with the model anticancer drug 5-fluorouracil. The loaded zeolites were characterized by means of SEM, XRD, DSC, XPS, N2 physisorption and FT-IR. Higher loading of 5-FU was observed for NaX-FAU than BEA. Release studies were carried out in HCl 0.1N. Release of 5-FU from NaX-FAU showed exponential-type behaviour with the drug fully released within 10 min. In the case of BEA, the kinetics of 5-FU shows a multi-step profile with prolonged release over time. Molecular dynamics simulations showed that diffusion of the drug molecule through the BEA framework is lower than for NaX-FAU due to increased van der Waals interaction between the drug and the framework. The effect of zeolitic particles on the viability of Caco-2 monolayers showed that the NaX-FAU particles cause a reduction of cell viability in a more pronounced way compared with the BEA particles. FROM THE CLINICAL EDITOR: This article describes zeolite-based nanoparticles in generating time-controlled release of 5-FU from zeolite preparations for anti-cancer therapy.

Linking ß-methylamino-L-alanine exposure to sporadic amyotrophic lateral sclerosis in Annapolis, MD.

Most amyotrophic lateral sclerosis (ALS) cases occur sporadically. Some environmental triggers have been implicated, including beta-methylamino-L-alanine (BMAA), a cyanobacteria produced neurotoxin. This study aimed to identify environmental risk factors common to three sporadic ALS patients who lived in Annapolis, Maryland, USA and developed the disease within a relatively short time and within close proximity to each other. A questionnaire was used to identify potential risk factors for ALS among the cohort of patients. One common factor among the ALS patients was the frequent consumption of blue crab. Samples of blue crab from the patients' local fish market were tested for BMAA using LC-MS/MS. BMAA was identified in these Chesapeake Bay blue crabs. We conclude that the presence of BMAA in the Chesapeake Bay food web and the lifetime consumption of blue crab contaminated with BMAA may be a common risk factor for sporadic ALS in all three patients.