New flavanones from the leaves of Cryptocarya chinensis and their antituberculosis activity

Four new flavanones, cryptoflavanones A-D (1-4, resp.), together with eight known compounds, were isolated from the leaves of Cryptocarya chinensis. The structures of these new compounds were determined by spectral analyses. Among the isolated compounds, pinocembrin (5) and cryptocaryone (6) exhibited antituberculosis activity against Mycobacterium tuberculosis H(37) Rv strain in vitro with MIC values of 3.5 and 25.0 μg/ml, respectively.     

Expression systems for heterologous production of antimicrobial peptides

Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.     

Lobophytones U - Z₁, biscembranoids from the Chinese soft coral Lobophytum pauciflorum

Chemical examination of a Chinese soft coral Lobophytum pauciflorum resulted in the isolation of seven new biscembranoids named lobophytones U–Z₁ ( 1 – 7 , resp.), together with methyl sartortuoate ( 8 ) and nyalolide ( 9 ). The structures of the new compounds were elucidated by 1D‐ and 2D‐NMR (COSY, HSQC, HMBC, and NOESY) spectroscopic analysis in association with MS and IR data. All compounds were tested against lipopolysaccharide (LPS)‐induced nitric oxide (NO) release in mouse peritoneal macrophage. Lobophytone Z ( 6 ) inhibited NO production with an IC₅₀ value of 2.6 μM . Lobophytone H ( 1 ) showed inhibitory activities against the bacteria S. aureus and S. pneumoniae.     

Cytotoxic withanolides from Physalis angulata L

Four new withanolides, physagulins L-O (1-4), were isolated from the MeOH extract of the aerial parts of Physalis angulata L. (Solanaceae), together with seven known withanolides, compounds 5-11. Their structures were determined by spectroscopic techniques, including 1H-, 13C-NMR (DEPT), and 2D-NMR (HMBC, HMQC, 1H,1H-COSY, NOESY) experiments, as well as by HR-MS. All eleven compounds were tested for their antiproliferative activities towards human colorectal-carcinoma (HCT-116) and human non-small-cell lung-cancer (NCI-H460) cells. Compound 5 exhibited the highest anticancer activity against the HCT-116 cell line, with an IC50 value of 1.64+/-0.06 microM. Compound 9 exhibited the highest cytotoxicity towards the NCI-H460 cell line, with an IC50 value of 0.43+/-0.02 microM.     

Background and overview of current sediment toxicity identification evaluation procedures

Laboratory bioassays can provide an integrated assessment of the potential toxicity of contaminated sediments to aquatic organisms; however, toxicity as a sole endpoint is not particularly useful in terms of identifying remedial options. To focus possible remediation (e.g., source control), it is essential to know which contaminants are responsible for toxicity. Unfortunately, contaminated sediments can contain literally thousands of potentially toxic compounds. Methods which rely solely on correlation to identify contaminants responsible for toxicity are limited in several aspects: (a) actual compounds causing toxicity might not be measured, (b) concentrations of potentially toxic compounds may covary, (c) it may be difficult to assess the bioavailability of contaminants measured in a sediment, and (d) interactions may not be accounted for among potential toxicants (e.g., additivity). Toxicity identification evaluation (TIE) procedures attempt to circumvent these problems by using toxicity-based fractionation procedures to implicate specific contaminants as causative toxicants. Phase I of TIE characterizes the general physio-chemical nature of sample toxicants. Phase II employs methods to measure toxicants via different analytical methods, and Phase III consists of techniques to confirm that the suspect toxicants identified in Phases I and II of the TIE actually are responsible for toxicity. These TIE procedures have been used to investigate the toxicity of a variety of samples, including sediments. Herein we present a brief conceptual overview of the TIE process, and discuss specific considerations associated with sediment TIE research. Points addressed include: (a) selection and preparation of appropriate test fractions, (b) use of benthic organisms for sediment TIE work, and (c) methods for the identification of common sediment contaminants.     

Metabolite profiling analysis of Methylobacterium extorquens AM1 by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry

Methylobacterium extorquens AM1 is a facultative methylotroph, which is a potential candidate to be used in commercial processes to convert simple one-carbon compounds to a variety of multicarbon chemicals and products. To better understand C(1) metabolism in M. extorquens AM1 at the systems level, metabolite profiling tools were developed and applied in this bacterium. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC x GC-TOFMS) was used to obtain metabolite profiles of M. extorquens AM1 (primarily organic acids) and to identify the metabolite differences between cells grown on methanol (C(1) substrate) and succinate (multicarbon substrate). In this study, a list of compounds that included amino acids and major intermediates of central C(1) and multicarbon metabolism were studied as target metabolites. For these, calibration curves were obtained for absolute quantification by spiking different amounts of standard mixtures to cell cultures. Parallel factor analysis (PARAFAC) was used for accurate peak quantification. Unknown chemical differences between cells grown on methanol and succinate were identified by applying Fisher ratio analysis at a selective mass channel (m/z 147). Thirty-six compounds were discovered to be statistically differentially expressed between C(1) and multicarbon metabolism. Among these, 13 were identified by matching to library mass spectra, and the rest were novel compounds that were not included in libraries. These differentially expressed compounds have provided clues to new pathways that are specifically linked to C(1) metabolism.     

D-ring-opened phragmalin-type limonoids from Chukrasia tabularis var. velutina

Five new D-ring-opened phragmalin-type limonoids, tabulalins A-E (1-5, resp.), were isolated from the stem bark of Chukrasia tabularis var. velutina. In the structures of these new isolates, the D-ring (C(16)/C(17) δ-lactone ring) of phragmalins was cleaved, and rare C(16)/C(30) δ-lactone ring in 1-3 or C(16)/C(8) γ-lactone ring in 4 and 5 were formed. The structures of these new compounds were elucidated based on extensive 1D- and 2D-spectroscopic analyses (HSQC, HMBC, and ROESY) and HR-ESI-MS. The major compounds, 2, 3, and 5, were evaluated for their inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a macrophage (RAW264.7) cell line with IC(50) values of 15.3±0.6, 13.0±0.5, and 17.1±0.7 μM, respectively.     

Three new cytotoxic Diels-Alder-type adducts from Morus australis

Three new natural products, australisines A-C (1-3, resp.), were isolated from the stem bark of Morus australis, together with eight related compounds, including mulberrofurans E-G, J, and Q, mongolicin C, chalcomoracin, and kuwanon G. Their structures were fully characterized by spectroscopic methods. Compounds 1-3, mulberrofuran G, mongolicin C, and chalcomoracin showed moderate cytotoxic activities against five human cancer cell lines, with IC50 values ranging from 4.6-9.2 microg/ml, as determined by MTT assay.     

The use of enzyme mixtures for complex biosyntheses

Biocatalysis is currently employed to produce known substances more economically and for the synthesis of new compounds. Increased production or novel product synthesis can be achieved through the use of mutant organisms, tailored enzymes or novel combinations of enzymes in reactors. These complex biosyntheses, once only in the realm of the biopharmaceutical industry, have now been embraced by the food and textile industries and are finding geochemical and environmental applications. New uses are dictating novel methods of manufacture that utilize knowledge of systems level biology. Increased understanding of the functional interaction of proteins and protein-protein networks is also altering the practice of in vitro biosynthesis.     

Acute Toxicity and Sublethal Effects of Terpenoids and Essential Oils on the Predator <Emphasis Type="Italic">Chrysoperla externa</Emphasis> (Neuroptera: Chrysopidae)

The search for new safer insecticides has increased in recent agriculture. Botanical compounds such as terpenoids and plant essential oils with insecticidal activity could represent important tools in pest management, and their risk assessment against non-target organisms is necessary since they may serve as a precursor for the synthesis of new insecticide active ingredients. For this study, the acute toxicity and sublethal effects of seven terpenoids and three essential oils with recognized insecticidal activity were evaluated on the predator Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) in laboratory bioassays. Results indicate that these compounds feature relative selectivity to the predator C. externa; however, sublethal effects on reproduction were recorded for some compounds. The phenolic monoterpenoids carvacrol and thymol were more acutely toxic than other terpenoids screened, with LD₅₀ <20,000 μg/g; however, they were less toxic than natural pyrethrins (toxicity standard) in these bioassays. Sublethal effects on fecundity and fertility were observed for R-(+)-limonene, while oregano oil only affected fecundity. The compounds evaluated here have potential to be used as insecticides and can serve as backbone for future synthesis of selective active ingredients; however, a complete risk assessment to C. externa and other non-target organisms is necessary for their incorporation in future crop protection paradigms.     

Predicting solvent and aggregation effects of peptides using group contribution calculations

Assembly of proteins and peptides into amyloid fibrils is characteristic of the pathology of a number of degenerative diseases, of which perhaps the best known is Alzheimer's. Neuronal death in Alzheimer's disease has been linked to aggregation of beta-amyloid peptide and deposition of the aggregates in brain tissue. Recent studies suggest that soluble intermediate oligomers rather than fully formed fibrils may be responsible for beta-amyloid's toxicity; consistent with this hypothesis, some compounds that inhibited beta-amyloid toxicity were found to accelerate aggregation of the peptide. The degree of increase of aggregation caused by these compounds was shown to correlate strongly with their effect on the surface tension of aqueous buffers (Gibson, T. J.; Murphy, R. M. Biochemistry 2005, 44, 8898-8907). In work reported here, we used group contribution methods to calculate infinite dilution activity coefficients of moieties contained in these compounds. We demonstrate that activity coefficients calculated in this manner are strongly predictive of effects of these peptides on solvent surface tension and on beta-amyloid aggregation. These results suggest that group contribution calculations can be used for a priori design of compounds that modulate aggregation of peptides through their effect on solvent properties.     

Reductive dehalogenation as a respiratory process

Anaerobic bacteria can reductively dehalogenate aliphatic and aromatic halogenated compounds in a respiratory process. Only a few of these bacteria have been isolated in pure cultures. However, long acclimation periods, substrate specificity, high dehalogenation rates, and the possibility to enrich for the dehalogenation activity by subcultivation in media containing an electron donor indicate that many of the reductive dehalogenations in the environment are catalyzed by specific bacteria. Molecular hydrogen or formate appear to be good electron donors for the enrichment of such organisms. Furthermore, systems have to be employed which supply the cultures with the halogenated compounds beyond their toxicity level. All bacteria that are presently available in pure culture and grow with a halogenated compound as electron acceptor are members of new genera. Based on experimental results with the membrane-impermeable electron mediator methyl viologen, a model of the respiration system ofDehalobacter restrictus, a tetrachloroethene-dechlorinating bacterium, is presented. Further studies of the biochemistry and energetics of respiratory-dehalogenating strains will help to understand the mechanisms involved and perhaps reveal the evolutionary origin of the dehalogenating enzyme systems.Abbreviations PCE tetrachloroethene - TCE trichloroethene - cis-1,2-DCE cis-1,2-dichloroethene - PCER tetrachloroethene reductase     

Peptide antibiotics, beta-lactams, and related compounds

In the field of natural peptides, beta-lactams, and related compounds, recent exciting developments are discussed. The increasing interest in this class of bioactive amino-acid derived structures has been attributed to the use of new directed screens (enzyme inhibition assays, beta-lactam detection, immunomodulator studies), new and improved applications (antibiotic, transplantation, and cancer chemotherapy), and advances in functional studies (DNA binding peptides, nucleotide complexones, cell wall and protein processing inhibitors). Peptides offer unique access to modifications and analog production by in vivo (directed biosynthesis) and in vitro procedures (enzymatic synthesis) due to their general linear precursors permitting point replacements. Of special interest are recent developments in the genetics of these compounds (cyclic peptides and beta-lactams), which will find applications in production methods in the near future.     

稀土抗菌效应及应用的研究进展

稀土元素具有多种生物效应, 除了对农作物的增产作用外, 在医药方面还具有抗菌的作用, 近年来, 不少学者针对稀土元素的抗菌效应展开了相关的研究。本文介绍了稀土在抗菌领域的研究及应用, 包括稀土化合物对微生物生长的Hormesis效应、稀土化合物与抗生素的协同作用、稀土配合物的合成、以及稀土在抗菌材料上的应用等几个方面的内容, 并对稀土化合物及其配合物的抗菌机理进行了探讨, 最后, 展望了稀土化合物及配合物在抗菌领域的应用前景及研究重要性。     

抗菌肽结构改造与人工智能研发策略

抗菌肽是一类广泛存在于生物体内的小分子肽,参与构成生物体先天免疫,可以有效抵抗病原微生物的入侵。抗菌肽具有广谱抗菌活性,且不易产生耐药性等特点,在治疗感染性疾病方面具有独特的优势,有望成为理想的抗感染药物。然而,由于部分抗菌肽尚存在稳定性差、毒性高等问题,限制了抗菌肽的广泛应用。由于人工智能算法能有效合成具有高稳定性、低毒性的抗菌肽,在探索天然抗菌肽中展现了巨大的潜力,因此本文简述了抗菌肽的抗菌机制、结构改造以及利用机器学习和深度学习等人工智能算法进行新型抗菌肽研发的优化策略,以期为抗菌肽结构优化及研发提供新思路。     

Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein ( approximately 33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP ( approximately 2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

New hepatoprotective coumarinolignoids from Mallotus apelta

Three new coumarinolignoids, malloapelins A-C (1-3, resp.), together with three known coumarinolignoids, cleomiscosin A (4), cleomiscosin B (5), and 5'-demethylaquillochin (6), were isolated from the roots of Mallotus apelta MUELL.-ARG. Compounds 1-6 are three pairs of regioisomeric coumarinolignoids. Their structures were elucidated on the basis of spectral evidence. Compounds 3 showed promising hepatoprotective activity against D-galactosamine-induced toxicity in WB-F344 rat hepatic epithelial stem-like cells.     

Connecting peptide physicochemical and antimicrobial properties by a rational prediction model

The increasing rate in antibiotic-resistant bacterial strains has become an imperative health issue. Thus, pharmaceutical industries have focussed their efforts to find new potent, non-toxic compounds to treat bacterial infections. Antimicrobial peptides (AMPs) are promising candidates in the fight against antibiotic-resistant pathogens due to their low toxicity, broad range of activity and unspecific mechanism of action. In this context, bioinformatics' strategies can inspire the design of new peptide leads with enhanced activity. Here, we describe an artificial neural network approach, based on the AMP's physicochemical characteristics, that is able not only to identify active peptides but also to assess its antimicrobial potency. The physicochemical properties considered are directly derived from the peptide sequence and comprise a complete set of parameters that accurately describe AMPs. Most interesting, the results obtained dovetail with a model for the AMP's mechanism of action that takes into account new concepts such as peptide aggregation. Moreover, this classification system displays high accuracy and is well correlated with the experimentally reported data. All together, these results suggest that the physicochemical properties of AMPs determine its action. In addition, we conclude that sequence derived parameters are enough to characterize antimicrobial peptides.     

Organotin compounds and their interactions with microorganisms.

Organotin compounds are ubiquitous in the environment. The general order of toxicity to microorganisms increases with the number and chain length of organic groups bonded to the tin atom. Tetraorganotins and inorganic tin have little toxicity. Because of their lipophilicity, organotins are regarded as membrane active. There is evidence that the site of action of organotins may be both at the cytoplasmic membrane and intracellular level. Consequently, it is not known whether cell surface adsorption or accumulation within the cell, or both is a prerequisite for toxicity. Biosorption studies on a fungus, cyanobacteria, and microalgae indicates that cell surface binding alone occurred in these organisms, while studies on the effects of TBT (tributyltin) on certain microbial enzymes indicated that in some bacteria TBT can interact with cytosolic enzymes. Microorganism-organotin interactions are influenced by environmental conditions. In aquatic systems, both pH and salinity can determine organotin speciation and therefore reactivity. These environmental factors may also alter selectivity for resistant microorganisms in polluted systems. Tin-resistant microorganisms have been identified, and resistance can be either plasmid or chromosomally mediated. In one TBT-resistant organism, an Altermonas sp., an efflux system was suggested as the resistance mechanism. Biotransformation of organotin compounds by debutylation or methylation has been observed. These reactions may influence the toxicity, mobility, and environmental fate of organotin compounds.     

Early stage efficacy and toxicology screening for antibiotics and enzyme inhibitors

The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Toward this end, we have established a microplate-based, dual-assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage development. The bacterial assay uses nonpathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with selected agents from the ASADH inhibitor fragment library show some compounds with antibiotic activity, but as expected, most of these early agents display higher than desired mammalian cell toxicity.