Simple Substrates Research Articles

Functional Diversity of Soil Microbial Community after Conversion of a Chestnut Forest to an Agricultural System

In the National park of Cilento, Vallo di Diano and Alburni (South Italy), a portion of a chestnut forest was converted in 2012 into an agricultural system in order to crop a local variety of bean. We investigated the effect over time of the conversion on the functional diversity of the soil microbial community by two different approaches: the catabolic response profile, based on the short time CO2 evolution induced by 25 simple organic substrates and the Biolog community level physiological profile (CLPP), based on the growth of microorganisms on 31 different substrates. The soils were sampled at 13, 17, 29, 41 and 49 months after the soil use change. The results showed that the soil use change did not produce evident modifications of the substrate utilization patterns, but rather a general decrease in the activity in the agricultural soils, as a consequence of the reduction in organic matter content. The results also showed seasonal effects on the substrate utilization profiles and on the calculated functional diversity indexes. The two approaches appeared to be complementary: Degens catabolic response profile was more able to discriminate between the two systems, whereas the Biolog was more able to highlight the variability among samplings.

P62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

Surface enhanced Raman scattering analysis with filter-based enhancement substrates: A mini review

Abstract Surface enhanced Raman is a powerful analytical tool with high sensitivity and unique specificity and promising applications in various branches of analytical chemistry. Despite the fabrication of ingenious enhancement substrate used in laboratory research, the development of simple, flexible, and cost-effective substrate is also great important for promoting the application of SERS in practical analysis. Recently, paper and filter membrane as support to fabricate flexible SERS substrates received considerable attentions. Paper-based SERS substrate has been reviewed but no summary on filter-based SERS substrate is available. Compared with paper, filter membrane has unique advantage in robust mechanics, diverse component, and tunable pore size. These characteristics endow the filter-based substrates great advantages for practical SERS analysis including simple and low-cost substrate preparation, high efficiency in preconcentration, separation and detection procedure. Therefore, filter-based substrates have shown great promise in SERS analysis in environment monitoring, food safety with high sensitivity and efficiency. As more and more work has been emerged, it is necessary to summarize the state of such a research topic. Here, the research on filter involved SERS analysis in the past eight years is summarized. A short introduction was presented to understand the background, and then the brief history of filter-based substrate is introduced. After that, the preparation of filter-based substrate and the role of filter are summarized. Then, the application of filter involved SERS substrate in analysis is presented. Finally, the challenges and perspective on this topic is discussed.

Easily accessible non-aromatic heterocycles with handles: 4-bromo-2,3-dihydrofurans from 1,2-dibromohomoallylic alcohols.

The first general preparation of 4-bromo-2,3-dihydrofurans is reported. These non-aromatic heterocycles containing a useful coupling handle are accessed via Cu-catalyzed intramolecular cyclization of 1,2-dibromohomoallylic alcohols, which are themselves available in just two steps from aromatic and aliphatic aldehydes and ketones. Molecular dynamics simulations using the simple substrates and key geometric parameters provide a rationale for the selectivities observed. The synthetic utility of the 4-bromodihydrofurans is also demonstrated.

Efficient unified synthesis of diverse bridged polycyclic scaffolds using a complexity-generating 'stitching' annulation approach.

Regioselective and stereospecific directed C-H arylation of simple amine substrates, and cyclisation, delivered 30 diverse, three-dimensional scaffolds. The unified approach significantly expanded the range of bridged ring systems that contain both a nitrogen atom and an aromatic ring.

Spotlight on Photoinduced Aryl Migration Reactions.

Rearrangement reactions are certainly one of the most useful approaches towards complex structures in organic chemistry. With efficient conditions, it is indeed possible to convert simple substrates into highly functionalized products. Moreover, combining this approach with an attractive initiation process, such as visible-light catalysis, makes these reactions particularly powerful. Recently, tremendous improvements have been made, owing to a better understanding of photoredox mechanisms. In this review, recent progress on visible-light aryl migration reactions is discussed, focusing especially on Smiles rearrangement and related reactions.

Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus

Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

Ratiometric fluorescence sensing of glutathione by using the oxidase-mimicking activity of MnO2 nanosheet

In this work, a facile ratiometric fluorescence sensor for GSH measurement was designed based on MnO2 nanosheet (NS), carbon dots (CDs), as well as a simple substrate o-phenylenediamine (OPD). Herein, MnO2 NS played triple essential roles in the sensing system. First, it could be reduced by GSH through a special reaction, and therefore served as GSH recognizer. Second, it played as a fluorescence nanoquencher to strongly quench the fluorescence of CDs. Third, it could directly oxidize OPD to yield a luminescent product 2, 3-diaminophenazine (DAP) via the intrinsic oxidase-like activity. It revealed that MnO2 NS could be reduced to Mn2+ in the presence of GSH. Thus its oxidase-like activity and fluorescence quenching abilities were inhibited, which then restricted the generation of DAP and recovered the fluorescence of CDs. Based on this phenomenon, a novel ratiometric fluorescence sensor for GSH determination was fabricated by measuring the ratio of fluorescent intensity of DAP to that of CDs. Besides, the constructed ratiometric fluorescent sensor, which could be facilely operated with single-wavelength excitation, exhibited high sensitivity and selectivity with a wider linear range and a lower detection limit.

Facile fabrication of three-dimensional gold nanodendrites decorated by silver nanoparticles as hybrid SERS-active substrate for the detection of food contaminants

Applicable surface enhanced Raman scattering (SERS) active substrate require not only high enhancement factor (EF), but also excellent reproducibility and facile fabrication process. Although some colloidal SERS substrates have advantages of high EF, random assembly causing the unstable signal remains a challenge. This study presented a simple, effective and reproducible SERS substrate for sensitive detection of melamine and thiram in food samples. A three-dimensional gold nanodendrites (AuNDs) decorated by silver nanoparticles (AgNPs) via electrodeposition has been synthesized as hybrid SERS-active substrate. The synthesis conditions were optimized with voltage power of −1.5 V and deposition time of 300 s, consequently the gold nanodendrites were obtained. The developed AgNPs decorated AuNDs (Ag-AuNDs) substrate showed excellent Raman enhancement effect because of high local electromagnetic (EM) field enhancement. Under the optimal synthesis condition, the SERS substrate was utilized for quantitative detection of melamine and thiram, and the linear relationship were in the range of 0.01–5 mg/L for melamine and 0.12–24 mg/L for thiram. The limit of detection were 7.38 μg/L and 86.1 μg/L for melamine and thiram, respectively. Finally, the substrate was successfully applied to detect melamine in milk and thiram in apple juice, indicating that Ag-AuNDs substrate could be a promising SERS detection platform for food safety assurance.

Crystal Structure and Mechanistic Molecular Modeling Studies of Mycobacterium tuberculosis Diterpene Cyclase Rv3377c.

Terpenes make up the largest class of natural products, with extensive chemical and structural diversity. Diterpenes, mostly isolated from plants and rarely prokaryotes, exhibit a variety of important biological activities and valuable applications, including providing antitumor and antibiotic pharmaceuticals. These natural products are constructed by terpene synthases, a class of enzymes that catalyze one of the most complex chemical reactions in biology: converting simple acyclic oligo-isoprenyl diphosphate substrates to complex polycyclic products via carbocation intermediates. Here we obtained the second ever crystal structure of a class II diterpene synthase from bacteria, tuberculosinol pyrophosphate synthase (i.e., Halimadienyl diphosphate synthase, MtHPS, or Rv3377c) from Mycobacterium tuberculosis (Mtb). This enzyme transforms (E,E,E)-geranylgeranyl diphosphate into tuberculosinol pyrophosphate (Halimadienyl diphosphate). Rv3377c is part of the Mtb diterpene pathway along with Rv3378c, which converts tuberculosinol pyrophosphate to 1-tuberculosinyl adenosine (1-TbAd). This pathway was shown to exist only in virulent Mycobacterium species, but not in closely related avirulent species, and was proposed to be involved in phagolysosome maturation arrest. To gain further insight into the reaction pathway and the mechanistically relevant enzyme substrate binding orientation, electronic structure calculation and docking studies of reaction intermediates were carried out. Results reveal a plausible binding mode of the substrate that can provide the information to guide future drug design and anti-infective therapies of this biosynthetic pathway.

Eliminating irreproducibility in SERS substrates

AbstractIrreproducibility in surface‐enhanced Raman spectroscopy (SERS) due to variability among substrates is a source of recurrent debate within the field. It is regarded as a major hurdle towards the widespread adoption of SERS as a sensing platform. Most of the literature focused on developing substrates for various applications considers reproducibility of lower importance. Here, we address and analyse the sources of this irreproducibility in order to show how these can be minimised. We apply our findings to a simple substrate demonstrating reproducible SERS measurements with relative standard deviations well below 1% between different batches and days. Identifying the sources of irreproducibility and understanding how to reduce these can aid in the transition of SERS from the lab to real‐world applications.

Total syntheses of (−)-emestrin H and (−)-asteroxepin

First total syntheses of (−)-emestrin H and (−)-asteroxepin are described. To find the appropriate protecting group on the amide nitrogen of the diketopiperazine core, we conducted model studies using a simple diketopiperazine derivative. As a result, allyloxymethyl (Allom) group was the most suitable protecting group, which tolerated Nicolaou’s sulfenylation conditions, and was easily cleavable under the mild conditions using Pd(PPh 3 ) 4 and N,N -dimethylbarbituric acid leaving methylthioethers intact. The general utility of Allom group for protection of amides was studied using simple substrates. Finally, the effectiveness of Allom group was proved by the accomplishment of the first total synthesis of (−)-emestrin H. Allom group was robust enough during installation of two methylthioethers to the diketopiperazine core and easily removed at the final step. The first total synthesis of (−)-asteroxepin was also completed by acylation of (−)-emestrin H.

Enzyme-Free Electrochemical Biosensor Based on Localized DNA Cascade Displacement Reaction and Versatile DNA Nanosheets for Ultrasensitive Detection of Exosomal MicroRNA.

MicroRNA existing in exosomes (exo-miRNA) is a crucial and reliable biomarker for cancer screening and diagnosis. However, accurate detection of ultralow exo-miRNA amounts in real samples remains a challenge. Herein, a robust and ultrasensitive electrochemical biosensor was developed based on localized DNA cascade displacement reaction (L-DCDR) and versatile DNA nanosheets (DNSs) for enzyme-free analysis of exo-miRNA. The target activated L-DCDR repeatedly by consecutive toehold-mediated strand displacement, which released plentiful P strands to hybridize with capture probes immobilized on the electrode surface and DNS tags, generating an amplified electrochemical signal for the detection of exo-miRNA. The DNS could label-free load various electroactive molecules. The electrochemical biosensor revealed high sensitivity ranging from 0.1 fM to 1 nM with a limit of detection of 65 aM and good specificity. The constructed biosensor was demonstrated to be able to detect exo-miRNA derived from gastric cancer cell line (SGC-7901) and gastric cancer patients. In addition, the developed biosensor possessed several considerable advantages including simple substrate assembly, improved reaction rate, and high signal-to-noise ratio. Therefore, this strategy has great potential in bioanalysis and clinical diagnostics.

Feeding on fungi: genomic and proteomic analysis of the enzymatic machinery of bacteria decomposing fungal biomass

Dead fungal biomass is an abundant source of nutrition in both litter and soil of temperate forests largely decomposed by bacteria. Here, we have examined the utilization of dead fungal biomass by the five dominant bacteria isolated from the in situ decomposition of fungal mycelia using a multiOMIC approach. The genomes of the isolates encoded a broad suite of carbohydrate-active enzymes, peptidases and transporters. In the extracellular proteome, only Ewingella americana expressed chitinases while the two Pseudomonas isolates attacked chitin by lytic chitin monooxygenase, deacetylation and deamination. Variovorax sp. expressed enzymes acting on the side-chains of various glucans and the chitin backbone. Surprisingly, despite its genomic potential, Pedobacter sp. did not produce extracellular proteins to decompose fungal mycelia but presumably feeds on simple substrates. The ecological roles of the five individual strains exhibited complementary features for a fast and efficient decomposition of dead fungal biomass by the entire bacterial community.

Rhodium(II)-catalyzed multicomponent assembly of \u03b1,\u03b1,\u03b1-trisubstituted esters via formal insertion of O\u2013C(sp3)\u2013C(sp2) into C\u2013C bonds

The direct cleavage of C(CO)−C single bonds, delivering otherwise inaccessible compounds, is a significant challenge. Although the transition metal-catalyzed insertion of functional groups into C(CO)−C bonds has been studied, strained ketone substrates or chelating assistance were commonly required. In this article, we describe a rhodium(II)-catalyzed three-component reaction of 1,3-diones, diazoesters, and N,N-dimethylformamide (DMF), leading to an unusual formal insertion of O–C(sp3)–C(sp2) into unstrained C(CO)−C bonds. This procedure provides a rapid entry to a gamut of otherwise inaccessible α,α,α-trisubstituted esters/amide from relatively simple substrates in a straightforward manner. 55 examples of highly decorated products demonstrate the broad functional group tolerance and substrate scope. The combination of control experiments and isotope-labeling reactions support that O, C(sp3), and C(sp2) units derive from 1,3-diones, diazoesters, and DMF, respectively.

A monodomain class II terpene cyclase assembles complex isoprenoid scaffolds.

Class II terpene cyclases, such as oxidosqualene and squalene-hopene cyclases, catalyze some of the most complex polycyclization reactions. They minimally exhibit a β,γ-didomain architecture that has been evolutionarily repurposed in a wide range of terpene-processing enzymes and likely resulted from a fusion of unidentified monodomain proteins. Although single domain class I terpene cyclases have already been identified, single domain class II terpene cyclases have not been previously reported. Here we report high-resolution X-ray structures of a monodomain class II cyclase, merosterolic acid synthase (MstE). With a minimalistic β-domain architecture, this cyanobacterial enzyme is able to construct four rings in cytotoxic meroterpenoids with a sterol-like topology. The structures with bound substrate, product, and inhibitor provide detailed snapshots of a cyclization mechanism largely governed by residues located in a noncanonical enzyme region. Our results complement the few known class II cyclase crystal structures, while also indicating that archaic monodomain cyclases might have already catalyzed complex reaction cascades.

A compact substrate integrated waveguide backed self‐quadruplexing antenna for C‐band communication

This article presents a simple, compact, and lightweight substrate integrated waveguide (SIW) backed self-quadruplexing antenna for quad-band applications. The design procedure is straightforward. Topside of the SIW cavity is modified to form four patches of different lengths which are fed separately by four 50-Ω microstrip feed lines and operate at 5.14, 5.78, 6.74, and 7.74. It attains 4.1, 4.96, 6.2, and 6.1 peak gain at the above frequencies, respectively. The observed front-to-back-ratio is more than 17.5 dB and the isolation level is above 28 dB. This antenna topology allows to redesign each resonant frequencies as per application requirement using a single parameter and without disturbing other performances. Design guidelines for developing the proposed antenna are provided. A prototype antenna is fabricated using RT-Duroid (5870) substrate and characterized for validation. The proposed antenna is suitable for handheld microwave devices for C-band communication.

Chemiluminescent Enzyme Immunoassay and Bioluminescent Enzyme Immunoassay for Tenuazonic Acid Mycotoxin by Exploitation of Nanobody and Nanobody-Nanoluciferase Fusion.

The isolation of nanobodies (Nbs) from phage display libraries is an increasingly effective approach for the generation of new biorecognition elements, which can be used to develop immunoassays. In this study, highly specific Nbs against the Alternaria mycotoxin tenuazonic acid (TeA) were isolated from an immune nanobody phage display library using a stringent biopanning strategy. The obtained Nbs were characterized by classical enzyme-linked immunosorbent assay (ELISA), and the best one Nb-3F9 was fused with nanoluciferase to prepare an advanced bifunctional fusion named nanobody-nanoluciferase (Nb-Nluc). In order to improve the sensitivity and reduce the assay time, two different kinds of luminescent strategies including chemiluminescent enzyme immunoassay (CLEIA) and bioluminescent enzyme immunoassay (BLEIA) were established, respectively, on the basis of the single Nb and the fusion protein Nb-Nluc for TeA detection. The two-step CLEIA was developed on the basis of the same nanobody as ELISA, only with simple substrate replacement from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol. In contrast with CLEIA, the novel BLEIA was conducted in one-step new strategy on the basis of Nb-Nluc and bioluminescent substrate coelenterazine-h (CTZ-h). Their half maximal inhibitory concentration (IC50) values were similar to 8.6 ng/mL for CLEIA and 9.3 ng/mL for BLEIA, which was a 6-fold improvement in sensitivity compared with that of ELISA (IC50 of 54.8 ng/mL). Both of the two assays provided satisfactory recoveries ranging from 80.1%-113.5% in real samples, which showed better selectivity for TeA analogues and other common mycotoxins. These results suggested that Nbs and Nb-Nluc could be used as useful reagents for immunodetection and that the developed CLEIA/BLEIA have great potential for TeA analysis.

Asymmetric Reductive Dicarbofunctionalization of Alkenes via Nickel Catalysis.

Alkenes are an appealing functional group that can be transformed into a variety of structures. Transition-metal catalyzed dicarbofunctionalization of alkenes can efficiently afford products with complex substitution patterns from simple substrates. Under reductive conditions, this transformation can be achieved while avoiding stoichiometric organometallic reagents. Asymmetric difunctionalization of alkenes has been underdeveloped, in spite of its potential synthetic utility. Herein, we present a summary of our efforts to control enantioselectivity for alkene diarylation with a nickel catalyst. This reaction is useful for preparing triarylethanes. The selectivity is enhanced by an N-oxyl radical additive.

Direct C(sp3)-N Radical Coupling: Photocatalytic C-H Functionalization by Unconventional Intermolecular Hydrogen Atom Transfer to Aryl Radical.

An unconventional approach for intermolecular direct C(sp3)-N radical coupling has been developed by photocatalytic C(sp3)-H activation of simple alkyl substrates using O-benzoyl oximes. The selective photocatalytic energy-transfer-driven homolysis followed by decarboxylation generates the persistent iminyl radical and aryl radical, which would undergo an unprecedented intermolecular hydrogen atom abstraction from the alkyl substrate to provide the key C(sp3) radical. Selective radical-radical C-N cross-coupling furnishes imines which are valuable amine building blocks.