Whole-cell spectroscopy is a convenient tool to assist molecular identification of cultivatable marine bacteria and to investigate their adaptive metabolism

Recent developments in whole-cell spectroscopic methods allow rapid characterization of microorganisms of interest to human health, but have yet to be widely applied to marine microbiological studies. In this study of bacteria associated with the kelp Laminaria digitata, we have isolated 18 epiphytic bacterial strains from several thalli, sequenced their 16S rDNA, built corresponding phylogenetic trees, and characterized them using spectroscopic methods. Molecular taxonomy revealed Gram⁺Actinobacteria and Gram⁻Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. Twelve marine reference strains (Gram⁺Firmicutes, and Gram⁻Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes) were treated accordingly. Whole-cell MALDI-TOF MS spectral profiles of 29 of the 30 strains were built into a database against which 16 replicate spectra of each strain were compared and categorized into groups. The proton HR-MAS NMR stack plots allowed visual delineation into taxonomic groups according to their most common peaks, in agreement with identifiable compounds from corresponding D₂O solution spectra. With both methods, these groups corresponded to taxa identified by 16S rDNA sequences, MALDI-TOF MS being more discriminative than HR-MAS NMR. Culture age did not influence the spectral signatures in both approaches. Most cells grown under minimal conditions (VNSS medium) afforded HR-MAS NMR profiles markedly different to those grown in enriched conditions (ZoBell medium), indicating different adaptive metabolic responses between the two media. Spectral signatures obtained under strictly controlled conditions can be used as rapid and reliable tools for taxonomic purposes and as markers of physiological status.     

Comparison of thrombus,gut, and oral microbiomes in Korean patients with ST-elevation myocardial infarction: a case–control study

ST-segment elevation myocardial infarction (STEMI) is characterized by thrombotic coronary artery occlusions caused by atherosclerotic plaque rupture. The gut microbiome potentially contributes to the pathogenesis of coronary artery diseases. This study investigated the microbial diversity and composition of coronary thrombi in STEMI patients and the composition of the thrombus microbiome relative to that of the oral and gut microbiomes. A case–control study was performed with 22 STEMI patients and 20 age- and sex-matched healthy controls. Coronary thrombi were acquired from STEMI patients via manual thrombus aspiration during primary coronary intervention. Oral swab and stool samples were collected from both groups, and 16S rRNA sequencing and metagenomic microbiome analyses were performed. Microbial DNA was detected in 4 of 22 coronary thrombi. Proteobacteria (p) and Bacteroidetes (p) were the most abundant phyla. The oral and gut microbiomes significantly differed between patients and healthy controls. The patient group presented microbial dysbiosis, as follows: a higher relative abundance of Proteobacteria (p) and Enterobacteriaceae (f) in the gut microbiome and a lower abundance of Firmicutes (p) and Haemophilus (g) in the oral microbiome. Furthermore, 4 significantly abundant genera were observed in the coronary thrombus in the patients: Escherichia, 1.25%; Parabacteroides, 0.25%; Christensenella, 0.0%; and Bacteroides, 7.48%. The present results indicate that the relative abundance of the gut and oral microbiomes was correlated with that of the thrombus microbiome.Subject terms: Genetics research, Myocardial infarction     

Comparative Study of Volatile Compounds and Expression of Related Genes in Fruit from Two Apple Cultivars during Different Developmental Stages

Aromatic volatile compounds are important contributors to fruit quality that vary among different cultivars. Herein, headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was used to determine changes in volatile compounds and related gene expression patterns in “Ruixue” and “Fuji” apples (Malus domestica Borkh.) during fruit development and maturation. Volatile compounds detected in the fruit of both cultivars exhibited similar trends across different developmental stages. In the early stages of “Ruixue” fruit development (60 days after full bloom), there were fewer volatile compounds, mainly aldehydes (87.0%). During fruit maturation (180 days after full bloom), the types and amounts of volatile compounds increased, mainly including esters (37.6%), and alkenes (23.2%). The total volatile concentration, the types of major volatile compounds, and their relative content in both cultivars varied across different stages. Gene expression analysis indicated that the upregulation of MdLOX, MdAAT2, and MdADH3 was associated with increased aroma compound content, especially esters, during fruit development in both cultivars. Changes in the expression of MdArAT, MdACPD, MdADH3, MdAAT2, and MdLOX may lead to differences in volatile compounds between apple cultivars.     

Microbiological Study in Petrol-Spiked Soil

The pollution of arable lands and water with petroleum-derived products is still a valid problem, mainly due the extensive works aimed to improve their production technology to reduce fuel consumption and protect engines. An example of the upgraded fuels is the BP 98 unleaded petrol with Active technology. A pot experiment was carried out in which Eutric Cambisol soil was polluted with petrol to determine its effect on the microbiological and biochemical properties of this soil. Analyses were carried out to determine soil microbiome composition—with the incubation and metagenomic methods, the activity of seven enzymes, and cocksfoot effect on hydrocarbon degradation. The following indices were determined: colony development index (CD); ecophysiological diversity index (EP); index of cocksfoot effect on soil microorganisms and enzymes (IF_G); index of petrol effect on soil microorganisms and enzymes (IF_P); index of the resistance of microorganisms, enzymes, and cocksfoot to soil pollution with petrol (RS); Shannon–Weaver’s index of bacterial taxa diversity (H); and Shannon–Weaver’s index of hydrocarbon degradation (ID_H). The soil pollution with petrol was found to increase population numbers of bacteria and fungi, and Protebacteria phylum abundance as well as to decrease the abundance of Actinobacteria and Acidobacteria phyla. The cultivation of cocksfoot on the petrol-polluted soil had an especially beneficial effect mainly on the bacteria belonging to the Ramlibacter, Pseudoxanthomonas, Mycoplana, and Sphingobium genera. The least susceptible to the soil pollution with petrol and cocksfoot cultivation were the bacteria of the following genera: Kaistobacter, Rhodoplanes, Bacillus, Streptomyces, Paenibacillus, Phenylobacterium, and Terracoccus. Cocksfoot proved effective in the phytoremediation of petrol-polluted soil, as it accelerated hydrocarbon degradation and increased the genetic diversity of bacteria. It additionally enhanced the activities of soil enzymes.     

Chlovalicin B,a Chlorinated Sesquiterpene Isolated from the Marine Mushroom Digitatispora marina

As part of our search for bioactive metabolites from understudied marine microorganisms, the new chlorinated metabolite chlovalicin B (1) was isolated from liquid cultures of the marine basidiomycete Digitatispora marina, which was collected and isolated from driftwood found at Vannøya, Norway. The structure of the novel compound was elucidated by spectroscopic methods including 1D and 2D NMR and analysis of HRMS data, revealing that 1 shares its molecular scaffold with a previously isolated compound, chlovalicin. This represents the first compound isolated from the Digitatispora genus, and the first reported fumagillin/ovalicin-like compound isolated from Basidiomycota. Compound 1 was evaluated for antibacterial activities against a panel of five bacteria, its ability to inhibit bacterial biofilm formation, for antifungal activity against Candida albicans, and for cytotoxic activities against malignant and non-malignant human cell lines. Compound 1 displayed weak cytotoxic activity against the human melanoma cell line A2058 (~50% survival at 50 µM). No activity was detected against biofilm formation or C. albicans at 50 µM, or against bacterial growth at 100 µM nor against the production of cytokines by the human acute monocytic leukemia cell line THP-1 at 50 µM.     

Aronia melanocarpa (Michx.) Elliot,Chaenomeles superba Lindl. and Cornus mas L. Leaf Extracts as Natural Preservatives for Pork Meat Products

The aim of this study was to investigate the possibility of using Aronia melanocarpa, Chaenomeles superba, and Cornus mas leaf extracts as natural preservatives for pork meat products. Pork sausages were stored in modified atmosphere packaging (MAP) (80% N₂ and 20% CO₂) at 4 °C for 29 days. The total psychrotrophic counts (TPC) were determined during the storage period, along with the numbers of Enterobacteriaceae and lactic acid bacteria (LAB). The extracts improved the microbial quality of the meat products but to a lesser extent than sodium nitrate (III). They reduced the amounts of Enterobacteriaceae and LAB. The A. melanocarpa leaf extract showed the strongest preservative effect. The bacterial biodiversity of the meat products was investigated based on high-throughput sequencing of the 16S rRNA gene. Two predominant bacteria phyla were identified, Proteobacteria and Firmucutes, mostly consisting of genera Photobacterium, Brochothrix, and Carnobacterium. The extracts also influenced microbial community in sausages decreasing or increasing bacterial relative abundance. The extracts significantly inhibited lipid oxidation and improved the water-holding capacity of the meat, with C. superba extract showing the strongest influence. In addition, A. melanocarpa and C. superba improved the redness (a*) of the sausages. The results of this study show that A. melanocarpa, C. superba, and C. mas leaf extracts can extend the shelf life of meat products stored in MAP at 4 °C.     

Miniaturized Salinity Gradient Energy Harvesting Devices

Harvesting salinity gradient energy, also known as “osmotic energy” or “blue energy”, generated from the free energy mixing of seawater and fresh river water provides a renewable and sustainable alternative for circumventing the recent upsurge in global energy consumption. The osmotic pressure resulting from mixing water streams with different salinities can be converted into electrical energy driven by a potential difference or ionic gradients. Reversed-electrodialysis (RED) has become more prominent among the conventional membrane-based separation methodologies due to its higher energy efficiency and lesser susceptibility to membrane fouling than pressure-retarded osmosis (PRO). However, the ion-exchange membranes used for RED systems often encounter limitations while adapting to a real-world system due to their limited pore sizes and internal resistance. The worldwide demand for clean energy production has reinvigorated the interest in salinity gradient energy conversion. In addition to the large energy conversion devices, the miniaturized devices used for powering a portable or wearable micro-device have attracted much attention. This review provides insights into developing miniaturized salinity gradient energy harvesting devices and recent advances in the membranes designed for optimized osmotic power extraction. Furthermore, we present various applications utilizing the salinity gradient energy conversion.     

Synthesis of Isothiocyanates Using DMT/NMM/TsO− as a New Desulfurization Reagent

Thirty-three alkyl and aryl isothiocyanates, as well as isothiocyanate derivatives from esters of coded amino acids and from esters of unnatural amino acids (6-aminocaproic, 4-(aminomethyl)benzoic, and tranexamic acids), were synthesized with satisfactory or very good yields (25–97%). Synthesis was performed in a “one-pot”, two-step procedure, in the presence of organic base (Et₃N, DBU or NMM), and carbon disulfide via dithiocarbamates, with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO⁻) as a desulfurization reagent. For the synthesis of aliphatic and aromatic isothiocyanates, reactions were carried out in a microwave reactor, and selected alkyl isothiocyanates were also synthesized in aqueous medium with high yields (72–96%). Isothiocyanate derivatives of L- and D-amino acid methyl esters were synthesized, under conditions without microwave radiation assistance, with low racemization (er 99 > 1), and their absolute configuration was confirmed by circular dichroism. Isothiocyanate derivatives of natural and unnatural amino acids were evaluated for antibacterial activity on E. coli and S. aureus bacterial strains, where the most active was ITC 9e.     

Secondary Metabolites of the Genus Amycolatopsis: Structures,Bioactivities and Biosynthesis

Actinomycetes are regarded as important sources for the generation of various bioactive secondary metabolites with rich chemical and bioactive diversities. Amycolatopsis falls under the rare actinomycete genus with the potential to produce antibiotics. In this review, all literatures were searched in the Web of Science, Google Scholar and PubMed up to March 2021. The keywords used in the search strategy were “Amycolatopsis”, “secondary metabolite”, “new or novel compound”, “bioactivity”, “biosynthetic pathway” and “derivatives”. The objective in this review is to summarize the chemical structures and biological activities of secondary metabolites from the genus Amycolatopsis. A total of 159 compounds derived from 8 known and 18 unidentified species are summarized in this paper. These secondary metabolites are mainly categorized into polyphenols, linear polyketides, macrolides, macrolactams, thiazolyl peptides, cyclic peptides, glycopeptides, amide and amino derivatives, glycoside derivatives, enediyne derivatives and sesquiterpenes. Meanwhile, they mainly showed unique antimicrobial, anti-cancer, antioxidant, anti-hyperglycemic, and enzyme inhibition activities. In addition, the biosynthetic pathways of several potent bioactive compounds and derivatives are included and the prospect of the chemical substances obtained from Amycolatopsis is also discussed to provide ideas for their implementation in the field of therapeutics and drug discovery.     

Short-chain reactive probes as tools to unravel the Pseudomonas aeruginosa quorum sensing regulon

In recent years, the world has seen a troubling increase in antibiotic resistance among bacterial pathogens. In order to provide alternative strategies to combat bacterial infections, it is crucial deepen our understanding into the mechanisms that pathogens use to thrive in complex environments. Most bacteria use sophisticated chemical communication systems to sense their population density and coordinate gene expression in a collective manner, a process that is termed “quorum sensing” (QS). The human pathogen Pseudomonas aeruginosa uses several small molecules to regulate QS, and one of them is N-butyryl-l-homoserine lactone (C₄-HSL). Using an activity-based protein profiling (ABPP) strategy, we designed biomimetic probes with a photoreactive group and a ‘click’ tag as an analytical handle. Using these probes, we have identified previously uncharacterized proteins that are part of the P. aeruginosa QS network, and we uncovered an additional role for this natural autoinducer in the virulence regulon of P. aeruginosa, through its interaction with PhzB1/2 that results in inhibition of pyocyanin production.

Short-chain reactive probes can be used as tools to shed new light on virulence mechanisms in bacterial pathogens.     

Effects of sludge retention time on membrane fouling and microbial community structure in a membrane bioreactor

In this study, the effect of sludge retention time (SRT) on membrane bio-fouling was investigated in a membrane bioreactor (MBR) equipped with a sequential anoxic/anaerobic reactor. Specific cake resistance (α), trans-membrane pressure (TMP), mixed liquor suspended solids (MLSS), particle sizes, extracellular polymer substances bound in sludge (bound-EPS) and their correlations with membrane bio-fouling were studied at different SRTs. As SRT decreased to 20 days, the bound-EPS per unit of biomass increased, and consequently, the value of α increased, which resulted in the rise of TMP. However, the reduction of the bound-EPS content was relatively small as compared to a significant decrease in the value of α at longer SRTs (above 60 days). These observations suggest that colloidal particles significantly contribute to membrane bio-fouling. In addition, the diversity of the microbial community structure of activated sludge in the MBRs was observed using the respiratory quinone profile. The ubiquinone species containing UQ-8, belonging to the class β-Proteobacteria type were the major constituents of the microbial community structure. The mole fraction of menaquinone MK-6, -7 and -8(H2) increased as SRT increased. Thus, the results of this study indicate that growth of microorganisms belonging to the δ- and ?-subclass of Proteobacteria as well as the members of the Cytophaga–Flavobacterium cluster increased at longer SRTs.     

Activity and Diversity of Microorganisms in Root Zone of Plant Species Spontaneously Inhabiting Smelter Waste Piles

The aim was to assess plant driven changes in the activity and diversity of microorganisms in the top layer of the zinc and lead smelter waste piles. The study sites comprised two types (flotation waste—FW and slag waste—SW) of smelter waste deposits in Piekary Slaskie, Poland. Cadmium, zinc, lead, and arsenic contents in these technosols were extremely high. The root zone of 8 spontaneous plant species (FW—Thymus serpyllum, Silene vulgaris, Solidago virgaurea, Echium vulgare, and Rumex acetosa; and SW—Verbascum thapsus; Solidago gigantea, Eupatorium cannabinum) and barren areas of each waste deposit were sampled. We observed a significant difference in microbial characteristics attributed to different plant species. The enzymatic activity was mostly driven by plant-microbial interactions and it was significantly greater in soil affected by plants than in bulk soil. Furthermore, as it was revealed by BIOLOG Ecoplate analysis, microorganisms inhabiting barren areas of the waste piles rely on significantly different sources of carbon than those found in the zone affected by spontaneous plants. Among phyla, Actinobacteriota were the most abundant, contributing to at least 25% of the total abundance. Bacteria belonging to Blastococcus genera were the most abundant with the substantial contribution of Nocardioides and Pseudonocardia, especially in the root zone. The contribution of unclassified bacteria was high—up to 38% of the total abundance. This demonstrates the unique character of bacterial communities in the smelter waste.     

Phylogenetic characterization of a mixed microbial community capable of degrading carbon tetrachloride

Two bacterial communities (NO92 and GBS) capable of degrading carbon tetrachloride (CT) were enriched from in-house CT-contaminated water. These communities are able to degrade CT in the presence of toluene. To characterize the community structure and diversity, one enrichment (NO92) was subjected to 16S ribosomal RNA (rRNA) gene-based molecular analysis. The 16S rRNA genes were amplified from the bulk genomic community DNA and cloned into plasmid vectors. Unique 16S rRNA gene clones, i.e., phylotypes, were detected by four tetrameric restriction enzymes. Together, 123 16S rRNA gene clones were obtained; thirty-one showed different restriction fragment length polymorphism (RFLP) patterns. About 73% of the clones belong to two dominant RFLP patterns. Phylogenetic analysis based on the partial 16S rRNA gene sequences of 10 major phylotypes showed that all the phylotypes that were sequenced were affiliated with the high G+C Gram-positive bacteria. Whereas seven of the phylotypes (∼80% of the clones) were closely related to Rhodococcus, the other three (∼5% of the clones) were related to Curtobacterium. These results suggest that this CT-degrading community is diverse but is predominated by closely related bacterial groups.     

Development of a Fast and Robust UHPLC Method for Apixaban In-Process Control Analysis

In-process control (IPC) is an important task during chemical syntheses in pharmaceutical industry. Despite the fact that each chemical reaction is unique, the most common analytical technique used for IPC analysis is high performance liquid chromatography (HPLC). Today, the so-called “Quality by Design” (QbD) principle is often being applied rather than “Trial and Error” approach for HPLC method development. The QbD approach requires only for a very few experimental measurements to find the appropriate stationary phase and optimal chromatographic conditions such as the composition of mobile phase, gradient steepness or time (tG), temperature (T), and mobile phase pH. In this study, the applicability of a multifactorial liquid chromatographic optimization software was studied in an extended knowledge space. Using state-of-the-art ultra-high performance liquid chromatography (UHPLC), the analysis time can significantly be shortened. By using UHPLC, it is possible to analyse the composition of the reaction mixture within few minutes. In this work, a mixture of route of synthesis of apixaban was analysed on short narrow bore column (50 × 2.1 mm, packed with sub-2 µm particles) resulting in short analysis time. The aim of the study was to cover a relatively narrow range of method parameters (tG, T, pH) in order to find a robust working point (zone). The results of the virtual (modeled) robustness testing were systematically compared to experimental measurements and Design of Experiments (DoE) based predictions.     

Carbene catalyzed umpolung of α,β-enals: a reactivity study of diamino dienols vs. azolium enolates,and the characterization of advanced reaction intermediates

Since their discovery by Bode and Glorius in 2004, N-heterocyclic carbene catalyzed conjugate umpolung reactions of α,β-enals have been postulated to involve the formation of diamino dienols (“homoenolates”) and/or azolium enolates (“enolates”), typically followed by addition to electrophiles, e.g. Michael-acceptors. In this article, we provide evidence, for the first time, for the postulated individual and specific reactivity patterns of diamino dienols (γ-C–C-bond formation) vs. azolium enolates (β-C–C-bond formation). Our study is based on the pre-formation of well defined diamino dienols and azolium enolates, and the in situ NMR monitoring of their reactivities towards enone electrophiles. Additionally, reaction intermediates were isolated and characterized, inter alia by X-ray crystallography.     

Determination of nanogram levels of lanthanoids in a marine macro-alga by neutron activation analysis combined with separation by selective precipitation

Preseparation of lanthanoids by substoichiometric precipitation of calcium oxalate and simple radiochemical separation of lanthanoids by lanthanum oxalate have been developed. They were combined with neutron activation analysis of a marine macro-alga (Laminaria religiosa, brown alga) sample. Quantitative recovery of lanthanoids throughout the procedure was examined by radiotracer technique. Eleven lanthanoids, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Er, Yb and Lu, at 0.7–140 ng g^–1 were determined with a relative standard deviation of 1–7% (n=3). Concentrations of lanthanoids in the marine macro-alga were compared with land plant and sea water.     

Molecular Umbrella as a Nanocarrier for Antifungals

A molecular umbrella composed of two O-sulfated cholic acid residues was applied for the construction of conjugates with cispentacin, containing a “trimethyl lock” (TML) or o-dithiobenzylcarbamoyl moiety as a cleavable linker. Three out of five conjugates demonstrated antifungal in vitro activity against C. albicans and C. glabrata but not against C. krusei, with MIC₉₀ values in the 0.22–0.99 mM range and were not hemolytic. Antifungal activity of the most active conjugate 24c, containing the TML–pimelate linker, was comparable to that of intact cispentacin. A structural analogue of 24c, containing the Nap-NH₂ fluorescent probe, was accumulated in Candida cells, and TML-containing conjugates were cleaved in cell-free extract of C. albicans cells. These results suggest that a molecular umbrella can be successfully applied as a nanocarrier for the construction of cleavable antifungal conjugates.     

Is a polymer semiconductor having a “perfect” regular structure desirable for organic thin film transistors?

This study utilized high temperature NMR and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry to reveal that appreciable amounts of structural defects are present in the diketopyrrolopyrrole (DPP)–quaterthiophene copolymers (PDQT) synthesized by the Stille coupling polymerization with Pd(PPh₃)₂Cl₂, Pd₂(dba)₃/P(o-tol)₃, and Pd(PPh₃)₄ catalyst systems. It was proposed that these structural defects were produced via homocoupling side reactions of the C–Br bonds and the organostannane species. Model Stille coupling reactions further substantiated that the amount of structural defects are catalyst-dependent following the order of Pd(PPh₃)₂Cl₂ > Pd₂(dba)₃/P(o-tol)₃ > Pd(PPh₃)₄. To verify the structural assignments, “perfect” structurally regular PDQT polymers were prepared using Yamamoto coupling polymerization. When compared to the structurally regular polymers, the polymers containing defects exhibited notable redshifts in their absorption spectra. Surprisingly, the “perfect” structurally regular polymers showed poor molecular ordering in thin films and very low charge transport performance as channel semiconductors in organic thin film transistors (OTFTs). On the contrary, all the “defected” polymers exhibited much improved molecular ordering and significantly higher charge carrier mobility.