Evaluation of the enantioselectivity of new chiral ligands based on imidazolidin-4-one derivatives.

The new chiral ligands I-III based on derivatives of imidazolidin-4-one were synthesised and characterised. The catalytic activity and enantioselectivity of their corresponding copper(II) complexes were studied in asymmetric Henry reactions. It was found that the enantioselectivity of these catalysts is overall very high and depends on the relative configuration of the ligand used; cis-configuration of ligand affords the nitroaldols with major enantiomer S- (up to 97% ee), whereas the application of ligands with trans-configuration led to nitroaldols with major R-enantiomer (up to 96% ee). The "proline-type" ligand IV was also tested in asymmetric aldol reactions. Under the optimised reaction conditions, aldol products with enantioselectivities of up to 91% ee were obtained.

Prevalence of SARS-CoV-2 variants in Prague wastewater determined by nanopore-based sequencing.

The early detection of upcoming disease outbreaks is essential to avoid both health and economic damage. The last four years of COVID-19 pandemic have proven wastewater-based epidemiology is a reliable system for monitoring the spread of SARS-CoV-2, a causative agent of COVID-19, in an urban population. As this monitoring enables the identification of the prevalence of spreading variants of SARS-CoV-2, it could provide a critical tool in the fight against this viral disease. In this study, we evaluated the presence of variants and subvariants of SARS-CoV-2 in Prague wastewater using nanopore-based sequencing. During August 2021, the data clearly showed that the number of identified SARS-CoV-2 RNA copies increased in the wastewater earlier than in clinical samples indicating the upcoming wave of the Delta variant. New SARS-CoV-2 variants consistently prevailed in wastewater samples around a month after they already prevailed in clinical samples. We also analyzed wastewater samples from smaller sub-sewersheds of Prague and detected significant differences in SARS-CoV-2 lineage progression dynamics among individual localities studied, e.g., suggesting faster prevalence of new variants among the sites with highest population density and mobility.

Evaluation of Membrane Integrity Monitoring Methods for Hollow Fiber Nanofiltration Membranes: Applicability in Gray Water Reclamation Systems.

Source-separated gray water reclamation using nanofiltration as an advanced post-treatment option has received substantial interest in meeting the growing water demand. During reclamation, membrane integrity is crucial to ensure the water's safety. This study evaluated several chemical and novel microbial indicators as indirect membrane integrity-monitoring methods for hollow fiber nanofiltration membranes in reclamation schemes. Under normal conditions, high retention of divalent ions and organic matter and near-complete removal of Escherichia coli (E. coli) were observed. Limited removal of the antibiotic gene (ARG) tetO was observed due to low feed concentrations and a higher detection limit (LOD). While 16S rRNA and ARG sul1 were not limited by their LODs, lower removals were observed, most likely due to free-floating DNA passing through the membranes. A broken fiber in a pilot-scale module reduced organic matter and microorganism removal substantially, while flux and ion rejection remained similar. Predictions made using the observed results and a previously proposed model allowed for the evaluation of the selected methods in upscaled reclamation systems. Based on these results, it was concluded that microorganisms could be employed as indicators in indirect membrane integrity-monitoring methods in large-scale reclamation schemes, while UV254nm absorbance (used in organic matter determination) could be a viable solution in pilot-scale systems.

Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environment.

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.

Immobilization of Trifluoromethyl-Substituted Pyridine-Oxazoline Ligand and Its Application in Asymmetric Continuous Flow Synthesis of Benzosultams.

This study presents an improved synthetic route to ligand (S)-4-(tert-butyl)-2-(5-(trifluoromethyl)pyridin-2-yl)-4,5-dihydrooxazole and its application as a highly active and enantioselective catalyst in the addition of arylboronic acids to cyclic N-sulfonylketimines. Immobilization of such a ligand was achieved using a commercially available starting material and a PS-PEG TentaGel S NH2 support, resulting in a stable heterogeneous catalyst. Although the anchored catalyst exhibited a slight reduction in enantioselectivity and a 4-fold decrease in reaction rate, it displayed remarkable stability, enabling 10 consecutive reaction cycles. Furthermore, the successful transition to a continuous flow system demonstrated even higher turnover numbers compared to batch arrangements. These findings provide valuable insights into the development of efficient flow reactors for continuous synthesis of benzosultams, further advancing the field of asymmetric catalysis.

Monitoring of monkeypox viral DNA in Prague wastewater.

Monkeypox virus (Mpxv) is a dsDNA virus that has become a global concern for human health in 2022. As both infected people and non-human hosts can shed the virus from their skin, faeces, urine and other body fluids, and the resulting sewage contains viral load representative of the whole population, it is highly promising to detect the spread of monkeypox virus in municipal wastewater. We established a methodology for sewage-based monitoring of Mpxv in Prague and analysed samples (n = 24) already early August-October of 2022 in a municipality with 1.4 million inhabitants that only reported 29 cumulative cases in this period. We isolated Mpxv DNA with the Wizard Enviro Total Nucleic Acid Kit, and thereafter detected Mpxv DNA using the EliGene® Monkeypox RT-PCR Kit. Prague wastewater was positive for Mpxv (in total 9 positive samples in periods with 1-9 new cases per week, coinciding with a weekly incidence of 0.07-0.64 per 100,000 inhabitants. The method for confirmation of wastewater positivity via semi-nested PCR and Sanger sequencing was successfully confirmed on positive controls including Mpxv particles and Mpxv-positive wastewater from the Netherlands. However, for Prague wastewater samples, amplification of Mpxv DNA via semi-semi-nested PCR was unsuccessful. This was probably due to extremely low case count, leading to the amplification of non-target bacterial DNA. Compared to other studies with much higher Mpxv prevalence, we show the outstanding sensitivity of our approach for monitoring the spread of monkeypox using wastewater.

Switchable cyclopalladation of substrates containing two directing groups: on the way to non-symmetrical [2.2]-dipalladaparacyclophanes.

Simple switching of the site-selectivity of C-H activation reactions of substrates containing multiple directing groups is particularly important for the so-called late stage functionalization synthetic approach. In this work, we verified the possibility of achieving this by adding acids of different strengths. Using a substrate containing two differently strong (and basic) directing groups, the influence of the addition of acids on the regioselectivity of the C-H activation step of the reaction with palladium acetate was thoroughly studied. The addition of no or weak acids results in cyclopalladation being controlled by a stronger directing group. However, the addition of a strong acid causes protonation of this group and the reaction is then controlled by a weaker directing group. Finally, this approach enables double C-H activation leading to a unique class of compounds: "non-symmetrical" [2.2]-dipalladaparacyclophanes.

Monitoring COVID-19 spread in Prague local neighborhoods based on the presence of SARS-CoV-2 RNA in wastewater collected throughout the sewer network.

Many reports have documented that the presence of SARS-CoV-2 RNA in the influents of municipal wastewater treatment plants (WWTP) correlates with the actual epidemic situation in a given city. However, few data have been reported thus far on measurements upstream of WWTPs, i.e. throughout the sewer network. In this study, the monitoring of the presence of SARS-CoV-2 RNA in Prague wastewater was carried out at selected locations of the Prague sewer network from August 2020 through May 2021. Various locations such as residential areas of various sizes, hospitals, city center areas, student dormitories, transportation hubs (airport, bus terminal), and commercial areas were monitored together with four of the main Prague sewers. The presence of SARS-CoV-2 RNA was determined by reverse transcription - multiplex quantitative polymerase chain reaction (RT-mqPCR) after the precipitation of nucleic acids with PEG 8,000 and RNA isolation with TRIzol™ Reagent. The number of copies of the gene encoding SARS-CoV-2 nucleocapsid (N1) per liter of wastewater was compared with the number of officially registered COVID-19 cases in Prague. Although the data obtained by sampling wastewater from the major Prague sewers were more consistent than those obtained from the small sewers, the correlation between wastewater-based and clinical-testing data was also good for the residential areas with more than 7,000 registered inhabitants. It was shown that monitoring SARS-CoV-2 RNA in wastewater sampled from small sewers could identify isolated occurrences of COVID-19-positive cases in local neighborhoods. This can be very valuable while tracking COVID-19 hotspots within large cities.

Effect of temperature on the compositions of ladderane lipids in globally surveyed anammox populations.

The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC-HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10-37 °C. "Candidatus Brocadia" appeared to be the dominant organism in all but two laboratory enrichments of "Ca. Scalindua" and "Ca. Kuenenia". At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. "Ca. Scalindua" and "Ca. Kuenenia" had distinct profile of ladderane lipids compared to "Ca. Brocadia" biomasses with potential implications for adaptability to low temperatures. "Ca. Brocadia" membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for "Ca. Scalindua" at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of "Ca. Scalindua" in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

Diphyllin Shows a Broad-Spectrum Antiviral Activity against Multiple Medically Important Enveloped RNA and DNA Viruses.

Diphyllin is a natural arylnaphtalide lignan extracted from tropical plants of particular importance in traditional Chinese medicine. This compound has been described as a potent inhibitor of vacuolar (H+)ATPases and hence of the endosomal acidification process that is required by numerous enveloped viruses to trigger their respective viral infection cascades after entering host cells by receptor-mediated endocytosis. Accordingly, we report here a revised, updated, and improved synthesis of diphyllin, and demonstrate its antiviral activities against a panel of enveloped viruses from Flaviviridae, Phenuiviridae, Rhabdoviridae, and Herpesviridae families. Diphyllin is not cytotoxic for Vero and BHK-21 cells up to 100 µM and exerts a sub-micromolar or low-micromolar antiviral activity against tick-borne encephalitis virus, West Nile virus, Zika virus, Rift Valley fever virus, rabies virus, and herpes-simplex virus type 1. Our study shows that diphyllin is a broad-spectrum host cell-targeting antiviral agent that blocks the replication of multiple phylogenetically unrelated enveloped RNA and DNA viruses. In support of this, we also demonstrate that diphyllin is more than just a vacuolar (H+)ATPase inhibitor but may employ other antiviral mechanisms of action to inhibit the replication cycles of those viruses that do not enter host cells by endocytosis followed by low pH-dependent membrane fusion.

Development and validation of novel PCR primers for identification of plasmid-mediated colistin resistance (mcr) genes in various environmental settings.

Antibiotic resistance is considered one of the biggest threats to public health and has become a major concern for governments and international organizations. Combating this problem starts with improving global surveillance of antibiotic resistance genes (ARGs) and applying standardized protocols, both in a clinical and environmental context, in agreement with the One Health approach. Exceptional efforts should be directed to controlling ARGs conferring resistance to Critically Important Antimicrobials (CIA). In this study, a systematic literature review to synthesize data on the identification of mcr genes using a PCR technique was performed. Additionally, a novel set of PCR primers for mcr-1 - mcr-9 genes detection was proposed. The developed primers were in silico and experimentally validated by comparison with mcr-specific PCR primers reported in the literature. This validation, besides being a proof-of-concept for primers' usefulness, provided insight into the distribution of mcr genes in municipal wastewater, clay and river sediments, glacier moraine, manure, seagulls and auks feces and daphnids from four countries. This analysis proved that commonly used primers may deliver false results, and some mcr genes may be overlooked in tested samples. Newly-developed PCR primers turned out to be relevant for the screening of mcr genes in various environments.

Adaptation of flocculent anammox culture to low temperature by cold shock: long-term response of the microbial population.

Partial nitritation-anammox (PN/A) process will substantially reduce the costs for the removal of nitrogen in the mainstream of municipal sewage. However, one of the mainstream PN/A challenges is to reduce the time necessary for the adaptation of anammox bacteria to lower temperatures in mild climates. In this study, we exposed anammox flocculent culture to cold shocks [35°C → 5°C (8 h) → 15°C] and evaluated long-term cold shock response. Over a post-shock period of 40 d at 15°C, the nitrogen removal rates in the shocked culture were significantly higher compared to control, with maximum rates up to 0.082 and 0.033 kg-N/kg-VSS/d or 0.164 and 0.076 kg-N/m3/d, for shocked culture and control, respectively. In the corresponding semi-batch cycles, the shocked culture was on average 136 ± 101% more active than the control, due to the negative effect of cold shock on side populations and more active anammox cells. Per FISH, Ca. Brocadia anammoxidans and Ca. Scalindua survived the shock and remained present throughout. Thus, both anammox microorganisms seem to respond favourably to cold shocks. In sum, we provide further evidence that cold shocks accelerate the adaptation of anammox to the mainstream of municipal WWTPs. Further, for the first time, we report the long-term adaptive response of anammox to cold shocks.

Recent advances in palladium-catalysed asymmetric 1,4-additions of arylboronic acids to conjugated enones and chromones.

The transition metal (palladium)-catalysed asymmetric 1,4-addition of arylboronic acids to conjugated enones belong to the most important and emerging strategies for the construction of C-C bonds in an asymmetric fashion. This review covers known catalytic systems used for this transformation. For clarity, we are using the type of ligand as a sorting criterion. Finally, we attempted to create a flowchart facilitating the selection of a suitable ligand for a given combination of enone and arylboronic acid.

Maximizing energy recovery from wastewater via bioflocculation-enhanced primary treatment: a pilot scale study.

Anaerobic digestion of municipal sewage sludge is widely used for harvesting energy from wastewater organic content. The more organic carbon we can redirect into the primary sludge, the less energy is needed for aeration in secondary treatment and the more methane is produced in anaerobic digesters. Bioflocculation has been proposed as a promising separation technology to maximize carbon capture in primary sludge. Thus far, only limited data on bioflocculation are available under real conditions, i.e. from pilot-scale reactors treating raw sewage. Moreover, no study has discussed yet the influence of bioflocculation on denitrification potential of sewage. Therefore, we performed bioflocculation of raw sewage in high-rate contact stabilization process in pilot-scale to investigate maximal primary treatment efficiency. During 100 days of operation at sludge retention time of only 2 days, the average removal efficiencies of chemical oxygen demand (COD), suspended solids and total phosphorus were 75%, 87% and 51%, respectively, using no chemicals for precipitation. Up to 76% of incoming COD was captured in primary sludge and 46% for subsequent anaerobic digestion, where energy recovery potential achieved 0.33-0.37 g COD as CH4 per g COD of influent. This study showed in real conditions that this newly adapted separation process has significant benefits over chemically enhanced primary treatment, enabling sewage treatment process to overcome energy self-sufficiency.

Disruption of Cell Adhesion and Cytoskeletal Networks by Thiol-Functionalized Silica-Coated Iron Oxide Nanoparticles.

One of the major obstacles that limits the use of magnetic nanoparticles in biomedical applications is their potential toxicity. In the present study, we evaluated the cytotoxic effects of thiol-functionalized silica-coated iron oxide (Fe3O4@SiO2-SH) nanoparticles using human lung epithelial cells A549. We investigated the effect of Fe3O4@SiO2-SH nanoparticles on the cell viability, proliferation, cell cycle distribution, adhesion, apoptosis, and the orientation of the cytoskeletal networks, as well as on expression of proteins involved in cell death, cell survival, and cell adhesion. We demonstrated that exposure of A549 cells to Fe3O4@SiO2-SH nanoparticles resulted in severe disruption of the actin microfilaments and microtubule cytoskeleton and reduced the size of focal adhesions. Furthermore, cell adhesion was significantly affected as well as the phosphorylation of focal adhesion kinase (FAK), extracellular-signal-regulated kinase (ERK), and p38. Our findings highlight the need for in-depth cytotoxic evaluation of nanoparticles supporting their safer use, especially in biomedical applications.

Micro-Oxygenation in Upflow Anaerobic Sludge Bed (UASB) Reactors Using a Silicon Membrane for Sulfide Oxidation.

Sulfide produced by sulphate-reducing bacteria in anaerobic reactors can seriously affect biogas quality. Microaeration has become a reliable way to remove sulfide, by promoting its oxidation. However, limited research is available regarding its application in upflow anaerobic sludge bed (UASB) reactors. In this research, silicon membranes were studied as a mechanism to dose oxygen in USAB reactors. Two configurations were tested: the membrane placed inside the reactor or in an external module. Our results show that the external membrane proved to be a more practical alternative, providing conditions for sulfide oxidation. This led to a reduction in its concentration in the liquid effluent and biogas. External membrane configuration achieved a sulfide conversion rate of 2.4 g-S m2 d-1. Since the membrane was not sulfide-selective, methane losses were observed (about 9%). In addition, excessive oxygen consumption was observed, compared to the stoichiometric requirement. As is the case for many membrane-based systems, membrane area is a key factor determining the correct operation of the system.

Partial integration of ADM1 into CFD: understanding the impact of diffusion on anaerobic digestion mixing.

Sufficient mixing is crucial for the proper performance of anaerobic digestion (AD), creating a homogeneous distribution of soluble substrates, biomass, pH, and temperature. The opaqueness of the sludge and mode of operation make it challenging to study AD mixing experimentally. Therefore, hydrodynamics modelling employing computational fluid dynamics (CFD) is often used to investigate this mixing. However, CFD models mostly do not include biochemical reactions and, hence, ignore the effect of diffusion-induced transport on AD heterogeneity. The novelty of this work is the partial integration of Anaerobic Digestion Model no. 1 (ADM1) into the CFD model. The aim is to better understand the effect of advection-diffusion transport on the homogenization of soluble substrates and biomass. Furthermore, AD homogeneity analysis in terms of concentration distribution is proposed rather than the traditional velocity distributions. The computed results indicate that including diffusion-induced transport affects the homogeneity of AD.

Silica coated iron oxide nanoparticles-induced cytotoxicity, genotoxicity and its underlying mechanism in human HK-2 renal proximal tubule epithelial cells.

Iron oxide nanoparticles (IONPs) have a great potential with regard to cell labelling, cell tracking, cell separation, magnetic resonance imaging, magnetic hyperthermia, targeted drug and gene delivery. However, a growing body of research has raised concerns about the possible unwanted adverse cytotoxic effects of IONPs. In the present study, the in vitro cellular uptake, antiproliferative activity, cytotoxicity, genotoxicity, prooxidant, microtubule-disrupting and apoptosis-inducing effect of Fe3O4@SiO2 and passivated Fe3O4@SiO2-NH2 nanoparticles on human renal proximal tubule epithelial cells (HK-2) have been studied. Both investigated silica coated IONPs were found to have cell growth-inhibitory activity in a time- and dose-dependent manner. Determination of cell cycle phase distribution by flow cytometry demonstrated a G1 and G2/M phase accumulation of HK-2 cells. A tetrazolium salt cytotoxicity assay at 24 h following treatment demonstrated that cell viability was reduced in a dose-dependent manner. Microscopy observations showed that both Fe3O4@SiO2 and Fe3O4@SiO2-NH2 nanoparticles accumulated in cells and appeared to have microtubule-disrupting activity. Our study also revealed that short term 1 h exposure to 25 and 100 µg/mL of silica coated IONPs causes genotoxicity. Compared with vehicle control cells, a significantly higher amount of γH2AX foci correlating with an increase in DNA double-strand breaks was observed in Fe3O4@SiO2 and Fe3O4@SiO2-NH2-treated and immunestained HK-2 cells. The investigated nanoparticles did not trigger significant ROS generation and apoptosis-mediated cell death. In conclusion, these findings provide new insights into the cytotoxicity of silica coated IONPs that may support their further safer use.

C-H Functionalizations by Palladium Carboxylates: The Acid Effect.

Finding optimal reaction conditions is usually complex, requires many experiments, and is therefore demanding in terms of human, financial, and environmental resources. This work provides a simple workflow for easier design of popular palladium-catalyzed C-H functionalization reactions, where the active palladium catalysts contain carboxylate ligands. The key factor for optimizing reaction conditions is to find a balance between two opposing effects of the carboxylic acid in the reaction mixture: generation of more reactive palladium catalyst versus deactivation of a substrate by its protonation.

A membrane aerated biofilm reactor for sulfide control from anaerobically treated wastewater.

A upflow anaerobic sludge blanket reactor was operated combined to a membrane aerated biofilm reactor for sulfate removal and for elemental sulfur reclamation. A commercial silicon tube was used as an oxygen delivery diffuser. The process achieved high rates of sulfide removal from the liquid phase (90%). The hydrogen sulfide removal was influenced by the pH value and at pH value of 7.5, 98% of the H2S was removed. The elemental sulfur was observed inside the membrane, with content in the biomass of 21%. Through the massive sequencing of the samples, the microbial community diversity and the stratification of biomass inside the silicon tube was demonstrated, confirming the presence of sulfide-oxidizing bacteria on the membrane wall. The most important genera found related to the sulfur cycle were Sulfuricurvum, Geovibrio, Flexispira and Sulforospirillum.