Nanoarchitectonics Design Strategy of Metal-Organic Framework and Bio-Metal-Organic Framework Composites for Advanced Wastewater Treatment through Adsorption.

Freshwater depletion is an alarm for finding an eco-friendly solution to treat wastewater for drinking and domestic applications. Though several methods like chlorination, filtration, and coagulation-sedimentation are conventionally employed for water treatment, these methods need to be improved as they are not environmentally friendly, rely on chemicals, and are ineffective for all kinds of pollutants. These problems can be addressed by employing an alternative solution that is effective for efficient water treatment and favors commercial aspects. Metal organic frameworks (MOFs), an emerging porous material, possess high stability, pore size tunability, greater surface area, and active sites. These MOFs can be tailored; thus, they can be customized according to the target pollutant. Hence, MOFs can be employed as adsorbents that effectively target different pollutants. Bio-MOFs are a kind of MOFs that are incorporated with biomolecules, which also possess properties of MOFs and are used as a nontoxic adsorbent. In this review, we elaborate on the interaction between MOFs and target pollutants, the role of linkers in the adsorption of contaminants, tailoring strategy that can be employed on MOFs and Bio-MOFs to target specific pollutants, and we also highlight the effect of environmental matrices on adsorption of pollutants by MOFs.

Laccase driven biocatalytic oxidation to reduce polymeric surface hydrophobicity: An effective pre-treatment strategy to enhance biofilm mediated degradation of polyethylene and polycarbonate plastics.

Plastic pollution is a major global environmental issue due to its structural complexity and poor biodegradability. Biological approaches are appropriate due to cost effectiveness and environmental friendliness, however effective polymer degradation is still in its infancy. As biological treatments are slower than physical and chemical approaches, they could be applied in conjunction with pre-treatment techniques such as photo-oxidation, heat treatment, and chemical treatments. But these processes lead to high energy consumption and hazardous secondary pollution. To address these concerns, an enzymatic pre-treatment strategy has been proposed in this study, with an aim of promoting surface oxidation on the plastics leading to improved hydrophilicity. This in turn, facilitates the surface attachment of microbes, ultimately, accelerating biodegradation. Scanning Electron Microscopy (SEM) and Fourier Transform Infrared (FT-IR) spectroscopy analyses confirmed the surface oxidation of the polyethylene (PE) and polycarbonate (PC) plastics mediated by the action of laccase enzyme. Contact angle measurement witnessed the increased hydrophilicity of the treated plastics. Following, a potential biofilm forming microbial consortium has been employed for the biodegradation of enzyme treated plastics. SEM analysis indicated the increased formation of corrosive pits and surface aberrations on the enzymatically pre-treated plastics and Confocal Laser Scanning microscopy (CLSM) analysis exhibited the enhanced biofilm formation and exopolysaccharide deposition on the pre-treated PE and PC. In addition, X-ray photoelectron spectroscopy (XPS) revealed the reduction in the elemental composition of carbon with an increment in the oxygen composition of plastics. Gel permeation chromatography (GPC) further confirmed the greater reduction in the molecular weights of the plastics subjected to integrated enzymatic and biofilm treatment than only biofilm treated plastics. This is the first report on the integration of enzymatic pre-treatment with the biofilm mediated microbial degradation to achieve enhanced treatment of plastics which demonstrated to be a promising technology for the effective mitigation of plastic pollution.

High-throughput bioamphiphile production by ethyl methane sulphonate induced mutant of hydrocarbonoclastic Enterobacter xiangfangensis STP-3: In depth structural elucidation and application to petroleum refinery oil sludge bioremediation.

The environmental release of noxious petroleum hydrocarbons (PHCs) from the petroleum refining industries is an intractable global challenge. Indigenous PHCs degrading microbes produce insufficient yield of amphiphilic biomolecules with trivial efficiency makes the bioremediation process ineffective. In this concern, the present study is focused on the production of high yield multi-functional amphiphilic biomolecule through the genetic modification of Enterobacter xiangfangensis STP-3 strain using Ethyl methane sulphonate (EMS) induced mutagenesis. Mutant M9E.xiangfangensis showed 2.32-fold increased yield of bioamphiphile than wild-type strain. Novel bioamphiphile produced by M9E.xiangfangensis exhibited improved surface and emulsification activities which ensure the maximum degradation of petroleum oil sludge (POS) by 86% than wild-type (72%). SARA, FT-IR, and GC-MS analyses confirmed the expedited degradation of POS and ICP-MS analysis indicated the enhanced removal of heavy metals in connection with the ample production of functionally improved bioamphiphile. FT-IR NMR, MALDI-TOF, GC-MS and LC-MS/MS analyses portrayed the lipoprotein nature of bioamphiphile comprising pentameric fatty acid moiety conjugated with the catalytic esterase moiety. Further, homology modelling and molecular docking revealed the stronger interaction of hydrophobic amino acids, leucine and isoleucine with the PHCs in the case of wild-type esterase moiety, whereas in the mutant, aromatic amino acids were majorly interacted with the long chain and branched chain alkanes, thereby exhibited better efficiency. This is the first report on the adoption of EMS induced mutagenesis strategy to ameliorate the amphiphilic biomolecules for their sustainable applications in diverse biotechnological, environmental and industrial arenas.

The novel pyridazine pyrazolecarboxamide insecticide dimpropyridaz inhibits chordotonal organ function upstream of TRPV channels.

BACKGROUND: Pyridazine pyrazolecarboxamides (PPCs) are a novel insecticide class discovered and optimized at BASF. Dimpropyridaz is the first PPC to be submitted for registration and controls many aphid species as well as whiteflies and other piercing-sucking insects. RESULTS: Dimpropyridaz and other tertiary amide PPCs are proinsecticides that are converted in vivo into secondary amide active forms by N-dealkylation. Active secondary amide metabolites of PPCs potently inhibit the function of insect chordotonal neurons. Unlike Group 9 and 29 insecticides, which hyperactivate chordotonal neurons and increase Ca2+ levels, active metabolites of PPCs silence chordotonal neurons and decrease intracellular Ca2+ levels. Whereas the effects of Group 9 and 29 insecticides require TRPV (Transient Receptor Potential Vanilloid) channels, PPCs act in a TRPV-independent fashion, without compromising cellular responses to Group 9 and 29 insecticides, placing the molecular PPC target upstream of TRPVs. CONCLUSIONS: PPCs are a new class of chordotonal organ modulator insecticide for control of piercing-sucking pests. Dimpropyridaz is a PPC proinsecticide that is activated in target insects to secondary amide forms that inhibit the firing of chordotonal organs. The inhibition occurs at a site upstream of TRPVs and is TRPV-independent, providing a novel mode of action for resistance management. © 2023 BASF Corporation. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

TRPV channel nanchung and TRPA channel water witch form insecticide-activated complexes.

We have previously shown that insect vanilloid-type transient receptor potential (TRPV) channels Nanchung (Nan) and Inactive (Iav) form complexes, which can be over-stimulated and eventually silenced by commercial insecticides, afidopyropen, pymetrozine and pyrifluquinazon. Silencing of the TRPV channels by the insecticides perturbs function of the mechano-sensory organs, chordotonal organs, disrupting sound perception, gravitaxis, and feeding. In addition to TRPV channels, chordotonal organs express an ankyrin-type transient receptor potential (TRPA) channel, Water witch (Wtrw). Genetic data implicate Wtrw in sound and humidity sensing, although the signaling pathway, which links Wtrw to these functions has not been clearly defined. Here we show that, in heterologous system, Nan and Wtrw form calcium channels, which can be activated by afidopyropen, pymetrozine and an endogenous agonist, nicotinamide. Analogous to Nan-Iav heteromers, Nan forms the main binding interface for afidopyropen, whereas co-expression of Wtrw dramatically increases its binding affinity. Pymetrozine competes with afidopyropen for binding to Nan-Wtrw complexes, suggesting that these compounds have overlapping binding sites. Analysis of Drosophila single-nucleus transcriptomic atlas revealed co-expression of nan and wtrw in audio- and mechanosensory neurons. The observation that Nan can form insecticide-sensitive heteromers with more than one type of TRP channels, raises a possibility that Nan may partner with some other TRP channel(s). In addition, we show that Wtrw can be activated by plant-derived reactive electrophiles, allyl isothiocyanate and cinnamaldehyde, defining new molecular target for these repellents.

Afidopyropen: New and potent modulator of insect transient receptor potential channels.

The commercial insecticides pymetrozine and pyrifluquinazon control plant-sucking pests by disturbing their coordination and ability to feed. We have previously shown that these compounds act by overstimulating and eventually silencing vanilloid-type transient receptor potential (TRPV) channels, which consist of two proteins, Nanchung and Inactive, that are co-expressed exclusively in insect chordotonal stretch receptor neurons. Here we show that a new insecticidal compound, afidopyropen, modulates chordotonal organs of American grasshoppers (Schistocerca americana) in the same fashion. Afidopyropen stimulated heterologously expressed TRPV channels from two different insect species - fruit fly (Drosophila melanogaster) and pea aphid (Acyrthosiphon pisum) - but did not affect function of the mammalian TRPV channel TRPV4. Activation of the insect TRPVs required simultaneous expression of both Nanchung and Inactive proteins. Tritium-labeled afidopyropen bound fruit fly TRPVs with higher affinity than pymetrozine and competed with pymetrozine for binding. Nanchung protein formed the main binding interface for afidopyropen, whereas co-expression of Inactive dramatically increased binding affinity. Another modulator of chordotonal organs, flonicamid, did not activate insect TRPV channels, nor did it compete with afidopyropen for binding, indicating that it has a different target site. These results define afidopyropen as a new, potent and specific modulator of insect TRPV channels, and provide insight into the unique binding mode of these compounds.

TRP Channels in Insect Stretch Receptors as Insecticide Targets.

Defining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

High-throughput automated confocal microscopy imaging screen of a kinase-focused library to identify p38 mitogen-activated protein kinase inhibitors using the GE InCell 3000 analyzer.

The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2-EGFP) and measured the subcellular distribution of the MK2-EGFP as a direct readout of p38 activation. The MK2-EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 microM and 156 were confirmed as inhibitors of p38-mediated MK2-EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50s less than 1 microM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.

Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors.

This chapter describes the generation and characterization of a stable MK2-EGFP expressing HeLa cell line and the subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 MAPK inhibitors. Mitogen-activated protein kinase activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase, and p38-induced phosphorylation of MK-2 induces a nucleus to cytoplasm translocation (Engel et al., 1998; Neininger et al., 2001; Zu et al., 1995). Through a process of heterologous expression of a MK2-EGFP fusion protein in HeLa cells using retroviral infection, antibiotic selection, and flow sorting, we were able to isolate a cell line in which the MK2-EGFP translocation response could be robustly quantified on the Cellomics ArrayScan platform using the nuclear translocation algorithm. A series of assay development experiments using the A4-MK2-EGFP-HeLa cell line are described to optimize the assay with respect to cell seeding density, length of anisomycin stimulation, dimethyl sulfoxide tolerance, assay signal window, and reproducibility. The resulting MK2-EGFP translocation assay is compatible with high-throughput screening and was shown to be capable of identifying p38 inhibitors. The MK2-EGF translocation response is susceptible to other classes of inhibitors, including nonselective kinase inhibitors, kinase inhibitors that inhibit upstream kinases in the p38 MAPK signaling pathway, and kinases involved in cross talk between different modules (ERKs, JNKs, and p38s) of the MAPK signaling pathways. An example of mining "high-content" image-based multiparameter data to extract additional information on the effects of compound treatment of cells is presented.

Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-Jun, and ERK activation.

This chapter describes the development and implementation of three independent imaging assays for the major mitogen-activated protein kinase (MAPK) signaling modules: p38, JNK, and ERK. There are more than 500 protein kinases encoded in the human genome that share an ATP-binding site and catalytic domain conserved in both sequence and structure. The majority of kinase inhibitors have been found to be competitive with ATP, raising concerns regarding kinase selectivity and potency in an environment of millimolar intracellular concentrations of ATP, as well as the potential for off-target effects via the many other cellular proteins that bind and/or utilize ATP. The apparent redundancy of the kinase isoforms and functions in the MAPK signaling modules present additional challenges for kinase inhibitor selectivity and potency. Imaging assays provide a method to address many of these concerns. Cellular imaging approaches facilitate analysis of the targets expressed in the context of their endogenous substrates and scaffolding proteins and in a complex environment for which subcellular localization, cross talk between pathways, phosphatase regulatory control, and intracellular ATP concentrations are relevant to the functions of the kinase. The assays described herein provide a strategy to profile kinase inhibitors for MAPK pathway selectivity while simultaneously providing information on cell morphology or toxicity. Results suggest that the MAPK pathways are indeed susceptible to nonselective kinase inhibitors such as staurosporin and inhibitors that inhibit upstream MAPK Kinase Kinases (MKKKs) and MAPK Kinases (MKKs) in the MAPK signaling pathway, especially those involved in cross talk between the pathways. However, selective MAPK inhibitors were identified that exhibited pathway selectivity as evidenced by significantly lower IC(50) values for their respective p38, JNK, or ERK signaling pathway assays.

Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform.

This chapter describes the conversion and assay development of a 96-well MK2-EGFP translocation assay into a higher density 384-well format high-content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well-substantiated hypothesis that mitogen-activated protein kinase-activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase and that p38-induced phosphorylation of MK-2 induces a nucleus-to-cytoplasm translocation. This chapter also presents a case history of the performance of the MK2-EGFP translocation assay, run as a "high-content" screen of a 32K kinase-biased library to identify p38 inhibitors. The assay performed very well and a number of putative p38 inhibitor hits were identified. Through the use of multiparameter data provided by the nuclear translocation algorithm and by checking images, a number of compounds were identified that were potential artifacts due to interference with the imaging format. These included fluorescent compounds, or compounds that dramatically reduced cell numbers due to cytotoxicity or by disrupting cell adherence. A total of 145 compounds produced IC(50) values <50.0 muM in the MK2-EGFP translocation assay, and a cross target query of the Lilly-RTP HTS database confirmed their inhibitory activity against in vitro kinase targets, including p38a. Compounds were confirmed structurally by LCMS analysis and profiled in cell-based imaging assays for MAPK signaling pathway selectivity. Three of the hit scaffolds identified in the MK2-EGFP translocation HCS run on the ArrayScan were selected for a p38a inhibitor hit-to-lead structure activity relationship (SAR) chemistry effort.

The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites.

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were approximately 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K(m) of 1.1 mmol/L and V(max) of 439 pmol/min/mg protein. Uptake of 250 micromol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 micromol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.