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Heterogeneous crystallization is a common occurrence during the formation of solid wastes. It leads to the encapsulation of valuable/hazardous metals within the primary phase, presenting significant challenges for waste treatment and metal recovery. Herein, we proposed a novel method involving the in-situ formation of a competitive substrate during the precipitation of jarosite waste, which is an essential process for removing iron in zinc hydrometallurgy. We observed that the in-situ-formed competitive substrate effectively inhibits the heterogeneous crystallization of jarosite on the surface of anglesite, a lead-rich phase present in the jarosite waste. As a result, the iron content on the anglesite surface decreases from 34.8% to 1.65%. The competitive substrate was identified as schwertmannite, characterized by its loose structure and large surface area. Furthermore, we have elucidated a novel mechanism underlying this inhibition of heterogeneous crystallization, which involves the local supersaturation of jarosite caused by the release of ferric and sulfate ions from the competitive substrate. The local supersaturation promotes the preferential heterogeneous crystallization of jarosite on the competitive substrate. Interestingly, during the formation of jarosite, the competitive substrate gradually vanished through a dissolution-recrystallization process following the Ostwald rule, where a metastable phase slowly transitions to a stable phase. This effectively precluded the introduction of impurities and reduced waste volume. The goal of this study is to provide fresh insights into the mechanism of heterogeneous crystallization control, and to offer practical crystallization strategies conducive to metal separation and recovery from solid waste in industries.

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Enhancing soil organic matter characteristics, ameliorating physical structure, mitigating heavy metal toxicity, and hastening mineral weathering processes are crucial approaches to accomplish the transition of tailings substrate to a soil-like substrate. The incorporation of biomass co-pyrolysis and plant colonization has been established to be a significant factor in soil substrate formation and soil pollutant remediation. Despite this, there is presently an absence of research efforts aimed at synergistically utilizing these two technologies to expedite the process of mining tailings soil substrate formation. The current study aimed to investigate the underlying mechanism of geochemical changes and rapid mineral weathering during the process of transforming tailings substrate into a soil-like substrate, under the combined effects of biomass co-smoldering pyrolysis and plant colonization. The findings of this study suggest that the incorporation of smoldering pyrolysis and plant colonization induces a high-temperature effect and biological effects, which enhance the physical and chemical properties of tailings, while simultaneously accelerating the rate of mineral weathering. Notable improvements include the amelioration of extreme pH levels, nutrient enrichment, the formation of aggregates, and an increase in enzyme activity, all of which collectively demonstrate the successful attainment of tailings substrate reconstruction. Evidence of the accelerated weathering was verified by phase and surface morphology analysis using X-ray diffraction and scanning electron microscopy. Discovered corrosion and fragmentation on the surface of minerals. The weathering resulted in corrosion and fragmentation of the surface of the treated mineral. This study confirms that co-smoldering pyrolysis of biomass, combined with plant colonization, can effectively promote the transformation of tailings into soil-like substrates. This method has can effectively address the key challenges that have previously hindered sustainable development of the mining industry and provides a novel approach for ecological restoration of tailings deposits.

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Background: Atrial fibrillation (AF) triggers atrial remodeling, impacting atrial function and ablation efficacy. This remodeling leads to atrial cardiomyopathy and dilatation, linked to mitral regurgitation, forming atrial functional mitral regurgitation (aFMR). Our study explores the relationship between early-stage-aFMR and the atrial electrical architecture, focusing on left atrial bipolar voltage and low-voltage areas (LVAs) in AF patients. Methods: We enrolled 282 patients undergoing redo-PVI after AF recurrence post-PVI. Echocardiography was performed prior to ablation, and only patients with no, mild, or mild-to-moderate aFMR were included. Ablation used radiofrequency and a 3D mapping system, with atrial voltage documented on each atrial wall. LVAs were calculated using high-density maps, and patients were followed for 15 months. Results: Significant differences in left atrial voltage and LVA extent were observed based on aFMR severity. Patients with aFMR 1 + had significantly lower atrial voltage compared to no-aFMR, but no significant increase in LVAs. Patients with aFMR 2 + showed lower voltage amplitudes in all atrial regions and larger LVAs compared to no-aFMR patients. AF recurrence was significantly higher in the aFMR group (62.9% vs. 48.3%, p = 0.027) within 1 year. aFMR was associated with AF recurrence after adjusting for sex, age, and AF types (HR: 1.517, 95% CI: 1.057-2.184, p = 0.025). Conclusion: aFMR in AF patients may indicate progressive atrial remodeling and left atrial cardiomyopathy, characterized by reduced atrial voltage and increased LVAs. aFMR is linked to PVI outcomes, suggesting its consideration in AF therapy decision-making.

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BACKGROUND: The docking protein IRS2 (insulin receptor substrate protein-2) is an important mediator of insulin signaling and may also regulate other signaling pathways. Murine hearts with cardiomyocyte-restricted deletion of IRS2 (cIRS2-KO) are more susceptible to pressure overload-induced cardiac dysfunction, implying a critical protective role of IRS2 in cardiac adaptation to stress through mechanisms that are not fully understood. There is limited evidence regarding the function of IRS2 beyond metabolic homeostasis regulation, particularly in the context of cardiac disease. METHODS: A retrospective analysis of an electronic medical record database was conducted to identify patients with IRS2 variants and assess their risk of cardiac arrhythmias. Arrhythmia susceptibility was examined in cIRS2-KO mice. The underlying mechanisms were investigated using confocal calcium imaging of ex vivo whole hearts and isolated cardiomyocytes to assess calcium handling, Western blotting to analyze the involved signaling pathways, and pharmacological and genetic interventions to rescue arrhythmias in cIRS2-KO mice. RESULTS: The retrospective analysis identified patients with IRS2 variants of uncertain significance with a potential association to an increased risk of cardiac arrhythmias compared with matched controls. cIRS2-KO hearts were found to be prone to catecholamine-sensitive ventricular tachycardia and reperfusion ventricular tachycardia. Confocal calcium imaging of ex vivo whole hearts and single isolated cardiomyocytes from cIRS2-KO hearts revealed decreased Ca²+ transient amplitudes, increased spontaneous Ca²+ sparks, and reduced sarcoplasmic reticulum Ca²+ content during sympathetic stress, indicating sarcoplasmic reticulum dysfunction. We identified that overactivation of the AKT1/NOS3 (nitric oxide synthase 3)/CaMKII (Ca2+/calmodulin-dependent protein kinase II)/RyR2 (type 2 ryanodine receptor) signaling pathway led to calcium mishandling and catecholamine-sensitive ventricular tachycardia in cIRS2-KO hearts. Pharmacological AKT inhibition or genetic stabilization of RyR2 rescued catecholamine-sensitive ventricular tachycardia in cIRS2-KO mice. CONCLUSIONS: Cardiac IRS2 inhibits sympathetic stress-induced AKT/NOS3/CaMKII/RyR2 overactivation and calcium-dependent arrhythmogenesis. This novel IRS2 signaling axis, essential for maintaining cardiac calcium homeostasis under stress, presents a promising target for developing new antiarrhythmic therapies.

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BsCotA laccase is a promising candidate for industrial application due to its excellent thermal stability. In this research, our objective was to enhance the catalytic efficiency of BsCotA by modifying the active site pocket. We utilized a strategy combining the diversity design of the active site pocket with molecular docking screening, which resulted in selecting five variants for characterization. All five variants proved functional, with four demonstrating improved turnover rates. The most effective variants exhibited a remarkable 7.7-fold increase in catalytic efficiency, evolved from 1.54 × 105 M-1 s-1 to 1.18 × 106 M-1 s-1, without any stability loss. To investigate the underlying molecular mechanisms, we conducted a comprehensive structural analysis of our variants. The analysis suggested that substituting Leu386 with aromatic residues could enhance BsCotA's ability to accommodate the 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonate (ABTS) substrate. However, the inclusion of charged residues, G323D and G417H, into the active site pocket reduced kcat. Ultimately, our research contributes to a deeper understanding of the role played by residues in the laccases' active site pocket, while successfully demonstrating a method to lift the catalytic efficiency of BsCotA. KEY POINTS: • Active site pocket design that enhanced BsCotA laccase efficiency • 7.7-fold improved in catalytic rate • All tested variants retain thermal stability.

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BACKGROUND: Although targeting atrial fibrillation (AF) drivers and substrates has been used as an effective adjunctive ablation strategy for patients with persistent AF (PsAF), it can result in iatrogenic scar-related atrial tachycardia (iAT) requiring additional ablation. Personalized atrial digital twins (DTs) have been used preprocedurally to devise ablation targeting that eliminate the fibrotic substrate arrhythmogenic propensity and could potentially be used to predict and prevent postablation iAT. OBJECTIVES: In this study, the authors sought to explore possible alternative configurations of ablation lesions that could prevent iAT occurrence with the use of biatrial DTs of prospectively enrolled PsAF patients. METHODS: Biatrial DTs were generated from late gadolinium enhancement-magnetic resonance images of 37 consecutive PsAF patients, and the fibrotic substrate locations in the DT capable of sustaining reentries were determined. These locations were ablated in DTs by representing a single compound region of ablation with normal power (SSA), and postablation iAT occurrence was determined. At locations of iAT, ablation at the same DT target was repeated, but applying multiple lesions of reduced-strength (MRA) instead of SSA. RESULTS: Eighty-three locations in the fibrotic substrates of 28 personalized biatrial DTs were capable of sustaining reentries and were thus targeted for SSA ablation. Of these ablations, 45 resulted in iAT. Repeating the ablation at these targets with MRA instead of SSA resulted in the prevention of iAT occurrence at 15 locations (18% reduction in the rate of iAT occurrence). CONCLUSIONS: Personalized atrial DTs enable preprocedure prediction of iAT occurrence after ablation in the fibrotic substrate. It also suggests MRA could be a potential strategy for preventing postablation AT.

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The ecological dynamics of particle-attached bacteria (PAB) were observed through changes in the core phytoplankton phycosphere, and were associated with the dynamics of free-living bacteria (FLB) using metabarcoding and microscopic analyses over 210 days (with weekly sampling intervals) in the Jangmok coastal ecosystem, South Korea. Cluster analysis and non-metric multidimensional scaling classified the phytoplankton community into six groups comprising core phytoplankton species, including the harmful algal species Akashiwo sanguinea (dinoflagellate) in late autumn, Teleaulax amphioxeia (cryptomonads) in early winter and spring, Skeletonema marinoi-dohrnii complex (diatom) in winter, Pseudo-nitzschia delicatissima (diatom) in early spring, and diatom complexes such as Chaetoceros curvisetus and Leptocylindrus danicus in late spring. We identified 59 and 32 indicators in PAB and FLB, respectively, which rapidly changed with the succession of the six core phytoplankton species. The characteristics of PAB were mainly divided into "Random encounters" or "Attraction of motivation by chemotaxis." When Akashiwo sanguinea bloomed, bacteria of the genera Kordiimonas and Polaribacter, which are commonly observed in PAB and FLB, indicated "Random encounter" characteristics. In addition, Sedimenticola of PAB was uniquely presented in Akashiwo sanguinea, exhibiting characteristics of "Attraction of motivation by chemotaxis." In contrast, FLB followed the strategy of "Random encounters" because it was not affected by specific habitats and energy sources. Thus, many common bacteria were PAB and FLB, thereby dictating the bacteria's strategy of "Random encounters." "Attraction of motivation by chemotaxis" has characteristics of the species-specific interactions between PAB and specific harmful algal species, and is potentially influenced by organic matter of core phytoplankton cell surface and/or EPS released from phytoplankton.

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BACKGROUND: Low voltage areas (LVA) are pivotal in atrial fibrillation (AF) pathogenesis, influencing local left atrial LA excitation and perpetuating AF occurrences. While pulmonary vein isolation (PVI) with cryo-balloon (CB) ablation is effective for AF, it doesn't provide insights into the LA substrate or detect LVA, which affects ablation success rates. This study examines whether LA voltage and LVAs can be anticipated by analyzing the voltage signal amplitude at the coronary sinus (CS) catheter, which is standard in CB and radiofrequency ablation procedures. METHODS: A retrospective analysis of 284 patients with recurrent AF undergoing RF catheter ablation was conducted at a high-volume EP center in Germany. The correlation between LA voltage and LVA with the CS signal was explored. RESULTS: The signal amplitude in the CS significantly correlated with voltage in LA walls, particularly in the proximal CS (correlation coefficient ρ = 0.81, p < 0.001). A CS signal cut-off of 1.155 mV effectively predicted severe atrial LVAs (>40%) with a sensitivity of 90.7% and a specificity of 100%. While a threshold of 1.945 mV identified patients with no significant atrial LVAs (<5%) with a sensitivity of 88% and a specificity of 50% (AUC: 0.81, 95% CI: 0.71-0.89, p < 0.001). CONCLUSION: The CS signal amplitude is associated with the LA voltage. Due to its potential as a diagnostic tool for atrial LVAs, the signal amplitude in the CS could provide valuable information about the LA substrate, especially when 3D mapping is not feasible.

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We recently found two α-L-glucosidases, which can hydrolyze p-nitrophenyl α-L-glucopyranoside (PNP L-Glc) rather than p-nitrophenyl α-L-fucopyranoside, in glycoside hydrolase family 29. This study evaluated their substrate specificity for p-nitrophenyl α-L-rhamnopyranoside (PNP L-Rha), α-L-quinovopyranoside (PNP L-Qui), and α-L-xylopyranoside (PNP L-Xyl), of which structure is similar to PNP L-Glc. The two α-L-glucosidases had little activity toward PNP L-Rha. They exhibited higher k cat/K m values for PNP L-Qui but smaller for PNP L-Xyl than for PNP L-Glc. The molecular docking studies indicated that these specificities were correlated well with the active-site structure of the α-L-glucosidases. The finding that α-L-quinovoside, which has been suggested to occur in nature, is also a substrate for α-L-glucosidases indicates that this enzyme are not solely dedicated to α-L-glucoside hydrolysis.

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Study of cell migration in cancer is crucial to the comprehension of the processes and factors that govern tumor spread. Cancer cells migrate invading tissues, causing alterations in cell adhesion, cytoskeleton, and signaling pathways. Little is known about the physical attributes of cancer cells that change when interacting with microenvironments. In this work, the local topography of the ECM has been mimicked through micropillar array substrates. MDA-MB-231 and MCF-7 breast cancer cells, exhibiting high and low metastatic potential, respectively, were analyzed. Differences in morphology and migration of the cells were investigated by examining the cell spreading area, circularity, aspect ratio, migration speed, and migration path. This work encountered that none of the studied cell lines have preferential orientation migrating on uniform patterns. In contrast, cell migration on graded patterns shows preferential orientation along the longitudinal direction from sparser to denser zones which is significantly influenced by substrate stiffness and indicates that both cell lines can sense the spacing gradient and respond to this topographical cue. The migration speed of the breast cancer cell lines significantly decreases from the sparse to medium to dense zones, registering higher values for the MDA-MB-231.

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Fine particulate matter (PM2.5) constituents are greatly affected by site-specific emission sources and are one of the main reasons for oxidative stress that leads to cardiovascular ailments. This study investigated the temporal, seasonal, and episodic variations in the oxidative potential (OP) of PM2.5 and its association with chemical components. Additionally, we have also examined the effect of filter substrates on OP. Dithiothreitol (DTT) and ascorbic acid (AA) acellular assays were used to estimate the formation of reactive oxygen species (ROS) in PM2.5 samples collected over a year from a regional site in India. PM2.5 morphology and functional groups were also analyzed. Results showed that OPDTTv was at the highest in winter (2.56 ±â€¯0.84 nmol min-1 m-3) and at the lowest during monsoon (0.79 ±â€¯0.65 nmol min-1 m-3). OPAAv exhibited the highest activity in post-monsoon (0.09 ±â€¯0.04 nmol min-1 m-3) and least in summer (0.05 ±â€¯0.04 nmol min-1 m-3). Biomass burning (BB) and open-field burning of crop residue during the rabi and kharif harvesting seasons were associated with significantly elevated PM2.5 toxicity, which is indicative of the contribution of combustion-derived particles. OPDTTv and OPAAv levels from BB in post-monsoon were 21 % and 67 % higher than the levels observed during BB in summer. Flaky irregular agglomerates and porous structures were observed during the BB period. Fourier-transformed infrared spectroscopy revealed that traffic-emitted organic hydrocarbons CH functional group was dominant across the season. Further, chemical species such as organics (OC and EC fractions) and ions (SO42-, NH4+, Cl-, NO3-) were found to be significantly associated with OP. Among the three filter substrates, the Teflon showed higher OP variability for both assays. This study emphasizes the impact of regional toxic aerosols across seasons and during episodic events. It contributes to our understanding of the toxicity of ambient PM2.5, which is crucial for developing targeted air-quality management strategies.

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Formycin A (FOR) and Pyrazofurin A (PYR) are nucleoside analogues with antiviral and antitumor properties. They are known to interfere with nucleic acid metabolism, but their direct effect on transcription is less understood. We explored how RNA polymerases (RNAPs) from bacteria, mitochondria, and viruses utilize FOR, PYR, and oxidized purine nucleotides. All tested polymerases incorporated FOR in place of adenine and PYR in place of uridine. FOR also exhibited surprising dual-coding behavior, functioning as a cytosine substitute, particularly for viral RNAP. In contrast, 8-oxoadenine and 8-oxoguanine were incorporated in place of uridine in addition to their canonical Watson-Crick codings. Our data suggest that the interconversion of canonical anti- and alternative syn-conformers underlies dual-coding abilities of FOR and oxidized purines. Structurally distinct RNAPs displayed varying abilities to utilize syn-conformers during transcription. By examining base pairings that led to substrate incorporation and the entire spectrum of geometrically compatible pairings, we have gained new insights into the nucleobase selection processes employed by structurally diverse RNAPs. These insights may pave the way for advancements in antiviral therapies.

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To achieve industrial-scale putrescine production, a high efficient bio-synthesis of putrescine involving arginase (ARG, EC 3.5.3.1) and L-ornithine decarboxylase was evaluated here. Among the four arginases tested, ARGBT from Bos Taurus showed the highest activity (1966 U/mg). Compared to the L-arginine decarboxylase (ADC) pathway, the strain expressing ARGBT and L-ornithine decarboxylase (SpeC) produced 28.7 g/L putrescine, a 38.6 % increase. Two pyridoxal phosphate (PLP) salvage pathways were evaluated, and the strain BL-PTac-PdxK co-expressed pyridoxal kinase (PdxK) performed better. D-Glucose was used as the co-substrate to improve the putrescine titer further. Under optimal conditions, 43.6 g/L putrescine was produced from 87.1 g/L L-arginine, and 76 g/L putrescine was synthesized on a 0.5 L scale. Using L-arginine fermentation broth (60 g/L) as the substrate, a titer of 30 g/L putrescine was achieved. This efficient biotransformation process presented here enables feasible industrial-scale putrescine production.

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Pectobacterium carotovorum subsp. carotovorum (PCC) is a notorious plant pathogen responsible for severe soft rot in kimchi cabbage, which results in significant economic losses. To detect PCC rapidly and accurately in kimchi cabbage, we developed a surface-enhanced Raman scattering (SERS) substrate on which silver nanospheres (AgNSs), nanowires (AgNWs), and nanoseeds are combined on a polydimethylsiloxane (PDMS) platform. The incorporation of Ag nanoseeds creates a higher density of hotspots, which ensures a low detection limit of 1.001 CFU/mL. Electron microscopy and spectroscopic analyses confirmed the successful fabrication of the substrate and its enhanced sensitivity. The SERS substrate exhibits excellent selectivity by effectively distinguishing PCC from other bacteria commonly found in kimchi cabbage. The substrate gives rise to strong Raman signals across PCC concentrations ranging from 101 to 106 CFU/mL. Additionally, a predictive model was developed for accurately detecting PCC in real kimchi cabbage samples, and the results were validated by polymerase chain reaction measurements. A sensitive, selective, and rapid approach for PCC detection in kimchi cabbage that offers a promising improvement over existing methodologies is presented.

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The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90ß, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1-/- cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

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2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products.

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Recognizing the critical need to elucidate the molecular determinants of this selectivity offers a pathway to engineer enzymes with broader and more versatile catalytic capabilities. Through integrated methods including phylogenetic analysis, molecular docking, and structural analysis, we identified a pivotal amino acid residue, αTrp116, linking the substrate binding pocket and the active site of a NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). This residue acts as a crucial determinant of substrate specificity within the NHase enzyme. The mutant αW116R modified the substrate specificity of PtNHase, significantly enhancing its catalytic efficiency towards aromatic substrates. The catalytic activity for aromatic compounds such as 3-Cyanopyridine was 14-fold that of the wild-type, whereas its activity for aliphatic substrates diminished to one-sixth. MD simulations revealed that replacing αTrp116 with Arg allowed aromatic nitrile substrates to achieve more favorable conformations within the active site. Based on the mutant αW116R, we further constructed a combinatorial variant Pt-4, tailored for aromatic substrates, which exhibited an enzyme activity 50 times that of the wild-type. These results highlight the critical influence of amino acid residues in the enzyme's active site on substrate specificity and offer fresh perspectives and approaches for the evolution of enzymes.

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A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.

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Among all the heavy metals, Pb, Cd, and As are the most harmful pollutants in the environment. They reach into the organisms via various levels of food chains i.e. air and water. Glutathione-s-transferase (GST, E.C. 2.5.1.18), a key enzyme of xenobiotics metabolism, plays an important role in the removal of several toxicants. The present study aimed to evaluate any inhibitory action of these heavy metals on the GST enzyme isolated from the hepatic tissues of rats. A 10 % (w/v) homogenate of rat liver was prepared in cold and centrifuged at 4 °C at 9000xg for 30 min. The supernatant was collected and kept frozen at -20 °C or used fresh for carrying out different experiments. The activity of GST was monitored spectrophotometrically at 340 nm using 220 µg of soluble protein with varying equal substrate concentrations (0.125-2 mM) in phosphate buffer (50 mM, pH 6.5). To assess the impact of heavy metals on the enzyme activity, different concentrations of Cd (0-0.6 mM) and Pb (0-2 mM) were added to the reaction mixture followed by monitoring the residual activity. The optimum temperature and pH of rat liver GST were found to be 37 °C and 6.5, respectively. The Km value for GST was 0.69 mM and the Vmax was found to be 78.67 U/mg. The Cd and Pb significantly altered the kinetic behaviour of the enzyme. The Vmax and Kcat/Km parameters of GST were recorded to be decreased after interaction with Cd and Pb individually and showed a mixed type of inhibition pattern suggesting that these inhibitors may have a greater binding affinity either for the free enzyme or the substrate-enzyme complex. These metals showed a time-dependent enzyme inhibition profile. Cd was found to be the most potent inhibitor when compared to other treated metals; the order of inhibitory effect of metal ions was Cd>Pb>As. The in silico ion docking analysis for determining the probable interactions of Cd and Pb with fragmented GST validated that Cd exhibited higher inhibition potential for the enzyme as compared to Pb. The results of the present study indicated that exposure of both the Cd and Pb may cause significant inhibition of hepatic GST; the former with higher inhibitory potential than the later. However, As proved to be least effective against the enzyme under the aforesaid experimental conditions.

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An electroactive polyazomethine was prepared directly on a transparent electrode by the polycondensation of bis(triphenylamine) dialdehyde and its complementary methoxytriphenylamine diamine. The spray-and-bake method of coating the electrode for preparing electrochromic layers could be upscaled to prepare working devices larger than standard test devices. The film prepared by thermally annealing the complementary monomers was both electroactive and switched its color with an applied potential. The yellow electrochromic polyazomethine could be electrochemically oxidized reversibly to obtain a blue film. The electrochromic test device fabricated from the polyazomethine was operated upwards of 1 h for performance assessment. The electrochromic response times of the electrochromic device were ca. 3.3 and 1.2 s for the coloration and bleaching, respectively. The upscaled device prepared by the straightforward coating approach had consistent metrics with the small-area test device.