Carbonic Anhydrase Activity Research Articles

Biologically driven isotope fractionation in ultrastructurally different shell portions of freshwater pearl mussels (Margaritifera margaritifera): Implications for stream water δ18O reconstructions

AbstractOxygen isotopes in stream water can serve as natural tracers of watershed dynamics. Freshwater pearl mussels provide δ18Owater estimates that overcome temporal and spatial limitations of instrumental records. The reliability of shell‐based δ18Owater reconstructions depends on understanding which shell layer biomineralizes closer to oxygen isotopic equilibrium with ambient water. To determine this, both the (outer) prismatic and (inner) nacreous sublayers of the outer shell layer were sampled. Over 2500 isotope values were obtained from shells collected from the Our River (Luxembourg) and from mussels cultured in tanks at constant temperature and monitored δ18Owater. Calculated δ18Owater from the prismatic portion was in excellent agreement with monitored δ18Owater, while δ18Oshell of the nacreous portion was systematically offset by +0.43‰, overestimating δ18Owater by +0.53‰. Although shell portions were formed simultaneously from the same extrapallial fluid, they underwent different fractionation mechanisms, presumably due to differences in carbonic anhydrase activity catalyzing mineralization processes.

Zinc-based organophyllosilicate nanosheets as novel carbonic anhydrase-like nanozyme for efficient CO2 fixation

The unparalleled catalytic activity of carbonic anhydrases (CAs) towards CO2 hydrolysis can potentially contribute to carbon capture, utilization, and storage technology aimed at global decarbonization; however, the high cost and low stability of CAs significantly limit their practical applications. Recently, the development of robust and effective nanozymes with CA-mimicking activity has been proposed as a promising solution to this challenge. Herein, a novel zinc-based organophyllosilicate (ZnAC) nanozyme with CA-like catalytic activity was synthesized by a facile one-pot sol–gel method for the first time, leading to a Zn-O coordination structure that mimicked the catalytic center fragments of natural CAs, with surface amino groups acting as the affinity sites and proton shuttles. The synergistic effect of the two components on the open surface of the layered structure resulted in excellent CA-like activity with a Michaelis–Menten constant (Km) of 4.071 mM, maximum velocity (Vmax) of 0.220 mM/s, and turnover number (kcat) of 11.213 /s at 25 °C and pH=7.5, which were superior to those of most previously reported CA-like nanozymes. The catalytic performance of ZnAC significantly improved with increasing pH and temperature; furthermore, ZnAC exhibited excellent stability, maintaining high catalytic activity after the exposure to extreme temperatures or pH values, eight reaction cycles, and 30 d of storage. Additionally, ZnAC significantly increased the rate of CO2 hydration and the extent of CO2 mineralization and precipitation. This work proposes a new strategy for designing and constructing efficient CA-like nanozymes, providing valuable insights into the structure–activity relationship and new perspectives for the implementation of CO2 fixation.

New Thiazole Derivatives: Carbonic Anhydrase I–II and Cholinesterase Inhibition Profiles, Molecular Docking Studies

AbstractThiosemicarbazone derivative was obtained by the addition of thiosemicarbazide to 5‐nitro‐thiophene‐2‐carbaldehyde. The addition–cyclization of the 2‐bromoacetophenone derivative to thiosemicarbazone derivative gave the new thiazole derivatives (2 a‐k). Acetylcholinesterase (AChE), butyrylcholinesterase (BChE), antioxidant, and human carbonic anhydrase (hCA) I and II isoform inhibitory activities of the thiazole derivatives 2 a‐k were investigated. The effects of thiazole derivatives on carbonic anhydrase I and II (hCA I–II) isoenzymes were examined in vitro using the esterase method. IC50 values for enzyme inhibition were found to be 2.661–22.712 μM for hCA I and 5.372–26.813 μM for hCA II. All derivatives reduced the activities of carbonic anhydrase I and II isoenzymes and were new potential inhibitor molecule candidates. These compounds were found to have minimal effects on AChE and BChE. The antioxidant properties of the target compounds were determined using DPPH and ferric ion‐chelating assays. In particular, compounds 2 k and 2 h had the highest antioxidant effect in the series with IC50 values of 30.11±0.008 μM and 30.21±0.006 μM, respectively. ADMET properties of the compounds found to be effective were evaluated and their interactions with the enzyme were determined by molecular docking.

A novel heat-inducible plasmid-driven T7 system for enhancing carbonic anhydrase in engineered Escherichia coli strains

Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial plsB on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 °C, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42 °C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38,702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27,521 WAU/mL at pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future.

The effect of sulthiame on potential biomarkers in moderate to severe obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is a common disease with breathing disturbances during sleep. Sulthiame (STM), a carbonic anhydrase (CA) inhibitor, was recently shown to reduce OSA in a significant proportion of patients. CA activity and hypoxia-inducible factor (HIF)-1α are two potential biomarkers reported in severe OSA and hypoxia. Both have been considered to play roles in the development of OSA comorbidities. This study investigated the effects of STM on these biomarkers in OSA. This was an exploratory analysis of a randomised, double-blind, placebo-controlled trial of STM in OSA. Patients with moderate to severe OSA, body mass index 20-35 kg·m-2, aged 18-75 years and not accepting positive airway pressure treatment were randomised to 4 weeks with placebo, STM 200 mg or STM 400 mg. CA activity (n=43) and HIF-1α concentration (n=53) were determined at baseline, after 4 weeks of treatment and 2 weeks after treatment completion. In the 400 mg group, both CA activity and HIF-1α concentration were reduced (median difference -26% (95% CI -32- -12%) and -4% (95% CI -8- -2%); both p<0.05 versus placebo). The reductions were sustained 2 weeks after treatment completion. In the 200 mg group, both CA activity and HIF-1α were numerically reduced. The STM-induced reductions in CA activity and HIF-1α correlated significantly (r=0.443, p=0.023). STM treatment in OSA induced a reduction of both CA activity and HIF-1α concentration. The effects remained 2 weeks after treatment completion, suggesting prolonged effects of STM in OSA.

A study of bacteria producing carbonic anhydrase enzyme for CaCO3 precipitation and soil biocementation

We study the carbonic anhydrase (CA) pathway using autochthonous CA-producing bacteria as a means of inducing calcite precipitation, which acts as a biocement to improve the engineering soil properties. Forty different microbial strains producing CA were isolated from the foundation soil of a railway embankment in Prickwillow, UK. Three of the best CA-producing strains were selected and identified by DNA sequencing as Bacillus licheniformis, Bacillus toyonensis and Bacillus pumilus with CA activity values respectively of 1.79 U/ml, 1.42 U/ml and 1.55 U/ml. To optimise the treatments, we investigated the effect of pH, temperature, zinc co-factor and cementation solution molarity on the growth and CA activity and bioprecipitates, with CO2 added in the form of bicarbonate. Scanning electron microscope (SEM) analysis of the bioprecipitates showed that these had characteristic morphologies of calcite and vaterite crystals. The formation of calcite was further corroborated by FT-IR and Raman analysis of bioprecipitates. The precultured bacteria were injected into the fine-grained soil together with cementation solution. Unconfined compressive strength in treated soil increased up to 1 MPa, and its calcium carbonate content increased by 2.78%. This, as well as the stability of the treated soil upon water immersion, proved the biocementation of the fine-grained soil. These findings suggest the potential of employing the CA biocementation route for soil stabilisation pending further development of the technique.

Carbonyl Sulfide (COS) in Terrestrial Ecosystem: What We Know and What We Do Not

Over the past six decades, carbonyl sulfide (COS) in terrestrial ecosystems has been extensively studied, with research focusing on exploring its ecological and environmental effects, estimating source–sink volume, and identifying influencing factors. The global terrestrial COS sink has been estimated to be about 1.194–1.721 Tg a−1, with the terrestrial sink induced by plants and soils 0.50–1.20 Tg a−1, accounting for 41%–69% of the total. Hence, the role of plants and soils as COS sinks has been extensively explored. Now we know that factors such as the activity of carbonic anhydrase (CA), leaf structural traits, soil microbial activity, and environmental factors play significant roles in the COS budget. Developments in observational techniques have also made important contributions to the COS budget. This paper provides an overview of the research progress made on COS based on a comprehensive review of the literature. Then, it highlights the current research hotspots and issues requiring further exploration. For instance, it has been demonstrated that there are still significant uncertainties in the estimation of COS sources and sinks, emphasizing the need for further exploration of COS measuring techniques. This review aims to provide comprehensive guidance for COS research in terrestrial ecosystems.

Elucidating the role of primary and secondary sphere Zn2+ ligands in the cyanobacterial CO2 uptake complex NDH-14: The essentiality of arginine in zinc coordination and catalysis

Inorganic carbon uptake in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). Specialized Type-1 NDH complexes function as a part of this mechanism to couple photosynthetic energy generated by redox reactions of the electron transport chain (ETC) to CO2 hydration. This active site of CO2 hydration incorporates an arginine side chain as a Zn ligand, diverging from the typical histidine and/or cysteine residues found in standard CAs. In this study, we focused on mutating three amino acids in the active site of the constitutively expressed NDH-14 CO2 hydration complex in Synechococcus sp. PCC7942: CupB-R91, which acts as a zinc ligand, and CupB-E95 and CupB-H89, both of which closely interact with the arginine ligand. These mutations aimed to explore how they affect the unusual metal ligation by CupB-R91 and potentially influence the unusual catalytic process. The most severe defects in activity among the targeted residues are due to a substitution of CupB-R91 and the ionically interacting E95 since both proved essential for the structural stability of the CupB protein. On the other hand, CupB-H89 mutations show a range of catalytic phenotypes indicating a role of this residue in the catalytic mechanism of CO2-hydration, but no evidence was obtained for aberrant carbonic anhydrase activity that would have indicated uncoupling of the CO2-hydration activity from proton pumping. The results are discussed in terms of possible alternative CO2 hydration mechanisms.

Co-removal and recycling of Ba2+ and Ca2+ in hypersaline wastewater based on the microbially induced carbonate precipitation technique: Overlooked Ba2+ in extracellular and intracellular vaterite

This study investigates the co-precipitation of calcium and barium ions in hypersaline wastewater under the action of Bacillus licheniformis using microbially induced carbonate precipitation (MICP) technology, as well as the bactericidal properties of the biomineralized product vaterite. The changes in carbonic anhydrase activity, pH, carbonate and bicarbonate concentrations in different biomineralization systems were negatively correlated with variations in metal ion concentrations, while the changes in polysaccharides and protein contents in bacterial extracellular polymers were positively correlated with variations in barium concentrations. In the mixed calcium and barium systems, the harvested minerals were vaterite containing barium. The increasing concentrations of calcium promoted the incorporation and adsorption of barium onto vaterite. The presence of barium significantly increased the contents of O-CO, N-CO, and Ba-O in vaterite. Calcium promoted barium precipitation, but barium inhibited calcium precipitation. After being treated by immobilized bacteria, the concentrations of calcium and barium ions decreased from 400 and 274 to 1.72 and 0 mg/L (GB/T15454–2009 and GB8978–1996). Intracellular minerals were also vaterite containing barium. Extracellular vaterite exhibited bactericidal properties. This research presents a promising technique for simultaneously removing and recycling hazardous heavy metals and calcium in hypersaline wastewater.

Micronized Zn Oxide on Carbonic Anhydrase Activity, Health, and Yield of Ratoon Sugarcane Under Tropical Conditions

Micronized Zn Oxide on Carbonic Anhydrase Activity, Health, and Yield of Ratoon Sugarcane Under Tropical Conditions

Gamma rays and sodium azide induced variations in bio-physiological and agronomical traits in linseed (Linum usitatissimum L.)

Linseed is a valuable oilseed crop with huge therapeutic importance due to its high content of omega-3 fatty acids in the form of Alpha-linolenic acid (ALA). It is a self-pollinated crop with a low-yielding potential that restricts its improvement endeavors. To overcome low-yielding potential, individual and combination treatments of gamma rays and sodium azide were employed in widely grown linseed varieties. The results revealed a dose-dependent decline in seed germination, seedling height, pollen fertility, chlorophyll, and carotenoid contents and a dose-independent decline in carbonic anhydrase activity. Bio-physiological parameters decreased substantially in combination treatments compared to individual treatments of gamma rays and sodium azide. In contrast, lower doses of gamma rays, sodium azide, and their combinations effectively increased mean values of yield and yield-attributing traits in a few putative mutants. Such putative mutants represent a valuable genetic resource that could be used in future breeding programs for the genetic improvement of linseed and related medicinal plants.

Mineralogical and environmental effects on the δ13C, δ18O, and clumped isotope composition of modern bryozoans

Extensive bryozoan fossil records date back to the early Ordovician, forming an important part of sedimentary archives, yet the applicability of classical 18‑oxygen thermometry to bryozoan carbonate is still a matter of debate, with mineralogical and biological issues still hindering paleoclimate reconstructions. Complementing other methods (i.e., δ18O, Mg/Ca), clumped-isotope thermometry (Δ47) could provide more accurate paleotemperature estimates and help identify potential biotic factors influencing the isotopic composition of bryozoans. Here we report on the first investigation of clumped-isotope thermometry applied to bryozoan carbonate, spanning a broad range of modern species living in different environments from two localities (Atlantic Ocean and Mediterranean Sea). We confirm that bryozoan δ13C and δ18O records are affected by biotic and abiotic factors susceptible to bias in growth condition estimates. Our Atlantic bryozoans yield Δ47 derived temperatures (T47) consistent with spring/fall seawater temperature (but only after correcting for minor mineralogical effects), reflecting seasonal growth bias or, more likely, moderate isotopic disequilibrium. By contrast, Mediterranean samples display large positive offsets from Δ47 equilibrium values. We propose that this stronger disequilibrium is related to the higher salinity at this site, decreasing Carbonic Anhydrase activity, favoring CO2 hydroxylation over hydration, and slowing down DIC (dissolved inorganic carbon) equilibration reactions at the precipitation site. Our findings highlight how “vital effects” in bryozoans is not only species-specific as often assumed for other biocalcifiers, but could also depend on mineralogy and environmental factors.

Linking red blood cell functional phenotypes to environmental tolerance in high-altitude adapted deer mice

Oxygen (O2) is essential for vertebrate life, and complex cardio-respiratory systems have evolved to transport the gas from the environment to each individual cell. Even short disruptions of this O2 flux can have deleterious effects that are linked to numerous disease states. Animals that have adapted to hypoxic environments, such as deer mice ( Peromyscus maniculatus) native to high altitude, can provide valuable insight into naturally evolved solutions to O2 deprivation. Previous work has shown that high-altitude deer mice have evolved a higher hemoglobin O2 affnity and other coordinated changes across the O2 transport cascade that enable higher metabolic rates in hypoxia. Red blood cells (RBC) are the functional unit of O2 and carbon dioxide transport in the blood and play central roles in matching O2 supply and demand in the microcirculation by releasing signaling molecules such as ATP and gasotransmitters; but how these cellular mechanisms respond to hypoxic environments has not been studied. We hypothesized that high-altitude adaptation in deer mice has improved the function of RBCs for cardiovascular gas transport in hypoxia. Lab-raised breeding colonies of deer-mice were established from wild mice caught at low altitude (~400 m in the Great Plains of Nebraska) and at high altitude (~4300 m in the Rocky Mountains of Colorado). Using a common-garden experimental design, third-generation deer mice from high- and low-altitude populations were acclimated to warm normoxia (21°C, 21 kPa O2) or cold hypobaric hypoxia (5°C, 12 kPa O2) for 8 weeks. Blood samples were collected for measurements of hematocrit, hemoglobin concentration, RBC volume, plasma erythropoietin concentration, RBC contents of membrane transport and channel proteins (anion exchanger, aquaporin 1 and rhesus associated glycoprotein) by immunocytochemistry and western blotting, and carbonic anhydrase activity using biochemical techniques. The release of ATP from RBCs was measured in tonometers at decreasing levels of O2 by luminometry, and the vascular sensitivity to ATP was determined by wire myography on second-order mesenteric arteries. Finally, bone marrow samples were collected from the femurs to measure gene expression levels in the erythropoietic tissue. Our experimental design allowed us to examine the interactive effects of cold hypoxic environments on RBC phenotype, by untangling environmentally-induced plasticity from the signatures of adaptation that are unique to high-altitude natives. This work is providing a better understanding of how RBC function participates in matching cardiovascular O2 supply and demand in extreme hypoxia, which has important applications in animal and human health. This work was supported by a NSERC Canada Banting Postdoctoral Fellowship to TSH and a Discovery Grant to GRS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation Effect of 2-β-hydroxy Manoyl Oxide Isolated from Sideritis perfoliata on Carbonic Anhydrase Isoenzymes I and II

Background: Sideritis species were used for the treatment of mental disorders such as Alzheimer’s and dementia traditionally in Turkey. Several in vivo studies report that the mid-polar extract of Sideritis species can develop the brain functions of mice. 2-β-hydroxy manoyl oxide, isolated from ethyl acetate extract of Sideritis perfoliata, was assayed in vitro and in silico on human erythrocytes CA I and CA II. The compound was found to be an activator on two isoenzymes. It has been reported that activators of carbonic anhydrases may be used as a novel approach to treating disorders such as Alzheimer’s and age-related diseases. This study aimed to investigate the activity effect of 2-β-hydroxy manoyl oxide in vitro and in silico on human erythrocytes CA I and CA II (hCA I and hCA II) and to elucidate its pharmacokinetic and physicochemical characteristics. Methods: The test compound was isolated from ethyl acetate extract of Sideritis perfoliata using chromatographic techniques and identified with spectroscopic evidence. Carbonic anhydrase activities were assayed using CO2 substrates. Docking studies were carried out with Molegro Virtual Docker. The compound underwent ADME-Tox prediction by using AdmetSAR and SwissADME software. Results: 2-β-hydroxy manoyl oxide was found to increase the hCA-l and hCAII activity with AC50 values 9 and 19 μM, respectively. These results were further confirmed in silico molecular modeling. It showed favorable pharmacokinetic and physicochemical characteristics as a new drug candidate. Conclusion: These findings demonstrated that 2-β-hydroxy manoyl oxide activated the hCA-l and hCA II. These results provide a novel and alternative activator for the carbonic anhydrase and confirm the traditional usage of the Sideritis perfoliata.

Effect of Ba2+ on the biomineralization of Ca2+ and Mg2+ ions induced by Bacillus licheniformis.

The effect of barium ions on the biomineralization of calcium and magnesium ions is often overlooked when utilizing microbial-induced carbonate precipitation technology for removing barium, calcium, and magnesium ions from oilfield wastewater. In this study, Bacillus licheniformis was used to bio-precipitate calcium, magnesium, and barium ions. The effects of barium ions on the physiological and biochemical characteristics of bacteria, as well as the components of extracellular polymers and mineral characteristics, were also studied in systems containing coexisting barium, calcium, and magnesium ions. The results show that the increasing concentrations of barium ions decreased pH, carbonic anhydrase activity, and concentrations of bicarbonate and carbonate ions, while it increased the contents of humic acids, proteins, polysaccharides, and DNA in extracellular polymers in the systems containing all three types of ions. With increasing concentrations of barium ions, the content of magnesium within magnesium-rich calcite and the size of minerals precipitated decreased, while the full width at half maximum of magnesium-rich calcite, the content of O-C=O and N-C=O, and the diversity of protein secondary structures in the minerals increased in systems containing all three coexisting ions. Barium ions does inhibit the precipitation of calcium and magnesium ions, but the immobilized bacteria can mitigate the inhibitory effect. The precipitation ratios of calcium, magnesium, and barium ions reached 81-94%, 68-82%, and 90-97%. This research provides insights into the formation of barium-enriched carbonate minerals and offers improvements for treating oilfield wastewater.

Comparative and interactive response of salicylic acid, 24–epibrassinolide or sodium nitroprusside against cadmium stress in Linum usitatissimum

AbstractConcerns regarding the effects of heavy metals (HMs) on agricultural productivity have grown over time. Because HM stress disrupts a number of the plants' physiological-biochemical and metabolic processes, it severely limits production. Phytohormones can effectively improve plants resistance to HM stress. This work was done to examine the comparative effectiveness of salicylic acid (SA), 24–epibrassinolide (EBL) and sodium nitroprusside (SNP) on photosynthetic attributes, growth, &amp; antioxidant enzymes activity in Linum usitatissimum cv. RLC–6 (flax) subjected to cadmium (Cd) stress during vegetative growth stages. Cd considerably decreases the length, biomass, leaf diameter, chlorophyll content, and photosynthetic traits; and further triggered ROS and MDA content in plant. Moreover, exogenous application of SA, EBL and SNP individually and in combination improved the antioxidant enzymatic machinery by increasing the levels of superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT) and decrease the superoxide, hydrogen peroxide, scavenges ROS and MDA accumulation. Furthermore, submission of phytohormones also caused proline to accumulate and the activities of carbonic anhydrase (CA) and nitrate reductase (NR) to be activated which were impaired due to Cd stress. Among the phytohormones, the most effective method for dropping the damaging impacts of Cd and promoting plant growth and development was EBL. However, combined application of all three phytohormones (SA + EBL + SNP) proved to be the best. Thus, it can be concluded that, these augmented activity of antioxidants and proline elicited by application of phytohormones, would have continued to be able to give Linum usitatissimum exposed to Cd stress resistance.

Effect of different microalgae-bacterium-fungus symbiont technologies on nutrient removal from aquaculture wastewater and biogas upgrading under a variety of mixed light wavelengths

To develop and characterize novel microalgae-based technology under different mixed light-emitting diode (LED) light wavelengths for simultaneous treatment of actual aquaculture tailwater and biogas upgrading, the performance of four microalgal cultivations in bamboo shrimp aquaculture tailwater treatment and biogas upgrading were systematically examined and analyzed. The results demonstrated that Chlorella vulgaris (C. vulgaris)-endophytic bacterium (S395-2)-Clonostachys rosea (C. rosea) symbiont had the highest growth rate, daily production rate and carbonic anhydrase activity at the 7th d treatment time-point under Red(5): Blue(5) LED mixed light source. After 10d treatment, the nutrient removal efficiency of C. vulgaris-S395-2-C. rosea symbiont under Red(5): Blue(5) LED mixed wavelengths reached up to 93.7% for total inorganic nitrogen, 97.4% for ammonium nitrogen, 96.9% for nitrate nitrogen and 98.8% for phosphate; carbon dioxide (CO2) removal rate maintained at 53.1% without significantly affecting methane (CH4) level. In summary, the present study demonstrates that Red (5): Blue(5) LED mixed wavelengths optimally increase the performance of C. vulgaris-S395-2-C. rosea symbiont in nutrient and CO2 removal without loss of CH4, providing a framework for future simultaneous performing actual aquaculture tailwater treatment and biogas upgrading using microalgae-endophytic bacteria-fungus symbiont.

Differential carbonic anhydrase activities control EBV-induced B-cell transformation and lytic cycle reactivation.

Epstein-Barr virus (EBV) contributes to ~1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naïve B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus, ectopic expression of EBNA2 and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

Novel chemotype NLRP3 inhibitors that target the CRID3-binding pocket with high potency.

The NLRP3 inflammasome plays a central role in various human diseases. Despite significant interest, most clinical-grade NLRP3 inhibitors are derived from sulfonylurea inhibitor CRID3 (also called MCC950). Here, we describe a novel chemical class of NLRP3-inhibiting compounds (NIC) that exhibit potent and selective NLRP3 inflammasome inhibition in human monocytes and mouse macrophages. BRET assays demonstrate that they physically interact with NLRP3. Structural modeling further reveals they occupy the same binding site of CRID3 but in a critically different conformation. Furthermore, we show that NIC-11 and NIC-12 lack the off-target activity of CRID3 against the enzymatic activity of carbonic anhydrases I and II. NIC-12 selectively reduces circulating IL-1ß levels in the LPS-endotoxemia model in mice and inhibits NLRP3 inflammasome activation in CAPS patient monocytes and mouse macrophages with about tenfold increased potency compared with CRID3. Altogether, this study unveils a new chemical class of highly potent and selective NLRP3-targeted inhibitors with a well-defined molecular mechanism to complement existing CRID3-based NLRP3 inhibitors in pharmacological studies and serve as novel chemical leads for the development of NLRP3-targeted therapies.

Changes in Paraoxonase, β-Glucosidase, and Carbonic Anhydrase Enzymes Related to Age and Scopolamine-Induced Memory Impairment in Rats

This study aimed to investigate the changes in Paraoxonase (PON), Carbonic Anhydrase (CA), and β-glucosidase levels of different aged rats and scopolamine-induced memory impairment rats. This study used young, adult, and middle-aged male Wistar Albino rats. Scopolamine was administered as a single dose/multiple doses and a Morris water maze (MWM) was used for spatial learning testing in rats. Enzyme-linked immunosorbent assays (ELISAs) were used to determine serum and liver PON and β-glucosidase levels. The CA enzyme activity was assayed following the hydration of CO2. As a result of the comparison of age-related and scopolamine-related changes in PON and β-glucosidase levels in liver and serum samples, no significant age-related and scopolamine-effective changes were observed in serum, while liver PON and liver β-glucosidase levels were found to change significantly. CA activity studies, on the other hand, showed that adults have the lowest CA activity compared to young and middle-aged groups and scopolamine inhibited CA activity in vivo. We found that adult rats modeled with memory impairment had statistically lower levels of liver PON and liver β-glucosidase. CA activity was also found to be significantly reduced. β-glucosidase and CA should be further investigated in terms of neurodegenerative disease risk factors, just like PON, whose importance has been determined by numerous studies in the literature.