Effects of down-regulated carbonic anhydrase 8 on cell survival and glucose metabolism in human colorectal cancer cell lines.

Carbonic anhydrase 8 (CA8) is a member of the α-carbonic anhydrase family but does not catalyze the reversible hydration of carbon dioxide. In the present study, we examined the effects of CA8 on two human colon cancer cell lines, SW480 and SW620, by suppressing CA8 expression through shRNA knockdown. Our results showed that knockdown of CA8 decreased cell growth and cell mobility in SW620 cells, but not in SW480 cells. In addition, downregulated CA8 resulted in a significant decrease of glucose uptake in both SW480 and SW620 cells. Interestingly, stable downregulation of CA8 decreased phosphofructokinase-1 expression but increased glucose transporter 3 (GLUT3) levels in SW620 cells. However, transient downregulation of CA8 fails to up-regulate GLUT3 expression, indicating that the increased GLUT3 observed in SW620-shCA8 cells is a compensatory effect. In addition, the interaction between CA8 and GLUT3 was evidenced by pull-down and IP assays. On the other hand, we showed that metformin, a first-line drug for type II diabetes patients, significantly inhibited cell migration of SW620 cells, depending on the expressions of CA8 and focal adhesion kinase. Taken together, our data demonstrate that when compared to primary colon cancer SW480 cells, metastatic colon cancer SW620 cells respond differently to downregulated CA8, indicating that CA8 in more aggressive cancer cells may play a more important role in controlling cell survival and metformin response. CA8 may affect glucose metabolism- and cell invasion-related molecules in colon cancer, suggesting that CA8 may be a potential target in future cancer therapy.

Systematic Analysis of the Relationship Between Elevated Zinc and Epilepsy.

Previous studies have indicated a potential relationship between zinc and epilepsy. The aim of this study is to investigate the causal relationship between zinc, zinc-dependent carbonic anhydrase, and gray matter volume in brain regions enriched with zinc and epilepsy, as well as explore the possible mechanisms by which zinc contributes to epilepsy. First, this study assessed the risk causality between zinc, carbonic anhydrase, and gray matter volume alterations in zinc-enriched brain regions and various subtypes of epilepsy based on Two-sample Mendelian randomization analysis. And then, this study conducted GO/KEGG analysis based on colocalization analysis, MAGMA analysis, lasso regression, random forest model, and XGBoost model. The results of Mendelian randomization analyses showed a causal relationship between zinc, carbonic anhydrase-4, and generalized epilepsy (p = 0.044 , p = 0.010). Additionally, carbonic anhydrase-1 and gray matter volume of the caudate nucleus were found to be associated with epilepsy and focal epilepsy (p = 0.014, p = 0.003 and p = 0.022, p = 0.009). A colocalization relationship was found between epilepsy and focal epilepsy (PP.H4.abf = 97.7e - 2). Meanwhile, the MAGMA analysis indicated that SNPs associated with epilepsy and focal epilepsy were functionally localized to zinc-finger-protein-related genes (p < 1.0e - 5). The genes associated with focal epilepsy were found to have a molecular function of zinc ion binding (FDR = 2.3e - 6). After the onset of epilepsy, the function of the gene whose expression changed in the rats with focal epilepsy was enriched in the biological process of vascular response (FDR = 4.0e - 5). These results revealed mechanism of the increased risk of epilepsy caused by elevated zinc may be related to the increase of zinc ion-dependent carbonic anhydrase or the increase of the volume of zinc-rich caudate gray matter.

Genome Sequence Analysis of Calcifying Bacteria Bacillus paranthracis CT5 and Its Biomineralization Efficacy to Improve the Strength and Durability Properties of Civil Structures.

Bacteria producing urea amidohydrolases (UA) and carbonic anhydrases (CA) are of great importance in civil engineering as these enzymes are responsible for microbially induced calcium carbonate precipitation (MICCP). In this investigation, genomic insights of Bacillus paranthracis CT5 and the expression of genes underlying in MICCP were studied. B. paranthracis produced a maximum level of UA (669.3 U/ml) and CA (125 U/ml) on 5th day of incubation and precipitated 197 mg/100 ml CaCO3 after 7 days of incubation. After 28 days of curing, compressive strength of bacterial admixed and bacterial cured (B-B) specimens was 13.7% higher compared to water-mixed and water-cured (W-W) specimens. A significant decrease in water absorption was observed in bacterial-cured specimens compared to water-cured specimens after 28 days of curing. For genome analysis, reads were assembled de novo producing 5,402,771 bp assembly with N50 of 273,050 bp. RAST annotation detected six amidohydrolase and three carbonic anhydrase genes. Among 5700 coding sequences found in genome, COG gene annotation grouped 4360 genes into COG categories with highest number of genes to transcription (435 genes), amino acid transport and metabolism (362 genes) along with cell wall/membrane/envelope biogenesis and ion transport and metabolism. KEGG functional classification predicted 223 pathways consisting of 1,960 genes and the highest number of genes belongs to two-component system (101 genes) and ABC transporter pathways (98 genes) enabling bacteria to sense and respond to environmental signals and actively transport various minerals and organic molecules, which facilitate the active transport of molecules required for MICCP.

Characterization of a novel γ-type carbonic anhydrase, Sjγ-CA2, in Saccharina japonica: Insights into carbon concentration mechanism in macroalgae.

Carbonic anhydrase (CA) is a crucial component of CO2-concentrating mechanism (CCM) in macroalgae. In Saccharina japonica, an important brown seaweed, 11 CAs, including 5 α-, 3 ß-, and 3 γ-CAs, have been documented. Among them, one α-CA and one ß-CA were localized in the periplasmic space, one α-CA was found in the chloroplast, and one γ-CA was situated in mitochondria. Notably, the known γ-CAs have predominantly been identified in mitochondria. In this study, we identified a chloroplastic γ-type CA, Sjγ-CA2, in S. japonica. Based on the reported amino acid sequence of Sjγ-CA2, the epitope peptide for monoclonal antibody production was selected as 165 Pro-305. After purification and specificity identification, anti-SjγCA2 monoclonal antibody was employed in immunogold electron microscopy. The results illustrated that Sjγ-CA2 was localized in the chloroplasts of both gametophytes and sporophytes of S. japonica. Subsequently, immunoprecipitation coupled with LC-MS/MS analysis revealed that Sjγ-CA2 mainly interacted with photosynthesis-related proteins. Moreover, the first 65 amino acids at N-terminal of Sjγ-CA2 was identified as the chloroplast transit peptide by the transient expression of GFP-SjγCA2 fused protein in tabacco. Real-time PCR results demonstrated an up-regulation of the transcription of Sjγ-CA2 gene in response to high CO2 concentration. These findings implied that Sjγ-CA2 might contribute to minimizing the leakage of CO2 from chloroplasts and help maintaining a high concentration of CO2 around Rubisco.

Uncovering the Dynamics of Urease and Carbonic Anhydrase Genes in Ureolysis, Carbon Dioxide Hydration, and Calcium Carbonate Precipitation.

The hydration of CO2 suffers from kinetic inefficiencies that make its natural trapping impractically sluggish. However, CO2-fixing carbonic anhydrases (CAs) remarkably accelerate its equilibration by 6 orders of magnitude and are, therefore, "ideal" catalysts. Notably, CA has been detected in ureolytic bacteria, suggesting its potential involvement in microbially induced carbonate precipitation (MICP), yet the dynamics of the urease (Ur) and CA genes remain poorly understood. Here, through the use of the ureolytic bacteriumSporosarcina pasteurii, we investigate the differing role of Ur and CA in ureolysis, CO2 hydration, and CaCO3 precipitation with increasing CO2(g) concentrations. We show that Ur gene up-regulation coincides with an increase in [HCO3-] following the hydration of CO2 to HCO3- by CA. Hence, CA physiologically promotes buffering, which enhances solubility trapping and affects the phase of the CaCO3 mineral formed. Understanding the role of CO2 hydration on the performance of ureolysis and CaCO3 precipitation provides essential new insights, required for the development of next-generation biocatalyzed CO2 trapping technologies.

Improved Solubility and Stability of a Thermostable Carbonic Anhydrase via Fusion with Marine-Derived Intrinsically Disordered Solubility Enhancers.

Carbonic anhydrase (CA), an enzyme catalyzing the reversible hydration reaction of carbon dioxide (CO2), is considered a promising biocatalyst for CO2 reduction. The α-CA of Thermovibrio ammonificans (taCA) has emerged as a compelling candidate due to its high thermostability, a critical factor for industrial applications. However, the low-level expression and poor in vitro solubility have hampered further utilization of taCA. Recently, these limitations have been addressed through the fusion of the NEXT tag, a marine-derived, intrinsically disordered small peptide that enhances protein expression and solubility. In this study, the solubility and stability of NEXT-taCA were further investigated. When the linker length between the NEXT tag and the taCA was shortened, the expression level decreased without compromising solubility-enhancing performance. A comparison between the NEXT tag and the NT11 tag demonstrated the NEXT tag's superiority in improving both the expression and solubility of taCA. While the thermostability of taCA was lower than that of the extensively engineered DvCA10, the NEXT-tagged taCA exhibited a 30% improvement in long-term thermostability compared to the untagged taCA, suggesting that enhanced solubility can contribute to enzyme thermostability. Furthermore, the bioprospecting of two intrinsically disordered peptides (Hcr and Hku tags) as novel solubility-enhancing fusion tags was explored, demonstrating their performance in improving the expression and solubility of taCA. These efforts will advance the practical application of taCA and provide tools and insights for enzyme biochemistry and bioengineering.

Detailed Analysis of ITPR1 Missense Variants Guides Diagnostics and Therapeutic Design.

BACKGROUND: The ITPR1 gene encodes the inositol 1,4,5-trisphosphate (IP3 ) receptor type 1 (IP3 R1), a critical player in cerebellar intracellular calcium signaling. Pathogenic missense variants in ITPR1 cause congenital spinocerebellar ataxia type 29 (SCA29), Gillespie syndrome (GLSP), and severe pontine/cerebellar hypoplasia. The pathophysiological basis of the different phenotypes is poorly understood. OBJECTIVES: We aimed to identify novel SCA29 and GLSP cases to define core phenotypes, describe the spectrum of missense variation across ITPR1, standardize the ITPR1 variant nomenclature, and investigate disease progression in relation to cerebellar atrophy. METHODS: Cases were identified using next-generation sequencing through the Deciphering Developmental Disorders study, the 100,000 Genomes project, and clinical collaborations. ITPR1 alternative splicing in the human cerebellum was investigated by quantitative polymerase chain reaction. RESULTS: We report the largest, multinational case series of 46 patients with 28 unique ITPR1 missense variants. Variants clustered in functional domains of the protein, especially in the N-terminal IP3 -binding domain, the carbonic anhydrase 8 (CA8)-binding region, and the C-terminal transmembrane channel domain. Variants outside these domains were of questionable clinical significance. Standardized transcript annotation, based on our ITPR1 transcript expression data, greatly facilitated analysis. Genotype-phenotype associations were highly variable. Importantly, while cerebellar atrophy was common, cerebellar volume loss did not correlate with symptom progression. CONCLUSIONS: This dataset represents the largest cohort of patients with ITPR1 missense variants, expanding the clinical spectrum of SCA29 and GLSP. Standardized transcript annotation is essential for future reporting. Our findings will aid in diagnostic interpretation in the clinic and guide selection of variants for preclinical studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

A Carbonic Anhydrase, ZmCA4, Contributes to Photosynthetic Efficiency and Modulates CO2 Signaling Gene Expression by Interacting with Aquaporin ZmPIP2;6 in Maize.

Carbonic anhydrase (CA) catalyzes the reversible CO2 hydration reaction that produces bicarbonate for phosphoenolpyruvate carboxylase (PEPC). This is the initial step for transmitting the CO2 signal in C4 photosynthesis. However, it remains unknown whether the maize (Zea mays L.) CA gene, ZmCA4, plays a role in the maize photosynthesis process. In our study, we found that ZmCA4 was relatively highly expressed in leaves and localized in the chloroplast and the plasma membrane of mesophyll protoplasts. Knock-out of ZmCA4 reduced CA activity, while overexpression of ZmCA4 increased rubisco activity, as well as the quantum yield and relative electron transport rate in photosystem II. Overexpression of ZmCA4 enhanced maize yield-related traits. Moreover, ZmCA4 interacted with aquaporin ZmPIP2;6 in bimolecular fluorescence complementation and co-immunoprecipitation experiments. The double-knock-out mutant for ZmPIP2;6 and ZmCA4 genes showed reductions in its growth, CA and PEPC activities, assimilation rate and photosystem activity. RNA-Seq analysis revealed that the expression of other ZmCAs, ZmPIPs, as well as CO2 signaling pathway homologous genes, and photosynthetic-related genes was all altered in the double-knock-out mutant compared with the wild type. Altogether, our study's findings point to a critical role of ZmCA4 in determining photosynthetic capacity and modulating CO2 signaling regulation via its interaction with ZmPIP2;6, thus providing insight into the potential genetic value of ZmCA4 for maize yield improvement.

Climate change: approach to intervention using expression vector for carbonic anhydrase via glycosylphosphatidylinositol.

Climate change is a change in the usual weather found in a place. The climate change has a major impact not only on natural disasters of the Earth but also on human health. The climate crisis is then no longer a future concern. It includes both the global warming driven by human emissions of greenhouse gases (GHG), and the resulting large-scale shifts in weather patterns. Global warming can occur from a variety of causes, both natural and human induced. The primary GHG in Earth's atmosphere, listed in decreasing order of average global mole fraction, are: water vapor (H2O), carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and ozone (O3). Today, scientists around the world continue to try and solve the puzzle of climate change. It is clear that to address climate change, the amount of CO2 released into the atmosphere by industrial process has to be reduced because once it is added to the atmosphere, it can continue to affect climate for thousands of years. For such a purpose, an approach to intervention using expression vectors for any protein targeting to the cell plasma membrane via the glycosylphosphatidylinositol, GPI, anchor is suggested. The resulting GPI-anchored proteins would be useful for studying intermolecular interactions, especially gene-environment interactions, in investigating the potential impact of any chemical compounds on any genes of interest and could be used for carbonic anhydrase (CA)-based CO2-capture (environmental application). This approach would be crucial not only for capturing CO2 via GPI and CA but also for the production of CA enzyme as well as its stabilization and therefore useful for combating the global warming of climate change.

Rational design engineering of a more thermostable Sulfurihydrogenibium yellowstonense carbonic anhydrase for potential application in carbon dioxide capture technologies.

Implementing hyperthermostable carbonic anhydrases into CO2 capture and storage technologies in order to increase the rate of CO2 absorption from the industrial flue gases is of great importance from technical and economical points of view. The present study employed a combination of in silico tools to further improve thermostability of a known thermostable carbonic anhydrase from Sulfurihydrogenibium yellowstonense. Experimental results showed that our rationally engineered K100G mutant not only retained the overall structure and catalytic efficiency but also showed a 3 °C increase in the melting temperature and a two-fold improvement in the enzyme half-life at 85 °C. Based on the molecular dynamics simulation results, rearrangement of salt bridges and hydrogen interactions network causes a reduction in local flexibility of the K100G variant. In conclusion, our study demonstrated that thermostability can be improved through imposing local structural rigidity by engineering a single-point mutation on the surface of the enzyme.

Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis.

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

Evolutionary analysis and quality assessment of ζ-carbonic anhydrase sequences from environmental microbiome.

Carbonic anhydrase (CA) is one of the most vital enzymes in living cells. This study has been performed due to the significance of this metalloenzyme for life and the novelty of some CA families like ζ-CA to evaluate evolutionary processes and quality check their sequences. In this study, bioinformatics methods revealed the presence of ζ-CA in some eukaryotic and prokaryotic microorganisms. Notably, it has not been previously reported in prokaryotes. The coexistence of ß- and ζ-CAs in some microorganisms is also a novel finding as well. Also, our analysis identified several CA proteins with 6-14 amino acid intervals between histidine and cysteine in the second highly conserved motif, which can be classified as the novel ζ-CA subfamily members that emerged under the Zn deficiency of aquatic ecosystems and selection pressure in these environments. There is also a possibility that the achieved results are rooted in the contamination of samples from the environmental microbiome genome with genomes of diatom species and the occurrence of errors was observed in the DNA sequencing outcomes. Combining of all results from evolutionary analysis to quality control of ζ-CA DNA sequences is the incentive motivation to explore more the hidden aspects of ζ-CAs.

An overview of novel antimicrobial carbonic anhydrase inhibitors.

INTRODUCTION: Four different genetic families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) are present in bacteria, α-, ß-, γ- and ι-CAs. They play relevant functions related to CO2, HCO3-/H+ ions homeostasis, being involved in metabolic biosynthetic pathways, pH regulation, and represent virulence and survival factors for bacteria in various niches. Bacterial CAs started to be considered druggable targets in the last decade, as their inhibition impairs survival, growth, and virulence of these pathogens. AREAS COVERED: Significant advances were registered in the last years for designing effective inhibitors of sulfonamide type for Helicobacter pylori α-CA, Neisseria gonorrhoeae α-CA, vacomycin-resistant enterococci (VRE) α- and γ-CAs, for which the in vivo validation has also been achieved. MIC-s in the range of 0.25-4.0 µg/mL for wild type and drug resistant N. gonorrhoeae strains, and of 0.007-2.0 µg/mL for VRE were observed for some 1,3,4-thiadiazole-2-sulfonamides, and acetazolamide was effective in gut decolonization from VRE. EXPERT OPINION: Targeting bacterial CAs from other pathogens, among which Vibrio cholerae, Mycobacterium tuberculosis, Brucella suis, Salmonella enterica serovar Typhimurium, Legionella pneumophila, Porphyromonas gingivalis, Clostridium perfringens, Streptococcus mutans, Burkholderia pseudomallei, Francisella tularensis, Escherichia coli, Mammaliicoccus (Staphylococcus) sciuri, Pseudomonas aeruginosa, may lead to novel antibacterials devoid of drug resistance problems.

Mechanistic and structural insights into the bifunctional enzyme PaaY from Acinetobacter baumannii.

PaaY is a thioesterase that enables toxic metabolites to be degraded through the bacterial phenylacetic acid (PA) pathway. The Acinetobacter baumannii gene FQU82_01591 encodes PaaY, which we demonstrate to possess γ-carbonic anhydrase activity in addition to thioesterase activity. The crystal structure of AbPaaY in complex with bicarbonate reveals a homotrimer with a canonical γ-carbonic anhydrase active site. Thioesterase activity assays demonstrate a preference for lauroyl-CoA as a substrate. The AbPaaY trimer structure shows a unique domain-swapped C-termini, which increases the stability of the enzyme in vitro and decreases its susceptibility to proteolysis in vivo. The domain-swapped C-termini impact thioesterase substrate specificity and enzyme efficacy without affecting carbonic anhydrase activity. AbPaaY knockout reduced the growth of Acinetobacter in media containing PA, decreased biofilm formation, and impaired hydrogen peroxide resistance. Collectively, AbPaaY is a bifunctional enzyme that plays a key role in the metabolism, growth, and stress response mechanisms of A. baumannii.

Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Frankia alni Carbonic Anhydrase Regulates Cytoplasmic pH of Nitrogen-Fixing Vesicles.

A phyloprofile of Frankia genomes was carried out to identify those genes present in symbiotic strains of clusters 1, 1c, 2 and 3 and absent in non-infective strains of cluster 4. At a threshold of 50% AA identity, 108 genes were retrieved. Among these were known symbiosis-associated genes such as nif (nitrogenase), and genes which are not know as symbiosis-associated genes such as can (carbonic anhydrase, CAN). The role of CAN, which supplies carbonate ions necessary for carboxylases and acidifies the cytoplasm, was thus analyzed by staining cells with pH-responsive dyes; assaying for CO2 levels in N-fixing propionate-fed cells (that require a propionate-CoA carboxylase to yield succinate-CoA), fumarate-fed cells and N-replete propionate-fed cells; conducting proteomics on N-fixing fumarate and propionate-fed cells and direct measurement of organic acids in nodules and in roots. The interiors of both in vitro and nodular vesicles were found to be at a lower pH than that of hyphae. CO2 levels in N2-fixing propionate-fed cultures were lower than in N-replete ones. Proteomics of propionate-fed cells showed carbamoyl-phosphate synthase (CPS) as the most overabundant enzyme relative to fumarate-fed cells. CPS combines carbonate and ammonium in the first step of the citrulline pathway, something which would help manage acidity and NH4+. Nodules were found to have sizeable amounts of pyruvate and acetate in addition to TCA intermediates. This points to CAN reducing the vesicles' pH to prevent the escape of NH3 and to control ammonium assimilation by GS and GOGAT, two enzymes that work in different ways in vesicles and hyphae. Genes with related functions (carboxylases, biotin operon and citrulline-aspartate ligase) appear to have undergone decay in non-symbiotic lineages.

Upregulation of carbonic anhydrase 1 beneficial for depressive disorder.

Carbonic Anhydrase 1 (CAR1) is a zinc-metalloenzyme that catalyzes the hydration of carbon dioxide, and the alteration of CAR1 has been implicated in neuropsychiatric disorders. However, the mechanism underlying the role of CAR1 in major depressive disorder (MDD) remains largely unknown. In this study, we report the decreased level of CAR1 in MDD patients and depression-like model rodents. We found that CAR1 is expressed in hippocampal astrocytes and CAR1 regulates extracellular bicarbonate concentration and pH value in the partial hilus. Ablation of the CAR1 gene increased the activity of granule cells via decreasing their miniature inhibitory postsynaptic currents (mIPSC), and caused depression-like behaviors in CAR1-knockout mice. Astrocytic CAR1 expression rescued the deficits in mIPSCs of granule cells and reduced depression-like behaviors in CAR1 deficient mice. Furthermore, pharmacological activation of CAR1 and overexpression of CAR1 in the ventral hippocampus of mice improved depressive behaviors. These findings uncover a critical role of CAR1 in the MDD pathogenesis and its therapeutic potential.

Arabidopsis stromal carbonic anhydrases exhibit non-overlapping roles in photosynthetic efficiency and development.

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO2 to HCO3 - . The Arabidopsis genome encodes members of the α-, ß- and γ-CA families, and it has been hypothesized that ßCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial ßCAs, ßCA1 and ßCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of ßCA5 induced the expression of ßCA1, supporting the existence of regulatory mechanisms to control the expression of stromal ßCAs. We also established that ßCA1 and ßCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that ßCA5 had a first-order rate constant ~10-fold lower than ßCA1, and that the loss of ßCA5 is detrimental to growth and could be rescued by high CO2 . Furthermore, we established that, while a ßCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of ßCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed ßCA1 does not compensate for the loss of a less active ßCA5, which in turn is involved in growth and photosynthesis at ambient CO2 levels. These results lend support to the hypothesis that, in Arabidopsis,ßCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal ßCA5 and a dispensable role for ßCA1.

Association between Carbonic Anhydrase VI Gene Copy Number Variations and Dental Caries Experience.

The current study examined the association between the carbonic anhydrase VI (CA VI) copy number variations (CNVs) and dental caries experience in adults. In total, 202 of 35-72 years old subjects participating in the Lithuanian National Oral Health Survey (LNOHS) agreed to provide saliva samples; thus, their data were included in the current study. Information about sociodemographic, environmental, and behavioural determinants was acquired via the self-administered World Health Organization (WHO) questionnaire. Fluoride levels in the drinking water were recorded based on information provided by water suppliers. Dental caries experience was recorded by one calibrated examiner using the WHO criteria for recording caries on smooth (including proximal, buccal, and oral) or occlusal surfaces. Caries experience was measured as the total number of decayed (D3), missing (M), filled (F) surfaces. DNA was extracted from saliva samples to examine CA VI CNVs using the QX200 Droplet Digital PCR system. Negative binomial regression and Poisson regression analyses were employed for data analyses. Based on multivariable regression analyses, higher copy number of CA VI were associated with higher caries experience on smooth surfaces (IRR 1.04, 95% CI: 1.005-1.08) and occlusal surfaces (IRR 1.02, 95% CI: 1.003-1.04). Positive associations between higher copy number of CA VI and higher caries experience on smooth and occlusal surfaces were found, suggesting that the CA VI coding gene may be associated with caries development. Future studies are needed to validate our results and to examine the underlying mechanisms of such associations.

Computational and biochemical methods to measure the activity of carboxysomes and protein organelles in vivo.

Cyanobacteria are photosynthetic microorganisms that play important ecological roles as major contributors to global nutrient cycles. Cyanobacteria are highly efficient in carrying out oxygenic photosynthesis because they possess carboxysomes, a class of bacterial microcompartments (BMC) in which a polyhedral protein shell encapsulates the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase and functions as the key component of the cyanobacterial CO2-concentrating mechanism (CCM). Elevated CO2 levels within the carboxysome shell as a result of carbonic anhydrase activity increase the efficiency of RuBisCO. Yet, there remain many questions regarding the flux or exclusion of metabolites across the shell and how the activity of BMCs varies over time. These questions have been difficult to address using traditional ensemble techniques due to the heterogeneity of BMCs extracted from their native hosts or with heterologous expression. In this chapter, we describe a method to film and extract quantitative information about carboxysome activity using molecular biology and live cell, timelapse microscopy. In our method, the production of carboxysomes is first controlled by deleting the native genes required for carboxysome assembly and then re-introducing them under the control of an inducible promoter. This system enables carboxysomes to be tracked through multiple generations of cells and provides a way to quantify the total biomass accumulation attributed to a single carboxysome. While the method presented here was developed specifically for carboxysomes, it could be modified to track and quantify the activity of bacterial microcompartments in general.