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Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of ∼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.

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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Prognostic significance and gene signatures associated with carbonic anhydrase IX (CAIX) was investigated in triple negative breast cancer (TNBC) patients., Methods: Immunohistochemistry (IHC) for CAIX was performed in tissue microarrays (TMAs) of 136 TNBC patients. In a subset of 52 patients Digital Spatial Profiler (DSP) was performed in tumour (pan-cytokeratin+) and stroma (pan-cytokeratin-). Differentially expressed genes (DEGs) with P&lt;0.05 and and log2 fold change (FC)>(±0.25 and ±0.3, for tumour and stromal compartment, respectively) were identified. Four genes were validated at the protein level., Result: Cytoplasmic CAIX expression was independently associated with poor recurrence free survival in TNBC patients [hazard ratio (HR)=6.59, 95% confidence interval (CI): 1.47-29.58, P=0.014]. DEG analysis identified 4 up-regulated genes (CD68, HIF1A, pan-melanocyte, and VSIR) in the tumour region and 9 down-regulated genes in the stromal region (CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27, and FAS) when low versus high CAIX expression was explored. Employing IHC, high CD68 and HIF-1α was associated with poorer prognosis and high BCL2 and CD3 was associated with good prognosis., Conclusions: DSP technology identified DEGs in TNBC. Selected genes validated by IHC showed involvement of CD3 and BCL2 expression within stroma and HIF-1α, and CD68 expression within tumour. However, further functional analysis is warranted., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Carboxysomes are protein-based organelles that serve as the centerpiece of the bacterial CO 2 concentration mechanism (CCM). They are present in all cyanobacteria and many chemoautotrophic proteobacteria and encapsulate the key enzymes for CO 2 fixation, carbonic anhydrase and the carboxylase Rubisco, within a protein shell. The CCM actively accumulates bicarbonate in the cytosol, which diffuses into the carboxysome where carbonic anhydrase rapidly equilibrates it to CO 2 . This creates a high CO 2 concentration around Rubisco, ensuring efficient carboxylation. In this chapter, we present a general method for purifying α-carboxysomes and measuring carbonic anhydrase activity within these purified compartments. We exemplify this with α-carboxysomes purified from the chemoautotroph Halothiobacillus neapolitanus c2, a model organism for the α-carboxysome based CCM. However, this purification protocol can be adapted for other species, such as carboxysomes from α-cyanobacteria or carboxysomes expressed in heterologous hosts. Further, we describe the Khalifah/pH indicator assay for measuring steady-state kinetics of carbonic anhydrase catalyzed CO 2 hydration. This method allows us to determine the kinetic parameters k cat , K M and k cat /K M for the purified α-carboxysomes. It uses a stopped-flow spectrometer for rapid mixing and detection, crucial for capturing the fast equilibrium between CO 2 and bicarbonate. The reaction progress is monitored by absorbance via a pH indicator that changes color due to the proton release. While the method specifically focuses on measuring carbonic anhydrase activity on carboxysomes, it can be used to measure activity on carbonic anhydrases from other contexts as well., (Copyright © 2024. Published by Elsevier Inc.)

Purpose: Hypoxia is a characteristic of many solid tumours and an adverse prognostic factor for cancer therapy. Hypoxia results in upregulation of carbonic anhydrase IX (CAIX) expression, a pH-regulating enzyme. Many human tissue studies have examined the prognostic value of CAIX expression in breast cancer but have yielded inconsistent results. Therefore, a systematic review and meta-analysis was undertaken to assess the prognostic value of CAIX expression for breast cancer patients., Methods: The electronic databases were systematically searched to identify relevant papers. The clinical outcomes included disease-free survival (DFS), recurrence-free survival (RFS) and overall survival (OS) in breast cancer patients. Review Manager version 5.4 was employed to analysis data from 23 eligible studies (containing 8390 patients)., Results: High CAIX expression was associated with poorer RFS [HR = 1.42, 95% CI (1.32-1.51), p < 0.00001], DFS [HR = 1.64, 95% CI (1.34-2.00), p < 0.00001], and OS [HR = 1.48, 95% CI (1.22-1.80), p < 0.0001]. Heterogeneity was observed across the studies. There was an effect of the CAIX antibody employed, scoring methods, and tumour localisation on CAIX expression., Conclusion: CAIX overexpression was significantly associated with poorer RFS, DFS, and OS in breast cancer patients. However, further work in high quantity tissue cohorts is required to define the optimal methodological approach., (© 2023. The Author(s).)

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., (© 2022 S. Karger AG, Basel.)

Purpose: Accurate identification of nodal status enables adequate neck irradiation for nasopharyngeal carcinoma (NPC). However, most conventional techniques are unable to pick up occult metastases, leading to underestimation of tumor extensions. Here we investigate the clinical significance of carbonic anhydrase IX (CAIX) in human NPC samples, and develop a CAIX-targeted imaging strategy to identify occult lymph node metastases (LNMs) and extranodal extension (ENE) in animal studies., Methods: A total of 211 NPC samples are performed CAIX staining, and clinical outcomes are analyzed. The metastatic murine models are generated by foot pad injection of NPC cells, and a CAIX-targeted imaging agent (CAIX-800) is intravenously administered. We adopt fluorescence molecular tomography and ultrasonography (US)-guided spectroscopic photoacoustic (sPA) imaging to perform in vivo studies. Histological and immunohistochemical characterization are carried out via node-by-node analysis., Results: For clinical samples, 90.1% (91/101) primary tumors, 73.3% (66/90) metastases, and 100% (20/20) local recurrences are CAIX positive. In metastases group, 84.7% (61/72) nodal metastases and 22.2% (4/18) organ metastases are CAIX positive. CAIX expression in primary tumors is significantly associated with NPC stage and prognosis. For animal studies, CAIX-800-based fluorescence imaging achieves 81.3% sensitivity and 93.8% specificity in detecting occult LNMs in vivo, with a minimum detectable diameter of 1.7 mm. Coupled with CAIX-800, US-guided sPA imaging could not only detect subcapsular deposits of metastatic cancer cells 2 weeks earlier than conventional techniques, but also successfully track pathological ENE., Conclusion: CAIX remarkably expresses in human NPCs and stratifies patient prognosis. In preclinical studies, CAIX-800-based imaging successfully identifies occult LNMs and tracks early stage of pathological ENE. This attractive method shows potential in clinic, allowing medical workers to longitudinally monitor nodal status and helping to reduce unnecessary nodal biopsy for patients with NPC. The schematic diagram for the study. CAIX, carbonic anhydrase IX; NPC, nasopharyngeal carcinoma; US, ultrasonography; sPA, spectroscopic photoacoustic., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Carbonic anhydrases (CA, EC 4.2.1.1) catalyze the hydration of carbon dioxide and take part in many essential physiological processes. In humans, 15 CAs are characterized, including the only secreted isoenzyme CA VI. CA VI has been linked to specific processes in the mouth, namely bitter taste perception, dental caries, and maintenance of enamel pellicle, and implicated in several immunity-related phenomena. However, little is known of the mechanisms of the above. In this study, we characterized human CA VI purified from saliva and milk with biophysical methods and measured their enzyme activities and acetazolamide inhibition. Size-exclusion chromatography showed peaks of salivary and milk CA VI corresponding to hexameric state or larger at pH 7.5. At pH 5.0 the hexamer peaks dominated. SDS- PAGE of milk CA VI protein treated with a bifunctional crosslinker further confirmed that a majority of CA VI is oligomers of similar sizes in solution. Mass spectrometry experiments confirmed that both of the two putative N-glycosylation sites, Asn67 and Asn256, are heterogeneously glycosylated. The attached glycans in milk CA VI were di- and triantennary complex-type glycans, carrying both a core fucose and 1 to 2 additional fucose units, whereas the glycans in salivary CA VI were smaller, seemingly degraded forms of core fucosylated complex- or hybrid-type glycans. Mass spectrometry also verified the predicted signal peptide cleavage site and the terminal residue, Gln 18, being in pyroglutamate form. Thorough characterization of CA VI paves way to better understanding of the biological function of the protein., (© 2022. The Author(s).)

We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity ( k cat 3.0 × 10 5  s -1 ) for the physiological reaction of CO 2 hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9-13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with K A values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.

In recent years, food fraud is a global issue that has raised wide public concern. Mass spectrometry techniques have a significant advantage of qualitatively and quantitatively analyzing food authenticity, especially for highly processed meat products. In this work, a simple and specific, rapid resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of pork content in processed meat products according to internal standard (ISTD) method. To improve the efficiency of sample preparation, simplified bead-beating and enzymolysis process were investigated. In contrast with different heat-stable and specific porcine-peptides, EPITVSSDQMAK, GGPLTAAYR, HDPSLLPWTASYDPGSAK from Carbonic anhydrase 3 proved to have an excellent quantitative ability, thus obtaining good linear relationship and satisfactory recovery. This method was successfully applied to different types of meat products, thus demonstrating that complex mixtures of pork content can be accurately quantified., (Copyright © 2020. Published by Elsevier Ltd.)

Metastatic progression is the leading cause of mortality in breast cancer. However, molecular mechanisms that govern this process remain unclear. In this study, we found that carbonic anhydrase 13 (CA13) plays a potential role in suppressing bone metastasis. iRFP713-labeled iCSCL-10A (iRFP-iCSCL-10A) breast cancer cells, which exhibit the hallmarks of cancer stem cells, exerted the ability of bone metastasis in hind legs after 5-week injections, whereas no metastasis was observed in control iRFP713-labeled MCF-10A (iRFP-MCF10A) cells. Transcriptome analysis indicated that the expression of several genes, including metabolism-related CA13, was reduced in bone metastatic iRFP-iCSCL-10A cells. In vitro and in vivo analyses demonstrated that overexpression of CA13 in iRFP-iCSCL-10A cells suppressed migration, invasion, and bone metastasis, together with the reduction of VEGF-A and M-CSF expression. Furthermore, we found that breast cancer patients with a low CA13 expression had significantly shorter overall survival and disease-free survival rates compared to those with higher CA13 expression. These findings suggest that CA13 may act as a novel prognostic biomarker and would be a therapeutic candidate for the prevention of bone metastasis in breast cancer., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables the separation of ions on the basis of their differential mobility in an asymmetric oscillating electric field. We, and others, have previously demonstrated the benefits of FAIMS for the analysis of peptides and denatured proteins. To date, FAIMS has not been integrated with native mass spectrometry of folded proteins and protein complexes, largely due to concerns over the heating effects associated with the high electric fields employed. Here, we demonstrate the newly introduced cylindrical FAIMS Pro device coupled with an Orbitrap Eclipse enables analysis of intact protein assemblies up to 147 kDa. No evidence for dissociation was detected suggesting that any field heating is insufficient to disrupt the noncovalent interactions governing these assemblies. Moreover, the FAIMS device was integrated into native liquid extraction surface analysis (LESA) MS of protein assemblies directly from thin tissue sections. Intact tetrameric hemoglobin (64 kDa) and trimeric reactive intermediate deiminase A (RidA, 43 kDa) were detected. Improvements in signal-to-noise of between 1.5× and 12× were observed for these protein assemblies on integration of FAIMS.

The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and β-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.

A combination between gel electrophoresis and smartphone technology is applied for quantifying proteins in a serum sample. Electrophoresis not only allows one to separate different proteins but also to build-up a calibration curve for those proteins. To expand its applicability, a smartphone allows one to capture a gel image, through its camera. Additionally, the treatment of the data extracted through each protein band is performed using a freely available program (PhotoMetrix), which is downloaded to one's own smartphone. Through this approach, the quantification of proteins such as albumin, immunoglobulin, and carbonic anhydrase is performed in a serum sample by acquiring images using 32 × 32 pixels for each image in the region of the protein bands. An LOQ from 1.9 to 2.4 μg and r 2  > 0.980 may be obtained. Comparing results through the analyses of the gels using Image Master 2D Platinum 6.0 software reflects good agreement (95% confidence level) between the results. SIGNIFICANCE STATEMENT: In many biological studies involving structural analysis or biophysical and biochemical characterization after purification process, or even in assays of protein:protein interaction, the estimation of protein quantity and protein purity is a fundamental step. However, even although many methodologies are proposed in the literature for quantifying proteins, they present some limitations, once are frequently expensive and require solution sample. In addition, they usually do not quantify the specific active concentration, but the total amount. Thus, it is necessary to develop an easy, fast and low-cost method that allows to quantify and to evaluate protein purity of reactional system, for example, a protein:protein interaction. In this way, we presented a simple strategy based on the integration of Smartphone technology and gel electrophoresis, where SDS-PAGE provides multiple information regarding the quality of the protein, such as the presence of degradation products, as well as if there was interaction reaction between proteins. Then, the smartphone detects the proteins in the SDS-PAGE gel, allowing to evaluate purity degree and the quantity, at microgram level, of the protein. We believe that the combination of these features may help to increase the productivity and accuracy of gel electrophoresis analysis, and the results obtained through a smartphone, easily achieved in our pockets., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is well-known that with Orbitrap-based Fourier-transform-mass-spectrometry (FT-MS) analysis, longer-time-domain signals are needed to better resolve species of interest. Unfortunately, increasing the signal-acquisition period comes at the expense of increasing ion decay, which lowers signal-to-noise ratios and ultimately limits resolution. This is especially problematic for intact proteins, including antibodies, which demonstrate rapid decay because of their larger collisional cross-sections, and result in more frequent collisions with background gas molecules. Provided here is a method that utilizes numerous low-ion-count spectra and single-ion processing to reconstruct a conventional m/ z spectrum. This technique has been applied to proteins varying in molecular weight from 8 to 150 kDa, with a resolving power of 677 000 achieved for transients of carbonic anhydrase (29 kDa) with a duration of only ∼250 ms. A resolution improvement ranging from 10- to 20-fold was observed for all proteins, providing isotopic resolution where none was previously present.

Determination of collisional cross sections (CCS) by travelling wave ion mobility mass spectrometry (TWIM-MS) requires calibration against standards for which the CCS has been measured previously by drift tube ion mobility mass spectrometry (DTIM-MS). The different extents of collisional activation in TWIM-MS and DTIM-MS can give rise to discrepancies in the CCS of calibrants across the two platforms. Furthermore, the conditions required to ionize and transmit large, folded proteins and assemblies may variably affect the structure of the calibrants and analytes. Stable hetero-oligomeric phospholipase A 2 (PDx) and its subunits were characterized as calibrants for TWIM-MS. Conditions for acquisition of native-like TWIM (Synapt G1 HDMS) and DTIM (Agilent 6560 IM-Q-TOF) mass spectra were optimized to ensure the spectra exhibited similar charge state distributions. CCS measurements (DTIM-MS) for ubiquitin, cytochrome c, holo-myoglobin, serum albumin and glutamate dehydrogenase were in good agreement with other recent results determined using this and other DTIM-MS instruments. PDx and its β and γ subunits were stable across a wide range of cone and trap voltages in TWIM-MS and were stable in the presence of organic solvents. The CCS of PDx and its subunits were determined by DTIM-MS and were used as calibrants in determination of CCS of native-like cytochrome c, holo-myoglobin, carbonic anhydrase, serum albumin and haemoglobin in TWIM-MS. The CCS values were in good agreement with those measured by DTIM-MS where available. These experiments demonstrate conditions for analysis of native-like proteins using a commercially available DTIM-MS instrument, characterize robust calibrants for TWIM-MS, and present CCS values determined by DTIM-MS and TWIM-MS for native proteins to add to the current literature database. Graphical Abstract ᅟ.

In all vertebrates studied to date, CO 2 excretion depends on the enzyme carbonic anhydrase (CA) that catalyses the rapid conversion of HCO 3 - to CO 2 at the gas-exchange organs. The largest pool of CA is present within red blood cells (RBCs) and, in some vertebrates, plasma-accessible CA (paCA) isoforms participate in CO 2 excretion. However, teleost fishes typically do not have paCA at the gills and CO 2 excretion is reliant entirely on RBC CA - a strategy that is not possible in icefishes. As the result of a natural knockout, Antarctic icefishes (Channichthyidae) are the only known vertebrates that do not express haemoglobin (Hb) as adults, and largely lack RBCs in the circulation (haematocrit <1%). Previous work has indicated the presence of high levels of membrane-bound CA activity in the gills of icefishes, but without determining its cellular orientation. Thus, we hypothesised that icefishes express a membrane-bound CA isoform at the gill that is accessible to the blood plasma. The CA distribution was compared in the gills of two closely related notothenioid species, one with Hb and RBCs ( Notothenia rossii ) and one without ( Champsocephalus gunnari ). Molecular, biochemical and immunohistochemical markers indicate high levels of a Ca4 isoform in the gills of the icefish (but not the red-blooded N. rossii ), in a plasma-accessible location that is consistent with a role in CO 2 excretion. Thus, in the absence of RBC CA, the icefish gill could exclusively provide the catalytic activity necessary for CO 2 excretion - a pathway that is unlike that of any other vertebrate., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129 Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129 Xe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129 Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.

We report the synthesis and application of a small molecule probe for carbonic anhydrase (CA) to track holo-CA in cell lysates and live-cell models of zinc dyshomeostasis. The probe displays a 12-fold increase in fluorescence upon binding to bovine CA and also responds to human CA isoforms.

This study provides the presence of carbonic anhydrase (CA) activity in waters of the Yangtze River basin, China, as well as the correlation of CA activity with HCO(3)(-) concentration and CO(2) sink flux. Different degrees of CA activity could be detected in almost all of the water samples from different geological eco-environments in all four seasons. The CA activity of water samples from karst areas was significantly higher than from non-karst areas (P<0.01), indicating that the geological type of river basin affected the CA activity of waters. Distinct seasonal changes in CA activity were found, and the variational trend differed among different sampling sites. Generally, CA activity in summer and autumn was higher than in spring (P<0.01) for karst areas. The correlation analysis showed that water CA activity was positively correlated with HCO(3)(-) [corrected] concentration (r=0.672, P<0.01), and that the annual average water CA activity was positively correlated with the CO(2) [corrected] sink flux (r=0.602, P=0.076) in karst areas. This suggests that CA in waters might have a promoting effect on carbon sinks for atmospheric CO(2) in karst river basins. In conditions of similar geological type, higher CA activity was generally detected in water samples taken from areas that exhibited better eco-environments, implying that the CA activity index of waters could be used as an indicator for monitoring ecological environments and protection of river basins. These findings suggest that the role of CA in waters in the karst carbon sink potential of river basins is worthy of further in-depth studies., (Copyright © 2017. Published by Elsevier B.V.)

Carbonic anhydrase plays important role in life. This study sought to demonstrate the feasibility of detecting carbonic anhydrase activity in the human brain in vivo. After oral administration of [U- 13 C 6 ]glucose, 13 C saturation transfer experiments were performed with interleaved control spectra and carbon dioxide saturation spectra. Proton nuclear Overhauser effect pulses were used to increase signal to noise ratio; no proton decoupling was applied. Results showed that the 13 C signal of bicarbonate was reduced by 72% ± 0.03 upon saturating carbon dioxide. The unidirectional dehydration rate constant of the carbonic anhydrase reaction was found to be 0.28 ± 0.02 sec -1 in the human brain. These findings demonstrate the feasibility of measuring carbonic anhydrase activity in vivo in the human brain, which makes it possible to characterize this important enzyme in patients with brain disorders.

CruCA4, a coral α-carbonic anhydrase (CA, EC 4.2.1.1) involved in the biomineralization process of the Mediterranean red coral, Corallium rubrum , was investigated for its activation with a panel of amino acids and amines. Most compounds showed considerable activating properties, with a rather well defined structure-activity relationship. The most effective CruCA4 activators were d-His, 4-H₂N-l-Phe, Histamine, Dopamine, Serotonin, 1-(2-Aminoethyl)-piperazine, and l-Adrenaline, with activation constants in the range of 8-98 nM. Other amines and amino acids, such as d-DOPA, l-Tyr, 2-Pyridyl-methylamine, 2-(2-Aminoethyl) pyridine and 4-(2-Aminoethyl)-morpholine, were submicromolar CruCA4 activators, with K A ranging between 0.15 and 0.93 µM. Since it has been shown that CA activators may facilitate the initial phases of in-bone mineralization, our study may be relevant for finding modulators of enzyme activity, which can enhance the formation of the red coral skeleton., Competing Interests: The authors declare no conflict of interest.

This work describes the synthesis of azidonaphthalimide carboxylic acids which act as fluorescent templates with a built-in photoreactive group and a linker thus simplifying the design of protein labeling agents. These were separately connected to selectivity hands like a sulfonamide and ampicillin for successful labeling/detection of HCAII and PBPs, respectively.

Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

Objective: This study aimed to analyze the concentration and activity of carbonic anhydrase (CA) VI in the saliva of school children. We investigated the relationship among caries, CA VI concentration/activity, flow rate, pH, and buffering capacity., Materials and Methods: Seventy-four school children were divided into a caries-free group and a caries group. Clinical examinations were conducted by one examiner according to World Health Organization criteria + early caries lesions. Salivary flow rate, pH, and buffering capacity were analyzed. Salivary CA VI concentration and activity were evaluated by ELISA and zymography, respectively. The data were analyzed using Student's t test and the Mann-Whitney test, and Pearson and Spearman correlation analyses were also done. In multivariate modeling, associations between variables were expressed as odds ratios., Results: The results showed that salivary flow rate, salivary pH, and BC were significantly higher in the saliva of caries-free children. Also, the salivary CA VI concentration was significantly higher in the saliva of caries-free children. The salivary CA VI activity was higher in children with caries. We found a negative correlation between BC and dental caries. Also, in the caries group we found a positive correlation between the concentration and the activity of CA VI and a negative correlation between BC and CA VI activity. A negative correlation between salivary pH and CA VI concentration was observed in the caries-free group. A high activity of CA and a low salivary flow rate were associated with dental caries., Conclusion: These results support the conclusion that dental caries is highly affected by the activity of CA VI in saliva as well as by the salivary flow rate., (© 2017 S. Karger AG, Basel.)

Although saliva has esterase activity, this activity has not been characterized or studied in individuals subjected to physical stress. The aim of this report was to develop and validate an automated spectrophotometric assay for total esterase activity measurement in human saliva, as well as to study the contribution of different enzymes on this activity and its behaviour under physical stress in healthy subjects. The assay used 4-nitrophenyl acetate as substrate and was precise, accurate and provided low limits of detection and quantification. Inhibition with diisopropylfluorophosphate showed that cholinesterase, carboxylesterase and cholesterol esterase contributions not represented more than 20% of total esterase. Addition of standards of lipase and albumin to saliva samples showed that both proteins significantly contributed to esterase activity only when equal or higher than 11.6 IU/L and 250 μg/mL, respectively. Western blot analyses showed absence of paraoxonase-1 and high amount of carbonic anhydrase-VI. The high affinity of purified carbonic anhydrase-VI for the substrate supported a major contribution of this enzyme. Total esterase activity and alpha-amylase was measured in saliva samples from 12 healthy male students before and after participation in an indoor football match. The activity significantly increased after match and positively correlated with salivary alpha-amylase. This method could be used as a biomarker of physical stress in humans, with carbonic anhydrase-VI being the esterase that contributed more to the activity of the assay.

We report the implementation of proton transfer reactions (PTR) and ion parking on an Orbitrap mass spectrometer. PTR/ion parking allows charge states of proteins to be focused into a single lower charge state via sequential deprotonation reactions with a proton scavenging reagent, in this case, a nitrogen-containing adduct of fluoranthene. Using PTR and ion parking, we evaluate the charge state dependence of fragmentation of ubiquitin (8.6 kDa), myoglobin (17 kDa), and carbonic anhydrase (29 kDa) upon higher energy collisional dissociation (HCD) or ultraviolet photodissociation (UVPD). UVPD exhibited less charge state dependence, thus yielding more uniform distributions of cleavages along the protein backbone and consequently higher sequence coverage than HCD. HCD resulted in especially prominent cleavages C-terminal to amino acids containing acidic side-chains and N-terminal to proline residues; UVPD did not exhibit preferential cleavage adjacent to acidic residues but did show enhancement next to proline and phenylalanine.

Objectives: The objective of the study was to investigate prognostic and predictive value of pretreatment soluble carbonic anhydrase IX (CA IX) blood serum concentration in patients with locally advanced head and neck cancer., Background: Increased expression of CA IX in tumor tissues has been associated with treatment resistance and worth prognosis. Soluble form of CA IX, released from tumor cells, is detectable in blood serum and could be a convenient predictive factor of treatment effectiveness that would enable treatment individualization., Methods: The prospective study evaluated 48 patients with locally advanced squamous cell carcinomas of head and neck, treated by radiotherapy or chemo-radiotherapy. Pretreatment soluble CA IX serum concentrations were examined using enzyme-linked immunosorbent assay., Results: Soluble CA IX serum concentration failed to predict radiotherapy effectiveness in the studied patient population (p = 0.26). However, high CA IX serum concentrations have been associated with shorter overall survival (p = 0.035) CONCLUSION: High pretreatment CA IX serum concentration is a negative prognostic factor in locally advanced head and neck cancer patients (Tab. 1, Fig. 2, Ref. 23).

Hypoxia-associated proteome changes have been shown to be associated with resistance to chemo- and radiotherapy. Our study evaluated the role of the hypoxia-inducible (HIF)-1 target gene carbonic anhydrase (CA) IX in the prediction of the response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer (stages II and III). A total of 29 pretreatment biopsy specimens were stained for CA IX by immunohistochemistry, converted to digital images and evaluated in a quantitative fashion using image analysis software. Contrary to our expectations, a trend towards a correlation between better tumor regression (>50%) and higher expression of CA IX (p=0.056) was found. CA IX was also present more frequently in pathological tumor stage T1 (pT1) tumors (p=0.048). Conversely, no association with lymph node metastasis was identified. In conclusion, as a single marker, CA IX expression is not able to identify a hypoxia-related treatment resistant phenotype in rectal cancer.

Carbonic anhydrases (CAs) are metalloenzymes, and classified into the evolutionarily distinct α, β, γ, δ, ζ, and η classes. α-CAs are present in many living organisms. β- and γ-CAs are expressed in most prokaryotes and eukaryotes, except for vertebrates. δ- and ζ-CAs are present in phytoplanktons, and η-CAs have been found in Plasmodium spp. Since the identification of α- and β-CAs in Caenorhabditis elegans, the nematode CAs have been considered as an emerging target in research focused on antiparasitic CA inhibitors. Despite the presence of α-CAs in both helminths and vertebrates, structural studies have revealed different kinetic and inhibition results. Moreover, lack of β-CAs in vertebrates makes this enzyme as an attractive target for inhibitory studies against helminthic infection. Some CA inhibitors, such as sulfonamides, have been evaluated against nematode CAs. This review article aims to present comprehensive information about the nematode CAs and their inhibitors as potential anthelminthic drugs.

Adrenocortical carcinomas (ACCs) are complex neoplasias that may present unexpected clinical behavior, being imperative to identify new biological markers that can predict patient prognosis and provide new therapeutic options. The main aim of the present study was to evaluate the prognostic value of metabolism-related key proteins in adrenocortical carcinoma. The immunohistochemical expression of MCT1, MCT2, MCT4, CD147, CD44, GLUT1 and CAIX was evaluated in a series of 154 adult patients with adrenocortical neoplasia and associated with patients' clinicopathological parameters. A significant increase in was found for membranous expression of MCT4, GLUT1 and CAIX in carcinomas, when compared to adenomas. Importantly MCT1, GLUT1 and CAIX expressions were significantly associated with poor prognostic variables, including high nuclear grade, high mitotic index, advanced tumor staging, presence of metastasis, as well as shorter overall and disease free survival. In opposition, MCT2 membranous expression was associated with favorable prognostic parameters. Importantly, cytoplasmic expression of CD147 was identified as an independent predictor of longer overall survival and cytoplasmic expression of CAIX as an independent predictor of longer disease-free survival. We provide evidence for a metabolic reprogramming in adrenocortical malignant tumors towards the hyperglycolytic and acid-resistant phenotype, which was associated with poor prognosis.

Background and Objective: Carbonic anhydrase IX (CAIX) is widely expressed in a variety of malignant tumors, including-lung cancer. Our previous study has shown that the serum level of soluble form of carbonic anhydrase IX (s-CAIX) was significantly higher in patients with lung cancer than that in the healthy group. The aim of this study is to detect the s-CAIX level in the pleural effusion and its sediment, and to evaluate the significance of CAIX detection in the diagnosis of lung cancer., Methods: The s-CAIX level in pleural effusion of 29 lung cancer patients and 27 patients with tuberculosis was detected by ELISA. The expression of CAIX in the pleural effusion sediment of 21 lung cancer patients with malignant pleural effusion and 6 patients with benign pleural effusion was examined by immunohistochemistry. With pathological diagnosis as the gold standard, receiver operating characteristic (ROC) curve of pleural effusion s-CAIX was established for the diagnosis of lung cancer with malignant pleural effusion., Results: The s-CAIX level in the malignant pleural effusion was significantly higher than that in the tuberculosis group (P<0.05). The AUC of pleural effusion s-CAIX level was 0.761. At a threshold level of 109.135 pg/mL, sensitivity and specificity were 92.3% and 58.3%, respectively. The CAIX expression in all samples of the benign pleural effusion sediment was negative. The positive rate of CAIX expression in malignant pleural effusion sediment was 66.67%., Conclusions: Detection of CAIX in the pleural effusion and its sediment exhibits high sensitivity and specificity, and is helpful in diagnosis of lung cancer with malignant pleural effusion.

Nuptial gifts produced by males and transferred to females during copulation are common in insects. Yet, their precise composition and subsequent physiological effects on the female recipient remain unresolved. Male decorated crickets Gryllodes sigillatus transfer a spermatophore to the female during copulation that is composed of an edible gift, the spermatophylax, and the ampulla that contains the ejaculate. After transfer of the spermatophore, the female detaches the spermatophylax and starts to eat it while sperm from the ampulla are evacuated into the female reproductive tract. When the female has finished consuming the spermatophylax, she detaches the ampulla and terminates sperm transfer. Hence, one simple function of the spermatophylax is to ensure complete sperm transfer by distracting the female from prematurely removing the ampulla. However, the majority of orally active components of the spermatophylax itself and their subsequent effects on female behavior have not been identified. Here, we report the first analysis of the proteome of the G. sigillatus spermatophylax and the transcriptome of the male accessory glands that make these proteins. The accessory gland transcriptome was assembled into 17,691 transcripts whilst about 30 proteins were detected within the mature spermatophylax itself. Of these 30 proteins, 18 were encoded by accessory gland encoded messages. Most spermatophylax proteins show no similarity to proteins with known biological functions and are therefore largely novel. A spermatophylax protein shows similarity to protease inhibitors suggesting that it may protect the biologically active components from digestion within the gut of the female recipient. Another protein shares similarity with previously characterized insect polypeptide growth factors suggesting that it may play a role in altering female reproductive physiology concurrent with fertilization. Characterization of the spermatophylax proteome provides the first step in identifying the genes encoding these proteins in males and in understanding their biological functions in the female recipient.

Carbonic anhydrase buffers tissue pH by catalyzing the rapid interconversion of carbon dioxide (CO2) and bicarbonate (HCO3 (-)). We assessed the functional activity of CAIX in two colorectal tumor models, expressing different levels of the enzyme, by measuring the rate of exchange of hyperpolarized (13)C label between bicarbonate (H(13)CO3(-)) and carbon dioxide ((13)CO2), following injection of hyperpolarized H(13)CO3(-), using (13)C-magnetic resonance spectroscopy ((13)C-MRS) magnetization transfer measurements. (31)P-MRS measurements of the chemical shift of the pH probe, 3-aminopropylphosphonate, and (13)C-MRS measurements of the H(13)CO3(-)/(13)CO2 peak intensity ratio showed that CAIX overexpression lowered extracellular pH in these tumors. However, the (13)C measurements overestimated pH due to incomplete equilibration of the hyperpolarized (13)C label between the H(13)CO3(-) and (13)CO2 pools. Paradoxically, tumors overexpressing CAIX showed lower enzyme activity using magnetization transfer measurements, which can be explained by the more acidic extracellular pH in these tumors and the decreased activity of the enzyme at low pH. This explanation was confirmed by administration of bicarbonate in the drinking water, which elevated tumor extracellular pH and restored enzyme activity to control levels. These results suggest that CAIX expression is increased in hypoxia to compensate for the decrease in its activity produced by a low extracellular pH and supports the hypothesis that a major function of CAIX is to lower the extracellular pH., (©2015 American Association for Cancer Research.)

Sleep disordered breathing (SDB) represents a spectrum of disorders, including habitual snoring and obstructive sleep apnea (OSA). Sleep disordered breathing is characterized by chronic intermittent hypoxia, airflow limitation, and recurrent arousals, which may lead to tissue hypoperfusion, hypoxia, and inflammation. In this study, we aimed to examine whether SDB during pregnancy was associated with histopathologic evidence of chronic placental hypoxia and/or uteroplacental underperfusion. The placentas of women with OSA (n  =  23) and habitual snoring (n  =  78) as well as nonsnorer controls (n  =  47) were assessed for histopathologic and immunohistochemical markers of chronic hypoxia and uteroplacental underperfusion. Fetal normoblastemia was significantly more prevalent in SDB placentas than in those of nonsnorer controls (34.6% and 56.5% in snorers and OSA, respectively, versus 6.4% in controls). Expression of the tissue hypoxia marker carbonic anhydrase IX (CAIX) was more common in OSA placentas than controls (81.5% and 91.3% in snorers and OSA, respectively, versus 57.5% in controls). Adjusting for confounders such as body mass index, diabetes mellitus, or chronic hypertension did not alter the results. The uteroplacental underperfusion scores were similar among the 3 groups. Our findings suggest that SDB during pregnancy is associated with fetoplacental hypoxia, as manifested by fetal normoblastemia and increased placental carbonic anhydrase IX immunoreactivity. The clinical implications and underlying mechanisms remain to be determined.

Hemangioblastomas are rare capillary-rich tumors predominantly found in the CNS. The histological appearance of these tumors varies across a broad spectrum. Several entities show considerable histomorphological similarities to hemangioblastomas. Therefore, morphological evaluation can be challenging. In this study, we evaluated the diagnostic utility of immunohistochemistry using antibodies against carbonic anhydrase IX and cytokeratin staining. Within our files, we identified 20 hemangioblastomas. A collection of 46 other tumors relevant to the differential diagnosis (12 pilocytic astrocytomas, 11 meningiomas, one pleomorphic xanthoastrocytoma, one angiomatous fibrous histiocytoma, 14 carcinoma metastases and seven gliomas grades II-IX) served as control. The pattern of strong, diffuse expression of carbonic anhydrase IX with membranous accentuation in combination with keratin negativity was considered diagnostic for hemangioblastomas. It was found in 18 out of 20 (90%) hemangioblastomas and in none of the control cases (P < 0.001). This resulted in a sensitivity of 90% and a specificity of 100%. The positive and negative predictive values were 100% and 96%, respectively. Carbonic anhydrase IX with cytokeratin is thus a highly sensitive and specific marker combination for hemangioblastomas. It is therefore very helpful in the diagnosis of these tumors and in their discrimination from other entities., (Copyright © 2015. Published by Elsevier GmbH.)

In view of the increase in cancer-related mortality rates in low- to middle-income countries (LMIC), there is an urgent need to develop economical therapies that can be utilized at minimal infrastructure institutions. Photodynamic therapy (PDT), a photochemistry-based treatment modality, offers such a possibility provided that low-cost light sources and photosensitizers are available. In this proof-of-principle study, we focus on adapting the PDT light source to a low-resource setting and compare an inexpensive, portable, battery-powered light-emitting diode (LED) light source with a standard, high-cost laser source. The comparison studies were performed in vivo in a xenograft murine model of human squamous cell carcinoma subjected to 5-aminolevulinic acid-induced protoporphyrin IX PDT. We observed virtually identical control of the tumor burden by both the LED source and the standard laser source. Further insights into the biological response were evaluated by biomarker analysis of necrosis, microvessel density, and hypoxia [carbonic anhydrase IX (CAIX) expression] among groups of control, LED-PDT, and laser-PDT treated mice. There is no significant difference in the percent necrotic volume and CAIX expression in tumors that were treated with the two different light sources. These encouraging preliminary results merit further investigations in orthotopic animal models of cancers prevalent in LMICs.

Carbonic anhydrase 9 (CA9), which regulates cellular proliferation and the acid-base balance, is known as prognostic factor in various types of cancer. The aim of this study is to investigate the clinical implications of CA9 expression in patients with hepatocellular carcinoma. Immunohistochemical staining for CA9 was performed on tissue microarrays of hepatocellular carcinoma and paired non-neoplastic liver tissue from a training cohort of 838 patients and a validation cohort of 225 patients. Membranous staining in more than 5 % of the tumor cells was considered to indicate CA9 positivity. The prognostic value of CA9 expression was statistically evaluated. In the training cohort, CA9 positivity (181 cases, 21.5 %) was significantly correlated with shorter overall survival (OS; p < 0.001) and recurrence-free survival (RFS; p = 0.004). In multivariate analysis, CA9 positivity was independently associated with reduced OS (p = 0.023), but not significantly associated with RFS (p = 0.384). These results were validated in an additional cohort (CA9 positivity in 35 cases, 15.6 %; OS, p = 0.015; RFS, p = 0.979). Pooled cohort analysis showed that this predictor was independently associated with higher mortality (OS; p < 0.001). These data indicate that CA9 expression is a poor prognostic factor in resectable hepatocellular carcinoma (HCC) patients.

Ligand-directed Ru(bpy)3 photocatalysts induce chromophore-assisted light inactivation (CALI) of target proteins under visible light irradiation in vitro and within cells. Here, histidine, methionine, and tryptophan residues were oxidized by the singlet oxygen ((1)O2) generated by Ru(bpy)3 with light. The addition of a tyrosyl radical trapper (TRT), such as N'-acyl-N,N-dimethyl phenylenediamine, inhibited peptide/protein oxidation and induced labeling on the tyrosine residue. This mechanistic study suggests that TRT scavenges (1)O2, concomitant with the coupling reaction to the tyrosyl radical generated by Ru(bpy)3. Both CALI and labeling can be regulated by the Ru(bpy)3 photocatalysts in the absence or presence of TRT. Ligand-conjugated Ru(bpy)3 photocatalysts (local environmental single-electron transfer catalysts: LSCs) were used not only for target-selective protein labeling, but also for protein knockdown by CALI.

Purpose: Carbonic anhydrase IX (CAIX) is a hypoxia-inducible enzyme with extracellular catalytic domain that is overexpressed in a variety of cancers including breast cancer and plays a crucial role in maintaining favourable intracellular pH and reducing extracellular pH. The purpose of the current study was to elucidate the prognostic significance of CAIX in the intrinsic subtypes of breast cancer and to characterise CAIX as a drug target in breast cancer., Methods: The prognostic significance of CAIX mRNA expression was interrogated in a cohort of 3,455 breast tumours by using an online tool, Kaplan-Meier plotter. The functional effects of stable CAIX depletion by shRNA in three breast cancer cell lines—MDA-MB-231, MCF7 and SKBR-3, representing basal-like, luminal A and HER2+ subtypes, respectively—were studied by proliferation, invasion, clonal spheroid formation and chemosensitivity assays under normoxia and hypoxia. Finally, the effect of pharmacological CA inhibition alone or in the combination with doxorubicin on self-renewal was assessed by spheroid-forming assay., Results: High CAIX mRNA expression was significantly associated with poor survival in patients with basal-like, luminal B and triple-negative breast cancer, but not luminal A and HER+ subtypes. Silencing of CAIX expression had no significant effect on the cell proliferation or viability upon treatment with doxorubicin in any of the cell lines studied, while it inhibited spheroid formation in hypoxic conditions. Furthermore, pharmacological inhibition of CAs using acetazolamide had a synergistic effect with doxorubicin on decreasing the spheroid-forming efficiency in MDA-MB-231 cells., Conclusions: Inhibition of CAIX reduces the self-renewal capacity of breast cancer cells, and the combination of doxorubicin and CAIX inhibition is an attractive therapeutic strategy in basal-like and triple-negative breast cancer, which warrants further investigations.

The aim of this study is to evaluate the expression of carbonic anhydrase IX (CA9) and to identify its prognostic significance in intrahepatic cholangiocarcinoma (IHCC). We performed immunohistochemistry (IHC) for CA9 in a total of 85 IHCCs. CA9 overexpression was observed in 38 of 85 (44.7%) IHCCs. CA9 overexpression was related to tumors with intraductal growth than mass forming or periductal infiltrative type. CA9 overexpression was more observed in tumors with well/moderate differentiation than poor differentiation and without lymph node metastasis. No significant correlation was observed in CA9 overexpression with tumor size, pT, stage and lymphovascular invasion. Intrahepatic cholangiocarcinomas with CA9 overexpression showed better overall survival than that without expression (P = 0.001). In multivariate analysis, lymph node metastasis (95% CI: 2.103 (1.167-3.791), P = 0.013) was an independent poor prognostic factor. IHCC with CA9 overexpression showed a 0.5-fold (95% confidence interval, 0.328-0.944) lower risk of death compared with those of no or weak expression. CA9 overexpression was related to histologic differentiation and an independent good prognostic factor.

Carbonic anhydrases (CAs; EC 4.2.1.1) are well known zinc metalloproteins involved in the catalysis of a very simple but essential physiological reaction: carbon dioxide hydration to bicarbonate and proton. These enzymes are of clinical relevance in cancer therapy as among the 15 isoforms known in humans, two cell surface CA isoforms, namely CA IX (almost exclusively associated with tumors) and CA XII (overexpressed in some tumor types) are involved in tumorigenesis. Targeting the tumor associated isoforms IX is now considered as a pertinent approach for the development of new cancer therapeutics against hypoxic tumors. Different approaches and new family of pharmacological agents were described in the last 5 years for obtaining compounds that specifically target CA IX over the ubiquitous cytosolic off-target isoforms CA I and CA II. The aim of this review is to give a comprehensive update on the reported discoveries in the field of CA IX inhibitors with an emphasis on the new families of compounds which reach in vivo/preclinical studies for their potential in cancer therapy.

Background: Carbonic anhydrase IX (CAIX) protein has been correlated with progression and survival in patients with renal cell carcinoma (RCC). The prognostic value of CAIX in RCC however, remains inconclusive according to published works. This study aimed to analyze CAIX as a biological marker to predict RCC patient prognosis., Methods: A literature search of the PubMed and Web of Knowledge databases was performed to retrieve original studies from their inception to December of 2013. Fifteen studies, collectively including a total of 2611 patients with renal cell carcinoma, were carefully reviewed. Standard meta-analysis methods were applied to evaluate the prognostic impact of CAIX expression on patient prognosis. The hazard ratio (HR) and its 95% confidence interval (CI) were recorded for the relationship between CAIX expression and survival, and the data were analyzed using Review Manager 5.2 software and Stata software 11.0., Results: In patients with RCC, low CAIX expression was associated with poor disease-specific survival (HR = 1.89, 95% CI: 1.20-2.98, P = 0.006), unfavorable progression-free survival (HR = 2.62, 95% CI: 1.14-6.05, P = 0.02) and worse overall survival (HR = 2.03, 95% CI: 1.28-3.21, P = 0.002). Furthermore, low CAIX expression was significantly associated with the presence of lymph node metastases (odds ratio (OR) = 0.31, 95% CI = 0.15-0.62, P = 0.0009) and distant metastases (OR = 0.66, 95% CI = 0.46-0.96, P = 0.03) and predicted a higher tumor grade (OR = 0.41, 95% CI = 0.31-0.54, P<0.00001)., Conclusions: Low CAIX expression most likely indicates poor prognosis in RCC patients. Moreover, low CAIX expression was significantly associated with unfavorable clinicopathological factors. To strengthen our findings, further well-designed prospective studies should be conducted to investigate the role of CAIX expression in RCC.

Purpose: Carbonic anhydrase IX (CA IX), a transmembrane glycoprotein, is known as an endogenous marker for hypoxia. Overexpressed in cancer-associated fibroblasts, CA IX has been reported to be associated with a poor outcome for a number of malignant tumors. Aim of this study was to investigate the role of CA IX in the tumor surrounding stroma of esophageal cancer., Methods/patients: Stromal expression of CA IX in 361 formalin-fixed, paraffin-embedded specimens of invasive esophageal cancers, 206 adenocarcinoma (AC) and 155 squamous cell carcinoma (SCC), was investigated., Results: In 42 cases (11.6 %), CA IX expression in the tumor surrounding stroma (AC 23 and SCC 19) was observed. Expression of CA IX correlated with the factors tumor stage (p < 0.001) and lymph node status (p = 0.008). Patients with CA IX expressed in the tumor surrounding stroma had a significant shorter disease-free survival (p = 0.007) and overall survival (p = 0.013)., Conclusion: In esophageal cancer, CA IX-expressing tumor stroma is associated with shorter survival. Inhibition of the tyrosine kinase CA IX might represent a new onset for therapies against esophageal cancer.

Aims: Heat shock protein 27 (Hsp27) is induced by cell stress conditions. In the presence of oxidative stress it functions as an antioxidant. To study the putative expression patterns and clinical significance of Hsp27, we assessed the associations between Hsp27, R132H mutation of Isocitrate dehydrogenase1 (IDH1-R132H), Hypoxia-inducible factor subunit alpha (HIF-1 alpha), Carbonic anhydrase IX (CA IX), and patient prognosis in astrocytic gliomas., Methods: Tissue micro-array samples of 295 grade II-IV astrocytomas were stained immunohistochemically for Hsp27, IDH1-R132H, HIF-1 alpha, and CA IX. We tested their relationship with clinicopathological features and patient survival., Results: There was a significant correlation between Hsp27 expression and increasing WHO grade (p<0.001). Hsp27 expression correlated significantly with IDH1 mutation when studied within the entire cohort (p<0.001) as well as separately in WHO grade II and III tumors (p=0.006 and 0.002, respectively). IDH1 mutation and HIF-1 alpha positive staining were detected simultaneously (p<0.001). In IDH1 mutated tumors, positive HIF-1 alpha staining correlated with CA IX expression (p=0.027), whereas no such correlation was found in IDH1 non-mutated tumors. IDH1 mutation was associated with a low cell proliferation index (p=0.001) and HIF-1 alpha with increasing proliferation (p = 0.003). Hsp27 expression was associated with a shorter rate of patient survival in univariate survival analysis (p=0.001). In multivariate survival analysis, patient age, IDH1 mutation and HIF-1 alpha appeared as independent prognostic factors (p<0.000, <0.000 and 0.011 respectively), Conclusions: Hsp27 expression is associated with increasing WHO grade and patient prognosis in astrocytic gliomas. The results suggest that IDH1 mutation may have an effect on the expression pathways of Hsp27 and CA IX.

Three tertiary benzenesulfonamide inhibitors 4a-c were radiolabeled with (18)F and evaluated for imaging carbonic anhydrase IX (CA IX) expression with positron emission tomography. All three inhibitors exhibit <10 nM affinity for CA IX with no measurable affinity for CA II. Despite good affinity/selectivity to CA IX and excellent stability in plasma, uptake of [(18)F]4a-c in CA IX-expressing HT-29 tumours was low without significant contrast. [(18)F]4a,b were excreted rapidly, while [(18)F]4c exhibited significant in vivo defluorination leading to high bone uptake. Due to minimal uptake in HT-29 tumours compared to normal organs/tissues, (18)F-labeled benzenesulfonamides [(18)F]4a-c are not suitable as CA IX imaging agents., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Adjuvant chemotherapy reduces recurrences of non-small-cell lung cancer (NSCLC). To determine which patients need adjuvant chemotherapy, we assessed factors associated with time to relapse (TTR)., Methods: In 230 resected stage I-II NSCLCs, we correlated immunohistochemistry scores for factors associated with cell growth rate, growth regulation, hypoxia, cell survival, and cell death with TTR., Results: With a median follow-up of 82 months (1-158) for those alive and relapse free at last follow-up, median time to recurrence was not reached. The 2- and 5-year probabilities of maintaining freedom from recurrence were 80.7% (95% confidence interval, 75.3%, 86.4%) and 74.6% (95% confidence interval, 68.6%, 81.2%), respectively. TTR curves flattened at an apparent cure rate of 70%. In multicovariate Cox models, factors correlating with shorter TTR were membranous carbonic anhydrase IX (mCAIX) staining (any versus none, hazard ratio = 2.083, p = 0.023) and node stage (N1 versus N0, hazard ratio = 2.591, p = 0.002). mCAIX scores correlated positively with tumor size, grade, squamous histology, necrosis, mitoses, Ki67, p53, nuclear DNA methyltransferase 1, and cytoplasmic enhancer-of-split-and-hairy-related protein, and they correlated inversely with papillary histology, epidermal growth factor receptor mutation (trend), copper transporter-1, and cytoplasmic hypoxia-inducible factor-1α, vascular endothelial growth factor, DNA methyltransferase 1, and excision repair cross-complementing rodent repair deficiency, complementation group 1., Conclusion: Nodal stage and mCAIX immunohistochemistry were the strongest independent predictors of shorter TTR in resected NSCLCs. mCAIX correlated with tumor size, markers of tumor proliferation and necrosis, and tumor genetic characteristics, and it paradoxically correlated inversely with the hypoxia markers, hypoxia-inducible factor-1α and vascular endothelial growth factor. Presence of mCAIX could help determine patients with high risk of recurrence who might require adjuvant chemotherapy.

The objective of this study was to compare the predictive ability of potential tissue biomarkers to known prognostic factors that predict renal cell carcinoma (RCC) recurrence using an automated system of immunohistochemical analysis. After institutional review board approval, a tissue microarray was constructed using tissue from patients who had partial or radical nephrectomy for RCC. Patients with metastatic disease were excluded. Immunohistochemical staining of the tissue microarray for Ki-67, C-reactive protein, carbonic anhydrase 9, and hypoxia-inducible factors 1α and 2α was analyzed using automated image analysis. Univariable and multivariable analyses were performed to evaluate the association of putative biomarkers and known prognostic factors. Of 216 patients who met the entrance criteria, 34 (16%) patients developed metastatic recurrence within a median follow-up interval of 60.9 (interquartile range, 13.9-87.1) months. RCC morphotypes analyzed in this study include clear cell (n = 156), papillary (n = 38), chromophobe (n = 16), and collecting duct/unclassified (n = 6). Univariate analysis identified that only increased Ki-67 was predictive of RCC recurrence among the proteins evaluated, in addition to other known clinicopathological prognostic factors. After multivariate analysis, Ki-67 was identified as an independently predictive risk factor for RCC recurrence (hazard ratio [HR], 3.73 [confidence interval {CI}, 1.60-8.68]). Other independent predictors of RCC recurrence included tumor diameter (HR, 1.20 [CI, 1.02-1.41]) and perinephric fat invasion (HR, 4.49 [CI, 1.11-18.20]). We conclude that Ki-67 positivity is independently predictive of RCC recurrence after surgery in nonmetastatic patients. Automated analysis of tissue protein expression can facilitate a more objective and expedient investigation of tissue biomarkers for RCC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objectives: To immunohistochemically evaluate the carbonic anhydrase IX (CA IX) expression on 1,551 cases of tumors and normal tissues from various organs., Methods: Immunohistochemical evaluation of the expression of CA IX was performed on 1,125 malignant tumors, 69 benign neoplasms, and 322 normal tissues on tissue microarray sections and 18 intrahepatic cholangiocarcinomas (ICCs) and 17 hepatocellular carcinomas (HCCs) on routine sections., Results: There was overexpression of CA IX in clear cell renal cell carcinoma (CRCC) (88%, 68/77). Twenty-six (90%) of 29 ICCs were positive. In contrast, only 5 (15%) of 34 HCCs were focally positive. No staining was seen in chromophobe renal cell carcinoma (ChRCC), oncocytoma, seminoma, or carcinomas of the breast, thyroid, or prostate. All normal renal tubules except one case showed no staining., Conclusions: These data demonstrate the diagnostic utility of CA IX in (1) differentiating CRCC from ChRCC and oncocytomas, (2) distinguishing low-grade CRCC from normal renal tubules in small samples, (3) separating ICC from HCC, and (4) identifying metastatic CRCC from other metastases with clear cell features.

In this work we developed a multiplexed analysis platform providing a simple high-throughput means to characterize solutions. Automated analyses, requiring less than 5 s per sample without carryover and 1 s per sample, accepting minor cross contamination, was achieved using multiplexed solvent assisted ionization inlet (SAII) mass spectrometry (MS). The method involves sequentially moving rows of pipet tips containing sample solutions in close proximity to the inlet aperture of a heated mass spectrometer inlet tube. The solution is pulled from the container into the mass spectrometer inlet by the pressure differential at the mass spectrometer inlet aperture. This sample introduction method for direct injection of solutions is fast, easily implemented, and widely applicable, as is shown by applications ranging from small molecules to proteins as large as carbonic anhydrase (molecular weight ca. 29,000). MS/MS fragmentation is applicable for sample characterization. An x,y-stage and common imaging software are incorporated to map the location of components in the sample wells of a microtiter plate. Location within an x,y-array of different sample solutions and the relative concentration of the sample are displayed using ion intensity maps.