The carbonic anhydrase 2 (Car2) gene encodes the primary isoenzyme responsible for aqueous humor (AH) production and plays a major role in the regulation of intraocular pressure (IOP). The CRISPR-Cas9 system, based on the ShH10 adenovirus-associated virus, can efficiently disrupt the Car2 gene in the ciliary body. With a single intravitreal injection, Car2 knockout can significantly and sustainably reduce IOP in both normal mice and glaucoma models by inhibiting AH production. Furthermore, it effectively delays and even halts glaucomatous damage induced by prolonged high IOP in a chronic ocular hypertension model, surpassing the efficacy of clinically available carbonic anhydrase inhibitors such as brinzolamide. The clinical application of CRISPR-Cas9 based disruption of Car2 is an attractive therapeutic strategy that could bring additional benefits to patients with glaucoma.

Myocardial infarction (MI) is a significant threat to human health and life. Xue-Fu-Zhu-Yu Decoction (XFZYD), a renowned traditional Chinese medicine prescription for treating myocardial infarction, is known to play a significant role in the management of MI. However, its mechanism of action remains unclear. Through network pharmacology analysis of compound-target interactions, we have identified Carbonic Anhydrase II (CA2) as a critical target for XFZYD in the treatment of MI. Subsequently, we will embark on a target-based drug design approach with a focus on CA2 as the key target: Pharmacophore modeling: Two pharmacophore models were developed and validated to screen for small molecules with CA2 inhibitory features. Virtual screening: Based on two pharmacophore models, small molecules with the property of binding to the CA2 target were screened from a virtual screening library. Molecular docking: Molecular docking was employed to identify small molecules with stable binding affinity to CA2. ADMET prediction: ADMET models were utilized to screen for small molecules with favorable pharmacological properties. Molecular dynamics: Molecular dynamics simulations were further conducted to analyze the binding modes of the selected small molecules with CA2, ultimately resulting in the identification of Ligand 3 and Ligand 5 as small molecule inhibitors targeting CA2. Finally, the mechanisms underlying the anti-MI effects were discussed. The primary objective of this article is to uncover the mechanism by which XFZYD acts on MI and utilize it for drug development. These findings provide novel avenues for the development of anti-MI drugs.Communicated by Ramaswamy H. Sarma.

UNASSIGNED: Vascular calcification is a hallmark of atherosclerosis (AS). We and others confirmed that carbonic anhydrase I (CA1) and CA2 increased expression and catalyzed calcium deposition in atherosclerotic aortas. Macrophages have been demonstrated to be strongly related to AS. This study aimed to clarify how and which macrophage subtypes regulate CA1 and CA2 expression to stimulate aortic calcification.
UNASSIGNED: THP-1 cells were induced to form M0, M1 and M2 macrophage subtypes. These cells and their culture supernatants were separately incubated with human vascular smooth muscle cells (VSMCs). Calcification was strongly increased in VSMCs treated with β-GP, a chemical inducer of cellular calcification, following incubation with M1 macrophages or their culture supernatants, and was much higher than that in VSMCs treated with β-GP alone. Meanwhile, the expression of CA1 and CA2, as well as calcification marker genes, including Runx2, BMP-2 and ALP, was increased in VSMCs during this process. TNF-α levels were also increased in the culture medium of M1 macrophages. M0 and M2 macrophages or their supernatants did not significantly stimulate calcification in VSMCs. Following transfection with anti-CA1 or CA2 siRNAs, β-GP-induced VSMCs showed decreased calcification, but the calcification level was partially increased when those VSMCs were incubated with the supernatants of M1 macrophages, while CA1 and CA2 expression as well as TNF-α levels were also elevated. When VSMCs were treated with TNF-α without β-GP induction, calcification and the expression of CA1 and CA2 were also significantly increased.
UNASSIGNED: The results of this study suggest that M1 macrophages can increase CA1 and CA2 expression to promote atherosclerotic calcification in VSMCs by secreting TNF-α.

Aphids are the most common insect vector transmitting hundreds of plant viruses. Aphid wing dimorphism (winged vs. wingless) not only showcases the phenotypic plasticity but also impacts virus transmission; however, the superiority of winged aphids in virus transmission over the wingless morph is not well understood. Here, we show that plant viruses were efficiently transmitted and highly infectious when associated with the winged morph of Myzus persicae and that a salivary protein contributed to this difference. The carbonic anhydrase II (CA-II) gene was identified by RNA-seq of salivary glands to have higher expression in the winged morph. Aphids secreted CA-II into the apoplastic region of plant cells, leading to elevated accumulation of H+. Apoplastic acidification further increased the activities of polygalacturonases, the cell wall homogalacturonan (HG)-modifying enzymes, promoting degradation of demethylesterified HGs. In response to apoplastic acidification, plants accelerated vesicle trafficking to enhance pectin transport and strengthen the cell wall, which also facilitated virus translocation from the endomembrane system to the apoplast. Secretion of a higher quantity of salivary CA-II by winged aphids promoted intercellular vesicle transport in the plant. The higher vesicle trafficking induced by winged aphids enhanced dispersal of virus particles from infected cells to neighboring cells, thus resulting in higher virus infection in plants relative to the wingless morph. These findings imply that the difference in the expression of salivary CA-II between winged and wingless morphs is correlated with the vector role of aphids during the posttransmission infection process, which influences the outcome of plant endurance of virus infection.

The role of carbonic anhydrase II (CAII) in intrahepatic cholangiocarcinoma (ICC) was investigated and a novel prognostic system combining CAII and preoperative carbohydrate antigen 19-9 (CA19-9) was established to predict the survival of patients with ICC after curative resection.
A total of 110 patients who underwent curative-intent resection for ICC between 2012 and 2020 were retrospectively analyzed. CAII in tumor and peritumor regions was examined by immunohistochemistry, and the relationships between clinicopathological factors and the prognostic value of CAII and CA19-9 were analyzed.
CAII was frequently downregulated in ICC tissues (p < .001). Multivariate analyses indicated that showed that both low CAII expression level and preoperative CA19-9 ≥236 U/ml were independent risk factors for overall survival (OS) and recurrence-free survival (RFS) in patients with ICC after radical resection. Survival analysis revealed that patients with high CAII and low CA19-9 were significantly associated with a better OS and RFS (p < .001). The time-dependent receiver operating characteristic curves showed that CAII + CA19-9 had better prognostic predictive ability than CAII or CA19-9 alone. The nomogram constructed on independent factors including T stage, lymph node metastasis, CA19-9 (continuous variable), and CAII achieved C-indexes of 0.754 (95% CI, 0.701-0.807) and 0.730 (0.674-0.785) for OS and RFS, respectively. The calibration curve revealed acceptable agreement between actual and predicted OS and RFS.
The combination of CAII and preoperative CA19-9 is a novel and useful prognostic tool for predicting the survival of patients with ICC after curative resection and guiding clinical decisions.
Carbonic anhydrase II (CAII) was frequently downregulated in intrahepatic cholangiocarcinoma (ICC) tissues. Survival analysis revealed that CAII is a novel independent factor for prognosis in patients with ICC after curative resection. CAII could be a useful prognostic marker for patients with ICC after surgery. The combination of CAII and preoperative carbohydrate antigen 19-9 is a novel and useful prognostic tool for predicting the survival of patients with ICC after curative resection and guiding clinical decisions.

A simple and time-saving colorimetric method was developed to quantify sulfonamides (SAAs) in milk via inhibition of the human carbonic anhydrase II (hCAII)-like activity of ZIF-8 that can hydrolyze p-nitrophenyl acetate (pNPA) to p-nitrophenol (pNP), following the color change from yellow to colorless. Effects of different reaction conditions, including pH, temperature, amount of ZIF-8, and incubation time, were investigated. The value of Michaelis-Menten constant (Km) is measured to be 0.15 mM, which exhibits high affinity to pNPA. The IC50 (0.17, 0.24, and 0.60 mM) and inhibition constant (Ki) (0.09, 0.13, and 0.33 mM) of sulfamethazine (SD), sulfadimethoxine (SDM), and sulfathiazole (ST) on ZIF-8 were measured, respectively. Moreover, the activity of ZIF-8 remains more than 90.0% of its initial activity after 30 days\' storage. The colorimetric method for SD, SDM, and ST determination was established at the linear ranges of 6.3-750.0 μM (1.75-208.75 mg/kg), 6.3-750.0 μM (1.96-232.75 mg/kg), and 5.0-1250.0 μM (1.28-319.15 mg/kg) with limit of detection of 4.3, 3.2, and 3.9 μΜ (1.2, 0.99, and 0.96 mg/kg), respectively. In addition, the spiked recoveries of SAAs in milk sample are in the range of 81.6%-106.7% with RSD less than 6.5%. In short, the developed colorimetric method can achieve rapid analysis of SAAs in milk with simple operations.

BACKGROUND: Dilated cardiomyopathy (DCM) is a complex type of cardiomyopathy that is affected by both genetic and non-genetic factors. It is characterized by an enlargement of the left ventricle or bi-ventricle, and is often accompanied by cardiac systolic dysfunction. The main results include arrhythmia, heart failure (HF), and sudden death. The prognosis of this disease is usually poor, and the 5-year survival time is about 50%. Early diagnosis is very important for the treatment of DCM. Studies have shown that primary prevention after discovering the disease effectively reduces the mortality rate of the disease. However, there is currently no effective biomarker for the early diagnosis of DCM. The rapid development of omics in protein has promoted the \"precise\" study of modern medical research. In this article, the potential biomarkers for predicting and diagnosing DCM-related HF were studied by a plasma protein omics analysis.
METHODS: Tandem mass tag-labeled quantitative proteomic studies were performed in 20 patients, comprising 10 DCM-associated HF patients, and 10 control patients who without clinical HF events. Further validation research was conducted by enzyme-linked immunosorbent assay (ELISA) with an expanded cohort (control group =40; HF group =48).
RESULTS: Among the 854 identified proteins, the expression of 86 proteins was significantly upregulated, while the expression of 21 proteins was downregulated (with an expression difference >1.5-fold; P<0.05) in the 2 groups. The Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway enrichment, and protein-protein interaction (PPI) networks analyses indicated that the bicarbonate transport process played a critical role in HF. Importantly, carbonic anhydrase 2 (CA2) and 3 (CA3), which play central roles in regulating the transport of bicarbonate, were highly expressed in the HF group. The ELISA validation results showed that the expression levels of CA2 and CA3 at admission were remarkably higher (P<0.0001 and P=0.0157) in the plasma of the HF patients than that of the control patients.
CONCLUSIONS: The present study showed that two molecules (i.e., CA2 and CA3) are involved in the bicarbonate transport pathway, and are risk factors and potential biomarkers for the diagnosis of DCM patients with HF.

The sulfonamide-based thiadiazole derivatives (STDs) with different hydrophobic/hydrophilic substitutions were synthesized to investigate their potentials in carbonic anhydrase inhibition (CAI). The CAI activity of the STDs (4a-4h) and the mechanism of the inhibition kinetics were determined. STD 4f contained both methoxy and Cl groups at benzene ring in STD 4f showed the lowest IC50 value. The molecular docking study confirmed that STDs bind strongly with the active sites of the target protein PDBID 1V9E. With the help of Lineweaver-Burk plots, inhibition kinetics of PDBIR 1V9E protein with STDs were determined. Cytotoxicity was checked against human keratinocyte cell lines and the anticancer properties were determined against MCF-7 cell lines. The electrochemical method was used to investigate the binding study with DNA and CA enzymes. Anticancer studies showed that STDs have weak bonding ability to DNA and strong binding ability with CA. It is concluded that anticancer activity is through CAI rather than by DNA binding.

A series of N-((4-sulfamoylphenyl)carbamothioyl)alkanamides (5a-j) were synthesized by the reaction of sulphanilamide in dry acetone with freshly prepared alkyl and acyl isothiocyanates (5a-j). The structures of products were confirmed by IR, 1 H, and 13 C NMR. The synthesized compounds were screened as inhibitors of the bovine erythrocyte carbonic anhydrase isoform II (bCA II) and 15-lipoxygenase enzyme (15-LOX). Most of the derivatives showed significant activity against bCA-II while only few compounds were found active against 15-LOX. Molecular docking studies of most active compounds were carried out against bCA II as well as 15-LOX to rationalize the binding mode and interactions of compound in the active sites. Additionally, the pharmacokinetic properties of the compounds were predicted through computational tools, which reflect that these compounds possess acceptable pharmacokinetic profile and good drug-likeness.

In this paper, a new class of novel sulfonamides incorporating aminosaccharide tails were designed and synthesized based on the sugar-tail approach. Then, all the novel compounds were evaluated for their inhibitory activities against three carbonic anhydrase (CA, EC 4.2.1.1) isoenzymes (hCA I, hCA II and hCA IX). Interestingly, effective inhibition of these three CA isoforms were observed, especially the glaucoma associated isoform hCA II. It is worth noting that these glycoconjugated sulfonamide derivatives also showed better CA inhibitory effects compared to the initial segment carzenide. Among them, compound 8d was the most effective inhibitor with IC50 of 60 nM against hCA II. Subsequent physicochemical properties studies showed that all compounds have good water solubility and neutral pH values in solutions. And these important physicochemical properties make target compounds acquire obvious advantages in the preparation of topical and nonirritating antiglaucoma drugs. Moreover, the target compounds showed lower corneal cytotoxicity than acetazolamide (AAZ) and good metabolic stability in vitro. In addition, molecular docking studies confirmed the interactions between aminosaccharide fragment and hydrophilic subpocket of hCA II active site were crucial for the enhanced CA inhibitory activity. Taken together, these results suggested 8d would be a promising lead compound for the development of topical antiglaucoma CAIs.