Ca II Research Articles

Novel anti-tumor effect of natural products from Aloe vera resin and their in-vitro/in-silico targeting mechanism of carbonic anhydrase-II and IX.

Human carbonic anhydrase (hCA) plays a vital role in the development and progression of tumors in hypoxic conditions. Herein we report the hCA-II and hCA-IX activities of natural products isolated from Aloe vera (L.) Burm.f., to know their potential in tumors. These isolated compounds (1-10) displayed varying degrees of inhibition against hCA-II and hCA-IX. All the compounds showed potent activity against hCA-IX with IC50 values in the range of 2.9 - 29.1 µM. While for hCA-II, compounds 1, 2, 5-10 exhibited IC50 in the range of 4.7 - 23.4 µM. The most effective hCA IX and II inhibitors, 2 and 5, were chosen for in vitro mechanism studies, revealing that they are competitive inhibitors. Furthermore, when tested for their cytotoxic effect on BJ (normal) cell line, all the compounds showed no cytotoxic behavior, while on Prostate cancer cells (PC-3), compounds 1, 3, 5, 7, and 9 exhibited significant antiproliferative activity. Molecular docking was also conducted within the hCA IX and hCA-II active sites to observe their binding capability. Compounds 1, 5, 7, and 9 were active against both isozymes of hCA and in the PC-3 cell line, therefore these are the best choices for further in vivo studies..

Exploration of new thiazolidinones derived from natural verbenone: Design, synthesis, characterization, and in silico evaluation of alkaline phosphatase and carbonic anhydrase-II inhibition activity

Hybrid compounds featuring isoxazole and thiazolidinone nuclei, alongside verbenone, have become pivotal in pharmacological research due to their versatile properties and straightforward synthesis methods. In this study, the hybrid compounds 3–5 were thoroughly analyzed using high-resolution mass spectrometry (HRMS) and 1H- and 13C NMR spectroscopy, confirming the presence of the isoxazoline and thiazolidinone cores. Additionally, text of in vitro cytotoxicity, computational molecular docking and dynamics studies were performed to study the cytotoxic profile and the modes of possible interactions between the synthesized compounds and the biological targets. The cytotoxicity test indicated that product 5 exhibited the highest activity, with IC50 values ranging from 25.87±3.21 to 28.12±3.54 µM against MDA-MB-231 and A-549 cancer cell lines. From a theoretical point of view, the comparative analysis of compounds 3–5 against the references in the in silico study revealed significant interactions for these compounds with the isoenzymes of alkaline phosphatase and carbonic anhydrase.

359 Optimization of calcium inclusion levels in diets fed to growing and finishing beef cattle

Abstract Calcium (Ca) is a mineral that has an essential role in many biochemical pathways and functions of beef cattle. Limestone is the primary supplemental source of Ca in diets fed to growing and finishing cattle in the United States. Preliminary data indicated that inclusion of dietary Ca above the NASEM (2016) requirements as well as mean and modal concentrations reported by the most recent survey of consulting nutritionists may improve average daily gain (ADG) and feed efficiency (gain to feed ration; G:F) of finishing beef cattle. Thus, the objective of this experiment was to determine the optimal concentration of Ca in growing and finishing diets when limestone was the supplemental source of added Ca. A database was constructed containing 89 treatment means from 23 experiments published from 1963 to 2024 where dietary Ca inclusion via limestone represented treatments. Search parameters to construct the database included a combination of Ca, limestone, performance, efficiency, ADG, G:F, feedlot cattle, finishing cattle, growing cattle, and backgrounding cattle. Data were analyzed in JMP Pro 17 using a mixed model to determine linear and quadratic relationships between dietary Ca and ADG, DMI, or G:F with citation as a random effect. An economic sensitivity analysis was conducted to determine variables most influential to the dietary Ca concentration projected to minimize cost of gain using the above models. Concentration of dietary calcium was selected as the explanatory variable for the models rather than limestone because a greater proportion of variation was accounted for in the response variables prior to the citation adjustment (Ca R2 0.05 to 0.25; limestone R2 0.06 to 0.21). Diet Ca concentration and DMI were unrelated (P ≥ 0.75), but diet Ca was related to both ADG (R2 = 0.84; RMSE = 0.36; P ≤ 0.03) and G:F (R2 = 0.74; RMSE = 0.01; P < 0.01) in a quadratic fashion. Maximum ADG occurred when Ca was included at 1.20% of diet DM; approximately 2.6-fold greater than Ca requirements estimated by NASEM (2016). Cost of gain was minimized when Ca was 1.35% of diet DM. Sensitivity analysis revealed Ca concentration to minimize cost of gain was sensitive to changes in DMI, ADG, days on feed, ration cost and cost of yardage. These results provide further evidence that it may be beneficial to feed a greater amount of Ca than currently recommended by the NASEM (2016) for growing and finishing cattle. Future pen-scale feeding trials are warranted to validate the results of this meta-analysis and to determine the dose response of cattle fed graded levels of Ca. Future research should also be conducted to determine the mechanism of action responsible for the observed improvements in animal performance and efficiency resulting from feeding an increased amount of dietary Ca.

Exploring the aroma profile and biomedical applications of Scutellaria nuristanica Rech. F.: A new insight as a natural remedy

BackgroundThe Scutellaria genus has promising therapeutic capabilities as an aromatherapy. Based on that and local practices of S. nuristanica Rech. F. The essential oil was studied for the first time for its diverse biomedical applications. PurposeThis study aimed to evaluate and validate their therapeutic capabilities by screening the essential oil ingredients and examining their antimicrobial, antioxidant, carbonic anhydrase, and antidiabetic using further In silico assessment and In vivo anti-inflammatory and analgesic capabilities to devise novel sources as natural remedies alternative to the synthetic drugs. MethodsEssential oil was obtained through hydrodistillation, and the constituents were profiled using GC–MS. The antimicrobial assessment was conducted using an agar well diffusion assay. Free radical scavenging capabilities were determined by employing DPPH and ABTS assay. The carbonic anhydrase-II was examined using colorimetric assay, while the antidiabetic significance was performed using α-Glucosidase assay. The anti-inflammatory significance was examined through carrageenan-induced paw edema, and the analgesic features of the essential oil were determined using an acetic acid-induced writhing assay. ResultsFifty constituents were detected in S. nuristanica essential oil (SNEO), contributing 95.93 % of the total EO, with the predominant constituents being 24-norursa-3,12-diene (10.12 %), 3-oxomanoyl oxide (9.94 %), methyl 7-abieten-18-oate (8.85 %). SNEO presented significance resistance against the Gram-positive bacterial strains (GPBSs), Bacillus atrophaeus and Bacillus subtilis, as compared to the Salmonella typhi and Klebsiella pneumoniae, Gram-negative bacterial strains (GNBSs) as well as two fungal strains Aspergillus parasiticus and Aspergillus niger associated with their respective standards. Considerable free radical scavenging capacity was observed in DPPH compared to the ABTS assay when correlated with ascorbic acid. In addition, when equated with their standards, SNEO offered considerable in vitro carbonic anhydrase II and antidiabetic capabilities. Additionally, the antidiabetic behavior of the 9 dominant compounds of SNEO was tested via In silico techniques, such as molecular docking, which assisted in the assessment of the significance of binding contacts of protein with each chemical compound and pharmacokinetic evaluations to examine the drug-like characteristics. Molecular dynamic simulations at 100 ns and binding free energy evaluations such as PBSA and GBSA models explain the molecular mechanics and stability of molecular complexes. It was also observed that SNEO depicted substantial anti-inflammatory and analgesic capabilities. ConclusionHence, it was concluded that the SNEO comprises bioactive ingredients with biomedical significance, such as anti-microbial, antioxidant, CA-II, antidiabetic, anti-inflammatory, and analgesic agents. The computational validation also depicted that SNEO could be a potent source for the discovery of anti-diabetic drugs.

Chitosan functionalized iron oxide nanocomposites as a novel carbonic anhydrase nano-inhibitor

Carbonic anhydrases (CA) are metalloenzymes involved in various physiological and pathological processes, with carbonic anhydrase II (CAII) being the predominant physiological isoform.This study investigates the interaction between CAII and chitosan functionalized iron oxide nanoparticles (C-INPs) as a novel approach to inhibit CAII activity. Iron oxide nanoparticles were functionalized with chitosan of high (HC-INPs), medium (MC-INPs), and low (LC-INPs) molecular weights. The synthesized nanocomposites were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), zeta potential analysis, Fourier-transform infrared spectroscopy (FTIR), and vibrating sample magnetometry (VSM). The average diameters of HC-INPs, MC-INPs, and LC-INPs were approximately 4 nm, 11.5 nm, and 20 nm, respectively, with corresponding Zeta potential values of 39.2 ± 3.9 mV, 36.7 ± 4.1 mV, and 15.5 ± 4.6 mV. Fluorescence quenching assays and enzyme kinetics activity results demonstrated that the interaction of CAII with the nanocomposites induced conformational changes in CAII, inhibiting its enzymatic activity. The inhibitory potential was significantly influenced by the surface properties of the nanoparticles, with HC-INPs showing the most potent effect (Ki = 58.388 ng/ml), followed by MC-INPs Ki = 390.87 ng/ml) and LC-INPs (Ki = 1213.00 ng/ml). These findings suggest that Chitosan-functionalized iron oxide nanoparticles, particularly HC-INPs, represent a promising class of nano-inhibitors for CAII, opening new avenues for treating disorders associated with CAII overexpression.

Identification, crystallization, and first X-ray structure analyses of phenyl boronic acid-based inhibitors of human carbonic anhydrase-II

Human carbonic anhydrases (hCAs) play a central role in various physiological processes in the human body. HCAs catalyze the reversible hydration of CO2 into HCO3−, and hence maintains the fluid and pH balance. Overexpression of CA II is associated with diseases, such as glaucoma, and epilepsy. Therefore, CAs are important clinical targets and inhibition of different isoforms, especially hCA II is used in treatment of glaucoma, altitude sickness, and epilepsy. Therapeutically used CA inhibitors (CAI) are sulfonamide-based, such as acetazolamide, dichlorphenamide, methazolamide, ethoxzolamide, etc. However, they exhibit several undesirable effects such as numbness, tingling of extremities, malaise, metallic taste, fatigue, renal calculi, and metabolic acidosis. Therefore, there is an urgent need to identify safe and effective inhibitors of the hCAs. In this study, different phenyl boronic acids 1–5 were evaluated against bovine (bCA II) and hCA II. Among all, compound 1 (4-acetylphenyl boronic acid) was found to be active against bCAII and hCA II with IC50 values of 246 ± 0.48 and 281.40 ± 2.8 μM, respectively, while the remaining compounds were found in-active. Compound 1 was identified as competitive inhibitor of hCA II enzyme (Ki = 283.7 ± 0.002 μM). Additionally, compound 1 was found to be non-toxic against BJ Human fibroblast cell line. The X-ray crystal structure for hCA II in-complex with compound 1 was evaluated to a resolution of 2.6 Å. In fact, this the first structural analysis of a phenyl boron-based inhibitor bound to hCA II, allowing an additional structure-activity analysis of the compounds. Compound 1 was found to be directly bound in the active site of hCA II by interacting with His94, His119, and Thr199 residues. In addition, a bond of 3.11 Å between the zinc ion and coordinated boron atom of the boronic acid moiety of compound 1 was also observed, contributing to binding affinity of compound 1 for hCA II. PDB ID8IGF.

Pregnenolone derivatives for the treatment of Alzheimer's disease: synthesis, and in vitro inhibition of amyloid β1-42 peptide aggregation, acetylcholinesterase and carbonic anhydrase-II.

The amyloid state, which is a specific conformation of proteins, offers valuable information about both functional protein structures and the pathological assemblies associated with various diseases. One of the major hallmarks of Alzheimer's disease includes primarily the extracellular build-up of a peptide known as amyloid-β, which has a sequence consisting of 39 to 42 amino acid residues, and the formation of intracellular neurofibrillary tangles mostly consisting of hyperphosphorylated tau protein. Drugs that are expected to reduce Aβ production, prevent Aβ aggregation, and promote Aβ clearance are promising approaches for treating AD. Current work is focused on identifying the compounds that have balanced even mild biological activities against multiple targets instead of finding one-target compound with high potency. We synthesized pregnenolone derivatives and evaluated their potential against inhibition of eeAChE/eqBChE, hCA-II and self-mediated Aβ1-42 peptide aggregation. Our synthesized derivatives 23, and 25-27 exhibited concomitant inhibition of all the tested macromolecular targets. All the active compounds were found to be BBB penetrants in the PAMPA assay. Furthermore, these selected compounds were found to be non-neurotoxic in the MTT assay on neuroblastoma SH-SY5Y cells. Docking studies support dual binding site (PAS and CAS) inhibition of AChE which showed Aβ1-42 aggregation and AChE inhibition. Moreover, docking studies carried out on the 3D crystallographic structure of Aβ1-42 peptide (PDB ID = 1IYT) showed significant interactions with amino acid residues Asp 23 and Lys 28, and hydrophobic interactions with the Phe19, Phe20, and Ala 30 effectively impeding the formation of β-sheet structures.

Design, synthesis, and in vitro and in silico studies of morpholine derived thiazoles as bovine carbonic anhydrase-II inhibitors.

Carbonic anhydrase CA-II enzyme is essential for maintaining homeostasis in several processes, including respiration, lipogenesis, gluconeogenesis, calcification, bone resorption, and electrolyte balance due to its vital function within cellular processes. Herein, we screened 25 newly synthesized thiazole derivatives and assessed their inhibitory potential against the zinc-containing carbonic anhydrase CA-II enzyme. Intriguingly, derivatives of thiazole exhibited varying degrees of inhibitory action against CA-II. The distinctive attribute of these compounds is that they can attach to the CA-II binding site and block its action. Morpholine based thiazoles can be strategically modified to improve bovine CA-II inhibitor binding affinity, selectivity, and pharmacokinetics. Thiazole and morpholine moieties can boost inhibitory efficacy and selectivity over other calcium-binding proteins by interacting with target bovine CA-II binding sites. The derivatives 23-26 exhibited greater affinity when compared to the standard acetazolamide. Furthermore, kinetic study of the most potent compound 24 was performed, which exhibited concentration dependent inhibition with a K i value of 9.64 ± 0.007 μM. Molecular docking, MD simulation and QSAR analysis was also carried out to elucidate the interactions, orientation, and conformational changes of these compounds within the active site of the enzyme. Moreover, pharmacokinetic assessments showed that most of the compounds possess attributes conducive to potential drug development.

Commikuanoid D: A New Potent Carbonic Anhydrase-II Inhibitor from the Resin of Commiphora kua

Commikuanoid D: A New Potent Carbonic Anhydrase-II Inhibitor from the Resin of Commiphora kua

Fluconazole-Induced Protein Changes in Osteogenic and Immune Metabolic Pathways of Dental Pulp Mesenchymal Stem Cells of Osteopetrosis Patients.

Osteopetrosis is a rare inherited disease caused by osteoclast failure, resulting in increasing bone density in humans. Patients with osteopetrosis possess several dental and cranial complications. Since carbonic anhydrase II (CA-II) deficiency is a major cause of osteopetrosis, CA-II activators might be an attractive potential treatment option for osteopetrosis patients. We conducted comprehensive label-free quantitative proteomics analysis on Fluconazole-treated Dental Pulp Mesenchymal Stem/Stromal Cells from CA-II-Deficient Osteopetrosis Patients. We identified 251 distinct differentially expressed proteins between healthy subjects, as well as untreated and azole-treated derived cells from osteopetrosis patients. Twenty-six (26) of these proteins were closely associated with osteogenesis and osteopetrosis disease. Among them are ATP1A2, CPOX, Ap2 alpha, RAP1B and some members of the RAB protein family. Others include AnnexinA1, 5, PYGL, OSTF1 and PGAM4, all interacting with OSTM1 in the catalytic reactions of HCO3 and the Cl- channel via CAII regulation. In addition, the pro-inflammatory/osteoclast regulatory proteins RACK1, MTSE, STING1, S100A13, ECE1 and TRIM10 are involved. We have identified proteins involved in osteogenic and immune metabolic pathways, including ERK 1/2, phosphatase and ATPase, which opens the door for some CA activators to be used as an alternative drug therapy for osteopetrosis patients. These findings propose that fluconazole might be a potential treatment agent for CAII- deficient OP patients. Altogether, our findings provide a basis for further work to elucidate the clinical utility of azole, a CA activator, as a therapeutic for OP.

Chemical composition and biomedical effects of essential oil from Commiphora foliacea Sprague stem

Commiphora foliacea Sprague and related species are renowned for the expensive perfumes, cosmetics, and aromatherapy due to its pleasant smell of essential oils used to promote relaxation, reduce stress, and improve memory loss. The chemical ingredients from the stem of C. foliacea Sprague essential oil (EO) were evaluated for GC-MS analysis, in vitro (antioxidant, antimicrobial, antidiabetic, and carbonic anhydrase-II (CA-II)) and in vivo (anti-analgesic and anti-inflammatory) studies. The EO encompasses 56 compounds contributed 93.58% of the total with leading compounds of camphene (14.02%) followed by eremophilene (11.46%). Maximum activity was depicted via DPPH and ABTS protocols compared to the standards. The EO was found effective against B. subtilis (gram-positive bacteria), while maximum antifungal inhibition was observed against A. parasiticus. The substantial antidiabetic and CA-II potential was noted compared with standards. The EO presented considerable potential against breast cancer (MDA-MB-231) and normal (MCF-10A) cell lines with an IC50 of 108.4 and 300 μg/mL, correspondingly and it has been assumed that the essential oil did not deliver much damage to the normal cell lines. Moreover, among the in vivo, the EO revealed the substantial anti-inflammatory potential to reduce paw edema with 63.81% inhibition in comparison to the standard Diclofenac. EO also depicted a significant reduction in analgesic potential (62.05%) compared to the standard aspirin. Furthermore, up to the best of our knowledge, this is the first report on the GC-MS analysis, in vitro, anti-analgesic, and anti-inflammatory potential of the EO isolated from the stem of C. foliacea.

Carbonic anhydrase II deficiency syndrome with amelogenesis imperfecta linked to a homozygous CA2 deletion.

We performed a study to present a phenotypic and genotypic characterization of a patient clinically diagnosed with carbonic anhydrase II (CAII) deficiency syndrome. Medical records were reviewed, and oral examination was performed. Sanger sequencing was undertaken for molecular diagnosis. The patient presented with osteopetrosis, renal tubular acidosis, cerebral calcification, blindness, deafness, and development delay. The oral manifestations included anterior open bite, posterior crossbite, tooth eruption impairment, and hypoplastic amelogenesis imperfecta (AI). Molecular analysis revealed a CA2 homozygous deletion (c.753delG, p.Asn252Thrfs*14) and confirmed the clinical diagnosis. This study suggests that AI can be another feature of CAII deficiency syndrome. For the first time, a CA2 disease-causing variant is reported to be associated with syndromic AI.

Salivary carbonic anhydrase II in winged aphid morph facilitates plant infection by viruses

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.

GC-MS Profiling and Biomedical Applications of Essential Oil of Euphorbia larica Boiss.: A New Report

The present study explored Euphorbia larica essential oil (ELEO) constituents for the first time, obtained via hydro-distillation by means of Gas Chromatography-Mass Spectrometry (GC-MS) profiling. The essential oil was screened in vitro against breast cancer cells, normal cell lines, α-glucosidase, carbonic anhydrase-II (CA-II), free radical scavenging and in vivo analgesic and anti-inflammatory capabilities. The GC-MS screening revealed that the ELEO comprises sixty compounds (95.25%) with the dominant constituents being camphene (16.41%), thunbergol (15.33%), limonene (4.29%), eremophilene (3.77%), and β-eudesmol (3.51%). A promising antidiabetic capacity was noticed with an IC50 of 9.63 ± 0.22 μg/mL by the ELEO as equated to acarbose with an IC50 = 377.71 ± 1.34 μg/mL, while a 162.82 ± 1.24 μg/mL inhibition was observed against CA-II. Regarding breast cancer, the ELEO offered considerable cytotoxic capabilities against the triple-negative breast cancer (MDA-MB-231) cell lines, having an IC50 = 183.8 ± 1.6 μg/mL. Furthermore, the ELEO was also tested with the human breast epithelial (MCF-10A) cell line, and the findings also presumed that the ELEO did not produce any damage to the tested normal cell lines. The ELEO was effective against the Gram-positive bacteria and offered a 19.8 ± 0.02 mm zone of inhibition (ZOI) against B. atrophaeus. At the same time, the maximum resistance with 18.03 ± 0.01 mm ZOI against the fungal strain Aspergillus parasiticus was observed among the tested fungal strains. An appreciable free radical significance was observed via the DPPH assay with an IC50 = 133.53 ± 0.19 µg/mL as equated to the ABTS assay having an IC50 = 154.93 ± 0.17 µg/mL. The ELEO also offered a substantial analgesic capacity and produced 58.33% inhibition in comparison with aspirin, a 68.47% decrease in writhes, and an anti-inflammatory capability of 65.54% inhibition, as equated to the standard diclofenac sodium having 73.64% inhibition. Hence, it was concluded that the ELEO might be a natural source for the treatment of diabetes mellitus, breast cancer, analgesic, inflammatory, and antimicrobial-related diseases. Moreover, additional phytochemical and pharmacological studies are needed to isolate responsible chemical ingredients to formulate new drugs for the examined activities.

Profiling Sulfur(VI) Fluorides as Reactive Functionalities for Chemical Biology Tools and Expansion of the Ligandable Proteome.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (SVI-Fs) as reactive groups for chemical biology applications. SVI-Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of SVI-Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of SVI-Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the SVI-F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of SVI-F used. This work highlights how SVI-F reactivity can be used as a tool to expand the liganded proteome.

A novel prognostic system combining carbonic anhydrase II and preoperative CA19-9 for intrahepatic cholangiocarcinoma after curative resection.

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.

Synthesis, Biochemical Characterization, and in-Silico Investigations of Acyl-3-(Ciprofloxacinyl) Thioureas as Inhibitors of Carbonic Anhydrase-II

A new series of 1-(acyl/aroyl)-3-(ciprofloxacinyl) thioureas (5a–o) was synthesized by the reaction of ciprofloxacin (4) in dry acetone with acyl isothiocyanate intermediate (3). The synthesized compounds were characterized by FT-IR, 1H-NMR, and 13C-NMR. Furthermore, the synthesized compounds were tested for inhibitory potential against carbonic anhydrase (CA-II) and 15 lipoxygenase (LOX) enzymes. The tested compounds exhibited maximum inhibition of CA-11 with moderate anti-inflammatory potential, at the same concentration, i.e. 100 µM, therefore, can be considered as the selective inhibitors of CA-II. Among the tested derivatives 5g showed remarkable inhibitory activity against CA enzyme with IC50 value of 0.97 ± 0.11 µM and this derivative exhibited approximately 40% inhibition of 15-LOX, indicating that compound has moderate anti-inflammatory properties. The molecular docking studies, density functional theory (DFT) calculations, and molecular dynamic (MD) simulation studies were performed to evaluate their binding affinities, stability, and chemical reactivity within the active pocket of the targeted enzymes. The docking analysis and MD simulation studies revealed that the most active inhibitor 5g showed important interactions within the binding pockets of CA-II and may be responsible for the inhibitory activity of the compound toward the targeted enzymes. Therefore, the screened derivatives provided an outstanding platform for further development of CA inhibitors.

Elucidating Protein-Ligand Interactions in Cell Lysates Using High-Throughput Hydrogen-Deuterium Exchange Mass Spectrometry with Integrated Protein Thermal Depletion.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is a powerful technique for the characterization of protein-ligand interactions. Currently, there is a growing need for breakthroughs in the application of HDX-MS analysis to protein-ligand interactions in highly complex biological samples such as cell lysates. However, HDX-MS analysis in such systems suffers from extreme spectral complexity as a result of high sample complexity and limited LC separation power due to the traditional use of short LC gradients. Here, we introduced protein thermal depletion (PTD) to reduce protein complexity in E. coli cell lysate for our subzero-temperature long gradient UPLC-HDX-MS platform (PTD-HDX-MS) to facilitate high-throughput analysis of protein-ligand interactions in cell lysates. We spiked bovine carbonic anhydrase II (CaII) and its inhibitor acetazolamide (AZM) into E. coli cell lysate as a model system in our study. We demonstrated that PTD at 60 °C greatly reduces protein complexity in cell lysates, while the AZM-targeted CaII complex remains in solution due to improved thermal stability upon binding. Using both PTD to reduce sample complexity and subzero-temperature long gradient UPLC to boost LC separation power, we successfully elucidated the interaction sites between AZM and CaII in E. coli cell lysate from the high-throughput HDX-MS analysis of thousands of deuterated peptides from hundreds of proteins. Our results highlight the great promise of the PTD-HDX-MS platform for the identification of ligand targets and characterization of protein-ligand interactions in highly complex biological samples such as cell lysates.

Differential antigen expression between human apocrine sweat glands and eccrine sweat glands

Bromhidrosis has a great negative impact on personal occupation and social psychology. It is not yet clear whether bromhidrosis is caused by apocrine sweat glands or the co-action of apocrine sweat glands and eccrine sweat glands. To distinguish between apocrine sweat glands and eccrine sweat glands, specific antigen markers for apocrine sweat glands and eccrine sweat glands must be found first. In the study, we detected the expression of K7, K18, K19, Na+-K+-2Cl- cotransporter 1 (NKCC1), carbonic anhydrase II (CAII), Forkhead transcription factor a1 (Foxa1), homeobox transcription factor engrailed homeobox1 (En1), gross cystic disease fluid protein-15 (GCDFP-15), mucin-1 (MUC-1), cluster of differentiation 15 (CD15) and apolipoprotein (APOD) in eccrine sweat glands and apocrine sweat glands by immunofluorescence staining. The results showed that K7, K18, K19, Foxa1, GCDFP-15 and MUC-1 were expressed in both apocrine and eccrine sweat glands, CD15 and APOD were only expressed in apocrine sweat glands, and CAII, NKCC1 and En1 were only expressed in eccrine sweat glands. We conclude that CD15 and APOD can serve as specific markers for apocrine sweat glands, while CAII, NKCC1 and En1 can serve as specific markers for eccrine sweat glands to differentiate the two sweat glands.

Synthesis, characterization, molecular docking and network pharmacology of bioactive metallic sulfonamide-isatin ligands against promising drug targets

A series of sulfonamide and isatin based Schiff bases, (S1) and (S2), and their metal (Co2+, Ni2+, Cu2+ and Zn2+) complexes (1)-(8) were synthesized and characterized by spectroscopic (UV, IR, MS, 1H and 13C-NMR), elemental, magnetic and physical techniques. The non-electrolytic character of Co2+, Ni2+, and Zn2+ compounds and electrolytic nature of Cu2+ was established by their conductance studies. The energies of Frontier Molecular Orbitals (FMOs) were also used to explore various global and quantum chemical qualities. To find the activity and molecular targets in curing Alzheimer's Disease (AD) and Carbonic Anhydrase II (CA-II) inhibition, Network Pharmacology modeling was used. The prospective targets were predicted using the Swiss Target PredictionR online facility. The Gene CardsR database has been used to find genes linked to AD and CA-II. We also conducted Gene OntologyR (GO) analysis on the intersecting genes targets on active targets of synthesized compounds by DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatics Services using the CytoscapeR program. The in vitro enzyme inhibition assays were done against protease, amylase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) while their antimicrobial studies were performed against pathogenic bacterial and fungal species. The antioxidant values, evaluated as 2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing assay power (FRAP) (%) ranged between 51.0±0.11-68.1±0.11% with IC50 ranging 146.84-196.08 µL/mol.