Expression Of Carbonic Anhydrase Research Articles

Value of [68Ga]Ga-NYM046 PET/CT, in Comparison with 18F-FDG PET/CT, for Diagnosis of Clear Cell Renal Cell Carcinoma.

This study aimed to investigate the diagnostic efficacy of [68Ga]Ga-NYM046 PET/CT in animal models and patients with clear cell renal cell carcinoma (ccRCC) and to compare its performance with that of 18F-FDG PET/CT. Methods: The invivo biodistribution of [68Ga]Ga-NYM046 was evaluated in mice bearing OS-RC-2 xenografts. Twelve patients with ccRCC were included in the study; all completed paired [68Ga]Ga-NYM046 PET/CT and 18F-FDG PET/CT. The diagnostic efficacies of these 2 PET tracers were compared. Moreover, the positive rate of carbonic anhydrase IX in the pathologic tissue sections was compared with the SUVmax obtained by PET/CT. Results: The tumor accumulation of [68Ga]Ga-NYM046 at 1 h after injection in OS-RC-2 xenograft tumor models was 7.21 ± 2.39 injected dose per gram of tissue. Apart from tumors, the kidney and stomach showed high-uptake distributions. In total, 9 primary tumors, 96 involved lymph nodes, and 147 distant metastases in 12 patients were evaluated using [68Ga]Ga-NYM046 and 18F-FDG PET/CT. Compared with 18F-FDG PET/CT, [68Ga]Ga-NYM046 PET/CT detected more primary tumors (9 vs. 1), involved lymph nodes (95 vs. 92), and distant metastases (137 vs. 127). In quantitative analysis, the primary tumors' SUVmax (median, 13.5 vs. 2.4; z = -2.668, P = 0.008) was significantly higher in [68Ga]Ga-NYM046 PET/CT. Conversely, the involved lymph nodes' SUVmax (median, 5.9 vs. 7.6; z = -3.236, P = 0.001) was higher in 18F-FDG PET/CT. No significant differences were found for distant metastases (median SUVmax, 5.0 vs. 5.0; z = -0.381, P = 0.703). Higher [68Ga]Ga-NYM046 uptake in primary tumors corresponded to higher expression of carbonic anhydrase IX, with an R 2 value of 0.8274. Conclusion: [68Ga]Ga-NYM046 PET/CT offers a viable strategy for detecting primary tumors, involved lymph nodes, and distant metastases in patients with ccRCC.

Application of carbonic anhydrase IX in sporadic hemangioblastoma of the central nervous system and hemangioblastoma associated with von Hippel-Lindau disease.

This research aims to examine the expression of carbonic anhydrase IX (CAIX) protein in hemangioblastoma of the central nervous system and its potential application in pathological diagnosis and differential diagnosis. Immunohistochemistry was used to identify the expression of CAIX and the α-inhibin protein. The sensitivity and specificity of CAIX and α-inhibin for identifying hemangioblastoma of the central nervous system were compared. In addition, 86 patients with meningiomas were gathered to detect CAIX protein expression. Hemangioblastoma and angiomatous, microcystic the two subtypes of meningiomas, were compared for CAIX and EMA protein expression. In hemangioblastoma, there were significant differences in the median positive percentage and staining intensity of CAIX and α-inhibin (p < 0.05). There was no discernible difference in the expression of the CAIX protein between sporadic hemangioblastoma of the central nervous system and those linked to von Hippel‒Lindau disease. In comparison to angiomatous and microcystic meningiomas, the positive rate of CAIX in hemangioblastomas was substantially greater (p < 0.001). The expression of EMA in microcystic meningioma (6/6) and angiomatous meningioma (17/17) was significantly different from hemangioblastoma (0/30) (p < 0.0001). Hemangioblastoma might be diagnosed with high specificity and sensitivity through CAIX immunohistochemistry. The combination of CAIX with EMA is useful for the diagnosis and differential diagnosis of hemangioblastoma.

Expression of Carbonic Anhydrase IX as a Novel Diagnostic Marker for Differentiating Pleural Mesothelioma from Non-small Cell Lung Carcinoma

Expression of Carbonic Anhydrase IX as a Novel Diagnostic Marker for Differentiating Pleural Mesothelioma from Non-small Cell Lung Carcinoma

Proteomic Dynamics of Multidrug Resistance Mechanisms in Lucena 1 Cell Line.

The Lucena 1 cell line, derived from the human chronic myeloid leukemia cell line K562 under selective pressure of vincristine supplementation, exhibits multidrug resistance (MDR). This study aims to explore and elucidate the underlying mechanisms driving MDR in the Lucena 1 cell line. A proteomic analysis comparing K562 and Lucena 1 revealed qualitative differences, with a focus on the ATP-dependent efflux pump, Translocase ABCB1, a key contributor to drug resistance. Tubulin analysis identified two unique isoforms, Tubulin beta 8B and alpha chain-like 3, exclusive to Lucena 1, potentially influencing resistance mechanisms. Additionally, the association of Rap1A and Krit1 in cytoskeletal regulation and the presence of STAT1, linked to the urea cycle and tumor development, offered insights into Lucena 1's distinctive biology. The increased expression of carbonic anhydrase I suggested a role in pH regulation. The discovery of COP9, a tumor suppressor targeting p53, further highlighted the Lucena 1 complex molecular landscape. This study offers new insights into the MDR phenotype and its multifactorial consequences in cellular pathways. Thus, unraveling the mechanisms of MDR holds promise for innovating cancer models and antitumor targeted strategies, since inhibiting the P-glycoprotein (P-gp)/ABCB1 protein is not always an effective approach given the associated treatment toxicity.

Ca2+-controlled Mn(II) removal process in Aurantimonas sp. HBX-1: Microbially-induced carbonate precipitation (MICP) versus Mn(II) oxidation

The application of manganese-oxidizing bacteria (MnOB) to produce manganese oxides (MnOx) has been widely studied, but often overlooking the concurrent formation of MnCO3. In this study, we found Ca2+ plays a crucial role in controlling Mn(II) removal in the bacterium Aurantimonas sp. HBX-1. Under conditions with 6.8 mM Ca2+ and without adding Ca2+, 100 μM Mn(II) was removed by 96.96 % and 38.28 % within 8 days, respectively. X-ray photoelectron spectroscopy (XPS) showed that adding Ca2+ increased the average oxidation state (AOS) of the solid products from 2.05 to 2.37. X-ray absorption fine structure (XAFS) analysis revealed the product proportions as follows: under Ca2+-supplemented condition, the ratio of MnOx (1 < x ≤ 2) to MnCO₃ was 52 % to 28.1 %, while under Ca2+-free condition, the ratio shifted to 4.6 % for MnOx (1 < x ≤ 2) and 55.2 % for MnCO₃. Urease activity assay and proteomic analysis confirmed the expression of urease and carbonic anhydrase, leading to the formation of MnCO3. Additionally, animal heme peroxidase (AHP) in strain HBX-1 was found to be responsible for Mn(II) oxidation through superoxide production, with Ca2+ addition promoting its expression level. Given the widespread presence of Ca2+ in wastewater, its potential impact on the biogeochemical Mn(II) cycle driven by bacteria should be reconsidered.

Roles of eyestalk in salinity acclimatization of mud crab (Scylla paramamosain) by transcriptomic analysis

Salinity acclimatization refers to the physiological and behavioral adjustments made by crustaceans to adapt to varying salinity environments. The eyestalk, a neuroendocrine organ in crustaceans, plays a crucial role in salinity acclimatization. To elucidate the molecular mechanisms underlying eyestalk involvement in mud crab (Scylla paramamosain) acclimatization, we employed RNA-seq technology to analyze transcriptomic changes in the eyestalk under low (5 ppt) and standard (23 ppt) salinity conditions. This analysis revealed 5431 differentially expressed genes (DEGs), with 2372 upregulated and 3059 downregulated. Notably, these DEGs were enriched in crucial biological pathways like metabolism, osmoregulation, and signal transduction. To validate the RNA-seq data, we further analyzed 15 DEGs of interest using qRT-PCR. Our results suggest a multifaceted role for the eyestalk: maintaining energy homeostasis, regulating hormone synthesis and release, PKA activity, and downstream signaling, and ensuring proper ion and osmotic balance. Furthermore, our findings indicate that the crustacean hyperglycemic hormone (CHH) may function as a key regulator, modulating carbonic anhydrase expression through the activation of the PKA signaling pathway, thereby influencing cellular osmoregulation, and associated metabolic processes. Overall, our study provides valuable insights into unraveling the molecular mechanisms of mud crab acclimatization to low salinity environments.

#102 Increased expression of carbonic anhydrase 4 in megalin deficiency

Abstract Background and Aims Megalin is an endocytic receptor located in the apical membrane of proximal tubule (PT) cells. It prevents low molecular-weight proteinuria by reuptake of a variety of proteins from the filtrate. Megalin is required for normal uptake of Cubilin-bound proteins, but it is unknown if the receptor is important for other apical PT proteins. Carbonic anhydrase 4 (CA4) is an enzyme crucial for PT bicarbonate reuptake. Our aim was to investigate the relationship between the expression patterns of these two proteins. Method A mouse model of kidney-specific mosaic megalin-knockout (M-KO). Liquid chromatography-mass spectrometry (LC-MS) on mouse kidney cortex. Immunohistochemistry (IHC), electron microscopy (EM) and Western blotting (WB) of mouse kidney. Biochemical analysis of mouse blood and urine. Results LC-MS revealed a 4.23-fold increase in CA4 abundance in M-KO mice together with smaller but also statistically significant alterations in other acid-base-associated proteins. IHC shows that the increase in CA4 abundance specifically was in the cells lacking megalin, while cells with megalin retained normal abundance. The same pattern was observed in a biopsy from a patient lacking megalin. WB revealed increased shedding of CA4 in urine from M-KO mice and EM showed mislocalization of CA4. Conclusion We show a hitherto unknown relationship where the expression and subcellular localization of CA4 is altered in PT-cells lacking megalin and show that M-KO mice have altered acid-base handling. Comparable blood parameters at baseline are maintained despite differences in urinary excretion and when challenged with acetazolamide the urinary excretion of the groups becomes alike, however blood parameters become different. While the mechanisms and full extend are unknown, this links megalin to the acid-base regulating systems of the PT.

Developing rainbow trout (Oncorhynchus mykiss) lose branchial plasma accessible carbonic anhydrase expression with hatch and the transition to pH-sensitive, adult hemoglobin polymorphs.

Salmonids possess a unique respiratory system comprised of three major components: highly pH-sensitive hemoglobins, red blood cell (RBC) intracellular pH (pHi) protection, and a heterogeneous distribution of plasma accessible carbonic anhydrase (paCA), specifically with absence of paCA at the gills. These characteristics are thought to have evolved to enhance oxygen unloading to the tissues while protecting uptake at the gills. Our knowledge of this system is detailed in adults, but little is known about it through development. Developing rainbow trout (Oncorhynchus mykiss) express embryonic RBCs containing hemoglobins that are relatively insensitive to pH; however, availability of gill paCA and RBC pHi protection is unknown. We show that pre-hatch rainbow trout express gill paCA, which is lost in correlation with the emergence of highly pH-sensitive adult hemoglobins and RBC pHi protection. Rainbow trout therefore exhibit a switch in respiratory strategy with hatch. We conclude that gill paCA likely represents an embryonic trait in rainbow trout and is constrained in adults due to their highly pH-sensitive hemoglobins.

Multi-omics revealed the mechanism of feed efficiency in sheep by the combined action of the host and rumen microbiota

This study was conducted to investigate potential regulatory mechanisms of feed efficiency (FE) in sheep by linking rumen microbiota with its host by the multi-omics analysis. One hundred and ninety-eight hybrid female sheep (initial body weight = 30.88 ± 4.57 kg; 4-month-old) were selected as candidate sheep. Each test sheep was fed in an individual pen for 60 days, and the residual feed intake (RFI) was calculated. The ten candidate sheep with the highest RFI were divided into the Low-FE group, and the ten with the lowest RFI were divided into the High-FE group, all selected for sample collection. The RFI, average daily gain and average daily feed intake were highly significantly different between the two experimental groups (P < 0.05). Compared with Low-FE group, the insulin-like growth factor-1 and very low-density lipoprotein in serum and the propionate in rumen significantly increased in High-FE group (P < 0.01), but the acetate:propionate ratio in rumen significantly decreased in High-FE group (P = 0.034). Metagenomics revealed Selenomonas ruminantium, Selenomonas sp. and Faecalibacterium prausnitzii were key bacteria, and increased abundance of the genes encoding the enzymes for cellulose degradation and production of propionate in High-FE group. The results of proteomics and section showed the rumen papilla length (P < 0.001) and expression of carbonic anhydrase and Na+/K+-ATPase were significantly higher in High-FE group (P < 0.05). On the other hand, the acetyl-CoA content significantly increased in the liver of High-FE group (P = 0.002). The relative expression levels of insulin-like growth factor-1 and apolipoprotein A4 genes were significantly up-regulated in the liver of High-FE group (P < 0.01), but relative expression level of monoacylglycerol O-acyltransferase 3 gene was significantly down-regulated (P = 0.037). These findings provide the mechanism by which the collaborative interaction between rumen microbiota fermentation and host uptake and metabolism of fermentation products impacts feed efficiency traits in sheep.

Abstract 4004: Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma

Abstract On-target off-tumor toxicity (OTOT) is one of the major hurdles preventing tumor-associated antigen (TAA) targeted therapy from effectively treating patients with solid tumors due to the shared presence of the same TAA on normal tissues. We have previously demonstrated high expression of carbonic anhydrase IX (CAIX) in ccRCC patient samples and low-density expression on healthy bile duct epithelium. In 2006, a clinical trial was stopped due to OTOT bile duct toxicity observed in patients infused with anti-CAIX CAR T cell therapy. To address the issue of OTOT in the bile duct, we performed affinity/avidity fine-tuning of the anti-CAIX CAR G36 to specifically target ccRCC tumor cells with high CAIX expression while sparing cholangiocytes with low CAIX expression. We generated a complementarity-determining region (CDR) single variant mutagenesis library of G36 and displayed the library on yeast for screening. Using flow cytometry, we were able to identify six populations based on antigen-binding affinity: high, med-high, med, med-low, low, and loss of binding. These libraries were sequenced via PacBio, a next generation sequencing (NGS) platform that excels in long-read sequencing, providing a powerful tool to detect single mutations in the scFv. The results were analyzed to identify enriched mutations compared to the wild-type G36, and inspected using in silico modeling and docking. Furthermore, enriched mutants from med-low and low populations were tested in CAR-T format for their cytotoxic capacity on tumor cells and bile duct cholangiocytes, as well as their avidity. We demonstrate that mutations in CDR2 and CDR3 of the heavy chain successfully mitigate OTOT toxicity on cholangiocytes with physiological levels of CAIX expression while maintaining tumor-killing capacity, shedding light on CDR mutagenesis as a useful tool to mitigate OTOT toxicity. Citation Format: Stephen Hsien-Chi Yuan, Yufei Wang, Rabia Abbas, Peterson Mathenge, Charles Chien, Christian D. Coherd, Branson Bajoua, Matthew R. Chang, Nithyassree Murugan, Gabriella M. Kastrunes, Guilin Wang, Song-My Hoang, Wayne A. Marasco. Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4004.

Vasohibin-1 Expression Can Predict Pathological Complete Remission of Advanced Bladder Cancer with Neoadjuvant Chemotherapy.

Neoadjuvant chemotherapy (NAC) is a well-established standard practice in invasive bladder cancer (BCa), however patient selection remains challenging. High expression of vasohibin-1 (VASH1), an endogenous regulator of angiogenesis, has been reported in high-grade and advanced BCa; however, its prognostic value for chemotherapy outcomes remains unexplored. In this study, we sought to identify biomarkers of chemotherapy response focusing on the relationship between angiogenesis and tissue hypoxia. Forty Japanese patients with BCa who underwent NAC and radical cystectomy were included in the present analysis. We compared the immunohistochemical expression of CD34, VASH1, and carbonic anhydrase 9 (CA9) between patients who achieved tumor clearance at operation (ypT0) and those with residual disease after cystectomy. There were 19 patients in the ypT0 group, while the remaining 21 patients had residual tumors at operation. Patients in the ypT0 group had high microvessel density (p=0.031), high VASH1 density (p<0.001), and stronger CA9 staining (p=0.046) than their counterparts. Multivariate analysis identified microvessel and VASH1 density as independent predictive factors for pathological ypT0 disease (p=0.043 and 0.002, respectively). The 5-year recurrence-free survival rate was higher in the high VASH1 density group than in the low VASH1 density group (66.3% vs. 33.3%, p=0.036). VASH1 density is a potential therapeutic biomarker for chemotherapy response in BCa.

Surface expression of carbonic anhydrase on E. coli as a sustainable approach for enzymatic CO2 capture

The utilisation of carbonic anhydrase (CA) in CO2 sequestration is becoming prominent as an efficient, environment friendly and rapid catalyst for capturing CO2 from industrial emissions. However, the application of CA enzyme in soluble form is constrained due to its poor stability in operational conditions of CO2 capture and also production cost of the enzyme. Addressing these limitations, the present study focuses on the surface display of CA from Bacillus halodurans (BhCA) on E coli aiming to contribute to the cost-effectiveness of carbon capture through CA technology. This involved the fusion of the BhCA-encoding gene with the adhesion molecule involved in diffuse adherence (AIDA-I) autotransporter, resulting in the efficient display of BhCA (595 ± 60 U/gram dry cell weight). Verification of the surface display of BhCA was accomplished by conjugating with FITC labelled anti-his antibody followed by fluorescence-activated cell sorting (FACS) and cellular fractionation in conjunction with zymography. Biochemical characterisation of whole-cell biocatalyst revealed a noteworthy enhancement in thermostability, improvement in the thermostability with T1/2 of 90 ± 1.52 minutes at 50 ˚C, 36 ± 2.51 minutes at 60 ˚C and18 ± 1.52 minutes at 80˚C. Surface displayed BhCA displayed remarkable reusability retaining 100% activity even after 15 cycles. Surface displayed BhCA displayed highly alkali stable nature like free counterpart in solution. The alkali stability of the surface-displayed BhCA was comparable to its free counterpart in solution. Furthermore, the study investigated the impact of different metal ions, modulators, and detergents on the whole-cell biocatalysts. The present work represents the first report on surface display of CA utilising the AIDA-1 autotransporter.

Downregulation of carbonic anhydrase IX expression in mouse xenograft nasopharyngeal carcinoma model via doxorubicin nanobubble combined with ultrasound.

The purpose of this study was to investigate whether doxorubicin nanobubbles (DOX-NB) combined with diagnostic ultrasound (DUS) irradiation could downregulate the expression of carbonic anhydrase IX (CAIX) in mouse xenograft nasopharyngeal carcinoma (NPC) model. In this study, the prepared DOX-NB was round and well dispersed. The average diameter of DOX-NB was 250.9 ± 50.8 nm, with an average polydispersity of 0.321 ± 0.05. The cumulative release of DOX in the DOX-NB + DUS group was significantly higher compared with that of the DOX-NB group (p < 0.05). DOX-NB combined with DUS irradiation could significantly inhibit cell viability (p < 0.05). The expression of CAIX and microvessel density (MVD) in the xenografted tumors was the lowest in the DOX-NB + DUS group compared with that of other groups (p < 0.05). In conclusion, DOX-NB combined with DUS irradiation could improve DOX-NB drug release and synergistically inhibit NPC cell activity. DOX-NB combined with DUS irradiation can downregulate the expression of CAIX in mouse xenograft NPC model. This may be due to the synergistic effect of DUS combined with DOX-NB in reducing MVD in NPC.

Synchronous Double Primary Malignant Tumors and their Possible Shared Genes: A Rare Clinical Entity.

This study sought to analyze the 18F-FDG PET/CT and contrast-enhanced computed tomography (CT) images of synchronous colorectal cancer (CRC) and renal clear cell carcinoma (ccRCC) and identify the shared genes between these two types of cancer through bioinformatic analysis. A retrospective analysis was conducted on a patient with synchronous CRC and ccRCC who underwent 18F-FDG PET/CT and contrast-enhanced CT before treatment. Databases were analyzed to identify differentially expressed genes between CRC and ccRCC, and co-expression genes were extracted for RCC and CRC. 18F-FDG PET/CT revealed intense metabolic activity in the primary colorectal lesion (SUVmax 13.2), while a left renal mass (diameter = 35 mm) was observed with no significant uptake. Contrast-enhanced CT during the arterial phase showed heterogeneous intense enhancement of the renal lesion, and the lesion washed out earlier than in the renal cortex in the nephrographic and excretory phases, indicating ccRCC. The histopathological results confirmed synchronous double primary malignant tumors. Our bioinformatic analysis results showed that synchronous occurrence of CRC and ccRCC may correlate with simultaneous expression of Carbonic Anhydrase 9 (CA9), integrin-binding sialoprotein (IBSP), and Fibrinogen γ chain (FGG). 18F-FDG PET/CT combined with contrast-enhanced CT is an effective diagnostic tool in evaluating synchronous CRC and RCC. By analyzing this clinical case and conducting bioinformatic analysis, we improved our current understanding of the mechanisms underlying synchronous tumors.

Effects of Mulberry Leaf Extract on the Liver Function of Juvenile Spotted Sea Bass (Lateolabrax maculatus).

In order to explore the effect of mulberry leaf extract (ELM) on the liver function of spotted sea bass, 360 fish with healthy constitution (average body weight 9.00 ± 0.02 g) were selected and randomly divided into six groups with three repetitions, and six groups of fish were randomly placed into 18 test tanks (200 L) with 20 fish per tank for the 52-day feeding test. Every day, the fish were fed the experimental feed with different concentrations (0, 3, 6, 9, 12, 15 g/kg) to the level of apparent satiation, with a crude protein content of 48.0% and a crude fat content of 8.6%. And the water temperature was maintained at 25-28°C with a salinity of 0.5%-1‰. After feeding, five fish were randomly selected to collect their livers and serum for detection of indicators. The results showed that, compared with the control group, ELM significantly increased the activities of lipase (LPS) and trypsin (TRS) in the liver, and reached the highest level when the amount of ELM added was 6 g/kg (P < 0.05). ELM significantly increased the activities of lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT) involved in the metabolic process in liver tissue, and GOT activity reached the highest when ELM was added at 9 g/kg, and LDH activity reached the highest when ELM was added at 15 g/kg (P < 0.05). ELM had no significant effect on liver antioxidant enzymes (P > 0.05), but the content of malondialdehyde was significantly reduced (P < 0.05). Compared with the control group, ELM significantly increased the activities of AKP and ACP in the liver, and the AKP activity reached the highest when the ELM addition amount was 3 g/kg, and the ACP activity reached the highest when the ELM addition amount was 9 g/kg (P < 0.05). Through comparative transcriptomic analysis, it was indicated that ELM enhanced the hepatic lipids and carbohydrates metabolism ability, as manifested in the upregulation of expression of phosphatidate phosphatase, glucuronosyltransferase, inositol oxygenase, carbonic anhydrase, and cytochrome c oxidase subunit 2. ELM can also increase the expression of signal transducer and activator of transcription 1, ATP-dependent RNA helicase and C-X-C motif chemokine 9 involved in the immune process. The above results show that the ELM can enhance the digestion, metabolism, and immunity of the liver by increasing the activity of digestive enzymes, metabolic enzymes, and the expression of metabolism and immune regulation genes. This study provides a theoretical basis for the application of ELM in the cultivation of spotted sea bass by exploring the effect of ELM on the liver function of spotted sea bass.

Effects of Dietary Betaine on the Laying Performance, Antioxidant Capacity, and Uterus and Ovary Function of Laying Hens at the Late Stage of Production.

Betaine has been found to alleviate oxidative stress, inflammation, and apoptosis. However, whether dietary betaine can protect late-laying hens against these adverse effects is unknown. Here, 270 65-week-old Jinghong-1 laying hens were randomly divided into the Control, 0.1% Betaine, and 0.5% Betaine groups and fed a basal diet, 0.1%, and 0.5% betaine supplemented diet, respectively. The trial lasted for seven weeks. Birds that consumed 0.5% betaine laid more eggs with thicker eggshells. Accordingly, uterine reduced glutathione (GSH), glutathione peroxidase (GSH-PX), and ovarian superoxide dismutase (SOD) contents were increased. The uterine calcium ion content and the mRNA expression of ovalbumin, ovotransferrin, and carbonic anhydrase two were increased. Moreover, ovarian IL-1β, Caspase-1, Caspase-8, and Caspase-9 mRNA expressions were decreased; luteinising hormone receptor (LHR) and follicle-stimulating hormone receptor mRNA expressions were increased. Furthermore, dietary betaine decreased the ovaries' mRNA expression of DNA methyltransferase 1 (DNMT)1, DNMT3a, and DNMT3b. The methylation level at the promoter region of ovarian LHR decreased. These results indicated that dietary betaine consumption with a concentration of 0.5% could increase the laying rate and the eggshell thickness during the late-laying period. The underlying mechanism may include antioxidative, anti-apoptosis, and hormone-sensitivity-enhancing properties.

Arenobufagin increases the sensitivity of gastric cancer to cisplatin via alkaliptosis

BackgroundGastric cancer is the third leading cause of cancer-related death worldwide, for which several novel therapeutic strategies have been developed. Cisplatin (CDDP) mainly exerts its anti-gastric cancer effects; however, drug resistance limits its use. Thus, the development of drugs that can augment their antitumor effects is necessary. Arenobufagin (ArBu) is a novel anticancer drug, and the effects of ArBu in combination with CDDP on gastric cancer have not yet been studied. AimsTo identify a possible synergistic effect between ArBu and CDDP in gastric cancer and investigate the underlying mechanism. MethodsCell viability, colony formation, migration, apoptosis, cell cycle, western blotting, immunofluorescence, and reverse-transcription polymerase chain reaction (RT-PCR) were analyzed in vitro. Western blotting, RT-PCR, hematoxylin and eosin (H&E) staining and blood biochemistry were carried out to examine in vivo. ResultsWe found that ArBu, in combination with CDDP, effectively inhibited the proliferation and migration of gastric cancer cells, promoted apoptosis, and downregulated the expression of carbonic anhydrase 9 (CA9), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9). In addition, treatment with ArBu in combination with CDDP increased the level of inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), E-cadherin, and nuclear factor kappa-B/p65 (NF-κB/p65). Furthermore, the combination of ArBu and CDDP inhibited tumor growth in xenograft nude mice with no obvious side effects. ConclusionsArBu synergizes with CDDP to inhibit tumor growth both in vivo and in vitro by inducing alkaliptosis. This indicated that ArBu combined with CDDP may serve as a potential agent for the treatment of gastric cancer.

Hypoxia-Induced Changes in L-Cysteine Metabolism and Antioxidative Processes in Melanoma Cells.

This study was performed on human primary (WM115) and metastatic (WM266-4) melanoma cell lines developed from the same individual. The expression of proteins involved in L-cysteine metabolism (sulfurtransferases, and cystathionine β-synthase) and antioxidative processes (thioredoxin, thioredoxin reductase-1, glutathione peroxidase, superoxide dismutase 1) as well as the level of sufane sulfur, and cell proliferation under hypoxic conditions were investigated. Hypoxia in WM115 and WM266-4 cells was confirmed by induced expression of carbonic anhydrase IX and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 by the RT-PCR and Western blot methods. It was shown that, under hypoxic conditions the inhibition of WM115 and WM266-4 melanoma cell proliferation was associated with decreased expression of thioredoxin reductase-1 and cystathionine β-synthase. These two enzymes may be important therapeutic targets in the treatment of melanoma. Interestingly, it was also found that in normoxia the expression and activity of 3-mercaptopyruvate sulfurtransferase in metastatic WM266-4 melanoma cells was significantly higher than in primary melanoma WM115 cells.

First-in-human clinical trial design of a first-in-class theranostic approach with a peptide-based radioligand targeting CA IX-expressing tumors.

TPS3160 Background: Expression of carbonic anhydrase IX (CA IX), a cell surface glycoprotein, can be induced by a state of hypoxia or by mutation of the Von Hippel-Lindau tumor suppressor gene. CAIX expression in tumors is often associated with progressive disease and overall poor outcomes in various solid cancers. Notably, high CA IX expression has been observed in 83%, 40%, and 29% of clear cell renal cell carcinoma (ccRCC), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC) specimens, respectively1. CA IX may therefore represent a valuable therapeutic target. DPI-4452 is a first-in-class cyclic peptide that binds with high affinity to CA IX. Radiolabeling DPI-4452 with gallium-68 ([68Ga]Ga-DPI-4452) or lutetium-177 ([177Lu]Lu-DPI-4452) is an innovative theranostic approach for identifying and treating patients (pts) with CA IX-expressing tumors. Pre-clinical data has shown that [177Lu]Lu-DPI-4452 is taken up by tumors and induces dose-dependent growth inhibition in ccRCC and CRC xenograft mouse models1,2. Methods: We present the design of a Phase I/II study (NCT05706129) of [68Ga]Ga-DPI-4452 and [177Lu]Lu-DPI-4452 in pts with unresectable locally advanced or metastatic ccRCC, CRC or PDAC. The study is comprised of three phases: imaging (Part A), dose-escalation (Part B), and expansion (Part C). In Part A, pts will receive a single intravenous (IV) 185 MBq dose of [68Ga]Ga-DPI-4452. Safety, tolerability, pharmacokinetics (PK), dosimetry and imaging characteristics of [68Ga]Ga-DPI-4452 will be evaluated using positron emission tomography/computed tomography (CT) at four timepoints on Day 1. All pts will be followed for seven days post-injection for safety observations. In Part B, based on [68Ga]Ga-DPI-4452 uptake, pts will receive sequential, escalating doses of IV [177Lu]Lu-DPI-4452 on Day 1 of each 28-day cycle for up to eight cycles. During Cycle 1, each pt will undergo up to four whole-body planar imaging and single photon emission CT/CT analyses for dosimetry over seven days. In Part B, the recommended Phase 2 dose of [177Lu]Lu-DPI-4452 will be determined, guided by a Bayesian Logistic Regression model. Safety, PK, and dosimetry will be assessed to minimize any radiation-induced toxicity and to optimize absorbed radiation dose in the tumor. Upon completion of Part B, Part C will enroll up to three cohorts of pts with CA IX-positive ccRCC, CRC or PDAC to assess preliminary efficacy of [177Lu]Lu-DPI-4452, with a primary endpoint of objective response rate, as measured by RECIST v1.1. References Wiedemann N et al. J Nucl Med 2022;63(Suppl 2):4048. Attinger A et al. Eur J Nucl Med Mol Imaging 2022;49(Suppl 1):S1–S751. Clinical trial information: NCT05706129 .

Molecular characterization and DNA methylation analysis of carbonic anhydrase (Sp-CA) in the mud crab Scylla paramamosain: Its potential osmoregulation role under carbonate alkalinity stress

Saline–alkali water represents a huge underdeveloped resource due to its harsh properties of high pH, high salinity, and complex ionic composition. In our previous research, the mud crab, Scylla paramamosain could adapt to the saline–alkali environment, which may bring dual benefits to high-quality aquaculture and the utilization of saline–alkali water. Yet, no systematic study related to S. paramamosain's ability to carbonate alkali, the key gene responsible for adaptation to the harsh environment, has been reported. In this study, we investigated the mud crab's response to a wide range of carbonate–alkali waters, verifying the specific gill tissue with the highest carbonic anhydrase (CA) expression. Afterward, we cloned the CA gene in S. paramamosain (Sp-CA) for the first time. The cDNA sequence is 1085 bp, with a 55-bp 5′-untranslated region (UTR) and 214-bp 3′-UTR. The open reading frame (ORF) of 816-bp nucleotides encodes a polypeptide of 271 amino acids (AAs). In silico analysis and phylogenetic analysis of crustacea's CAs were performed, elucidating the unique function and evolutionary relation of Sp-CA. We also conducted short-term and long-term stress experiments with mud crabs in a saline–alkali environment, with the relative mRNA expression and DNA methylation profile of Sp-CA under such stress having been clarified, bringing new insight into the understanding of mud crabs' adaptation and tolerance to a saline–alkali environment.