Elevation Of Ca2 Research Articles

Calcium regulates the interactions between dissolved organic matter and planktonic bacteria in Erhai Lake, Yunnan Province, China

In recent years, the global carbon cycle has garnered significant research attention. However, details of the intricate relationship between planktonic bacteria, hydrochemistry, and dissolved organic matter (DOM) in inland waters remain unclear, especially their effects on lake carbon sequestration. In this study, we analyzed 16S rRNA, chromophoric dissolved organic matter (CDOM), and inorganic nutrients in Erhai Lake, Yunnan Province, China. The results revealed that allochthonous DOM (C3) significantly regulated the microbial community, and that autochthonous DOM, generated via microbial mineralization (C2), was not preferred as a food source by lake bacteria, and neither was allochthonous DOM after microbial mineralization (C4). Specifically, the correlation between the fluorescence index and functional genes (FAPRPTAX) showed that the degree of utilization of DOM was a critical factor in regulating planktonic bacteria associated with the carbon cycle. Further examination of the correlation between environmental factors and planktonic bacteria revealed that Ca2+ had a regulatory influence on the community structure of planktonic bacteria, particularly those linked to the carbon cycle. Consequently, the utilization strategy of DOM by planktonic bacteria was also determined by elevated Ca2+ levels. This in turn influenced the development of specific recalcitrant autochthonous DOM within the high Ca2+ environment of Erhai Lake. These findings are significant for the exploration of the stability of DOM within karst aquatic ecosystems, offering a new perspective for the investigation of terrestrial carbon sinks.

Altered circadian rhythm, sleep, and rhodopsin 7–dependent shade preference during diapause in Drosophila melanogaster

To survive adverse environments, many animals enter a dormant state such as hibernation, dauer, or diapause. Various Drosophila species undergo adult reproductive diapause in response to cool temperatures and/or short day-length. While flies are less active during diapause, it is unclear how adverse environmental conditions affect circadian rhythms and sleep. Here we show that in diapause-inducing cool temperatures, Drosophila melanogaster exhibit altered circadian activity profiles, including severely reduced morning activity and an advanced evening activity peak. Consequently, the flies have a single activity peak at a time similar to when nondiapausing flies take a siesta. Temperatures ≤15 °C, rather than photoperiod, primarily drive this behavior. At cool temperatures, flies rapidly enter a deep-sleep state that lacks the sleep cycles of flies at higher temperatures and require high levels of stimulation for arousal. Furthermore, we show that at 25 °C, flies prefer to siesta in the shade, a preference that is virtually eliminated at 10 °C. Resting in the shade is driven by an aversion to blue light that is sensed by Rhodopsin 7 outside of the eyes. Flies at 10 °C show neuronal markers of elevated sleep pressure, including increased expression of Bruchpilot and elevated Ca2+ in the R5 ellipsoid body neurons. Therefore, sleep pressure might overcome blue light aversion. Thus, at the same temperatures that cause reproductive arrest, preserve germline stem cells, and extend lifespan, D. melanogaster are prone to deep sleep and exhibit dramatically altered, yet rhythmic, daily activity patterns.

Diagnostic Performance of F-18 FDG PET/CT in the Detection of Recurrent Colorectal Cancer: Correlation with Biochemical Markers and Conventional Imaging Modalities.

Background/Objectives: Although the role of PET/CT imaging is well established in oncology, its diagnostic value in routine monitoring for recurrent colorectal cancer (CRC) is still controversial. The aim was to evaluate the diagnostic value of F-18 FDG PET/CT in detecting recurrent CRC in correlation with CEA, CA 19-9 levels, and conventional imaging modalities (CIM). Methods: Between 2009 and 2023, a retrospective study was performed including 134 CRC patients referred for PET/CT imaging on the suspicion of recurrence, based on elevated CEA and/or CA 19-9 and/or equivocal CIM findings. According to our institution's Tumor Board CRC protocol, after the initial treatment, which was dependent on the TNM stage (neoadjuvant therapy, primary resection, or adjuvant treatment), patients underwent a standard 5-year surveillance including CEA and CA 19-9 measurements, CIM, and colonoscopy, every six months. The statistics, including univariate and multivariate analyses were conducted using the IBM SPSS 20.0 statistical software. p-values < 0.05 were considered statistically significant. Results: Recurrent CRC was confirmed in 54/134 (40.3%) patients with elevated tumor markers. PET/CT showed high diagnostic performance in detecting recurrent CRC with sensitivity, specificity, PPV, NPV, and accuracy of 94.4%, 82.5%, 78.5%, 95.7%, and 87.3%, respectively. The CEA showed a high sensitivity of 98.1% but both low specificity and accuracy of 15% and 48.5%, respectively. The sensitivity, specificity, and accuracy for CA 19-9 and CIM for diagnosis of CRC recurrence were 44.4%, 67.5%, 58.2%, and 51.9%, 98.8%, 79.9%, respectively. The AUC for PET/CT, elevated CEA levels, CIM, and elevated CA 19-9 levels was 0.885 (95% CI: 0.824-0.946; p < 0.001), 0.844 (95% CI: 0.772-0.916; p < 0.001), 0.753 (95% CI: 0.612-0.844; p < 0.001), and 0.547 (95% CI: 0.442-0.652; p = 0.358), respectively. Univariate analysis showed that both PET/CT and CIM positive results were highly associated with CRC recurrence (p < 0.001 and p < 0.001, respectively). At the same time, gender, mucinous tumor type, presence of initial lymph node metastasis (N+), and presence of initial distant metastasis (M+) had no significance (p = 0.211, p = 0.158, p = 0.583, and p = 0.201, respectively). Our multivariate analysis showed that independent predictors for CRC recurrence are positive PET/CT scans (p < 0.001), positive CIM results (p = 0.001), and elevated CA 19-9 levels (p = 0.023). Although CA 19-9 was not detected as a statistically significant predictor in the univariate analysis (p = 0.358), in a multivariate analysis it was recognized as a significant predicting factor in detecting the CRC recurrence (p = 0.023). Conclusions: F-18 FDG PET/CT showed high diagnostic efficacy in CRC recurrence detection, in correlation with CEA levels, CA 19-9 levels, and CIM. This imaging modality should be routinely integrated into the post-operative follow-op in patients with elevated tumor markers.

Peritoneal pseudomyxoma in a patient affected by appendicular mucocele: a case report

Pseudomyxoma peritonei (PMP) is an uncommon clinical condition, characterized by mucinous ascites, generally associated with a rupture of an epithelial neoplasm of the appendix. Some authors also use the term PMP to indicate mucinous dissemination after rupture of mucin-producing tumors of other sites (i.e. colon, ovary). Clinical presentation is variable and depends on the progressive accumulation of mucinous ascites (obstructive symptoms); weight loss, elevated Ca 19.9 and Ca 125 levels can be present. Here, we report a case of a 58-year-old patient with a history of appendicular mucocele and HCV-related hepatopathy, who accessed to the emergency department of our institute for the onset of epigastric pain associated with food vomiting for 5 days. Blood tests demonstrated a mild leukocytosis, a mild anemia (Hb 12.8 g/dL), and increased transaminases and cholestasis indices. A contrast-enhanced CT (CECT) scan was performed: a peritoneal cavity filled with a neoformation with lobulated margins and over-water density leading to multiple incisions of the hepatic and splenic parenchyma was documented. Appendix had an increased caliber (4 cm) with coprolite near its origin. Hence, the suspicion of PMP was raised and later confirmed on surgical exploration.

Differential impact of incrementally elevated CA 19-9 levels on prognosis of resected pancreatic ductal adenocarcinoma

BackgroundCA 19–9 is an extremely useful biomarker for pancreatic ductal adenocarcinomas (PDACs). However, the optimal cut-off and prognostic significance at higher cut-offs are yet to be determined. MethodsRetrospective analysis included patients with PDAC who underwent curative resection from January 2010 to May 2020 at Tata Memorial Centre, Mumbai. The pretherapy CA 19–9 was dichotomized using various cut-off levels and analysed. ResultsIn 244 included patients, the median overall survival (OS) for those with CA19-9 level (IU/ml) < or >78, 200, 500, 1000, and 2000 was 27, 24, 23, 22, 21 months versus 18, 16, 15, 14, 13 months; respectively, and was statistically significant (p-value- 0.002, 0.001, 0.002, 0.002 and 0.004, respectively). The number of recurrences and mortality had significant correlation with CA 19–9 cut-offs. On multivariate analysis, adjuvant treatment completion (p-0.004) and decreasing or stable CA19-9 after Neoadjuvant therapy (NAT) (p- 0.031) were associated with improved OS. ConclusionThe prognostic significance of CA 19–9 was observed at all the cut-off levels examined, beyond mere elevated value as per the standard cut-off level. In patients with high CA19-9 level, surgery should be offered if technically and conditionally feasible, only when a response in CA19-9 level to NAT is achieved.

Disruption of P2Y2 signaling promotes breast tumor cell dissemination by reducing ATP-dependent calcium elevation and actin localization to cell junctions.

The tumor microenvironment and wound healing after injury both contain extremely high concentrations of the extracellular signaling molecule, adenosine triphosphate (ATP) compared to normal tissue. P2Y2 receptor, an ATP-activated purinergic receptor, is typically associated with pulmonary, endothelial, and neurological cell signaling. Here we report its role and importance in breast epithelial cell signaling and how it is altered in metastatic breast cancer. In response to ATP activation, P2Y2 receptor signaling causes an increase of intracellular Ca 2+ in non-tumorigenic breast epithelial cells, while their tumorigenic and metastatic counterparts have significantly reduced Ca 2+ responses. The non-tumorigenic cells respond to increased Ca 2+ with actin polymerization and localization to cell edges, while the metastatic cells remained unaffected. The increase in intracellular Ca 2+ after ATP stimulation was blunted using a P2Y2 antagonist, which also prevented actin mobilization and caused cell dissemination from spheroids in non-tumorigenic breast epithelial cells. Furthermore, the lack of Ca 2+ concentration changes and actin mobilization in the metastatic breast cancer cells could be due to reduced P2Y2 expression, which correlates with poorer overall survival in breast cancer patients. This study elucidates rapid changes that occur after elevated intracellular Ca 2+ in breast epithelial cells and how metastatic cancer cells have adapted to evade this cellular response.

Resectability rate of pancreatic cancer after neoadjuvant therapy.

e16348 Background: Pancreatic Cancer is one of the leading causes of cancer death in Saudi Arabia. Surgery is the mainstay of treatment in resectable non-metastatic cases. Working on improving the resectability rate to achieve R0 resection is the standard of care. Methods: Unresectable cases of pancreatic adenocarcinoma seen from 2004 till 2018 in three hospitals in Saudi Arabia were reviewed to know the conversion rate from unresectable or borderline resectable to resectable after neoadjuvant chemotherapy and to explore the possible prognostic factors. The characteristics and outcomes of these patients were reviewed, with more attention on the known prognostic factors and their impact on the outcome. Notably, performance status, CA19-9, Neutrophil Lymphocyte Ration (NLR), and Platelet Lymphocyte Ratio (PLR). Overall response to neoadjuvant therapy, R0 resection, relapse free survival and overall survival were all reported. Results: Two hundred seventy-three captured cases of all types of pancreatic tumors from the tumor registry in the years between 2004 and 2018. 39 cases were unresectable or borderline resectable cases. The median age was 60 years (range:29-79), and 25 patients were males. 30 patients had an ECOG PS of 0-1and 6 had an ECOG PS of 2. The head of the pancreas was the primary tumor site in 31 patients and the body or tail was in 8 patients. 21 patients (54%) presented with weight loss ( &gt; 5kg), and 21 patients (54%) presented with obstructive jaundice. Neoadjuvant chemotherapy protocols were FOLFORINOX (13 patients, 33%), Gemcitabine/Capecitabine (10 patients), single agent Gemcitabine (10 patients), and other regimens in 6 patients. The median number of chemotherapy cycles was 6 (range: 1-14). 17 patients had dose reduction. 6 patients had preoperative radiation. Response to chemotherapy: ORR: 36%, SD: 26% and PD: 38% .Surgery was done after neoadjuvant therapy in 12 patients (31%). Only 4 out 12 had confirmed R0. Median Overall Survival (MS) and Progression-Free survival (PFS) in all patients were 30 mo (95% CI: 17.75 - 42.56) and 8 mo (95% CI: 5.65 - 11.89) respectively. MS in the surgery and the non-surgery groups was 30 and 25 mo (p- value = 0.26) respectively. The median PFS in the surgery and the non-surgery groups was 19.6 and 5.8 mo (p- value = 0.008) respectively. Multivariate regression analysis done for the following variables: ECOG PS of 2, elevated CA 199, high NLR &gt; 3, high PLR &gt; 225, weight loss and results showed numerical difference of both MS and PFS with no statistical significance (Type 2 error). Conclusions: The conversion rate from unresectable or borderline resectable to resectable after neoadjuvant therapy is 31% which is consistent with the international data. There is PFS improvement in those who underwent surgery (19.6 mo vs. 5.8 mo) with statistical significance (p:0.008), and there is numerical improvement of MS (30 mo vs. 25 mo) that did not reach statistical significance.

TRPA1 Covalent Ligand JT010 Modifies T Lymphocyte Activation.

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel involved in sensitivity to a plethora of irritating agents and endogenous mediators of oxidative stress. TRPA1 influences neuroinflammation and macrophage and lymphocyte functions, but its role is controversial in immune cells. We reported earlier a detectable, but orders-of-magnitude-lower level of Trpa1 mRNA in monocytes and lymphocytes than in sensory neurons by qRT-PCR analyses of cells from lymphoid organs of mice. Our present goals were to (a) further elucidate the expression of Trpa1 mRNA in immune cells by RNAscope in situ hybridization (ISH) and (b) test the role of TRPA1 in lymphocyte activation. RNAscope ISH confirmed that Trpa1 transcripts were detectable in CD14+ and CD4+ cells from the peritoneal cavity of mice. A selective TRPA1 agonist JT010 elevated Ca2+ levels in these cells only at high concentrations. However, a concentration-dependent inhibitory effect of JT010 was observed on T-cell receptor (TcR)-induced Ca2+ signals in CD4+ T lymphocytes, while JT010 neither modified B cell activation nor ionomycin-stimulated Ca2+ level. Based on our present and past findings, TRPA1 activation negatively modulates T lymphocyte activation, but it does not appear to be a key regulator of TcR-stimulated calcium signaling.

Contrasting water-use strategies to climate warming in white birch and larch in a boreal permafrost region.

The effects of rising atmospheric CO2 concentrations (Ca) with climate warming on intrinsic water-use efficiency and radial growth in boreal forests are still poorly understood. We measured tree-ring cellulose δ13C, δ18O, and tree-ring width in Larix dahurica (larch) and Betula platyphylla (white birch), and analyzed their relationships with climate variables in a boreal permafrost region of northeast China over past 68years covering a pre-warming period (1951-1984; base period) and a warm period (1985-2018; warm period). We found that white birch but not larch significantly increased their radial growth over the warm period. The increased intrinsic water-use efficiency in both species was mainly driven by elevated Ca but not climate warming. White birch but not larch showed significantly positive correlations between tree-ring δ13C, δ18O and summer maximum temperature as well as vapor pressure deficit in the warm period, suggesting a strong stomatal response in the broad-leaved birch to temperature changes. The climate warming-induced radial growth enhancement in white birch is primarily associated with a conservative water-use strategy. In contrast, larch exhibits a profligate water-use strategy. It implies an advantage for white birch over larch in the warming permafrost regions.

Using portable X-ray fluorescence elemental analysis to explore porous skeletal lesions: Interplay of sex, age at death, and cause of death.

Search for possible associations between bone elemental concentration and the presence of porous skeletal lesions (PSLs), considering the sex, age, and cause of death (COD) of the individuals. The sample comprised 107 non-adult individuals (56 females, 51 males) aged 0-20 (x̄ = 13.2, SD = 5.8) from the Coimbra and Lisbon Identified Skeletal Collections. Cribra cranii, orbitalia, humeralis, and femoralis were recorded as present/absent, and elemental concentrations were assessed by portable x-ray fluorescence (pXRF). A multivariate statistical approach was applied. Well-preserved skeletons with minimal diagenesis showed no sex-related elemental variations or PSL associations. In contrast, age-at-death correlated with elevated Ca, P, Sr, and Pb levels. Cribra cranii increased with age while other cribra declined post-adolescence. Higher concentrations of Fe and lower of S were linked to cribra cranii. Respiratory infections as COD increased the odds of expressing cribra femoralis (OR = 5.25, CI = 1.25-15.14), cribra cranii (OR = 2.91, CI = 0.97-8.69), and cribra orbitalia (OR = 2.76, CI = 1.06-7.24). Feasible pXRF results and low cribra intraobserver error assure replicability. Elevated Ca, P, and Sr in older individuals may relate to skeletal growth, while increased Pb suggests bioaccumulation. Cribra's increase with age reflects different rates of marrow conversion and bone remodeling. Higher Fe and lower S in individuals with cribra cranii possibly reflects poor nutrition, early alcohol use, and sideroblastic anemia, aligning with 19th-20th-century Portugal's living conditions. Respiratory infections increased cribra expression, revealing intricate interplays among inflammation, anemia(s), marrow expansion, and diet. This research highlights a complex scenario and blazes a new path for cribra interpretation.

Non-neuronal cell-derived acetylcholine, a key modulator of the vascular endothelial function in health and disease.

Vascular endothelial cells play an important role in regulating peripheral circulation by modulating arterial tone in the microvasculature. Elevated intracellular Ca2+ levels are required in endothelial cells to induce smooth muscle relaxation via endothelium-dependent mechanisms such as nitric oxide production, prostacyclin, and endothelial cell hyperpolarization. It is well established that exogenous administration of acetylcholine can increase intracellular Ca2+ concentrations, followed by endothelium-dependent vasodilation. Although endogenous acetylcholine's regulation of vascular tone remains debatable, recent studies have reported that endogenously derived acetylcholine, but not neuronal cell-derived acetylcholine, is a key modulator of endothelial cell function. In this minireview, we summarize the current knowledge of the non-neuronal cholinergic system (NNCS) in vascular function, particularly vascular endothelial cell function, which contributes to blood pressure regulation. We also discuss the possible pathophysiological impact of endothelial NNCS, which may induce the development of vascular diseases due to endothelial dysfunction, and the potential of endothelial NNCS as a novel therapeutic target for endothelial dysfunction in the early stages of metabolic syndrome, diabetes, and hypertension.

Evaluation of enrofloxacin in the Chinese soft-shelled turtle (Pelodiscus sinensis) based on the biochemical, histopathological and intestinal microbiota responses

Enrofloxacin (ENR) is a veterinary medicine extensively used in aquatic farming, the abuses of antibacterials can lead to drug residues and toxicity to aquatic animals. Nevertheless, its toxicological effects on the Pelodiscus sinensis remain unclear. The objective of this work was to study the toxic effects of ENR on male P. sinensis following multiple oral administration. For hematological, antioxidant status, histological and ultrastructural analyzed, male P. sinensis were orally administrated with ENR for 5 consecutive days, each at the dose of 15 (recommended dose, 1×), 45 (3 times the recommended dose, 3×), and 75 (5 times the recommended dose, 5×) mg/kg body weight (BW) daily. The effect of ENR on P. sinensis by changes in the intestinal microbiota using high-throughput sequencing technology was also investigated. Samples of blood and fresh tissue (liver, spleen, intestine, muscle, spermary, testes, heart, lung, and kidney) were collected after 24 h of drug withdrawal. The results showed that multiple oral administration of ENR induced immunosuppression (lowered total proteins, alkaline phosphatase, and albumin), hepatic-renal dysfunction (elevated aspartate transaminase, alanine aminotransferase, urea nitrogen, K+ and Ca2+), and lowering the hepatic and splenic antioxidant functions (higher MDA, and declines in the activities of SOD, CAT, and NOS) in 3× ENR-dosed or 5× ENR-dosed groups. Noteworthy, histopathological and ultrastructural examination of tissue samples revealed adverse effects with increasing oral dosage of ENR. The 16S sequencing analysis showed that the ENR treatment can greatly change the intestinal microbial diversity and abundance in P. sinensis, but more probiotics such as Cetobacterium, Lactococcus, and Paraclostridium were retained in the short-term period. The findings may provide new insights into the toxicity of ENR to P. sinensis, and help to determine the dose to ensure the safety of aquatic food products.

Algal Oil Mitigates Sodium Taurocholate-Induced Pancreatitis by Alleviating Calcium Overload, Oxidative Stress, and NF-κB Activation in Pancreatic Acinar Cells.

Acute pancreatitis (AP) is characterized by elevated intracellular Ca2+ concentrations, mitochondrial dysfunction, and oxidative stress in pancreatic acinar cells. Algal oil (AO) has demonstrated antioxidant and anti-inflammatory properties. This study aims to explore the effects of algal oil on the microenvironment of AP. Rat pancreatic acinar AR42J cells were pretreated with AO containing 0, 50, 100, or 150 μM of docosahexaenoic acid (DHA) 2 h prior to AP induction using sodium taurocholate (STC). After 1 h of STC treatment, AR42J cells exhibited a significant increase in intracellular Ca2+ concentration and the production of amylase, lipase, reactive oxygen species, and pro-inflammatory mediators, including tumor necrosis factor-α and interleukin-6. These STC-induced increases were markedly reduced in cells pretreated with AO. In comparison to cells without AO, those treated with a high dose of AO before STC exposure demonstrated a significant increase in mitochondrial membrane potential and a decrease in lipid peroxidation. Furthermore, STC-activated nuclear factor kappa-B (NF-κB) was attenuated in AO-pretreated cells, as evidenced by a significant decrease in activated NF-κB. In conclusion, AO may prevent damage to pancreatic acinar cells by alleviating intracellular Ca2+ overload, mitigating mitochondrial dysfunction, reducing oxidative stress, and attenuating NF-κB-targeted inflammation.

Rotavirus non-structural protein 4 usurps host cellular RIPK1-RIPK3 complex to induce MLKL-dependent necroptotic cell death

The dynamic interface between invading viral pathogens and programmed cell death (PCD) of the host is a finely regulated process. Host cellular demise at the end of the viral life cycle ensures the release of progeny virions to initiate new infection cycles. Rotavirus (RV), a diarrheagenic virus with double-stranded RNA genome, has been reported to trigger different types of PCD such as apoptosis and pyroptosis in a highly regulated way to successfully disseminate progeny virions. Recently our lab also showed that induction of MLKL-driven programmed necroptosis by RV. However, the host cellular machinery involved in RV-induced necroptosis and the upstream viral trigger responsible for it remained unaddressed. In the present study, the signalling upstream of MLKL-driven necroptosis has been delineated where the involvement of Receptor interacting serine/threonine kinase 3 (RIPK3) and 1 (RIPK1) from the host side and RV non-structural protein 4 (NSP4) as the viral trigger for necroptosis has been shown. Interestingly, RV-NSP4 was found to be an integral component of the necrosome complex by interacting with RIPK1, thereby bypassing the requirement of RIPK1 kinase activity. Subsequently, NSP4-driven elevated cytosolic Ca2+ concentration and Ca2+-binding to NSP4 lead further to RHIM domain-dependent RIPK1-RIPK3 interaction, RIPK3-dependent MLKL phosphorylation, and eventual necroptosis. Overall, this study presents the interplay between RV-NSP4 and the host cellular necrosome complex to induce necroptotic death of host cells.

Abstract PO4-03-03: Conditional Use of Serial Serum Tumor Markers CA 15.3 and CEA as Predictors of Disease Relapse in Patients with High-Risk Early-Stage Breast Cancer

Abstract Background: Because of the diagnosis at an earlier stage, and the wider availability of more effective therapies, treatment outcomes of patients with breast cancer are getting much better resulting in an increasing proportion of patients surviving their disease, however, a significant percentage of patients with early-stage disease at initial diagnosis may eventually relapse. Residual tumor cells can remain dormant for many years before causing tumor recurrence. Physical examination and mammography are strongly recommended in surveillance guidelines, but data on routine blood tests or imaging is lacking. The revolution made in recent years, following the approval of novel targeted and immunotherapy agents, may have better outcome if started earlier in the disease course with the lowest tumor burden and in patients with good performance status and adequate organs’ function. Additionally, early detection of oligometastatic disease can give a chance for cure for a subset of these patients. Levels of CA15.3 and CEA are used in decision making in the metastatic setting but their role in surveillance is still controversial. We aim to investigate whether serial measurement of tumor markers, when it correlates with tumor bulk (conditional), can detect early asymptomatic recurrence among subset of patients with high-risk early-stage breast cancer. Methods: Patients with high-risk early-stage breast cancer were invited to participate. High-risk features include any of the following: large tumor size (T3/4), grade-3, node-positive, triple-negative, HER2-positive or hormone receptor (HR)-negative disease. Patients were considered eligible if they have elevated CA15.3 and/or CEA at baseline, that subsequently normalize 6 weeks after surgical resection; upfront or following neoadjuvant chemotherapy. Serial testing of the elevated CA15.3 and/or CEA is done every 2 months thereafter, samples are cryopreserved and will not be processed until disease relapse. As such results will not be used for clinical decisions. The study is still ongoing, we here present an interim analysis evaluating the correlation between certain tumor characteristics and elevated serum marker(s) levels. Results: Since the launch of the study, 367 patients deemed high-risk by the above criteria, accepted to participate. Abnormal baseline CA15.3 and/or CEA was found in 110 (30%); 65 (18%) patients had elevated CA 15.3 while elevated CEA was found in 45 (12%) patients. Abnormal CA 15.3 was found in 27% of cases with T3/4 disease compared with 13% in T1/2 disease (p=0.001), and in 21% of node positive disease versus 7% in node negative disease (p=0.002), in 9% of HR negative disease versus 20% in HR-positive (P=0.038). Tumor grade and HER2 status were not associated with significant difference. Regarding CEA, abnormal levels were detected in 24% of HER2-positive disease compared to 5% of HER2-negative disease (P&amp;lt; 0.0001). Other tumor features were not associated with significant difference. Conclusions: Only a third of patients with high-risk early-stage breast cancer had high level of serum tumor markers at initial diagnosis and thus such markers may correlate with tumor presence. Large tumors (T3/4) and node positive disease were associated with high CA15.3 but not CEA. HER2-stutus and tumor grade had no effect on CA15.3 level, while HER2 positive disease correlated well with high CEA. Citation Format: Sarah Edaily, Baha' Sharaf, Maher Sughayer, Lina Yousef, Hala Abu-Fares, Sarah Abdel-Razeq, Hala Abu-Jaish, Mahmoud Abunasser, Suhaib Khater, Anas Zayed, Osama El Khatib, Rahaf Rahal, Hazem Abdulelah, Maria Shraim, Hikmat Abdel-Razeq. Conditional Use of Serial Serum Tumor Markers CA 15.3 and CEA as Predictors of Disease Relapse in Patients with High-Risk Early-Stage Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-03.

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

CalDAG-GEFIII controls Ca2+ entry via Rap1 and H-Ras, triggering distinct signaling pathways

Objective: Small GTPase Rap1 plays key functions in endothelium, including controlling Ca2+-dependent endothelial function (NO release). Our recent study suggests the Rap1A isoform restricts Ca2+ entry, to control eNOS activity. The activity of Rap1, a GDP to GTP switch, is controlled by Guanine Nucleotide Exchange Factors (GEFs). However, how Rap1 is activated in the context of Ca2+ signaling and regulation of eNOS activity is unknown. A Ca2+ and diacylglycerol (DAG)-activated CalDAG-GEFIII can activate Ha-Ras, R-Ras, and Rap1, but its functions in endothelial cells (ECs), and the signaling specificity of CalDAG-GEFIII through Rap1 and Ras pathways are not well understood. Our study aims to elucidate the role of CalDAG-GEFIII in regulating Ca2+ signaling in ECs. Approach and Results: To determine relative expression of Rap1 GEFs in ECs in different tissues, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data from eleven publicly available scRNA-seq datasets. We found RasGRP3 (encoding CalDAG-GEFIII) was highly expressed compared to other Rap1-GEFs in endothelial cells from different organs. To examine the involvement of CalDAG-GEFIII Rap1 and Ras activation in heart ECs, we determined the effect of on Vascular Endothelial Growth Factor (VEGF)-induced Rap1-GTP and Ras-GTP loading using pull-down assay. Knockdown of RasGRP3 blocked Rap1 and Ras activation downstream from VEGFR2, suggesting CalDAG-GEFIII acts as a GEF (activator) of both Rap1 and Ras in endothelial cells. To examine the involvement of CalDAG-GEFIII and H-Ras in Ca2+ signaling in ECs, we measured VEGF-induced intracellular Ca2+ using Fura2 dye, an intracellular Ca2+ indicator, in siRasGRP3, siH-Ras (most highly expressed Ras isoform) and siControl HUVECs. We found that Ca2+ release unchanged, but Ca2+ entry was increased in siRasGRP3 and siRas ECs, similarly to what we had found in siRap1A ECs. This suggests that Ras, like Rap1, limits ATP and TG-induced Ca2+ entry in HUVECs and that CalDAG-GEFIII may act upstream from both Rap1 and Ras. The elevated Ca2+ entry in siRap1A, siRasGRP3 ECs was partially normalized by exogenous expression of constitutively activated Rap1A or activation of Rap1 A by cAMP-dependent GEF, Epac. This indicates that Rap1A controls Ca2+ entry both acutely via a signaling mechanism, and at the transcript level. To determine the involvement of CalDAG-GEFIII in signaling by Rap1 and Ras, we determined the impact of siRNA knockdown of siRap1, siRas and siRasGRP3 on Ser1177 eNOS phosphorylation and Thr202/Tyr204 ERK phosphorylation in response to VEGF stimulation. Knockdown of RasGRP3 or Rap1 attenuated VEGF-induced Ser-1177 eNOS phosphorylation, while knockdown of RasGRP3 or Ras, but not Rap1, inhibited ERK phosphorylation in response to VEGF. Conclusions: Rap1 and Ras, activated downstream from CalDAG-GEFIII in response to VEGF activation, limit agonist-induced Ca2+ entry in endothelial cells. CalDAG-GEFIII promotes eNOS phosphorylation through Rap1B and promotes ERK phosphorylation via Ras, independent of Rap1. This suggests that CalDAG-GEFIII acts in two separate pathways, that are Rap1 and Ras dependent. NIH NHLBI: R01HL111582. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ASSESSMENT OF ANGIOTENSIN II-INDUCED CALCIUM SIGNALS IN PRIMARY HUMAN ADRENOCORTICAL CELLS

Objective: Primary aldosteronism (PA), a common form of secondary hypertension, is characterized by an increase of cytosolic intracellular Ca2+ concentrations [Ca2+]i. Our aim is to investigate transients and oscillations of [Ca2+]i in a human model of isolated CD56+ aldosterone-producing adenoma (APA) cells and normal surrounding adrenocortical (NAC) cells. Design and method: We monitored [Ca2+]i, in CD56+ NAC and APA cells in basal condition and after Angiotensin II (Ang II) stimulation using Fura-2 dye and live confocal microscopy, and we developed a custom-made pipeline to analyze calcium signals in terms of frequency and peak parameters like amplitude, full width at half maximum (FWHM) and area under the curve (AUC). To investigate the potential role of the two-pore domain K+ TASK- 2 channel in regulating Ca2+ signals, TASK-2 channels of NAC cells were pharmacologically inhibited using Quinidine (20 microM) 20’ prior to [Ca2+]i imaging acquisition. Results: Resting [Ca2+]i basal levels in NAC (n=11) were higher (0.76 a.u.+- 0.04) than in APA (n=12) (0.56 a.u.+- 0.05, p= 0.0087). NAC TASK-2 inhibition decreased resting [Ca2+]i to 0.58 a.u. +- 0.07 (p &gt; 0.05). Spontaneous Ca2+ activity was higher in NAC with significant higher amplitude and AUC parameters compared to APA cells. Quinidine further increased spontaneous activity in NAC while decreased Amplitude and AUC. For both cell types stimulation with 2 doses of Ang II concentrations [10 -8 and 10 -9 mM], higher Ang II doses promoted higher Ca2+ peak parameters, amplitude and AUC, and a significant decrease in events frequency. Comparison between APA and NAC revealed that, regardless of Ang II dose, first Ca2+ transient parameters are intensified in NAC. However, APA showed consistent elevated subsequent Ca2+ oscillations parameters (Amplitude and AUC), frequency, and degree of oscillation compared to NAC cells stimulated with similar Ang II doses. NAC TASK-2 inhibited cells, when stimulated with [10 -9 mM] Ang II, displayed only increased subsequent peak parameters. Conclusions: The bias-free approach designed to quantitatively assess [Ca2+]i signals, revealed significant differences in [Ca2+]i dynamics of human NAC and APA cells.

The MAP kinase FvHog1 regulates FB1 synthesis and Ca2+ homeostasis in Fusarium verticillioides

The high osmolarity glycerol 1 mitogen-activated protein kinase (Hog1-MAPK) cascade genes are important for diverse biological processes. The activated Hog1 upon multiple environmental stress stimuli enters into the nucleus where it directly phosphorylates transcription factors to regulate various physiological processes in phytopathogenic fungi. However, their roles have not been well-characterized in Fusarium verticillioides. In this study, FvHog1 is identified and functionally analyzed. The findings reveal that the phosphorylation level and nuclear localization of FvHog1 are increased in Fumonisin B1 (FB1)-inducing condition to regulate the expression of FB1 biosynthesis FUM genes. More importantly, the deletion mutants of Hog1-MAPK pathway show increased sensitivity to Ca2+ stress and elevated intracellular Ca2+ content. The phosphorylation level and nuclear localization of FvHog1 are increased with Ca2+ treatment. Furthermore, our results show that FvHog1 can directly phosphorylate Ca2+-responsive zinc finger transcription factor 1 (FvCrz1) to regulate Ca2+ homeostasis. In conclusion, our findings indicate that FvHog1 is required for FB1 biosynthesis, pathogenicity and Ca2+ homeostasis in F. verticillioides. It provides a theoretical basis for effective prevention and control maize ear and stalk rot disease.

Mitochondrial Ca2+-coupled generation of reactive oxygen species causes endothelial dysfunction in Cantú syndrome

Cantú syndrome is a multisystem disorder caused by gain-of-function (GOF) mutations in KCNJ8 and ABCC9, the genes encoding the pore-forming inward rectifier Kir6.1 and regulatory sulfonylurea receptor SUR2B subunits of vascular ATP-sensitive K+ channels (KATP). In this study, we investigated the effects of Cantú syndrome on the vascular endothelium using mutant mice that model the disease. We found that endothelium-dependent dilation was impaired in small mesenteric arteries from Cantú mice. Loss of endothelium-dependent vasodilation led to increased vasoconstriction in response to intraluminal pressure and to the adrenergic receptor agonist phenylephrine. We also found that KATP GOF and acute activation of KATP channels with pinacidil increased the amplitude and frequency of wave-like Ca2+ events that were generated in the endothelium in response to the vasodilator agonist carbachol (CCh). Elevated cytosolic Ca2+ signaling activity in arterial endothelial cells from Cantú mice was associated with high mitochondrial [Ca2+], reactive oxygen species (ROS) generation, and peroxynitrite levels. Scavenging intracellular and mitochondrial ROS restored endothelium-dependent vasodilation in the arteries of mice with KATP GOF mutations. We conclude that mitochondrial Ca2+ overload and ROS generation cause endothelial dysfunction in mice with Cantú syndrome. This study was supported by grants from NHLBI (R35HL155008) and NIGMS (P20GM130459) to S.E., and NIH (R35HL140024) to C.G.N., and NIH (R00HL150277) to C.M. The Transgenic Genotyping and Phenotyping Core and the High Spatial and Temporal Resolution Imaging Core at the COBRE Center for Molecular and Cellular Signaling in the Cardiovascular System, University of Nevada, Reno is maintained by grants from NIH/NIGMS (P20GM130459). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.