Stable Marker Research Articles

TMIC-80. HIGH-DIMENSIONAL HISTOPATHOLOGIC EVALUATION OF THE HYPOXIC TUMOR MICROENVIRONMENT IN GLIOBLASTOMA

Abstract Gliobastoma (GBM) is a highly aggressive solid tumor of the brain, characterized by a hypoxic tumor microevironment (TME) leading to histopathological features of necrosis, microvascular proliferation and deregulated hypervascularization. The heterogeneous nature of GBM results in a gradient of intra-tumoral hypoxia that causes molecular changes to specific cell populations within the bulk of the tumor. Our study is the first clinical study to utilize the exogenous oxygen-independent marker pimonidazole (PIMO) to identify the hypoxic TME using high-dimensional spatial and single-cell proteomics of specific cell populations within GBM. A cohort of 35 patients with primary GBM, recurrent GBM or IDH-mutant glioma were administered PIMO prior to surgical resection of tumor tissue for downstream bulk proteomic analysis of bulk tissue by serial immunohistochemistry and imaging mass cytometry (IMC) and single-cell analysis of dissociated tissue by Cytometry by time-of-flight (CyTOF). We utilized high-resolution imaging analyses to validate PIMO as a sensitive and stable marker for hypoxia against a panel of transiently expressed HIF-target genes and examine the inter- and intra-tumoral heterogeneity of hypoxia within each tumor subtype. A custom CyTOF marker panel was developed for further single-cell analyses of T-cell subsets and their functional states, myeloid cell subpopulations, natural killer cells, glial cells, and tumor cells in the hypoxic TME. We demonstrate the utility of PIMO to effectively study the hypoxic TME through spatial and single cell methods and further elucidate mechanisms of hypoxia-driven treatment resistance.

The role of DNA methylation in chondrogenesis of human iPSCs as a stable marker of cartilage quality

BackgroundLack of insight into factors that determine purity and quality of human iPSC (hiPSC)-derived neo-cartilage precludes applications of this powerful technology toward regenerative solutions in the clinical setting. Here, we set out to generate methylome-wide landscapes of hiPSC-derived neo-cartilages from different tissues-of-origin and integrated transcriptome-wide data to identify dissimilarities in set points of methylation with associated transcription and the respective pathways in which these genes act.MethodsWe applied in vitro chondrogenesis using hiPSCs generated from two different tissue sources: skin fibroblasts and articular cartilage. Upon differentiation toward chondrocytes, these are referred to as hFiCs and hCiC, respectively. Genome-wide DNA methylation and RNA sequencing datasets were generated of the hiPSC-derived neo-cartilages, and the epigenetically regulated transcriptome was compared to that of neo-cartilage deposited by human primary articular cartilage (hPAC).ResultsMethylome-wide landscapes of neo-cartilages of hiPSCs reprogrammed from two different somatic tissues were 85% similar to that of hPACs. By integration of transcriptome-wide data, differences in transcriptionally active CpGs between hCiC relative to hPAC were prioritized. Among the CpG-gene pairs lower expressed in hCiCs relative to hPACs, we identified genes such as MGP, GDF5, and CHAD enriched in closely related pathways and involved in cartilage development that likely mark phenotypic differences in chondrocyte states. Vice versa, among the CpG-gene pairs higher expressed, we identified genes such as KIF1A or NKX2-2 enriched in neurogenic pathways and likely reflecting off target differentiation.ConclusionsWe did not find significant variation between the neo-cartilages derived from hiPSCs of different tissue sources, suggesting that application of a robust differentiation protocol such as we applied here is more important as compared to the epigenetic memory of the cells of origin. Results of our study could be further exploited to improve quality, purity, and maturity of hiPSC-derived neo-cartilage matrix, ultimately to realize introduction of sustainable, hiPSC-derived neo-cartilage implantation into clinical practice.

Characterization of the Biological Variability of the Angiome Biomarkers over Time in Healthy Subjects.

Biomarker analyses are an integral part of cancer research. Despite the intense efforts to identify and characterize biomarkers in cancer patients, little is known regarding the natural variation of biomarkers in healthy populations. Here we conducted a clinical study to evaluate the natural variability of biomarkers over time in healthy participants. The angiome multiplex array, a panel of 25 circulating protein biomarkers, was assessed in 28 healthy participants across 8 timepoints over the span of 60 days. We utilized the intraclass correlation coefficient (ICC) to quantify the reliability of the biomarkers. Adjusted ICC values were calculated under the framework of a linear mixed-effects model, taking into consideration age, sex, body mass index (BMI), fasting status, and sampling factors. ICC was calculated to determine the reliability of each biomarker. HGF was the most stable marker (ICC=0.973), while PDGF-BB was the most variable marker (ICC=0.167). In total, ICC analyses revealed that 22 out of 25 measured biomarkers display good (≥0.4) to excellent (>0.75) ICC values. Three markers (PDGF-BB, TGF-1, PDGF-AA) had ICC values <0.4. Greater age was associated with higher IL-6 (p=0.0114). Higher BMI was associated with higher levels of IL-6 (p=0.0003) and VEGF-R3 (p=0.0045). Of the 25 protein biomarkers measured over this short time period, 22 markers were found to have good or excellent ICC values, providing additional validation for this biomarker assay. This data further supports the validation of the angiome biomarker assay and its application as an integrated biomarker in clinical trial testing.

Changes in P300 amplitude to negative emotional stimuli correlate with treatment responsiveness to sertraline in adolescents with depression

ObjectiveAdolescents with depression is characterized by high rates of recurrence and functional impairment, with a significant association with suicide risk. Antidepressants are commonly prescribed to treat depression, yet few reproducible neurobiological markers for depression and antidepressant treatment response have been identified. Therefore, discovering a stable and reliable neurobiological marker holds significant value for both the clinical diagnosis and treatment of depression in adolescents. MethodsOne hundred and seven patients with major depressive disorder (MDD group, 30 males, 77 females, mean age: 14.80 years), and 25 healthy subjects (HC group, 13 males, 12 females, mean age: 15.72 years) were recruited to perform a two-choice oddball task related to negative emotional cues. All participants completed a self-administered questionnaire to gather demographic information. A trained psychiatrist administered the Hamilton Depression Scale (HAMD-17) to assess depression severity. Of the 107 adolescents with depression, 61 received antidepressant medication for 8 weeks, and 61 of these patients were followed up. Multichannel EEG was recorded continuously from 64 scalp electrodes using the Curry 8 system. EEG signal preprocessing and analysis was performed offline using the EEGLAB toolbox in MATLAB. The ERP component characteristics associated with emotional processing were extracted from the difference waves and statistically analyzed. ResultsAdolescents with depression exhibited significantly larger P300 amplitudes than healthy controls in response to both neutral and negative emotional cues. Following sertraline treatment, both depression scores and P300 amplitudes decreased significantly in adolescents with depression. Moreover, a strong positive correlation was observed between changes in depression scores and changes in P300 amplitude in response to negative emotional cues before and after treatment. ConclusionsChanges in neural reactivity to negative emotional stimuli among adolescents with depression can be selectively modulated by sertraline and are significantly associated with improvements in depressive symptoms. SignificanceChanges in P300 amplitude to negative emotional stimuli significantly correlate with treatment responsiveness to sertraline in adolescents with depression.

Neuroprotective Actions of Hydrogen Sulfide-Releasing Compounds in Isolated Bovine Retinae

Background: We have evidence that hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H2S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H2S-releasing compounds against hydrogen peroxide (H2O2)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H2S biosynthesis called L-cysteine, with the fast H2S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H2S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H2O2 (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H2S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H2S-releasing compounds. Results: Exposure of the isolated retinas to H2O2 produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p &lt; 0.001; n = 12) prevented H2O2-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H2O2 damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H2O2 alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H2O2-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H2S-releasing compounds and the substrate, L-cysteine, can prevent H2O2-induced lipid peroxidation in an isolated bovine retina.

CardioGuard: AI-driven ECG authentication hybrid neural network for predictive health monitoring in telehealth systems

The increasing integration of telehealth systems underscores the importance of robust and secure methods for patient data management. Traditional authentication methods, such as passwords and PINs, are prone to breaches, underscoring the need for more secure alternatives. Therefore, there is a need for alternative approaches that provide enhanced security and user convenience. Biometric-based authentication systems uses individuals unique physical or behavioral characteristics for identification, have emerged as a promising solution. Specifically, Electrocardiogram (ECG) signals have gained attention among various biometric modalities due to their uniqueness, stability, and non-invasiveness. This paper presents CardioGaurd, a deep learning-based authentication system that leverages ECG signals—unique, stable, and non-invasive biometric markers. The proposed system uses a hybrid Convolution and Long short-term memory based model to obtain rich characteristics from the ECG signal and classify it as authentic or fake. CardioGaurd not only ensures secure access but also serves as a predictive tool by analyzing ECG patterns that could indicate early signs of cardiovascular abnormalities. This dual functionality enhances patient security and contributes to AI-driven disease prevention and early detection. Our results demonstrate that CardioGaurd offers superior performance in both security and potential predictive health insights compared to traditional models, thus supporting a shift towards more proactive and personalized telehealth solutions.

Clinical experience on switching trientine tetrahydrochloride to trientine dihydrochloride in Wilson disease patients.

This study evaluates the effectiveness and safety of trientine dihydrochloride (TETA 2-HCl) in patients with Wilson disease (WD) following a switch from trientine tetrahydrochloride (TETA 4-HCl). A total of 30 WD patients with stable copper metabolism were identified for treatment with TETA 2-HCl (Cufence™) after prior use of TETA 4-HCl (Cuprior™). Biochemical markers including urinary copper, non-ceruloplasmin bound copper (NCC) and liver function were analyzed at baseline and followed up over 12 months. Safety was assessed based on reported adverse events (AEs). Urinary copper levels and NCC remained stable across all follow-ups, indicating adequate copper metabolism control. Reported AEs during TETA 2-HCl treatment were mostly gastrointestinal discomfort (n = 6). In two patients, progressive elevation of transaminases occurred (despite stable copper metabolism). AEs led to discontinuation of treatment in five cases. Median baseline dose per day was 10.2 mg TETA 4-HCl/kg bodyweight, whereas median baseline dose after therapeutic switch to TETA 2-HCl was 12.8 mg/kg bodyweight. Median daily dose at 12 months did not differ significantly from TETA 2-HCl dose at switching timepoint, with stable biochemical markers and markers of copper metabolism in most (25/30) of the patients. Transitioning from TETA 4-HCl to TETA 2-HCl maintained stable copper parameters and liver function in most of analyzed patients. TETA 2-HCl treatment was generally well tolerated, suggesting that switching medications is safe and effective. In our real-life cohort, adjustment factor of ~1.25× for the switch of TETA 4-HCl to TETA 2-HCl resulted in adequate copper metabolism control.

Motor-evoked potentials as biomarkers for sexual arousal?

Motor cortex excitability may represent the neuronal endpoint of motivational processes and was shown to be modulated by both sexual arousal and deceptive behavior. This is the first study to investigate the influence of lying and sex in heterosexual women and men based on motor-evoked potentials (MEPs) measured while viewing sexually arousing pictures. Sixteen heterosexual couples were shown 360 trials consisting of pictures displaying both almost naked females and males and neutral control images. In a subsequent forced-choice question about wanting to see the respective pictures fully naked, they were instructed to either answer in agreement with or opposite to their sexual preference. Participants went through 2 blocks of answering truthfully and 2 blocks of lying, with these 4 blocks being shown in a randomized alternating order. To measure cortical excitability, MEPs were used, evoked by single transcranial magnetic stimulation pulses between image presentation and response. In normalized MEPs, women and men showed higher amplitudes for preferred over non-preferred sexual stimuli, but only on a descriptive level. Planned contrasts showed higher non-normalized MEPs for lying in all picture categories. Direct comparisons to a preliminary study showed overall lower effect sizes. Both sexes tend to show higher MEPs in response to their sexually preferred stimuli. MEPs are not stable markers for willful volitionally controlled deception although lying does increase cortical excitability. The present experimental design does not seem valid enough to serve as a diagnostic marker for sexual preference or paraphilia and malingering. This is the first study investigating whether sexual motivational stimuli modulate MEPs in women, while also examining the influence of lying for both sexes. The sample was too small for some found effects to be significant. Also, the experimental setup may have been less suited for female participants in comparison to male ones. The operationalization of sexual motivation via MEPs seems to highly depend on different experimental factors including the sex of the participants, induced motivation, and lying.

Circulating Exosomes Studied by Label-free Proteomics Analysis Reveal Transition Signatures from Diabetes Mellitus to Diabetic Kidney Disease

Background: Diabetic kidney disease (DKD) is a common microvascular complication of diabetic mellitus (DM). At present, the early diagnosis of DKD mainly depends on microalbuminuria, which is prone to be affected by confounding factors such as urinary tract infections. Methods: To identify the more stable early diagnosis markers, the whole proteome in the circulating exosomes from controls, DM patients, and DKD patients was quantified by label-free proteomics analysis and then validated with parallel reaction monitoring. Results: Three hundred ninety-one quantitative proteins were detected, and the expression trends of 7 proteins in the validation phase were consistent with that in the discovery phase. The expression level assessment results revealed that the expression of EFEMP1 and ApoA4 in the DKD group was significantly higher than those in DM and controls. Correlation analysis showed that EFEMP1 and APOA4 were positively correlated with urinary microalbumin and urinary albumin creatinine ratio and had excellent diagnostic values for distinguishing DKD from DM and controls. Conclusions: ApoA4 and EFEMP1 could serve as the early diagnosis markers of DKD. These findings provide a possibility for the development of a clinical diagnostic index that can efficiently distinguish DKD from DM in the near future.

(Sensor Division Early Career Award) Portable Diagnostics for Cancer Management at Printed Strips

Point-of-care testing (PoC) is revolutionizing the healthcare sector improving patient care in daily hospital practice and allowing reaching even remote areas. In the frame of cancer management, the design and validation of PoC enabling the non-invasive, rapid detection of cancer markers is urgently required to implement liquid biopsy. However, despite substantial advances in sensing technologies, the development, preparation, and use of self-testing devices is still confined to specialist laboratories and users. Decentralized analytical devices will enormously impact daily lives, enabling people to analyze diverse clinical, environmental, and food samples, evaluate them and make predictions to improve quality of life, particularly in remote, resource-scarce areas. In recent years, paper-based analytical tools have attracted a great deal of attention; the well-known properties of paper, such as abundance, affordability, lightness, and biodegradability, combined with features of printed electrochemical sensors, have enabled the development of sustainable devices that drive (bio)sensors beyond the state of the art. Their blindness toward colored/turbid matrices (i.e., blood, soil), their portability, and the capacity of paper to autonomously filter/purge/react with target species make such devices powerful in establishing point-of-need tools for use by non-specialists. Depending on analytical requisites, different types of paper (filter, office) and configurations (1D, 2D, 3D) can be adopted. Focusing on stable blood-based markers with high-specificity, such as miRNAs, is of crucial importance. A wide overview regarding application ranging from DNA to miRNA detection will be provided, with the aim in showing the potentialities of portable electrochemical biosensors for improving society involvement in monitoring circulating nucleic acids associated to diverse conditions, i.e. cancer, Alzheimer’s disease, HIV.

Association between glucose levels and all-cause mortality in cancer survivors: findings from NHANES 1999–2018

BackgroundHyperglycemia is a rapidly increasing risk factor for cancer mortality worldwide. However, the dose‒response relationship between glucose levels and all-cause mortality in cancer survivors is still uncertain.MethodsWe enrolled 4,491 cancer survivors (weighted population 19,465,739) from the 1999–2019 National Health and Nutrition Examination Survey (NHANES). Cancer survivors were defined based on the question of whether they had ever been diagnosed with cancer by a doctor or a health professional. Hemoglobin A1c (HbA1c) was selected in this study as a stable marker of glucose level. Mortality was ascertained by linkage to National Death Index records until December 31, 2019. Cox proportional hazard, Kaplan‒Meier survival curves and Restricted cubic spline regression models were used to evaluate the associations between HbA1c and all-cause mortality risk in cancer survivors.ResultsIn NHANES, after adjusting for confounders, HbA1c had an independent nonlinear association with increased all-cause mortality in cancer survivors (nonlinear P value < 0.05). The threshold value for HbA1c was 5.4%, and the HRs (95% CI) below and above the threshold value were 0.917 (0.856,0.983) and 1.026 (1.010,1.043), respectively. Similar associations were found between fasting glucose and all-cause mortality in cancer survivors, and the threshold value was 5.7 mmol/L.ConclusionsHbA1c was nonlinearly associated with all-cause mortality in cancer survivors, and the critical value of HbA1c in decreased mortality was 5.4%, suggesting optimal glucose management in cancer survivors may be a key to preventing premature death in cancer survivors.

Chemical composition combined with network pharmacology and quality markers analysis for the quality evaluation of Qing-fei-da-yuan granules.

Qing-fei-da-yuan granules (QFDYGs) had been proved to be an effective TCM prescription for treating coronavirus disease 2019 (COVID-19), which are composed of a variety of TCMs, and characterized by multiple components, multiple targets and overall regulation. It is meaningful to further study the chemical composition and pharmacology of QFDYGs for quality evaluation. However, due to the complexity of the components of QFDYGs, there are no reliable and simple analytical methods for current quality evaluation. In this work, antipyretic activity assessment of QFDYGs in the LPS-induced New Zealand rabbit model was carried out to verify the efficacy firstly. It was proved that QFDYGs can be used to relieve fever to help preventing or controlling the prevalence of influenza and pneumonia. Subsequently, UHPLC-ESI-QTOF-MS/MS combined with network pharmacology, quality markers and fingerprint analysis were used to establish the quality control condition. The chemical compositions were analyzed by UHPLC-ESI-QTOF-MS/MS, and 79 of them were identified, such as arecoline, mangiferin, paeoniflorin, etc. Then, the network pharmacology strategy based on 45 candidate components (CCs) in conjunction with influenza and pneumonia diseases was employed to screen the potential active ingredients. According to the drug-CCs-genes-diseases (D-CCs-G-D) networks, baicalein, honokiol, baicalin, paeoniflorin, saikosaponin A, glycyrrhizic acid and hesperidin were selected as quality markers. And a method for content determination of the 7 quality markers was established by optimizing extraction methods, chromatographic conditions and methodological verification. Finally, the quality of 15 batches of QFDYGs was evaluated by using the 7 quality markers combined with fingerprints and principal component analysis (PCA). The analyzed results showed that baicalin, paeoniflorin, glycyrrhizic acid and hesperidin were the high content and stable quality markers. QFDYGs were characterized by overall consistency and individual ingredient differences among the 15 batches. Our quality evaluation study will provide reference for the further development and research of QFDYGs.

Prostate cancer cancer-associated fibroblasts with stable markers post-androgen deprivation therapy associated with tumor progression and castration resistant prostate cancer.

The specificity and clinical relevance of cancer-associated fibroblasts (CAFs) in prostate cancer (PCa), as well as the effect of androgen deprivation therapy (ADT) on CAFs, remain to be fully elucidated. Using cell lineage diversity and weighted gene co-expression network analysis (WGCNA), we pinpointed a unique CAF signature exclusive to PCa. The specificity of this CAF signature was validated through single-cell RNA sequencing (scRNA-seq), cell line RNA sequencing, and immunohistochemistry. This signature associates CAFs with tumor progression, elevated Gleason scores, and the emergence of castration resistant prostate cancer (CRPC). Using scRNA-seq on collected samples, we demonstrated that the CAF-specific signature is not altered by ADT, maintaining its peak signal output. Identifying a PCa-specific CAF signature and observing signaling changes in CAFs after ADT lay essential groundwork for further PCa studies.

Comprehensive in vitro and in silico Evaluation of Safety and Probiotic Potential of Lactiplantibacillus plantarum strain LFN10

ABSTRACT The study aimed to assess the safety and probiotic potential of strain Lactiplantibacillus plantarum LFN10, isolated from Algerian fermented milk “Lben,” using genome-sequencing and in vitro approaches. Safety assessments revealed LFN10 as negative for virulence and antibiotic resistance. With favorable physiological and biochemical traits, a versatile carbohydrate metabolism and good exopolysaccharide production abilities, it qualifies as nonstarter culture for food fermentation. The strain displayed resilience to acidic and bile salt stress and exhibited strong cholesterol-lowering capabilities. The moderate interaction with pathogens suggests its potential ability to inhibit them. The strain also demonstrated inhibitory effects on foodborne pathogens. Whole genome sequencing and bioinformatics analyses facilitated taxogenomic classification and confirmed the presence of stable chromosomal marker genes associated with probiotic and technological functional qualities. Overall, LFN10 appears as a promising candidate for application as probiotics, which requires further in vivo studies to confirm its safety and efficacy as well as to optimize its application in food and as nutraceuticals.

A Flow Cytometry-Based Method for Assessing CAR Cell Binding Kinetics Using Stable CAR Jurkat Cells.

Chimeric antigen receptors (CARs) are synthetic fusion proteins that can reprogram immune cells to target specific antigens. CAR-expressing T cells have emerged as an effective treatment method for hematological cancers; despite this success, the mechanisms and structural properties that govern CAR responses are not fully understood. Here, we provide a simple assay to assess cellular avidity using a standard flow cytometer. This assay measures the interaction kinetics of CAR-expressing T cells and targets antigen-expressing target cells. By co-culturing stably transfected CAR Jurkat cells with target positive and negative cells for short periods of time in a varying effector-target gradient, we were able to observe the formation of CAR-target cell doublets, providing a readout of actively bound cells. When using the optimized protocol reported here, we observed unique cellular binding curves that varied between CAR constructs with differing antigen binding domains. The cellular binding kinetics of unique CARs remained consistent, were dependent on specific target antigen expression, and required active biological signaling. While existing literature is not clear at this time whether higher or lower CAR cell binding is beneficial to CAR therapeutic activity, the application of this simplified protocol for assessing CAR binding could lead to a better understanding of the proximal signaling events that regulate CAR functionality. Key features • Determines CAR receptor cellular interaction kinetics using a Jurkat cell model. • Can be used for a wide variety of CAR target antigens, including both hematological and solid tumor targets. • Experiments can be performed in under two hours with no staining using a standard flow cytometer. • Requires stable CAR Jurkat cells and target cells with stable fluorescent marker expression for optimal results.

Sodium-Glucose Cotransporter 2 Inhibitor Combined with Conventional Diuretics Ameliorate Body Fluid Retention without Excessive Plasma Volume Reduction.

We previously reported that sodium-glucose cotransporter 2 (SGLT2) inhibitors exert sustained fluid homeostatic actions through compensatory increases in osmotic diuresis-induced vasopressin secretion and fluid intake. However, SGLT2 inhibitors alone do not produce durable amelioration of fluid retention. In this study, we examined the comparative effects of the SGLT2 inhibitor dapagliflozin (SGLT2i group, n = 53) and the combined use of dapagliflozin and conventional diuretics, including loop diuretics and/or thiazides (SGLT2i + diuretic group, n = 23), on serum copeptin, a stable, sensitive, and simple surrogate marker of vasopressin release and body fluid status. After six months of treatment, the change in copeptin was significantly lower in the SGLT2i + diuretic group than in the SGLT2i group (-1.4 ± 31.5% vs. 31.5 ± 56.3%, p = 0.0153). The change in the estimated plasma volume calculated using the Strauss formula was not significantly different between the two groups. Contrastingly, changes in interstitial fluid, extracellular water, intracellular water, and total body water were significantly lower in the SGLT2i + diuretic group than in the SGLT2i group. Changes in renin, aldosterone, and absolute epinephrine levels were not significantly different between the two groups. In conclusion, the combined use of the SGLT2 inhibitor dapagliflozin and conventional diuretics inhibited the increase in copeptin levels and remarkably ameliorated fluid retention without excessively reducing plasma volume and activating the renin-angiotensin-aldosterone and sympathetic nervous systems.

#2548 In vitro evaluation of proenkephalin kinetics in hemofiltration and hemodialysis

Abstract Background and Aims Proenkephalin A 119-159 (penKid) is a stable surrogate marker for enkephalins (endogenous opioids regulating kidney function). PenKid plasma concentration correlates to GFR and increases in AKI, for this reason it can be used for prediction and diagnosis of AKI (even subclinical forms) and need of RRT. Furthermore, it seems to be also a reliable biomarker to predict the successful liberation from RRT and may therefore aid in the successful weaning of patients from RRT. PenKid has the perk of maintaining stable its concentration during RRT, suggesting that is barely filtered, in contrast to other biomarkers. However, with its small size of 4.5 kDa, penKid can be assumed to be freely filtered through the glomerulus and dialysis membranes. To address the issue about stable penKid plasma concentrations in RRT (no extracorporeal removal or increased production in response to impaired kidney function), we planned ex vivo experiments simulating different conditions of RRT. The aim of this study is to determine the sieving coefficient and the diffusive clearance of the penKid molecule in conditions of in vitro continuous veno-venous hemofiltration (CVVH) and continuous veno-venous hemodialysis (CVVHD), respectively. Method The lyophilized peptide was solved in 20 mM Dipotassium phosphate, 6 mM Disodium EDTA [pH 8], avoiding concentrations higher than 1 mg/mL. The compound was diluted in 50 mL of plasma to ensure homogeneous distribution. For each experimental modality, a batch of blood of 1000 mL with adjusted hematocrit at 30% and albumin concentration of 3 g% was utilized. Blood was spiked with lyophilized synthetic penKid to achieve two target concentrations of 150 and 500 pmol/L in CVVH and CVVHD modalities, respectively. Blood was adjusted for a volume of 1000 mL, maintained at 37° and continuously stirred during the experiments. CVVH was performed with blood flow (QB) = 150 mL/min and ultrafiltration rate (QUF) = 20 mL/min to reproduce a filtration fraction of 20%. The ultrafiltrate was reinfused in the venous line to maintain initial volume. This allowed to define convective clearance and solute sieving coefficient in pure convective mode. CVVHD was performed with QB = 150 mL/min and dialysate flow (QD) = 35 mL/min (Net QUF = 0 mL/min). This allowed the calculation of diffusive clearance. A polysulfone dialyzer (AV1000 FMC, Bad Homburg, Germany) was utilized. Samples were taken at 10, 30, 60 minutes from the initiation of the circulation, in both modalities. Results Initial penKid concentrations in blood corresponded to the desired targets in all the experiments. Sieving coefficients displayed values between 0.69 and 1.5 and they appear to remain stable over time (Fig. 1). Diffusive clearance displayed values between 35 and 15.7 mL/min. Values appear to present a slight decrease over time (Fig. 2). The observed variability can be attributed to several factors: difference in plasma protein interference in various moments of the experiments, difference in concentration of penKid in plasma water versus plasma or blood, variability in the sample measurements. Conclusion Significant removal of penKid can be obtained by extracorporeal therapies both in convection (CVVH) and diffusion (CVVHD). Further investigation is needed to assess if these findings are confirmed during in vivo treatments in which penKid kinetics is affected by generation, elimination and changes in distribution volume.

#2512 Behaviour of Human Proenkephalin A 119-159 (penKid) in an in vitro model of hemoadsorption

Abstract Background and Aims Proenkephalin A 119-159 (penKid) is a stable surrogate marker for enkephalins (endogenous opioids regulating kidney function). Different clinical studies have shown severity-dependent increased penKid levels in patients with sepsis-associated acute kidney injury (AKI), indicating that penKid can be used for prediction and diagnosis of AKI and need of renal replacement therapy (RRT) and even subclinical forms of AKI. In the absence of targeted therapies, organ support in terms of RRT is the ultimate treatment in severe AKI cases. Hemoadsorption is utilized as an adjunctive therapy in septic patients. The aim of this study is to define the penKid removal ratio in conditions of in vitro hemoadsorption (HA) using a synthetic microporous hemo-compatible resin. Method The lyophilized peptide was solved in 20 mM Dipotassium phosphate, 6 mM Disodium EDTA [pH 8], avoiding concentrations higher than 1 mg/mL. The compound was diluted in 50 mL of plasma to ensure homogeneous distribution. A batch of blood of 1000 mL with adjusted hematocrit at 30% and albumin concentration of 3 g% was utilized. Blood was spiked with lyophilized synthetic penKid to achieve the target concentration of 600 pmol/L. Blood was adjusted for a volume of 1000 mL, maintained at 37° and continuously stirred during the experiment. HA was performed with QB = 150 mL/min in a closed-loop configuration. A sorbent minimodule of HA380 (Jafron, Zhuhai, People Republic of China) was utilized. Samples were taken at 5, 15, 30, 60 and 120 minutes from the initiation of the circulation. Total removal and reduction ratios were calculated to assess the adsorption. This procedure was repeated three times. Results Initial penKid concentrations in blood corresponded to the desired target. In the three experiments penKid concentrations displayed a consistent decrease that approached 75% removal ratio at 120 minutes (Figs 1 and 2). Minimal variability was observed confirming a consistent behavior of the interaction between the sorbent and the molecule. Conclusion The results suggest that penKid is effectively removed during hemoadsorption and its levels should be evaluated in light of these results when in vivo hemoadsorption is carried out in critically ill patients.

Time-restricted feeding improves aortic endothelial relaxation by enhancing mitochondrial function and attenuating oxidative stress in aged mice

Age-related endothelial dysfunction is a pivotal factor in the development of cardiovascular diseases, stemming, at least in part, from mitochondrial dysfunction and a consequential increase in oxidative stress. These alterations are central to the decline in vascular health seen with aging, underscoring the urgent need for interventions capable of restoring endothelial function for preventing cardiovascular diseases. Dietary interventions, notably time-restricted feeding (TRF), have been identified for their anti-aging effects on mitochondria, offering protection against age-associated declines in skeletal muscle and other organs. Motivated by these findings, our study aimed to investigate whether TRF could similarly exert protective effects on endothelial health in the vasculature, enhancing mitochondrial function and reducing oxidative stress. To explore this, 12-month-old C57BL/6 mice were placed on a TRF diet, with food access limited to a 6-h window daily for 12 months. For comparison, we included groups of young mice and age-matched controls with unrestricted feeding. We evaluated the impact of TRF on endothelial function by measuring acetylcholine-induced vasorelaxation of the aorta. Mitochondrial health was assessed using fluororespirometry, and vascular reactive oxygen species (ROS) production was quantified with the redox-sensitive dye dihydroethidium. We also quantified 4-hydroxynonenal (4-HNE) levels, a stable marker of lipid peroxidation, in the aorta using ELISA. Our findings demonstrated that aged mice on a standard diet exhibited significant impairments in aortic endothelial relaxation and mitochondrial function, associated with elevated vascular oxidative stress. Remarkably, the TRF regimen led to substantial improvements in these parameters, indicating enhanced endothelial vasorelaxation, better mitochondrial function, and reduced oxidative stress in the aortas of aged mice. This investigation establishes a vital foundation, paving the way for subsequent clinical research aimed at exploring the cardiovascular protective benefits of intermittent fasting.

An experimental estimation method of diffusion coefficients in ternary and multicomponent systems from a single diffusion profile

Until recently, it was textbook knowledge that the diffusion coefficients could not be estimated in a multi-component system following the widely practised diffusion couple method. The recently proposed constrained diffusion couple method largely solved the problems. The need to produce two intersecting diffusion paths in a multi-component space with very well-controlled compositions of the diffusion couple end members may be tricky in certain situations, depending on the complications of diffusion paths. In this study, we have shown the estimation of all types of diffusion coefficients from a single diffusion profile without neglecting Onsager's cross terms in concentrated ternary Ni-Co-Fe, Fe-rich quaternary Fe-Ni-Co-Cr and concentrated Ni-Co-Fe-Cr-Al quinary alloys. This is applicable in ternary and multicomponent component systems (n≥3) at a stable Kirkendall marker plane position. One can even estimate the impurity diffusion coefficients utilizing the composition profiles at the ends of the diffusion couples following the Hall method, which has been rarely practised in multicomponent systems until now. We have further demonstrated the importance of estimating the tracer and intrinsic diffusion coefficients in a concentrated or multi-principal element alloy in which interdiffusion coefficients can be vague and misleading for understanding the elements' diffusional interactions and relative mobilities. We have also shown the importance of considering the vacancy wind effect in concentrated alloys. The method proposed in this study will help to generate a mobility database with relative ease in various multicomponent systems important for various applications, which was considered impossible until recently.