Serum Samples Research Articles

The Predictive Value of Estimation of N Terminal - Pro B- Type Natriuretic Peptide in Patients with ST Elevation Myocardial Infarction Undergoing Primary Percutaneous Intervention for the Outcome of Myocardial Reperfusion

Abstract Background Primary PCI is considered to be the preferred reperfusion strategy for STEMI. The goal of reperfusion is to resume normal blood flow to the cardiac tissues, and not just to achieve an open epicardial artery. Aim of the work The aim of this study was to evaluate the predictive value of NT-pro BNP before primary PCI in patients presenting with STEMI, for myocardial reperfusion assessed by the degree of ST resolution in the ECG 90 minutes post primary PCI, the TIMI flow and the MBG post primary PCI. Patients and Methods This study included 40 patients who presented to emergency departments of Ain Shams University hospitals and Misr University for Sciences and Technology hospital from September 2021 till June 2022, diagnosed with STEMI and had been revascularized by primary PCI as per the recommendations of The European society of cardiology guidelines for diagnosis and management of ST elevation myocardial infarction published in 2017. Serum samples for NT-pro BNP level were obtained before primary PCI to be correlated with the TIMI flow grade, the MBG, and the ST resolution post primary PCI. Results Our study had demonstrated that a higher baseline NT-pro BNP level is significantly associated with higher risk of poor myocardial reperfusion following primary PCI expressed by ST resolution less than 50%, TIMI flow grade less than 3 as well as MBG less than 3 and was also associated with higher incidence of No Reflow. Conclusion Initial NT-pro BNP level in STEMI patients correlates with myocardial reperfusion following primary PCI. This supports the potential use of NT-pro BNP as an early marker for risk stratification of patients with STEMI. High risk patients will be in need of more aggressive complementary treatment strategies to improve microvascular perfusion and optimization of the medical therapies to help decrease the development of heart failure.

Correlation Between High-sensitive Troponin Level, Neutrophil Lymphocyte ratio and SYNTAX score in Acute Myocardial Infarction patients undergoing Coronary Angiography

Abstract Background Acute myocardial infarction is one of the leading causes of death in the developed world. The prevalence of the disease approaches three million people worldwide. Acute myocardial infarction divided into two categories, non-ST-segment elevation MI (NSTEMI) and ST-segment elevation MI (STEMI). Objective To assess the value of using high sensitive troponin and the neutrophil lymphocyte ratio as marker to predict the complexity of angiographic coronary anatomy in AMI patients assessed by SYNTAX Score. Patients and Methods this study included 100 patients who presented to emergency department of Ain Shams University Hospitals and Misr University for Science and Technology (MUST) hospital, between November/2021 and May/2022 diagnosed with AMI and underwent coronary angiography with or without stent implantation. Serum sample for differential CBC count was obtained to assess NLR as will as zero point high sensitive troponin to be correlation SYNTAX score. Results The study showed that there was a significant correlation between the NLR and the complexity of coronary lesions assessed by SYNTAX score, but the study couldn’t found any correlation between the high sensitive troponin level and the complexity of coronary lesions. Also the study showed there were no correlation between NLR and either TIMI follow or MBG. The conducted patients in the study were categorized in a tripartite fashion according to their SYNTAX score (low: 0 to 22, intermediate: 23 to 32, high: >32). Conclusion An increased in neutrophil lymphocyte ratio on admission is a strong and independent predictor of the complexity of the coronary lesions, but there is no significant correlation between High sensitive troponin level and the complexity of coronary lesion .

A double boronic acid affinity "sandwich" SERS biosensor based on magnetic boronic acid controllable-oriented imprinting for high-affinity biomimetic specific recognition and rapid detection of target glycoproteins.

Transferrin (TRF), recognized as a glycoprotein clinical biomarker and therapeutic target, has its concentration applicable for disease diagnosis and treatment monitoring. Consequently, this study developed boronic acid affinity magnetic surface molecularly imprinted polymers (B-MMIPs) with pH-responsitivity as the "capture probe" for TRF, which have high affinity similar to antibodies, with a dissociation constant of (3.82 ± 0.24) × 10-8M, showing 7 times of reusability. The self-copolymerized imprinted layer synthesized with dopamine (DA) and 3-Aminophenylboronic acid (APBA) as double monomers avoided nonspecific binding sites and produced excellent adsorption properties. Taking the gold nanostar (AuNS) with a branch tip "hot spot" structure as the core, the silver-coated AuNS functionalized with the biorecognition element 4-mercaptophenylboronic acid (MPBA) was employed as a surface-enhanced Raman scattering (SERS) nanotag (AuNS@Ag-MPBA) to label TRF, thereby constructing a double boronic acid affinity "sandwich" SERS biosensor (B-MMIPs-TRF-SERS nanotag) for the highly sensitive detection of TRF. The SERS biosensor exhibited a detection limit for TRF of 0.004ng/mL, and its application to spiked serum samples confirmed its reliability and feasibility, demonstrating significant potential for clinical TRF detection. Moreover, the SERS biosensor designed in this study offers advantages in stability, detection speed (40min), and cost efficiency. The portable Raman instrument for SERS detection fulfills the requirements for point-of-care testing.

P-296 The relationship between serum HE4 and ultrasound findings of adenomyosis in infertility patients and its correlation with implantation and pregnancy outcomes in those undergoing IVF-ET

Abstract Study question Are there correlations between HE4 levels and the presence of ultrasonographic findings of adenomyosis in infertile women, and how does this relationship effect pregnancy outcomes? Summary answer The presence of subendometrial linear lines as marker of endo-myometrial invasion, is correlated with serum HE4 levels and negatively impacts pregnancy outcomes. What is known already Adenomyosis observed in women of reproductive age can present with infertility, particularly as implantation is adversely affected in junctional zone abnormalities. The endo-myometrial invasion in the pathogenesis of adenomyosis has been variously defined based on ultrasound criteria outlined in the MUSA (Morphological Uterus Sonographic Assessment) consensus. In patients with adenomyosis, while implantation rates may remain unchanged in some studies, an increased rate of first-trimester miscarriages has been reported in others. This study was conducted based on the hypothesis that HE4 (Human Epididymis Protein 4) marker may play a role in this potential invasiveness in adenomyosis and negatively affect embryo implantation. Study design, size, duration 82 infertile patients diagnosed with adenomyosis based on the MUSA consensus ultrasound criteria were included in our 10-month prospective study. HE4 testing was performed on serum samples collected from the patients. Our primary outcome focused on the association between ultrasonographic findings and the serum HE4 levels. The secondary outcomes comprised assessing the relationship between HE4 levels and the outcomes of implantation and pregnancy in the patients who underwent embryo transfer. Participants/materials, setting, methods In this study, infertile patients aged 20-40 diagnosed with adenomyosis via ultrasonography were included. Patients with a history of myoma or myomectomy, high-dose biotin intake, chronic organ failure, and a history of malignancy were excluded from the study. Ultrasonographic features were documented, and 39 patients underwent frozen embryo transfer. Pregnancy outcomes were categorized as clinical pregnancy or miscarriage for analysis. Main results and the role of chance As a primary outcome of the study, a statistically significant correlation between serum HE4 levels and subendometrial linear lines, as indicated in the MUSA consensus ultrasound criterias is found (p = 0.015). Logistic regression analysis showed that HE4 independently increased subendometrial linear striations [OR (95% CI) = 1.05 (1.01-1.10); model significance p = 0.020]. ROC analysis suggested that serum HE4 levels above the 37.4 pmol/L cut-off value may be associated with the presence of subendometrial linear striations. Although no relationship was found between HE4 levels and implantation rates, statistically significant higher levels of HE4 were observed in cases of miscarriage compared to ongoing pregnancies (p = 0.038). Limitations, reasons for caution Study limitations include reliance on 2D-TVUSG without 3D and Doppler. Enhanced evaluation using 3D-TVUSG and Doppler for JZ and subendometrial areas is recommended. Larger sample sizes could yield more impactful findings. Wider implications of the findings HE4 correlates with observed subendometrial linear lines, suggesting a link to endo-myometrial invasion. While it doesn’t impact transfer cycle implantation rates, it may be linked to higher early pregnancy losses. Trial registration number not applicable

Effect of Central Dialysis Fluid Delivery System (CDDS) using high flux dialyzer versus Regular Water Treatment Stations on Endotoxemia and Inflammatory Markers among Prevalent Patients on Regular Hemodialysis

Abstract Background and Objectives Translocated endotoxins derived either from intestinal bacteria or backfiltration, have a wide range of adverse effects on patients. Most end stage renal disease (ESRD) patients have systemic inflammation which increases the risk of morbidity and mortality. The central dialysis fluid delivery system (CDDS) has been used exclusively in Japan since 1960s, and now is being used in Ain Shams University. Our aim of the study is to evaluate the effect of (CDDS) versus single patient dialysis fluid delivery system (SPDDS) on endotoxemia and inflammatory markers among prevalent patients on regular hemodialysis (HD). Methods Eighty Patients were randomly selected from HD units, then divided into two groups, (group I) doing HD using CDDS water purification system at El Demerdash Hospital, (group II) using regular water purification system at Ain Shams University Specialized Hospital. Pre- dialysis and post-dialysis serum samples were obtained to measure endotoxin levels of both groups, as well as postdialysis serum samples were obtained to measure interleukin 6 (IL6) level and highly sensitive Creactive protein (Hs CRP). Results Circulating endotoxemia was most notable in group II, The mean predialysis endotoxin level in group I was 0.07 ± 0.05 Eu/mL, while in group II was 0.20 ± 0.07 Eu/mL and mean of post-dialysis endotoxin level in group I was 0.04 ± 0.02 Eu/mL, while in group II was 0.15 ± 0.03 Eu/mL. IL6 as well as Hs CRP levels were higher in group II in comparison to group I. Conclusion Our data showed significant reduction in circulating endotoxins in CDDS group.

P-353 Perfluorooctanoic acid (PFOA) impacts murine ovarian follicle development through apoptotic signaling pathways and exhibits transgenerational effects

Abstract Study question How does Perfluorooctanoic Acid (PFOA) exposure impact ovarian function and the physiological and reproductive health of subsequent offspring? Summary answer PFOA exposure leads to ovarian dysfunction, characterized by follicular depletion and hormonal imbalances, and adversely affects the reproductive health of offspring. What is known already Mass spectrometry reveals higher perfluorinated compound levels in premature ovarian failure patients compared to healthy individuals. PFOA, an environmental contaminant, is known for its endocrine-disrupting effects, but its specific impact on ovarian function and transgenerational health remains less explored. This study addresses the gap by examining PFOA’s direct ovarian effects and its consequential impacts on offspring, using both in vivo and in vitro models. Study design, size, duration This study employs mouse models and ovarian granulosa cells to assess PFOA’s impact. Mice were administered PFOA (50mg/Kg) and evaluated for ovarian function and offspring health. Ovarian tissue underwent histological analysis and hormone assays. RNA transcriptome sequencing identified activated apoptotic pathways post-PFOA exposure. Offspring physiological and reproductive parameters were also examined. Parallel in vitro experiments with ovarian granulosa cells at 50uM PFOA concentration provided insights into cellular apoptosis. Participants/materials, setting, methods Clinically, we collected serum samples from approximately 100 Premature Ovarian Insufficiency (POI) patients and 100 healthy individuals, analyzed using mass spectrometry. In murine experiments, we simulated human environmental exposure to PFOA (particularly in high-exposure professions) using a 50mg/Kg dose administered via gavage. Transcriptome sequencing analyzed potential signaling pathways in both experimental and control mice. Offspring underwent physiological metabolic assessments and fertility evaluations, including fertilization and blastocyst rates. Main results and the role of chance Mass spectrometry results from human samples revealed significantly higher levels of perfluorinated compounds in POI patients compared to healthy controls, suggesting a potential link between PFOA exposure and ovarian dysfunction. In murine models, PFOA administration at 50mg/Kg resulted in notable ovarian changes. Histological analysis showed a reduction in follicle counts and abnormalities in AMH and FSH levels, indicative of premature ovarian failure. Transcriptome sequencing of these mice highlighted the activation of apoptotic pathways, especially the PI3K/Akt/mTOR signaling, post-PFOA exposure. This suggests a mechanistic pathway through which PFOA may induce ovarian dysfunction. Furthermore, offspring of PFOA-exposed mice displayed significant reproductive health challenges. They exhibited irregularities in estrous cycles and a decrease in oocyte fertilization rates, pointing to potential transgenerational effects of PFOA exposure. These findings in murine models align with the elevated perfluorinated compound levels observed in human POI patients, reinforcing the hypothesis of PFOA’s detrimental impact on ovarian health and subsequent generational fertility. The consistency of results across human clinical data and animal models strengthens the argument for a causal relationship between PFOA exposure and reproductive health risks. Limitations, reasons for caution While these findings highlight PFOA’s detrimental effects on ovarian function and offspring health, translating results from animal models to humans necessitates caution. The specific mechanisms and potential variability in human response to environmental PFOA exposure require further investigation. Wider implications of the findings This research underscores the far-reaching impacts of environmental contaminants like PFOA on reproductive health. It emphasizes the need for stringent regulation of such pollutants and further studies to understand their long-term effects on human health, particularly regarding fertility and intergenerational wellbeing. Trial registration number not applicable

P-591 Per- and Polyfluoroalkyl Substances Serum Concentrations and Polycystic Ovary Syndrome among Women Attending a Fertility Clinic

Abstract Study question What is the association between serum per- and poly-fluoroalkyl substances (PFAS) concentrations and polycystic ovary syndrome (PCOS) risk in populations with low-level PFAS exposure. Summary answer Higher serum concentrations of perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) were associated with elevated risk of PCOS among women attending a U.S. fertility clinic. What is known already Previous studies reported that exposure to high-levels of PFAS exposure was associated with elevated risk of PCOS. However, studies evaluating PCOS risk in populations with exposure to lower levels of PFAS are limited. Study design, size, duration A cohort study of 502 females who sought fertility evaluation for medically assisted reproduction in a U.S. fertility clinic and had similar PFAS exposure level as the general U.S. population. Participants/materials, setting, methods Nine PFAS were quantified in non-fasting serum samples collected at study entry. Diagnosis of PCOS was based on the Rotterdam criteria after study entry before treatment. We used logistic regression to examine the odds ratio (OR) of PCOS in relation to PFAS concentrations (continuous and by tertiles). We applied quantile g-computation (QGC) and Bayesian Kernel Machine Regression (BKMR) to examine the joint associations of PFAS mixture with PCOS. Main results and the role of chance A total of 38 (7.6%) females were diagnosed with PCOS. Doubling of serum PFOS and PFHxS concentrations were associated with higher odds of PCOS [OR (95%CI): 1.70 (1.06, 2.81) and 1.45 (1.02, 2.08), respectively]. There was a dose-response relationship of PFOS with PCOS risk (p of trend by tertiles = 0.07). Both QGC and BKMR identified PFOS as the most important contributor among the PFAS mixture to PCOS risk. No clear joint effect was found for other examined PFAS nor for PFAS as a total mixture on PCOS risk. Limitations, reasons for caution Findings in this subfertile population may not be directly generalizable to reproductive aged women in the general population. Wider implications of the findings Our findings are consistent with existing evidence from populations with higher concentrations of some of the evaluated PFAS and supports the potential adverse effects of PFAS on female reproductive function. Larger prospective studies are needed to validate the findings. Trial registration number NA

Decreased Serum Apolipoprotein CIII in the Acute Phase of Kawasaki Disease.

Plasma exchange is an effective treatment for Kawasaki disease (KD), suggesting that plasma from patients with KD bears its causative agents. The aim of this study was to use mass spectrometry to identify candidate agents in patient sera. Serum samples were obtained from 17 KD patients. In six patients, samples were collected in each of three phases: the acute phase prior to acetylsalicylic acid (ASA) and intravenous immunoglobulin administration (Phase A1), the remission phase with ASA (Phase A2), and the remission phase without any medication (Phase A3). Sera from the remaining 11 patients were collected during Phases A1 and A2. The study also included two age- and gender-matched control groups, one with eight afebrile children and one with eight febrile children diagnosed with infectious disease. Patients in Phase A1 and febrile controls did not differ in body temperature, white blood cell counts, or C-reactive protein levels. Mass spectrometry analysis revealed that the intensity levels of m/z 9416, identified as apolipoprotein CIII (Apo CIII), were lower in Phase A1 samples compared with samples from patients in Phases A2 and A3, and from febrile controls (all comparisons, p < 0.01). Serum Apo CIII levels were also lower in Phase A1 samples compared with samples from Phase A2 patients and afebrile controls (both p < 0.01), but samples from patients in Phase A2 did not differ significantly from those of the afebrile controls (p = 0.55). This study demonstrated that serum Apo CIII level was decreased in the acute phase of KD.

Clinical serum lipidomic profiling revealed potential lipid biomarkers for early diabetic retinopathy

Diabetic retinopathy (DR) is a serious complication of diabetes featuring abnormal lipid metabolism. However, the specific lipid molecules associated with onset and progression remain unclear. We used a broad-targeted lipidomics approach to assess the lipid changes that occur before the proliferative retinopathy stage and to identify novel lipid biomarkers to distinguish between patients without DR (NDR) and with non-proliferative DR (NPDR). Targeted lipomics analysis was carried out on serum samples from patients with type I diabetes, including 20 NDRs and 20 NPDRs. The results showed that compared with the NDR group, 102 lipids in the NPDR group showed specific expressions. Four lipid metabolites including TAG58:2-FA18:1 were obtained using the Least Absolute Shrink And Selection Operator (LASSO) and Support Vector Machine Recursive Feature Elimination (SVM-RFE) methods. The four-lipid combination diagnostic models showed good predictive ability in both the discovery and validation sets, and were able to distinguish between NDR patients and NPDR patients. The identified lipid markers significantly improved diagnostic accuracy within the NPDR group. Our findings help to better understand the complexity and individual differences of DR lipid metabolism.

Tofacitinib Regulates Endostatin via Effects on CD147 and Cathepsin S

Angiogenesis is critical for rheumatoid arthritis (RA) progression. The effects of tofacitinib, a JAK-STAT inhibitor used for RA treatment, on angiogenesis in RA are unclear. We, therefore, evaluated the levels of angiogenic factors in two systems of a human co-culture of fibroblast (HT1080) and monocytic (U937) cell lines treated with tofacitinib and in serum samples from RA patients before and after six months of tofacitinib treatment. Tofacitinib reduced CD147 levels, matrix metalloproteinase-9 (MMP-9) activity, and angiogenic potential but increased endostatin levels and secreted proteasome 20S activity. In vitro, tofacitinib did not change CD147 mRNA but increased miR-146a-5p expression and reduced STAT3 phosphorylation. We recently showed that CD147 regulates the ability of MMP-9 and secreted proteasome 20S to cleave collagen XVIIIA into endostatin. We show here that tofacitinib-enhanced endostatin levels are mediated by CD147, as CD147-siRNA or an anti-CD147 antibody blocked proteasome 20S activity. The correlation between CD147 and different disease severity scores supported this role. Lastly, tofacitinib reduced endostatin’ s degradation by inhibiting cathepsin S activity and recombinant cathepsin S reversed this in both systems. Thus, tofacitinib inhibits angiogenesis by reducing pro-angiogenic factors and enhancing the anti-angiogenic factor endostatin in a dual effect mediated partly through CD147 and partly through cathepsin S.

MUC16 as a serum-based prognostic indicator of prometastatic gastric cancer

Metastatic gastric cancer (GC) presents significant clinical challenges due to its poor prognosis and limited treatment options. To address this, we conducted a targeted protein biomarker discovery study to identify markers predictive of metastasis in advanced GC (AGC). Serum samples from 176 AGC patients (T stage 3 or higher) were analyzed using the Olink Proteomics Target panels. Patients were retrospectively categorized into nonmetastatic, metastatic, and recurrence groups, and differential protein expression was assessed. Machine learning and gene set enrichment analysis (GSEA) methods were applied to discover biomarkers and predict prognosis. Four proteins (MUC16, CAIX, 5’-NT, and CD8A) were significantly elevated in metastatic GC patients compared to the control group. Additionally, GSEA indicated that the response to interleukin-4 and hypoxia-related pathways were enriched in metastatic patients. Random forest classification and decision-tree modeling showed that MUC16 could be a predictive marker for metastasis in GC patients. Additionally, ELISA validation confirmed elevated MUC16 levels in metastatic patients. Notably, high MUC16 levels were independently associated with metastatic progression in T3 or higher GC. These findings suggest the potential of MUC16 as a clinically relevant biomarker for identifying GC patients at high risk of metastasis.

Expression levels and clinical significance of serum miR-19a/CCL20 in patients with acute cerebral infarction.

Acute cerebral infarction (ACI) is a lethal disease whose early diagnosis is critical for treatment. microRNA (miR)-19a targets CC chemokine ligand 20 (CCL20) in myocardial infarction. We investigated the expression patterns of serum miR-19a and CCL20 of ACI patients and assessed their clinical values. Serum samples of 50 healthy subjects and110 ACI patients were collected. Serum levels of miR-19a, CCL20 mRNA, and biochemical indexes were assessed. miR-19a downstream target gene and the binding relationship between miR-19a and CCL20 were predicted and verified. miR-19a and CCL20 mRNA were subjected to correlation and diagnostic efficiency analysis. miR-19a was poorly expressed in the serum of ACI patients, especially in patients with unstable plaque and large infarction. tumor necrosis factor-α, low-density lipoprotein, and platelet/lymphocyte ratio negatively correlated with serum miR-19a level and positively correlated with CCL20. Dual-luciferase assay revealed that miR-19a could negatively regulate CCL20 expression. CCL20 was highly expressed in the serum of ACI patients. The area under receiver-operating characteristic curve of miR-19a combined with CCL20 was 0.9741 (98.00% specificity, 90.91% sensitivity), higher than their single diagnosis. Collectively, miR-19a had high diagnostic value for ACI and could target to restrain CCL20. The combination of miR-19a and CCL20 improved diagnostic value for ACI.

Integrative multi-omics analysis unravels the host response landscape and reveals a serum protein panel for early prognosis prediction for ARDS

BackgroundThe multidimensional biological mechanisms underpinning acute respiratory distress syndrome (ARDS) continue to be elucidated, and early biomarkers for predicting ARDS prognosis are yet to be identified.MethodsWe conducted a multicenter observational study, profiling the 4D-DIA proteomics and global metabolomics of serum samples collected from patients at the initial stage of ARDS, alongside samples from both disease control and healthy control groups. We identified 28-day prognosis biomarkers of ARDS in the discovery cohort using the LASSO method, fold change analysis, and the Boruta algorithm. The candidate biomarkers were validated through parallel reaction monitoring (PRM) targeted mass spectrometry in an external validation cohort. Machine learning models were applied to explore the biomarkers of ARDS prognosis.ResultsIn the discovery cohort, comprising 130 adult ARDS patients (mean age 72.5, 74.6% male), 33 disease controls, and 33 healthy controls, distinct proteomic and metabolic signatures were identified to differentiate ARDS from both control groups. Pathway analysis highlighted the upregulated sphingolipid signaling pathway as a key contributor to the pathological mechanisms underlying ARDS. MAP2K1 emerged as the hub protein, facilitating interactions with various biological functions within this pathway. Additionally, the metabolite sphingosine 1-phosphate (S1P) was closely associated with ARDS and its prognosis. Our research further highlights essential pathways contributing to the deceased ARDS, such as the downregulation of hematopoietic cell lineage and calcium signaling pathways, contrasted with the upregulation of the unfolded protein response and glycolysis. In particular, GAPDH and ENO1, critical enzymes in glycolysis, showed the highest interaction degree in the protein–protein interaction network of ARDS. In the discovery cohort, a panel of 36 proteins was identified as candidate biomarkers, with 8 proteins (VCAM1, LDHB, MSN, FLG2, TAGLN2, LMNA, MBL2, and LBP) demonstrating significant consistency in an independent validation cohort of 183 patients (mean age 72.6 years, 73.2% male), confirmed by PRM assay. The protein-based model exhibited superior predictive accuracy compared to the clinical model in both the discovery cohort (AUC: 0.893 vs. 0.784; Delong test, P < 0.001) and the validation cohort (AUC: 0.802 vs. 0.738; Delong test, P = 0.008).InterpretationOur multi-omics study demonstrated the potential biological mechanism and therapy targets in ARDS. This study unveiled several novel predictive biomarkers and established a validated prediction model for the poor prognosis of ARDS, offering valuable insights into the prognosis of individuals with ARDS.

Verification of Outer Hair Cell Motor Protein, Prestin, as a Serological Biomarker for Mouse Cochlear Damage

The motor protein prestin, found in the inner ear’s outer hair cells (OHCs), is responsible for high sensitivity and sharp frequency selectivity in mammalian hearing. Some studies have suggested that prestin could be a serological biomarker for cochlear damage, as OHCs are highly vulnerable to damage from various sources. However, the reported data are inconsistent and lack appropriate negative controls. To investigate whether prestin can be used as a serological biomarker for cochlear damage or stress, we measured prestin quantities in the bloodstreams of mice using ELISA kits from different companies. Wildtype (WT) mice were exposed to different ototoxic treatments, including noise exposure and ototoxic reagents that rapidly kill OHCs. Prestin-knockout (KO) mice were used as a negative control. Our data show that some ELISA kits were not able to detect prestin specifically. The ELISA kit that could detect the prestin protein from cochlear homogenates failed to detect prestin in the bloodstream, despite there being significant damage to OHCs in the cochleae. Furthermore, the optical densities of the serum samples, which correlate to prestin quantities, were significantly influenced by hemolysis in the samples. In conclusion, Prestin from OHCs is not a sensitive and reliable serological biomarker for detecting cochlear damage in mice using ELISA.

Ionic-liquid/Carbowax 20M functionalized capsule phase microextraction platform for the extraction of phosphodiesterase-5 inhibitors from human serum and urine prior to their determination by LC-MS

Capsule phase microextraction (CPME) is an efficient bioanalytical technique that streamlines the sample preparation by integrating the filtration and stirring mechanism directly into the device. A novel composite sorbent designed to be selective towards the target analytes consisting of mixed-mode sorbent chemistry synthesized by sol-gel technology is found promising and superior to the conventional C18 sorbents. Herein we describe the encapsulation of an ionic liquid (IL)/Carbowax 20M-functionalized sol-gel sorbent (sol-gel IL/Carbowax 20M) in the lumen of porous polypropylene tubes for the capsule phase microextraction of three phosphodiesterase-5 inhibitors namely avanafil, sildenafil, and tadalafil in human serum and urine samples. The CPME device was characterized by Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR). The experimental parameters of CPME procedure (e.g. sample pH and ionic strength, extraction time, stirring rate, elution solvent and volume) were carefully optimized to achieve the highest possible extraction efficiency for the analytes. Method validation was conducted in terms of precision, linearity, accuracy, matrix effect, lower limits of quantification, and limits of detection (LOD). The method linearity was investigated in the range of 50 – 1000 ng mL−1 for all analytes while the precision was less than 11.8% in all cases. For all analytes, the LOD values were 17 ng mL−1. The IL/CW 20M-functionalized microextraction capsules could be reused at least 25 times both for urine and serum samples. The green character and the applicability of the proposed method were evaluated using the ComplexGAPI and BAGI indexes. The optimized CPME protocol exhibited reduced consumption of organic solvent and generation of waste, cost-effectiveness, and simplicity. Finally, the proposed method was successfully applied to the analysis of sildenafil in human urine after administration of drug-containing formulation.

Molecular allergen IgE tests show neosensitizaions occurring during house dust mite specific immunotherapy

Objective:Neosensitizations may be occur during the allergen specific immunotherapy（AIT） due to the differences between allergen vaccine's content and a patient's molecular sensitization profile. This study investigates whether AIT with HDM extract changes the sensitization profile, whether de novo sensitization occurs, and the clinical importance of the neosensitization. Methods:Fifty-three patients with HDM allergic rhinitis ,with/without asthma, patients were received one year HDM subcutaneous AIT . Fourteen patients were recruited as control group and received only necessary medications. Serum samples were collected at baseline, 6thmoths and 12thof AIT, respectively. Serum samples were tested specific IgE against Der p, Der p 1/2/3 and Der f, Der f 1/2/3, as well as IgG4 against Der p, Der p 1/2 and Der f, Der f 1/2. VAS were collected at the time-points as well. Results:In AIT group, Der p, Der p 1/3, and Der f 1/3 specific IgE levels were significantly higher after one-year treatment, especially for Der p 3. There were 69.2%（18/26） patients whose Der p 3 specific IgE below 0.35 kU/L at baseline but became positive（>0.35 kU/L） after treatment, that is, neosensitization occurred. All tested allergen specific IgG4 level significantly increased after one year AIT treatment and the VAS declined dramatically. However, for patients with neosensitization and without neosensitization, there were no significantly changes concerning to IgG4 level and VAS. Conclusion:Patients undergoing AIT might have a risk of neosensitization to the allergen components in the vaccines. However, the clinical importance of the neosensitization remains unclear and warrants further studies.

Influence of Bovine Pestivirus Heterogeneity on Serological Responses to 10 Different Commercial Vaccine Formulation

Bovine Pestivirus typically involves one or more organ systems, with clinical manifestations ranging from mild to severe fatal systemic illness that lead to significant reproductive, productive, and economic losses. Vaccines face the challenge of addressing the significant variability of pestiviruses, which affects the interaction between viral antigens and the immune system’s ability to provide protection. This study aimed to evaluate the serological responses against bovine viral diarrhea virus 1 (Pestivirus A) and Pestivirus B induced by 10 commercial vaccines, including one recombinant (vaccine E), two modified live (MLV multivalent, vaccine I, and MLV monovalent, vaccine J), and seven killed vaccines (KLV, vaccines A to H). Additionally, we evaluated the cross-reactivity between Pestivirus A and B from vaccines and HoBi-like pestivirus (Pestivirus H). In Phase 1, guinea pigs were used to screen for non-MLVs. They were divided into nine groups (n=6 each) and received two doses (⅕ of bovine dose) of eight different non-MLV on Days 0 and 21. Serum samples were collected on Days 0 and 30 for serological analyse. In Phase 2, Holstein × Gir heifers (n= 45) were divided into five groups, comprising 6 to 9 animals. They were vaccinated either once with MLVs or twice with the top non-MLVs screened in Phase 1. Serum samples were harvested on d0 (vaccination day) and d60 (60 days after the first dose) for MLV and non-MLV. Specific antibody titers were assessed virus neutralization (VN) and transformed in log2 for statistical analysis using PROC-MIXED. Significant effects were observed for vaccine groups, time points, and their interactions concerning neutralizing antibodies against Pestivirus A and B in both Guinea pigs and heifers. The Phase 1 study revealed serological responses against Pestivirus A exclusively in non-MLV D (85.33±13.49) and E (72.00±19.26). In the bovine study, the KLD vaccine D (72.00±15.10), recombinant vaccine E (90.66±25.85), and MLV I (170.66±28.22) resulted in an average of neutralizing antibodies against Pestivirus A that exceeded the protective threshold (≥ 60). However,individual analysis of heifers showed a higher frequency of animals presenting titers of Pestivirus A Ab surpassing 32 following vaccination with MLV I and J. None of the vaccine formulations in either study elicited a protective immune response against Pestivirus B or demonstrated cross-reactivity against Pestivirus H.

Dengue virus (DV) non-cross-reactive Omicron wave COVID-19 serums enhanced DV3 infectivity in vitro.

Introduction. In India, the SARS-CoV-2 Delta wave (2020-2021) faded away with the advent of the Omicron variants (2021-present). Dengue incidences were observed to be less in Southeast Asia during the active years of the pandemic (2020-2021). However, dengue virus type 3 (DV3) cases were increasingly reported in this region (including India) concurrent with the progression of the Omicron waves since 2022.Hypothesis. What could be the reason(s) behind this unusual DV3 surge after an overall dip in dengue incidences in many parts of Southeast Asia?Aim. We, therefore, investigated the current state of cross-reactivity of prevalent (Omicron era) SARS-CoV-2 serums with different DV serotypes and evaluated the impact of such serums on DV neutralization in cell culture.Methodology. Fifty-five COVID-19 serum samples (January-September 2022) and three pre-pandemic archived serum samples from apparently healthy individuals were tested for DV or SARS-CoV-2 IgM/IgG using the lateral flow immunoassays. DV1-4 virus neutralization tests (VNTs) were done with the SARS-CoV-2 antibody (Ab)-positive serums in Huh7 cells. DV3 envelope (env) gene was PCR amplified and sequenced for three archived DV isolates, one from 2017 and two from 2021.Results. SARS-CoV-2 Ab-positive samples constituted 74.5 % of the serums. Of these, 41.5 % were DV cross-reactive and 58.5 % were not. The DV cross-reactive serums neutralized all DV serotypes (DV1-4), as per previous results and this study. The DV non-cross-reactive serums (58.5 %) also cross-neutralized DV1, 2 and 4 but increased DV3 infectivity by means of antibody-dependent enhancement of infection as evident from significantly higher DV3 titres in VNT compared to control serums. The DV3 envelope was identical among the three isolates, including isolate 1 used in VNTs. Our results suggest that DV cross-reactivity of SARS-CoV-2 serums diminished with the shift from Delta to Omicron prevalence. Such COVID-19 serums (DV non-cross-reactive) might have played a major role in causing DV3 surge during the Omicron waves.Conclusion. Patients suspected of dengue or COVID-19 should be subjected to virus/antigen tests and serological tests for both the diseases for definitive diagnosis, prognosis and disease management.

Excessive palmitic acid disturbs macrophage α-ketoglutarate/succinate metabolism and causes adipose tissue insulin resistance associated with gestational diabetes mellitus

Abnormal polarization of adipose tissue macrophages (ATMs) results in low-grade systemic inflammation and insulin resistance (IR), potentially contributing to the development of diabetes. However, the underlying mechanisms that regulate the polarization of ATMs associated with gestational diabetes mellitus (GDM) remain unclear. Thus, we aimed to determine the effects of abnormal fatty acids on macrophage polarization and development of insulin resistance in GDM. Levels of fatty acids and inflammation were assessed in the serum samples and adipose tissues of patients with GDM. An in vitro cell model treated with palmitic acid was established, and the mechanisms of palmitic acid in regulating macrophage polarization was clarified. The effects of excessive palmitic acid on the regulation of histone methylations and IR were also explored in the high-fat diet induced GDM mice model. We found that pregnancies with GDM were associated with increased levels of serum fatty acids, and inflammation and IR in adipose tissues. Increased palmitic acid could induce mitochondrial dysfunction and excessive ROS levels in macrophages, leading to abnormal cytoplasmic and nuclear metabolism of succinate and α-ketoglutarate (αKG). Specifically, a decreased nuclear αKG/succinate ratio could attenuate the enrichment of H3K27me3 at the promoters of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, leading to cytokine secretion. Importantly, GDM mice treated with GSK-J4, an inhibitor of histone lysine demethylase, were protected from abnormal pro-inflammatory macrophage polarization and excessive production of pro-inflammatory cytokines. Our findings highlight the importance of the metabolism of αKG and succinate as transcriptional modulators in regulating the polarization of ATMs and the insulin sensitivity of adipose tissue, ensuring a normal pregnancy. This novel insight sheds new light on gestational fatty acid metabolism and epigenetic alterations associated with GDM.

Epidemiological survey and risk factors associated with Sarcoptes scabiei in wild lagomorphs in Spanish Mediterranean ecosystems

Although mortality caused by Sarcoptes scabiei has been reported in European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis), there is a lack of detailed information regarding the exposure of wild lagomorph species to this parasite. Here, we aimed to determine the seroprevalence and potential risk factors associated with S. scabiei exposure in European wild rabbits and Iberian hares in Mediterranean ecosystems of southern Spain. Between 2018/2019 and 2021/2022 hunting seasons, serum samples from 464 wild rabbits and 132 Iberian hares were collected from 100 hunting grounds in Andalusia (southern Spain). Sera were tested using an in-house indirect ELISA to detect specific anti-S. scabiei antibodies based on the immunodominant protein Ssλ20ΔB3. The overall apparent individual seroprevalence was 15.9% (95/596; 95%CI: 13.0–18.9). Antibodies against S. scabiei were detected in 11.6% (54/464; 95%CI: 8.7–14.5) of the European wild rabbits and 31.1% (41/132; 95%CI: 23.2–39.0) of the Iberian hares. Species (Iberian hare), age (adults) and geographical area (western Andalusia) were identified as risk factors potentially associated with S. scabiei exposure using generalized estimating equation analysis. By applying spatial analysis, two significant cluster of high seropositivity were detected in western and central Andalusia, respectively. The seroprevalence values obtained provide evidence of endemic, widespread and heterogeneous exposure to S. scabiei among wild lagomorph populations in Spanish Mediterranean ecosystems. Our findings underscore the importance of implementing integrated surveillance programs for sarcoptic mange in wild lagomorphs as well as in other sympatric species.