Fluid Samples Research Articles

A methodology to globally assess ectodomain shedding using soluble fractions from the mouse brain.

Ectodomain shedding (ES) is a fundamental process involving the proteolytic cleavage of membrane-bound proteins, leading to the release of soluble extracellular fragments (shed ectodomains) with potential paracrine and autocrine signaling functions. In the central nervous system (CNS), ES plays pivotal roles in brain development, axonal regulation, synapse formation, and disease pathogenesis, spanning from cancer to Alzheimer's disease. Recent evidence also suggests its potential involvement in neurodevelopmental conditions like autism and schizophrenia. Past investigations of ES in the CNS have primarily relied on cell culture supernatants or cerebrospinal fluid (CSF) samples, but these methods have limitations, offering limited insights into how ES is modulated in the intact brain parenchyma. In this study, we introduce a methodology for analyzing shed ectodomains globally within rodent brain samples. Through biochemical tissue subcellular separation, mass spectrometry, and bioinformatic analysis, we show that the brain's soluble fraction sheddome shares significant molecular and functional similarities with in vitro neuronal and CSF sheddomes. This approach provides a promising means of exploring ES dynamics in the CNS, allowing for the evaluation of ES at different developmental stages and pathophysiological states. This methodology has the potential to help us deepen our understanding of ES and its role in CNS function and pathology, offering new insights and opportunities for research in this field.

Unveiling the Pathophysiology of Osteoarthritis in Joint Anatomy

Background: Osteoarthritis (OA) is a prevalent and debilitating joint disorder characterized by the degeneration of articular cartilage and underlying bone. Understanding the pathophysiology of OA is essential for developing targeted therapies and improving patient outcomes. Objective: To explore the underlying pathophysiological mechanisms of osteoarthritis within the context of joint anatomy, focusing on cartilage degradation, synovial inflammation, and subchondral bone changes. Methods: This prospective study was conducted at private hospitals in Karachi from June 2022 to December 2022. Eighty patients aged 45 to 70 years, diagnosed with OA, were included. Detailed clinical evaluations were performed, including pain assessment using the Visual Analog Scale (VAS) and functional status assessment using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Radiographic analysis was conducted using the Kellgren-Lawrence grading scale. Biochemical analysis of serum and synovial fluid was performed to measure levels of collagen type II cleavage products (C2C), cartilage oligomeric matrix protein (COMP), and inflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). Synovial fluid and tissue samples were collected during arthroscopy or joint replacement surgery for molecular pathway analysis. Data were analyzed using SPSS version 25, with correlations and regression analyses performed to identify predictors of disease progression. Results: OA was associated with significant pain and functional impairment, with a mean pain score of 7.2 ± 1.3 on the VAS and WOMAC scores averaging 55 ± 10 for physical function and 30 ± 5 for stiffness. Radiographic analysis showed 25% of patients classified as grade II, 50% as grade III, and 25% as grade IV. Biochemical markers indicated elevated levels of C2C (serum: 150 ± 20 ng/mL; synovial fluid: 200 ± 25 ng/mL) and COMP (serum: 10 ± 2 µg/mL; synovial fluid: 15 ± 3 µg/mL). Inflammatory cytokines were also elevated (IL-1β: 50 ± 5 pg/mL; TNF-α: 75 ± 10 pg/mL; IL-6: 100 ± 12 pg/mL). A strong positive correlation (r = 0.85, p < 0.01) was observed between VAS pain scores and synovial IL-1β levels. Conclusion: The pathophysiology of osteoarthritis involves a complex interplay of biomechanical, biochemical, and cellular factors. The findings from this study enhance the understanding of OA mechanisms and provide a foundation for developing more effective therapeutic strategies aimed at halting or reversing the progression of OA.

BIOM-01. CARBONIC ANHYDRASE IX INTERPLAY WITH LOCAL AND SYSTEMIC IMMUNE RESPONSE IN PEDIATRIC BRAIN TUMORS

Abstract BACKGROUND Brain tumors (BT) remain the leading cause of death among childhood cancers. There is an urgent need to identify novel targets in these patients. BT progression depends on the adaptive responses to hypoxia. Carbonic anhydrase IX (CAIX) helps cancer cells survive in a hypoxic environment and can be potentially as a therapeutic target. The role of CAIX in the interplay between the tumor and immune cells requires in-depth investigation to design effective therapies. METHODS Fresh BT, blood, and intraoperative cerebrospinal fluid (iCSF) samples were collected prospectively from pediatric patients, who underwent brain tumor surgery at Vilnius University Hospital Santaros Klinikos from March 2022 to December 2023. All tumor types were included. Immunohistochemistry (IHC) and flow cytometry (FC) were performed to analyze the expression of CAIX and selected immunological markers in the fresh tumor, iCSF and peripheral blood mononuclear cells (PBMCs). Soluble CAIX content in serum and iCSF was assessed by ELISA. In addition, a retrospective analysis of previously diagnosed pediatric glioma specimens was performed by IHC. RESULTS Twelve patients with heterogeneous BT types have been enrolled prospectively and 17 patients with glioma were reviewed retrospectively. Glioma accounted for 72% (21/29) of all samples. IHC showed that 57% of gliomas were positive for CAIX as well as a moderate correlation between the CAIX score and the expression of CD44, CD206, and CCR2 markers. PD-1/PD-L1 expression was negligible. In the prospective cohort, 83% (10/12) of BT tissue was positive for CAIX by IHC and FC. Soluble CAIX was detectable in 40% of iCSF samples, but not in serum. We found a correlation trend of CAIX expression in the fresh tumor cells and iCSF with increased CCR2 expression on CD45+ PBMCs. CONCLUSIONS This interim analysis showed a correlation between CAIX tumor expression and immune biomarkers, prompting further investigation by enrolling more patients.

STEM-08. ANGIOGENESIS IN PEDIATRIC CHOROID PLEXUS PAPILLOMAS

Abstract BACKGROUND Choroid plexus papillomas (CPP) are rare brain tumors that tend to occur in very young children. Although most of them are curative with surgical resection, a subset of them has propensity for tumor recurrence, progression, and metastasis. Moreover, surgery can be technically difficult due to their deep locations and high vascularity. In-depth mechanisms of their biology remain poorly understood. Separately, the process of angiogenesis has been implicated in several cancers. To date, there has been no previous study focused on CPP and angiogenesis. This study aims to explore and ascertain the existence of angiogenesis in CPP. METHODS This is a study approved by the hospital ethics review board. Cerebrospinal fluid (CSF) and CPP tumor samples are collected at the time of surgery. A multiplex immunoassay panel is used to measure cytokine concentrations in the CSF samples. Identified cytokines of interest are input into online platforms to assess for protein-protein interaction pathways. Concurrently, patient-derived primary cell cultures and their supernatants are derived from CPP samples. A targeted proteome blot array and HUVEC tubule formation assays are used to validate clinical and in silico findings. RESULT CSF profiling showed higher expression of MCP-1, MMP-1, IL-2, TNF-ɑ, TRAIL and CD40-L in CPP patient samples versus non-tumor controls. Next, in silico assessment via STRING and BioGRID platforms report that these cytokines are associated with endothelial cell regulation. This is clinically relevant as we are aware that the endothelium has important functions in the regulation of angiogenesis. Using an angiogenesis-focused approach, CPP-derived cell lines and supernatants showed congruently higher expression of MCP-1, MMP-1 and TNF-ɑ. Next, tubule formation was observed in HUVEC cultures where conditioned CPP cell culture media was added. CONCLUSIONS Based on our preliminary findings, this proof-of-concept study demonstrates potential to explore the role of angiogenesis in CPP for better disease understanding.

Epigenetic age acceleration in follicular fluid and markers of ovarian response among women undergoing IVF.

Are markers of epigenetic age acceleration in follicular fluid associated with outcomes of ovarian stimulation? Increased epigenetic age acceleration of follicular fluid using the Horvath clock, but not other epigenetic clocks (GrimAge and Granulosa Cell), was associated with lower peak estradiol levels and decreased number of total and mature oocytes. In granulosa cells, there are inconsistent findings between epigenetic age acceleration and ovarian response outcomes. Our study included 61 women undergoing IVF at an academic fertility clinic in the New England area who were part of the Environment and Reproductive Health Study (2006-2016). Participants provided a follicular fluid sample during oocyte retrieval. DNA methylation of follicular fluid was assessed using a genome-wide methylation screening tool. Three established epigenetic clocks (Horvath, GrimAge, and Granulosa Cell) were used to predict DNA-methylation-based epigenetic age. To calculate the age acceleration, we regressed epigenetic age on chronological age and extracted the residuals. The association between epigenetic age acceleration and ovarian response outcomes (peak estradiol levels, follicle stimulation hormone, number of total, and mature oocytes) was assessed using linear and Poisson regression adjusted for chronological age, three surrogate variables (to account for cellular heterogeneity), race, smoking status, initial infertility diagnosis, and stimulation protocol. Compared to the median chronological age of our participants (34 years), the Horvath clock predicted, on an average, a younger epigenetic age (median: 24.2 years) while the GrimAge (median: 38.6 years) and Granulosa Cell (median: 39.0 years) clocks predicted, on an average, an older epigenetic age. Age acceleration based on the Horvath clock was associated with lower peak estradiol levels (-819.4 unit decrease in peak estradiol levels per standard deviation increase; 95% CI: -1265.7, -373.1) and fewer total (% change in total oocytes retrieved per standard deviation increase: -21.8%; 95% CI: -37.1%, -2.8%) and mature oocytes retrieved (% change in mature oocytes retrieved per standard deviation increase: -23.8%; 95% CI: -39.9%, -3.4%). The age acceleration based on the two other epigenetic clocks was not associated with markers of ovarian response. Our sample size was small and we did not specifically isolate granulosa cells from follicular fluid samples so our samples could have included mixed cell types. Our results highlight that certain epigenetic clocks may be predictive of ovarian stimulation outcomes when applied to follicular fluid; however, the inconsistent findings for specific clocks across studies indicate a need for further research to better understand the clinical utility of epigenetic clocks to improve IVF treatment. The study was supported by grants ES009718, ES022955, ES000002, and ES026648 from the National Institute of Environmental Health Sciences (NIEHS) and a pilot grant from the NIEHS-funded HERCULES Center at Emory University (P30 ES019776). RBH was supported by the Emory University NIH Training Grant (T32-ES012870). N/A.

(008) VAGINAL TESTOSTERONE ABSORBANCE AFTER SEXUAL AROUSAL

Abstract Introduction While topical (exogenous) estrogens have been a focus of treatment for vaginal pain, recent work has shown testosterone may also revitalize weakened vaginal tissue. This research has documented that vaginal tissue responds well to exogenous testosterone treatment, supporting the hypothesis that endogenous testosterone may play a significant role in vaginal health. However, is it not known how the dynamics of vaginal arousal may contribute to activity of endogenous testosterone within the vaginal environment. Specifically, there is reason to expect that vaginal sexual arousal may activate mechanisms within the vaginal tissue that increase absorption of testosterone from the mucosal layer into the tissue beneath. Objective This project aims to characterize the influence of vaginal arousal on the presence of testosterone in the vaginal mucosa both pre- and post-arousal, and the within-person stability of these effects over time (3 weeks). Methods Analyses are drawn from an ongoing clinical trial in which participants complete 3 in-lab sessions spaced 3 weeks apart, in which they provided samples of cervicovaginal fluid before and after watching an erotic video (~7 minutes). For each fluid sample, participants insert a disposable menstrual cup, which sits loosely around the cervix, for about 10 minutes. After the post-arousal menstrual cup is removed, participants complete a survey battery that includes demographics and measures of sexual function and sexual desire. Results For the present analysis, data were drawn from 68 participants that had pre- and post-arousal samples for at least one study session. Preliminary tests revealed that, controlling for changes in fluid volume, testosterone in the vaginal mucosa decreased significantly between pre- and post-vaginal sexual arousal (F(1, 197.26) = 67.29, p < .0001). Additional data regarding the association between absorption of testosterone during arousal and self-reported sexual functioning will also be presented. Conclusions Though further work is needed to confirm testosterone is absorbed into the surrounding vaginal tissue, our findings suggest a significant movement of testosterone out of cervicovaginal fluid during sexual arousal. Likely, the increased blood flow that accompanies vaginal sexual arousal allows for greater absorption of testosterone originally present in the mucosa into the vaginal tissue. If vaginal sexual arousal allows for greater testosterone absorption, it may provide a unique window of time during which application of exogenous testosterone may be most advantageous. Further, if endogenous testosterone absorption happens during masturbation or sexual activity, this may function over the longer term as a protective factor for tissue integrity. Disclosure No.

The influence of intraoral cryotherapy on postoperative pain and substance P in symptomatic apical periodontitis: randomized clinical study

Cryotherapy is widely utilized in medicine, particularly for pain management. This randomized clinical trial aimed to assess the effect of intraoral cold pack application (cryotherapy) on postoperative pain (POP) and the level of Substance P (SP) in patients with symptomatic apical periodontitis (SAP). Enrolled patients were randomly assigned to either cryotherapy or control group. After adequate anesthesia, access cavity, and biomechanical preparation of the root canal system were completed, the first apical fluid (AF) sample (S1) was obtained. A custom-made intraoral ice-gel pack was applied for 30 min in the cryotherapy group, while no intervention was performed in the control group. The second AF sample (S2) was collected 30 min later in both groups. Patients were asked to complete the Visual Analogue Scale (VAS) questionnaire to assess their POP. Quantification of SP in AF samples was performed using the enzyme-linked immunosorbent assay (ELISA) test. Data were analyzed statistically, revealing a significant reduction in POP and SP levels in the cryotherapy group compared to the control group (P ≤ 0.05). Furthermore, a moderate positive correlation was observed between SP levels and POP (P ≤ 0.05). In conclusion, intraoral cryotherapy represents a simple and cost-effective option for controlling POP and reducing inflammation levels in patients with SAP.

Fabrication of ternary hybrid composites of Co-MOFs/MoS2/PEDOTs for sensitive detection of dopamine and norepinephrine in biological samples

In this study, a simple and multifunctional application for Co-based organic frameworks (Co-MOFs) with molybdenum disulfide (MoS2) and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate (PEDOTs:PSS) is presented. The Co-MOFs with MoS2 exhibit excellent peroxidase mimicking and catalytic activities, making them suitable for colorimetric dopamine sensors. From an electrochemical standpoint, fouling-free and selective sensing of dopamine (DA) and norepinephrine (NE) is highly challenging. However, the synergistic effect of morphological tunability of Co-MOFs with hybrid composites of MoS2 and PEDOTs:PSS offers a wide detection concentration range of 2 nM-350 µM for DA and 20 nM-1000 µM for NE, high sensitivity, excellent anti-fouling, facile kinetic rate reaction, and rapid signal response. A high selectivity rate was achieved in the presence of twenty times higher concentrations of a mixture of ascorbic acid, uric acid, and other bio-chemical co-interferents. Interestingly, ultrasensitive detection limits of 0.23 nM and 2.18 nM (S/N = 3) were achieved for DA and NE, enabling the real-time precise detection of cellular concentrations in PC12 living cells, and biological fluid samples with simple dilution pretreatments. Moreover, the sensor demonstrated satisfactory reproducibility and stability. Finally, the morphological and composite activity-based probes can detect neurotransmitter concentrations in real samples beyond the concentration limit, suggesting a potential clinical sensor tool.

Macrophage-Mimicking Cellular Nanoparticles Scavenge Proinflammatory Cytokines in Specimens of Patients with Inflammatory Disorders.

Effectively neutralizing inflammatory cytokines is crucial for managing a variety of inflammatory disorders. Current techniques that target only a subset of cytokines often fall short due to the intricate nature of redundant and compensatory cytokine networks. A promising solution to this challenge is using cell membrane-coated nanoparticles (CNPs). These nanoparticles replicate the complex interactions between cells and cytokines observed in disease pathology, providing a potential avenue for multiplex cytokine scavenging. While the development of CNPs using experimental animal models has shown great promise, their effectiveness in scavenging multiple cytokines in human diseases has yet to be demonstrated. To bridge this gap, this study selected macrophage membrane-coated CNPs (MФ-CNPs) and assessed their ability to scavenge inflammatory cytokines in serum samples from patients with COVID-19, sepsis, acute pancreatitis, or type-1 diabetes, along with synovial fluid samples from patients with rheumatoid arthritis. The results show that MФ-CNPs effectively scavenge critical inflammatory cytokines, including interleukin (IL)-6, IL-8, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, in a dose-dependent manner. Overall, this study demonstrates MФ-CNPs as a multiplex cytokine scavenging formulation with promising applications in clinical settings to treat a range of inflammatory disorders.

Fine-needle aspiration technique under endoscopic ultrasound guidance: A technical approach for RNA profiling of pancreatic neoplasms.

Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) has been a longstanding challenge. The prognosis of patients with PDAC depends on the stage at diagnosis. It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis. Most molecular studies are done using paraffin-embedded blocks; however, the integrity of DNA and RNA is often compromised in this format. Moreover, RNA isolated from human pancreatic tissue samples is generally of low quality, in part, because of the high concentration of endogenous pancreatic RNAse activity present. To assess the potential of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling, including next-generation sequencing (NGS). Thirty-four EUS-FNA samples were included in this study: PDAC (n = 15), chronic pancreatitis (n = 5), pancreatic cysts (n = 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia) n = 7, serous cystic neoplasms n = 5, and pseudocysts n = 2. Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy (TTNB). Samples were stored at -80 °C until analysis. RNA purity (A260/230, A260/280 ratios), concentration, and integrity (RIN) were assessed. Real-time polymerase chain reaction was conducted on all samples, and small RNA libraries were prepared from solid mass samples. RNA was successfully extracted from 29/34 (85%) EUS-FNA samples: 100% pancreatic adenocarcinoma samples, 100% chronic pancreatitis samples, 70% pancreatic fluid cyst samples, and 50% TTNB samples. The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples (n = 29) including low-quality RNA specimens. Low concentration and nonoptimal RIN values (no less than 3) of RNA extracted from EUS-FNA samples did not prevent NGS library preparation. The suitability of cyst fluid samples for RNA profiling varied. The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7 (5.0-8.2), which was compatible with that from solid neoplasms [6.2 (0-7.8)], whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0. The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens.

A novel approach to cancer screening using the nematode Caenorhabditis elegans-based detection assays.

Early diagnosis of cancer is essential for its effective treatment. Currently, established screening tests are cancer-specific and require screening for each type of cancer separately. The primary objective of cancer research is to develop methods that can detect multiple types of tumors from a single body fluid sample. Multicancer early detection tests aim to detect fragments of circulating tumor DNA, cell-free DNA, circulating microRNAs, or proteins released by cancer cells in the patient's body fluids. However, these tests are not suitable for routine cancer prevention due to their high cost. Therefore, in recent years, cancer screening tests have been developed to detect volatile organic compounds in urine using living organisms, such as nematodes, Caenorhabditis elegans. Measuring only 1 mm in length, C. elegans has the potential to offer a new, efficient, cost-effective, quick, and painless method to detect the presence of tumor. The purpose of this review is to present an overview of the literature on the development and validation of C. elegans-based cancer detection methods. The potential benefits of these assays are significant, as they could become a valuable tool for the early identification and diagnosis of cancer, even though this research is still in its initial stages of development.

Nanopore sequencing for smear-negative pulmonary tuberculosis—a multicentre prospective study in China

PurposeIn this prospective study, the diagnosis accuracy of nanopore sequencing-based Mycobacterium tuberculosis (MTB) detection was determined through examining bronchoalveolar lavage fluid (BALF) samples from pulmonary tuberculosis (PTB) -suspected patients. Compared the diagnostic performance of nanopore sequencing, mycobacterial growth indicator tube (MGIT) culture and Xpert MTB/rifampin resistance (MTB/RIF) assays.MethodsSpecimens collected from suspected PTB cases across China from September 2021 to April 2022 were tested then assay diagnostic accuracy rates were compared.ResultsAmong the 111 suspected PTB cases that were ultimately diagnosed as PTB, the diagnostic rate of nanopore sequencing was statistically significant different from other assays (P < 0.05). Fleiss’ kappa values of 0.219 and 0.303 indicated fair consistency levels between MTB detection results obtained using nanopore sequencing versus other assays, respectively. Respective PTB diagnostic sensitivity rates of MGIT culture, Xpert MTB/RIF and nanopore sequencing of 36.11%, 40.28% and 83.33% indicated superior sensitivity of nanopore sequencing. Analysis of area under the curve (AUC), Youden’s index and accuracy values and the negative predictive value (NPV) indicated superior MTB detection performance for nanopore sequencing (with Xpert MTB/RIF ranking second), while the PTB diagnostic accuracy rate of nanopore sequencing exceeded corresponding rates of the other methods.ConclusionsIn comparison with MGIT culture and Xpert MTB/RIF assays, BALF’s nanopore sequencing provided superior MTB detection sensitivity and thus is suitable for testing of sputum-scarce suspected PTB cases. However, negative results obtained using these assays should be confirmed based on additional evidence before ruling out a PTB diagnosis.

Plasmapheresis and IVIG for Treatment of Non-Tumor Anti-Tr/DNER Antibody-Associated Ataxia: A Case Report.

Autoimmune cerebellar ataxia (ACA) is a condition characterized by progressive ataxia resulting from an immune-mediated attack on cerebellar structures. The presence of anti-Tr/DNER antibodies, strongly associated with Hodgkin lymphoma, has been identified in ACA. However, cases with no underlying malignancy are rare. We report the case of a 49-year-old woman presenting with progressive ataxia, slurred speech, and dizziness over three months. The patient exhibited significant cerebellar symptoms, including dysarthria and limb ataxia, without signs of other systemic illnesses. Comprehensive investigations, including imaging, lumbar puncture, and autoantibody testing, were performed. The cerebrospinal fluid (CSF) sample revealed positivity for Tr/DNER antibodies, leading to a diagnosis of autoimmune cerebellar ataxia. The patient underwent nine sessions of plasmapheresis, followed by six doses of intravenous immunoglobulin (IVIG), resulting in significant clinical improvement. Despite extensive cancer screening, no underlying malignancy was detected, suggesting a non-tumor origin of anti-Tr/DNER antibodies. The patient's gait improved, ataxia resolved, and cerebellar tests normalized following treatment. The patient was further managed with rituximab treatment every six months. This case represents a presentation of anti-Tr/DNER-associated autoimmune cerebellar ataxia without malignancy. The successful treatment with plasmapheresis and IVIG suggests that these interventions may be effective in managing autoimmune cerebellar ataxia associated with anti-Tr/DNER antibodies. Further research is needed to understand the underlying mechanisms of this condition and to determine the optimal treatment strategies.

Diagnostic potential of endothelin-1 in peri-implant diseases: a cross-sectional study

PurposeThis study aimed to evaluate the potential of Endothelin-1 (ET-1), a peptide derived from vascular endothelial cells, as a biomarker for diagnosing peri-implant diseases.MethodsA cohort of 29 patients with a total of 76 implants was included in this study and subsequently divided into three groups based on peri-implant clinical parameters and radiographic examination: healthy (peri-implant health) (n = 29), mucositis (n = 22), and peri-implantitis (n = 25) groups. The levels of ET-1 (ρg/site) and interleukin (IL)-1β (ρg/site) in peri-implant sulcus fluid (PISF) samples were determined using enzyme immunoassay. Statistical analyses were conducted using Kruskal–Wallis and Steel–Dwass tests. Logistic regression and receiver operating characteristic (ROC) curve analyses were performed to evaluate the diagnostic performance of the biomarkers.ResultsET-1 levels were significantly elevated in the peri-implantitis group compared to those in the healthy group, and were highest in the peri-implant mucositis group. Additionally, IL-1β levels were significantly higher in the peri-implantitis group than those in the healthy group. ROC curve analysis indicated that ET-1 exhibited superior area under the curve values, sensitivity, and specificity compared to those of IL-1β.ConclusionsOur findings suggest that the presence of ET-1 in PISF plays a role in peri-implant diseases. Its significantly increased expression in peri-implant mucositis indicates its potential for enabling earlier and more accurate assessments of peri-implant inflammation when combined with conventional examination methods.

Changes in neuroinflammatory biomarkers correlate with disease severity and neuroimaging alterations in patients with COVID-19 neurological complications

COVID-19 induces acute and persistent neurological symptoms in mild and severe cases. Proposed concomitant mechanisms include direct viral infection and strain, coagulopathy, hypoxia, and neuroinflammation. However, underlying molecular alterations associated with multiple neurological outcomes in both mild and severe cases are majorly unexplored. To illuminate possible mechanisms leading to COVID-19 neurological disease, we retrospectively investigated in detail a cohort of 35 COVID-19 mild and severe hospitalized patients presenting neurological alterations subject to clinically indicated cerebrospinal fluid (CSF) sampling. Clinical and neurological investigation, brain imaging, viral sequencing, and cerebrospinal CSF analyses were carried out. We found that COVID-19 patients presented heterogeneous neurological symptoms dissociated from lung burden. Nasal swab viral sequencing revealed a dominant strain at the time of the study, and we could not detect traces of SARS-CoV-2's spike protein in patients' CSF by multiple reaction monitoring analysis. Patients presented ubiquitous systemic hyper-inflammation and broad alterations in CSF proteomics related to inflammation, innate immunity, and hemostasis, irrespective of COVID-19 severity or neuroimaging alterations. Elevated CSF interleukin-6 (IL6) correlated with disease severity (sex-, age-, and comorbidity-adjusted mean Severe 24.5 pg/ml, 95% confidence interval (CI) 9.62–62.23 vs. Mild 3.91 pg/mL CI 1.5–10.3 patients, p = 0.019). CSF tumor necrosis factor-alpha (TNFα) and IL6 levels were higher in patients presenting pronounced neuroimaging alterations compared to those who did not (sex-, age-, and comorbidity-adjusted mean TNFα Pronounced 3.4, CI 2.4–4.4 vs. Non-Pronounced 2.0, CI 1.4–2.5, p = 0.022; IL6 Pronounced 33.11, CI 8.89–123.31 vs Non-Pronounced 6.22, CI 2.9–13.34, p = 0.046). Collectively, our findings put neuroinflammation as a possible driver of COVID-19 acute neurological disease in mild and severe cases.

Bacterial endometritis-induced changes in the endometrial proteome in mares: Potential uterine biomarker for bacterial endometritis

Equine endometritis is one of the main causes of subfertility in the mare. Unraveling the molecular mechanisms involved in this condition and pinpointing proteins with biomarker potential could be crucial in both diagnosing and treating this condition. This study aimed to identify the endometritis-induced changes in the endometrial proteome in mares and to elucidate potential biological processes in which these proteins may be involved. Secondly, biomarkers related to bacterial endometritis (BE) in mares were identified. Uterine lavage fluid samples were collected from 28 mares (14 healthy: negative cytology and culture, and no clinical signs and 14 mares with endometritis: positive cytology and culture, in addition to clinical signs). Proteomic analysis was performed with a UHPLC-MS/MS system and bioinformatic analysis was carried out using Qlucore Omics Explorer. Gene Ontology enrichment and pathway analysis (PANTHER and KEGG) of the uterine proteome were performed to identify active biological pathways in enriched proteins from each group. Quantitative analysis revealed 38 proteins differentially abundant in endometritis mares when compared to healthy mares (fold changes >4.25, and q-value = 0.002). The proteins upregulated in the secretome of mares with BE were involved in biological processes related to the generation of energy and REDOX regulation and to the defense response to bacterium. A total of 24 biomarkers for BE were identified using the biomarker workbench algorithm. Some of the proteins identified were related to the innate immune system such as isoforms of histones H2A and H2B involvement in neutrophil extracellular trap (NET) formation, complement C3a, or gelsolin and profilin, two actin-binding proteins which are essential for dynamic remodeling of the actin cytoskeleton during cell migration. The other group of biomarkers were three known antimicrobial peptides (lysosome, equine cathelicidin 2 and myeloperoxidase (MPO)) and two uncharacterized proteins with a high homology with cathelicidin families.Findings in this study provide the first evidence that innate immune cells in the equine endometrium undergo reprogramming of metabolic pathways similar to the Warburg effect during activation. In addition, biomarkers of BE in uterine fluid of mares including the new proteins identified, as well as other antimicrobial peptides already known, offer future lines of research for alternative treatments to antibiotics.

Potential biomarkers for multiple sclerosis stage from targeted proteomics and microRNA sequencing.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system. There is a need for new circulating biomarkers for multiple sclerosis, in particular, markers that differentiate multiple sclerosis subtypes (relapsing-remitting, secondary progressive and primary progressive multiple sclerosis), as this can help in making treatment decisions. In this study, we explore two classes of potential multiple sclerosis biomarkers-proteins and microRNAs-circulating in the cerebrospinal fluid and serum. Targeted medium-throughput proteomics (92 proteins) and microRNA sequencing were performed on serum samples collected in a cross-sectional case-control cohort (cohort I, controls n = 30, multiple sclerosis n = 75) and a prospective multiple sclerosis cohort (cohort II, n = 93). For cohort I, we also made these measurements in paired cerebrospinal fluid samples. In the cohort I cerebrospinal fluid, we observed differences between multiple sclerosis and controls for 13 proteins, including some previously described to be markers for multiple sclerosis [e.g. CD27, C-X-C motif chemokine 13 (CXCL13) and interleukin-7 (IL7)]. No microRNAs were significantly differentially expressed between multiple sclerosis and controls in the cerebrospinal fluid. In serum, 10 proteins, including angiopoietin-1 receptor (TIE2), and 16 microRNAs were significantly different between relapsing-remitting multiple sclerosis and secondary progressive multiple sclerosis after performing a meta-analysis combining both cohorts. In the prospective part of the study, participants with relapsing-remitting multiple sclerosis were followed for around 3 years, during which time 12 participants converted to secondary progressive multiple sclerosis. In these longitudinally collected serum samples, we observed a peak in granzyme B, A and H proteins around the time of conversion. Single-sample enrichment analysis of serum microRNA profiles revealed that the peak in granzyme B levels around conversion coincides with enrichment for microRNAs that are enriched in CD4+, CD8+ and natural killer cells (e.g. miRNA-150). We identified several proteins and microRNAs in serum that represent potential biomarkers for relapsing-remitting and secondary progressive multiple sclerosis. Conversion to secondary progressive disease is marked by a peak in granzyme B levels and enrichment for immune-related microRNAs. This indicates that specific immune cell-driven processes may contribute to the conversion of relapsing-remitting multiple sclerosis to secondary progressive multiple sclerosis.

Detection of α-thalassemia South-East Asian deletion based on a fully integrated digital polymerase chain reaction system DropXpert S6

ABSTRACT Objectives: This study aimed to establish a droplet digital polymerase chain reaction (ddPCR) assay for South-East Asian (SEA) deletion based on a fully integrated digital PCR system DropXpert S6. Methods: A total of 151 whole blood samples, 10 chorionic villus samples, and 17 amniotic fluid samples were collected, including 106 SEA heterozygotes, 43 normal individuals, 10 Hb Bart's hydrops details, and 19 SEA deletions combined with other genotypes.Genotypes of these samples were determined by the Gap-PCR method. We perform a series of optimizations of the ddPCR system to ensure the performance of the entire ddPCR reaction, such as droplet stability, fluorescence clustering, sensitivity, and accuracy. Results: Our assay exhibited 99.4% (177/178) accuracy compared with the Gap-PCR method, and the minimum detection limit of DNA was 0.1 ng/μL.Both targets have reliable linearity, R2 = 0.9999 for the α-thalassemia SEA deletion allele and R2 = 1 for the wild-type allele. The coefficient of variation for α-thalassemia SEA deletion allele detection at 2 and 10 ng/μL concentrations was 5.42% and 1.91%, respectively. In contrast, the coefficient of variation for wild-type allele detection was 4.06% and 1.83%, demonstrating its high quantitative accuracy. In addition, the DropXpert S6 PCR system showed some advantages over other ddPCR instruments, such as reducing testing costs, simplifying and automating the workflow. Conclusions: The DropXpert S6 PCR system provided a highly accurate diagnosis for α-thalassemia SEA deletion and can be used to detect α-thalassemia as an alternative method.

Proton Nuclear Magnetic Resonance (1H NMR) Metabolomics Study in Serum, Urine, and Cystic Fluid for Differentiating Fertility and Staging of Intra-abdominal Hydatid Cyst in Adults.

Cystic echinococcosis (CE) is a parasitic zoonosis caused by the tapeworm Echinococcus granulosus. Over the past few years, a lot of research has been done on liver illnesses using metabolomics techniques to identify biomarkers which could identify the diseases in its early stages. The present study was done to explore biomarkers in serum, urine, and cystic fluid which would help in differentiating, staging, and assessing fertility of intra-abdominal hydatid cyst by using proton nuclear magnetic resonance (1H NMR) metabolomics. In the study, 28 subjects (16 cases and 12 controls) were enrolled. Staging of hydatid cysts was performed using ultrasonography. In patients complying with case and control definition, blood, urine, and cystic fluid were collected for complete blood count, urine culture, Echinococcus IgG enzyme-linked immunosorbent assay (ELISA), and metabolomic analysis. The 17, 15, and 11 metabolites in serum, urine, and cystic fluid samples were quantified, respectively, to differentiate between case and control group. In this study, we observed that there was a significant downregulation of succinate metabolite in urine samples of cases, down-regulation of five metabolites (isoleucine, valine, histidine, tyrosine and formate) and upregulation of alanine in cystic fluid of cases. Current study demonstrates that metabolomics can be used non-invasively for rapid diagnosis of CE. This is one of the very few studies, which used 1H NMR spectroscopy, to analyze the profile of metabolites in serum, urine, and cystic fluid in cases of CE and controls. Raj N, Pandey A, Roy R, et al. Proton Nuclear Magnetic Resonance (1H NMR) Metabolomics Study in Serum, Urine, and Cystic Fluid for Differentiating Fertility and Staging of Intra-abdominal Hydatid Cyst in Adults. Euroasian J Hepato-Gastroenterol 2024;14(1):30-34.

MPXV DNA kinetics in bloodstream and other body fluids samples

Since spring 2022, the global epidemiology of the monkeypox virus (MPXV) has changed. The unprecedented increase of human clade II MPXV cases worldwide heightened concerns about this emerging zoonotic disease. We analysed the positivity rates, viral loads, infectiousness, and persistence of MPXV DNA for up to 4 months in several biological samples from 89 MPXV-confirmed cases. Our data showed that viral loads and positivity rates were higher during the first two weeks of symptoms for all sample types. Amongst no-skin-samples, respiratory specimens showed higher MPXV DNA levels and median time until viral clearance, suggesting their usefulness in supporting MPXV diagnosis, investigating asymptomatic patients, and monitoring viral shedding. Infectious virus was cultured from respiratory samples, semen, and stools, with high viral loads and collected within the first 10 days. Notably, only one saliva and one semen were found positive for viral DNA after 71 and 31 days from symptoms, respectively. The focus on bloodstream samples showed the best testing sensitivity in plasma, reporting the overall highest MPXV DNA detection rate and viral loads during the 3-week follow-up as compared to serum and whole-blood. The data here presented can be useful for MPXV diagnostics and a better understanding of the potential alternative routes of its onward transmission.