Normal Plasma Research Articles

Simplified Method for Removing Direct Oral Anticoagulant Interference in Mechanical Coagulation Test Systems-A Proof of Concept.

Direct oral anticoagulants (DOACs) cause unwanted interference in various hemostasis assays, including lupus anticoagulant (LA) testing, where false positive and false negative identification may occur. DOAC Stop (DS) is an activated charcoal (AC) product used to specifically and effectively adsorb DOACs from test plasma. This process normally requires plasma treatment, centrifugation and plasma separation prior to tests, but inexperienced operators may also inadvertently transfer residual AC particles, thereby potentially adversely affecting clot detection. We hypothesized that residual DS might not be problematic for mechanical clot detection. We therefore investigated the potential impact of DS and a new DS liquid (DS-L) product on clotting tests using a mechanical clot detection system. Varying concentrations of DS were added to normal and abnormal plasmas with and without DOAC presence. Clotting tests including PT, APTT and dRVVT were performed directly in the analyzer without plasma/DS centrifugation. DS up to double the recommended treatment level had only minor effects on all test results, despite completely obscuring visibility in the plasma/reagent mix. This confirms that the centrifugation step may be able to be omitted when using mechanical detection systems. Should DS carryover into treated plasmas occur, this should not cause issues with testing performed on mechanical clot-sensing devices. Moreover, we hypothesize that DS can be used directly in these systems, without the need for centrifugation, thereby simplifying its many potential applications.

Comparison of fluoroscopy guided inter-laminar epidural platelet-rich plasma versus steroid injection in patients with lumbar radicular pain

Background: Radicular back pain is one of the prevalent causes for low back pain. Objective: The aim of work is to compare the efficacies of fluoroscopic guided inter-laminar epidural injection of platelet-rich plasma (PRP) and epidural steroids in improving lumbar radicular pain. Settings and Design: This study was a prospective randomized controlled clinical trial. Methods: Forty-eight patients were enrolled in this study and divided into two groups. Steroid group (S); A 24 patient, that received fluoroscopic guided inter-laminar epidural injection of 1ml methylprednisolone (40mg/ml) + 4 ml normal saline and platelet rich Plasma group (p); A 24 patients, that received fluoroscopic guided inter-laminar epidural injection of 4.5 ml PRP + 0.5 ml PRP activator (calcium gluconate). Patients were followed up at one week, 4 weeks, and 3 months after the procedure. Results: No differences between study groups as regard pain scores at pretreatment, after 1 and 4 weeks and 3 months of treatment and disability scores at pretreatment, after 4 weeks and 3 months of treatment.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. N/A. This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Decreased Thrombin Generation is Associated with Increased Thrombin Generation Biomarkers and Blood Cellular Indices in Pulmonary Embolism.

Pulmonary embolism (PE) is a heterogenous condition with variable clinical presentations. Thrombin generation potential (TGP) and biomarkers, and blood cellular indices can reflect the underlying pathophysiology and risk stratification of PE. This case-control study analyzed TGP in 209 PE patients from Loyola University, Pulmonary Embolism Response Team program compared to normal human plasma (NHP) controls. The present study evaluates TGP and biomarkers, and cellular indices in relation to PE severity, according to the European Society of Cardiology (ESC) guidelines. Statistical analysis including median with interquartile range (IQR), 2-tailed Wilcoxon Mann-Whitney test, Chi-square test, and Spearman Correlational analysis were performed. There were 209 patients with PE, with an almost equal distribution between sex, and a median age of 63 years. Significant downregulation in peak thrombin and endogenous thrombin potential (ETP), as well as upregulation in lag time, were observed in PE patients versus controls. Biomarker analysis revealed pronounced elevations, with D-dimer demonstrating the most significant increase. Blood cellular indices also rose in PE patients, correlating with disease severity. PE severity was associated with higher TGP and biomarker levels. Mortality rates differed significantly across risk categories and were highest in patients with elevated cellular indices. TGP and biomarkers are intricately linked to PE severity and can aid in risk stratification. Elevated cellular indices are associated with increased mortality, highlighting their potential as prognostic markers. These findings could enhance the precision of PE management strategies.

The diagnostic potential of miR-196a-1 in colorectal cancer

BackgroundColorectal cancer (CRC) is a common malignancy worldwide. MicroRNAs (miRNAs) are important epigenetic alterations that notably impact various physiological and pathological processes by acting as negative regulators of gene expression. Furthermore, they have a vital function in different types of cancers, including CRC. In this research, we evaluated, for the very first time, the expression levels of miR-196a-1 in the tissue and plasma of patients with CRC and also homeobox D8 (HOXD8) as the target gene.Materials and methodsThis study included a collection of 220 plasma and tissue samples from 55 patients diagnosed with CRC, as well as 55 healthy individuals matched by age and sex. Total RNA was extracted from plasma and tissue samples, and then polyadenylation and cDNA synthesis were performed. The expression levels of miR-196a-1 and HOXD8 as target gene was evaluated by quantitative RT-PCR (qRT-PCR) assay. We compared the diagnostic value of plasma miR-196a-1 with that of the circulating tumor markers CA19-9 and CEA using a Receiver Operating Characteristics (ROC) analysis. The association of miR-196a-1 with clinicopathological characteristics was assessed in tissue and plasma samples from patients with CRC.ResultsOur data demonstrated that the expression levels of miR-196a-1 in the tissue and plasma samples of CRC patients were 11.426- and 11.655-fold higher, respectively than those in adjacent normal tissue and plasma samples from normal subjects (p < 0.001). Through ROC curve analysis, it was identified that the sensitivity and specificity of miR-196a-1 for tissue samples, with an AUC of 0.925, were 89% and 98%, respectively. In addition, the sensitivity and specificity for plasma samples with an AUC of 0.801 were 70% and 98%, respectively. These findings reveal that miR-196a-1 is a useful biomarker for discriminating cases from controls. Furthermore, the expression of HOXD8 was not significantly altered in tumor tissue samples compared to adjacent normal tissues (P > 0.05).ConclusionsThese results show that miR-196a-1 has an oncogenic impact and plays a significant role in CRC development. The results also indicate that miR-196a-1 could serve as a novel noninvasive biomarker for the detection of CRC.

Nucleosome profiling at AR binding sites (ARBS) to provide a prognostic tool for metastatic castration-resistant prostate cancer (mCRPC).

42 Background: Prostate cancer is the second leading cause of cancer death for men in the U.S., and metastatic castration-resistant prostate cancer (mCRPC) is the most advanced form. In previous work, we have reported prognostic significance of PTEN-PI3K-AKT pathway and DNA damage response aberrations detected in cell-free DNA (cfDNA) of mCRPC. Here we utilized nucleosome profiling pattern at androgen receptor (AR) binding sites to further investigate AR signaling pathway dysregulation of mCRPC. Methods: Using the next-generation sequencing PredicineScore liquid biopsy assay (low-pass whole genome sequencing, LP-WGS), we profiled cfDNA fragment coverage centered around AR binding sites (ARBS). For 118 mCRPC patient samples, we quantified sample-level ARBS nucleosome profiling abnormality score (ARBS score) by comparing its centric fragment coverage with normal plasma background using standard Z-score. ARBS score was correlated with clinical outcomes including PSA response rate (PSA RR), progression-free survival and overall survival (OS). Results: By comparing ARBS nucleosome profiling and normal plasma background, we found that mCRPC plasma samples had significant ARBS nucleosome depletion, reflective of transcription factor binding and persistent AR signaling. Notably, ARBS nucleosome depletion was prostate cancer specific, and did not occur in other examined cancer types. ARBS score was highly correlated with tumor fraction (R=0.82), indicating that high tumor fraction mCRPC sample had higher AR binding. Importantly, we found that high ARBS score (&gt;2) in pre-treatment samples (n=86) was associated with significantly shorter OS and lower PSA RR compared with low ARBS (≤2) group for patients receiving AR pathway inhibitor (ARPI) or taxane chemotherapy (13.4 vs. 19.4 months, p=7.3×10-3, 45.7% vs. 77.5%, p=5.2×10-3). By integrating genome-wide DNA methylation profiling, we further found that ARBS was hypo-methylated in mCRPC patients, and identified a strong negative correlation between DNA methylation beta value and ARBS score (R=-0.83). Conclusions: ARBS nucleosome profiling abnormality inferred from LP-WGS can serve as a key prognostic tool for mCRPC patients. Patients with high ARBS score had worse clinical outcomes, highlighting the potential clinical utility of cfDNA fragmentomics analysis for mCRPC patient stratification. Integration of mutation, copy number variation and fragmentomics profiling of ARBS may further enhance our understanding of molecular aberrations and treatment outcomes in mCRPC.

SRRM2 may be a potential biomarker and immunotherapy target for multiple myeloma: a real-world study based on flow cytometry detection

Serine/arginine repetitive matrix 2 (SRRM2) has been implicated in tumorigenesis, cancer development, and drug resistance through aberrant splicing; however, its correlation with multiple myeloma (MM) has not been reported. We investigated the potential of SRRM2 as a biomarker and immunotherapeutic target in MM by examining its expression in MM cells using flow cytometry. Our study included 95 patients with plasma cell disease, including 80 MM cases, and we detected SRRM2 expression on plasma cells and normal blood cells to analyze its relationship with clinical profiles. We found widespread positive expression of SRRM2 on plasma cells with little expression on normal blood cells, and its expression on abnormal plasma cells was higher than that on normal plasma cells. Comparative analysis with clinical data suggests that SRRM2 expression on plasma cells correlates with MM treatment response. MM patients with high SRRM2 expression had higher levels of serum β2-mg and LDH, ISS staging, and plasma cell infiltration, as well as high-risk mSMART 3.0 stratification and cytogenetic abnormalities, particularly 1q21 amplification. In patients with previous MM, high SRRM2 expression on plasma cells was associated with higher plasma cell infiltration, high-risk mSMART 3.0 risk stratification, cytogenetic abnormalities, more relapses, and fewer autologous stem cell transplant treatments. In summary, SRRM2 may serve as a novel biomarker and immunotherapeutic target for MM. Its expression level on plasma cells can help in risk stratification of MM and monitoring of treatment response.

Elevated levels of citrullinated fibrinogen in patients with cancer.

Neutrophil released peptidyl arginine deiminase 4 (PAD4) converts arginine residues on plasma proteins into citrulline. Here, we developed an assay to quantify citrullinated fibrinogen. We employed a biotin-conjugated phenylglyoxal (biotin-phenylglyoxal (PG)) compound that selectively labels citrulline. Patient samples were derived from a multicenter prospective cohort study that aimed to identify cancer patients at high risk for venous thromboembolism (VTE). Our data show that cancer patients have higher (median 2-fold increased) citrullinated fibrinogen levels when compared to normal human plasma and a cohort of healthy donors. Our results show that citrullination of fibrinogen is a common posttranslational modification in patients with cancer.

Effect Of Integrated Naturopathic and Yoga Therapies in a Management of Diabetic Foot -A Case Study

A 55-year-old male patient of a security job had a right sole ulcer in his leg and increased sugar level diagnosed as Diabetic foot ulcer (DFU). Hydrotherapy and diet were the main treatments. Yoga therapy is the perfect treatment to reduce stress and mood. The combined effect of Naturopathy in the form of water-based packs, sun-therapy, and nutrition avoided the Amputation. After 4 months of treatment size of the ulcer decreased and the patient was able to walk independently. The blood test relieved normal Hba1c and plasma glucose.

β2-glycoprotein I promotes the clearance of circulating mitochondria.

β2-glycoprotein I (β2-Gp1) is a cardiolipin-binding plasma glycoprotein. It is evolutionarily conserved from invertebrates, and cardiolipin-bound β2-Gp1 is a major target of antiphospholipid antibodies seen in autoimmune disorders. Cardiolipin is almost exclusively present in mitochondria, and mitochondria are present in circulating blood. We show that β2-Gp1 binds to cell-free mitochondria (CFM) in the circulation and promotes its phagocytosis by macrophages at physiological plasma concentrations. Exogenous CFM had a short circulation time of less than 10 minutes in mice. Following infusion of CFM, β2-Gp1-deficient mice had significantly higher levels of transfused mitochondria at 5 minutes (9.9 ± 6.4 pg/ml versus 4.0 ± 2.3 pg/ml in wildtype, p = 0.01) and at 10 minutes (3.0 ± 3.6 pg/ml versus 1.0 ± 0.06 pg/ml in wild-type, p = 0.033, n = 10). In addition, the splenic macrophages had less phagocytosed CFM in β2-Gp1-deficient mice (24.4 ± 2.72% versus 35.6 ± 3.5 in wild-type, p = 0.001, n = 5). A patient with abnormal β2-Gp1, unable to bind cardiolipin, has increased CFM in blood (5.09 pg/ml versus 1.26 ± 1.35 in normal) and his plasma induced less phagocytosis of CFM by macrophages (47.3 ± 1.6% versus 54.3 ± 1.3, p = 0.01) compared to normal plasma. These results show the evolutionarily conserved β2-Gp1 is one of the mediators of the clearance of CFM in circulation.

A comparative in vitro study of the anticoagulant effect of branded versus generic rivaroxaban

BackgroundSeveral generic formulations of rivaroxaban were recently marketed to be used interchangeably with their branded equivalent. However, there have been no previously published studies that directly compared the in vitro anticoagulant effect of branded vs. generic rivaroxaban. The aim of this in vitro study was to compare the effects of three raw rivaroxaban materials, obtained from the branded (Xarelto®) and two generic (Rivarolto® and Rivaroxaban Sandoz®) rivaroxaban formulations on an array of coagulation assays. MethodsA pool of normal plasma was spiked with several concentrations of the three rivaroxaban (range 50–750 ng/ml). The concentrations were assessed with a rivaroxaban calibrated anti-Xa assay and confirmed by ultra-high-performance liquid chromatography-mass spectrometry coupled with tandem mass spectrometry (UHPLC-MS/MS). The following assays were performed: Prothrombin time (PT), activated Partial Thromboplastin time (aPTT), Diluted Russell's Viper Venom Test (dRVVT), Thrombin time (TT), Clauss Fibrinogen, Factor VII, VIII and IX assays, and thromboelastography. ResultsThe results obtained by the three rivaroxaban at similar concentrations were comparable. Increasing concentrations of the three rivaroxaban showed a strong positive correlation with the PT, aPTT and dRVVT assays (r > 0.95, p < 0.01 for all), and a strong negative correlation with the Factors assays (r < −0.95, p < 0.01 for all). TT and Clauss Fibrinogen were not affected by rivaroxaban. No significant difference was identified in the mean assays' results obtained by the three rivaroxaban. ConclusionThis study showed that the branded and generic rivaroxaban exert an identical in vitro anticoagulant effect across a wide range of concentrations.

P-43 Pancreatic insulinoma with delayed challenging diagnosis by 68Ga-DOTATATE PET/CT scan: Case report

Abstract Introduction Insulinoma (I) is most common neuroendocrine pancreatic tumor with an incidence of 0.1-0.3/100000(1). It is faintly more in females and can develop at any age(2). Usually benign, sporadic, less than 2 cm, solitary and in 10% of cases is multiple and happens as a component of multiple endocrine neoplasia syndrome type I (MEN–I)(3). Nonspecific symptoms may lead to delayed or misdiagnosis. Improper insulin secretion causes clinical features of hypoglycemia(2). Laboratory verification, localization followed by surgical removal are the main steps management. Localization may be difficult and delayed as here. Clinical Case A female of 58 years old with episodes of sweating, palpitation and tremor improved by drinking sweets, weight gain and denied any drugs intake. No personal or family history of diabetes or antidiabetic drugs. Body weight was 87 kg. Investigations during the attack showed low plasma glucose of 36 mg/dL, high serum insulin of 94.8 mIU/L (1.7 - 31), elevated C-peptide of 10.6 ng/mL (0.9-4). Cortisol and thyroid function are normal. Abdominal ultrasound (US), CT scan and MRI are normal. Due to failure of localization of Insulinoma, treatment by diazoxide started with no response. Octreotide long-acting repeatable (Octreotide LAR) 20 mg monthly injection was initiated and increased to 30 mg monthly with good improvement and less attacks. After three years, the patient developed abdominal pain and abdominal US showed multiple gall stones. The patient was advised to do 68Ga-DOTATATE PET/CT Scan, which revealed distal pancreatic mass of 26 mm (Figure1A and B). Distal pancreatectomy and cholecystectomy had been done with smooth postoperative period normal normal plasma glucose, insulin and c-peptide with no further hypoglycemia, histopathological examination confirmed Insulinoma (Figure 2A and B). Hypoglycemic symptoms of I, whether neuroglycopenic or sympathoadrenal are precipitated by fasting or exercise and could precede diagnosis by months and even years(3). Insulinoma, should be suggested when patient has typical Whipple's triad, high insulin, c-peptide and proinsulin during the attack(3). Weight gain noted in about 18% of cases(4). Modalities used to localize Insulinoma include trans-abdominal US, CT scan, MRI, EUS (endoscopic ultrasound), intraoperative US, intraoperative palpation and arteriography. An American study considered 68Ga-DOTATATE PET/CT as an adjuvant tool for diagnosis with 90% detection rate (5). A 90-95% of cases are benign, patients have high survival with complete resolution of symptoms after excision with only 5.4% recurrences(6). Malignant I limited to pancreas and responsive to chemotherapy has 5 years survival rate after resection and 19 months for recurrent tumors(6). Conclusion Insulinoma should be considered in patients with typical clinical and laboratory finding. Follow up, assessment and different imaging techniques are main steps in management with teamwork.Figure 1.68Ga-DOTATATE PET/CT Scan68Ga-DOTATATE PET/CT Scan revealed distal pancreatic mass of 26 mm

O-03 Adrenalectomy for Cushing's syndrome during pregnancy

Abstract Introduction Cushing's syndrome (CS) is rare in pregnancy because CS itself is a cause of anovulatory infertility due to excess cortisol secretion. Moreover it is also difficult to diagnose CS in pregnancy because placental production of corticotropin releasing and adrenocorticotropic hormone (ACTH), increased maternal production of estrogen induced corticosteroid binding globulin leads to physiological hypercortisolism during pregnancy. So physicians face a clinical overlap in many features of CS such as weight gain, fatigue, edema, hypertension, hyperglycemia, stretch marks, mood changes that can also be attributed to pregnancy. Thus their coexistence represents a diagnostic challenge. Herein we report a case of CS that was diagnosed and successfully treated during pregnancy. Clinical Case A 43-year-old gravida 3 female was consulted at the 21st week of gestation with persistent high blood pressure and hyperglycemia for a month. She had an obstetric history of 2 spontaneous first term abortion, 2 healthy labor 18 years ago and she was healthy after. She was in such a depressive mood that she was consulted with psychiatry many times. On physical examination edema, proximal weakness of lower extremities and hirsutism (modified Ferriman–Gallwey score of 9) was detected. Initial laboratory tests were all within normal range except potassium level (2.7 mmol/L), the results were not consistent with preeclampsia and there was a suspicious adrenal mass detected on ultrasound. She was currently receiving high dose antihypertensive treatment with methyldopa, calcium channel blockers, rapid acting and detemir insulin for diabetes and i.v. potassium replacement. We ruled out conns syndrome with normal plasma aldosterone renin ratio and found catecholamine levels normal. Hormonal analyses revealed with high plasma cortisol levels, increased nocturnal cortisolemia, a suppressed plasma ACTH and dehydroepiandrosterone sulfate level and distinctly elevated 24-hour urinary cortisol greater than 3-fold the upper normal reference range. Non contrast magnetic resonance imaging demonstrated a 47×32 mm left adrenal mass. Laparoscopic adrenalectomy was performed in the 26th week of gestation and the following histopathology revealed an adrenocortical adenoma. Antihypertensive medication was progressively reduced, glycemic control and hypokalemia reversal were achieved. She was followed up with appropriate glucocorticoid replacement. At 34 weeks of gestation, after sudden labor pain and regular uterine contractions a 2586 g male infant with Apgar scores of 8 was born by cesarean section. They were discharged in good health. Conclusion CS should be kept in mind especially in the case of hypertension and diabetes that started before the 20th gestational age. Analysis of the urinary free cortisol level and circadian rhythm of cortisol is necessary to diagnose. Despite the diagnostic challenge we can achieve better maternal and fetal outcomes with proper management.Figure 1.Non-contrast magnetic resonance imaging of the patient Table 1.Patient's laboratuary test results Table 2.Patient's laboratory test results

Salivary Cortisol and Cortisone Can Circumvent Confounding Effects of Oral Contraceptives in the Short Synacthen Test.

Adrenal insufficiency (AI) is usually diagnosed by low plasma cortisol levels following a short Synacthen test (SST). Most plasma cortisol is bound to corticosteroid-binding globulin, which is increased by estrogen in combined estrogen-progestin oral contraceptives (COCs). Women with AI using COCs are therefore at risk of having an apparently normal plasma cortisol level during SST, which would not adequately reflect AI. This work aimed to test whether salivary cortisol or cortisone during SST is more robust against the COC effect and to calculate the lower reference limits (LRLs) for these to be used as tentative diagnostic cutoffs to exclude AI. Forty-one healthy women on COCs and 46 healthy women without exogenous estrogens underwent an SST with collection of plasma and salivary samples at 0, 30, and 60 minutes after Synacthen injection. The groups were compared using regression analysis with age as covariate and the LRLs were calculated parametrically. SST-stimulated plasma cortisol levels were significantly higher in the COC group vs controls, while mean salivary cortisol and cortisone levels were slightly lower in the COC group. Importantly, COC use did not significantly alter LRLs for salivary cortisol or cortisone. The smallest LRL difference between groups was seen for salivary cortisone. Salivary cortisol and especially salivary cortisone are considerably less affected by COC use than plasma cortisol during SST. Due to similar LRLs, a common cutoff for salivary cortisol and cortisone during SST can be used to exclude AI in premenopausal women irrespective of COC use.

A Case of Haemorrhages caused by Multiple Myeloma Induced Factor Deficiencies: A Bleeding Enigma

A 74-year-old male patient presented with anaemia, thrombocytopenia, and extensive haemorrhages over both thighs and back. A coagulation work-up revealed markedly elevated Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT). Reduced functional values of fibrinogen as determined by clot-based assay (126.4 mg/dL), as well as factor V (17.5%) and factor X (27.8%), were noted. These abnormalities were corrected by mixing studies with normal plasma. Neoplastic plasma cells were seen in the bone marrow biopsy, while abdominal fat biopsies showed no amyloid deposition. Serum electrophoresis and immunofixation were positive for monoclonal gammopathy. The patient was diagnosed with multiple myeloma complicated by multiple acquired factor deficiencies. The patient showed a complete response within six months of treatment, which included bortezomib and cyclophosphamide. Multiple myeloma is a monoclonal proliferation of plasma cells that results in increased production of free immunoglobulin light chains, resulting in increased paraproteins. These paraproteins bind with and inhibit various clotting factors, resulting in their inactivation and depletion. Factor X deficiency in these cases is usually attributed to concomitant amyloidosis, whose fibrils bind to Factor X. However, these deficiencies can occur in the absence of amyloidosis as well. While various reports of this entity have highlighted isolated factor deficiencies including factors V, VII, IX, and X, multiple factor deficiency is a rare occurrence, with no other cases being reported from southern India.

Analysis of the role of dihydromyricetin derived from vine tea (Ampelopsis grossedentata) on multiple myeloma by activating STAT1/RIG-I axis.

Multiple myeloma (MM) is a plasma cell malignancy and remains incurable as it lacks effective curative approaches; thus, novel therapeutic strategies are desperately needed. The study aimed to explore the therapeutic role of dihydromyricetin (DHM) in MM and explore its mechanisms. Human MM and normal plasma samples, human MM cell lines, and normal plasma cells were used for in vitro experiments. Cell counting kit-8 (CCK-8), flow cytometry, and trans-well assays were performed for the assessment of cell viability, apoptosis, migration, and invasion, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the mRNA expression of signal transducer and activator of transcription 1 (STAT1) and retinoic acid-inducible gene I (RIG-I). Western blotting was employed to assess E-cadherin, N-cadherin, signal transducer, STAT1, p-STAT1, and RIG-I protein expression. A tumor xenograft model was used for in vivo experiments. Here, dihydromyricetin (DHM) dose-dependently restrained viability, apoptosis, migration, and invasion, and facilitated apoptosis of U266 cells. After DHM treatment, the E-cadherin level was increased and the N-cadherin level was decreased in U266 and RPMI-8226 cells, suggesting the inhibitory effects of DHM on epithelial-mesenchymal transition (EMT) in MM. Besides, the levels of p-STAT1/STAT1 and RIG-I were down-regulated in MM. However, the STAT1 inhibitor fludarabine undid the suppressive effect of DMH on the malignant characteristics of U266 cells. Also, DHM inhibited MM tumor growth and EMT, and activated STAT1/RIG-I pathway in vivo. Collectively, this study first revealed that DHM can restrain EMT and tumor growth in MM by activating STAT1/RIG-I signaling, which provides a novel drug for the treatment of MM.

Factor H-related protein 1 in systemic lupus erythematosus.

Factor H (FH) is a major soluble inhibitor of the complement system and part of a family comprising five related proteins (FHRs 1-5). Deficiency of FHR1 was described to be linked to an elevated risk of systemic lupus erythematosus (SLE). As FHR1 can partially antagonize the functionality of FH, an altered FHR1/FH ratio could not only enhance SLE vulnerability but also affect the disease expression. This study focuses on the analysis of FH and FHR1 at a protein level, and the occurrence of anti-FH autoantibodies (anti-FH) in a large cohort of SLE patients to explore their association with disease activity and/or expression. We assessed FH and FHR1 levels in plasma from 378 SLE patients compared to 84 healthy controls (normal human plasma, NHP), and sera from another cohort of 84 healthy individuals (normal human serum, NHS), using RayBio® CFH and CFHR1 ELISA kits. Patients were recruited by the Swiss SLE Cohort Study (SSCS). Unmeasurable FHR1 levels were all confirmed by Western blot, and in a subgroup of patients by PCR. Anti-FH were measured in SLE patients with non-detectable FHR1 levels and matched control patients using Abnova's CFH IgG ELISA kit. Overall, FH and FHR1 levels were significantly higher in healthy controls, but there was no significant difference in FHR1/FH ratios between SLE patients and NHPs. However, SLE patients showed a significantly higher prevalence of undetectable FHR1 compared to all healthy controls (35/378 SLE patients versus 6/168 healthy controls; p= 0.0214, OR=2.751, 95% CI = 1.115 - 8.164), with a consistent trend across all ethnic subgroups. Levels of FH and FHR1, FHR1/FH ratios and absence of FHR1 were not consistently associated with disease activity and/or specific disease manifestations, but absence of FHR1 (primarily equivalent to CFHR1 deficiency) was linked to the presence of anti-FH in SLE patients (p=0.039). Deficiency of FHR1 is associated with a markedly elevated risk of developing SLE. A small proportion of FHR1-deficient SLE patients was found to have autoantibodies against FH but did not show clinical signs of microangiopathy.

Effect of Microfluidic and Density gradient techniques to select Frozen-thawed Epididymal Sperm from Bighorn rams.

Gamete preservation of wild species is a priority in conservation programs. However, field techniques to recover sperm from deceased animals may impair its final quality.The cryopreservation process also has its challenges, and the minimal required quality parameters may differ depending on the intended use of the semen.Often, postmortem harvested and cryopreserved sperm may need to undergo a selection process to improve the final quality of the sample. Certain sperm selection techniques that employ centrifugal forces can increase the risk of damaging the cells. In contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage.This study aims to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm using two commercial selection techniques, VetCount Harvester® device (MFD) and BovipureTM (NidaCon International AB, Möndal, Sweden) density gradient centrifugation (DGC).Sperm parameters associated with quality were, progressive motile sperms (PMS), straight velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linearity (LIN), straightness (STR), beat cross frequency (BCF), amplitude of lateral head deviation (ALH) and plasma membrane integrity (PMI). Computer Assisted Sperm Analyzer was used for the semen analysis. Eosin/nigrosine and Spermac stains were used to assess morphology and acrosome integrity, respectively. Two veterinarians counted the total number of morphologically normal sperm with complete acrosome.Mean sperm concentration was higher (P < 0.01) before filtration (129.56 ±53.9 ×106/ml) than DCG (68.42 ±29.18 ×106/ml) and MFD (52.94 ±28.76 ×106/ml). PMS was higher (P <0.01) after selection using the DGC (79.70 ± 11.11) and MFD (81.23 ±5.64) compared to the no sperm-selected controls (45.36 ±14.67). Sperm kinetics parameters (VCL, VSL, VAP, BCF, LIN, and STR) were higher in the DGC (P<0.01). However, ALH (P<0.01) was significantly higher in the MFD and Control. Morphological normal sperm, acrosome integrity, and plasma membrane integrity were higher (P<0.01) in samples processed with DGC and MFD.In conclusion, microfluidics and single-layer centrifugation improved motion parameters, viability, and proportion of morphologically normal sperm with intact acrosomes from frozen-thawed sperm harvested from Bighorn epididymis. The VetCount Harvester® microfluidic device was comparable to DGC and could be an alternative option for sperm selection. Further research is needed to explore the effect of selected sperm using this technique on artificial insemination, re-cryopreservation, and fertility outcomes.

Effects of Renal Denervation on Ouabain‐Induced Hypertension in Rats

Background. Ouabain, a Na+, K+‐ATPase inhibitor, is elevated in hypertensive patients. Evidence suggests ouabain contributes to hypertension mainly through activation of the sympathetic nervous system (SNS). Renal nerves play a vital role in the regulation of SNS activity, so we hypothesize that renal denervation may attenuate the development of ouabain‐induced hypertension. Methods and Results. Forty Sprague‐Dawley rats were divided into following groups (n = 10 each): control group (sham surgery plus intraperitoneal saline injection), RDN group (renal denervation (RDN) plus intraperitoneal saline injection), ouabain group (sham surgery plus intraperitoneal ouabain injection), and ouabain + RDN group (RDN plus intraperitoneal ouabain injection). After eight weeks, compared with the control group, rats in the ouabain group exhibited elevated blood pressure (P &lt; 0.05), increased plasma epinephrine, norepinephrine, angiotensin II, and aldosterone levels (P &lt; 0.05). These indexes could be significantly ameliorated by RDN. RDN also reduced the thickening of aortic tunica media and downregulated the expression of proliferating cell nuclear antigen (PCNA) in the thoracic aorta induced by ouabain. Masson staining and echocardiography showed that myocardial fibrosis and increased left ventricular mass in the ouabain group could be attenuated by RDN. Conclusions. The present study reveals that renal nerves play an important role in the development of ouabain‐induced hypertension. RDN could inhibit the pressor effect and the myocardial remodeling induced by ouabain potentially via inhibiting catecholamine release and vascular smooth muscle cell proliferation. Clinical studies are needed to explore whether RDN may exhibit better antihypertensive effects on hypertensive patients with high plasma ouabain levels as compared to those with normal plasma ouabain levels.

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bβ and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.