Enzyme-linked Immunosorbent Assay-based Method Research Articles

Performance of novel collagen turnover biomarkers to detect increased liver stiffness in MASLD.

Cleavage products from collagen formation and degradation hold potential as first-line biomarkers for the risk of advanced fibrosis in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). Here, we evaluated the performance of PRO-C3, PRO-C6, C4M, PRO-C18L, and the clinical score ADAPT (age, diabetes, PRO-C3, and platelet count) to detect patients with an LSM >8kPa or >12kPa in comparison to the Fibrosis-4 Index (FIB-4). Serum from patients with MASLD (n=269) from six Swedish University Hospitals was analyzed using enzyme-linked immunosorbent assay-based methods. Liver stiffness measurement (LSM) by vibration-controlled transient elastography was performed. The area under the curve (AUC), calibration curves, and net benefit analysis were used. An LSM >8kPa was found in 108 (40.1%) patients. PRO-C3, PRO-C6, C4M, and PRO-C18L had AUCs ranging from 0.48 to 0.62. ADAPT had the highest AUC (0.73, 95% confidence interval [CI]=0.67-0.79) to detect patients >8kPa, compared to FIB-4 (0.71, (95%CI=0.64-0.77, p=0.35), and had a higher net benefit compared to FIB-4 from a probability threshold of 15%. FIB-4 and ADAPT performed equally well to detect patients with an LSM >12kPa, AUC 0.76 versus 0.76, p=0.93. ADAPT seems to be marginally better than FIB-4 in identifying patients with an LSM >8kPa. However, the clinical utility of ADAPT as a first line test is uncertain, especially in low-risk populations. The overall performance of FIB-4 was similar to that of ADAPT in detecting patients with an LSM of >12kPa. Altogether, the results suggest that ADAPT might be useful to detect earlier stages of fibrosis in MASLD, but that FIB-4 remains a first-line test for advanced fibrosis.

Serum level of interleukin-6, interleukin-17A, and interferon-α in hospitalized COVID-19 patients and their clinical correlation: A hospital-based case–control study from North-East India

BACKGROUND AND OBJECTIVES: Dysregulation in the homeostasis of the cytokine pool induces severity in COVID-19 disease, which may result in poor clinical outcomes in severely ill patients. The main objective of this study was to evaluate serum interleukin-6 (IL-6), IL-17A, and interferon-α (IFN-α) in hospitalized COVID-19 patients and its association with the disease severity and clinical outcome. MATERIALS AND METHODS: Fifty-one hospitalized COVID-19 patients and 30 age- and sex-matched healthy volunteers were enrolled in this study. Serum IL-6, IL-17A, and IFN-α were quantified by enzyme-linked immunosorbent assay-based method. Receiver operating characteristic curve (ROC) analysis was performed for the cytokines. RESULTS: Serum IL-6, IL-17A, and IFN-α were significantly higher in COVID-19 patients than healthy control volunteers. Across severity groups, serum IL-6 was significantly higher in the severe group, whereas IFN-α was significantly higher in the mild–moderate group. Serum IL-6 was significantly higher in the deceased group compared to the recovered group. ROC analysis suggests that serum IL-6 can be used as a diagnostic and prognostic marker for severity and clinical outcome, respectively. CONCLUSION: Elevated levels of serum IL-6, IL-17A, and IFN-α in COVID-19 patients indicate cytokine dysregulation following severe acute respiratory syndrome coronavirus 2 infection. Elevated serum IL-6 was associated with disease severity and poor clinical outcome. Elevated IFN-α in mild–moderate group compared to the severe group signifies the antiviral effect of IFN-α may help in limiting disease severity in COVID-19 disease. Our data suggest that IL-6 can be used as a diagnostic and prognostic marker for disease severity and clinical outcome, respectively.

Assessing rupture risk by hemodynamics, morphology and plasma concentrations of the soluble form of tyrosine kinase receptor Axl in unruptured intracranial aneurysms

ObjectiveThis study investigated the relationship between morphology, hemodynamic parameters, and plasma concentrations of the soluble form of tyrosine kinase receptor Axl (sAxl) and their potential role in assessing the intracranial aneurysm rupture risk. MethodsThirty-nine patients were retrospectively recruited and these patients were divided into low and high rupture risk groups based on the PHASES score. Plasma levels of sAxl were measured using an enzyme-linked immunosorbent assay-based method. Computational fluid dynamics were used to calculate the morphological and hemodynamic parameters. Differences between clinical data, morphological-hemodynamic parameters and sAxl level were initially determined using univariate analysis. The variables (p ＜ 0.05) were included in a logistic regression model, and the specificity and sensitivity of the selected parameters were evaluated both graphically and statistically using receiver operating characteristic (ROC) curve methods. ResultsAneurysm size ratio (p = 0.023), and normalized wall shear stress (WSS) (p = 0.02) showed significant differences between the two groups. Plasma concentrations of sAXL with a high rupture risk were significantly higher than the low rupture risk (8.47 ± 4.43 ng/ml vs. 5.37 ± 3.21 ng/ml; p = 0.016). Binary logistic regression analysis indicated that the concentration of sAxl was an independent determinant of high rupture risk (odds ratio=1.41, 95%CI=1.08–1.83, p = 0.011). The combination of sAxl + size ration (SR) + WSS achieved the highest area under the curve (0.849) for predicting rupture risk. ConclusionsUnruptured intracranial aneurysms with a higher rupture risk had a larger SR, lower WSS, and higher plasma sAxl concentration. Combining sAxl, SR, and WSS could help estimate the rupture risk of intracranial saccular aneurysm.

Non-Enzymatic Glycation and Formation of Advanced Glycation End-Products Alters the Activity and Related Kinetic Properties of Aldose Reductase

Aldose reductase was incubated with and without either fructose or glucose for 42 days to initiate the glycation process. The concentrations of fructosamine were measured on every 7th day using the standard nitroblue tetrazolium reagent assay. Carboxymethyllysine formed was determined using enzyme linked immunosorbent assay-based methods, fluorescent end-products were measured using spectrofluorometric methods. Activities were assayed by measuring the absorbance of co-factor nicotinamide adenine dinucleotide phosphate hydrogen at 340 nm. Fructosamine, carboxymethyllysine protein adducts and fluorescent end-products were significantly higher (p < 0.001) when aldose reductase was incubated with fructose or glucose than without. Although the glycation of aldose reductase did not result in the alteration of both the optimum pH and temperature of the enzyme, both the activity and Vmax were increased, whereas Km was decreased. Non-enzymatic glycation of aldose reductase increases both its activity and Vmax, while decreasing its Km. Additionally, glycation did not affect the pH of enzyme and temperature optima.

Bioactive Plasma Mitochondrial DNA Is Associated With Disease Progression in Scleroderma-Associated Interstitial Lung Disease.

Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is characterized by variable clinical outcomes, activation of innate immune pattern-recognition receptors (PRRs), and accumulation of α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs Toll-like receptor 9 (TLR-9) and cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. Human lung fibroblasts (HLFs) from normal donors and SSc-ILD explants were treated with synthetic CpG DNA and assayed for α-SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in 2 independent SSc-ILD cohorts and demographically matched controls. The ability of SSc-ILD and control plasma to induce TLR-9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin-6 (IL-6), and oxidized DNA were measured using electrochemiluminescence and enzyme-linked immunosorbent assay-based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. Normal HLFs and SSc-ILD fibroblasts developed increased α-SMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc-ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR-9 and cGAS/STING activation as well as type I IFN and IL-6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. These findings demonstrate a previously unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

Aptamer‐based ellipsometric sensor for ultrasensitive determination of aminoglycoside group antibiotics from dairy products

Residual antibiotics taken along with food consumed through the food chain are the main cause of the super-bacteria and may damage organs such as liver and kidney. Therefore, monitoring residual antibiotic levels of products in the food chain is both important and a requirement. Maximum residual limits for kanamycin and neomycin are 150 ng mL-1 and 500 ng mL-1 respectively, which are challenging for most sensor platforms. In this paper, a novel method is presented for the determination of antibiotics residues in animal-derived foods. Aptamer-based kanamycin and neomycin biosensors based on the spectroscopic ellipsometer and the surface plasmon resonance-enhanced total internal reflection ellipsometer methods as transducing element were developed. Detection limits of both sensor platforms were in the 0.1-1 nmol L-1 ranges, and the detection range was between the detection limit and 1000 nmol L-1 . Both ellipsometry-based aptasensors can be used as an alternative to the existing enzyme-linked immunosorbent assay-based method in terms of assay time (10 min), detection limit (0.22 ng mL-1 for neomycin and 0.048 ng mL-1 for kanamycin), and detection range. © 2020 Society of Chemical Industry.

An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection.

Hantaviruses, etiologic pathogens responsible for two severe human diseases, exist in areas ranging from Eurasia to America and remain global public health concerns. Conventionally, plaque formation assays have been used for hantavirus titering. However, hantaviruses replicate slowly within cells and produce minimal cytopathic effects, making this technique difficult to master. The improved enzyme-linked immunosorbent assay-based antigen detection method is easier to perform but is still time consuming. Here, we established an enzyme-linked focus formation assay (FFA) for Hantaan virus titering that is twice as fast as traditional assays. Moreover, using this method, we evaluated the effects of favipiravir (T-705) and another influenza virus drug, baloxavir acid (BXA), on hantavirus replication. We found that the endonuclease inhibitor BXA exerted similar anti-hantavirus effects as T-705. Overall, we developed a time-saving method for hantavirus titering and suggest BXA as a potential treatment choice for hantavirus-exposed individuals.

Thrombogenic indices in an evaluation of pregnant Nigerian women with pregnancy loss

Background: Pregnancy losses (PLs) are usually a source of pain and psychological stress to the expectant couples. The association between ABO blood groups and some thrombogenic markers with PL among Nigerian women is mostly unknown. Aim: This study investigates the association between ABO blood group, deficiencies of protein C (PC), and protein S (PS) and PL. Patients and Methods: A cross-sectional study involving 170 pregnant women grouped into two, those with or without a history of clinically- or ultrasonographically recognizable PL. ABO blood groups using the tile method, plasma concentrations of free protein S (fPS) antigen, protein C antigen (PCAg) by the enzyme-linked immunosorbent assay-based method, and PC activity (PCAc) by PROTAC method was determined. Results: There was no difference in mean values between the two groups for PCAg, PCAc, FPS, and blood group (P > 0.05). The chances of PL were; non-O blood group (AOR 1.29; 95% CI 0.65--2.54), deficient PCAg (AOR 1.75; 95% CI 0.87--3.54), and deficient PCAc (AOR 1.05;95%CI 0.25--4.13). There was a very poor correlation of miscarriage with FPS (rho 0.04), PCAg (rho 0.09), and PCAc (rho = 0.05). Conclusion: There was no significant association between PLs and ABO blood group phenotypes, PCAg, PCAc, fPS.

Measurement of the IgM and IgG Autoantibody Immune Responses in Human Serum has High Predictive Value for the Presence of Colorectal Cancer

IntroductionColorectal cancer is a major public health issue, with incidences continuing to rise owing to the growing and aging world population. Current screening strategies for colorectal cancer diagnosis suffer from various limitations, including invasiveness and poor uptake. Consequently, there is an unmet clinical need for a minimally invasive, sensitive, and specific method for detecting the presence of colorectal cancer and pre-malignant lesions. Patients and MethodsAn indirect enzyme-linked immunosorbent assay was used to measure the primary (IgM) and secondary (IgG) adaptive humoral immune responses to a panel of previously identified cancer antigens in the sera of normal and adenoma samples, and sera from patients with colorectal cancer. ResultsAn optimal panel of 7 biomarkers capable of identifying patients with colorectal cancer as distinct from both normal and adenoma samples is identified. The cumulative sensitivity and specificity of the assay are 70.8% and 86.5%, respectively. The positive and negative predictive values of the cohort are 77.3% and 82.1%. This assay was not able to accurately discriminate between normal and adenoma samples. Patients whose serum was positive for the presence of anti-ICLN IgM autoantibodies had a significantly poorer 5-year survival than patients whose serum was negative (P = .004). ConclusionThis study describes a novel minimally invasive enzyme-linked immunosorbent assay-based method, capable of identifying patients with colorectal cancer as distinct from both normal and adenoma samples. Patients are likely to be far more amenable to a blood-based test such as the one described herein, rather than a fecal-based test, likely leading to increased patient uptake.

Elevated thrombopoietin and platelet indices confirm active thrombopoiesis but fail to predict clinical severity of puumala hantavirus infection.

We evaluated the mechanisms of thrombocytopenia and procoagulant changes in relation with clinical variables in a cohort of patients with acute hantavirus disease.Blood samples of 33 prospectively recruited, consecutive, hospitalized patients with acute Puumala virus–induced hemorrhagic fever with renal syndrome (HFRS) were collected acutely and at the recovery visit (control). Serum thrombopoietin (TPO) and activity of plasma microparticles (MPs) from various cell sources were measured with enzyme-linked immunosorbent assay-based methods. The results were related to data on platelet indices and functions, coagulation variables, and clinical disease.Serum TPO was nearly 4-fold higher acutely compared with the control (median 207 pg/mL, range 56–1258 pg/mL vs. median 58 pg/mL, range 11–241 pg/mL, P < 0.001) and coincided with high mean platelet volume (MPV) and immature platelet fraction (IPF%). Prothrombin fragments and D-dimer were high acutely compared with the control (F1 + 2 median 704 pmol/L, range 284–1875 pmol/L vs. median 249 pmol/L, range 118–556 pmol/L, P < 0.001; d-dimer median 2.8 mg/L, range 0.6–34.0 mg/L vs. median 0.4 mg/L, range 0.2–1.1 mg/L, P < 0.001), and associated with low platelet count and severe acute kidney injury (AKI). MPs’ procoagulant activity was high acutely only among patients with mild AKI (plasma creatinine below the median at the time of the measurement).Upregulated TPO together with high MPV and IPF% confirm active thrombopoiesis, but do not predict severity of HFRS. Simultaneously, elevated prothrombin fragments and d-dimer suggest increased consumption of platelets in patients with severe AKI. Activity of platelet-derived MPs in HFRS should be studied with flow cytometry in a larger cohort of patients.

Elevated matrix metalloproteinase-9 expression may contribute to the pathogenesis of bladder cancer.

The present study investigated the potential association between matrix metalloproteinase-9 (MMP-9) expression and the pathogenesis of bladder cancer. The present study reviewed previous studies published in Chinese and English using predefined selection criteria, which identified high-quality studies concerning MMP-9 and bladder cancer. Statistical analyses of the data were conducted using Comprehensive Meta-Analysis software version 2.0. In total, 23 case-control studies were selected, which consisted of 1,040 bladder cancer patients and 244 healthy controls. The expression rates and protein levels of MMP-9 were significantly increased in bladder cancer patients compared with the healthy controls, which was demonstrated using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay-based methods. Furthermore, the expression rate of MMP-9 in histological G1/G2 grade bladder cancer tumors was significantly decreased compared with G3 tumors. Subgroup analysis based on ethnicity demonstrated that the rate of MMP-9 protein expression between bladder cancer patients and healthy controls was significantly different in African, Asian and Caucasian patients, which was identified using IHC. The MMP-9 protein levels in bladder cancer patients and healthy controls were significantly different between Asian and Caucasian patients, but not African patients. The differences between MMP-9 expression in ethnic groups were also evident in the expression rate of MMP-9 identified in histological G1/G2 grade tumors in Asian and Caucasian patients compared with G3 grade tumors, which was not evident in African patients. In conclusion, the present meta-analysis results markedly indicate that MMP-9 expression is associated with clinicopathological features of bladder cancer, suggesting that MMP-9 may be a useful biomarker in the diagnosis and clinical management of bladder cancer, and may be a valuable therapeutic target.

Phylogenetic analysis, fumonisin production and pathogenicity ofFusarium fujikuroistrains isolated from rice in the Philippines

Fusarium fujikuroi Nirenberg is a maize and rice pathogen causing important agricultural losses and produces fumonisins - mycotoxins which pose health risk to humans and farm animals. However, little information is available about the phylogenetics of this species and its ability to produce fumonisins in rice. We studied 32 strains isolated from rice in the Philippines and performed a phylogenetic analysis using the partial sequence of Elongation Factor 1 alpha (EF-1α) including isolates belonging to closely related species. Fumonisin B1 (FB1 ) production was analyzed in 7-day-old cultures grown in fumonisin-inducing medium by an enzyme-linked immunosorbent assay-based method and by real-time reverse transcriptase-polymerase chain reaction using primers for FUM1 gene, a key gene in fumonisin biosynthesis. Nucleotide diversities per site (π) were 0.00024 ± 0.00022 (standard deviation) for the 32 F. fujikuroi strains from the Philippines and 0.00189 ± 0.00143 for all 34 F. fujikuroi strains, respectively. F. fujikuroi isolates grouped into one cluster separated from the rest of isolates belonging to the closely related F. proliferatum and showed very low variability, irrespective of their geographic origin. The cluster containing strains of F. proliferatum showed higher intraspecific variability than F. fujikuroi. Thirteen of the 32 strains analyzed were FB1 producers (40.62%), with production ranging from 0.386 to 223.83 ppm. All isolates analyzed showed FUM1 gene expression above 1 and higher than the CT value of the non-template control sample. Both seedling stunting and elongation were induced by the isolates in comparison with the control. F. fujikuroi are distinct from F. proliferatum isolates based on phytogenetic analysis and are potential fumonisin producers because all are positive for FUM1 gene expression. No relationship between fumonisin production and pathogenicity could be observed.

Pregnancy-induced changes in immune protection of the genital tract: defining normal

Both the state of pregnancy as well as disruption of vaginal flora and immune mediators may increase the risk of human immunodeficiency virus-1 acquisition. The objective of this study was to define immune changes in lower genital and systemic immunity associated with normal pregnancy. This prospective cohort enrolled low-risk pregnant and nonpregnant women ages 18-35 years. Pregnant women at <14 weeks and nonpregnant women in follicular phase of the menstrual cycle were included. Cervical and vaginal fluid was collected. Concentrations of immune mediators were measured using enzyme-linked immunosorbent assay-based methods or multiplex immunoassay. Samples were inoculated onto various culture media allowing for growth of Lactobacillus species, Gardnerella vaginalis, Escherichia coli, Enterococcus species, anaerobic gram-negative rods, Candida, Staphylococcus aureus, Ureaplasma species, and Mycoplasma hominis. Concentrations of immune mediators and vaginal colonization frequencies were compared between the pregnant and nonpregnant groups. Genital tract concentration of interleukin-1β was higher during pregnancy compared to nonpregnant participants. Serum C-reactive protein concentrations were higher in all trimesters of pregnancy. Concentrations of secretory leukocyte protease inhibitor did not differ between groups. Lactobacillus was more commonly isolated from vaginal cultures of nonpregnant participants (100% vs 70.2%, P = .02). Identification of Candida, G vaginalis, M hominis, and S aureus was common and not different between groups. Ureaplasma species was isolated from >60% pregnant participants. The proinflammatory cytokine, interleukin-1β, as well as the systemic marker of inflammation, C-reactive protein, are increased during pregnancy. The impact of these proinflammatory changes during pregnancy deserves further study.

Analyses of diversity among fungicidal Anabaena strains

This study characterized 28 Anabaena strains exhibiting fungicidal activity using different molecular approaches. Based on 16S rDNA and internal transcribed spacer region sequences, all the Anabaena strains isolated from India were found genetically diverse from the other reference strains in the National Center for Biotechnology Information database. Out of these, six belonging to five different species were found to produce microcystin toxins as confirmed by polymerase chain reaction amplification of microcystin synthase gene and an enzyme-linked immunosorbent assay-based method. All the sequenced Anabaena strains with the fungicidal trait formed a separate group in both the trees, when compared to other reference Anabaena strains. Thus, it can be hypothesized that the antifungal trait has led to evolutionary divergence among these strains. This study illustrates the diversity among the Anabaena strains with fungicidal activity and their uniqueness vis a vis Anabaena strains available globally.

Enzyme-Linked Immunosorbent Assay-Based Method to Quantify the Association of Small Molecules with Aggregated Amyloid Peptides

This paper describes a simple enzyme linked immunosorbent assay (ELISA) protocol for quantifying the binding of small molecules to aggregated β-amyloid (Aβ) peptides. Amyloid-targeting small molecules have attracted wide interest as potential agents for the treatment or diagnosis of neurodegenerative disorders such as Alzheimer's disease. The lack of general methods to evaluate small molecule-amyloid binding interactions, however, has significantly limited the number of amyloid-targeting molecules that have been studied to date. Here, we demonstrate a general method to quantify small molecule-amyloid binding interactions via a modified quantitative ELISA protocol. A key feature of this protocol is the treatment of commercial ELISA plates with an air plasma to help maintain the desired β-sheet content of the aggregated Aβ upon immobilization of these peptides on to the polystyrene surface. We developed an ELISA-based competition assay on these air plasma-treated plates and evaluated the binding of five previously known amyloid-binding small molecules to aggregated Aβ. We show that this general ELISA-based competition assay can be used to quantify small molecule-amyloid binding interactions in the low nanomolar to low micromolar range, which is the typical range of affinities for many amyloid-targeting diagnostic agents under current development. This simple protocol for quantifying the interaction of small molecules with aggregated Aβ peptides overcomes many limitations of previously reported spectroscopic or radioactivity assays and may, therefore, facilitate the screening and evaluation of a more structurally diverse set of amyloid-targeting agents than had previously been possible.

Hyperosmolar glucose induces vasoconstriction through Rho/Rho‐kinase pathway in the rat aorta

Rho/Rho-kinase signalling pathway plays a substantial role in vascular contractions. In this study, we investigated any roles of Rho/Rho-kinase pathway in the vasoconstriction of the rat conductance and capacitance vessels by hyperosmolar glucose solution. Isolated aortic, mesenteric and renal rings were suspended and exposed to hyperosmolar glucose, sucrose and NaCl in the organ chambers filled with Krebs solution gassed with 95% O2 and 5% CO2 and maintained at 37 °C. The effect of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10(-5) M), was tested on the contraction induced by hypertonic solutions. Endothelial integrity was also assessed after hyperosmolar glucose exposure. Moreover, the activity and expression of Rho-kinase (ROCK-2) as well as RhoA translocation were detected by Western blotting and enzyme-linked immunosorbent assay-based RhoA activity detection method detection kit. The vessels produced substantial contractions in response to hyperosmolar solutions. Y-27632 significantly reduced hyperosmolarity-induced vasoconstrictions (P < 0.05). Phosphorylation of myosin-phosphatase target 1 increased after hyperosmolar glucose exposure, and this phosphorylation was significantly decreased by Y-27632 (P < 0.05) in the aorta. Furthermore, RhoA translocation but not ROCK-2 expression markedly increased by hyperosmolar glucose solution. These results may indicate that hyperosmolarity could induce vasoconstriction through Rho/Rho-kinase signalling.

Electrochemical detection of tyrosine derivatives and protein tyrosine kinase activity using boron-doped diamond electrodes

In this report, we determined protein tyrosine kinase (PTKs) activity in human epidermoid carcinoma cells (A431) by employing a novel electrochemical method using boron-doped diamond (BDD) electrodes that enables the electrochemical oxidation of tyrosine (Tyr), phosphorylated Tyr (Tyr-P) and sulfated Tyr (Tyr-S) in water-based solutions. Cyclic voltammetry for Tyr, Tyr-P and Tyr-S showed well-defined oxidation peaks at 0.8 V for Tyr, 1.4 V for Tyr-P and 1.7 V for Tyr-S, respectively. Very little work has been reported previously on the detection of Tyr-P and Tyr-S, probably due to their high oxidation potentials. We utilized electrochemical methods for the detection of kinase activity in connection with poly(Glu-Tyr) modified magnetic beads. Linear-sweep voltammograms for the electrochemical detection of PTKs activity were carried out using BDD electrodes consisting of peptide-modified magnetic beads. Without phosphorylation of the peptide-modified magnetic beads using PTKs, we observed clear oxidation peaks for Tyr oxidation and no significant electrochemical responses for Tyr-P oxidation at 1.4 V for the background. On the other hand, with phosphorylation of the beads using PTKs, the peak oxidation current at 1.4 V clearly increased, while the peak oxidation current for Tyr oxidation decreased. This indicates that PTKs activity could be successfully detected by using electrochemical methods employing BDD electrodes. This method was utilized for the in vitro kinase activity detection of human cell lysate, and the electrochemical measurements were compatible with the Enzyme-Linked ImmunoSorbent Assay based method. Our results indicate that the electrochemical method can be applied to real samples such as cell lysate.

Relaxin's Involvement in Extracellular Matrix Homeostasis

Burgeoning evidence suggests that the hormone relaxin modulates collagen in the extracellular matrix of diverse tissues. In separate lines of study, we provide further substantiation of this hypothesis. Immunofluorescence was used to probe isolated fibroblasts derived from volar oblique ligament explant culture for vimentin, actin, RXFP1, and estrogen receptor beta. Ligaments were obtained as surgical waste from thumb reconstruction patients. Four specimens have been examined to date. Cells derived from these patients expressed vimentin and actin, consistent with fibroblast morphology. Putative fibroblasts derived from two of three female patients expressed RXFP1 receptors; the solitary male was negative. Given the small sample, however, the data are considered preliminary. Immunohistochemistry was used on frozen sections from 26 skin biopsies obtained from children undergoing genitoplasty. A subset of samples was also probed for transforming growth factor (TGF-beta1) and TGF-beta3. Appropriate controls were used. Finally, a subset of patient blood was assayed for relaxin by using an enzyme-linked immunosorbent assay-based method. The results showed RXFP1 receptor expression in the cells that populate the basement membrane in 96% of patients, regardless of gender. Most tissue expressed TGF-beta. Finally, serology suggested that relaxin was detectable in these children. Our two lines of research provide additional evidence for the diverse tissue tropism of relaxin. In particular, connective tissues as diverse as ligaments and basal lamina keratinocytes express RXFP1. These data lend support to our contention that relaxin affects ligament integrity and wound healing.

Plasma 1,25 Dihydroxy Vitamin D3 Level and Expression of Vitamin D Receptor and Cathelicidin in Pulmonary Tuberculosis

Vitamin D(3), which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D(3) levels and increased risk of tuberculosis have been reported. Plasma 1,25 dihydroxy vitamin D(3) levels (1,25(OH)(2) D(3)) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D(3). VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. 1,25(OH)(2) D(3) were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)(2) D(3) levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D(3)-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). The present study suggests that PTB patients may have increased 1,25(OH)(2) D(3) levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)(2) D(3) might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.

Global Epiproteomic Signatures Distinguish Embryonic Stem Cells from Differentiated Cells

Complex organisms contain a variety of distinct cell types but only a single genome. Therefore, cellular identity must be specified by the developmentally regulated expression of a subset of genes from an otherwise static genome. In mammals, genomic DNA is modified by cytosine methylation, resulting in a pattern that is distinctive for each cell type (the epigenome). Because nucleosomal histones are subject to a wide variety of post-translational modifications (PTMs), we reasoned that an analogous "epiproteome" might exist that could also be correlated with cellular identity. Here, we show that the quantitative evaluation of nucleosome PTMs yields epiproteomic signatures that are useful for the investigation of stem cell differentiation, chromatin function, cellular identity, and epigenetic responses to pharmacologic agents. We have developed a novel enzyme-linked immunosorbent assay-based method for the quantitative evaluation of the steady-state levels of PTMs and histone variants in preparations of native intact nucleosomes. We show that epiproteomic responses to the histone deacetylase inhibitor trichostatin A trigger changes in histone methylation as well as acetylation, and that the epiproteomic responses differ between mouse embryonic stem cells and mouse embryonic fibroblasts (MEFs). ESCs subjected to retinoic acid-induced differentiation contain reconfigured nucleosomes that include increased content of the histone variant macroH2A and other changes. Furthermore, ESCs can be distinguished from embryonal carcinoma cells and MEFs based purely on their epiproteomic signatures. These results indicate that epiproteomic nucleosomal signatures are useful for the investigation of stem cell identity and differentiation, nuclear reprogramming, epigenetic regulation, chromatin dynamics, and assays for compounds with epigenetic activities. Disclosure of potential conflicts of interest is found at the end of this article.