Disposable label-free electrochemical immunosensor based on gold nanoparticles-Prussian blue for neutrophil gelatinase-associated lipocalin detection in urine samples.

Neutrophil gelatinase-associated lipocalin (NGAL) is a remarkable biomarker for assessing acute kidney injury. In this study, we developed a novel label-free NGAL electrochemical immunosensor based on gold nanoparticles (AuNPs) and Prussian blue (PB) without an external mediator. The AuNPs-PB based immunosensor was fabricated on a custom gold-electrode (AuE)-based polypropylene (PP) substrate. We systematically assessed and optimized key experimental parameters, including the process of AuNPs-PB electrodeposition, antibody concentration, and incubation time. The immunosensor response toward NGAL was determined using differential pulse voltammetry, where the decrease in the oxidation current response of the PB redox probe correlating with the increase in NGAL concentration. Our results demonstrated that the synergistic benefits of both AuNPs and PB significantly improved electrochemical activity for NGAL detection and provided a highly stable sensor across a range of pH values. The label-free immunosensor exhibited two linear ranges: 0.10-1.40 ng mL-1 and 1.40-25.0 ng mL-1, with a low detection limit of 0.094 ng mL-1. The developed NGAL immunosensor displayed high selectivity and excellent reproducibility. Furthermore, NGAL detection was completed within 30 min and the immunosensor exhibited storage stability for six weeks. Notably, NGAL levels determined in human urine samples using this developed label-free immunosensor showed good agreement with the results obtained from the enzyme-linked immunosorbent assay. This novel label-free NGAL immunosensor provides great potential in developing NGAL point-of-care testing applications.

Glucose Interference in Serum and Urine Samples with Various Creatinine Concentrations Measured by the Jaffe Kinetic Method.

Background: The effect of glucose interference on creatinine measurement by Jaffe kinetic method differs between serum and urine specimens. We investigated the effects of creatinine concentration and specimen dilution on glucose interference with urine creatinine measurement. Methods: Leftover serum and urine specimens were collected and stored at -20°C until study. Serum specimens were mixed to make 5 glucose concentrations ranging from <5.6 to 27.8 mmol/L, each group consisting of 5 levels of creatinine concentration ranging from <45 to 354 µmol/L. Urine specimens were divided into 5 groups of creatinine concentration ranging from <1,769 to >7956 µmol/L, each sample was spiked with glucose powder to produce 5 aliquots with glucose concentrations ranging from 0 to 666 mmol/L. Urine samples were automatically diluted 1:20 before analysis. Percent interference of creatinine measurement by Jaffe kinetic method was calculated using enzymatic method as the reference. Results: A total of 148 serum samples and 335 urine samples were analyzed. In serum, glucose interference with Jaffe creatinine measurement was found if creatinine concentrations were 177 µmol/L or less, corresponding to 3,540 µmol/L or less in urine specimens prior to 1:20 dilution. The degree of interference was greater when glucose concentration was higher or creatinine concentration was lower. Conclusions: When creatinine concentration and specimen dilution were considered, the effects of glucose interference on Jaffe creatinine measurement were similar in serum and urine specimens, and was found when creatinine concentrations in serum or diluted urine were 177 µmol/L or less.

Levels of blood pressure, cardiovascular biomarkers and their correlations in women with previous pre-eclamptic pregnancy within 7 years postpartum: a cross-sectional study in Thailand.

OBJECTIVE: To assess the levels of blood pressure, cardiovascular biomarkers and their correlations measured within 7 years postpartum in women with previous pre-eclamptic pregnancies compared with women with previous normotensive pregnancies. DESIGN: Cross-sectional study. SETTING: Two tertiary hospitals in the southern region of Thailand. PARTICIPANTS: Women with pre-eclamptic and normotensive pregnancies in the past 7 years were enrolled from 1 October 2019 to 30 April 2021. Eligible women were interviewed, examined for body mass index (BMI) and blood pressure, and donated morning spot urine and blood samples. PRIMARY OUTCOME MEASURES: Serum high-sensitivity C reactive protein, creatinine, fasting blood glucose (FBS), glycated haemoglobin (HbA1c), low-density lipoprotein (LDL) cholesterol, urine microalbumin to creatinine ratio (UACR) and sodium were measured. Group differences in biomarkers were tested using unpaired t-test, Wilcoxon rank-sum test or χ2 test. The levels of blood pressure and biomarkers between the two study groups at <2 years, 2-4 years and >4 years were also compared. The correlations between blood pressure and biomarkers were analysed using Pearson's correlation and partial correlation methods. RESULTS: From 206 women included in the analysis, 88 had pre-eclamptic pregnancies and 118 had normotensive pregnancies. Compared with women with previous normotensive pregnancies, women with previous pre-eclamptic pregnancies had significantly increased rates of hypertension (31.8% vs 7.6%, p<0.001) and obesity (55.7% vs 40.7%, p=0.038), as well as higher serum levels of FBS (p<0.001), HbA1c (p<0.001), LDL cholesterol (p=0.03), creatinine (p<0.001) and UACR (p<0.001). Correlation coefficients of BMI, serum creatinine and UACR with blood pressure ranged from 0.27 to 0.31. CONCLUSION: The risk of hypertension after a pre-eclamptic pregnancy increased. Blood pressure measurement combined with BMI, serum creatinine and UACR screening at least once during 7 years postpartum is suggested for early detection of cardiovascular risk.

Relationship of anthropometric measurements with glycated hemoglobin and 1-h blood glucose after 50 g glucose challenge test in pregnant women: A longitudinal cohort study in Southern Thailand.

AIMS: To assess correlations of anthropometric measurements with glycated hemoglobin (HbA1c) and 1-h blood glucose after a 50 g glucose challenge test during the first and late second trimesters and explore their relationships of anthropometric measurements with neonatal birth weight. METHODS: A longitudinal study was conducted among pregnant Thai women with gestational age ≤14 weeks. Anthropometric measurements, using body mass index, body compositions, and circumferences, and skinfold thickness, were measured at four-time points: ≤14, 18-22, 24-28, and 30-34 weeks of gestation. HbA1c and 1-h blood glucose were examined at ≤14 and 24-28 weeks. Neonatal birth weight was recorded. RESULTS: Of 312 women, HbA1c was more correlated with anthropometric measurements during pregnancy than 1-h blood glucose. At 24-28 weeks, women with high/very high body fat percentage were more likely to have higher HbA1c. Women with high subscapular skinfold thickness were more likely to have higher 1-h blood glucose at ≤14 and 24-28 weeks. High hip circumference significantly increased neonatal birth weights. CONCLUSION: Anthropometric measurements were longitudinally correlated with HbA1c and 1-h blood glucose, higher in the late second than first trimesters, as well as neonatal birth weight. The mechanisms to explain the relationship of different anthropometric measurements are required to be further studied.

Associations between HbA1c-derived estimated average glucose and fasting plasma glucose in patients with normal and abnormal hemoglobin patterns.

Fasting plasma glucose (FPG) and HbA1c are well-known tests for monitoring short and long-term glycemic control, respectively. Estimated average glucose (eAG) is derived from the HbA1c for expression in the same units as FPG. The present study aimed to evaluate the association between eAG and FPG values. This retrospective study was performed on 1285 patients who measured the FPG and HbA1c (capillary electrophoresis method) values on the same day. They were initially divided into 3 hemoglobin-pattern groups, normal, HbE, and high HbA2, and then these 3 groups were further subdivided into whole group, subgroup 1 (HbA1c ≤ 7%) and subgroup 2 (HbA1c >7%) for analytical comparisons. The eAG values were calculated using Nathan's equation. The differences, agreements, and correlations between eAG and FPG were evaluated. Good agreements and strong positive correlations between eAG and FPG values were observed in all 3 whole groups. In subgroup analysis, the degrees of agreement and correlation depended on the level of glycemic control, as all associations became stronger with better glycemic control. Additionally, the HbA1c levels and different eAG-FPG values between the normal and other groups were not significantly different in either whole group or subgroup comparisons. Our study found good agreements and strong positive correlations between eAG and FPG in all groups, indicating that high HbA2 or HbE did not affect the HbA1c levels or thus the associations between eAG and FPG. Reporting the eAG together with the HbA1c value should help improve the understanding of glycemic status in patients, leading to improved blood glucose control.

The Direct Spectrophotometric Method Is Reliable for Initial Assessment of Total Bilirubin in Neonatal Venous Plasma.

OBJECTIVE: To determine the consistency of bilirubin values between the direct spectrophotometric (DS) and colorimetric diazo (diazo) methods in neonatal venous plasma specimens. METHODS: We measured the total bilirubin via the DS and diazo methods in 255 neonatal venous plasma specimens and compared the overall and subgroup results. RESULTS: Slight underestimation of total bilirubin values in most specimens using the DS method was observed, with higher mean biases found in higher concentrations. Significantly high positive correlations were found in all groups in which most of the different values were within the limits of agreement. DS cutoff of > 12 mg/dL showed 100% for all predictive indices in comparison with the diazo cutoff > 15 mg/dL. CONCLUSIONS: Measurement of total bilirubin in neonatal venous plasma using the DS method had favorable agreement and high correlation with the diazo method. Therefore, the direct spectrophotometric method can be used as a reliable screening method.

Comparison of the complete filtration method using an automated feces analyzer with three manual methods for stool examinations.

Conventional diagnostic techniques using manual methods for stool examination have important limitations. Hence there is a need for improved technologies in routine clinical practice. This study aimed to compare detection rates, agreements, and diagnostic performances for stool examinations in all parameters of the complete filtration method using the Sciendox Feces Analysis System-50 automated feces analyzer with three manual methods, the direct smear, Kato's thick smear, and formalin ethyl concentration techniques. The 252 routine stool samples were examined for parasites, white blood cells (WBCs), red blood cells (RBCs), fat globules, and yeast cells using the four methods indicated above, and the complete filtration detection rates, Cohen's kappa (κ), and diagnostic performances were evaluated and compared. The detection rates of RBCs, fat globules, and yeast cells examined by the complete filtration automated method were comparable to the manual methods, but the detection rates of parasites and WBCs were significantly lower. Most methods detected the same seven parasite species, Ascaris lumbricoides, hookworm, Trichuris trichiura, Strongyloides stercoralis, Entamoeba histolytica/dispar, Blastocystis spp., and Giardia intestinalis. Pairwise agreements between the complete filtration and other methods were good to very good for all parameters showing κ values of 0.74 to 0.89. The diagnostic performances against the combined results showed complete filtration method sensitivities of 70%, 81.82%, 77.27%, 100%, and 95% for parasites, WBCs, RBCs, fat globules, and yeast cells, respectively, while the complete filtration negative predictive values (NPVs) and accuracies showed higher than 95% for all parameters. The complete filtration method using the automated feces analyzer showed high NPVs and accuracies, and good agreements with the three tested manual methods for stool examination in all parameters.

Correlations of spot urinary sodium and potassium with blood pressure in pregnant women: A longitudinal study in Southern Thailand.

OBJECTIVE: To assess the changes of spot urinary sodium and potassium and blood pressure (BP) throughout pregnancy and their correlations in southern Thailand. METHODS: A longitudinal study was conducted in southern Thailand from March 2018 to November 2019. Spot urinary excretion of sodium per creatinine (U[Na+ ]/[Cr]), potassium per creatinine (U[K+ ]/[Cr]) and U[Na+ ]/[K+ ] ratios, and BP were measured at four time points throughout pregnancy. A one-way analysis of variance with repeated measures and Bonferroni correction with post hoc analysis was used to identify significant differences between time points. The correlations were measured using Pearson's correlation coefficients. RESULTS: A total of 327 pregnant women were included. Both systolic and diastolic BPs decreased gradually from up to 14 weeks of pregnancy to 18-22 weeks and then increased until 30-34 weeks. Mean spot U[Na+ ]/[Cr] ratios did not significantly change during the study period. Mean spot U[K+ ]/[Cr] ratios gradually increased, and spot U[Na+ ]/[K+ ] ratios gradually decreased. The correlation of spot U[Na+ ]/[Cr] and U[K+ ]/[Cr] ratios with BPs was weakly negative at all four time points. CONCLUSIONS: Spot U[Na+ ]/[Cr] and U[K+ ]/[Cr] ratios are inversely correlated with BPs; weak correlations are found among pregnant women in southern Thailand. Further research in different populations is required to confirm its correlation and broader use.

Strong correlation and high comparability of capillary electrophoresis and three different methods for HbA1c measurement in a population without hemoglobinopathy.

The present study aimed to investigate the comparability of capillary electrophoresis (CE) and three different methods for HbA1c measurement. The 270 whole blood samples with normal hemoglobin profiles were analyzed for HbA1c values by the Roche turbidimetric inhibition immunoassay (TINIA), the Mindray enzymatic assay (EA), the Arkray high-performance liquid chromatography (HPLC) in comparison with the Sebia CE. The within-laboratory coefficient of variation of all four methods was within the accepted goal (<2%), showing good performance of all of these methods. Pairwise comparisons of HbA1c values obtained by CE and other methods were determined in both total group and subgroups (HbA1c levels of <6.5%, 6.5-8% and >8%). Mean differences of HbA1c values in all groups were very small in which the mean HbA1c value measured by EA was lower while those by TINIA and HPLC were higher than that of CE. The majority of different values were within the limits of agreement in Bland-Altman analysis, indicating good agreement between CE and the others. Less than 5% of percentage differences were out of the total allowable error limit in all groups, showing that differences of HbA1c values between CE and other methods were not clinically significant. Pairwise comparisons of HbA1c values of CE and the others in Passing-Bablok regression and Spearman rank correlation studies displayed high concordance and strong correlation in all groups. In conclusion, the present study showed strong correlation, high comparability and consistent results for HbA1c measurement between capillary electrophoresis and the other three different methods.

The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor.

Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from Bacillus thuringiensis has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct ß-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant (kd) comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.

Significant Changes in Plasma Alpha-Synuclein and Beta-Synuclein Levels in Male Children with Autism Spectrum Disorder.

Alpha-synuclein (α-synuclein) and beta-synuclein (ß-synuclein) are presynaptic proteins playing important roles in neuronal plasticity and synaptic vesicle regulation. To evaluate the association of these two proteins and autism spectrum disorder (ASD), we investigated the plasma α-synuclein and ß-synuclein levels in 39 male children with ASD (2 subgroups: 25 autism and 14 pervasive developmental disorder-not otherwise specified (PDD-NOS)) comparing with 29 sex- and age-matched controls by using enzyme-linked immunosorbent assay (ELISA). We first determined the levels of these two proteins in the ASD subgroups and found that there were no significant differences in both plasma α-synuclein and ß-synuclein levels in the autism and PDD-NOS groups. Thus, we could combine the 2 subgroups into one ASD group. Interestingly, the mean plasma α-synuclein level was significantly lower (P < 0.001) in the ASD children (10.82 ± 6.46 ng/mL) than in the controls (29.47 ± 18.62 ng/mL), while the mean plasma ß-synuclein level in the ASD children (1344.19 ± 160.26 ng/mL) was significantly higher (P < 0.05) than in the controls (1219.16 ± 177.10 ng/mL). This is the first study examining the associations between α-synuclein and ß-synuclein and male ASD patients. We found that alterations in the plasma α-synuclein and ß-synuclein levels might be implicated in the association between synaptic abnormalities and ASD pathogenesis.

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin: MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS.

The insecticidal feature of the three-domain Cry δ-endotoxins from Bacillus thuringiensis is generally attributed to their capability to form oligomeric pores, causing lysis of target larval midgut cells. However, the molecular description of their oligomerization process has not been clearly defined. Here a stable prepore of the 65-kDa trypsin-activated Cry4Ba mosquito-specific toxin was established through membrane-mimetic environments by forming an ∼200-kDa octyl-ß-D-glucoside micelle-induced trimer. The SDS-resistant trimer caused cytolysis to Sf9 insect cells expressing Aedes-mALP (a Cry4Ba receptor) and was more effective than a toxin monomer in membrane perturbation of calcein-loaded liposomes. A three-dimensional model of toxin trimer obtained by negative-stain EM in combination with single-particle reconstruction at ∼5 nm resolution showed a propeller-shaped structure with 3-fold symmetry. Fitting the three-dimensional reconstructed EM map with a 100-ns molecular dynamics-simulated Cry4Ba structure interacting with an octyl-ß-D-glucoside micelle showed relative positioning of individual domains in the context of the trimeric complex with a major protrusion from the pore-forming domain. Moreover, high-speed atomic force microscopy imaging at nanometer resolution and a subsecond frame rate demonstrated conformational transitions from a propeller-like to a globularly shaped trimer upon lipid membrane interactions, implying prepore-to-pore conversion. Real-time trimeric arrangement of monomers associated with L-α-dimyristoylphosphatidylcholine/3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid bicelle membranes was also envisaged by successive high-speed atomic force microscopy imaging, depicting interactions among three individual subunits toward trimer formation. Together, our data provide the first pivotal insights into the structural requirement of membrane-induced conformational changes of Cry4Ba toxin monomers for the molecular assembly of a prepore trimer capable of inserting into target membranes to generate a lytic pore.