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Artemisia argyi is an herbaceous plant of the genus Artemisia. Its young and mature leaves are used as food and medicine, respectively. Glandular trichomes (GTs) are distributed on the leaf surface in A. argyi and are generally considered the location of flavonoid biosynthesis and accumulation. However, the mechanism of flavonoid biosynthesis and accumulation in A. argyi remains unclear. In this study, the coregulatory genes involved in flavonoid biosynthesis and trichome development in this species were screened and evaluated, and the biosynthetic pathways for key flavonoids in A. argyi were uncovered. AaMYB1 and AaYABBY1 were screened using weighted gene co-expression network analysis, and both genes were then genetically transformed into Nicotiana tabacum L. cv. K326 (tobacco). Simultaneously, AaYABBY1 was also genetically transformed into Arabidopsis thaliana. The total flavonoid and rutin contents were increased in tobacco plants overexpressing AaMYB1 and AaYABBY1 , and the expression levels of genes participating in the flavonoid synthesis pathway, such as PAL , FLS , and F3H , were significantly up-regulated in plants overexpressing these genes. These results indicated that AaMYB1 and AaYABBY1 promote flavonoid biosynthesis in tobacco. Furthermore, compared to that in the wild-type, the trichome density was significantly increased in tobacco and A. thaliana plants overexpressing AaYABBY1. These results confirm that AaYABBY1 might be involved in regulating trichome formation in A. argyi. This indicates the potential genes involved in and provides new insights into the development of trichome cellular factories based on the "development–metabolism" interaction network and the cultivation of high-quality A. argyi. [Display omitted] • AaMYB1 and AaYABBY1 were identified from Artemisia argyi. • Overexpression of AaMYB1 and AaYABBY1 promoted flavonoid biosynthesis in transgenic tobacco. • AaYABBY1 increased trichome density by enhancing the expression of TTG1 , TTG2 , MYB5 and MYB23 in transgenic Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

A candidate reference measurement procedure (RMP) for measurement of unconjugated estriol in human serum has been developed and validated. The proposed method is highly reliable and uses isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and requires no derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotopically labeled internal standard. Unconjugated estriol and its internal standard were extracted from serum matrix using liquid-liquid extraction prior to reversed-phase LC-MS/MS. Calibrator bracketing was used to give higher specificity and accuracy for assigning serum level. The accuracy of the candidate RMP was validated by split-sample comparison to established RMPs. The lowest limit of detection (LLoD) and lowest limit of quantification (LLoQ) for developed RMP was estimated to be 0.14 nmol/L and 0.35 nmol/L, respectively. Both intra- and inter-assay imprecisions were ≤2.19% at 1.39, 17.34 and 69.35 nmol/L, respectively. Recoveries were 98.54% to 100.34% and linear response ranged from 0.35 to 173.38 nmol/L. No interference was observed. Biases were 5.6% and 2.8% against the targets of RELA2015A (3.87 nmol/L) and RELA2015B (40.62 nmol/L), respectively. Moreover, the candidate RMP was successfully applied to measure level of unconjugated estriol in serum samples of pregnant women (n = 3) and compared with two immunoassays in clinical laboratory. Our developed method is simple, accurate, and can be used as a candidate RMP to determine total unconjugated estriol level in human serum. Further improvement of certain immunoassays in accuracy and precision is needed.Selected ion chromatograms by LC-MS/MS using a C18 column for uE3 from a serum sample [ABSTRACT FROM AUTHOR]

Serum cystatin C (CysC) is a reliable and ideal endogenous marker for accurately assessing early changes in glomerular filtration rate (GFR), surpassing the limitations of creatinine-based estimated GFR. To improve the precision of GFR calculation, the development of strategies for accurately measuring serum CysC is crucial.In this study, the full-length CysC pure product and fully recombinant 15N-labeled CysC internal standard were subjected to protein cleavage. Subsequently, an LC-MS/MS method was developed for the absolute quantification of serum CysC. The traceability of the method was assigned calibrator using the amino acid reference measurement procedure (RMP). It involved calibrating the instrument using an amino acid reference material with known amino acid concentrations for calibration and comparison purposes.The total imprecision of the method was determined to be ≤8.2 %, and a lower functional limit of quantification (LLoQ) was achieved. The recoveries ranged from 97.36 to 103.26 %. The relative bias between this candidate RMP for measurement of ERM-DA471-IFCC and the target value was 1.74 %. The linearity response was observed within the concentration range of 0.21–10.13 mg/L, with a high R2 value of 0.999. The results obtained using our method was consistent with those obtained using other certified RMPs.With the establishment of this highly selective and accurate serum CysC measurement method, it is now possible to assess the correlation between immunoassay results of serum CysC and the intended target when discrepancies are suspected in the clinical setting. [ABSTRACT FROM AUTHOR]

A candidate reference measurement procedure (RMP) for serum theophylline via isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. With a single-step precipitation pretreatment and a 6-min gradient elution, the method achieved baseline separation of theophylline and its analogs on a C18-packed column. A bracketing calibration method was used to ensure repeatable signal intensity and high measurement precision. The intra-assay and inter-assay imprecisions were 1.06%, 0.84%, 0.72% and 0.47%, 0.41%, 0.25% at concentrations of 4.22 µg/mL (23.40 µmol/L), 8.45 µg/mL (46.90 µmol/L), and 15.21 µg/mL (84.43 µmol/L), respectively. Recoveries ranged from 99.35 to 102.34%. The limit of detection (LoD) was 2 ng/mL, and the lowest limit of quantification (LLoQ) was 5 ng/mL. The linearity range extended from 0.47 to 60 µg/mL (2.61–333.04 µmol/L). No ion suppression and carry-over (< 0.68%) were observed. The relative bias for this candidate RMP that participated in 2023 External Quality Control for Reference Laboratories (RELA) conducted by the International Federation of Clinical Chemistry (IFCC) was within a range of 0.17 to 0.93%. Furthermore, two clinical immunoassay systems were compared with this candidate RMP, demonstrating good correlations. The results of the Trueness Verification Plan indicate significant differences among routine systems, highlighting the need for standardization efforts. The developed candidate RMP for serum theophylline serves as a precise reference baseline for standardizing clinical systems and assigning values to reference materials. [ABSTRACT FROM AUTHOR]

Background: The underlying pathogenic genes and effective therapeutic agents of Alzheimer's disease (AD) are still elusive. Meanwhile, abnormal copper metabolism is observed in AD brains of both human and mouse models. Objective: To investigate copper metabolism-related gene biomarkers for AD diagnosis and therapy. Methods: The AD datasets and copper metabolism-related genes (CMGs) were downloaded from GEO and GeneCards database, respectively. Differentially expressed CMGs (DE-CMGs) performed through Limma, functional enrichment analysis and the protein-protein interaction were used to identify candidate key genes by using CytoHubba. And these candidate key genes were utilized to construct a prediction model by logistic regression analysis for AD early diagnosis. Furthermore, ROC analysis was conducted to identify a single gene with AUC values greater than 0.7 by GSE5281. Finally, the single gene biomarker was validated by quantitative real-time polymerase chain reaction (qRT-PCR) in AD clinical samples. Additionally, immune cell infiltration in AD samples and potential therapeutic drugs targeting the identified biomarkers were further explored. Results: A polygenic prediction model for AD based on copper metabolism was established by the top 10 genes, which demonstrated good diagnostic performance (AUC values). COX11, LDHA, ATOX1, SCO1, and SOD1 were identified as blood biomarkers for AD early diagnosis. 20 agents targeting biomarkers were retrieved from DrugBank database, some of which have been proven effective for the treatment of AD. Conclusions: The five blood biomarkers and copper metabolism-associated model can differentiate AD patients from non-demented individuals and aid in the development of new therapeutic strategies. [ABSTRACT FROM AUTHOR]

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 β-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 β-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0 pmol/L, and 2017012 was 761.9 ± 35.5 pmol/L. [ABSTRACT FROM AUTHOR]

Cardiac ischemia/reperfusion(I/R) induced-cardiac vascular endothelial injury is an important pathological process that appears in the early stage of cardiac I/R injury. The autophagy-lysosomal pathway is essential for the maintenance of cellular homeostasis. However, in cardiac I/R injury, the role of the autophagy-lysosomal pathway is controversial. The present study aimed to use oxygen-glucose deprivation/oxygen-glucose resupply(OGD/OGR) in human coronary artery endothelial cells(HCAECs) with I/R injury to assess the role of the autophagy-lysosomal pathway in I/R-induced endothelial injury. The results revealed lysosomal dysfunction and impaired autophagic flux in endothelial cells exposed to OGD/OGR. Meanwhile, our data showed that the levels of cathepsin D(CTSD) decreased time-dependently. Knockdown of CTSD caused lysosomal dysfunction and impaired autophagic flux. Conversely, restoration of CTSD levels protected HCAECs against OGD/OGR induced-defects in autophagy-lysosomal function and cellular damage. Our findings indicated that I/R induced-impaired autophagic flux, rather than excessive autophagic initiation, mediates endothelial cells injury. The maintenance of autophagy-lysosomal function is critical to protect endothelial cells against I/R injury, and CTSD is a key regulator. Thus, strategies focused on restoring CTSD function are potentially novel treatments for cardiac reperfusion injury. • Oxygen-glucose deprivation/oxygen-glucose resupply impaired autophagy degradation. • Oxygen-glucose deprivation/oxygen-glucose resupply caused lysosomal dysfunction. • Autophagic clearance dysfunction was one of the causes of endothelial injury. • CTSD knockdown mimicked these effects on autophagy-lysosomal function. • CTSD restoration protected endothelial cells against autophagy-lysosomal dysfunction. [ABSTRACT FROM AUTHOR]

The immune landscape of hepatocellular carcinoma (HCC) following transarterial chemoembolisation (TACE) remains to be clarified. This study aimed to characterise the immune landscape following TACE and the underlying mechanism of HCC progression. Tumour samples from five patients with treatment-naive HCC and five patients who received TACE therapy were collected and subjected to single-cell RNA sequencing. Another 22 paired samples were validated using immunofluorescence staining and flow cytometry. To clarify the underlying mechanisms, in vitro co-culture experiments and two types of TREM2-KO/WT mouse models, namely, an HCC cell orthotopic injection model and a spontaneous HCC model, were used. A reduced number of CD8+ T cells and an increased number of tumour-associated macrophages (TAMs) were observed in the post-TACE microenvironment. TACE therapy reduced the cluster CD8_C4, which was highly enriched with tumour-specific CD8+ T cells of pre-exhausted phenotype. TREM2 was found to be highly expressed in TAMs following TACE, which was associated with a poor prognosis. TREM2+ TAMs secreted less CXCL9 but more galectin-1 than did TREM2- TAMs. Galectin-1 promoted PD-L1 overexpression in vessel endothelial cells, impeding CD8+ T cell recruitment. TREM2 deficiency also increased CD8+ T cell infiltration, which inhibited tumour growth in both in vivo HCC models. More importantly, TREM2 deficiency enhanced the therapeutic effect of anti-PD-L1 blockade. This study shows that TREM2+ TAMs play an important role in suppressing CD8+ T cells. TREM2 deficiency increased the therapeutic effect of anti-PD-L1 blockade by enhancing antitumour activity of CD8+ T cells. These findings explain the reasons for recurrence and progression after TACE and provide a new target for HCC immunotherapy after TACE. Studying the immune landscape in post-TACE HCC is important to uncover the mechanisms of HCC progression. By using scRNA sequencing and functional assays, we discovered that both the number and function of CD8+ T cells are compromised, whereas the number of TREM2+ TAMs is increased in post-TACE HCC, correlating with worse prognosis. Moreover, TREM2 deficiency dramatically increases CD8+ T cell infiltration and augments the therapeutic efficacy of anti-PD-L1 blockade. Mechanistically, TREM2+ TAMs display lower CXCL9 and increased Gal-1 secretion than do TREM2- TAMs, with Gal-1 mediating the overexpression of PD-L1 in vessel endothelial cells. These results suggest that TREM2 could be a novel immunotherapeutic target for patients treated with TACE in HCC. This provides an opportunity to break the plateau of limited therapeutic effect. This study has the value of understanding the tumour microenvironment of post-TACE HCC and thinking a new strategy of immunotherapy in the field of HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology. [Display omitted] • TACE therapy decreased the number and antitumour function of CD8+ T cells. • TACE therapy results in an increased number of TREM2+ TAMs, correlating with a worse clinical prognosis. • Compared with TREM2- TAMs, TREM2+ TAMs showed low secretion of CXCL9 but increased release of Gal-1. • Gal-1 upregulated PD-L1 on vascular endothelial cells and impaired CD8+ T-cell recruitment. • TREM2 deficiency enhanced the therapeutic efficacy of anti-PD-L1 blockade in HCC. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to describe the vitamin D status and its distribution in different age groups, sexes, seasons, and provinces of a large Chinese population. Methods: This study retrospectively analyzed 1,528,685 results of serum 25-hydroxyvitamin D (25(OH)D) in the central laboratory of KingMed Diagnostics. The samples were from the individuals aged 0–119 years old in 30 provinces of China. Serum 25(OH)D was measured by an accurate commercial liquid chromatography-tandem mass spectrometry (LC–MS/MS) method from January 2017 to December 2019. The subjects were stratified by age, sex, the season of blood collection, and the province of residence. Results: The median 25(OH)D concentration was 25.5 ng/mL (interquartile range (IQR) 18.7–32.7 ng/mL) in males and 20.8 ng/mL (IQR 14.4–28.2 ng/mL) in females. Overall, the median 25(OH)D concentration decreased with age in both males and females. Males had a 0.2–2.4 ng/mL higher median 25(OH)D concentration than females in different age groups. Vitamin D deficiency (25(OH)D < 15 ng/mL for the individuals under 14 years old; < 20 ng/mL for the individuals over 14 years old) was found in 21.3% of males and 43.6% of females. Significant seasonal variation of serum 25(OH)D concentrations was repeatedly observed in 3 years, with median concentration higher in summer (25.3 ng/mL (IQR 19.3–31.9 ng/mL)) and lower in winter (18.5 ng/mL (IQR 12.3–26.6 ng/mL)). Vitamin D status varied by province. The median 25(OH)D concentration was the highest in Hainan (31.0 ng/mL (IQR 24.9–39.2 ng/mL)) and the lowest in Qinghai (14.4 ng/mL (IQR 9.6–20.0 ng/mL)). 25(OH)D2 was detected in 12.2% of the results, and no significant seasonal variation was observed. Conclusion: In China, vitamin D deficiency is prevalent in the population participating in clinical vitamin D measurement. Age and sex differences in vitamin D levels were observed in our study. Seasonal variation and provincial differences are important aspects of serum vitamin D status. 25(OH)D2 cannot be ignored entirely in clinical measurement practice in China. [ABSTRACT FROM AUTHOR]

Saffron floral bio‐residues (SFB) are a valuable natural source of antioxidants and dyes that can contribute to the development of new food products and cosmetic products. Color change was usually observed during SFB storage, which may result in quality degradation of SFB or even cause potential hazard to human health. In order to clarify the mechanism of color change of SFB sample, the chemical differences among SFB samples stored under different conditions were analyzed using ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry and chemometrics methods, from which differential flavonols and anthocyanins were screened and their kinetic variations during sample storage summarized. In addition, the color change of the SFB sample was digitalized using an electronic eye (E‐eye), which was found to be related to the content of delphinidin‐3,5‐di‐O‐glucoside (DDG). Moreover, the degradation kinetic parameters of DDG under different storage conditions were studied. In conclusion, the variation of kaempferol‐, isorhamnetin‐, and quercetin‐type flavonol, and delphinidin‐ and petunidin‐type anthocyanin resulted in the color change of SFB sample, and anthocyanin was found more unstable than flavonol. Practical Application: Saffron floral bio‐residues (SFB) are popular natural sources of antioxidants and colorants that can be used in food and cosmetic products. Color change usually occurs during SFB storage period. Clarifying the mechanism of the color change of SFB will help us to ensure the quality of SFB. [ABSTRACT FROM AUTHOR]

Venom-induced thrombocytopenia (VIT) is one of the most important hemotoxic effects of a snakebite, which is often associated with venom-induced consumptive coagulopathy (VICC). Refractory thrombocytopenia without significant coagulation abnormalities has also been reported after envenomation by some viperid snakes; however, the mechanisms are not well understood and therapeutic strategies are lacking. Here, we found that patients injured by Daboia siamensis or Agkistrodon halys snakes, who were resistant to standard antivenom treatment, had developed coagulopathy-independent thrombocytopenia. Venoms from these viperid snakes, rather than from the elapid snake (Bungarus multicinctus), induced platelet surface expression of neuraminidase-1 (NEU-1), and significantly increased the desialylation of the glycoproteins on human platelets. The desialylated platelets caused by viperid snake venoms were further internalized by macrophages, which resulted in reduced platelet numbers in peripheral blood. Importantly, neuraminidase inhibitor significantly decreased viper venom-induced platelet desialylation, therefore inhibiting platelet phagocytosis by macrophages, and alleviating venom-induced thrombocytopenia. Collectively, these findings support an important role for desialylated platelet clearance in the progression of viper envenomation-induced, coagulopathy-independent thrombocytopenia. Our study demonstrates that the neuraminidase inhibitor may be a potential therapy or adjuvant therapy to treat snakebite-induced thrombocytopenia. [ABSTRACT FROM AUTHOR]

[Display omitted] • The first design of DNA-peptide probes targeting lncRNA with high specificity and efficient detection. • A novel method for the quantitative analysis of lncRNAs is proposed, utilizing LC-MS/MS and DNA-peptide probes. • The first application of mass spectrometry in conjunction with DNA-peptide probe technology for liver cancer diagnosis. Liver cancer, a prominent contributor to cancer-related deaths, necessitates timely identification for successful intervention. In recent years, long non-coding RNAs (lncRNAs) have gained attention as potential biomarkers for liver cancer, and accurate quantification of lncRNA is essential for early diagnosis of liver cancer. However, existing methods for lncRNA detection rely on amplification and labeling techniques, resulting in semi-quantitative or qualitative outcomes. DNA-peptide technology offers a promising alternative. It has successfully translated nucleic acid quantification into peptide quantification, particularly in the context of miRNA analysis. However, the intricate structure of long-chain lncRNAs presents a challenge not encountered with miRNAs, making the design and acquisition of specific DNA-peptide probes for direct quantification more difficult. In this study, a strategy for lncRNA quantification was developed using a DNA-peptide probe coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quasi-targeted proteomics. The inherent structural complexity of long-chain lncRNAs was overcome by designing DNA-peptide probes based on their unique sequence segments, enabling direct quantification without the need for amplification. Highly upregulated in liver cancer (HULC) was identified as a target lncRNA through bioinformatics analysis using GEO2R online software. Subsequently, the target lncRNA (i.e., HULC), which had been biotinylated and bound to streptavidin agarose beforehand, was hybridized with the probe. Following trypsin digestion, the reporter peptide was released and quantified using LC-MS/MS. Finally, this method was applied to analyze HULC levels in liver cancer cell lines and serum samples, enabling quantitative assessment and early diagnosis. The results demonstrated that liver cancer diagnostics could be enhanced through the integration of mass spectrometry and DNA-peptide probe technology for lncRNA quantification. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5 %. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64 % - 102.22 % and 98.41 % - 100.97 % for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625 ng/mL and 3.906 ng/mL, respectively; the quantification limits were 62.5 ng/mL and 7.813 ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125 ng/mL to 20 μg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogs. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma. • The cRMP has been developed for the measurement of urine HVA and VMA. • A multiple-site method comparison with clinical samples is presented. • The newly established cRMP displayed desirable method performance. [ABSTRACT FROM AUTHOR]

Patient-based real-time quality control (PBRTQC) has gained increasing attention in the field of laboratory quality management in recent years. However, PBRTQC has not been reported for use in molecular diagnostics. This study introduces PBRTQC to quantitative hepatitis B virus (HBV) DNA test using moving rate (MR) of positive and negative patient results. In contrast to the MR protocols described in other literature, MR protocol for HBV-DNA test has an additional logarithmic transformation and binary conversion steps before using a common statistical process control algorithm, such as the MR. We used all patient test results of HBV-DNA assay from August 2018 to August 2021 at the Second Affiliated Hospital of Guangzhou University of Chinese Medicine, for parameters setting, optimization, and performance validation. The false rejection rate, error detection curves and validation charts were used to assess the MR protocols. The false rejection rates of two MR protocols were both <0.7%. The optimal block sizes for positive and negative errors in each cut-off value were not the same, so we first proposed a combined protocol that used different block size to detect negative and positive errors. It turned out that the combined protocols outperformed the simple protocols for each cut-off value, especially detecting positive errors. The performances of MR protocols using positive or negative patient results to detect constant errors of HBV-DNA test could meet laboratory requirements. Therefore, we have provided an effective alternative tool for internal quality control in the field of molecular diagnostics. [ABSTRACT FROM AUTHOR]

ObjectiveTo evaluate the photometry technical and analytical performance of a newly launched Mindray BS-2000M1 clinical chemistry system (BS-2000M1). Design and Methods The photometric parameters were evaluated according to the China Food and Drug Administration (CFDA) Automatic Chemistry Analyzer Guideline. The precision, accuracy, linearity and interference were evaluated according to CLSI protocols EP5-A2, EP9-A2, EP6-A and EP7-A2 respectively. The trueness verification on Ca2 +, Mg2 +, P− and Cl− was conducted by comparing with the reference methods using fresh samples. Results The photometer accuracy, precision, linearity, stability and stray light at 340nm were acceptable. The within-run coefficients of variation (CVs) ranged from 0.16% to 2.13% and the total CV ranged from 0.64% to 4.12%. A good correlation (R>0.95) of method comparison between BS-2000M1 and our reference system was observed for most of the parameters tested with exception of Ca2 + (R=0.85), Mg2 + (R=0.71), P− (R=0.96, Slope=0.88), Cl− (R=0.93), and ASO (R=0.94, Slope=0.93, intercept=− 8.81). The trueness verification on Ca2 +, Mg2 +, P− and Cl− showed acceptable results on both BS-2000M1 and our reference systems. Linearity study showed acceptable linearity range for all parameters. Significant interferences occurred for some evaluated parameters, but were identical to the manufacturer statement. Conclusions Mindray BS-2000M1 achieved the desirable photometry technical and analytical performance, and is therefore suitable for its utilization in modern clinical laboratories. [ABSTRACT FROM AUTHOR]

• We investigated the important role of mitochondrial dysfunction and immune infiltration and explored their crosstalk in diabetic nephropathy (DN). • Six mitochondrial-related hub genes, EFHD1, CASP3, AASS, MPC1, NT5DC2, and BCL2A1, have been identified as potential biomarkers for DN. • A diagnostic model constructed based on the six genes demonstrated good diagnostic efficacy. • Spearman correlation analysis revealed that the six hub genes were significantly associated with the infiltration of immune cells. • Ten potential therapeutic drugs for DN were predicted from the CMAP database. Diabetic nephropathy (DN) is a common and severe microvascular complication of diabetes. Mitochondrial dysfunction and immune inflammation are important factors in the pathogenesis of DN. However, the specific mechanisms and their intricate interactions in DN remain unclear. Besides, there are no effective specific predictive or diagnostic biomarkers for DN so far. Therefore, this study aims to elucidate the role of mitochondrial-related genes and their possibility as predictive or diagnostic biomarkers, as well as their crosstalk with immune infiltration in the progression of DN. Based on the GEO database and limma R package, the differentially expressed genes (DEGs) of DN were identified. Mitochondrial-related DEGs (MitoDEGs) were then obtained by intersecting these DEGs with mitochondria-related genes from the MitoCarta 3.0 database. Subsequently, the candidate hub genes were further screened by gene co-expression network analysis (WGCNA), and verified mRNA levels of these genes by real-time quantitative PCR (qRT-PCR) in high-glucose-treated human proximal tubular (HK-2) cells. The verified hub genes were utilized to construct a combined diagnostic model for DN, with its diagnostic efficacy assessed across the GSE30122 and GSE96804 datasets. Additionally, the immune infiltration pattern in DN was assessed with the CIBERSORT algorithm, and the Nephroseq v5 database was used to analyze the correlation between hub genes and clinical features of DN. Seven mitochondria-related candidate hub genes were screened from 56 MitoDEGs. Subsequently, the expression levels of six of them, namely EFHD1, CASP3, AASS, MPC1, NT5DC2, and BCL2A1, exhibited significant inter-group differences in the HK-2 cell model. The diagnostic model based on the six genes demonstrated good diagnostic efficacy in both training and validation sets. Furthermore, correlation analysis indicated that EFHD1 and AASS, downregulated in DN, are positively correlated with eGFR and negatively with serum creatinine. Conversely, CASP3, NT5DC2, and BCL2A1, upregulated in DN, show opposite correlations. In addition, spearman analysis revealed that the six hub genes were significantly associated with the infiltration of immune cells, including M1 and M2 macrophages, mast cells, resting NK cells, gamma delta T cells, and follicular helper T cells. This study elucidated the characteristics of mitochondria-related genes and their correlation with immune cell infiltration in DN, providing new insights for exploring the pathogenesis of DN and facilitating the identification of new potential biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Critical events in Alzheimer's disease (AD) involve an imbalance between the production and clearance of amyloid-β (Aβ) peptides from the brain. The ratio of Aβ42 to Aβ40 in plasma was useful for evaluating AD, but quantification is limited by factors including preanalytical analyte loss and insufficient sensitivity. The availability of a targeted UPLC-MS/MS method with adequate analytical sensitivity and accurate values traceable to the SI units is essential for implementing a strategy for assay standardization. A targeted UPLC-MS/MS method for plasma Aβ42 and Aβ40 quantification was developed based on selected characteristic peptides spiked by 15N-labeled Aβ. The calibrator was assigned using an amino acid analysis reference method trace to SI units. UPLC-MS/MS conditions and sample preparation procedures were assessed. 59 plasma samples comparison were used to evaluate immunoassays. Additionally, two clinical cohorts were selected for diagnostic performance evaluation. The LOQ of Aβ42 and Aβ40 is 10 pg mL−1 and 20 pg mL−1, respectively. The linear range was 10–500 pg mL−1 for Aβ42 and 20–1000 pg mL−1 for Aβ40, recoveries between 95.3 % and 108.2 % for Aβ42, 93.2 % and 104.1 % for Aβ40, imprecisions were <7 %. The accuracy of method was validated by analysis of a certified reference material. Clinical cohorts for diagnostic performance evaluation shown that the area under the curve (AUC) for plasma Aβ42 and Aβ42/Aβ40 to differentiate between AD and CN were 0.767 and 0.799, respectively. A robust UPLC-MS/MS method was developed and demonstrated that suitable for a wide range of plasma Aβ42 and Aβ40. Applied to the investigation of clinically discrepant results, this method can act as an arbiter of the concentration of plasma Aβ42 and Aβ40 present. • An optimized UPLC-MS/MS method for human plasma Aβ42 and Aβ40 measurement was developed and needed for many clinical scenarios. • The measurement value was traceable to SI unit and the accuracy of method was validated by certified reference material. • The method was successfully applied to measure plasma Aβ42 and Aβ40 for verifying immunoassays performance. • Two clinical cohorts were used to diagnostic performance evaluation of plasma Aβ42/Aβ40 in AD diagnosis. [ABSTRACT FROM AUTHOR]

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020. [ABSTRACT FROM AUTHOR]

Background. Traditional Chinese medicine Smilax is the rhizome of liliaceous plant Smilax china L., which is used to treat pelvic inflammatory disease and anxieties. Purpose. To investigate the mechanism of anti-inflammatory activity of the extract from Smilax china L. (ES). Methods. The components of ES were identified by UPLC-QTOF-MS/MS. The anti-inflammatory activities were evaluated in xylene-induced ear oedema and egg white-induced plantar swelling test. Cell viability was examined by CCK-8 assay. The inflammatory mediators, proinflammatory cytokines, and MAPK and NF-κB signals in LPS-stimulated THP-1 cells were determined using ELISA, real-time PCR, and Western blot, respectively. Results. 20 compounds of ES were confirmed by comparing with the reference substance. ES displayed more prominent anti-inflammatory activity than the positive control "Jin Gang Teng" capsule in the in vivo acute inflammatory model. ES suppressed the expression of PGE2 and 6-Keot-PGF1α, and the ratio of IC50 (COX-1)/IC50 (COX-2) of ES was 3.15, which indicated that ES could selectively inhibit COX-2. ES dose-dependently (12.5, 25, and 50 mg/L) decreased the production and mRNA levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α. Furthermore, ES significantly decreased LPS-induced phosphorylation of p38, JNK, ERK1/2, and p65, inhibiting the expression of IKKα and the degradation of IκBα. Conclusion. The results suggested that ES could selectively inhibit the activity of COX-2, and the anti-inflammatory effect of ES was associated with the inhibition of IL-1β, IL-6, and TNF-α via negative regulation of MAPK and NF-κB signaling pathways in LPS-induced THP-1 cells. [ABSTRACT FROM AUTHOR]

Influenza A, influenza B, severe acute respiratory syndrome coronavirus 2, adenovirus, respiratory syncytial virus, Mycoplasma pneumoniae, and Chlamydophila pneumoniae are common pathogens that can cause severe pneumonia and other symptoms, resulting in acute lower respiratory tract infections. The objective of this study was to design and evaluate a sensitive and specific multiplex one-step reverse transcription PCR (RT-PCR)–dipstick chromatography method for simultaneous rapid detection of these seven pathogens. Streptavidin-coated blue latex particles were used to read out a positive signal. Based on the DNA–DNA hybridization of oligonucleotide sequences (Tag) for forward primer with the complementary oligonucleotide sequence (cTag) on the dipstick and biotin–streptavidin interactions, PCR products were able to be illuminated visually on the dipstick. The specificity and the limit of detection (LOD) were also evaluated. Moreover, the clinical performance of this method was compared with Sanger sequencing for 896 samples. No cross reaction with other pathogens was found, confirming the high specificity of this method. The LOD was 10 copies/µL for each of the tested pathogens, and the whole procedure took less than 40 min. Using 896 samples, the sensitivity and specificity were shown to be no lower than 94.5%. The positive predictive value was higher than 82.1%, and the negative predictive value was higher than 99.5%. The kappa value between the PCR–dipstick chromatography method and Sanger sequencing ranged from 0.869 to 0.940. In summary, our one-step RT-PCR–dipstick chromatography method is a sensitive and specific tool for rapidly detecting multiplex respiratory pathogens. [ABSTRACT FROM AUTHOR]

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11–101.61%. The relative bias between this candidate RMP and the established RMP was 2.76–1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV) infection is one of the main leading causes of hepatocellular carcinoma (HCC) worldwide. However, it remains uncertain how the reverse-transcriptase (rt) gene contributes to HCC progression.Methods: We enrolled a total of 307 patients with chronic hepatitis B (CHB) and 237 with HBV-related HCC from 13 medical centers. Sequence features comprised multidimensional attributes of rt nucleic acid and rt/s amino acid sequences. Machine-learning models were used to establish HCC predictive algorithms. Model performances were tested in the training and independent validation cohorts using receiver operating characteristic curves and calibration plots.Results: A random forest (RF) model based on combined metrics (10 features) demonstrated the best predictive performances in both cross and independent validation (AUC, 0.96; accuracy, 0.90), irrespective of HBV genotypes and sequencing depth. Moreover, HCC risk scores for individuals obtained from the RF model (AUC, 0.966; 95% confidence interval, .922-.989) outperformed α-fetoprotein (0.713; .632-.784) in distinguishing between patients with HCC and those with CHB.Conclusions: Our study provides evidence for the first time that HBV rt sequences contain vital HBV quasispecies features in predicting HCC. Integrating deep sequencing with feature extraction and machine-learning models benefits the longitudinal surveillance of CHB and HCC risk assessment. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae colonization in pregnant women can cause postpartum intrauterine infections and life-threatening neonatal infections. To formulate strategies for the prevention and treatment of S. agalactiae infections, we performed a comprehensive analysis of antibiotic resistance and a molecular-based epidemiological investigation of S. agalactiae in this study. Seventy-two S. agalactiae strains, collected from pregnant women, were subjected to antibiotic susceptibility tests; then, the screened erythromycin and clindamycin nonsusceptible isolates were used for macrolides and clindamycin resistance genes detection, respectively. Detection of resistance genes, serotyping, and determination of virulence genes were performed by polymerase chain reaction. The clonal relationships among the colonized strains were evaluated by multilocus sequence typing. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) mass peak analysis was performed to discriminate the specific sequence types (STs). In our study, 69.4% and 47.2% of the strains were nonsusceptible to erythromycin and clindamycin, respectively; the multidrug resistance rate was 66.7%. All erythromycin nonsusceptible strains harbored resistance genes, whereas only 52.9% of the clindamycin nonsusceptible strains possessed the linB gene. Erythromycin resistance was mainly mediated by the ermB or mefA/E genes. Four serotypes were identified, and the most common serotype was serotype III (52.8%), followed by Ib (22.2%), Ia (18.0%), and II (4.2%). All the strains were divided into 18 STs that were assigned to nine clonal complexes. Most of the major STs were distributed into specific serotypes, including ST19/serotype III, ST17/serotype III, ST485/serotype Ia, ST862/serotype III, and ST651/serotype III. Analysis of virulence genes yielded seven clusters, of which bca-cfb-scpB-lmb (61.6%) was the predominant virulence gene cluster. Among all ST strains distributed in this region, only the ST17 strains had a mass peak at 7620 Da. The outcomes of this study are beneficial for the epidemiological comparison of colonized S. agalactiae in different regions and may be helpful for developing the strategies for the prevention of S. agalactiae infection in Guangzhou. Furthermore, our results show that MALDI-TOF MS can be used for the rapid identification of the ST17 strains. [ABSTRACT FROM AUTHOR]

Objective: We aimed to describe the 25-hydroxyvitamin D (25(OH)D) status of southern Chinese individuals by a high-accuracy liquid chromatography tandem mass spectrometry (LC-MS/MS) method which can trace to reference measurement procedure. Materials and methods: From January 2018 to June 2019, a total of 4775 southern Chinese individuals were evaluated in our study. The serum levels of parathyroid hormone (PTH) were detected simultaneously in 162 cases. 25(OH)D was determined by LC-MS/MS, and PTH was detected using routine automated analysers. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D in males and females of different age groups were studied. Results: The mean 25(OH)D concentration in our study was 32.57 ng/mL (4.20–101.40 ng/mL). The global 25(OH)D concentration in males was higher than that in females of different age group. The prevalence of vitamin D deficiency (< 20 ng/mL) in females (16.65%) was higher than that in males (6.83%). The prevalence of vitamin D deficiency (< 20 ng/mL) was most common in winter (22.98% of all women and 15.49% of all men). 25(OH)D concentrations were higher in those from whom blood samples were collected in summer and autumn than in winter and spring. 25(OH)D2 was detected in 672 serum samples (14.07%). In addition, there was a negative correlation between the concentrations of 25(OH)D and serum PTH (r = − 0.149, P < 0.05). Conclusion: Our study demonstrated that the average serum 25(OH)D concentration in southern Chinese individuals was higher than that in other Chinese cohorts by a high-accuracy LC-MS/MS method. The global 25(OH)D concentration in males was higher than that in females of different ages, and the prevalence of vitamin D deficiency in females was higher than that in males. Seasonal change was an important aspect of 25(OH)D concentration in young and middle-aged people but became less relevant for that in older subjects. 25(OH)D2 detection was of minor practical significance in our study. In addition, we also found that there was a negative correlation between the serum levels of 25(OH)D and PTH in southern Chinese individuals. [ABSTRACT FROM AUTHOR]

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography–tandem mass spectrometry (ID–LC–MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid–liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID–LC–MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. [ABSTRACT FROM AUTHOR]

Highlights • This paper describes the development and evaluation of a candidate reference measurement procedure (cRMP) for serum urea that uses a LC-MS/MS method. This novel cRMP directly detects urea without derivatization, which makes the pre-processing of samples very simple compared to the reference methods now recognized by the JCTLM. • The performance of this cRMP is well characterized, with good demonstrated precision and accuracy. • This cRMP could facilitate the implementation and standardization of procedures and testing results for serum urea among clinical laboratories. Abstract Urea is an important indicator of liver and kidney disease, and very frequently determined in clinical chemistry. The reference measurement procedures (RMPs) for serum urea recognized by the Joint Committee for Traceability in Laboratory Medicine are spectrophotometry, and isotope dilution (ID)-mass spectrometry (MS) coupled with gas chromatography. This study investigated a candidate RMP (cRMP) for detecting serum urea directly via ID-liquid chromatography (LC)-MS/MS, without derivatization, which simplifies pre-processing samples. The cRMP was developed and evaluated relative to the recognized RMP, inter- and intra-laboratories. The intra-precisions were 1.35%, 1.98% and 1.47% at 4.95, 24.74 and 31.36 mmol/L, respectively; inter-precision was 2.10%, 2.60% and 2.10%. The relative bias for the measurement of standard human serum (SRM 909c) was –0.49%. The relative biases were 0.41% and 0.02% for IFCC-RELA (International Federation of Clinical Chemistry and Laboratory Medicine-External Quality Assessment Scheme [EQAS] for reference laboratories) 2015A and 2015B. The linearity response between 2.4 mmol/L and 53.7 mmol/L was R 2 = 0.9989. No carryover, ion suppression, or interference was detected. Correlation was acceptable with the reference spectrophotometry (R 2 = 0.9985, P < 0.0001). Between our laboratory and other reference laboratories, the absolute deviation range for IFCC-RELA 2016A and 2016B was from –0.63 to 1.52 mmol/L. This well-characterized cRMP for urea can provide a base of accuracy for the traceability of clinical systems. [ABSTRACT FROM AUTHOR]

Introduction: Hepatocellular carcinoma (HCC) is a major contributor to cancer-related deaths due to its often late stage diagnosis. Our previous study showed that dermcidin (DCD) may have the potential to be used as a serum biomarker for HCC for more timely diagnoses. Materials and Methods: In this study, we measured serum DCD and alpha-fetoprotein (AFP) levels in 87 HCC patients; 33 liver cirrhosis (LC); and 44 normal controls (NC), evaluated the relationship between DCD levels and clinicopathological parameters. Results: Serum DCD levels in HCC patients (27.03 ng/mL) were significantly higher than in LC patients (24.78 ng/mL, p < 0.05), and NC subjects (18.98 ng/mL, p < 0.001). The optimum cutoff values were 25.75 ng/mL for DCD and 9.86 ng/mL for AFP. DCD had a greater area under the receiver operating characteristic curve (AUC) for differentiating HCC from the controls than AFP (AUC = 0.769 vs. 0.729, respectively). Importantly, our cohort revealed that serum DCD levels were positively correlated with metastasis in HCC patients versus HCC patients without metastatic disease (32.31 vs. 23.95, p < 0.001). Western blot results showed that DCD expression was significantly upregulated in seven tumor tissues compared with the noncancerous adjacent tissues. Immunohistochemistry performed in four paired samples confirmed the upregulation of DCD expression in the tumor tissues. Conclusions: Our results showed that serum DCD levels were significantly increased in HCC patients and in cancerous tissue. DCD could potentially be used as a biomarker for the diagnosis of HCC. [ABSTRACT FROM AUTHOR]

Blood glucose is an important monosaccharide functioning as the main source of energy for the human body. The accurate measurement of blood glucose is crucial for the screening, diagnosis, and monitoring of diabetes and diabetes-associated diseases. To assure the reliability and traceability of blood glucose measurements, we developed a reference material (RM) for use in human serum at two different concentrations, which were certified by the National Institute of Metrology (NIM) as GBW(E)091040 and GBW(E)091043. Raw serum samples were collected from residual samples after clinical testing, filtered, and repackaged under mild stirring. The homogeneity and stability of the samples were examined according to ISO Guide 35: 2017. Commutability was evaluated in compliance with CLSI EP30-A. Value assignment was carried out in six certified reference laboratories using the JCTLM-listed reference method for serum glucose. Moreover, the RMs was further applied in a trueness verification program. The developed RMs was homogeneous and commutable enough for clinical use. They were also stable for 24 h at 2–8 ℃ or 20–25 ℃ and for at least 4 years at − 70 ℃. The certified values were 5.20 ± 0.18 mmol/L and 8.18 ± 0.19 mmol/L (k = 2) for GBW(E)091040 and GBW(E)091043, respectively. The pass rates were evaluated by bias, coefficient of variation (CV), and total error (TE) for 66 clinical laboratories in the trueness verification program were 57.6%, 98.5%, and 89.4% of GBW(E)091040, and 51.5%, 98.5%, and 90.9% of GBW(E)091043, respectively. The developed RM could be used for the standardization of reference and clinical systems with satisfactory performance and traceable values, providing strong support for the accurate measurement of blood glucose. • The details of the development method, collaborative assignment and performance verification for reference material of blood glucose are described in this paper. • The developed reference material has sufficient homogeneity, stability, and commutability for clinical use, as well as traceable to SI units. • The developed reference material was applied to a trueness verification program to facilitate the harmonization of glucose assays in clinical laboratories. [ABSTRACT FROM AUTHOR]

Estrogen measurements are important in the assessment of female reproductive function and have expanding roles in other fields. A simple, accurate, highly sensitive and specific isotope-dilution liquid chromatography-tandem mass spectrometry method was developed and evaluated to simultaneously measure three endogenous estrogens in serum: estrone (E), 17β-estradiol (E), and estriol (E). Chromatographic separation was achieved on a C column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode. The sample preparation in this assay requires no derivatization and extraction by liquid-liquid extraction. After optimization of the extraction conditions, the final extraction efficiency of E, E, and E was 83.8%, 78.9%, and 77.3% respectively. The metabolites and structural analogs that have the same molecular masses as the estrogens were separated under the optimized liquid chromatography conditions. Method validation showed satisfactory linearity over the concentration range of 20-10000 pg mL for all three estrogens ( r > 0.997). The limits of quantification were 5, 10, and 10 pg mL for E, E, and E respectively, and their recoveries ranged from 94.7% to 103.5%. The accuracy of the proposed method was further evaluated with use of certified reference materials BCR-576, BCR-577, and BCR-578 for E and 2014 International Federation of Clinical Chemistry and Laboratory Medicine External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine samples for E, whose certified values were determined by reference methods. Great agreement was observed between the measured values and the certified values. Satisfactory precision (coefficients of variation less than 7.44%) was also obtained for the three estrogens. Moreover, the proposed method was successfully applied to measure the three estrogens in serum samples of pregnant women in the second trimester and to assess the accuracy of chemiluminescent immunoassays in clinical laboratories by determination of E and unconjugated E in serum samples. [ABSTRACT FROM AUTHOR]

Haemoglobinopathies may interfere with the haemoglobin A1c (HbA1c) measurement, leading to incorrect diagnosis and inappropriate treatment. It is essential that HbA1c assays are capable of identifying haemoglobinopathies. We report two cases of haemoglobin New York (HbNY) discovered through HbA1c analysis using capillary electrophoresis (Capillarys 2 Flex Piercing [C2FP], Sebia). We used these samples to evaluate the ability of three other HbA1c assays to identify this variant: ion-exchange high-performance liquid chromatography (Variant II Turbo [VII-T], Bio-Rad); boronate affinity high-performance liquid chromatography (Ultra2, Trinity Biotech) and immunoassay (Cobas c501 Tina-quant Generation 3, Roche Diagnostics). Each method was used for HbA1c assay of in samples from two cases of heterozygous haemoglobinopathy: β0-thalassemia/HbNY (Case 1) and HbA/NY (Case 2). Only the C2FP system detected HbNY (an additional peak appeared between HbA1c and HbA0). Clinical laboratories should be aware of the limitations of their HbA1c assay methods especially in geographic areas, where haemoglobinopathy prevalence is high. [ABSTRACT FROM AUTHOR]

Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease in which monocytes/macrophages infiltration and skewed T helper type (Th) 1 and Th17 cell responses participate in the development of the disease. Human peripheral blood monocytes are heterogeneous and can be divided into classical CD14CD16, intermediate CD14CD16, and nonclassical CD14CD16 monocyte subsets. Compared to classical monocytes, CD16 monocytes are generally termed pro-inflammatory monocytes and play an important pathogenic role in autoimmune diseases. However, little is known about the immunophenotype and immunopathogenic role of peripheral blood CD16 monocytes in PBC. Thus, we investigated the phenotype and function of these circulating monocyte subsets from PBC patients. The frequencies of circulating CD14CD16 and CD14CD16 subpopulation were increased in disease compared with healthy controls. Among them, CD14CD16 monocyte subset positively correlated with disease progress, liver damage indicators and serum C-reactive protein, respectively. Furthermore, the frequencies of Th1 and Th17 cells were upregulated and CD14CD16 monocyte subset was also positively associated with Th1 cell frequency in PBC. Using a vitro coculture model, we further found that CD14CD16 monocytes promoted Th1 cell polarization compared to classical monocytes. Interleukin-12 (IL-12) and direct contact of patient CD4T cell and CD14CD16 monocytes, were responsible for CD14CD16 monocytes promotion of Th1 cells polarization in PBC. Our study demonstrated that the enhanced CD14CD16 monocyte subset participated in fostering liver damage and inflammatory responses, and promoted Th1 cells skewing in PBC. [ABSTRACT FROM AUTHOR]

Uric acid is a precipitating factor for gout and renal disease, and regular monitoring of uric acid concentration is essential for health management. Hence, accurate measurement is indispensable for clinical diagnosis. Reference measurement procedure (RMP) is an essential element to ensure accuracy and comparability of tested results. In this study, the optimization of a candidate RMP (cRMP) adopting isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for human serum uric acid measurement has been developed. The accuracy of this cRMP was validated by measuring certified reference materials (CRMs) and participating in the external quality assessment (EQA) scheme initiated by International Federation of Clinical Chemistry (IFCC) and National Center for Clinical Laboratory (NCCL). Biases were −0.64-1.51%, which were within the required equivalent limit. Excellent precision was obtained with intra- and inter-assay CVs of 0.42–0.73% and 0.20–0.43%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) was determined to be 0.24 and 1.19 μmol/L, respectively. A good liner response was obtained ranged from 30.0 to 895 μmol/L. Compared with the previous RMP for uric acid, the much easier one-step protein precipitation method without further evaporation, the simplified isocratic elution of chromatographic separation condition, and the shorter analysis running time in only 5 min allowed simpler and faster analysis. Moreover, this optimized cRMP was also applied for value assignment for clinical serum samples covered a wide concentration range of uric acid and obtained a decent calibration and measurement capability (CMC). [Display omitted] • The optimization of a candidate RMP (cRMP) adopting ID-LC-MS/MS method for serum uric acid measurement has been developed. • The method performance was fully validated. • The method was applied to clinical samples and compared with routine methods. [ABSTRACT FROM AUTHOR]

Background: Complete blood count (CBC) reference intervals are important to diagnose diseases, screen blood donors, and assess overall health. However, current reference intervals established by older instruments and technologies and those from American and European populations are not suitable for Chinese samples due to ethnic, dietary, and lifestyle differences. The aim of this multicenter collaborative study was to establish CBC reference intervals for healthy Han Chinese adults. Methods: A total of 4,642 healthy individuals (2,136 males and 2,506 females) were recruited from six clinical centers in China (Shenyang, Beijing, Shanghai, Guangzhou, Chengdu, and Xi’an). Blood samples collected in K2EDTA anticoagulant tubes were analyzed. Analysis of variance was performed to determine differences in consensus intervals according to the use of data from the combined sample and selected samples. Results: Median and mean platelet counts from the Chengdu center were significantly lower than those from other centers. Red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) values were higher in males than in females at all ages. Other CBC parameters showed no significant instrument-, region-, age-, or sex-dependent difference. Thalassemia carriers were found to affect the lower or upper limit of different RBC profiles. Conclusion: We were able to establish consensus intervals for CBC parameters in healthy Han Chinese adults. RBC, HGB, and HCT intervals were established for each sex. The reference interval for platelets for the Chengdu center should be established independently. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

Background: Reference intervals of Liver function tests are very important for the screening, diagnosis, treatment, and monitoring of liver diseases. We aim to establish common reference intervals of liver function tests specifically for the Chinese adult population. Methods: A total of 3210 individuals (20–79 years) were enrolled in six representative geographical regions in China. Analytes of ALT, AST, GGT, ALP, total protein, albumin and total bilirubin were measured using three analytical systems mainly used in China. The newly established reference intervals were based on the results of traceability or multiple systems, and then validated in 21 large hospitals located nationwide qualified by the National External Quality Assessment (EQA) of China. Results: We had been established reference intervals of the seven liver function tests for the Chinese adult population and found there were apparent variances of reference values for the variables for partitioning analysis such as gender(ALT, GGT, total bilirubin), age(ALP, albumin) and region(total protein). More than 86% of the 21 laboratories passed the validation in all subgroup of reference intervals and overall about 95.3% to 98.8% of the 1220 validation results fell within the range of the new reference interval for all liver function tests. In comparison with the currently recommended reference intervals in China, the single side observed proportions of out of range of reference values from our study for most of the tests deviated significantly from the nominal 2.5% such as total bilirubin (15.2%), ALP (0.2%), albumin (0.0%). Most of reference intervals in our study were obviously different from that of other races. Conclusion: These used reference intervals are no longer applicable for the current Chinese population. We have established common reference intervals of liver function tests that are defined specifically for Chinese population and can be universally used among EQA-approved laboratories located all over China. [ABSTRACT FROM AUTHOR]

Microtubule target agents (MTAs) are widely-used clinical anti-cancer drugs for decades, but the acquired drug resistance severely restricted their application. Thioredoxin reductases (TrxR) was reported to be overexpressed in most tumors and closely related to high risk of cancer recurrence and drug resistance, making it a potential target for anticancer drug discovery. Multi-target-directed ligands (MTDLs) by a single molecule provide a logical and alternative approach to drug combinations. In this work, based on the structure-activity relationships obtained in our previous study, some structure modifications were performed. On one hand, the retained skeleton structure of MTAs endowed its tubulin polymerization inhibition activity, on the other hand, the selenium-containing structure and α,β-unsaturated ketone moiety endowed the TrxR inhibition activity. As results, the newly obtained compounds exhibited superior anti-proliferative activities towards various human cancer cells and drug-resistance cells, and displayed high selectivity towards various human normal cells. The mechanism study revealed that the dual effect of cell cycle arrest triggered by targeting tubulin and the abnormal accumulation of ROS caused by TrxR inhibition eventually lead to cell apoptosis. Notably, compared with the MTA agents CA-4P, and the TrxR inhibitor Ethaselen, the optimized compound 14c , which served as dual-targeting inhibitor of tubulin and TrxR, exerted greatly improved in vivo anti-tumor activity. In summary, 14c deserved further consideration for cancer therapy. [Display omitted] • Structure modifications of chalcone derivatives as tubulin and TrxR dual targeting agents were performed. • The optimized compound 14c exerted high cytotoxicity towards both sensitive and resistant cancer cells. • Compound 14c disturbed tubulin-microtubule equilibrium and thus inducing cell cycle arrest. • 14c inhibited TrxR activity, induced ROS accumulation and MMP dysfunction and eventually led to cell apoptosis. [ABSTRACT FROM AUTHOR]

Rapid, sensitive, and specific diagnostic methods are indispensable for sexually transmitted infections (STIs). In this study, a multiplex PCR-dipstick DNA chromatography assay for diagnosis of four STI pathogens, namely Neisseria gonorrhoeae (N. gonorrhoeae), Mycoplasma hominis (M. hominis) , Ureaplasma (U. urealyticum and U. parvum), and Chlamydia trachomatis (C. trachomatis), was established and evaluated. Based on the hybridization of probes and interaction between streptavidin and biotin, PCR products were visualized through hybridization of specific probes and enzymatic color generation. The sensitivity and specificity of all four pathogens were evaluated. Clinical performance of the test was evaluated using 295 specimens, and comparisons among results were determined via culture or colloidal gold assay. No cross-reactions were observed, confirming the high specificity of this method. The limit of detection (LOD) of the four STI pathogens was 100 copies/μL. The sensitivity between PCR-dipstick DNA chromatography and culture or colloidal gold assay ranged from 84.6% to 100%. The specificity was between 93.5% and 96.6%, positive predictive value ranged from 53.6% to 86.7%, negative predictive value was over 98.3%, kappa value ranged from 0.676 to 0.864 (Cohen's kappa coefficient test), and the agreement rate was over 93.5%. In conclusion, PCR-dipstick DNA chromatography serves as a rapid, sensitive, and specific method for simultaneous diagnosis of four STI pathogens. • PCR-dipstick DNA chromatography was rapid, sensitive, and specific. • This assay was able to simultaneously detect four sexually transmitted pathogens. • PCR products were visualized through hybridization of probes and color generation. [ABSTRACT FROM AUTHOR]

Glycocholic acid (GCA) is an important identified biomarker for various hepatobiliary diseases. However, results obtained from routine clinical measurement significantly varied due to the interference from the extremely similar structure analogues, which cause confusion to clinical diagnosis. In this study, a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method was developed. The established method exhibited good performance criteria through the adoption of isotope dilution, calibrator bracketing, and gravimetric measurements. The satisfactory precision (0.16%–2.01%) and good recoveries were achieved at five spiked levels (98.0–100.9%). The limit of detection (LOD) and limit of quantification (LOQ) were 0.01 ng/mL and 0.05 ng/mL, respectively. No interference, matrix effect, and carryover were observed. The sample preparation process was easier to operate, without further solvent evaporation and time-saving. The optimized method was successfully applied to quantify GCA with a wide range of concentrations in clinical human serum samples and also compared with clinical routine systems. [Display omitted] • An isotope-dilution based LC-MS/MS method for serum GCA determination was developed. • The method performance was validated. • The method was applied to clinical samples and compared with routine methods. [ABSTRACT FROM AUTHOR]

Natural products are a large source of clinically effective antitumor drugs. Millepachine, a natural product derived from leguminous plants, was reported to display antitumor activity. In this study, the novel compound, (1H-indol-5-yl) (5-methoxy-2,2-dimethyl-2H-chromen-8-yl)methanone (MIL-1), was designed and synthesized by fusing millepachine and indole rings. MIL-1 exerted much better antitumor activity than millepachine, manifesting as a 24- to 201-fold increase in vitro cytotoxicity and a 2.4-fold increase in in vivo antitumor activity in hepatocellular cell lines-derived models. The immunofluorescence and HPLC detection revealed that MIL-1 was a potent microtubule targeting agent by interfering with the equilibrium of tubulin-microtubule dynamics and irreversibly binding to tubulin. MIL-1 displayed remarkable antitumor activity with an IC 50 of 31–207 nM towards various human cancer cell lines derived from various organs and tissues, and it exerted no evidence of toxicity against normal cells. Mechanistic studies showed that MIL-1 arrested the cell cycle at G2/M phase and induced apoptosis by activating caspase-3 activity and reactive oxygen species (ROS) accumulation. Moreover, the superior antitumor effect of MIL-1 is worthy of further detailed study for the treatment of hepatocellular carcinoma (HCC). • MIL-1 derived from natural product millepachine were designed and synthesized. • MIL-1 exerted a 24–201 folds increase in cytotoxicity than millepachine. • MIL-1 exerted a 2.36-fold increase in vivo antitumor activity. • MIL-1 targeted tubulin system through binding irreversibly to β-tubulin. • MIL-1 arrested cell cycle at G2/M phase and induced HepG2 cell apoptosis. [ABSTRACT FROM AUTHOR]

Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain 15N isotope-labeled recombinant Cys C (15N-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli , and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and 15N-Cys C was expressed in M9 medium using 15N as the only nitrogen source. 15N-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from 15N-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of 15N-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m / z of 15N-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)2+ was the parent ion of 15N-Cys C and that the secondary ions of 15N-labeled peptides from y+5 to y+9 were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of 15N-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS. • The 15N isotope labeled Cys C was successfully expressed and purified, and the purity was more than 95% for the first time. • The codon and GC content of gene were optimized to improve the express yield. • The purified 15N labeled Cys C was confirmed by SDS-PAGE and MALDI-TOF MS. [ABSTRACT FROM AUTHOR]

• A highly reliable, sensitive, accurate, and precise ID-LC–MS/MS-based candidate reference measurement procedure for E 2 measurement was developed and validated. • Bracketing calibration method (BCM) compared to classical calibration curve. • BCM has demonstrated to be higher accurate and precise and in good agreement with the need of RMPs. • Sample preparation requires no derivatization, and appropriate retention of E 2 and high resolution of structural analogs were achieved within a short time span. • The validated method was successfully applied to measure the E 2 level in serum samples and to assess the accuracy of three immunoassays in a clinical laboratory. A highly accurate liquid chromatography tandam mass spectrometry (LC–MS/MS) based candidate reference measurement procedure (cRMP) for the determination of human serum 17β-estradiol (E 2) was developed. A bracketing calibration method (BCM) based on internal standard (IS) concentrations for each sample used to best match the analyte concentration was compared to the classical calibration curve-based method with a constant IS concentration. Precision was analyzed according to the inter-assay reproducibility (n = 15) and intra-assay repeatability (n = 9). Accuracy was confirmed using certified reference materials (CRMs) and further validated by comparison to the established RMPs. Sixty patient serum samples were collected, and the E 2 levels were measured by the BCM isotope dilution (ID) procedure coupled with the LC–MS/MS method to evaluate three immunoassays commonly used in clinical laboratories in China. Limits of quantification (LoQ) as low as 5 pg/mL (18 pmol/L) were achieved. The isomer of E2 (17α-estradiol) was successfully separated by optimizing the LC method. The results of method validation showed that the intra- and inter-assay imprecisions were 1.84 ∼ 2.91% vs 2.08 ∼ 9.53%, while the analytical recoveries were 98.73 ∼ 100.77% vs 93.72 ∼ 102.39% for the BCM and classical calibration curve quantification method, and the BCM demonstrated a higher agreement with CRMs and established RMPs. Bland-Altman plots of the E 2 results revealed concentration-dependent biases. In conclusion, a precise, facile, and reliable ID-LC–MS/MS method is developed in this study. It is demonstrated that the use of the BCM could achieve higher precision and accuracy, which better meet the needs of RMPs. [ABSTRACT FROM AUTHOR]

Adenosine deaminase (ADA) is a key enzyme of adenosine metabolism. There are currently various kits and systems available for ADA measurement, and all yield variable results. This study optimized a reference measurement procedure (RMP) for serum ADA for the standardization of routine methods. ADA coupled with purine-nucleoside-phosphorylase, xanthine-oxidase and peroxidase was selected as the basic method and was optimized using Response Surface Methodology. Then the performance was validated and the results were compared after replication by 3 other reference laboratories. A reference interval was also developed. In addition, this optimized method was applied to calibrate a routine system. The intra-assay precision was 0.44% at both concentrations of 29.8 and 100.4 U/L, and inter-assay precision was 1.01% and 0.95% at 30.1 and 100.3 U/L, respectively. The linearity was up to 351.9 U/L (R2 = 0.9998), with no significant interference or carryover (<5%). A Comparison among 4 reference laboratories showed good reproducibility (R2 ≥ 0.9975). The procedure proved valid for a reference interval of 11.7–38.5 U/L. The mean relative deviation for a routine system was −55.9% and −3.7% before and after calibration. This candidate RMP for serum ADA can potentially be used for standardization of clinical systems. Image 1 • This candidate reference method for serum ADA will facilitate the implementation and standardization of clinical systems. • The enzyme coupling method used in this work is simple, easy, fast, non-toxic and has the potential for automation. • Measurement conditions were optimal, determined by response surface methodology. [ABSTRACT FROM AUTHOR]

• Thermal degradation of moxa floss was studied with TG/DSC and TG-FTIR. • The mass loss rate was relatively low, indicating it burned mildly and slowly. • Combustion heat was released continuously and steadily in a long temperature period. • Infrared radiation spectra emitted by burning moxa floss was similar to human skin. The combustion of moxa floss was investigated by thermogravimetric/differential scanning calorimeter (TG/DSC), thermogravimetric/Fourier transform infrared spectroscopy (TG-FTIR) and an infrared radiation spectrum-detecting system. The average mass loss rate at the major stage of combustion was 1.08%/min with a peak at 7.86%/min. Moxa floss burning released the integral heat of 9188 mJ/mg from the temperature of 200 °C to 500 °C. The peak heat flow during char combustion stage was higher than those of other stages. The results indicated the combustion of moxa floss was a mild and slow process, releasing heat continuously and steadily. The infrared radiation spectra of burning moxa stick were in a range of 2.5–13.0 µm and showed double peaks at 3.75 µm and 8.75 µm. Thus, this study provided insight into why moxa floss biomass is the optimal material for thermal therapy and is beneficial for the development of electric thermal stimulation alternative. [ABSTRACT FROM AUTHOR]