Altered neopterin and IDO in kynurenine metabolism based on LC-MS/MS metabolomics study: Novel therapeutic checkpoints for type 2 diabetes mellitus.

BACKGROUND: This study assessed the alternations of kynurenine pathway (KP) and neopterin in type 2 diabetes mellitus (T2DM) and explored possible differential metabolites. METHODS: A fresh residual sera panel was collected from 80 healthy control (HC) individuals and 72 T2DM patients. Metabolites/ratios of interest including tryptophan (TRP), kynurenine (KYN), 5-hydroxytryptamine (5HT), kynurenic acid (KA), xanthurenic acid (XA), neopterin (NEO), KA/KYN ratio and KYN/TRP ratio were determined using a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics approach, and the difference between groups was assessed. Supervised orthogonal partial least squares-discriminant analysis and differential metabolite screening with fold change (FC) were performed to identify distinct biomarkers. The diagnostic performance of KP metabolites in T2DM was evaluated. RESULTS: Significant decreases of TRP, 5HT, KA, XA, and KA/KYN and increases of KYN/TRP and NEO in T2DM compared to HC group were observed (P < 0.05). The KP metabolites panel significantly changed between T2DM and HC groups (Q2: 0.925, P < 0.005). 5HT (FC: 0.63, P < 0.01) and NEO (FC: 3.27, P < 0.01) were proven to be distinct differential metabolites. A combined testing of fasting plasma glucose and KYN/TRP showed good value in the prediction of T2DM (AUC: 0.904, 95% CI 0.843-0.947). CONCLUSIONS: The targeted LC-MS/MS metabolomics study is a powerful tool for evaluating the status of T2DM. This study facilitated the application of KP metabolomics into future clinical practice. 5HT and NEO are promising biomarkers in T2DM. KYN/TRP was highly associated with the development of T2DM and may serve as a potential treatment target.

Up-to-date quality survey and evaluation of neonatal screening programs in China.

AIMS: To thoroughly evaluate the quality of the entire process of neonatal screening (NBS) in China. METHODS: We collected survey questionnaires from 54.4% (135/248) of NBS institutions in China and conducted on-site visits to 20 of these facilities to validate the data. The quality performance of the institutions was evaluated, and differences across various factors were analysed. RESULTS: Merely 62.5% of the provinces had acceptable performance in neonatal screening. Institutions with limited staff were more prone to organizational management shortcomings. Institutions in provinces with a per capita GDP below 10,000 USD exhibited lower quality control levels than those with a per capita GDP between 10,000 and 15,000 USD. Obstetrics departments have a lower awareness of quality control compared to other blood collection facilities. CONCLUSIONS: A nationwide, comprehensive quality control system for continuous enhancements in quality management, screening, diagnosis, and treatment is imperative to ensure prompt diagnosis and intervention.

Valsartan Attenuated Homocysteine-Induced Impaired Autophagy and ER Stress in Human Umbilical Vein Endothelial Cells.

Hyperhomocysteinemia is a risk factor for various cardiovascular diseases. However, the mechanism underlying homocysteine- (Hcy-) induced vascular injury remains unclear. The purpose of the present study was to examine a potential mechanism by which Hcy induced injury in human umbilical vascular endothelial cells (HUVEC). The protein abundance of autophagy-related markers was markedly decreased after Hcy treatment, which was associated with endoplasmic reticulum (ER) stress and apoptosis in HUVECs. Protein expression level of angiotensin II type 1 receptor (AT1 receptor) was dramatically increased in response to Hcy. Valsartan, an AT1 receptor blocker, improved autophagy and prevented ER stress and apoptosis in HUVECs treated with Hcy. Consistent with this, silence of AT1 receptor with siRNA decreased the protein abundance of ER stress markers, prevented apoptosis, and promoted autophagy in HUVECs. Inhibition or knockdown of AT1 receptor was shown to be associated with suppression of p-GSK3ß/GSK3ß-p-mTOR/mTOR signaling pathway. Additionally, inhibition of autophagy by 3-MA aggravated Hcy-induced apoptosis, while amelioration of ER stress by 4-PBA prevented Hcy-induced injury in HUVECs. Hcy-induced HUVEC injury was likely attributed to AT1 receptor activation, leading to impaired autophagy, ER stress, and apoptosis.

Abnormal kynurenine-pathway metabolites in gout: Biomarkers exploration based on orthogonal partial least squares-discriminant analysis.

BACKGROUND: This study aims to investigate serological characteristics of kynurenine pathway (KP) metabolites in healthy controls (HC) and gout patients and explore possible differential metabolites. METHODS: A total of 191 individual fresh residual sera was collected from 129 HC and 62 gout patients. A liquid chromatography-tandem mass spectrometry method was fully validated to measure 6 metabolites, including tryptophan (TRP), kynurenine (KYN), 5-hydroxytryptamine (5HT), kynurenic acid (KA), xanthurenic acid (XA), and neopterin (NEO). Supervised orthogonal partial least squares-discriminant analysis (OPLS-DA) and differential metabolite screening with fold change (FC) were performed to identify intrinsic variations and differential levels of KP metabolites between the HC and gout groups. Logistic regression was used to assess the contributions of KP metabolites to gout. RESULTS: There were significant decreases of TRP, 5HT, XA, and NEO and increases of KYN, KA, KA/KYN, and KYN/TRP in gout patients compared to the HC group (all p < 0.05). KP metabolites of the gout group showed good discrimination from those of the HC group (Q2: 0.892). Two distinct different metabolites were identified in gout, i.e., XA (FC: 0.56, p < 0.01) and NEO (FC: 0.34, p < 0.01). Of the KP metabolites, KYN was strongly associated with gout (OR: 7.91, p < 0.01). CONCLUSIONS: Abnormal levels of serum KP metabolites were observed in gout. XA and NEO are promising biomarkers that were relevant to the status of gout. The level of KYN could be an attractive checkpoint for the management of gout. Continuous monitoring of KP metabolism in gout provides new opportunities to predict therapeutic efficacy and prognosis.

Poor comparability of plasma renin activity measurement in determining patient samples: the status quo and recommendations for harmonization.

OBJECTIVES: This study aims to investigate and update the consistency and comparability of plasma renin activity (PRA) assays in measuring clinical samples. The contributions of recalibration, blank subtraction, and incubation strategies to interchangeability were also explored. METHODS: Five different laboratories were evaluated using forty-six individual plasma samples, including four liquid chromatography-tandem mass spectrometry (LC‒MS/MS) assays and one chemiluminescence immunoassay (CLIA). Spearman correlation coefficient (R), Passing-Bablok regression, and Bland‒Altman plot analyses were used to evaluate the consistency among assays. Consistency before and after recalibration, blank subtraction, and incubation strategy unification was compared. RESULTS: A good correlation was observed among all assays (R>0.93). None of the samples measured by all assays showed coefficient variation (CV) <10 %, and 37 % of samples showed overall CVs >20 %. The 95 % confidence intervals (CIs) for slopes did not contain 1 for most assay pairs. Large relative biases (-85.1-104.2 %) were found, and 76 % (52-93 %) of samples had unacceptable biases. Recalibration reduced the calibration bias. Ignoring blank subtraction improved the comparability across all assays while unifying incubation did not. CONCLUSIONS: The interchangeability of PRA measurement was unsatisfying. Harmonization on calibrator and ignoring blank were recommended. Unifying incubation strategy was unnecessary.

Development of a designed comparison method based on isotope dilution liquid chromatography-tandem mass spectrometry for determining plasma renin activity and its clinical assessment of renin activity stability in plasma.

Plasma renin activity (PRA) is recommended as the first screening indicator for primary aldosteronism. Immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for quantifying PRA, but the interchangeability across assays and laboratories was suboptimal, which predominantly related to the differences in the plasma incubation strategy. This study aims to establish and validate a designed comparison method based on LC-MS/MS. The sensitivity, matrix effect, precision, accuracy, and storage stability were validated according to the Clinical Laboratory Standard Institution (CLSI) C-62A guidelines. The plasma incubation procedure was optimized to achieve maximum PRA results. The short-term stability of PRA plasma was assessed at 4 °C and room temperature (RT) for specific time points. Differences from the baseline were calculated using a one-way analysis of variance. The designed comparison method for PRA measurement exhibits excellent performance characteristics. The results from the 2022 national external quality assessment scheme for PRA showed good consistency of the developed method with other LC-MS/MS methods (relative biases: -6.8% to 4.6%), which demonstrated the reliability of the established method. Two sets of generation buffers were optimized to maximize the renin activity. The acetate buffer was recommended to be used in laboratory practice due to better metrological sensitivity. PRA plasma is stable for one day at 4 °C and RT. In summary, a reliable, traceable, and reproducible LC-MS/MS method for determining PRA was well-established and validated. The recommended incubation protocol is hoped to reduce the discrepancy in Ang1 generation. The evaluated short-term stability for PRA plasma could provide flexibility in clinical practice.

A Rapid, Simple, Trace, Cost-Effective, and High-Throughput Stable Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry Method for Serum Methylmalonic Acid Quantification and Its Clinical Applications.

Background: Methylmalonic acid (MMA) is an essential indicator of vitamin B12 (VB12) deficiency and inherited metabolic disorders (IMDs). The increasing number of requests for MMA testing call for higher requirements for convenient MMA testing methods. This study aims to develop a convenient quantification method for serum MMA. Methods: The method was established based on the stable isotope-dilution liquid chromatography−tandem mass spectroscopy (ID-LC-MS/MS) technique. The LC-MS/MS parameters and sample preparation were optimized. Specificity, sensitivity, robustness, accuracy, and clinical applicability were validated according to CLSI C62-A guidelines. MMA levels in VB12-sufficient subjects and VB12-deficient subjects were measured. Results: MMA and its intrinsic isomer, i.e., succinic acid (SA), were completely separated. The average slope, intercept, and correlation relationship (R) with 95% confidence intervals, during the two months, were 0.992 (0.926−1.059), −0.004 (−0.012−0.004), and 0.997 (0.995−0.999), respectively. The limit of detection and quantification were <0.058 µmol/L and 0.085 µmol/L, respectively. Intra-run, inter-run, and total imprecisions were 1.42−2.69%, 3.09−5.27%, and 3.22−5.47%, respectively. The mean spiked recoveries at the three levels were 101.51%, 92.40%, and 105.95%, respectively. The IS-corrected matrix effects were small. The VB12-deficient subjects showed higher MMA levels than VB12-sufficient subjects. Conclusions: A convenient LC-MS/MS method for serum MMA measurement was developed and validated, which could be suitable for large-scale MMA testing and evaluating MMA levels in VB12-deficient patients.

The establishment of LC-MS/MS assays-specific reference intervals for serum folates and its application in evaluating FA-supplemented folate deficiency patients: Appeals for a suitable and individualized supplementation.

BACKGROUND: Little known about folates status in folate deficiency patients. This study aims to establish liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay-specific reference intervals (RIs) for serum 5-Methyltetrahydrofolate (5MeTHF), folic acid (FA), 5-Formyltetrahydrofolate (5FoTHF), and total folate (TFOL), and investigate the folates status in FA-supplemented folate deficient patients. METHODS: Sera from 120 reference subjects were selected and measured. An LC-MS/MS method for serum 5MeTHF, FA, and 5FoTHF was employed. RIs were derived based on the CLSI C28-A3. Serum folate levels of 38 FA-supplemented folate deficiency patients were analyzed. RESULTS: RIs (median) for 5MeTHF, FA, 5FoTHF, and TFOL were 3.83-62.33 nmol/L (12.27 nmol/L), 0.30-0.92 nmol/L (0.49 nmol/L), <0.73 nmol/L (0.00 nmol/L), and 4.17-63.47 nmol/L (12.66 nmol/L), respectively. Approximately 53 % (20/38), 74 % (28/38), and 63 % (24/38) of patients presented high levels of 5MeTHF, FA, and TFOL, respectively, which far exceeded the upper reference limit (URL) of the corresponding RIs. A half (18/38) of patients showed simultaneously higher 5MeTHF and FA levels which were beyond the URLs of RIs. Near one-third (11/38) of patients exhibited extremely high FA levels which exceeded the 100-fold URL of RIs. The highest levels can be 539 nmol/L for FA, 364 nmol/L for 5MeTHF, and 686 nmol/L for TFOL. CONCLUSIONS: LC-MS/MS assay-specific RIs were established for folates vitamers without age or gender partitioning. Abnormally high levels of unmetabolized FA and 5MeTHF were observed in quite a few FA-supplemented patients. Considering the adverse risks caused by folates excess, we appeal for a justifiable and individualized FA supplementation. We also recommend establishing LC-MS/MS assay-specific RIs for routine monitoring of folates status.

The spectrum of plasma renin activity and hypertension diseases: Utility, outlook, and suggestions.

BACKGROUND: Plasma renin activity (PRA) is one of the recommended screening indicators for primary aldosteronism (PA) diagnosis and had become increasingly important in hypertension identification, medication guidance, and endocrine disorder confirmation. METHODS: To provide an overview of the PRA measurement progress and clinical value, this review summarizes the main contributing factors related to PRA measurement and necessary precautions during the entire analysis process. We also outline the characteristics of PRA in different endocrine diseases and their clinical utility. RESULTS: Significant inconsistency was observed in PRA measurement methods, including immunoassay and isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS), which could be attributed to preanalytical, analytical, and postanalytical variations. Meanwhile, consensus about environmental and procedural factors during the entire analytical process, including storage temperature, incubation condition, blank subtraction, and standardized operational procedures across different self-developed assay laboratories, could be important to minimize analytical variations. Furthermore, commutable uniform calibrators should be prepared to improve consistency, ultimately achieving accurate and reliable measurement of PRA. CONCLUSION: This review summarizes the clinical utilization of PRA as a biomarker in multiple diseases, elaborating on routine detection methods and the key factors in the analytical process. We also provide feasible strategies for improving standardization and facilitating PRA assessment for larger-scale clinical applications.

A simple method for rapid screening and diagnosis of common organic acidemias: quantitative detection of serum and urine organic acid profiles based on liquid chromatography-tandem mass spectrometry.

Organic acid (OA) analysis is a specific test for inherited metabolic disorders (IMDs); however, the previous detection methods are laborious and costly. This study aims to develop a rapid method for the simultaneous quantification of serum and urine OA profiles. The method was established based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. The specificity, sensitivity, robustness, and accuracy of the established method were validated. Fifteen healthy subjects and nine IMD patients were measured for clinical validation. OAs with their intrinsic isomers were completely separated. The LC-MS/MS analysis time was 5.5 min. Calibration curves were linear within the ranges of 27.00 µg/g for all OAs. The average correlation relationship (R) varied from 0.9891 to 0.9998. The limit of detection and limit of quantification varied from 0.003 to 0.07 µg/g and 0.006 to 0.08 µg/g, respectively. No obvious carryover was observed. The intra-assay, inter-assay, and total imprecisions were 1.22-4.14%, 0.90-5.20%, and 1.67-5.90%, respectively. The mean spiked recovery at the three levels varied from 94.31 to 106.68%. The matrix effects can be compensated for by internal standard correction. Nine IMD patients were identified. A robust LC-MS/MS method for the rapid determination of serum and urine OA profiles without derivatization or liquid-liquid extraction was developed and validated. The analysis of five common OAs can be completed in short minutes. This innovative LC-MS/MS method for OA profiles may present its potential in future rapid screening and diagnosis of IMDs.

Comparison of four different immunoassays and a rapid isotope-dilution liquid chromatography-tandem mass spectrometry assay for serum folate.

OBJECTIVES: Accurate measurement of serum folate is essential for the diagnosis and management of various disorders. This study aims to investigate the between-method differences of four immunoassays and a rapid isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method. METHODS: Roche Cobas (USA), Abbott Alinity i2000 (USA), Beckman Coulter Access (USA), Mindray CL-6000i (China), and the ID-LC-MS/MS method were compared using 46 human serum samples. The results were analysed by Passing-Bablok regressions and Bland-Altman plots. A bias of 13.31% based on biological variation was used as the bias criterion. RESULTS: All the within-run and total coefficients of variation (CVs) met the specification. The folate concentrations determined by all the assays were significantly different (p=0.0028). All assays had correlation coefficients over 0.97 with each other. The 95% confidence intervals (CIs) for the slope seldom contained 1 and few 95% CIs for the intercept contained 0 in the regression equations. Compared to ID-LC-MS/MS, the biases of all assays ranged from -20.91 to 13.56 nmol/L, and the mean relative biases ranged from -9.85 to 40.33%. The predicted mean relative biases at the medical decision levels rarely met the criterion. CONCLUSIONS: Assays for serum folate had good correlations with each other but lacked good agreement. The accuracy and consistency of assays for serum folate should be measured and assessed routinely. Standardization work to improve the accuracy of serum folate assays, such as the extension of traceability to reference methods or materials, calibration standardization efforts, and assay-adjusted cut-offs should be promoted.

Prevalence of methylmalonic acidemia among newborns and the clinical-suspected population: a meta-analyse.

IMPORTANCE: Knowing the scale of rare inborn errors is important for screening and resource allocation. Evidence on the prevalence of methylmalonic acidemia (MMA) among newborns and the clinical-suspected population from large-scale screening programs needs to be systematically synthesized. OBJECTIVE: To estimate the worldwide prevalence of MMA for newborns and the clinical-suspected population and explore the differences in different regions, periods, and diagnostic technologies. DATA SOURCES: MEDLINE, Embase, CRD, Cochrane Library, Scopus, CINAHL, and PROSPERO. Study Selection: All studies reporting the epidemiology characteristics of MMA were selected. DATA EXTRACTION AND SYNTHESIS: Characteristics of study, subjects, and epidemiology were extracted, random-effect models were used for meta-analyses. MAIN OUTCOME AND MEASURE: Pooled prevalence of MMA. RESULTS: This study included 111 studies. The pooled prevalence of MMA worldwide was 1.14 per 100,000 newborns (1516/190,229,777 newborns, 95% CI: 0.99-1.29) and 652.11 per 100,000 clinical-suspected patients (1360/4,805,665 clinical-suspected individuals, CI: 544.14-760.07). Asia and Africa got a higher pooled prevalence of MMA. The prevalence of MMA in newborns increased through the years, while that in the clinical-suspected population decreased. Collecting blood ≥ 72 h after birth had a higher pooled prevalence of MMA than collecting during 24 h-72 h after birth. The combining-use of MS/MS and GC/MS had a higher pooled prevalence than the single-use of MS/MS or GC/MS. Prevalence of cbl C, mut, cbl B, cbl A, isolated MMA, combined MMA and homocystinuria, vitamin B12-responsive MMA was synthesized. CONCLUSIONS AND RELEVANCE: Prevalence of MMA among newborns was extremely low, but considerably high in the clinical-suspected population, indicating the need for more efficient newborn screening strategies and closer monitoring of the high-risk population for the early signs of MMA. Asia and Africa should attach importance to the high prevalence of MMA. Further diagnostic tests were recommended for the combining-use vs single-use of MS/MS and GC/MS and for collecting blood after 72 h vs during 24-72 h after birth.

M1 Bone Marrow-Derived Macrophage-Derived Extracellular Vesicles Inhibit Angiogenesis and Myocardial Regeneration Following Myocardial Infarction via the MALAT1/MicroRNA-25-3p/CDC42 Axis.

Myocardial infarction (MI) is a severe cardiovascular disease. Some M1 macrophage-derived extracellular vesicles (EVs) are involved in the inhibition of angiogenesis and acceleration dysfunction during MI. However, the potential mechanism of M1 phenotype bone marrow-derived macrophages- (BMMs-) EVs (M1-BMMs-EVs) in MI is largely unknown. This study sought to investigate whether M1-BMMs-EVs increased CDC42 expression and activated the MEK/ERK pathway by carrying lncRNA MALAT1 and competitively binding to miR-25-3p, thus inhibiting angiogenesis and myocardial regeneration after MI. After EV treatment, the cardiac function, infarct size, fibrosis, angiogenesis, and myocardial regeneration of MI mice and the viability, proliferation and angiogenesis of oxygen-glucose deprivation- (OGD-) treated myocardial microvascular endothelial cells (MMECs) were assessed. MALAT1 expression in MI mice, cells, and EVs was detected. MALAT1 downstream microRNAs (miRs), genes, and pathways were predicted and verified. MALAT1 and miR-25-3p were intervened to evaluate EV effects on OGD-treated cells. In MI mice, EV treatment aggravated MI and inhibited angiogenesis and myocardial regeneration. In OGD-treated cells, EV treatment suppressed cell viability, proliferation, and angiogenesis. MALAT1 was highly expressed in MI mice, OGD-treated MMECs, M1-BMMs, and EVs. Silencing MALAT1 weakened the inhibition of EV treatment on OGD-treated cells. MALAT1 sponged miR-25-3p to upregulate CDC42. miR-25-3p overexpression promoted OGD-treated cell viability, proliferation, and angiogenesis. The MEK/ERK pathway was activated after EV treatment. Collectively, M1-BMMs-EVs inhibited angiogenesis and myocardial regeneration following MI via the MALAT1/miR-25-3p/CDC42 axis and the MEK/ERK pathway activation.

A machine learning model for the prediction of down syndrome in second trimester antenatal screening.

BACKGROUND: Down syndrome (DS) is the most common human chromosomal abnormality. About 1200 laboratories carry out antenatal screening for DS in second trimester pregnancies in China. Their prenatal assessment of DS pregnancy risk is based on biometric calculations conducted on maternal serum biochemical markers and ultrasonic markers of fetal growth. However, the performance of this triple test for DS in second trimester pregnancies has a false positive rate of 5%, and a detection rate of about 60%∼65%. METHOD: A total of 58,972 pregnant women, including 49 DS cases, who had undergone DS screening in the second trimester were retrospectively included and a machine learning (ML) model based on random forest was built to predict DS. In addition, the model was applied to another hospital data set of 27,170 pregnant women, including 27 DS cases, to verify the predictive efficiency of the model. RESULTS: The ML model gave a DS detection rate of 66.7%, with a 5% false positive rate in the model data set. In the external verification data set, the ML model achieved a DS detection rate of 85.2%, with a 5% false positive rate . In comparison with the current laboratory risk model, the ML model improves the DS detection rate with the same false positive rate, while the difference has no significance. CONCLUSIONS: The ML model for DS detection described here has a comparable detection rate with the same false positive rate as the DS risk screening software currently used in China. Our ML model exhibited robust performance and good extrapolation, and could function as an alternative tool for DS risk assessment in second trimester maternal serum.

Identification of rare heterozygous linkage R965C-R1309H mutations in the pore-forming region of SCN5A gene associated with complex arrhythmia.

BACKGROUND: We examined the genetic background of a Chinese Han family in which some members presented with complex arrhythmias including sick sinus syndrome, progressive conduction block, atrial fibrillation, atrial standstill and Brugada syndrome. The possible underlying mechanism associated with the genetic mutation was explored. METHODS: Targeted capture sequencing was conducted in the probands in the coding and splicing regions of genes implicated in inherited arrhythmias. Stable cell lines overexpressing wild type (WT) or mutant SCN5A were generated in HEK293T cells. Whole-cell recording was performed to evaluate the functional changes in sodium channels. RESULTS: The rare heterozygous linkage mutations, SCN5A R965C and R1309H, were found in these patients with complex familial arrhythmias. Compared to WT, R965C or R1309H, the peak current of sodium channel was dramatically reduced in HEK293T cell with linkage R965C-R1309H mutation when testing potentials ranging from -45 to 15 mV. Notably, the maximum peak current of sodium channels with R1309H and linkage R965C-R1309H displayed significant decreases of 31.5% and 73.34%, respectively, compared to WT. Additionally, compared to R965C or R1309H alone, the linkage mutation R965C-R1309H demonstrated not only a more obvious depolarisation-shifted activation and hyperpolarisation-shifted inactivation, but also a more significant alteration in the time constant, V1/2 and the slope factor of activation and inactivation. CONCLUSIONS: The linkage mutation SCN5A R965C-R1309H led to a more dramatically reduced current density, as well as more significant depolarisation-shifted activation and hyperpolarisation-shifted inactivation in sodium channels than R965C or R1309H alone, which potentially explain this complex familial arrhythmia syndrome.

Multiple roles of Ca2+ in the interaction of ciprofloxacin with activated sludge: Spectroscopic investigations of extracellular polymeric substances.

Calcium ion is an important cation influencing the binding of recalcitrant organic contaminants with activated sludge during wastewater treatment process, but there is still unknown about its role in amphoteric fluoroquinolones binding. Binding experiments show that Ca2+ markedly inhibited binding of ciprofloxacin (CIP) onto sludge, causing 7-203 times of CIP release. Multi-spectroscopic examinations indicate that tryptophan-like and tyrosine-like proteins in extracellular polymeric substances (EPS) were dominant components for CIP binding by static quenching and forming CIP-proteins complexes. Addition of Ca2+ into EPS and CIP binding systems induced increase of association constants (from 0.024-0.064 to 0.027-0.084 L/µmol) and binding constants (from 0.002-0.039 to 0.012-0.107) and decrease of binding sites number (from 0.893-2.007 to 0.721-1.386). Functional groups of EPS and secondary structure of proteins were remarkably changed upon reactions with CIP and Ca2+. Calcium ion interacted with EPS and CIP binding system in two distinct ways: Ca2+ shielded CO in amide I in EPS for CIP binding, whereas strengthened binding between CIP and functional groups including CO in carboxyl groups in extra-microcolony polymers and OH in extra-cellular polymers by forming ternary complexes. Cation competition for CO in amide I is responsible for Ca2+ induced CIP release from the sludge. Results suggest the highly potential release of CIP from high saline wastewater and cation-conditioned sludge which needs further monitoring and evaluation.

Acute cardiac injury in adult hospitalized COVID-19 patients in Zhuhai, China.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has already became a public health emergency of international concern. COVID-19 related cardiac injury remains largely unclear. METHODS: We retrospectively analyzed demographic, clinical, laboratory and cardiovascular imaging data of all consecutively admitted adult COVID-19 patients in Zhuhai, China from January 17th, 2020 to February 18th, 2020. RESULTS: A total of 93 patients were included in the study. Acute cardiac injury was found in 9 (9.7%) COVID-19 patients with median level of hypersensitive cardiac troponin I (hs-cTnI) to be 0.085 µg/L (IQR 0.027-0.560 µg/L). Compared with patients without cardiac injury, the median age of patients with cardiac injury was significantly older (65.0 vs. 44.0, P<0.05), hypertension was significantly more common (44.4% vs. 14.3%, P<0.05), and the proportion of severe-critical cases were greater (77.8% vs. 17.9%, P<0.05). Patients with cardiac injury were more likely have elevation of N-terminal proBNP (NT-proBNP) in comparison (66.7% vs. 10.0%, P<0.05). There was no significant difference in echocardiographic parameters between patients with and without cardiac injury. Multivariable logistic regression analysis indicated that older age (OR: 1.093, 95% CI: 1.011-1.182) and increased NT-proBNP (OR: 10.979, 95% CI: 2.024-59.555) were independent risk factors for cardiac injury. Cardiac magnetic resonance (CMR) imaging performed on three patients at around one month after they underwent significant hs-cTnI elevation showed that they had underlying cardiovascular comorbidities. CONCLUSIONS: Acute cardiac injury was seen in the minority of hospitalized COVID-19 patients in Zhuhai, China. Older age and increased NT-proBNP were associated with acute cardiac injury. REGISTRATION NUMBER: ChiCTR2000030952.

Ku80 promotes melanoma growth and regulates antitumor effect of melatonin by targeting HIF1-α dependent PDK-1 signaling pathway.

Melanoma is one of the most malignant and aggressive cancers with high cancer-related deaths. However, it is unclear whether Ku80 regulates tumor growth in human melanoma. In this study, we screened a siRNA library targeting 6024 human genes and identified Ku80 as a potential therapeutic target in melanoma cells. Knockdown of Ku80 significantly suppressed melanoma cell proliferation and induced apoptosis, as well as enhanced the antitumor effect of melatonin in melanoma in vitro and in vivo. Overexpression of Ku80, however, promoted melanoma growth and increased the insensitivity of melanoma cells to melatonin. Mechanistically, we found that Ku80 bound to the PDK1 promoter and activated the transcription of PDK1. Moreover, we showed that the binding of Ku80 at the PDK-1 promoter was HIF1-α dependent, and melatonin degraded HIF1-α in melanoma cells. Furthermore, clinical data revealed that the expression of Ku80 and PDK-1 proteins were positively correlated and elevated in the tumor tissues of melanoma patients, and high expression of Ku80 predicted a poor prognosis in melanoma. Collectively, our study demonstrated that Ku80 promoted melanoma growth and regulated antitumor activity of melatonin by targeting HIF1-α dependent PDK-1 signaling pathway, suggesting that Ku80 may be a potential molecular target for melanoma treatment.