Determination of cotinine and 3-hydroxynicotinine in human serum by liquid chromatography-tandem mass spectrometry and its application.

In this study, we developed a method for determining cotinine and 3-hydroxycotinine in human serum and established a methodology for an in-depth study of tobacco exposure and health. After the proteins in the human serum samples were precipitated with acetonitrile, they were separated on a ZORBAX SB-Phenyl column with a mobile phase of methanol encompassing 0.3% formic acid-water encompassing 0.15% formic acid. The measurement was performed on an API5500 triple quadrupole mass spectrometer in the multiple reaction monitoring mode. Cotinine, 3-hydroxycotinine, and cotinine-d3 isotope internal standards were held for 2.56 minutes, 1.58 minutes, and 2.56 minutes, respectively. In serum, the linear range was 0.05 to 500 ng·mL-1 for cotinine and 0.50 to 1250 ng·mL-1 for 3-hydroxycotinine. The lower limit of quantification (LLOQ) was 0.05 ng·mL-1 and 0.5 ng·mL-1 for cotinine and 3-hydroxycotinine, respectively. The intra-day and inter-day relative standard deviations were <11%, and the relative errors were within ±â€…7%. Moreover, the mean extraction recoveries of cotinine and 3-hydroxycotinine were 98.54% and 100.24%, respectively. This method is suitable for the rapid determination of cotinine and 3-hydroxycotinine in human serum because of its rapidity, sensitivity, strong specificity, and high reproducibility. The detection of cotinine levels in human serum allows for the identification of the cutoff value, providing a basis for differentiation between smoking and nonsmoking populations.

Gestational diabetes mellitus is associated with distinct folate-related metabolites in early and mid-pregnancy: A prospective cohort study.

AIMS: This study aimed to evaluate the association between gestational diabetes mellitus (GDM) and circulating folate metabolites, folic acid (FA) intake, and the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genotype. MATERIALS AND METHODS: A prospective pregnancy cohort study was conducted in Beijing, China, from 2022 to 2023. Circulating folate metabolites, including red blood cell (RBC) 5-methyltetrahydrofolate (5-MTHF), 5, 10-methylene-tetrahydrofolate (5,10-CH2-THF), 5- formyltetrahydrofolate (5-CHO-THF), and unmetabolised folic acid (UMFA), and plasma homocysteine (HCY), 5-MTHF, and methylmalonic acid (MMA), were determined at 6-17 weeks and 20-26 weeks of gestation. FA intake and the MTHFR and MTRR genotype were also examined. GDM was diagnosed between 24 and 28 weeks of pregnancy by a 75-g oral glucose tolerance test (OGTT). The association between the folate status and GDM was ascertained using multivariate generalised linear models, logistic regression models, and restricted cubic spline regression, adjusting for potential confounders. RESULTS: The study included 2032 pregnant women, of whom 392 (19.29%) developed GDM. UMFA above the 75th percentile (≥P75) [adjusted OR (aOR) (95% confidence interval [CI]) = 1.36 (1.01-1.84)], UMFA ≥ P90 [aOR (95% CI) = 1.82 (1.23-2.69)], and HCY ≥ P75 [aOR (95% CI) = 1.40 (1.04-1.88)] in early pregnancy, and RBC 5-MTHF [aOR (95% CI) = 1.48 (1.10-2.00)], RBC 5,10-CH2-THF [aOR (95% CI) = 1.55 (1.15-2.10)], and plasma 5-MTHF [aOR (95% CI) = 1.36 (1.00-1.86)] in mid-pregnancy ≥ P75 are associated with GDM. Higher UMFA levels in early pregnancy show positive associations with the 1-h and 2-h glucose levels during the OGTT, and higher HCY levels are associated with increased fasting glucose levels during the OGTT. In comparison, RBC 5- MTHF and 5,10-CH2-THF, and plasma 5- MTHF in mid-pregnancy are positively associated with the 1-h glucose level (p < 0.05). The MTHFR and MTRR genotype and FA intake are not associated with GDM. CONCLUSIONS: Elevated levels of UMFA and HCY during early pregnancy, along with elevated RBC 5-MTHF and 5,10-CH2-THF and plasma 5-MTHF during mid-pregnancy, are associated with GDM. These findings indicate distinct connections between different folate metabolites and the occurrence of GDM.

Occurrence and health risk of pesticide residues in Chinese herbal medicines from Shandong Province, China.

Pesticide residue was one of the stress factors affecting quality and safety of Chinese herbal medicines (CHMs). The present study was designed to investigate the occurrence and dietary exposure of 70 pesticide residues in 307 samples of CHMs, including 104 American ginseng, 100 Ganoderma lucidum (G. lucidum), and 103 Dendrobium officinale (D. officinale) in Shandong Province, China. The study revealed that a total of 29 pesticides were detected in the majority (92.5%) of samples, and the pesticide residues of 85 (27.7%) samples exceeded the maximum residue levels (MRLs). Particularly, the maximum concentration of chlorpyrifos was 23.8 mg kg-1, almost 50 times of the MRLs in food in GB 2763-2021, while there's no standard restrictions specified in CHMs in China. The chronic, acute, and cumulative risk assessment results indicated that risk exposure of the three types of CHMs were unlikely to pose a health risk to consumers. However, more attention should be paid to the multiple residues with the presence of four or more pesticides in one sample and high over-standard rate of pesticides. The pesticide users and the government should pay more attention to the pesticides used in CHMs and regularly monitor the presence of these compounds. The study recommended the MRLs of these pesticides in CHMs should be established and perfected by the relevant departments in China.

Simultaneous detection of dual microRNAs related to EV71 using ICP-MS based on metal nanoparticle labeling with hybridization chain reaction.

BACKGROUND: Hand, foot, and mouth (HMFD) disease caused by enterovirus 71 (EV 71), is closely associated with severe clinical manifestations and can be deadly. Early detection of EV 71 can be achieved by detecting the increment in miR296 and miR16 in the serum. Using HCR to amplify signals and convert biological signals into metal nanoparticle signals detectable by ICP-MS is a detection method that can collect more accurate and reliable information, compared with traditional methods, in the detection of biological samples. RESULTS: We described a strategy for the simultaneous detection of miR296 and miR16 by ICP-MS based on metal nanoparticles (NPs) labeling with HCR. Briefly, single-stranded DNA (ssDNA) and magnetic beads (MBs), as well as NPs and signal probes for miRNA (Sp-miR) were firstly conjugated via the streptavidin-biotin recognition system, constituting ssDNA-MBs and NPs-Sp-miR complex, respectively. The latter complex then hybridized with the former through HCR, generating the nanosensors for targets. Then, the targets were added and hybridized with ssDNA, and the HCR complex with NPs was released into the solution. Finally, the corresponding signals of the NPs were measured by ICP-MS. Results demonstrated that the developed method had good sensitivity and satisfactory selectivity and precision. Furthermore, when applied to biological samples with a complex matrix, the developed method also showed good recovery (88 % - 92 %) and reproducibility (RSD<10 %). SIGNIFICANCE: This method contributes to the early diagnosis of HFMD and opens up ideas for the further development of high-throughput biomarker detection. The strategy has practical potential for miR296 and miR16 detection in biological samples and provides a promising tool for multiple miRNA detection.

ICP-MS and fluorescence dual-mode detection of ZIKV-RNA based on quantum dot labeling with hybridization chain reaction.

The detection of Zika virus (ZIKV) is of great significance to human life and health. Herein, we presented an ICP-MS and fluorescent dual-mode sensor for quantitative analysis of Zika virus RNA fragments (ZIKV-RNA), which employed quantum dots (QDs) as signal tags and combined with hybridization chain reaction (HCR). The dual-mode sensor realized cross-checking of the analysis results and improved the assay accuracy. Firstly, the single-stranded DNA (ssDNA) was anchored on the surface of magnetic beads (MBs). Afterward, HCR was conducted with probe DNA-CdSe quantum dots conjugates (pDNA-QDs) and link DNA (lDNA), producing the MBs-ssDNA-[pDNA-QDs-lDNA]n conjugates. In the presence of target ZIKV-RNA, a strand displacement reaction occurred, leading to the dissociation of the [pDNA-QDs-lDNA]n labels from the conjugates into the solution. Finally, the signal intensity was detected using ICP-MS and fluorescence analysis, with achieved limits of detection of 131 pM and 152 pM, respectively. The inter-assay RSD values of fluorescence and ICP-MS were 3.94 % and 4.26 % at 10 nM level, respectively, showing that the method had good precision. This method showed high selectivity and was applied to the analysis of biological fluids. There was no significant difference between the results of ICP-MS modes and fluorescence mode. This method offers a new strategy for sensitivity analysis of ZIKV-RNA and exhibits promise in clinical applications for diagnosis.

Occurrence of 8 trace elements in Rhizoma Cibotii from China and exposure assessment.

The contamination of trace elements in Chinese edible herbs has attracted worldwide concern over the world. The objective of the present study was to investigate the occurrence and exposure assessment of eight trace elements in Rhizoma Cibotii from China. For this purpose, the method of inductively coupled plasma mass spectrometry was employed to detect the contamination levels of target trace elements in 58 Rhizoma Cibotii samples. The results demonstrated that the trace elements of Cr, Ni, Cu, Zn, and Pb were detected in all analyzed samples; the occurrence frequencies of As, Se, and Cd were 98.3%, 96.6%, and 98.3%, respectively. The highest mean levels were found in Zn (17.32 mg/kg), followed by Pb (8.50 mg/kg) and Cu (3.51 mg/kg). For a further step, one-way ANOVA was used to compare the difference of eight elements levels among groups, and Pearson's correlation analysis was used to explore the correlation between elements in Rhizoma Cibotii. A strong positive correlation between Zn and Cd was observed by Pearson's correlation analysis, which indicated that the possible presence of Cd contamination in Rhizoma Cibotii. Based on the contamination levels, the mean exposure of individual element and the health risks of eight trace elements in Rhizoma Cibotii were estimated by health risk assessment models. The calculated HQ values were less than 1, indicating that the contamination of trace elements in Rhizoma Cibotii did not pose significant health risks to human. In conclusion, the study provided baseline information on the contamination levels of trace elements in Rhizoma Cibotii. Moreover, it is necessary to monitor the trend of trace elements levels in Rhizoma Cibotii, which will be useful for ingredient control and human health protection.

Molecular basis of signal transduction mediated by the human GIPR splice variants.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, ß-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated ß-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).

Diverse associations observed between pregnancy complications and RBC or plasma folates determined by an in-house developed LC-MS/MS method.

BACKGROUND: As folates are essential for embryonic development and growth, it is necessary to accurately determine the levels of folates in plasma and red blood cells (RBCs) for clinical intervention. The aims of this study were to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitation of folates in plasma and RBCs and to examine the association between plasma and RBC folate concentrations and gestational diabetes mellitus (GDM), gestational hypertension (GH) and preeclampsia (PE). METHODS: With the in-house developed LC-MS/MS, a retrospective cross-sectional study was conducted. The healthy pregnant women of first- (n = 147), second- (n = 84) and third-trimester (n = 141) or the women diagnosed with GDM (n = 84), GH (n = 58) or PE (n = 23), that were aged between 22 and 46 years old and registered at our institute, were subjected for measurement of folic acid (FA) and 5-methyltetrahydrofolate (5-MTHF), followed by appropriate statistical association analysis. RESULTS: The assay for simultaneous quantitation of FA and 5-MTHF in plasma and RBCs was linear, stable, with imprecision less than 15% and recoveries within ±10%. The lower limits of quantification for FA and 5-MTHF measurement in whole blood were 0.57 and 1.09 nmol/L, and in plasma were 0.5 and 1 nmol/L, respectively. In the association analysis, the patients with lower RBC folate level (<906 nmol/L) presented higher risks of PE development (OR 4.861 [95% CI 1.411-16.505]) by logistic regression and restricted cubic spline (RCS) regression in a nonlinear fashion. In addition, higher level of plasma folates in pregnancy was significantly associated with GH risk but may be protective for the development of GDM. CONCLUSIONS: The in-house developed LC-MS/MS method for folates and metabolites in plasma or RBC showed satisfactory analytical performance for clinical application. Further, the levels of folates and metabolites were diversely associated with GDM, GH and PE development.

The Association between Macrosomia and Amino Acids' Levels in Maternal and Cord Sera: A Case-Control Study.

This study aims to explore the relationship between macrosomia and amino acids in maternal and cord sera. METHODS: In the case-control study, 78 pairs of mothers and newborns were recruited from December 2016 to November 2019. Participants were divided into the macrosomia group (BW ≥ 4000 g, n = 39) and the control group (BW between 2500 g and 3999 g, n = 39) according to the birth weight (BW) of newborns. Maternal vein blood samples were collected before delivery and cord vein blood samples were collected after birth. The levels of amino acids in maternal and cord sera were measured by liquid chromatography and mass spectrometry (LC-MS/MS) in the year 2021. The difference in amino acid levels in maternal and cord sera between the two groups was compared, and the contribution of each amino acid to the difference between the two groups was analyzed. Unconditional logistic regression analysis was used to test the relationship between macrosomia and amino acids. RESULTS: In maternal serum during the antepartum, the levels of asparagine, glutamine, methionine, alanine, and threonine in the macrosomia group were higher but arginine was lower than that in the control group (p < 0.05). In cord serum, the levels of lysine, histidine, phenylalanine, arginine, tryptophan, valine, isoleucine, glutamate, tyrosine, and total essential amino acid (EAA) in the macrosomia group were lower while glutamine was higher than that in the control group (p < 0.05). The ratios of EAA, valine, threonine, methionine, tryptophan, and alanine in maternal serum to those in cord serum were higher, while the ratio of glutamine was lower in the macrosomia group (p < 0.05). Arginine and threonine in maternal serum and glutamate, glutamine, and histidine in cord serum were associated with macrosomia (p < 0.05). CONCLUSION: Most of the amino acid levels in the maternal sera of the macrosomia group are higher than those in the control group, while most of the amino acids' levels in the cord sera of the macrosomia group are lower than those in the control group. The ratios of some amino acids in maternal serum to those in cord serum were different between the two groups. Arginine and threonine in maternal serum and glutamate, glutamine, and histidine in cord serum are closely related to macrosomia.

[Effect of different delivery modes on the level of cortical hormones in cord artery and vein blood].

OBJECTIVE: To investigate the difference of cortical hormones in cord artery and vein blood of newborns with different delivery modes. METHODS: A total of 65 pregnant women who delivered in the People's Hospital of Danyang City, Jiangsu Province from June to September 2021 were selected as the study subjects, including 26 cases of spontaneous delivery and 39 cases of cesarean section. The basic information of 65 pregnant women and newborns was collected by questionnaire survey. According to the mode of delivery, the levels of corticosteroids in umbilical vein and umbilical artery blood were determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS), including corticosterone, 11-desoxycorticosterone, aldosterone, cortisol, 11-deoxycortisol and cortisone. The data were statistically analyzed using IBM SPSS Statistics 26.0 statistical software. RESULTS: The levels of cortisol, 11-deoxycortisol, aldosterone, cortisol, 11-deoxycortisol and cortisone in the umbilical vein blood of the spontaneous delivery group were(2.44±1.87), (0.64±0.29), (0.49±0.35), (54.95±40.80), (3.20±1.23) and(142.27±57.42)ng/mL, respectively. The levels of corticosterone, 11-deoxycortisol, aldosterone, cortisol, 11-deoxycortisol and cortisone in umbilical artery blood were(4.51±4.47), (0.57±0.28), (0.42±0.29), (60.79±45.53), (2.69±1.25) and(123.10±46.32)ng/mL, respectively. The levels of corticosterone, 11-deoxycortisone, aldosterone, cortisol, 11-deoxycortisone and cortisone in umbilical vein blood of cesarean section group were(0.94±1.09), (0.47±0.14), (0.26±0.14), (22.63±19.82), (2.30±0.90) and(84.51±29.49)ng/mL, respectively. The levels of corticosterone, 11-deoxycortisol, aldosterone, cortisol, 11-deoxycortisol and cortisone in umbilical artery blood were(2.22±2.24), (0.43±0.17), (0.27±0.14), (30.09±25.93), (1.87±0.76) and(75.03±24.90)ng/mL, respectively. The levels of corticosterone, 11-desoxycorticosterone, aldosterone, cortisol, 11-deoxycortisol and cortisone in cord vein blood and cord artery blood in spontaneous labor group were significantly higher than those in cesarean section group(P<0.05). The levels of corticosterone and cortisol in cord vein blood were significantly lower in spontaneous labor group and cesarean section group than those in cord artery blood(P<0.05), the levels of 11-desoxycorticosterone, 11-deoxycortisol and cortisone in cord vein blood were significantly higher in spontaneous labor group and cesarean section group than those in cord artery blood(P<0.05). CONCLUSION: There are differences in the level of cortical hormones in cord artery and vein blood in different delivery modes.

Dissolution of lignocellulose with high lignin content in AlCl3/ZnCl2 aqueous system and properties of the regenerated cellulose film.

Herein, a novel method for dissolving lignocellulose at room temperature is proposed by combining deep eutectic solvents (DES) pretreatment and subsequent dissolution in AlCl3/ZnCl2 aqueous system. Results showed that DES pretreatment could significantly increase the dissolubility of lignin-containing cellulose (CL) samples in AlCl3/ZnCl2 aqueous system. The dissolution ratio of the CL sample with 15.6 % lignin content in AlCl3/ZnCl2·3H2O solvent was as high as 90 %. Besides, the mechanism for the remarkable dissolution of CL samples in low water AlCl3/ZnCl2 aqueous solvent was also proposed. Moreover, the dissolved CL sample was regenerated for the production of lignocellulose films, which have excellent ultraviolet (UV) blocking, hydrophobic, mechanical strength, and natural degradation properties. In particular, the films could be completely naturally degraded after 10 days, which provided a promising way to prepare biodegradable lignocellulose materials, and to encourage the potential utilization of renewable lignocellulose in packaging industry.

Clinical efficacy of intravesical gemcitabine combined with ubenimex in patients with non-muscle-invasive bladder carcinoma after transurethral resection of bladder tumor.

Objectives: To evaluate the clinical value of intravesical gemcitabine combined with immunotherapy in patients with non-muscle-invasive bladder carcinoma (NMIBC) after transurethral resection of bladder tumor (TURBT). Methods: Eighty patients with non-muscle-invasive urothelial carcinoma treated in Baoding No.1 Hospital from November 2016 to November 2019 were randomly divided into two groups, with 40 patients in each group. Both groups underwent TURBT. After surgery, the research group was treated with intravesical chemotherapy using gemcitabine combined with ubenimex, while the control group was given 40 mg pirarubicin by intravesical instillation. Postoperative condition was evaluated by cystoscopy every three months in both groups. The recurrence six months, one year and two years after treatment, the incidence of lower urinary tract symptoms such as dysuria, hematuria and frequent urination, general adverse drug reactions such as rashes, liver function damage and gastrointestinal reaction, as well as the changes in CD3+, CD4+, CD8+ and CD4+/CD8+ T lymphocyte subsets before and after treatment were comparatively analyzed between the two groups. Results: The recurrence rate showed no statistical significance between the two groups 6 months after treatment (p=0.17), but significant differences one year (p=0.04) and two years (p=0.03) after treatment, which were significantly lower in the research group than the control group. The incidence of adverse drug reactions was 22.5% in the research group and 7.5% in the control group, without significant difference (p=0.36). The incidence of lower urinary tract symptoms was 32.5% and 55%, respectively, in the research group and the control group. The incidence of lower urinary tract symptoms in the research group was significantly lower compared with the control group, with a statistically significant difference (p=0.04). After treatment, CD3+, CD4+ and CD4+/CD8+ levels in the research group increased significantly than those in the control group, with statistically significant differences (CD3+, p=0.01; CD4+, p=0.00; CD4+/CD8+, p=0.00). Conclusions: For NMIBC patients receiving bladder-preserving surgery, intravesical gemcitabine combined with immunotherapy can reduce the recurrence rate, relieve lower urinary tract symptoms, increase the tolerance of patients to intravesical chemotherapy and significantly improve the function of T lymphocytes, without obvious increase in adverse drug reactions. Therefore, it is safe and effective, and has certain clinical value.

Occurrence of quinolones in cultured fish from Shandong Province, China and their health risk assessment.

The residue levels of 6 quinolones in 160 cultured fish samples from Shandong Province, China were investigated using UPLC-MS/MS. The detection rate was 43.1% and enrofloxacin had the highest detection rate as well as the highest residue concentration. The violation rates were 2.50% for the sum of enrofloxacin and ciprofloxacin and 1.25% for ofloxacin. Among the 9 fish species, quinolone contamination problems should receive more attention in Carp, Grass carp, Crucian and Catfish. The health risk assessment showed that when calculated by the maximum concentration, the estimated daily intakes (EDIs) of Carp, Grass carp and Crucian for the high consumption group accounted for more than 10% of the acceptable daily intakes (ADIs), indicating that a large intake of these fish species might pose a potential health risk and health risk monitoring of quinolones in cultured fish should be continually performed.

Modulating effects of RAMPs on signaling profiles of the glucagon receptor family.

Receptor activity-modulating proteins (RAMPs) are accessory molecules that form complexes with specific G protein-coupled receptors (GPCRs) and modulate their functions. It is established that RAMP interacts with the glucagon receptor family of GPCRs but the underlying mechanism is poorly understood. In this study, we used a bioluminescence resonance energy transfer (BRET) approach to comprehensively investigate such interactions. In conjunction with cAMP accumulation, Gα q activation and ß-arrestin1/2 recruitment assays, we not only verified the GPCR-RAMP pairs previously reported, but also identified new patterns of GPCR-RAMP interaction. While RAMP1 was able to modify the three signaling events elicited by both glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), and RAMP2 mainly affected ß-arrestin1/2 recruitment by GCGR, GLP-1R and glucagon-like peptide-2 receptor, RAMP3 showed a widespread negative impact on all the family members except for growth hormone-releasing hormone receptor covering the three pathways. Our results suggest that RAMP modulates both G protein dependent and independent signal transduction among the glucagon receptor family members in a receptor-specific manner. Mapping such interactions provides new insights into the role of RAMP in ligand recognition and receptor activation.

Current status of therapeutic drug monitoring for methotrexate, imatinib, paclitaxel in China.

BACKGROUND: Accurate TDMs of plasma methotrexate, imatinib and paclitaxel assist in the development of optimal therapeutic regimes. This study aims to investigate the current status of methotrexate, imatinib and paclitaxel measurements in China and explore the suitable EQA materials for those drugs. METHODS: 4 processed plasma samples including 2 levels of frozen pooled plasma samples and 2 levels of lyophilized pooled plasma samples were measured in different laboratories using different measurement systems. The inter-laboratory %CV and intra-measurement-system %CV of laboratories were calculated to assess the status of methotrexate, imatinib and paclitaxel measurements. The short-term stability and homogeneity of those processed samples were studied and compared. The relative differences (%) between the results of those two kinds of processed samples were also calculated to determine whether there were significant differences in their matrix effects for various measurement systems. RESULTS: The mean inter-laboratory %CVs ranged from 12.8% to 15.3%, 14.7% to 19.6% and 56.8% to 81.6% for methotrexate, imatinib and paclitaxel, respectively. The intra-measurement %CV of homogeneous commercial measurement systems was better than other measurement systems. The lyophilized samples were more stable than frozen samples and there were no obvious differences in their matrix effects for most measurement systems. CONCLUSIONS: The agreement among the results of methotrexate, imatinib, and especially paclitaxel from different laboratories was not satisfactory. Currently, the lyophilized samples were the more suitable EQA material for methotrexate, imatinib and paclitaxel than frozen samples.

High-Throughput Screening Campaign Identified a Potential Small Molecule RXFP3/4 Agonist.

Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.

Detection and phylogenetic analysis of a novel tick-borne virus in Haemaphysalis longicornis ticks and sheep from Shandong, China.

Dabieshan tick virus (DTV) was first identified in Haemaphysalis longicornis from Hubei Province, China in 2015. However, its pathogenic potential to animals and human remains to be further explored. In this study, a total of 170 engorged ticks and 22 sheep serum samples were collected from Taian and Yantai city, Shandong Province to investigate the presence of DTV. The results of qRT-PCR revealed the positive rate of 13.6% (3/22) in sheep serum and 8.2% (14/170) in attached ticks, respectively. Phylogenetic analysis demonstrated a close evolutionary relationship among those DTV isolates from animal and ticks, and DTV might be relatively conservative in evolution. These findings are the first to demonstrate molecular evidence of DTV in domestic animals. Nonetheless, whether or not causing disease in animals, DTV deserves further investigation.

Constitutive signal bias mediated by the human GHRHR splice variant 1.

Alternative splicing of G protein-coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone-releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to ß-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus ß-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward ß-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

Engineering a probiotic strain of Escherichia coli to induce the regression of colorectal cancer through production of 5-aminolevulinic acid.

Bacterial vectors can be engineered to generate microscopic living therapeutics to produce and deliver anticancer agents. Escherichia coli Nissle 1917 (Nissle 1917) is a promising candidate with probiotic properties. Here, we used Nissle 1917 to develop a metabolic strategy to produce 5-aminolevulinic acid (5-ALA) from glucose as 5-ALA plays an important role in the photodynamic therapy of cancers. The coexpression of hemAM and hemL using a low copy-number plasmid led to remarkable accumulation of 5-ALA. The downstream pathway of 5-ALA biosynthesis was inhibited by levulinic acid (LA). Small-scale cultures of engineered Nissle 1917 produced 300 mg l-1 of 5-ALA. Recombinant Nissle 1917 was applied to deliver 5-ALA to colorectal cancer cells, in which it induced the accumulation of antineoplastic protoporphyrin X (PpIX) and specific cytotoxicity towards colorectal cancer cells irradiated with a 630 nm laser. Moreover, this novel combination therapy proved effective in a mouse xenograft model and was not cytotoxic to normal tissues. These findings suggest that Nissle 1917 will serve as a potential carrier to effectively deliver 5-ALA for cancer therapy.

Occurrence of polycyclic aromatic hydrocarbons in fried and grilled fish from Shandong China and health risk assessment.

Diet is the main way for the human body to ingest polycyclic aromatic hydrocarbons (PAHs). In this study, the occurrence, dietary exposure, and health risks of 15 PAHs in 31 fried and grilled fish samples were investigated, which were collected from the Shandong Province of China. The results showed that benzo[a]pyrene (BaP) of 5 samples exceeded the European Union (EU) limit value. Naphthalene (NaP) and fluorene (Fle) were present in all samples, and the average concentration of ∑15PAHs was 91.1 µg/kg, with light PAHs dominated. The average contamination level of ∑15PAHs in fried and grilled fish was distributed differently, and there seemed to be more PAH contamination in the grilled samples. The results of the margin of exposure (MOE) suggested that PAH ingestion through fried and grilled fish did not imply significant toxicological concern for consumers in Shandong. The incremental lifetime cancer risk (ILCR) values for the consumption of fried and grilled fish were higher than 1 × 10-6, indicating a potential health risk in the adult population. The study provides baseline health information on PAH intake by residents due to dietary exposure to fried and grilled fish food products, suggesting that health risk monitoring of PAHs in such foods should be continually performed.