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).

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.

Ultrasensitive detection of the H5N1 nucleic acid fragment by ICP-MS using DNA dendrimer-carried silver nanoparticle labeling.

The importance of avian influenza virus (AIV) detection in clinical diagnosis and prognosis has been deeply recognized. In this study, the ultrasensitive detection of AIV subtype H5N1 was achieved by ICP-MS combined with DNA dendrimer-carried silver nanoparticle (AgNP) labeling. First, a magnetic control system was constructed by anchoring double-strand DNAs (dsDNAs) which contained a complementary sequence of H5N1 and two locked triggers on the surface of magnetic beads (MBs). When H5N1 was present, the two triggers were released and initiated dendrimer hybridization chain reactions which led to the generation of DNA dendrimer-carried AgNPs on the surface of the MBs. Finally, the AgNPs were collected via magnetic separation, digested by nitric acid, and tested using ICP-MS. The signal intensities of 107Ag were positively correlated with the concentrations of H5N1. Notably, the DNA dendrimer assembly contributed to significant signal amplification and good sensitivity with the limit of detection as low as 2.0 × 10-11 mol L-1. Moreover, the method displayed favorable selectivity against mismatched H5N1 and good recoveries in human serum samples. It is a promising analytical tool for the H5N1 virus and other subtypes of AIV, and has potential value in clinical diagnosis and prognosis of infectious diseases.

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.

Comparative Proteomics and Metabonomics Analysis of Different Diapause Stages Revealed a New Regulation Mechanism of Diapause in Loxostege sticticalis (Lepidoptera: Pyralidae).

Histone acetylation is an important epigenetic mechanism that has been shown to play a role in diapause regulation. To explore the physiological and molecular mechanisms of histone deacetylase in the diapause process, LC-MS/MS analysis was used to perform TMT proteomic and metabolomic analysis on non-diapause (ND), pre-diapause (PreD), diapause (D), cold treatment (CT), and post-diapause (RD) stages of the meadow moth. A total of 5367 proteins were identified by proteomics, including 1179 differentially expressed proteins. We found 975 (602 up-regulated and 373 down-regulated), 997 (608 up-regulated and 389 down-regulated), 1119 (726 up-regulated and 393 down-regulated), 1179 (630 up-regulated and 549 down-regulated), 94 (51 up-regulated and 43 down-regulated), 111 (63 up-regulated and 48 down-regulated), 533 (243 up-regulated and 290 down-regulated), 58 (31 up-regulated and 27 down-regulated), and 516 (228 up-regulated and 288 down-regulated) proteins in ND and PreD, ND and D, ND and CT, ND and RD, PreD and D, PreD and CT, PreD and RD, D and CT, D and RD, and CT and RD stages, respectively. A total of 1255 differentially expressed metabolites were annotated by metabolomics. Through KEGG analysis and time series analysis of differentially expressed metabolites, we found that phospholipids were annotated in significantly different modules, demonstrating their important role in the diapause process of the meadow moth. Using phospholipids as an indicator for weighted gene co-expression network analysis, we analyzed the most relevant differentially expressed proteins in the module and found that ribosomal 40s and 60s subunits were the most relevant proteins for diapause. Because there have been studies that have shown that histone deacetylase is associated with the diapause of meadow moths, we believe that histone deacetylase regulates the 40s and 60s subunits of ribosomes, which in turn affects the diapause of meadow moths. This finding expands our understanding of the regulation of meadow moth diapause and provides new insights into its control mechanism.

[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.

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.

Cryo-electron microscopy structure of the glucagon receptor with a dual-agonist peptide.

Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-Gs complex and compared this structure to our recently published structure of the GCGR-Gs complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.

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.

Less polar ginsenosides have better protective effects on mice infected by Listeria monocytogenes.

Listeria monocytogenes widely exists in the natural environment and does great harm, which can cause worldwide public safety problem. Infection with L. monocytogenes can cause rapid death of Kupffer cell (KCs) in liver tissue and liver damage. American ginseng saponins is a natural compound in plants, which has great potential in inhibiting L. monocytogenes infection. Therefore, American ginseng stem-leaf saponins (AGS) and American ginseng heat-transformed saponins (HTS) were used as raw materials to study their bacteriostatic experiments in vivo and in vitro. In this experiment, female Kunming mice were randomly divided into five groups: control group, negative group, AGS group, HTS group (10 mg/kg/day in an equal volume via gastric administration) and penicillin group, each group containing six mice. Profiles AGS and HTS components were evaluated by high-performance liquid chromatography (HPLC) analysis. The bacteriostatic effect of AGS and HTS on L. monocytogenes was evaluated by inhibition zone test, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The bacteriostatic effect of AGS and HTS pretreatment on mice infected with L. monocytogenes were studies by animal experimental. The results showed that the content of polar saponins in AGS was 0.81 ± 0.003 mg/mg, less polar saponins was 0.08 ± 0.02 mg/mg, the content of polar saponins in HTS was 0.10 ± 0.01 mg/mg, less polar saponins was 0.76 ± 0.02 mg/mg. The in vitro bacteriostatic diameter of HTS (16.6 ± 0.8 mm) is large than that of AGS (10.2 ± 1.2 mm). AGS and HTS pretreatment could reduce the colony numbers in the livers of mice infected with Listeria monocytogenes. The levels of alanine aminotransferase (ALT), IL-1ß, IL-6, TNF-α and IFN-γ in the livers of mice in the pretreatment group were significantly lower than those in the negative group. There were obvious leukoplakia, calcification and other liver damage on the liver surface in the negative control group, and obvious inflammatory cell infiltration in HE sections. AGS and HTS pretreatment can reduce liver injury caused by L. monocytogenes and protect the liver. Compared with AGS, HTS has higher content of less polar saponins and better bacteriostatic effect in vitro. The count of bacterial in liver tissue of HTS group was significantly lower, the survival rate was significantly higher than that of AGS group. Less polar saponins had better bacteriostatic effect. Collectively, less polar saponins pretreatment has a protective effect on mice infected with L. monocytogenes, to which alleviated liver damage, improved anti-inflammatory ability and immunity of the body, protected liver may contribute.

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.

Publisher Correction: Integrating yeast chemical genomics and mammalian cell pathway analysis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Effect of a beta-cypermethrin and emamectin benzoate pesticide mixture on reproductive toxicity in male mice in a greenhouse environment.

With the widespread use of pesticides, the resistance to pesticides of pests has gradually increased, caused mixed pesticides to become even more widely used for practical applications. To investigate the effects of mixed pesticides on reproductive health in an occupational greenhouse environment, the greenhouse environment and the characteristics of the actual application were constructed, and then the male mice were comprehensively exposed to a mixture of the beta-cypermethrin and emamectin benzoate environmental. Additionally, the effect of the beta-cypermethrin and emamectin benzoate mixture on the reproductive health of male mice was known. The results showed that with the prolongation of exposure duration, the activities of Glutathione Peroxidase (GSH-Px), Total Superoxide Dismutase (T-SOD), Lactate dehydrogenase (LDH) and Acid phosphatase (ACP) in the testes of mice gradually decreased and the activity of Malondialdehyde (MDA) gradually increased. It was also found that the apoptosis rate of murine testicular cells increased and that DNA damage occurred with prolonged exposure duration. Therefore, it can be inferred that exposure to a mixture of the pesticides beta-cypermethrin and emamectin benzoate in the greenhouse environment may have adverse effects on the reproductive health of male mice.

Improving long-term stability of retinol in dried blood spots and quantification of its levels via a novel LC-MS/MS method.

Vitamin A deficiency (VAD) is a major micronutrient deficiency in children. Although plasma and serum retinol levels are proposed as the key indicators of VAD, collecting and transporting plasma and serum are difficult and inconvenient in field studies. Dried blood spot (DBS) retinol has been used as an alternative to plasma retinol in several epidemiological and clinical studies. A limitation of methods that use DBS retinol is the instability and apparent loss of retinol in DBSs. Therefore, an accurate, reliable method for stabilizing retinol in DBSs and quantifying and comparing DBS retinol concentrations with equivalent plasma retinol levels is required. In this study, antioxidants on paper combined with vacuum treatment were found to greatly increase the stability of DBS retinol during 120 min of air drying and 30 days of room-temperature storage. A surrogate matrix of whole blood prepared using a mixture of human erythrocytes and 2% BSA in PBS was firstly used in DBS retinol determination based on the fact that retinol is excluded from erythrocytes. The method was linear in the concentration range of 0.04-300 µg/mL. Both the between-run (n = 5) and within-run (n = 6) precision (relative standard deviations, RSD%) were below 8.42%. The spiked recoveries at 3 concentrations ranged from 86.48 to 98.13%. The internal standard (IS)-normalized matrix factor (MF) was 99.72% with a RSD% of 10.50% (n = 3). The accuracy was calibrated using two National Institute of Standards and Technology (NIST) serum-generated calibrants at concentrations of 0.1962 and 0.3948 g/mL, and relative errors (RE% values) of 0.07% and 4.95% were found, respectively. A simple calibration model was first developed to convert DBS retinol concentration to the equivalent plasma retinol concentration, thereby enabling comparisons with clinical reference ranges and with studies using serum or plasma samples. Graphical abstract.

Integrating yeast chemical genomics and mammalian cell pathway analysis.

Chemical genomics has been applied extensively to evaluate small molecules that modulate biological processes in Saccharomyces cerevisiae. Here, we use yeast as a surrogate system for studying compounds that are active against metazoan targets. Large-scale chemical-genetic profiling of thousands of synthetic and natural compounds from the Chinese National Compound Library identified those with high-confidence bioprocess target predictions. To discover compounds that have the potential to function like therapeutic agents with known targets, we also analyzed a reference library of approved drugs. Previously uncharacterized compounds with chemical-genetic profiles resembling existing drugs that modulate autophagy and Wnt/ß-catenin signal transduction were further examined in mammalian cells, and new modulators with specific modes of action were validated. This analysis exploits yeast as a general platform for predicting compound bioactivity in mammalian cells.

[Comparison and analysis of 5 metal elements in serum between laboratories].

OBJECTIVE: To evaluate the ability for the detection of 5 metal elements in serum in the laboratories of disease prevention and control system. METHODS: The samples for calcium, magnesium, iron, copper and zinc detection were distributed to 48 laboratories of disease prevention and control system. Inductively coupled plasma mass spectrometry( ICP-MS) analysis or self-selected determination method were allowed to use during detection for each laboratory. The results were analyzed by robust statistical analysis and Z value was used to evaluate the detection ability. RESULTS: Of the laboratories involved in the study, 40 reported results of metal elements detection. Among them, 29 laboratories had satisfactory results, and 11 laboratories had unsatisfactory or suspicious results. The laboratory pass rate of this inter-laboratory comparison was60. 4%. CONCLUSION: The detection level of calcium, magnesium, iron, copper and zinc in serum in disease prevention and control system is generally satisfactory, but the detection ability of some laboratories needs to be further improved.

Transcriptome Analysis of Transiently Reversible Cell Vacuolization Caused by Excessive Serum Concentration in Scophthalmus maximus.

As an important research tool, cell lines play a vital role in life science research, medical research, and drug development. During the culture of the Scophthalmus maximus head kidney (TK) cell line, we found a phenomenon of cell vacuolization caused by excessive serum concentration. Moreover, the vacuolization of the cells gradually disappeared after passage by trypsin digestion. In clarifying the formation mechanism of this reversible cellular vacuolation, transcriptomics was utilized to explore the mechanism of cell vacuolization caused by excessive serum concentration. Transcriptome analysis indicated that excessive serum concentration could cause the up-regulated expression of PORCN and other genes to promote cell proliferation. Compared with cells whose vacuolization disappeared after trypsin digestion and passage, the expression of mitosis-related genes (BUB1, ttk, Mad2, Cdc20, CDK1, CCNB1), nuclear stability-related genes LMNB1 and tissue stress and repair-related genes HMMR in vacuolated cells caused by excessive serum concentration was significantly up-regulated. There is a regulatory system related to adaptation and stress repair in the cells, which can maintain cell stability to a certain extent. This study provides a theoretical basis for the stable culture of fish cell lines and the solution to the problem of cell vacuolation.

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.

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.

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.