Free and bioavailable 25-hydroxyvitamin D thresholds for bone metabolism and their associations with metabolic syndrome in Chinese women of childbearing age.

Objective: The free hormone hypothesis suggests that free and bioavailable 25-hydroxyvitamin D [25(OH)D] may better reflect vitamin D bioactivity. This study aimed to determine the free and bioavailable 25(OH)D characteristics, estimate their thresholds based on parathyroid hormone (PTH) and bone turnover markers (BTMs), assess their associations with the risk of metabolic syndrome (MetS), and evaluate their potential advantages. Methods: A cross-sectional study was conducted using a nationally representative database (n = 1,505, female, 18-45 years). Serum total 25(OH)D, vitamin D-binding protein, albumin, PTH, and BTMs [osteocalcin, ß-CrossLaps of type 1 collagen containing cross-linked C-telopeptide (ß-CTX), and procollagen type 1 N-terminal propeptide (P1NP)] were measured. Free 25(OH)D and bioavailable 25(OH)D were calculated. The threshold associations of 25(OH)D with PTH and BTMs were analyzed. The relationship between 25(OH)D and MetS risk was examined. An intervention study was then performed in 39 women (18-47 years) to assess the associations of increasing 25(OH)D with PTH and BTMs after vitamin D supplementation. Results: In the cross-sectional study, the three forms of 25(OH)D were found to have similar distribution characteristics. Free and bioavailable 25(OH)D correlated well with total 25(OH)D. Significant total 25(OH)D cutoffs were observed for PTH (14.19 ng/mL and 18.03 ng/mL), osteocalcin (15.14 ng/mL), ß-CTX (14.79 ng/mL), and P1NP (15.08 ng/mL). Free and bioavailable 25(OH)D cutoffs were only found for P1NP (3.47 pg/mL and 1.66 ng/mL, respectively). A total 25(OH)D of <15.14 ng/mL was marginally associated with a higher risk of reduced high-density lipoprotein cholesterol (HDL-C) [odd ratios (OR) = 1.371 (0.991-1.899)]. The ORs of higher versus lower free and bioavailable 25(OH)D levels for reduced HDL-C were 0.770 (0.621-0.956) and 0.772 (0.622-0.958), respectively. The results of the intervention study indicated that PTH and BTMs responded more sensitively to total 25(OH)D than to free or bioavailable 25(OH)D. Conclusion: Free and bioavailable 25(OH)D only had a threshold effect on P1NP. The active 25(OH)D thresholds could be used for risk assessment of reduced HDL-C. However, no superiority of free or bioavailable 25(OH)D was found based on the response of PTH and BTMs to changes in 25(OH)D in Chinese women of childbearing age following vitamin D supplementation. Clinical trial registration: http://www.chictr.org.cn, ChiCTR2200058290.

[Association of vitamin D gene polymorphisms and serum 25-hydroxyvitamin D in Chinese women of childbearing age].

OBJECTIVE: To analyze the relationship between vitamin D(VitD)-related single nucleotide polymorphism(SNP) and 25-hydroxyvitamin D(25(OH) D) levels and VitD nutritional status. METHODS: A total of 1507 women of childbearing age aged 18-45 were selected from the sample bank of "2015 Chinese adult chronic disease and nutrition monitoring". Basic information(including region, season, age, height, weight, etc. ) of the subjects was collected. The SNPs related to VitD metabolism were screened, and the improved multiple ligase detection reaction was used for SNP testing. Liquid chromatography tandem mass spectrometry was used to determine the serum 25(OH)D concentration. The effects of genotypes on 25(OH)D level and VitD deficiency were analyzed by generalized linear model and binary logistic regression model, respectively. RESULTS: After adjusting for latitude, region, region type, season and age, CYP2R1 rs12794714, GC rs2282679, GC rs7041 and VDR rs2228570 were associated with serum 25(OH)D levels in women of childbearing age. The risk of VitD deficiency in individuals carrying GG genotype at rs2282679 was significantly higher than that in individuals carrying TT genotype(OR=2.466, 95%CI 1.690-3.598, P<0.001), and the risk of VitD deficiency in individuals carrying A allele at rs2228570 was lower than that in individuals carrying G allele(OR_(AA)=0.625, 95%CI 0.446-0.876, P_(AA)=0.006;OR_(GA)=0.661, 95%CI 0.502-0.869, P_(GA)=0.003). CONCLUSION: The genotype distribution of CYP2R1 rs12794714, GC rs2282679, GC rs7041 and VDR rs2228570 may be related to serum 25(OH)D level or VitD nutritional status of Chinese women of childbearing age.

Association of Serum 25(OH)D with Metabolic Syndrome in Chinese Women of Childbearing Age.

Objective: To analyze the associations between serum 25(OH)D levels and the risk of metabolic syndrome (MetS) and its components, and the related genetic and non-genetic factors in non-diabetic women of childbearing age in China. Methods: Subjects were randomly selected from the 2015 Chinese Adult Chronic Disease and Nutrition Surveillance. The data of sociodemographic characteristics and lifestyle factors were obtained through questionnaire survey. Anthropometry was measured by trained interviewers, and fasting blood was collected to test 25-hydroxyvitamin D [25(OH)D], total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting blood glucose (FBG), and other related parameters. Generalized linear mode and multivariate logistic analysis were performed to analyze the associations between serum 25(OH)D and MetS and its components, adjusting for the possible confounders. Results: Body mass index (BMI), serum alanine aminotransferase (ALT), hypersensitive C-reactive protein (hs-CRP), 25(OH)D, phosphorus (P), and parathyroid hormone (PTH) levels were associated with the number of MetS's components. G allele carriers of GC rs2282679 had higher diastolic blood pressure (DBP) and FBG levels compared with the TT genotypes, while higher genetic risk score (GRS) seemed to be associated with reduced HDL-C level. The odds ratio (OR) for MetS in lowest group of 25(OH)D was 1.533 (0.980−2.399) after adjusting for season, district, area type, latitude, age, BMI, PTH, P, ALT, CRE, interleukin-6 (IL-6), and hs-CRP, compared with the median group, but the association was not significant. An insufficient 25(OH)D concentration (<14.22 ng/mL) was significantly related to the risk of elevated waist circumference (WC) (OR = 1.612 (1.014−2.561)) and TG (OR = 2.210 (1.318−3.706)), and reduced HDL-C (OR = 1.639 (1.206−2.229)) after adjusting for the confounders among these women. Moreover, these relationships were not affected by vitamin D metabolism-related gene polymorphisms. Conclusion: After comprehensively considering various influencing factors, significant associations between insufficient serum 25(OH)D and MetS's components, including elevated WC, TG, and reduced HDL-C, were observed. However, MetS, hypertension, and hyperglycemia were not found independently associated with 25(OH)D levels.

[Relationship between rs7041 polymorphism of GC gene and serum vitamin D status in Chinese women of childbearing age].

OBJECTIVE: To investigate the association of rs7041 polymorphism of GC gene that encodes the vitamin D-binding protein with serum vitamin D status in Chinese women of childbearing age. METHODS: A total of 1812 plasma samples of women childbearing aged 18-44 years old were selected by stratified random sampling technology from the established biological samples bank of Chinese Chronic Diseases and Nutrition Survey(CCDNS, 2015-2018). The serum 25(OH)D status was detected by enzyme-linked immunosorbent assay. The genotypes of rs7041 in the GC gene were analyzed by improved multiple ligase detection reaction method. RESULTS: A total of 1812 childbearing women aged 18-49 years were included in this study. The frequency of rs7041 genotypes in the study were distributed according to the Hardy-Weinberg equilibrium, indicating sufficient representativeness of our sample. The median serum 25(OH)D status was 16. 69(12. 04, 21. 69)ng/mL. The higher 25(OH)D levels was detected in the overall sample, southern women or women with normal vitamin D status with the CC genotype than the AA genotype(P<0. 05). Before and after correction, the risk of vitamin D insufficiency in the women carrying the CC genotype was decreased significantly compared with the women carrying the AA genotype(OR=0. 571, 95%CI 0. 373-0. 873). And the CC genotype of rs7041 was associated with a significant decrease in risk of 25(OH)D deficiency(in the subgroup of southern childbearing women, OR=0. 284, 95%CI 0. 144-0. 560 and in the subgroup of northern childbearing women, OR=0. 109, 95%CI 0. 015-0. 798). CONCLUSION: The GC rs7041 with A/C polymorphism are significantly correlated with 25(OH)D status in Chinese childbearing women, mutant CC genotype is a protective factor for vitamin D non-normal status risks.

A pH-responsive colorimetric detection of human telomerase RNA based on a three-dimensional DNA amplifier.

Human telomerase RNA (hTR), one of the essential components of telomerase, serves as a reverse template to add repeated segments of (TTAGGG)n to the 3' end of telomere DNA for maintaining the length of telomere DNA, endowing cells indefinite proliferation capability. Expression level of hTR displays a close relationship with tumor grade. Inspired by the mechanism of urease hydrolyzing urea to release ammonia and elevate the pH value of the sample solution, we developed a facile and novel pH-responsive colorimetric strategy for hTR detection by incorporating catalyzed hairpin assembly (CHA) onto the magnetic beads (MBs). The CHA process was initiated by target hTR and recycled via toehold binding and branch migration, thereby abundant urease being anchored on the surface of MBs. After separated by an external magnetic field, the assembled urease catalyzed the hydrolysis of urea to release a large amount of ammonia, which gave rise to a remarkable pH signal. Thus, quantification of hTR was achieved by measuring the solution pH via a hand-held pH meter or visualizing the solution color with the assistance of the pH indicator phenol red. The proposed sensing platform exhibits excellent performance toward hTR with a detection limit as low as 41 pM and a remarkable sequence selectivity, being able to differentiate a single mismatch in the target DNA. The pH-responsive colorimetric sensing platform contributes to introducing pH-related portable strategies into the detections of numerous universal biomarkers such as nucleic acids and proteins.

Sensitive multicolor visual detection of telomerase activity based on catalytic hairpin assembly and etching of Au nanorods.

Detection of telomerase activity is crucial for the telomerase-related early diagnosis of cancer and drug screening. Herein, a multicolor visual telomerase detection method with high sensitivity was developed based on the etching of hexadecyl trimethyl ammonium bromide-stablized Au nanorods (Au NRs) by the oxidation state 3,3',5,5'-tetramethylbenzidine sulfate (TMB2+). In order to meet the demand of bare-eye inspection, an enzyme-free signal amplification strategy of catalytic hairpin assembly (CHA) was incorporated. After the introduction of telomerase, telomerase extension products specifically triggered cyclic CHA and led to the successive formation of G-quadruplex/hemin DNAzyme, which catalyzed the H2O2-mediated oxidation of TMB. The Au NRs were gradually etched as the concentration of catalysate TMB2+ increased, resulting in the decrease of aspect ratio of the Au NRs. Correspondingly, the longitudinal localized surface plasmon resonance peak of Au NRs was blue-shifted, with the concomitant generation of a series of color transition. Under optimal conditions, a highly sensitive detection toward telomerase was realized down to 15 HeLa cells. Compared to previous colorimetric method for telomerase determination, multiple colors corresponding to telomerase activity was the most attractive virtue of our approach. More strikingly, telomerase-induced cyclic CHA significantly enhanced the accumulation of G-quadruplex/hemin DNAzyme, thus the signal amplification was effectively realized. Our approach exhibited excellent sensitivity and convenient signal readout, which is expected to provide great potential application in cancer diagnosis and therapy.

A signal-on split aptasensor for highly sensitive and specific detection of tumor cells based on FRET.

We present here a signal-on fluorescence biosensor for highly sensitive and specific detection of tumor cells with a split aptamer based on fluorescence resonance energy transfer (FRET). This sensor holds considerable potential for simple, rapid, sensitive and specific tumor cell detection in early clinical diagnosis.

Single-walled carbon nanotubes (SWCNTs)-assisted cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) for improving screening efficiency.

Separating the specific from the nonspecific bound single-strand DNA (ssDNA) is the most important step to improve the efficiency of selection procedure. However, most cell-SELEX protocols (where SELEX = systematic evolution of ligands by exponential enrichment) use simply washing only, which leads to incomplete separation. It is well-established that ssDNAs can be adsorbed on single-walled carbon nanotubes (SWCNTs). Based on this, herein, we developed a modified cell-SELEX approach termed "SWCNTs-assisted cell-SELEX". In our approach, SWCNTs are applied in the separation step, during which the unbound or the nonspecific ssDNAs are adsorbed onto SWCNTs, while the bound ssDNAs still remain on the cell surface, because of the stronger interaction between ssDNA and target. The cells can then be centrifuged to enrich the specifically binding aptamers. As a proof of concept, two nasopharyngeal carcinoma (NPC) cell lines-CNE2 cell and HONE cell-were used as the target cell and the negative cell, respectively. The result show that it takes only 6 cycles to enrich the aptamer pool through the SWCNTs-assisted cell-SELEX, which is much shorter than 15 cycles in the conventional cell-SELEX, thus improving the screening efficiency. Moreover, the achieved aptamers show high specificity and affinity with CNE2 cells, which are highly attractive for clinical diagnosis and biomedicine applications.

Proximity-dependent protein detection based on enzyme-assisted fluorescence signal amplification.

In this paper, we develop a sensitive fluorescence method for protein detection based on proximity extension and enzyme-assisted signal amplification. In this novel method, pairs of proximity probes are designed, and the recognition elements are integrated into the proximity probes. Then proteins are detected by transforming aptamer or antibody-protein binding signals into DNA detection based on proximity effect. In addition, nick sites are introduced into the proximity probes to amplify the detectable signal. As proof of concept, detection of human α-thrombin and human IgG are demonstrated in this study. The aptamers and antibodies are coupled in the proximity probes as recognition elements for human α-thrombin and human IgG respectively. In the presence of target protein, aptamer or antibody-protein binding signals are transformed into detectable signals by the proximity effect, and can be further amplified by enzyme-assisted strand displacement. The above mentioned strategies consequently bring the limit of detection (LOD) to as low as 1 pM for human α-thrombin and 6 pM for human IgG. Furthermore, this method might be extended to sensitive detection of other proteins by changing recognition elements of proximity probes.