Objective: This study employed next-generation sequencing (NGS) for large-scale genotyping of 42 blood group systems in blood donors to evaluate its applicability in multi-system blood group identification and rare blood type repository construction, while exploring the complementary value of third-generation sequencing (TGS) in haplotype phasing and variant capture in regions with insufficient sequencing depth for ABO ambiguous samples. Methods: A total of 562 regular blood donors (median donation frequency 11 times) from a blood center were enrolled. NGS was used for genotyping of 42 blood group systems, with a focused analysis on 12 systems essential for rare blood type repository construction: MNS, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Colton, Gerbich, Ok, H, and I. TGS was employed to resolve haplotype phasing (i. e., determining whether different mutation sites are located on the same chromosome) in 12 ABO samples with discrepancies between forward and reverse typing. Serological and genotyping results were compared for MNS, Duffy, Kidd, Lewis, and ABO systems. Results: NGS-based genotyping revealed that Fy (a-b+) in the Duffy system accounted for 0.87% (4/458), and Di (a+b+) in the Diego system accounted for 10.62% (31/292). The Lutheran, Kell, Yt, Colton, H, Ok, I, and Gerbich systems exhibited near-uniform phenotype distributions. Among the 12 ABO ambiguous samples, NGS yielded multiple possible genotype combinations in 5 cases due to inability to phase alleles, whereas TGS uniquely resolved the genotypes through long-read sequencing. In one case, NGS detected only 2 mutation sites due to insufficient sequencing depth, while TGS identified all 11 sites and assigned the A1 phenotype. Concordance rates between serology and genotyping were: Duffy 96.55% (140/145), ABO 96.43% (513/532), Kidd 94.17% (97/103), MNS 85.07% (57/67), and Lewis 68.18% (75/110). In the MNS system, 7 of 8 samples serologically typed as M+N+ but genotyped as M-N+ carried GYPB variants. Lewis discrepancies predominantly featured genotype Le (a-b+) with serological phenotypes of Le (a+b-), Le (a+b+), or Le (a-b-). The NGS platform completed sequencing of 192 samples within 2 weeks. Conclusion: The tiered genotyping strategy combining NGS, TGS, and serology enables multi-system blood group identification in large-scale blood donor populations. TGS provides complementary value in phasing ambiguous ABO samples and detecting variants in regions with insufficient sequencing depth. This study provides a technical framework and baseline frequency data for the expansion of a local rare blood type repository. However, standardization and cost-effectiveness of this strategy require further validation with expanded sample sizes, and serological confirmation should be retained for secretion status-related systems such as Lewis.

Objective:To identify the development trajectories of self-neglect behavior in older adults and explore the associated influencing factors.Methods:A fixed cohort was constructed based on the data from three surveys of Chinese longitudinal healthy longevity survey (CLHLS) from 2011 to 2018. A total of eight variables from 4 dimensions including living environment, lifestyle, social interaction, and health care were selected to evaluate self-neglect. Group-based trajectory model was used to identify the development trajectory of self-neglect behavior in the older adults, and polynomial Logistic regression model was used to explore its influencing factors by Stata 16.1.Results:Finally, 2 754 older adults aged 60 and above were included.The development trajectory of self-neglect behavior in older adults, based on the group-based trajectory model, can be classified into stable-low group ( n=268, 9.7%), descending-moderate group ( n=2 224, 80.8%), and decreasing-high group ( n=262, 9.5%). Polynomial Logistic regression showed that, compared with stable-low group, living in rural areas ( B=1.116, OR=3.053, 95% CI= 2.278-4.091) and higher activities of daily living scores( B=0.137, OR=1.147, 95% CI=1.046-1.258) were the risk factors of descending-moderate group. Education levels with 1-6 years( B=-0.398, OR=0.672, 95% CI=0.469-0.963), >6 years( B=-1.072, OR=0.342, 95% CI=0.229-0.513), being married( B=-0.476, OR=0.621, 95% CI=0.444-0.870), self-reported good health( B=-0.808, OR=0.446, 95% CI= 0.213-0.932), improved health status( B=-0.704, OR=0.495, 95% CI=0.320-0.766), self-reported average economic status( B=-1.065, OR=0.345, 95% CI=0.148-0.802), self-reported good economic status( B=-1.634, OR=0.195, 95% CI=0.082-0.467), and a higher cognition score( B=-0.142, OR=0.867, 95% CI=0.798-0.942) served as protective factors of descending-moderate group. In addition to the above factors, being in the age group of 75-89 years( B=0.481, OR=1.617, 95% CI=1.057-2.473) was a risk factor for decreasing-high group compared to stable-low group. Conclusions:Three types of self-neglect behavior trajectories among older adults were identified in this study, suggesting that physical health and economy are the influencing factors of the development trajectory of self-neglect of the elderly.

This study aimed to elucidate the effect of tryptophan (Trp) on gut hormone secretion as well as the roles of the calcium-sensing receptor (CaSR) and its downstream signaling pathway in gut hormone secretion by assessing swine duodenal perfusion in vitro. Swine duodenum was perfused with Krebs-Henseleit buffer as a basal solution. Various concentrations (0, 10, and 20 mM) of Trp were applied to investigate its effect on gut hormone secretion. A CaSR antagonist was used to detect the involvement of CaSR and its signal molecules. The 20 mM Trp concentration promoted the secretion of cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP), elevated the mRNA level of CaSR, and upregulated the protein levels of CaSR, protein kinase C (PKC), and inositol trisphosphate receptor (IP3R). However, NPS 2143, an inhibitor of CaSR, attenuated the CCK and GIP release, reduced the mRNA level of CaSR, and decreased the protein levels of CaSR, PKC, and IP3R with 20 mM Trp perfusion. The results indicate that CCK and GIP secretion can be induced by Trp in swine duodenum in vitro, and the effect is mediated by CaSR and its downstream signal molecules PKC and IP3R.
Cholecystokinin ; Duodenum ; Gastric Inhibitory Polypeptide ; In Vitro Techniques ; Inositol ; Perfusion ; Protein Kinase C ; Receptors, Calcium-Sensing ; RNA, Messenger ; Swine ; Tryptophan

Objective To investigate the apoptosis induction effects and the possible mechanism of YM155 on triple negative breast cancer MDA-MB-231cells.Methods MDA-MB-231 cells were treated with dif-ferent concentrations of YM 155, and the survival rate of the cells was determined by CCK-8 assay and the half maximal inhibitory concentration ( IC50 ) value of YM155 was calculated .The apoptosis rate was examined by An-nexin V-FITC/PI double staining.mRNA expression of survivin and bcl-2 in MDA-MB-231cells was detected by RT-PCR.The protein expression of survivin , bcl-2, caspase-3, and PARP were detected by Western blot .Re-sults YM155 significantly inhibited the growth of MDA-MB-231 cells in a dose-and-time-dependenct way .IC50 was(1.749 ±0.265) ng/ml and(0.823 ±0.125) ng/ml respectively at 24 and 48 hours.The apoptosis rate of cells treated with 0.5 ng/ml, 1.0 ng/ml, and 1.5 ng/ml YM155 was (10.93 ±0.94)%,(31.10 ±1.51)%, and(46.83 ±2.92)%respectively, which had significant difference compared to that of the control group (6.4 ± 1.2)%(P<0.01).YM155 could significantly decrease mRNA and protein expression of surviving , besides, it reduced bcl-2 expression and increased caspase-3 and PARP protein expression .Conclusions YM155 can ef-fectively induce the apoptosis of MDA-MB-231 cells by downregulating survivin and activating caspase pathway . Bcl-2 might play a role in the apoptosis .

OBJECTIVETo investigate the clinical value of multiplex nested reverse transcription PCR (RT-nPCR) in screening acute myeloid leukemia(AML)fusion genes.

METHODSA novel multiplex RT-nPCR assay was developed to detect 16 AML-related fusion genes (AML1-EVI1, AML1-ETO, AML1-MDS1, AML1-MTG16, MLL-AF9, MLL-AF6, MLL-AF10, MLL-ENL, MLL-MLL, PML-RARα, PLZFRARα, NPM1-RARα, CBFB-MYH11, DEK-CAN, SET-CAN and TLS-ERG) according to 2008 WHO classification of AML. The chromosome reciprocal translocations of 356 AML cases were detected by multiplex RT-nPCR and karyotyping. The positive samples were further confirmed by split- out PCR and FISH.

RESULTSThe fusion genes were detected in 172 patients with the positive detection rate of 48.31%(172/356), which was higher than that of karyotyping (31.46%) (χ²=70.314, P<0.01). Multiplex RT-nPCR is superior to karyotyping and FISH in identifying the rare, cryptic chromosome translocation (χ²=96.074, P<0.01).

CONCLUSIONThe multiplex RT-nPCR used in this study can quickly, effectively and accurately screen the fusion genes in AML patients, which can provide important evidence for assessing diagnosis and treatment, and also provide necessary information for minimal residual disease (MRD) and prognosis.

Adolescent ; Adult ; Aged ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Multiplex Polymerase Chain Reaction ; methods ; Oncogene Proteins, Fusion ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Young Adult