Objective: To reveal the molecular biological mechanism of a rare Dc-variant individual using PacBio third-generation sequencing technology. Methods: ABO and Rh blood type identification, DAT, unexpected antibody screening and D antigen enhancement test were conducted by serological testing. The absorption-elution test was used to detect the e antigen. RHCE gene typing was performed by PCR-SSP, and the 1-10 exons of RHCE were sequenced by Sanger sequencing. The full-length sequences of RHCE, RHD and RHAG were detected by PacBio third-generation sequencing technology. Results: Serological findings: Blood type O, Dc-phenotype, DAT negative, unexpected antibody screening negative; enhanced D antigen expression; no detection of e antigen in the absorption-elution test. PCR-SSP genotyping indicated the presence of only the RHCE c allele. Sanger sequencing results: Exons 5-9 of RHCE were deleted, exon 1 had a heterozygous mutation at c. 48G/C, and exon 2 had five heterozygous mutations at c. 150C/T, c. 178C/A, c. 201A/G, c. 203A/G and c. 307C/T. Third-generation sequencing results: RHCE genotype was RHCE 02N. 08/RHCE-D(5-9)-CE; RHD genotype was RHD 01/RHD 01; RHAG genotype was RHAG 01/RHAG 01 (c. 808G>A and c. 861G>A). Conclusion: This Dc-individual carries the allele RHCE 02N. 08 and the novel allele RHCE-D(5-9)-CE. The findings of this study provide data support and a theoretical basis for elucidating the molecular mechanisms underlying RhCE deficiency phenotypes.

Randomized controlled trial(RCT) is considered to represent the gold standard for evaluating the efficacy of interventions and has been widely used to evaluate the clinical efficacy of traditional Chinese medicine(TCM). However, there are unique challenges in implementing RCT in TCM. Patients seeking TCM treatment often have preferences for TCM due to the unsatisfactory therapeutic effect of western medicine, their personal intolerance, and their rejection of certain drugs, medical devices, or surgery. Patients are generally reluctant to be randomly assigned to a group, making it challenging to enroll patients in the control group of western medicine during the implementation of RCT in TCM. This has become a prominent problem restricting the implementation of RCT in TCM and needs to be solved urgently. Therefore, this paper introduced commonly used research designs used in solving the problem of enrolling patients in control group during the implementation of RCT in TCM, including Zelen design, partially randomized patient preference trial(PRPP), single-arm objective performance criteria(OPC), cohort studies, single-arm clinical trials using real world data(RWD) alone as the external control group, and the design method based on RWD-augmented control group samples in RCT. The paper outlined the definitions and principles of these methods, evaluated their advantages, disadvantages, and applicable scenarios, and explored their applications in the TCM field, so as to offer insights for solving the difficulty in enrolling patients in the control group during the implementation of RCT in TCM.
Humans ; Medicine, Chinese Traditional/methods* ; Randomized Controlled Trials as Topic/methods* ; Research Design ; Patient Selection ; Drugs, Chinese Herbal/therapeutic use* ; Control Groups

Neuropathic pain, a debilitating condition caused by dysfunction of the somatosensory nervous system, remains difficult to treat due to limited understanding of its molecular mechanisms. Bioinformatics analysis identified cerebellin 2 (CBLN2) as highly enriched in human and murine proprioceptive and nociceptive neurons. We found that CBLN2 expression is persistently upregulated in dorsal root ganglia (DRG) following spinal nerve ligation (SNL) in mice. In addition, transcription factor SOX11 binds to 12 cis-regulatory elements within the Cbln2 promoter to enhance its transcription. SNL also induced SOX11 upregulation, with SOX11 and CBLN2 co-localized in nociceptive neurons. The siRNA-mediated knockdown of Sox11 or Cbln2 attenuated SNL-induced mechanical allodynia and thermal hyperalgesia. High-throughput sequencing of DRG following intrathecal injection of CBLN2 revealed widespread gene expression changes, including upregulation of numerous NF-κB downstream targets. Consistently, CBLN2 activated NF-κB signaling, and inhibition with pyrrolidine dithiocarbamate reduced CBLN2-induced pain hypersensitivity, proinflammatory cytokines and chemokines production, and neuronal hyperexcitability. Together, these findings identified the SOX11/CBLN2/NF-κB axis as a critical mediator of neuropathic pain and a promising target for therapeutic intervention.
Animals ; Neuralgia/metabolism* ; Ganglia, Spinal/metabolism* ; Up-Regulation ; Mice ; NF-kappa B/metabolism* ; SOXC Transcription Factors/genetics* ; Male ; Neuroinflammatory Diseases/metabolism* ; Mice, Inbred C57BL ; Nerve Tissue Proteins/genetics* ; Hyperalgesia/metabolism* ; Signal Transduction ; Spinal Nerves

Objective To optimize the screening methods for antiallergic activity of two Chinese medicines in vitro and compare the antiallergic activity of five medicine pairs in vitro.Methods The degranulation assay of RBL-2H3 cells was optimized,and the activity of the five drugs on inhibiting mast cell degranulation was compared by toluidine blue staining and the release of β-HEX and histamine(HIS).The hyaluronidase inhibition test was optimized to compare the hyaluronidase inhibition effect of five Chinese medicine pairs.Results In the experiment of degranulation of RBL-2H3 cells,5 medicine pairs could inhibit β-HEX release from model cells to different degrees(P<0.05),including Schizonepetae Herba-Saposhnicovia divaricata,Ephedrae Herba-Asarum,Saposhnicovia divaricata-Radix Angelicae dahuricae,Rhizoma Chuanxiong-Asarum.The β-HEX release rate in the supernatant of degranulated model cells was significantly increased(P<0.05)after coculture with Flos Magnoliae-Fructus Xanthii.All the five medicine pairs could reduce HIS release in the supernatant of degranulated model cells.In the hyaluronase inhibition rate test,the hyaluronase inhibition rate of each medicine pair was Rhizoma Chuanxion-Asarum>Saposhnicovia divaricate-Radix Angelicae dahuricae>Schizonepetae Herba-Saposhnicovia divaricate>Ephedrae Herba-Asarum,among which Rhizoma Chuanxiong-Asarum had the strongest anti-allergic activity.Conclusion The trend of antiallergic activity of different drugs obtained by the two methods is consistent in vitro,indicating that the two methods can be used for screening antiallergic activity in vitro.

Talents are the main force for the development of traditional Chinese medicine（TCM）， and the construction of TCM talents and the reformation of talent evaluation system are essential to promote the inheritance and innovation of TCM. At present， we are still exploring and developing in the fields of the formulation， implementation and evaluation indicators of TCM talent evaluation system. However， there are shortcomings and difficulties. For instance， insufficient stratification in the evaluation， excessive emphasis on the quantity of achievements， neglecting the quality of the achievements and the actual contribution， imperfect assessment indicators， and the weak characteristics of TCM. Therefore， national ministries and commissions have jointly issued a document requesting to break the four only and set a new standard， in order to promote the construction of a scientific and technological talent evaluation system oriented by innovation value， ability and contribution. For the evaluation of TCM clinical talents， China Association for Science and Technology commissioned China Association of Chinese Medicine to build the China Clinical Cases Library of TCM（CCCL-TCM）， which aims at collecting the most authoritative and representative TCM clinical cases and exploring the advantages of applying clinical cases as masterpiece of achievement in TCM clinical talents evaluation. CCCL-TCM can promote the construction of a talent evaluation system that is more in line with the development characteristics of TCM industry， and to carry out relevant pilot in TCM colleges and institutions across the country in order to promote the reformation of TCM talent evaluation system.

Objective To investigate the mechanism of morin-induced autophagy in non-small cell lung cancer A549 cells based on mTOR/STAT3 signaling axis.Methods A549 cells were divided into blank group and 30,60,90,120 and 150 μg·mL-1 of morin groups.After 24,48 and 72 hours of culture,the cell proliferation activity was detected by CCK-8 method,and the cell inhibition rate was calculated.A549 cells were divided into blank group and 30,90,150 μg·mL-1 morin groups.After 14 days of culture,the cell proliferation was detected by colony formation assay.After 24 hours of culture,the cell proliferation ability was detected by BeyoClickTM EdU-488.Apoptosis was detected by flow cytometry;acridine orange staining was used to detect cell autophagy;the formation of autophagosomes was observed by transmission electron microscopy.Western Blot was used to detect the expression levels of apoptosis,autophagy and mTOR/STAT3 signaling axis-related proteins in cells.A549 cells were divided into blank group,blank group + chloroquine(10 μg·mL-1)group,morin(30,150 μg·mL-1)group,morin(30,150 μg·mL-1)+ chloroquine(10 μg·mL-1)group.After 48 hours of intervention,the cell activity was detected by CCK-8 method,and the cell survival rate was calculated.Results Compared with the blank group,the inhibition rate of A549 cells in 60,90,120,150 μ g·mL-1 of morin group was significantly increased after 24 hours of intervention(P＜0.05,P＜0.001).The inhibition rates of A549 cells in 30,60,90,120 and 150 μg·mL-1 of morin groups were significantly increased after 48 and 72 hours of intervention(P＜0.001).The number of A549 cell colonies and the number of green fluorescent proliferation positive cells in the 30,90,150 μg·mL-1 of morin groups were significantly decreased(P＜0.01,P＜0.001),the apoptosis rate was significantly increased(P＜0.01,P＜0.001),and the protein expression level of cleaved-PARP was significantly increased(P＜0.001).The protein expression levels of p-P38/P38 MAPK in A549 cells of 90 and 150 μg·mL-1 of morin groups were significantly increased(P＜0.01,P＜0.001).Different degrees of orange fluorescence appeared in A549 cells of 30,90 and 150 μg·mL-1 of morin groups,and the orange fluorescence of 90 and 150 μg·mL-1 of morin groups was significant.Autophagosomes and autolysosomes appeared in the cytoplasm of A549 cells in 150 μg·mL-1 of morin group,respectively.The protein expression of LC3-Ⅱ in A549 cells of 150 μg·mL-1 of morin group was significantly up-regulated(P＜0.05).The protein expression of Atg16L1-Ⅱ in A549 cells of 90,150 μg·mL-1 of morin group was significantly up-regulated(P＜0.001),and the protein expressions of p-mTOR/mTOR and p-STAT3/STAT3 were significantly down-regulated(P＜0.001).Compared with the morin(150 μg·mL-1)group,the survival rate of A549 cells in the morin(150 μg·mL-1)+chloroquine(10 μg·mL-1)group was significantly increased(P＜0.05).Conclusion Morin can promote the apoptosis of A549 cells and induce autophagy in A549 cells,and the mechanism may be related to mTOR/STAT3 axis.

Objective:To investigate the onset time distribution and influential factors in patients with acute myocardial infarction (ACI) with different body mass index (BMI) levels.Methods:A cross-sectional study was conducted to collect clinical data from 1 000 patients with AMI who received treatment at Heilongjiang Provincial Hospital from January 2016 to November 2022. The patients were divided into groups based on different BMI levels: < 18.5 kg/m 2 group ( n = 49), 18.5-24 kg/m 2 group ( n = 369), > 24-28 kg/m 2 group ( n = 338), and > 28 kg/m 2 group ( n = 244). The incidence of AMI was analyzed among patients with different BMI levels as per diurnal variation, seasonality, and weekday. Results:A total of 1 000 patients were included in this study, including 648 men and 352 women. The mean age of these patients was 65 years (range 56-74 years). The median body mass was 70 kg (range 60-76 kg), the median height was 1.69 m (range 1.60-1.72 m), and the median BMI was 24.49 kg/m 2 (range 22.22-26.79 kg/m 2). The onset time of AMI differed significantly among patients with different BMI levels in terms of the period from 0:00 to 5:59, winter, and Wednesday ( P = 0.047, 0.029, 0.005). Among all samples, the number of patients with a BMI of 18.5-24 kg/m 2 was the highest, reaching 369 cases. Conclusion:The incidence of AMI in patients with different BMI levels exhibits a regular distribution as per diurnal variation, seasonality, and weekday.

Objective To explore the effect of circular RNA(circRNA)transcription factor 25(TCF25)-targeting microRNA-128b(miR-128b)on the proliferation and apoptosis of MCF-7 human breast cancer cells.Methods MCF-7 cells were cultured until the loga-rithmic growth stage.Cells were randomly divided into the blank(untransfected),si-NC(transfected si-NC),si-circRNA TCF25(transfected si-circRNA TCF25),pcDNA-circRNA TCF25(transfected pcDNA-circRNA TCF25),miR-NC(transfected miR-NC),miR-128b mimic(transfected miR-128b mimic),miR-128b inhibitor(transfected miR-128b inhibitor),and pcDNA-circRNA TCF25+miR-128b mimic(transfected with pcDNA-circRNA TCF25 and miR-128b mimic)groups.Each group included six multiple pores.Forty-eight hours after transfection,the expression of circRNA TCF25 and miR-128b in each group was determined using real-time reverse transcription-quanti-tative polymerase chain reaction(RT-qPCR).An RNase R enzyme digestion assay was used to identify circular RNA.Subcellular locali-zation of circRNA TCF25 was determined through cytoplasmic-nuclear separation.Cell proliferative activity was measured using the 2,5-diphenyl-2H-tetrazolium bromide(MTT)assay.Apoptosis was detected using flow cytometry.RT-qPCR was performed to determine the mRNA expression levels of phosphatase and tensin homolog(PTEN),proliferating nuclear antigen 67(Ki-67),andcaspase-3.Western blotting was performed to measure PTEN,Ki-67,caspase-3,and cleaved caspase-3 protein expression.The dual-luciferase reporter(DLR)assay was performed to analyze the relationship between circRNA TCF25 and miR-128b.Results Compared to the control group,the relative expression of circRNA TCF25 did not exhibit significant changes after RNase R enzyme treatment(P＞0.05),whereas that of linear TCF25 decreased after RNase R enzyme treatment(P＜0.05).The relative expression of circRNA TCF25 in the cytoplasm was higher than that in the nucleus(P＜0.05).Compared with the blank and si-NC groups,the cell proliferation activity of the si-circRNA TCF25 group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3mRNA and protein expression increased.In addition,cell proliferation activity increased and apoptosis rate decreased in the pcDNA-circRNA TCF25 group.Ki-67 mRNA and protein expression were increased,and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression decreased,with statistical significance(all P＜0.05).Compared with the blank and miR-NC groups,the cell proliferation activity of the miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleaved caspase-3 mRNA and protein expression were increased,whereas the cell proliferation activity increased and apoptosis rate decreased in the miR-128b inhibitor group.Ki-67mRNA and protein expression were increased,and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression decreased,with statistical significance(all P＜0.05).Compared with the pcDNA circRNA TCF25 group,the cell proliferation activity of the pcDNA circRNA TCF25+miR-128b mimic group decreased;the apoptosis rate increased;Ki-67mRNA and protein expression decreased;and PTEN,caspase-3,and cleavedcaspase-3mRNA and protein expression increased,with statistical significance(all P＜0.05).The DLR assay results confirmed that circRNA TCF25 targets miR-128b.Conclusion CircRNAs may play a key role in promoting the proliferation of MCF-7 human breast cancer cells and inhibiting their apoptosis by targeting miR-128b expression;promoting Ki-67 expression;and inhibiting PTEN,caspase-3,and cleaved caspase-3 expression.

Primary hypertrophic osteoarthropathy(PHO)is a rare disease also known as pachydermo-periostosis.We reported a painless case whose diagnosis was confirmed by genetic test.A 24-year-old male presented a series of symptoms that first began at 14.He suffered from progressive clubbed-fingers accompa-nied by swelling of the wrist and ankle joints.Facial skin concentric thickening and alar nose broadening ap-peared simultaneously and increased progressively.He was also prone to acne and hyperhidrosis.X-rays showed thickening of the metacarpal and phalangeal bones,as well as symmetrical periosteal ossification of both the tibia and fibula.Clinical diagnosis of PHO is difficult because of the variable features.With acromeg-aly excluded,the diagnosis was confirmed by a genetic test.Whole exome sequencing revealed a heterozygous SLCO2A1 c.611C＞T(p.Ser204Lue)and SLCO2A1 c.1602C＞A(p.Asn534Lys)mutation from each par-ent.It suggests that primary hypertrophic osteoarthropathy should be considered for young limb hypertrophic patients especially when periosteal thickening signs were showed in X-ray.A confirmatory diagnosis can be made through the genetic test.

Aim To investigate the effect of Xuefu Zhuyu decoction on transforming growth factor-β1(TGF-β1 ) -induced endothelial-to-mesenchymal transition (EndMT) of pulmonary microvascular endothelial cells ( PMVEC), and further analyze the mechanism related to the TGF-β1/Smad signaling pathway. Method To construct an EndMT cell model, PMVEC was treated with TGF-β1 (5 μg · L