This article summarizesd Professor Li Zhigang's experience in treating limb dysfunction after acute disseminated encephalomyelitis(ADEM)by acupuncture.Prof.Li respected the classical theory of Chinese medicine,which is to seek the root cause of the disease,clarified the disease mechanism of"deficiency of Yangming leads to longitudinal tendons and unfavorable belt veins",and followed the principle of"treating flaccidity syndrome only by taking advantage of Yangming",and combined with the morbidity characteristics of ADEM that occurs in children,emphasized the important influence of regulating the root of innate and acquired in the treatment of the disease.The importance of regulating the essence of congenital and acquired constitution in the treatment of this disease was emphasized,taking into account the importance of regulating the mind and spirit.The main acupoints of Yangming Stomach Meridian,Large Intestine Meridian,Spleen Meridian,and Governor Vessel,together with the five main points of the lower limbs and the main points of water-inducing,all together play the role of tonifying the middle energizer,benefiting the essence and filling in the marrow,and strengthening the muscles and bones.

This article summarizesd Professor Li Zhigang's experience in treating limb dysfunction after acute disseminated encephalomyelitis(ADEM)by acupuncture.Prof.Li respected the classical theory of Chinese medicine,which is to seek the root cause of the disease,clarified the disease mechanism of"deficiency of Yangming leads to longitudinal tendons and unfavorable belt veins",and followed the principle of"treating flaccidity syndrome only by taking advantage of Yangming",and combined with the morbidity characteristics of ADEM that occurs in children,emphasized the important influence of regulating the root of innate and acquired in the treatment of the disease.The importance of regulating the essence of congenital and acquired constitution in the treatment of this disease was emphasized,taking into account the importance of regulating the mind and spirit.The main acupoints of Yangming Stomach Meridian,Large Intestine Meridian,Spleen Meridian,and Governor Vessel,together with the five main points of the lower limbs and the main points of water-inducing,all together play the role of tonifying the middle energizer,benefiting the essence and filling in the marrow,and strengthening the muscles and bones.

Objective:To preliminarily investigate the applicability of a poliovirus pseudovirus-based neutralization assay in evaluating the immunogenicity of recombinant poliovirus vaccines and their in vivo potency in rats. Methods:Serum samples from rats immunized with recombinant poliovirus vaccines were tested using both the pseudovirus neutralization assay and the live-virus neutralization assay with Sabin strain. The consistency and correlation of the two methods were analyzed using the Kappa test and Spearman′s rank correlation.Results:For the neutralizing antibodies against typeⅠ, Ⅱ, and Ⅲ polioviruses, the Kappa values for consistency analysis of the two methods were 0.914, 1.000, and 0.751, respectively ( P<0.001), and the correlation coefficients ( R values) were 0.833, 0.927, and 0.859, respectively ( P<0.001). Conclusions:The test results of the two methods are consistent and show a good correlation, indicating that the pseudovirus neutralization assay can be applied to evaluating the immunogenicity of poliovirus vaccines and also can be used in rat potency tests.

The theory of"upper spine"and"below the navel"comes from the"Inner Cannon of Yellow Emperor":"The treatment of diseases of the dumai is on the bone,or even below the navel".It is an important principle of acupuncture for treating diseases of the dumai,and spinal cord injury belongs to the category of dumai diseases,and the treatment of spinal cord injury based on this theory as the principle of matching points contains rich theoretical connotation and clinical significance.In this paper,we firstly discuss the concepts and body positions of"upper spine"and"below the navel",and then we start from the concepts of"upper spine"as the channel for the medulla,"upper spine"with"below the navel"to harmonise yin and yang,and"below the navel"contains the"life gate"and"qi".The three aspects of this theory are discussed to prove that the treatment of spinal cord injury by acupuncture has certain theoretical basis.At the same time,we analyse the application of this theory in ancient Chinese medical texts in order to provide useful reference for the treatment of spinal cord injury with acupuncture.

The theory of"upper spine"and"below the navel"comes from the"Inner Cannon of Yellow Emperor":"The treatment of diseases of the dumai is on the bone,or even below the navel".It is an important principle of acupuncture for treating diseases of the dumai,and spinal cord injury belongs to the category of dumai diseases,and the treatment of spinal cord injury based on this theory as the principle of matching points contains rich theoretical connotation and clinical significance.In this paper,we firstly discuss the concepts and body positions of"upper spine"and"below the navel",and then we start from the concepts of"upper spine"as the channel for the medulla,"upper spine"with"below the navel"to harmonise yin and yang,and"below the navel"contains the"life gate"and"qi".The three aspects of this theory are discussed to prove that the treatment of spinal cord injury by acupuncture has certain theoretical basis.At the same time,we analyse the application of this theory in ancient Chinese medical texts in order to provide useful reference for the treatment of spinal cord injury with acupuncture.

As a reversible and dynamic epigenetic marker, N⁶-adenylate methylation (m⁶A) modification is the most common mRNA modification in eukaryotes. This paper briefly described how m⁶A can influence RNA splicing, stability, and translation after transcription, and then participate in a variety of signaling pathways and biological and pathological processes, regulating cell proliferation, apoptosis, epithelial mesenchymal transformation (EMT) processes, and tumor invasion and metastasis. In addition, according to current studies, m⁶A methyltransferases (writers) are believed to promote EMT and tumor development, and readers and erasers both promote and inhibit EMT in different research objects. In this review, we summarized the mechanism of m⁶A modification and its role in cell transformation, and pointed out the direction of disease treatment.

BACKGROUND: Cancer stem-like cells (CSCs) are a small subset of cells in tumors that exhibit self-renewal and differentiation properties. CSCs play a vital role in tumor formation, progression, relapse, and therapeutic resistance. B7-H3, an immunoregulatory protein, has many protumor functions. However, little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer (GC) stemness. Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism. METHODS: GC stemness influenced by B7-H3 was detected both in vitro and in vivo . The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction, Western blotting, and flow cytometry. Sphere formation assay was used to detect the sphere-forming ability. The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments. The signaling pathway (Protein kinase B [Akt]/Nuclear factor erythroid 2-related factor 2 [Nrf2] pathway) of B7-H3 on the regulation of glutathione (GSH) metabolism was examined by Western blotting assay. Multi-color immunohistochemistry (mIHC) was used to detect the expression of B7-H3, cluster of differentiation 44 (CD44), and Nrf2 on human GC tissues. Student's t -test was used to compare the difference between two groups. Pearson correlation analysis was used to analyze the relationship between two molecules. The Kaplan-Meier method was used for survival analysis. RESULTS: B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo . Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells, which was further confirmed by the experimental results. Meanwhile, stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism. Furthermore, Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway, and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness. mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2. Importantly, GC patients with high expression of B7-H3, CD44, and Nrf2 had worse prognosis ( P = 0.02). CONCLUSIONS B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway. Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.
Humans ; Cell Line, Tumor ; Neoplasm Recurrence, Local ; NF-E2-Related Factor 2/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Stomach Neoplasms

Background Occupational and environmental particulate matter may cause fibrosis, accompanied by RNA m⁶A modification changes. Neodymium oxide (Nd₂O₃) can cause mouse lung fibrosis, which contains a large number of fibroblasts. Objective To investigate m⁶A modification of tumor necrosis factor receptor-associated protein 6/nuclear factor-κB (TRAF6/NF-κB) signaling pathway in fibrosis of human embryonic lung fibroblasts induced by Nd₂O₃, and identify the key m⁶A modification sites of TRAF6. Methods Designed concentrations of Nd₂O₃ (0, 1.563, 3.125, 6.25, 12.5, 25, 50, 100, and 200 mg∙L⁻¹) were infected with HELF cells for 24 and 48 h, and cell viability was detected to determine exposure time and dose. Measurements included indicators of fibrosis [hydroxyproline (HYP) and transforming growth factor-β1 (TGF-β1)], m⁶A methylation level, methyltransferases (METTL3 and METTL14), demethylases (FTO and ALKBH5), reading proteins (YTHDC2 and YTHDF2), fibrosis-associated genes (collagen-І, vimentin, and α-SMA), and proteins related to signaling pathway (TRAF6, NFKB1, P65, and P-P65). The enrichment of m⁶A in TRAF6 mRNA was measured by methylated RNA immunoprecipitation-quantitative real-time PCR (MeRIP-qPCR). Results The results of cell viability indicated that 6.25, 12.5, 25 mg∙L⁻¹ Nd₂O₃ and 48 h exposure time were used for subsequent experiments. After 48 h exposure, compared with the control group, the HYP level in the 25 mg∙L⁻¹ Nd₂O₃ group was increased, and the levels of TGF-β1 in the 6.25, 12.5, and 25 mg∙L⁻¹ Nd₂O₃ groups were increased (P<0.05); the overall m⁶A methylation levels of HELF cells in the 12.5 and 25 mg∙L⁻¹ Nd₂O₃ groups were increased (P<0.05). At mRNA level, compared with the control group, the mRNA expression levels of methyltransferases METTL3 and METTL14 (6.25, 12.5, and 25 mg∙L⁻¹ Nd₂O₃) were increased (P<0.05); the mRNA expression level of reading protein YTHDF2 (6.25, 12.5, and 25 mg∙L⁻¹ Nd₂O₃) was increased (P<0.05), while the mRNA expression level of YTHDC2 (25 mg∙L⁻¹ Nd₂O₃) was decreased (P<0.05); the mRNA expression levels of demethylases FTO (12.5 and 25 mg∙L⁻¹ Nd₂O₃) and ALKBH5 (25 mg∙L⁻¹ Nd₂O₃) were decreased (P<0.05); the mRNA expression levels of fibrosis-related genes vimentin, α-SMA, and collagen-Ⅰ (6.25, 12.5, and 25 mg∙L⁻¹ Nd₂O₃) were increased (P<0.05); the mRNA expression levels of pathway-related genes TRAF6 (25 mg∙L⁻¹ Nd₂O₃) and NFKB1 (12.5 and 25 mg∙L⁻¹ Nd₂O₃) were increased (P<0.05). At protein level, compared with the control group, the expression levels of methyltransferases METTL3 (25 mg∙L⁻¹ Nd₂O₃) and METTL14 (12.5 and 25 mg∙L⁻¹ Nd₂O₃) were increased (P<0.05); the expression level of reading protein YTHDF2 (12.5 and 25 mg∙L⁻¹ Nd₂O₃) was increased, while the expression level of YTHDC2 (25 mg∙L⁻¹ Nd₂O₃) was decreased (P<0.05); the expression level of demethylase FTO (25 mg∙L⁻¹ Nd₂O₃) was decreased (P<0.05); the expression level of fibrosis-associated protein vimentin was increased at 25 mg∙L⁻¹ Nd₂O₃, and the expression levels of α-SMA and collagen-Ⅰ were increased at 12.5 and 25 mg∙L⁻¹ Nd₂O₃ (P<0.05); the expression levels of TRAF6 and P-P65 were increased at 25 mg∙L⁻¹ Nd₂O₃ (P<0.05). The MeRIP-qPCR results showed that compared with the control group, the concentrations of m⁶A in all Nd₂O₃ groups were significantly increased (P<0.05). Conclusions Upon exposure of HELF cells to Nd₂O₃, the alterations in fibrosis-related indexes increase the expression of some m⁶A methylases and decrease the expression of demethylases, thereby increasing the m⁶A methylase level, and may promote the progression of fibrosis by activating the TRAF6/NF-κB signaling pathway.

Objective:To investigate the RHD genotypes of RhD-negative pregnant women and explore the optimum strategy for fetal RHD screening among this population in the region. Methods:This prospective study recruited 33 cases of RhD-negative singleton pregnancies at ≥12 weeks of gestation in Nanjing Drum Tower Hospital from March to November 2021. On the basis of RHD genotyping, quantitative real-time polymerase chain reaction (PCR) was used to amplify the exons 5 and 10 of RHD gene in the circulating cell-free DNA of RhD-negative pregnant women harboring whole RHD gene deletion and RHD-CE(2-9)- D. High-throughput sequencing was performed to detect chr1:25648453 locus from circulating cell-free DNA in plasma of RhD-negative pregnant women harboring RHD 1227A mutation to screen the fetal RhD blood group. Neonatal umbilical cord blood samples were collected for verifying fetal RHD genotyping. Descriptive statistical analysis was used. Results:Whole RHD gene deletion homozygous genotype ( n=20, 60.6%), RHD-CE(2-9) -D/whole RHD gene deletion heterozygous genotype ( n=5, 21.2%), RHD 1227A/whole RHD gene deletion heterozygous genotype ( n=7, 15.2%) and RHD 711delC/whole RHD gene deletion heterozygous genotype ( n=1) were identified in the 33 RhD-negative pregnant women. In the 25 cases with whole RHD gene deletion homozygous genotype or RHD-CE(2-9)- D/whole RHD gene deletion heterozygous genotype, 22 fetuses were RhD-positive and three were RhD-negative based on prenatal screening, which were confirmed by the neonatal serological test results after birth. In the seven cases carrying RHD 1227A/whole RHD gene deletion heterozygous genotype, all fetuses were RhD-positive, which were consistent with the results of serological detection after delivery. The case harboring RHD 711delC/whole RHD gene deletion heterozygous genotype did not receive fetal RHD screening. Conclusions:This study suggests that whole RHD gene deletion homozygous genotype is the most common allele in RhD-negative population in this area, followed by RHD 1227A/whole RHD gene deletion heterozygous genotype and RHD- CE(2-9)- D/whole RHD gene deletion heterozygous genotype. For women with whole RHD gene deletion homozygous genotype, RHD- CE(2-9)- D, or RHD 1227A mutation, fetal RHD screening with quantitative real-time PCR and high-throughput sequencing are important for the management of RhD-negative pregnant women.

Background Arsenic can be toxic to human by triggering oxidative stress, which is companied by epigenetic modifications. Objective To investigate the modification of N⁶-methyladenosine (m⁶A) in human embryonic lung fibroblasts (HELF) during oxidative stress induced by sodium arsenite (NaAsO₂). Methods HELF cells were treated by designed concentrations of NaAsO₂ (0, 2.5, 5, 10, and 20 μmol·L⁻¹) for 48 h. Cell viability was detected by 3-(4,5-dimethylthia zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) method; the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) as well as the content of malondialdehyde (MDA) were detected with corresponding kits; the level of m⁶A methylation in total RNA was detected by enzyme-linked immunosorbent assay; the mRNA expressions of m⁶A modified enzymes were detected by real-time fluorescence quantitative PCR, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms' tumor 1-associated protein (WTAP), fat mass and obesity-associated protein (FTO), alkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases 5 (ALKBH5), YTH domain containing protein 2 (YTHDC2), YTH domain family protein 2 (YTHDF2), and YTH domain family protein 3 (YTHDF3); the protein expressions of METTL3, FTO, YTHDC2, YTHDF3, and nuclear factor erythroid 2-related factor 2 (NRF2) were detected by Western blotting. The enrichment of m⁶A in NRF2 mRNA was detected by RNA methylated immunoprecipitation combined with real-time fluorescence quantitative PCR (MeRIP-qPCR). Results After the 0, 2.5, 5, 10, and 20 μmol·L⁻¹ NaAsO₂ treatment, the MTS results showed that compared with the control group, the cell viability of the 20 μmol·L⁻¹ group decreased to 84% (P<0.05). The colorimetry results showed that compared with the control group, the activities of T-SOD in the 10 and 20 μmol·L⁻¹ groups decreased (P<0.05); the activities of GSH-Px in the 2.5 and 10 μmol·L⁻¹ groups decreased (P<0.05); the contents of MDA in the 10 and 20 μmol·L⁻¹ groups increased. The results of enzyme-linked immunosorbent assay showed that the overall m⁶A methylation levels in the 0, 2.5, 5, 10, and 20 μmol·L⁻¹ groups were (0.193 ± 0.023)%, (0.247 ± 0.021)%, (0.253 ± 0.006)%, (0.233 ± 0.006)%, and (0.262 ± 0.010)%, respectively, and compared with the control group, the m⁶A methylation levels in all the NaAsO₂ treated groups increased (P<0.05). The real-time fluorescence quantitative PCR results showed that compared with the control group, the mRNA relative expression level of METTL3 decreased in the 2.5, 10, and 20 μmol·L⁻¹ groups (P<0.05); the mRNA relative expression level of FTO decreased in the 20 μmol·L⁻¹ group; the mRNA relative expression level of YTHDC2 increased in the 10 and 20 μmol·L⁻¹ groups (P<0.05); the mRNA relative expression level of YTHDF3 increased in the 2.5, 10, and 20 μmol·L⁻¹ groups (P<0.05). The Western blotting results showed that compared with the control group, the relative protein expression of METTL3 decreased in the 10 and 20 μmol·L⁻¹ groups; the relative protein expression of FTO decreased in the 5 and 20 μmol·L⁻¹ groups; the relative protein expression of YTHDC2 decreased in the 20 μmol·L⁻¹ group (P<0.05); the relative nuclear protein expression of NRF2 decreased in the 10 and 20 μmol·L⁻¹ groups (P<0.05). The MeRIP-qPCR results showed that m⁶A enrichment was significantly increased in the 20 μmol·L⁻¹ NaAsO₂ exposure group compared with the control group (P<0.05). After over-expression of FTO, the mRNA and protein relative expression levels of FTO and the relative expression level of nuclear protein of NRF2 in the FTO group were higher than those in the control group (P<0.05); the mRNA and protein relative expression levels of FTO in the NaAsO₂ + FTO group and the nuclear protein expression level of NRF2 were higher than those in the NaAsO₂ group (P<0.05). Conclusion In the process of oxidative stress induced by NaAsO₂, m⁶A methylation level, m⁶A modified enzymes, m⁶A modification of NRF2 mRNA, and NRF2 expression could change in HELF cells.