Objective:To investigate the effects of moxibustion,filiform needle acupuncture,and electroacupuncture on synovial hypoxia and expression of synovial fibrosis markers[transforming growth factor(TGF)-β and collagen alpha-1(Ⅰ)chain(COL1A1)]in knee osteoarthritis(KOA)rats.Methods:Fifty Sprague-Dawley rats were randomly divided into a normal group,a model group,a moxibustion group,an acupuncture group,and an electroacupuncture group,with 10 rats in each group.Except for the normal group,rats in the other four groups were injected with sodium iodoacetate into the right knee joint cavity to establish KOA models.The right Futu(ST32)and Zusanli(ST36)were selected in the moxibustion,acupuncture,and electroacupuncture groups to perform the mild moxibustion with moxa sticks,filiform needle acupuncture,or electroacupuncture,respectively.Each intervention lasted 15 min,once every other day,and continued for 4 weeks,with a total of 14 interventions.The Lequesne score was used to evaluate the degree of knee dysfunction before and after intervention,and the rat's right knee joint diameter was measured to evaluate the degree of knee swelling.Morphological changes of the right knee joint synovial tissue were observed using hematoxylin-eosin staining.Hypoxia probe immunofluorescence staining was used to observe the degree of synovial hypoxia in rats.Western blotting was used to detect the protein expression levels of hypoxia-inducible factor(HIF)-1α,TGF-β,and COL1A1 in the synovial tissue of the right knee joints.Results:Before intervention,the Lequesne score and the right knee joint diameter in the other four groups increased significantly compared to the normal group(P<0.01).After intervention,the Lequesne score and the right knee joint diameter increased significantly in the model group compared to the normal group(P<0.01)together with worsened cartilage deformation and osteophytes,inflammatory cell infiltration and fibrosis in synovial tissue,and hypoxia degree,and the expression levels of HIF-1α,TGF-β,and COL1A1 proteins in the knee joint synovial tissue of rats increased(P<0.01).The Lequesne score and the right knee joint diameter decreased significantly(P<0.01 or P<0.05),the cartilage morphology was normal without obvious osteophytes,and the hypoxia degree reduced in the three intervention groups compared to the model group.Among them,the moxibustion group had the most notable improvement in hypoxia,synovial tissue fibrosis,angiogenesis,and inflammatory cell infiltration.The expression levels of HIF-1α,TGF-β,and COL1A1 proteins decreased in the moxibustion and electroacupuncture groups(P<0.01),and the expression levels of HIF-1α and TGF-β proteins were decreased in the acupuncture group(P<0.01).Conclusion:Moxibustion,filiform needle acupuncture,and electroacupuncture improve knee joint function and reduce knee joint swelling in KOA rats.The mechanism may be to improve synovial fibrosis of the knee joint by regulating HIF-1α;moxibustion has the best effect on improving hypoxia among the three interventions,but the effect of filiform needle acupuncture on COL1A1 is not significant.

Objective To investigate the relationship between the threonine and tyrosine kinase(TTK)gene and eyelid basal cell carcinoma(BCC).Methods Bioinformatics methods were used to screen the core gene(namely,TTK)associated with the occurrence and development of BCC from the Gene Expression Omnibus(GEO)database.Surgically removed eyelid BCC tissue specimens(BCC cells were divided into BBC Grade Ⅰ,Ⅱ and Ⅲ groups by tumor grade)and be-nign tumor tissue specimens(Control group)were collected from Ziyang Central Hospital for subsequent experiments.Cel-lular immunofluorescence assay(CIA)was used to detect the expression of the TTK gene in benign and malignant eyelid tumor cells.After knocking down TTK in BCC cells through transfection with lentiviruses(the cells transfected with LV-TTK-shRNA were taken as the TTK-shRNA group,and those transfected with LV-BBC-shRNA were taken as the BBC nega-tive control group),CIA was used to detect the expression of key proteins Bcl-2 and Bax in the apoptotic signaling pathway of each group of cells.Results The bioinformatics analysis showed that the TTK gene was the core gene associated with the occurrence and development of eyelid BCC.CIA detection results revealed that the fluorescence signal intensities in the tumor cytoplasm of Control,BCC Grade Ⅰ,Ⅱ,and Ⅲ groups were 1.03±0.07,1.28±0.11,1.58±0.13 and 1.92±0.17,respectively.The fluorescence signal intensity gradually increased,and the difference in fluorescence signal intensity among the four groups was statistically significant(all P＜0.05).Compared with that in the Control group(1.02±0.05),the cell fluorescence intensity was increased in the BCC negative control group(1.74±0.12)and decreased in the TTK-shRNA group(1.31±0.09)(P＜0.05).The difference in cell fluorescence intensity was significant among the Control,BCC nega-tive control and TTK-shRNA groups(all P＜0.05).The fluorescence intensity of the anti-apoptotic protein Bcl-2 was 1.04±0.12 in the Control group,2.12±0.23 in the BCC negative control group,and 1.43±0.15 in the TTK-shRNA group.The fluorescence intensity of the pro-apoptotic protein Bax was 1.02±0.08 in the Control group,0.64±0.11 in the BCC negative control group,and 1.47±0.16 in the TTK-shRNA group.After TTK knockdown,the expression level of BcL-2 in BCC cells decreased,and that of Bax increased.The fluorescence intensities of BcL-2 and Bax were significantly different among the Control,BCC negative control and TTK-shRNA groups(all P＜0.05).Conclusion The TTK gene plays a role in the regulation of eyelid BCC cell proliferation,and this effect is closely related to the PI3K-AKT-Bcl-2/Bax signaling path-way.

Objective:To investigate the effects of moxibustion,filiform needle acupuncture,and electroacupuncture on synovial hypoxia and expression of synovial fibrosis markers[transforming growth factor(TGF)-β and collagen alpha-1(Ⅰ)chain(COL1A1)]in knee osteoarthritis(KOA)rats.Methods:Fifty Sprague-Dawley rats were randomly divided into a normal group,a model group,a moxibustion group,an acupuncture group,and an electroacupuncture group,with 10 rats in each group.Except for the normal group,rats in the other four groups were injected with sodium iodoacetate into the right knee joint cavity to establish KOA models.The right Futu(ST32)and Zusanli(ST36)were selected in the moxibustion,acupuncture,and electroacupuncture groups to perform the mild moxibustion with moxa sticks,filiform needle acupuncture,or electroacupuncture,respectively.Each intervention lasted 15 min,once every other day,and continued for 4 weeks,with a total of 14 interventions.The Lequesne score was used to evaluate the degree of knee dysfunction before and after intervention,and the rat's right knee joint diameter was measured to evaluate the degree of knee swelling.Morphological changes of the right knee joint synovial tissue were observed using hematoxylin-eosin staining.Hypoxia probe immunofluorescence staining was used to observe the degree of synovial hypoxia in rats.Western blotting was used to detect the protein expression levels of hypoxia-inducible factor(HIF)-1α,TGF-β,and COL1A1 in the synovial tissue of the right knee joints.Results:Before intervention,the Lequesne score and the right knee joint diameter in the other four groups increased significantly compared to the normal group(P<0.01).After intervention,the Lequesne score and the right knee joint diameter increased significantly in the model group compared to the normal group(P<0.01)together with worsened cartilage deformation and osteophytes,inflammatory cell infiltration and fibrosis in synovial tissue,and hypoxia degree,and the expression levels of HIF-1α,TGF-β,and COL1A1 proteins in the knee joint synovial tissue of rats increased(P<0.01).The Lequesne score and the right knee joint diameter decreased significantly(P<0.01 or P<0.05),the cartilage morphology was normal without obvious osteophytes,and the hypoxia degree reduced in the three intervention groups compared to the model group.Among them,the moxibustion group had the most notable improvement in hypoxia,synovial tissue fibrosis,angiogenesis,and inflammatory cell infiltration.The expression levels of HIF-1α,TGF-β,and COL1A1 proteins decreased in the moxibustion and electroacupuncture groups(P<0.01),and the expression levels of HIF-1α and TGF-β proteins were decreased in the acupuncture group(P<0.01).Conclusion:Moxibustion,filiform needle acupuncture,and electroacupuncture improve knee joint function and reduce knee joint swelling in KOA rats.The mechanism may be to improve synovial fibrosis of the knee joint by regulating HIF-1α;moxibustion has the best effect on improving hypoxia among the three interventions,but the effect of filiform needle acupuncture on COL1A1 is not significant.

Objective To investigate the relationship between the threonine and tyrosine kinase(TTK)gene and eyelid basal cell carcinoma(BCC).Methods Bioinformatics methods were used to screen the core gene(namely,TTK)associated with the occurrence and development of BCC from the Gene Expression Omnibus(GEO)database.Surgically removed eyelid BCC tissue specimens(BCC cells were divided into BBC Grade Ⅰ,Ⅱ and Ⅲ groups by tumor grade)and be-nign tumor tissue specimens(Control group)were collected from Ziyang Central Hospital for subsequent experiments.Cel-lular immunofluorescence assay(CIA)was used to detect the expression of the TTK gene in benign and malignant eyelid tumor cells.After knocking down TTK in BCC cells through transfection with lentiviruses(the cells transfected with LV-TTK-shRNA were taken as the TTK-shRNA group,and those transfected with LV-BBC-shRNA were taken as the BBC nega-tive control group),CIA was used to detect the expression of key proteins Bcl-2 and Bax in the apoptotic signaling pathway of each group of cells.Results The bioinformatics analysis showed that the TTK gene was the core gene associated with the occurrence and development of eyelid BCC.CIA detection results revealed that the fluorescence signal intensities in the tumor cytoplasm of Control,BCC Grade Ⅰ,Ⅱ,and Ⅲ groups were 1.03±0.07,1.28±0.11,1.58±0.13 and 1.92±0.17,respectively.The fluorescence signal intensity gradually increased,and the difference in fluorescence signal intensity among the four groups was statistically significant(all P＜0.05).Compared with that in the Control group(1.02±0.05),the cell fluorescence intensity was increased in the BCC negative control group(1.74±0.12)and decreased in the TTK-shRNA group(1.31±0.09)(P＜0.05).The difference in cell fluorescence intensity was significant among the Control,BCC nega-tive control and TTK-shRNA groups(all P＜0.05).The fluorescence intensity of the anti-apoptotic protein Bcl-2 was 1.04±0.12 in the Control group,2.12±0.23 in the BCC negative control group,and 1.43±0.15 in the TTK-shRNA group.The fluorescence intensity of the pro-apoptotic protein Bax was 1.02±0.08 in the Control group,0.64±0.11 in the BCC negative control group,and 1.47±0.16 in the TTK-shRNA group.After TTK knockdown,the expression level of BcL-2 in BCC cells decreased,and that of Bax increased.The fluorescence intensities of BcL-2 and Bax were significantly different among the Control,BCC negative control and TTK-shRNA groups(all P＜0.05).Conclusion The TTK gene plays a role in the regulation of eyelid BCC cell proliferation,and this effect is closely related to the PI3K-AKT-Bcl-2/Bax signaling path-way.

Objective:To explore potential predictors of refractory Mycoplasma pneumoniae pneumonia (RMPP) in early stage. Methods:The prospective multicenter study was conducted in Zhejiang, China from May 1 st, 2019 to January 31 st, 2020. A total of 1 428 patients with fever >48 hours to <120 hours were studied. Their clinical data and oral pharyngeal swab samples were collected; Mycoplasma pneumoniae DNA in pharyngeal swab specimens was detected. Patients with positive Mycoplasma pneumoniae DNA results underwent a series of tests, including chest X-ray, complete blood count, C-reactive protein, lactate dehydrogenase (LDH), and procalcitonin. According to the occurrence of RMPP, the patients were divided into two groups, RMPP group and general Mycoplasma pneumoniae pneumonia (GMPP) group. Measurement data between the 2 groups were compared using Mann-Whitney U test. Logistic regression analyses were used to examine the associations between clinical data and RMPP. Receiver operating characteristic (ROC) curves were used to analyse the power of the markers for predicting RMPP. Results:A total of 1 428 patients finished the study, with 801 boys and 627 girls, aged 4.3 (2.7, 6.3) years. Mycoplasma pneumoniae DNA was positive in 534 cases (37.4%), of whom 446 cases (83.5%) were diagnosed with Mycoplasma pneumoniae pneumonia, including 251 boys and 195 girls, aged 5.2 (3.3, 6.9) years. Macrolides-resistant variation was positive in 410 cases (91.9%). Fifty-five cases were with RMPP, 391 cases with GMPP. The peak body temperature before the first visit and LDH levels in RMPP patients were higher than that in GMPP patients (39.6 (39.1, 40.0) vs. 39.2 (38.9, 39.7) ℃, 333 (279, 392) vs. 311 (259, 359) U/L, both P<0.05). Logistic regression showed the prediction probability π=exp (-29.7+0.667×Peak body temperature (℃)+0.004×LDH (U/L))/(1+exp (-29.7+0.667×Peak body temperature (℃)+0.004 × LDH (U/L))), the cut-off value to predict RMPP was 0.12, with a consensus of probability forecast of 0.89, sensitivity of 0.89, and specificity of 0.67; and the area under ROC curve was 0.682 (95% CI 0.593-0.771, P<0.01). Conclusion:In MPP patients with fever over 48 to <120 hours, a prediction probability π of RMPP can be calculated based on the peak body temperature and LDH level before the first visit, which can facilitate early identification of RMPP.

Objective:To investigate the application value of ultrasound-guided multimodal examinations in the diagnosis of lymph node mycobacterial infection.Methods:The clinical data of 42 patients with suspected lymph node mycobacterial infection who were initially diagnosed at the Affiliated Hospital of Shaoxing University from January 2019 to December 2020 were retrospectively analyzed. All patients underwent an ultrasound-guided lymph node-negative pressure puncture. Acid-fast staining, bacterial culture, pathological examination or their combination were used to screen lymph nodes for mycobacterial infection. The results were compared with those of acid-fast staining and bacterial culture of sputum and bronchoalveolar lavage fluid smears.Results:The combined application of acid fast staining, bacterial culture, and pathological examination for the puncture fluid smear showed a positive rate of 71.4% (30/42), which was significantly higher than the positive rate [26.2% (11/42)] for acid fast staining of the puncture fluid smear, the positive rate [42.9% (18/42)] for bacterial culture of the puncture fluid, and the positive rate [50.0% (21/42)] of pathological examination ( χ2 = 17.20, 7.00, 4.04, P < 0.001, P < 0.01, P = 0.040). The positive rate for sputum smear and bacterial culture was 21.4% (9/33). The positive rate for acid fast staining and bacterial culture of the bronchoalveolar lavage fluid was 28.6% (12/30). The differences were statistically significant ( χ2 = 21.11, 15.43, both P < 0.001). Conclusion:Ultrasound-guided negative pressure aspiration and puncture biopsy of lymph nodes combined with acid fast staining, bacterial culture, and pathological examinations can markedly increase the detection rate and diagnostic rate of mycobacterial infection.

Objective:To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing (PGT) of an ATR-X syndrome pedigree with chromosomal microduplication.Methods:The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022. After chromosome copy number variation sequencing (CNV Seq) detection, it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region, which involved partial sequences of the ATRX gene. The third generation long read sequencing technology was used to detect the female genome sequence, determine the physical location of the insertion of the above repeat into the genome, clarify the pathogenicity of the repeat, and obtain a single nucleotide polymorphism (SNP) haplotype linked to the above micro repeat inheritance. After the couple's full informed consent, PGT was performed to assist pregnancy. One haploid embryo without pathogenic microduplication was selected for transfer. To verify the consistency with PGT test results, amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy, and the fetus was followed up after birth. Results:The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX: 76804463-76804464 (GRCh37/hg19), which is an intra tandem repeat of the ATRX gene, and it is predicted that it may cause damage to the normal function of the ATRX protein. After one cycle of PGT treatment, 27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection (ICSI). Through genetic testing, it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication. After thawing and transferring one of the blastocysts, the pregnancy was achieved, and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results. In November 2023, at 39 +5 weeks of pregnancy, a female live baby was delivered by natural delivery, and she is in good health. Conclusion:The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics. It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity, but also obtain SNP haplotypes that are linked to the target mutation, thus preparing for subsequent embryo detection.

Objective:To explore the clinical application value of third-generation sequencing technology in preimplantation genetic testing (PGT) of an ATR-X syndrome pedigree with chromosomal microduplication.Methods:The study selected a pedigree with a suspected ATR-X syndrome child at Assisted Reproductive Center of Jiangxi Maternal and Child Health Hospital in October 2022. After chromosome copy number variation sequencing (CNV Seq) detection, it was found that the female carried a 550 kb heterozygous microrepeat with unclear clinical significance in the Xq21.1 region, which involved partial sequences of the ATRX gene. The third generation long read sequencing technology was used to detect the female genome sequence, determine the physical location of the insertion of the above repeat into the genome, clarify the pathogenicity of the repeat, and obtain a single nucleotide polymorphism (SNP) haplotype linked to the above micro repeat inheritance. After the couple's full informed consent, PGT was performed to assist pregnancy. One haploid embryo without pathogenic microduplication was selected for transfer. To verify the consistency with PGT test results, amniocentesis prenatal diagnosis was performed in the second trimester after successful pregnancy, and the fetus was followed up after birth. Results:The results of the third generation long read sequencing and Sanger sequencing verification showed that the Xq21.1 microrepeat carried by the female was inserted into the genome chrX: 76804463-76804464 (GRCh37/hg19), which is an intra tandem repeat of the ATRX gene, and it is predicted that it may cause damage to the normal function of the ATRX protein. After one cycle of PGT treatment, 27 oocytes were obtained and 13 blastocysts were successfully developed after intracytoplasmic sperm injection (ICSI). Through genetic testing, it was found that two blastocysts were haploid embryos without carrying the aforementioned pathogenic microduplication. After thawing and transferring one of the blastocysts, the pregnancy was achieved, and the prenatal diagnosis results of amniocentesis in the second trimester were consistent with the PGT results. In November 2023, at 39 +5 weeks of pregnancy, a female live baby was delivered by natural delivery, and she is in good health. Conclusion:The third-generation sequencing technology has significant advantages in PGT detection of clinically ambiguous microreplicates with functional deficiency due to its long read length characteristics. It can not only determine the location of microreplicates inserted into the genome and determine their pathogenicity, but also obtain SNP haplotypes that are linked to the target mutation, thus preparing for subsequent embryo detection.

Objective:To study the prevalence and distribution of adult thyroid diseases in urban and rural areas of Hebei Province.Methods:A multi-stage stratified cluster sampling method was used to select Renqiu City and Licun Town, Luquan City of Hebei Province as the urban and rural survey sites, respectively. Questionnaire survey, physical examination and thyroid B ultrasound examination were conducted on local permanent residents (≥ 5 years of residencies) over 18 years old. The fasting venous blood sample was collected to determine the serum thyroid function indicaters.Results:A total of 2 650 adults were surveyed, including 1 393 urban residents and 1 257 rural residents (1 357 males and 1 293 females). A total of 435 patients with thyroid diseases were diagnosed, the detection rate was 16.42%. There were seven thyroid diseases, including subclinical hypothyroidism (60.92%, 265/435), Hashimoto's thyroiditis (34.02%, 148/435), hypothyroidism (4.83%, 21/435), simple goiter (3.22%, 14/435), hyperthyroidism (2.53%, 11/435), subclinical hyperthyroidism (2.53%, 11/435), and thyroid cancer (1.84%, 8/435). The detection rates of thyroid diseases in urban and rural areas were 21.18% (295/1 393) and 11.14% (140/1 257), respectively. The detection rates of thyroid diseases in males and females were 11.42% (155/1 357) and 21.66% (280/1 293), respectively. The detection rates of thyroid diseases in 18-< 30, 30-< 40, 40-< 50, 50-< 60 and ≥60 years old were 13.46% (91/676), 14.81% (81/547), 15.42% (89/577), 20.94% (85/406) and 20.05% (89/444), respectively. There were statistically significant differences between different areas, gender and age groups (χ 2 = 48.54, 50.53, 14.68, P < 0.05). Conclusions:The detection rate of subclinical hypothyroidism in adults in urban and rural areas of Hebei Province is relatively high, followed by Hashimoto's thyroiditis. Attention should be paid to the screening, evaluation, and intervention of thyroid function among urban female populations.