ObjectiveTo make clear exercise combined with Shenghui Tang interferes in acetylcholine receptor （M1AChR） to improve mitochondrial autophagy and enhance cognition of Alzheimer's disease （AD） model rats through the adenylate activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway. MethodsForty-eight male SD rats of SPF grade were randomly divided into a blank group， a model group， a Shenghui Tang group （9.3 g·kg^-1）， an exercise group， an exercise + Shenghui Tang group （9.3 g·kg^-1）， and a rapamycin group （1.5 mg·kg^-1）. Except for the blank group， the rat model of AD was constructed by injecting amyloid beta （Aβ_1-42） into hippocampus stereotaxically. The exercise group received treadmill exercise for 4 weeks， while the Shenghui Tang group received intragastric administration for 4 weeks， and the exercise + Shenghui Tang group received treadmill exercise and intragastric administration of Shenghui Tang for 4 weeks simultaneously. After the intervention， the Morris water maze test was used to detect the learning and memory ability. Spontaneous behavior was observed in the open field test. The pathological structure of hippocampal neurons was observed by NISSl staining. The expression level of M1AChR in hippocampus was detected by immunohistochemistry （IHC）. The autophagy ultrastructure of hippocampal neurons was observed by transmission electron microscopy. The apoptosis rate was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL）. The expression of Beclin1 and microtubule-associated protein light chain 3β （LC3β） was detected by immunofluorescence （IF）. The protein expression of M1AChR， AMPK， p-AMPK， mTOR， Beclin1， LC3β， and chelate 1 （SQSTM1/p62） in hippocampus was detected by Western blot. ResultsCompared with the blank group， the model group exhibited significantly increased platform escape latency on the fifth day （P<0.01） and significantly decreased activity distance in the target quadrant and times of crossing the platform （P<0.01）. The total movement distance in the open field， the time of movement in the central area， and the average speed obviously decreased （P<0.05）. The arrangement of nerve cells in hippocampus CA1 region was dispersed， and the numbers of Nissl bodies and M1AChR positive cells significantly decreased （P<0.01）. The expression of TUNEL positive cells was significantly increased （P<0.01）. The typical autophagic lysosomal structure decreased. The protein expression of M1AChR， p-AMPK/AMPK， p-mTOR/mTOR， Beclin1， LC3Ⅱ/Ⅰ in hippocampus was significantly decreased （P<0.01）， and the protein expression of p62 was significantly increased （P<0.01）. Compared with the model group， the exercise + Shenghui Tang group exhibited obviously improved space exploration and positioning navigation ability （P<0.05， P<0.01）. The total movement distance in the open field， the time of movement in the central area， and the average speed of movement significantly increased （P<0.01）. The number of Nissl bodies significantly increased （P<0.01）. The number of M1AChR positive cells in hippocampus was significantly increased （P<0.01）. The expression of TUNEL positive cells was significantly decreased （P<0.01）. The protein expression of M1AChR， p-AMPK/AMPK， p-mTOR/mTOR， Beclin1， LC3Ⅱ/Ⅰ in hippocampus was significantly increased （P<0.01）， while the protein expression of p62 was significantly decreased （P<0.01）. Compared with the exercise + Shenghui Tang group， the Shenghui Tang group and the exercise group showed significantly increased platform escape latency on the fifth day （P<0.01） and obviously decreased activity distance in the target quadrant and times of crossing the platform （P<0.05， P<0.01）. The total movement distance in the open field， the time of movement in the central area， and the average speed of movement significantly decreased （P<0.01）. The number of Nissl bodies and the number of M1AChR positive cells significantly decreased （P<0.01）. The expression of TUNEL positive cells was obviously increased （P<0.05）. Ultrastructure of the hippocampal region showed decreased autophagy level. The protein expression of M1AChR， p-AMPK/AMPK， p-mTOR/mTOR， LC3Ⅱ/Ⅰ in the hippocampus was obviously decreased in the Shenghui Tang group （P<0.05， P<0.01）， while the protein expression of p62 was significantly increased （P<0.01）. In the exercise group， the protein expression of M1AChR， p-AMPK/AMPK， Beclin1， and LC3Ⅱ/Ⅰ was obviously decreased （P<0.05， P<0.01）， while the protein expression of p-mTOR/mTOR and p62 was significantly increased （P<0.01）. ConclusionExercise combined with traditional Chinese medicine can enhance the expression of M1AChR in the hippocampus of AD model rats， induce autophagy through the AMPK/mTOR signaling pathway， and improve the learning and memory ability of AD rats.

Objective To evaluate the patterns of sleep duration by integrating nocturnal and daytime sleep,and to explore their asso-ciations with activities of daily living(ADL)in middle-aged and older adults.Methods The data of sleep and ADL were obtained from 11 085 subjects aged 45 and older,which were collected from the China Health and Retirement Longitudinal Study(CHARLS).At enrollment in 2011,data on nocturnal and daytime sleep duration were collected through questionnaires.Firstly,subjects were divided into three groups based on the 33rd and 66th percentiles of nocturnal sleep duration,assigned scores of 1,2 and 3,respectively.Subsequently,subjects were divided into three groups based on the 33rd and 66th percentiles of daytime sleep du-ration,assigned scores of 3,2 and 1,respectively.Finally,the scores for nocturnal and daytime sleep were summed to create a total sleep score ranging from 2 to 6.A total sleep score of 6 was defined as sleep duration pattern 1,indicating a longer nocturnal sleep duration with no or shorter daytime sleep duration.Similarly,total sleep scores of 5,4,3 and 2 were defined as sleep duration patterns 2,3,4 and 5,respectively,with sleep duration pattern 5 indicating a shorter nocturnal sleep duration and a longer daytime sleep duration.The status of ADL was assessed using the Activities of Daily Living Scale(Katz edition)at baseline and during follow-up in 2013,2015 and 2018.The association between sleep duration patterns and the risk of ADL disability was evaluated us-ing a Cox proportional hazards model.Results A total of 63 015 person-years were followed,with 11 085 subjects,during which 3 239 individuals experienced ADL disability.The 33rd and 66th percentiles of the nocturnal sleep duration in the study population were 6.00 hours and 7.00 hours,respectively;while the 33rd and 66th percentiles of the daytime sleep duration were 0.00 hours and 1.00 hours,respectively.Of those,1 522 were classified into sleep duration pattern 1,2 196 into sleep duration pattern 2,4 299 into sleep duration pattern 3,2 304 into sleep duration pattern 4,and 764 into sleep dura-tion pattern 5.Compared to sleep duration pattern 1,the risk of ADL disability of patterns 3,4 and 5 were higher(P<0.05),with P-value for the trend less than 0.001,after adjusting for age,sex,body mass index,marital sta-tus,educational level,residence,smoking,drinking,history of chronic diseases,depression status and season.No interaction effect between gender,age and season,and sleep duration patterns was observed(P>0.05).Com-pared to subjects with nocturnal sleep duration≥9 hours and daytime sleep duration<2 hours,those with noctur-nal sleep duration<7 hours and daytime sleep duration≥2 hours had a higher risk of ADL disability(P<0.05).Conclusion Older adults who sleep less at night but take longer naps during the day are at a higher risk of experiencing limitations in their ADL.Sleep patterns may influence ADL among middle-aged and older populations,and man-aging their sleep duration patterns could help prevent the onset of ADL limitations.

Objective To evaluate the patterns of sleep duration by integrating nocturnal and daytime sleep,and to explore their asso-ciations with activities of daily living(ADL)in middle-aged and older adults.Methods The data of sleep and ADL were obtained from 11 085 subjects aged 45 and older,which were collected from the China Health and Retirement Longitudinal Study(CHARLS).At enrollment in 2011,data on nocturnal and daytime sleep duration were collected through questionnaires.Firstly,subjects were divided into three groups based on the 33rd and 66th percentiles of nocturnal sleep duration,assigned scores of 1,2 and 3,respectively.Subsequently,subjects were divided into three groups based on the 33rd and 66th percentiles of daytime sleep du-ration,assigned scores of 3,2 and 1,respectively.Finally,the scores for nocturnal and daytime sleep were summed to create a total sleep score ranging from 2 to 6.A total sleep score of 6 was defined as sleep duration pattern 1,indicating a longer nocturnal sleep duration with no or shorter daytime sleep duration.Similarly,total sleep scores of 5,4,3 and 2 were defined as sleep duration patterns 2,3,4 and 5,respectively,with sleep duration pattern 5 indicating a shorter nocturnal sleep duration and a longer daytime sleep duration.The status of ADL was assessed using the Activities of Daily Living Scale(Katz edition)at baseline and during follow-up in 2013,2015 and 2018.The association between sleep duration patterns and the risk of ADL disability was evaluated us-ing a Cox proportional hazards model.Results A total of 63 015 person-years were followed,with 11 085 subjects,during which 3 239 individuals experienced ADL disability.The 33rd and 66th percentiles of the nocturnal sleep duration in the study population were 6.00 hours and 7.00 hours,respectively;while the 33rd and 66th percentiles of the daytime sleep duration were 0.00 hours and 1.00 hours,respectively.Of those,1 522 were classified into sleep duration pattern 1,2 196 into sleep duration pattern 2,4 299 into sleep duration pattern 3,2 304 into sleep duration pattern 4,and 764 into sleep dura-tion pattern 5.Compared to sleep duration pattern 1,the risk of ADL disability of patterns 3,4 and 5 were higher(P<0.05),with P-value for the trend less than 0.001,after adjusting for age,sex,body mass index,marital sta-tus,educational level,residence,smoking,drinking,history of chronic diseases,depression status and season.No interaction effect between gender,age and season,and sleep duration patterns was observed(P>0.05).Com-pared to subjects with nocturnal sleep duration≥9 hours and daytime sleep duration<2 hours,those with noctur-nal sleep duration<7 hours and daytime sleep duration≥2 hours had a higher risk of ADL disability(P<0.05).Conclusion Older adults who sleep less at night but take longer naps during the day are at a higher risk of experiencing limitations in their ADL.Sleep patterns may influence ADL among middle-aged and older populations,and man-aging their sleep duration patterns could help prevent the onset of ADL limitations.

Objective:To investigate the etiological characteristics of hand, foot and mouth disease (HFMD) and the VP1 gene evolution of coxsackievirus A6 (CVA6) in Qingdao from 2020 to 2022.Methods:Total enterovirus (EV), enterovirus 71 (EV71), coxsackievirus A16 (CVA16), CVA6 and coxsackievirus A10 (CVA10) were detected by fluorescent RT-PCR in throat swab samples from HFMD patients in Qingdao from 2020 to 2022. The full-length sequence amplification of VP1 gene and gene sequence determination of CVA6 isolates were performed, and the phylogenetic and molecular characteristics were analyzed by MEGA-X software.Results:The overall positive rate of enteroviruses in 1 645 samples collected during 2020-2021 was 80.73%(1 328/1 645). The proportions of EV71, CVA16, CVA6, CVA10 and other enteroviruses were 0.08%(1/1 328), 19.65%(261/1 328), 68.15%(905/1 328), 0.98%(13/1 328) and 11.14%(148/1 328), respectively. Children under 5 years old accounted for 75.68% (1 245/1 645) of all cases and 100% (2/2) of severe cases. A phylogenetic tree was constructed based on the VP1 region sequences of 45 CVA6 strains showing all strains belonging to the D3a subtype. There were 10 non-synonymous mutations of amino acids in the VP1 protein by gene evolution analysis.Conclusions:CVA6 D3a subtype is the main epidemic strain causing HFMD in Qingdao from 2020 to 2022, especially in children under 5 years old.

Objective:To investigate the molecular features of hand, foot and mouth disease (HFMD) caused by coxsackievirus A10 (CVA10) in Qingdao and analyze the clinical features of mild and severe cases.Methods:A total of 6 677 cases of HFMD routinely monitored by Qingdao Women and Children Hospital from 2014 to 2021 were enrolled. Throat swab samples were collected. Clinical data of these cases were retrospectively analyzed. Virus nucleic acid was extracted from the samples and the serotypes of enteroviruses were identified. The VP1 genes of CVA10 strains were amplified and sequenced. A phylogenetic tree based on the VP1 gene sequences was constructed using MEGA7. 0 software. SPSS23.0 software was used for statistical analysis.Results:There were 285 cases positive for CVA10, including 183 males and 102 females, and children under five years old accounted for 89.8%. Most of CVA10 infection occurred between the months of April to September. The count of white blood cells, the percentage of neutrophils, the concentration of hemoglobin, and the levels of aspartate aminotransferase and alanine transaminase were significantly higher in severe patients than in mild patients. Besides, chest radiography and brain CT revealed more abnormalities in severe patients, and the duration of ECG monitoring was longer in them. Compared with mild cases, severe cases developed rash early than fever with rash mostly on buttocks ( P<0.05). Phylogenetic analysis showed that most of the CVA10 strains circulating in Qingdao between 2014 and 2021 belonged to clade Ⅰ, and there were two variations A23V and I283V in the amino acid sequence of clade Ⅰ. Conclusions:This study showed that children of all ages were susceptible to CVA10, especially those under five years old. CVA10 showed complex and diverse epidemic trends in different regions and years.

Objective: To illuminate the gene characteristics and clinical characterization of Coxsackievirus B5 (CV-B5) strains isolated from patients with sevre hand, foot and mouth disease (HFMD) in Qingdao city. Methods: A total of 1 844 patients of HFMD were consecutively admitted to Qingdao Women and Children's Hospital from 2013 to 2014. Information of the study population described above was collected retrospectively. The samples were collected from at least 1 site (throat swab, cerebrospinal fluid), which viral nucleic acid extracted and the entire VP1 gene sequences of CV-B5 isolates were amplified and sequenced, then the homology and phylogeny analysis were conducted by MEGA7.0. The prototype Faulkner strain and other VP1 amino acid sequences were derived from the GenBank database. Results: A total of 8 CV-B5 positive cases were obtained, including 4 males and 4 females; 6 severe hospitalized cases and 2 outpatients. The age of 6 hospitalized patients ranged from 3 to 48 months, with a median of 26 months. For the six inpatients, fever, convulsions vomiting, diarrhea and rash were the main clinical manifestation, and all combined with viral encephalitis. Compared with the prototype strain Faulkner, in the VP1 region,the nucleotide and the amino acid homologies was 77.3%-78.8% and 95.5%-97.0% respectively. Five out of the six severe cases with substitution of serine (S) to asparagine (N) at amino acid site 95 in the VP1 region. The sequences of 8 CV-B5 strains were classified into genogroup D. Conclusion Hand, foot and mouth disease associated with CV-B5 virus infection can result in nervous system involvement and the main complication was viral encephalitis. The CV-B5 strains associated with severe hand, foot and mouth disease had high nucleotide homology and present a certain regional aggregation.

Objective To investigate the effect of aldehyde-free fixation solution and aldehyde fixation solution on DNA and cytoplasm of nucleus.Methods The DNA quantification (IOD),discrete coefficient (CV),DNA index (DI) and nucleus area (area) were measured by DNA quantification system (ICM) in 8 different fixation solution;combined with cytological staining,the effects of different fixative solution on nuclear DNA and cytoplasmic staining were analyzed.Results Aldehyde fixation solution was better than aldehyde free fixation solution,the formaldehyde in fixtion solution has important influence on Feulgen-eosin staining.Conclusion Aldehyde fixation solution combined with cytological staining can obtained good dyeing effect,which provides the basis for the subsequent multi-technique joint diagnosis.

Objective To discuss the value of Fisher discriminant analysis of serum progesterone and the growing rate of β-human chorionic gonadotropin in the prediction of early ectopic pregnancy. Methods 66 patients with ectopic pregnancy (11 cases were successfully treated expectantly and 55 cases were treated surgically including 40 cases of rupture of fallopian tube and 15 cases of tubal abortion) and 55 patients with intrauterine pregnancy and 50 patients with threatened abortion were chosen. Serum progesterone,β-HCG,48 hβ-HCG and the 48 h growing rate of β-HCG in each group were measured and a Fisher discriminant analysis was used. Results The serum progester-one was (30.27± 18.20) nmol/L in ectopic pregnancy group,( 108.44±23.27 ) nmol/L in intrauterine pregnancy group and (91.68±34.90) nmol/L in threatened abortion group. The first β-HCG was ( 3767.63 ± 3530.38 ) U/L in ectopie pregnancy group,(29 028.65 ± 10 874.01 )U/L in intrauterine pregnancy group and (13 457.47±16 367.65)U/L in threatened abortion group. The second β-HCG was (4349.24±3536.22)U/L in ectopic pregnancygroup,(56 139.46 ± 23 296.87 ) U/L in intrauterine pregnancy group and (23 270.63 ± 23 811.68 ) U/L in threat-ened abortion group. The growing rate of β-HCG ( β-HCG/the first serum β-HCG) was 1.29 ± 0.28 in ectopic preg-nancy group,1.93 ± 0.36 in intrauterine pregnancy group and 1.97±0.28 in threatened abortion group. There was significant difference in serum progesterone,the first β-HCG and the second β-HCG as well as the growing rate of β-HCG among the groups(P<0.05 or <0.01). Fisher discriminant analysis of combing progesterone and the growing rate of β-HCG were connected with diagnosis of ectopic pregnancy,however,the only one serum β-HCG was not con-nected with diagnosis of ectopic pregnancy. 98.5% of ectopic pregnancy,65.6% of intrauterine pregnancy and 64.0% of threatened abortion were correctly classified in the Fisher discfiminant analysis,with overall correct rate of 77.8%. Conclusion Fisher discriminant analysis of combing progesterone and the growing rate of β-HCG can bet-ter predict the early ectopic pregnancy.