Epilepsy is a chronic neurological disorder that often begins in childhood and can extend over the entire life course; therefore, it is necessary to establish a long-term, systematic life-course management model. Whole life-cycle disease management emphasizes interventions at key points in time and focuses on diagnosis and treatment points at all stages of life and disease processes. Guided by the concept of holism and preventive treatment, this article discussed the application strategies of TCM in the prevention, control, treatment, health and management of epilepsy in children at different stages. In the prevention period, the disease should be prevented first, and early screening should be carried out; in the control period, the disease should be prevented from changing, and the early intervention of Chinese materia medica should be carried out; during the treatment period, the combination of disease and syndrome and dynamic syndrome differentiation by stages were adopted; during the rehabilitation period, the treatment of different diseases with the same treatment should be emphasized to manage the comorbidity and improve the prognosis; in the long-term management period, the prevention and recovery after stroke should be focused on, and the comprehensive management scheme of TCM in the transition period from children to adults should be put forward. This article systematically expounded the role of TCM in improving seizure control rate and prognosis in the whole life-cycle management of childhood epilepsy, and actively explored the construction of the whole process health management system of chronic diseases.

Epileptogenesis is a dynamic process of gradual progression from normal developing brain to pathological epileptic brain， which is the latent and budding stage of epilepsy. Combining the understanding of epileptogenesis in children from Western medicine and traditional Chinese medicine （TCM）， we proposed that the viewpoints of constitutional transformation， phlegm pathogen inducing epilepsy， and brain collateral damage， which correspond to key pathological mechanisms， namely gene polymorphism， immunoinflammation， and microvascular dysfunction of the blood-brain barrier， respectively. Based on these insights， strategies for prevention and treatment of epileptogenesis in children， as well as potential research directions are explored.

Objective:To explore the mechanism of miR-15b-5p involving in depression-like behavior in Parkinson's disease (PD).Methods:(1) Eighteen C57BL/6N mice were randomly divided into PD group, intervention group and control group ( n=6). PD models in PD group were established by stereotaxically injecting 0.25 mg/kg rotenone into the right striatum; mice in the intervention group were injected with 0.25 mg/kg rotenone and miR-15b-5p inhibitor lentivirus, while mice in the control group were injected with equal volume of PBS. Four weeks after that, open field test and rotarod test were performed to evaluate the motor ability, and sucrose preference and forced swimming tests were performed to evaluate the depression-like behaviors. And then, proteins and miRNAs in the substantia nigra were extracted; real-time fluorescent quantitative reverse transcription PCR (qRT-PCR) was used to detect the miR-15b-5p expression,and Western blotting and immunofluorescent staining were used to detect the tyrosine hydroxylase (TH), brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), and postsynaptic density protein 95 (PSD95) protein expressions. (2) The 100 nmol/L miR-15b-5p mimic/inhibitor and their negative control sequences were transfected into SH-SY5Y cells on 6-well plates (named miR-15b-5p mimic group, miR-15b-5p mimic control group, miR-15b-5p inhibitor group and miR-15b-5p inhibitor control group, respectively); 48 h after that, BDNF, TrkB and PSD95 protein expressions were detected by Western blotting and miR-15b-5p expression by qRT-PCR. (3) The 100 ng BDNF 3'-UTR wild-type or mutant luciferase reporter vector plasmids and 100 nmol/L miR-15b-5p mimic/inhibitor or their negative control sequences were co-transfected into SH-SY5Y cells on 24-well plates, and luciferase reporter activity assay was performed 48 h after co-transfection to detect the luciferase activity. Results:(1) Compared with the control group, the PD group had significantly reduced movement speed, shortened rotarod drop latency, decreased percentage of sucrose preference, and prolonged immobility time ( P<0.05); compared with the PD group, the intervention group had significantly increased movement speed, prolonged rotarod drop latency, increased percentage of sucrose preference, and shortened immobility time ( P<0.05). (2) Compared with the miR-15b-5p mimic control group, the miR-15b-5p mimic group had significantly increased miR-15b-5p expression, and decreased BDNF, TrkB and PSD95 protein expressions (100.00±5.75 vs. 66.79±5.90; 100.00±5.95 vs. 84.46±5.77; 100.00±7.02 vs. 80.43±3.25, P<0.05). Compared with the miR-15b-5p inhibitor control group, the miR-15b-5p inhibitor group had significantly decreased miR-15b-5p expression, and increased BDNF, TrkB and PSD95 expressions (100.00±6.81 vs. 119.90±5.66; 100.00±2.88 vs. 110.10±4.15; 100.00±2.19 vs. 124.60±11.69, P<0.05). Compared with the control group, PD group had significantly increased miR-15b-5p expression, and significantly decreased TH, BDNF, TrkB and PSD95 expressions and BDNF fluorescent intensity (100.00±9.20 vs. 63.60±12.80; 100.00±9.88 vs. 71.95±10.00; 100.00±5.16 vs. 70.37±8.43; 100.00±7.01 vs. 68.12±10.22; 100.00±12.99 vs. 48.23±12.58) in the substantia nigra ( P<0.05); compared with the PD group, the intervention group had significantly lower miR-15b-5p expression and increased TH, BDNF, TrkB and PSD95 expressions and BDNF fluorescent intensity (63.60±12.80 vs. 90.69±9.84; 71.95±10.00 vs. 93.31±4.50; 70.37±8.43 vs. 88.11±4.10; 68.12±10.22 vs. 89.59±5.93; 48.23±12.58 vs. 83.65±10.52) in the substantia nigra ( P<0.05). (3) Compared with the BDNF 3'-UTR wild-type+miR-15b-5p mimic control group, the BDNF 3'-UTR wild-type+miR-15b-5p mimic group had significantly decreased luciferase activity (100.00±5.07 vs. 90.59±1.75, P<0.05); compared with the BDNF 3'-UTR wild-type+miR-15b-5p inhibitor control group, the BDNF 3'-UTR wild-type+miR-15b-5p inhibitor group had significantly increased luciferase activity (100.00±5.08 vs. 152.20±31.87, P<0.05). Conclusion:MiR-15b-5p is involved in depression-like behavior in PD by down-regulating the BDNF/TrkB/PSD95 expressions.

Objective:To observe the effect of α-synuclein (α-Syn) aggregates incubated with plasma from patients with Parkinson's disease (PD) and multiple system atrophy (MSA) on mitochondrial and lysosomal functions, and further clarify the role of α-Syn in pathological mechanism of PD and MSA.Methods:Human neuroblastoma cells (SH-SY5Y) were treated with 10 μg/mL α-Syn aggregates incubated with phosphate buffered solution (PBS), and plasma from healthy controls (HCs), PD patients, and MSA patients, respectively; and the untreated SH-SY5Y cells were used as control group. Mito-Tracker Red CMXRos fluorescent staining was used to detect the mitochondrial membrane potential, Western blotting was used to detect the cytochrome C (Cyt C) expression in the mitochondria and cytoplasm, and adenosine triphosphate (ATP) test kit was used to detect the intracellular ATP level. In addition, the lysosomal associated membrane protein-1 (LAMP1) expression was detected by Western blotting, and cathepsin B activity was detected by Magic Red fluorescent staining.Results:(1) Effects of different α-Syn aggregates on mitochondrial function damage: compared with the control group, the PBS, HC, PD and MSA groups had significantly decreased relative fluorescent intensity (Mito-Tracker Red CMXRos staining) in the SH-SY5Y intracellular mitochondria ( P<0.05); the PD group and MSA group had significantly decreased relative fluorescent intensity compared with PBS group and HC group ( P<0.05); the MSA group had significantly decreased relative fluorescent intensity compared with the PD group (41.27%±5.97% vs. 60.05%±4.24%, P<0.05). Compared with the control group, the HC, PD and MSA groups had significantly decreased mitochondrial Cyt C protein expression, while the PBS, HC, PD and MSA groups had significantly increased cytoplasmic Cyt C protein expression ( P<0.05); the PD group and MSA group had significantly decreased mitochondrial Cyt C protein expression compared with the PBS group and HC group ( P<0.05); the MSA group had significantly increased cytoplasmic Cyt C protein expression compared with the PBS group ( P<0.05); the MSA group had significantly decreased mitochondrial Cyt C protein expression (51.50%±4.22%) and increased cytoplasmic Cyt C protein expression (199.40%±6.73%) compared with the PD group (65.52%±2.18% and 174.00%±16.18%, P<0.05). Compared with the control group, the PBS group, HC group, PD group and MSA group had significantly decreased ATP level ( P<0.05); the PD group and MSA group had significantly decreased ATP level compared with the PBS group and HC group ( P<0.05); the MSA group had significantly decreased ATP level (1.81±0.13 nmol/mg) compared with PD group (2.96±0.29 nmol/mg, P<0.05). (2) Effects of different α-Syn aggregates on lysosomal functions: compared with the control group, the PD group and MSA group had significantly decreased LAMP1 protein expression ( P<0.05); the MSA group had significantly decreased LAMP1 protein expression (60.74%±7.94%) compared with the PBS group, HC group, and PD group (79.70%±4.34%, P<0.05). Compared with the control group, the PBS, HC, PD and MSA groups had significantly enhanced relative fluorescent intensity of lysosomal cathepsin B ( P<0.05); the PD group and MSA group had significantly enhanced relative fluorescent intensity of lysosomal cathepsin B compared with PBS group and HC group ( P<0.05); the MSA group had significantly increased relative fluorescent intensity of lysosomal cathepsin B compared with PD group (399.00%±35.54% vs. 305.90%±24.87%, P<0.05). Conclusion:The α-Syn aggregates incubated with plasma from PD and MSA patients have damage to mitochondrial and lysosome functions, especially those incubated with plasma from MSA patients.

Objective:To observe the effect of α-synuclein (α-Syn) aggregates incubated with plasma from patients with Parkinson's disease (PD) and multiple system atrophy (MSA) on mitochondrial and lysosomal functions, and further clarify the role of α-Syn in pathological mechanism of PD and MSA.Methods:Human neuroblastoma cells (SH-SY5Y) were treated with 10 μg/mL α-Syn aggregates incubated with phosphate buffered solution (PBS), and plasma from healthy controls (HCs), PD patients, and MSA patients, respectively; and the untreated SH-SY5Y cells were used as control group. Mito-Tracker Red CMXRos fluorescent staining was used to detect the mitochondrial membrane potential, Western blotting was used to detect the cytochrome C (Cyt C) expression in the mitochondria and cytoplasm, and adenosine triphosphate (ATP) test kit was used to detect the intracellular ATP level. In addition, the lysosomal associated membrane protein-1 (LAMP1) expression was detected by Western blotting, and cathepsin B activity was detected by Magic Red fluorescent staining.Results:(1) Effects of different α-Syn aggregates on mitochondrial function damage: compared with the control group, the PBS, HC, PD and MSA groups had significantly decreased relative fluorescent intensity (Mito-Tracker Red CMXRos staining) in the SH-SY5Y intracellular mitochondria ( P<0.05); the PD group and MSA group had significantly decreased relative fluorescent intensity compared with PBS group and HC group ( P<0.05); the MSA group had significantly decreased relative fluorescent intensity compared with the PD group (41.27%±5.97% vs. 60.05%±4.24%, P<0.05). Compared with the control group, the HC, PD and MSA groups had significantly decreased mitochondrial Cyt C protein expression, while the PBS, HC, PD and MSA groups had significantly increased cytoplasmic Cyt C protein expression ( P<0.05); the PD group and MSA group had significantly decreased mitochondrial Cyt C protein expression compared with the PBS group and HC group ( P<0.05); the MSA group had significantly increased cytoplasmic Cyt C protein expression compared with the PBS group ( P<0.05); the MSA group had significantly decreased mitochondrial Cyt C protein expression (51.50%±4.22%) and increased cytoplasmic Cyt C protein expression (199.40%±6.73%) compared with the PD group (65.52%±2.18% and 174.00%±16.18%, P<0.05). Compared with the control group, the PBS group, HC group, PD group and MSA group had significantly decreased ATP level ( P<0.05); the PD group and MSA group had significantly decreased ATP level compared with the PBS group and HC group ( P<0.05); the MSA group had significantly decreased ATP level (1.81±0.13 nmol/mg) compared with PD group (2.96±0.29 nmol/mg, P<0.05). (2) Effects of different α-Syn aggregates on lysosomal functions: compared with the control group, the PD group and MSA group had significantly decreased LAMP1 protein expression ( P<0.05); the MSA group had significantly decreased LAMP1 protein expression (60.74%±7.94%) compared with the PBS group, HC group, and PD group (79.70%±4.34%, P<0.05). Compared with the control group, the PBS, HC, PD and MSA groups had significantly enhanced relative fluorescent intensity of lysosomal cathepsin B ( P<0.05); the PD group and MSA group had significantly enhanced relative fluorescent intensity of lysosomal cathepsin B compared with PBS group and HC group ( P<0.05); the MSA group had significantly increased relative fluorescent intensity of lysosomal cathepsin B compared with PD group (399.00%±35.54% vs. 305.90%±24.87%, P<0.05). Conclusion:The α-Syn aggregates incubated with plasma from PD and MSA patients have damage to mitochondrial and lysosome functions, especially those incubated with plasma from MSA patients.

Objective:To explore the role of α-synuclein (α-Syn) in pathogenesis of Parkinson's disease (PD) and multiple system atrophy (MSA) by observing the ability of α-Syn aggregates incubated with PD and MSA patients' plasma to destroy cell membrane.Methods:Peripheral blood samples were collected from 5 PD patients and 5 MSA patients diagnosed in Department of Neurology, Affiliated Hospital of Guilin Medical University from January 2018 to January 2022, as well as 5 physical examination healthy control subjects (HCs) during the same period. The α-Syn was dissolved in 0.01 mol/L PBS and then incubated with PBS, and plasma from HCs, PD patients and MSA patients at 37 ℃ for 4 d, respectively (named as PBS group, HC group, PD group and MSA group). Small unilamellar vesicles (SUVs) containing calcein were prepared with acidic phospholipid, 1-palmityl-2-oleoyl-Sn-glycerol-3-phospho-L-serine (POPS), by membrane dispersion-ultrasonic method; the particle size and morphology of SUVs were analyzed by Malvern Zetasizer Nano ZS and transmission electron microscope. SUVs and human neuroblastoma cells (SH-SY5Y) were treated with different concentrations of α-Syn aggregates (0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 μmol/L). The abilities of α-Syn aggregates formed under different conditions to destroy liposomes and cell membrane were evaluated by measuring the calcein relative release and intracellular calcein fluorescent value after dialysis.Results:(1) Destructive effect of α-Syn aggregates on SUVs: calcein induced by α-Syn aggregates in each group increasingly released with the increase of protein concentration; at protein concentration of 8 μmol/L, the calcein released from SUVs in PD and MSA groups was significantly higher than that in PBS and HC groups, and the calcein released from SUVs in MSA group was further significantly higher than that in the PD group ( P<0.05). (2) Destructive effect of α-Syn aggregates on cell membrane in SH-SY5Y: the calcein fluorescent value in each group decreased with the increase of protein concentration; at protein concentration of 8 μmol/L, the intracellular calcein fluorescent value in PD and MSA groups were significantly decreased compared with that in PBS and HC groups ( P<0.05); the intracellular calcein fluorescent value in MSA group was further significantly decreased compared with that in PD group ( P<0.05). (3) Effect of α-Syn aggregates on SH-SY5Y cell survival: at protein concentration of 8 μmol/L, the viability of SH-SY5Y cells in each group decreased obviously; PD and MSA groups had significantly decreased cell viability compared with PBS and HC groups ( P<0.05); and the viability in MSA group was further statistically decreased compared with that in PD group ( P<0.05). Conclusion:The ability of α-Syn aggregates incubated with PD and MSA patients' plasma to destroy cell membrane is greater than that with HCs' plasma, especially those with MSA patients' plasma.

Objective:To investigate the stability of α-synuclein (α-Syn) aggregates formed by incubation with blood plasma of patients with Parkinson's disease (PD).Methods:Peripheral blood samples were collected from 10 patients diagnosed as having PD in our hospital from June 2020 to December 2020 and 10 healthy control subjects (HC) at the same time period. The 1 mg recombinant human α-Syn was dissolved in 140 μL 0.01 mol/L phosphate buffer solution (PBS), and then, incubated with plasma from HC and PD patients and PBS at 37 ℃ for 7 d (HC group, PD group and PBS group). Preformed fiber (PFF) group was used for subsequent experiment with 10 μg PFF. After digestion with different concentrations of trypsin (concentration ratios of trypsin/α-Syn=1:80, 1:40, and 1:20) and protease K (PK, 1.0, 1.5, and 2.0 μg/mL), the differences of α-Syn levels before and after digestion were detected by Western blotting.Results:(1) Effect of trypsin on PFF digestion: PFF gradually decreased with the increase of trypsin doses; when trypsin/α-Syn ratio=1:20, α-Syn aggregates with molecular weight greater than 35 000 were almost completely digested, and its digestion was significantly different as compared with that in other concentration ratios ( P<0.05). (2) Effect of trypsin on digestion of α-Syn aggregates: at the relative molecular weight<25 000, when the concentration ratio of trypsin/α-Syn=1:20, as compared with HC and PD groups, the PBS group had significantly more obvious decrease ( P<0.05). At the relative molecular weight 35 000-40 000, the α-Syn levels in PBS, HC and PD groups were significantly decreased as compared with those before digestion at all concentration ratios; as compared with HC and PD groups, the PBS group had significantly more obvious decrease ( P<0.05). At molecular weight>40 000, α-Syn decreased significantly in PD group only when the concentration ratio of trypsin/α-Syn=1:20, and the degrees of digestion was PBS group>HC group>PD group, with significant differences among groups ( P<0.05). (3) Effect of PK on PFF digestion: when PK concentration was 1.0, 1.5 or 2.0 μg/mL, α-Syn was basically digested at relative molecular weight>35 000, and α-Syn monomer was reduced and small fragment appeared at relative molecular weight<25 000; as compared with negative controls (0 μg/mL PK), these changes in groups of PK concentration of 1.0, 1.5 or 2.0 μg/mL were significantly different ( P<0.05). (4) Effect of PK on digestion of α-Syn aggregates: at relative molecular weight<25 000, α-Syn in PBS group was digested into smaller fragments while α-Syn in HC and PD groups was not digested; significant differences was noted among groups at the same concentration ( P<0.05). At relative molecular weight of 35 000-40 000, with the increase of PK concentration, the amount of α-Syn dimer in PBS group decreased (increased digestibility), that in HC group increased, however, that in PD group did not change obviously; significant difference was noted at the same concentration among the three groups ( P<0.05). At relative molecular weight>40 000, with the increase of PK concentration, α-Syn in PBS, HC and PD groups decreased to a certain extent, and significant difference was noted among groups at the same concentration ( P<0.05). Conclusion:The stability of α-Syn aggregates formed by incubation with plasma from PD patients is higher than that formed by incubation with HC plasma and PBS.

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.
Clostridium Infections ; diagnosis ; Clostridium difficile ; genetics ; Electrophoresis, Capillary ; Feces ; microbiology ; Genes, Bacterial ; Humans ; Polymerase Chain Reaction ; Ribotyping ; Sensitivity and Specificity

Objective To compare the reliability and effectiveness of anterolateral thigh flap with double vein anastomosis or one vein anastomosis for reconstruction of head and neck defects.Methods Two hundred and sixty four cases of anterolateral thigh flap transfers for head and neck reconstruction from January,2013 to September,2013 in the Second Xiangya Hospital of Central South University were reviewed.260 patients were randomly divided into 2 groups.In the experimental group,there were 138 patients 140 cases of anterolateral thigh flap with one vein one artery anastomosis.In the control group,there were 122 patients 124 cases of anterolateral thigh flap with double vein one artery anastomosis.Results Among 264 anterolateral thigh flaps,the overall success rate of free flap was 98.1％ (259/264),5 free flaps were lost.In the experimental group,there were 6 free flaps occurred venous thrombosis,two of them were lost.In the control group,there were 5 cases occurred venous thrombosis,three of them were lost.No arterial thrombosis occurred in both groups.The time of micromanipulation was 18 to 101 min,with the average of 47 min in the experimental group.In the control group,the time was 45 to 133 min,with the average of 71 min.(P =0.0003).Conclusion Anterolateral thigh flap with one vein one artery anastomosis for head and neck reconstruction did not affect the survival rate but it can absolutely reduce the operation time.

Objective To summarize the application of 909 anterolateral thigh myocutaneous flaps in the repair of oral and maxillofacial defects and to examine their benefits in maxillofacial reconstruction of these defects.Methods Patients were recruited from January 2004 to December 2012 in the Department of Oral and Maxillofacial Surgery of the Second Xiangya Hospital of Central South University.All patients underwent reconstructive surgery with anterolateral thigh myocutaneous flaps,and patient age ranged from 19 to 81 years with a mean of 51.2 years.There were 761 flaps showing single lobe and 148 flaps showing a multi-island pedicle.The largest area among the single flaps was 28 cm× 12 cm,and the smallest was 4 cm× 2 cm.Results Among the 909 transferred flaps,882 survived and 27 showed necrosis,with a survival rate of about 97.0％.The common complications at flap donor site were poor wound healing 9.6％(87/909),localized paresthesia 61.0％(500/820),and altered quadriceps force 15.0％(123/820).No case was presented with local serious complications,and 90％ of the patients achieved good functional recovery and aesthetically acceptable results after the reconstruction by anterolateral thigh myocutaneous flaps.Conclusions The anterolateral thigh myocutaneous free flaps are more suitable for oral and maxillofacial defects than other flaps and should be preferred.