Objective To construct the eukaryotic expression plasmids of Mycobacterium tuberculosis ESAT-6 antigen and to investigate its immunogenicity in mice. Methods The sequence encoding ESAT-6 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA. Then, the amplified fragment was sub-cloned into pVAC expression vector. The eukaryotic expression plasmid pVAC-ESAT-6 was transfected into Vero E6 cells with liposome. The mRNA expression of pVAC-esat-6 in Vero E6 cells was detected by RT-PCR and its expression product was analyzed by Western-blot. The plasmid pVAC-esat-6 was introduced into the mice by gene gun injection. The specific antibody to ESAT-6 and the level of IFN-? in supernatant of spleno-lymphocyte cultures were detected by ELISA methods. Results The recombinant plasmid pVAC-esat-6 was successfully constructed, and the expression of ESAT-6 was also detected in vitro. After vaccinated three times, the mice produced specific antibody and the level of IFN-? in supernatant of spleno-lymphocyte cultures in group of pVAC-esat-6 was significantly higher than group of vector alone (P

Objective:To study the clinical features and molecular genetic mechanisms of children with lissencephaly (LIS), as well as to analyze the relationship between genotypes and phenotypes of the disease.Methods:From October 2016 to December 2017, the clinical data and follow-ups of 21 LIS children were collected in the Department of Neurology, Shenzhen Children′s Hospital.Whole genome sequencing (WGS) was performed for genetic testing.Results:Among these 21 cases, 18 cases developed epilepsy (86%), and 3 cases were seizure free (14%). The onset age of children with epilepsy was relatively young, and 16 cases occurred within 1 year old (89%). Among these cases, 16 were pachygyria (76%), 3 cases were agyria combined with pachygyria (14%) and 2 cases were agyria (10%). Epileptic syndromes included 12 cases of West syndrome (67%), 2 cases of Ohtahara syndrome (11%), 2 cases of other epileptic encephalopathy (11%), and 2 cases of focal epilepsy (11%). Brain magnetic resonance imaging(MRI) demonstrated that most cases were pachygyria, among which diffuse pachygyria was more common (56%, 9/16 cases). The results of WGS: 13 pathogenic or likely pathogenic single nucleotide variants (SNV) and copy number variants (CNV) were detected.The total detection rate was 62%, of which 2 cases were frameshift, 1 case was nonsense and 1 case was missense variants of PAFAH1B1, 6 cases were chromosome 17p13.3 deletion syndrome, thus lea-ding to the whole gene deletion of PAFAH1B1, and 1 case was missense variant of DCX, frameshift variant of KIF2A, and missense variant of PIK3R2, respectively.Totally, 48% (10/21 cases) of the cases were variants or deletions of PAFAH1B1, which resulted in lissencephaly in the parietal-occipital region of the brain.Novel variants were PAFAH1B1: c.1067G>A, PAFAH1B1: c.897delT and KIF2A: c.2225delG. Conclusions:Most cases of LIS accompanied with epilepsy, in which West syndrome was relatively more common.Brain MRI showed that most cases were diffuse pachygyria.The variants and deletions of PAFAH1B1 was the main genetic cause of LIS.The identification of the novel variants expanded the genotypical spectrum of LIS.

Objective To assess the efficacy and safety of two different protocols of ketogenic diet (KD)-eating on demand or eating at regular intervals for refractory epilepsy in children.Methods Sixty children with refractory epilepsy were randomly divided into eating on demand group (n =30) and eating at regular intervals group (n =30) by random number table method.After taking the whole amount of KD,the capillary blood ketone and glucose level and urine ketone were monitored every 6 hours in 72 continuous hours.Seizure frequency and onset time were recorded.Antiepileptic efficacy and diet tolerability of the two groups were evaluated on 4 weeks,12 weeks,24 weeks and 48 weeks after initiating the diet.Adverse effects were monitored.Results After treatment of 4 weeks,the complete seizure remission rates of eating on demand group and eating at regular intervals group were 33.3％ (10/30) and 30.0％ (9/30) respectively,which suggested a comparable efficacy for two groups (P ＞ 0.05).The day when KD started to work was averaged (6.18 ± 2.42) d and (8.63 ± 2.63) d respectively.The group of eating on demand showed a faster onset of action (P ＜0.05).After treatment of 12 weeks,24 weeks and 48 weeks,complete seizure remission rates of eating on demand group were 30.0％ (9/30),34.8％ (8/23) and 36.8％ (7/19) respectively;the eating at regular intervals group were 33.3％ (10/30),30.4％ (7/23) and 44.4％ (8/18) respectively.The two groups had no significant difference (P ＞ 0.05).One year later,the treatment retention rates of the two groups were 63.3 ％ (19/30) and 60.0％ (18/30) respectively.There was no significant difference (P ＞ 0.05).The adverse effects mainly including transient gastrointestinal symptoms and metabolic disturbances were mostly tolerable and curable.Conclusion The two different protocols of KD-eating on demand and eating at regular intervals are both effective and well-tolerated for refractory epilepsy in children.While protocol of eating on demand is more easier to achieve ketotic state and the effect is more quickly,so it can be more easily received by children.Therefore in clinical practice,we can choose flexible eating time according to children's eating habits,which can improve the therapeutic compliance.

Objective To explore the efficacy of ketogenic diet( KD) in the treatment of status epi-lepticus( SE) and whether KD could protect the brain,and propose a new thought on SE patients′reasonably individualized treatment, brain protection and prognosis improvement. Methods From Sep 2013 to Jan 2015,all the patients diagnosed as SE were advised to apply KD treatment; the patients who refused KD treatment were included in the control group,while the patients who accepted KD treatment were included in the treatment group. Based on the SE treatment principles,the control group applied traditional anti-convulsive therapy,while the treatment group applied traditional therapy combined with KD treatment. Before the treat-ment and after the epilepsy control,the patients′ serum was collected to test neuron specific enolase( NSE) and S100βlevels,and the duration of epilepsy control was recorded. Results The treatment group included a total of 10 patients; 3 patients had a good efficacy and obtained seizure-free after the treatment; clinical seizures declined significantly in 6 patients. The treatment group′s overall response rate was 9/10,which was higher than that of the control group(5/8)(P<0. 01). The treatment group′s duration to gain efficacy was shorter than that of the control group[(5. 2 ± 2. 9) d vs. (9. 8 ± 1. 5) d,P<0. 01]. After the treatment,the patients′NSE and S100β in both groups were significantly decreased than those before the treatment ( P<0. 001 or P<0. 05). After the treatment,the serum NSE and S100β of the patients in the treatment group were lower than those in the control group,with statistically significant difference(P<0. 05). Conclusion Frequent epileptic seizures and SE would impair the patient′s brain. Controlling the epileptic seizures actively could lower the severity of brain injury. KD could effectively control the epileptic seizure and had neuropro-tective effects.

Objective To analyze the clinical manifestations of familial acute necrotizing encephalopathy (ANE)and to improve the recognition of this disease. Methods The clinical data of a 25 - month - old girl with fa-milial and recurrent ANE with evidence of mutation in the RANBP2 gene were collected and analyzed,and the gene examination of their family members was performed. Results A previously healthy girl experienced recurrent ANE epi-sodes at the ages of 8 months,18 months and 25 months,respectively. At each beginning of each episodes the patient presented with lethargy and tremor of limbs following febrile illness of 3 - 4 days,even developed coma and convulsions in the last time. Brain magnetic resonance imaging showed bilateral and high T2 signal changes in thalamus,cerebellum and hippocampus. Abnormal signals also appeared in the brainstem,claustrum,corpus scallosum and cortex(temporal, parietal and cingulate)also appeared abnormal signals. Spinal MRI showed spinal cord involvement. The girl recovered after her first episode;she could speak but could not walk steadily after the second time;after the third episode,al-though she regained consciousness from coma,she could no longer speak or walk. The patient's sister died of encephali-tis at the age of 18 months. Her paternal uncle had suffered from dysnoesia from meningitis at his 17 months of age. The patient and her grandmother,father,uncle and one of her aunts harbored a mutation(c. 1754C ﹥ T)in RANBP2 gene. Conclusions Familial ANE has typical clinical manifestations and characteristic MRI findings. The patient with recur-rent history,especially with positive family history,should have the mutation in RANBP2 gene detected earlier in order to clarify the diagnosis of ANE.

Objective To investigate the radiation level around Jiangzha hot spring,and to analyze the sources of pollution.Methods The radon and its progeny concentration,γ dose rate in hot spring living district and surrounding area were measured with ATD monitors,radon and WL continuous measurement devices,γ dose rate meter.Results The radon concentration in water was 23 -764 Bq/L.Radon concentration indoors,outdoor and in bathing place were 254 -876 799,688 -709 and 3590-15 299 Bq/m3,respectively.γ dose rate were 205 -28718 nGy/h indoor,4104- 18254 n Gy/h outdoors.Conclusions Jiangzha hot spring is an area with rare high radon and high nature radiation.Its radiation level and health effects are worthy for further attention.

Objective To compare the clinical and electroencephalogram(EEG)characteristics and therapeutic response prognosis of different age groups of children with epileptic spasm .Methods From January 2002 to October 2011 the clinical data ,EEG fea-tures of epileptic spasms children under 15 years old with unknown disease cause(cryptogenic) were retrospectively reviewed .74 of them were followed up for 12 to 92 months .All of them were divided into two groups on the basis of onset age :3 -12 months of onset as group infantile-onset spasms(group IOS ,n=60);and 12 months to 7 years old of onset as group late-onset epileptic spasms (group LOS ,n=14) .Clinical process ,seizure semiology and EEG features were compared between two groups .Results Semiologic features of two groups were similar ,but they showed differences in interictal EEG features including the background ,the location of discharges ;The response to drugs between the two groups are also different .Conclusion There are differences between group IOS and group LOS when comparing EEG features and response to drugs .

OBJECTIVE: To explore the effect of modified pedicled cranial periosteum compound flap in reconstructing the defect of anterior cranial fossa complicated with cerebrospinal fluid leakage. METHOD: Seven nasal and sinus tumor cases with defects and CSF were undertaken reconstructing surgery with the flap, of which 1 was benign and the others were malignancies. The transferred flap pedicled with the frontal branch of the superficial temporal artery and complemented with ipsilateral supraorbital and/or supratraochlear artery. RESULT: All cases healed without failure, one case with adjuvant radiotherapy experienced partial free frontal bone necrosis and healed by ways of drainage through temporal incision. The inner surface of the flap in the nasal cavity was smooth. CONCLUSION Frontal cranial periosteum compound flap have some advantages, such as adequate quantity, good blood supply and easily making,so it can be choose as an important method to reconstruct the defects of anterior cranial fossa and CSF leakage.
Adult ; Aged ; Cerebrospinal Fluid Rhinorrhea ; surgery ; Cranial Fossa, Anterior ; pathology ; Female ; Humans ; Male ; Middle Aged ; Periosteum ; transplantation ; Reconstructive Surgical Procedures ; methods ; Skull Base ; surgery ; Surgical Flaps ; Treatment Outcome

Objective To construct a prokaryotic expression system containing the dense granule protein 4(GRA4) of Toxoplasma gondii,purify the expressed protein and detect its immunogenicity.Methods The specific fragment of GRA4 gene was amplified by PCR.After subcloning the prokaryotic expression recombinant pET,GRA4,the expressed product was purified with His?BindTM affinity chromatography and analyzed by Western blot.BALB/c mice were immunized with the GRA4 recombinant protein,and the antibody IgG titer was detected by ELISA.Results The pET,GRA4 prokaryotic expression system was obtained.The MW of the expressed protein was Mr 40 000 and formed in inclusion body.After purification,the recombinant protein could be specifically recognized by the T.gondii infected rabbit serum.Mice immunized with the purified recombinant protein elicited high titer of IgG antibody.Conclusion The pET,GRA4 recombinant protein was successfully expressed and purified,which shows the immunogenicity.

OBJECTIVETo analyze the ultra microstructures and the expression of platelet peroxidase (PPO) of megakaryocytes from bone marrow, their clinical manifestations and laboratory characteristics in patients with acute megakaryoblastic leukemia (AMKL).

METHODSKaryocytes from bone marrow of 22 AMKL patients were divided into two parts by lymphocyte separation liquid, one part was used to prepare the ordinary transmission electron microscope specimens to observe the morphological structures of megakaryocytes, the other was used to prepare the histochemical specimens of platelet peroxidase to analyze the positive reaction of PPO in AMKL, which were coupled with the patients' data of with bone marrow morphology, cell chemistry, and chromosome karyotype examination.

RESULTSMegakaryocytes from 17 of 22 patients were in the first stage, less than 20 µm in diameter, the nucleis were round, the cytoplasm contained microtubules, membranous vesicles and minute dense granules, no demarcation membrane system and surface-connected canalicular system, less dense granules and α-granules; Megakaryocytes in 5 cases were mainly in the first stage, while containing second and third stage megakaryocytes; the positive rate of PPO in megakaryocytes of 22 patients was 0-80%. The primitive and naive megakaryocytes were found in bone marrow smears of 22 cases, CD41 staining of the megakaryocytes was detected in the primitive and naive megakaryocytes, and more complex chromosome karyotype anomalies were observed.

CONCLUSIONThe majority of megakaryocytes in AMKL patients were the first stage ones, the rest were second and third stage ones, and the positive PPO reaction was significantly different. CD41 staining of the megakaryocytes was specific with complex chromosome karyotypeswere.

Blood Platelets ; enzymology ; Bone Marrow ; pathology ; Cell Count ; Chromosome Aberrations ; Chromosome Disorders ; Humans ; Karyotyping ; Leukemia, Megakaryoblastic, Acute ; diagnosis ; pathology ; Megakaryocytes ; pathology ; Peroxidase ; metabolism ; Staining and Labeling