Objective To establish a rapid immunological detection method for MPT64 protein based on red microspheres and select highly-sensitive and highly-specific antibody pairs.Methods A His-tagged prokaryotic expression vector was constructed for expression of MPT64 protein that was used to immunize New Zealand white rabbits after purification and validation.Peripheral blood mononuclear cells(PBMCs)were isolated from the rabbits,and antigen-specific B cells expressing antibodies were sorted using single B-cell flow cytometry.mRNA in B cells was reverse-transcribed into cDNA,and paired antibody heavy-and light-chain sequences were amplified via nested PCR.Expression vectors were constructed,and recombinant antibodies were produced in Expi293F cells.Fluorescent immunochromatography was employed to screen for matched antibody pairs.The selected antibodies were used to establish a rapid detection method based on red microsphere immunochromatography.Results Ten high-affinity monoclonal antibodies against MPT64 were generated.Two antibody pairs were selected for MPT64 immunodetection that reached a sensitivity of 0.0125 ng/mL.Conclusion High-affinity rabbit monoclonal antibodies against MPT64 are obtained via single B-cell technology,and a rapid red microspheres-based immunodetection method is established,enabling highly sensitive detection of Mycobacterium tuberculosis MPT64 protein.

Objective To observe changes of white matter function in adolescent smoker(AS)with resting-state functional MRI(rs-fMRI)fractional amplitude of low-frequency fluctuation(fALFF)technique.Methods Forty-five adolescents(AS group)and 45 control subjects(control group)were prospectively enrolled,and brain rs-fMRI were acquired.Brain regions with fALFF being different between groups were observed,and the correlations with clinical indicators were analyzed.Results Compared with that in control group,fALFF of the right superior longitudinal fasciculus significantly elevated in AS group(FDR correct Q＜0.05),in which the peak of the cluster was positively correlated with score of Fagerstr?m test for nicotine dependence(FTND)(r=0.294,P=0.049).Conclusion White matter function changed in AS,presenting as significantly increased fALFF in right superior longitudinal fasciculus,which was positively correlated with nicotine dependence.

Objective To observe changes of white matter function in adolescent smoker(AS)with resting-state functional MRI(rs-fMRI)fractional amplitude of low-frequency fluctuation(fALFF)technique.Methods Forty-five adolescents(AS group)and 45 control subjects(control group)were prospectively enrolled,and brain rs-fMRI were acquired.Brain regions with fALFF being different between groups were observed,and the correlations with clinical indicators were analyzed.Results Compared with that in control group,fALFF of the right superior longitudinal fasciculus significantly elevated in AS group(FDR correct Q＜0.05),in which the peak of the cluster was positively correlated with score of Fagerstr?m test for nicotine dependence(FTND)(r=0.294,P=0.049).Conclusion White matter function changed in AS,presenting as significantly increased fALFF in right superior longitudinal fasciculus,which was positively correlated with nicotine dependence.

Objective:To explore the intervention effect of Jianpifuwei granule(JPFWG)on gastric mucosal injury in rats with precancerous lesions of gastric cancer(PLGC)by regulating the IL-6/JAK/STAT3 signaling pathway and its mechanism.Methods:The PLGC model rats were established by MNNG combined with composite factors,and randomly divided into 6 groups(20 rats/group):the blank group(untreated),model group(treated with normal saline),Weimeisu group(0.05 g/mL Weimeisu),JPFWG low-dose group(JPFWG-L,0.088 g/mL),JPFWG medium-dose group(JPFWG-M,0.176 g/mL)and JPFWG high-dose group(JPFWG-H,0.351 g/mL).After being treated with Weimeisu and different doses of JPFWG for 12 weeks,the animals were anesthetized and sacrificed,and the gastric tissues were collected.The pathological changes of gastric mucosa were observed by H-E staining,and the expression levels of IL-6-mediated JAK/STAT3 signaling pathway factors(including IL-6,JAK and STAT3)and the mRNA and proteins of their downstream target genes c-Myc and cyclin D1 in gastric mucosal tissues were detected by immunohistochemistry,qPCR and WB.Results:Compared with that in the model group,the gastric mucosal inflammatory cell infiltration was reduced in the Weimeisu group and the low,medium,and high dose groups of JPFWG;pathological condition was improved,most obviously in the high dose group of JPFWG;The protein expressions of IL-6,JAK1 and STAT3 decreased significantly(P<0.05 or P<0.01).The expressions of mRNA and proteins of c-Myc and cyclin D1 in the gastric mucosal tissues of the rats in the Weimeisu group and JPFWG-H group decreased significantly(P<0.05 or P<0.01).Conclusion:JPFWG can improve the histopathological changes of gastric mucosa in rats with PLGC,and its mechanism may be down-regulating the expressions of c-Myc and cyclin D1 through regulating IL-6/JAK/STAT3 signal pathway,thus blocking inflammation-cancer transformation process.

Objective To observe the effects of compound Dihuang Granules on striatum PINK1,Parkin and α-syn in rats with Parkinson disease yin-deficiency and wind movement syndrome;To explore the mechanism of compound Dihuang Granules in the treatment of Parkinson disease yin-deficiency and wind movement syndrome.Methods 6-hydroxydopamine was injected into the substantia nigra of the midbrain to prepare the model of PD yin-deficiency and wind movement syndrome.Totally 65 model rats were randomly divided into model group,Madopar(150 mg/kg)group,and TCM low-,medium-,and high-dosage(1.75,3.5,7 g/kg)groups.Another 26 rats were divided into normal control group and sham-operation group,with 13 rats in each group.Each administration group was given corresponding medication solution by gavage,the normal control group,sham-operation group and the model group were given equivalent volume of normal saline,once a day for 28 consecutive days.The behavioral changes of rats were detected,ELISA and biochemical assay kits were used to detect ATP content and COX Ⅳactivity in striatum,HE staining was used to observe the morphological changes of the striatum,immunohistochemistry staining,Western blot and RT-qPCR were used to detect the expression of PINK1,Parkin,α-syn protein and mRNA in striatal tissue.Results Compared with the normal control group and the sham-operation group,the rats in the model group showed increased number of rotations and pole climbing time,decreased swimming score(P<0.01),the ATP content and COX Ⅳ activity in striatum tissue decreased(P<0.01),with disordered arrangement and reduced number of neurons,cell swelling,partial nuclear shrinkage,formation of vacuoles,and blurred boundaries,the expressions of PINK1,Parkin protein and mRNA in striatal tissue decreased(P<0.01),and the expressions of α-syn protein and mRNA increased(P<0.01).Compared with the model group,the number of rotations and pole climbing time of rats in each medication group decreased,swimming scores increased(P<0.01),ATP content and COX Ⅳ activity in striatal tissue increased(P<0.05,P<0.01),the number of neurons increased,the arrangement tended to be neat,and the morphology improved,the expressions of PINK1,Parkin protein and mRNA in striatal tissue increased(P<0.05,P<0.01),the expressions of α-syn protein and mRNA decreased(P<0.05,P<0.01).The improvement was most significant in the Medopar group and TCM high-dosage group(P<0.05,P<0.01),and there was no significant difference between the two groups(P>0.05),and TCM groups showed dosage dependence(P<0.05,P<0.01).Conclusion Compound Dihuang Granules can improve the behavioral symptoms of Parkinson disease yin-deficiency and wind movement syndrome,possibly by regulating the expressions of PINK1,Parkin and α-syn in the striatum tissue,thereby alleviating mitochondrial dysfunction,protecting dopaminergic neurons,and playing a role in treating Parkinson disease.

OBJECTIVE To explore the molecular mechanism of Compound Dihuang Granule in treating Parkinson's disease(PD)with yin-deficiency and dynamic wind syndrome by regulating the changes of ubiquitin-proteasome system-related proteins.METHODS SPF male SD rats were randomly selected as normal control group and sham operation group,13 rats in each group.The remaining rats were treated with 6-hydroxydopamine lateral brain injection to damage the substantia nigra to establish PD yin-deficien-cy and dynamic wind syndrome rat model.The established rats were randomly divided into model group,madopar group(150 mg·kg-1),low,medium and high dose groups of Compound Dihuang Granule(1.75,3.5,7 g·kg-1),13 rats in each group.Each group was given drugs by intragastric gavage for 28 d.The general conditions and neurobehavioral manifestations of the rats were observed;the expression of ubiquitin(UB),ubiquitin activating enzyme(UBE1),ubiquitin conjugating enzyme(UBE2A)and ubiquitin carboxyl terminal hydrolase(UCH-L1)positive cells,protein and mRNA expression levels in the damaged lateral stria-tum of the brain of rats were detected by immunohistochemistry,Western blot and qPCR methods;changes of dopamine(DA)content in the damaged lateral striatum of the brain of rats were detected by ELISA.RESULTS There was no significant difference in general conditions between the normal control group and the sham operation group,and there was no significant difference in the number of ro-tations,suspension time and number of moving grids.Compared to the sham-op group,the model group rats showed poor spirit,irrita-bility,limb tremor,slow movement,dark yellow fur,less food intake,reduced body weight;the modeling increased number of rota-tions,decreased hanging time and number of moving grids(P<0.01),significantly decreased expression of UB,UBE1,UBE2A,and UCH-L1 positive cells,mRNA and protein expression levels in the damaged lateral striatum(P<0.01),and significantly decreased DA content(P<0.01).Compared with the model group,the general condition of the rats in the madopar group and Compound Di-huang Granule groups was improved,the number of rotations was reduced,the hanging time and number of moving grids were increased(P<0.05,P<0.01),the expression of UB,UBE1,UBE2A,and UCH-L1 positive cells,mRNA and protein expression levels were increased(P<0.05,P<0.01),and the DA content was increased(P<0.01);behavioral tests and the expression levels of various in-dicators were most significantly improved in the madopar group and the high-dose Compound Dihuang Granule group(P<0.05,P<0.01),and there was no statistical significance between the two groups.CONCLUSION Compound Dihuang Granule may play a role in treating PD yin-deficiency and dynamic wind syndrome by regulating the expression of ubiquitin-proteasome system-related proteins,reducing abnormal protein aggregation,and thus alleviating DA content deficiency,and the high-dose Compound Dihuang Granule has the best effect.

Objective:To investigate the efficacy of 3D-printed quantitative bone implants assisting second-stage Masquelet technique for the treatment of long-segment bone defect following Gustilo type IIIB and IIIC tibial fractures.Methods:A retrospective case series analysis was made on 26 patients with long-segment bone defect following Gustilo type IIIB and IIIC tibial fractures treated in Wuxi Ninth People′s Hospital from July 2015 to December 2020, including 20 males and 6 females; aged 19-63 years [(46.5±4.5)years]. Gustilo classification was type IIIB in 23 patients and type IIIC in 3. In the first stage, all patients had thoroughly emergent debridement, removal of all free bone pieces, restoration of the length and force line plus externally fixion, and vacuum sealing drainage (VSD) of the residual wound. After 2-7 days, the external fixation was removed and replaced by internal fixation, with the bone cement filling in the defect area and the free flap covering the wound. The length of tibial bone defect was 5-14 cm [(6.3±0.4)cm], and the tibial defect volume was 12.2-73.1 cm 3 [(33.6±9.2)cm 3]. In the second stage (6-19 weeks after injury), the bone cement was removed, followed by autologous bone grafting. Prior to bone grafting, digital technology was used to accurately calculate the bone defect volume, and an equal volume of bone harvesting area was designe to produce the 3D printed osteotomy template. Bone grafting was conducted after bone removal according to the osteotomy template during operation. The success rate of one-time iliac bone extraction, bone harvesting time, and bleeding volume were recorded. Pain in the bone extraction area was evaluated by visual analogue score (VAS) at 1 day and 1 month after operation and at the last follow-up. Wound healing, complications, and bone healing were observed. Life quality was evaluated by health survey brief form (SF-36) including scores of physical component summary (PCS) and mental component summary (MCS) before bone grafting and at the last follow-up. Results:All the patients were followed up for 13-53 months [(32.3±12.5)months]. One-time iliac bone extraction was successful in all the patients. Bone harvesting time was 15-30 minutes [(21.0±2.5)minutes]. The bleeding volume was 50-120 ml [(62.3±29.0)ml]. The VAS was 1-4 points [(1.2±0.9)points] at 1 day after operation, higher than these (0.0±0.0)points at 1 month after operation and at the last follow-up (all P<0.01). Totally, 25 patients obtained wound healing after operation, except for 1 patient with superficial wound infection after bone grafting that was healed by dressing change. There was 1 patient with bone infection after 3 months of bone grafting that was healed by repeated surgery with Masquelet technique in the first and second stage. Besides, 2 patients had symptoms of cutaneous nerve injury in the iliac donor area. The time of bone healing was 4-7 months [(5.8±0.8)months]. The scores of PCS and MCS in SF-36 at the last follow-up were (73.6±12.8)points and (83.6±13.2)points, significantly higher than those before bone grafting [(46.8±0.5)points, (60.7±2.0)points] (all P<0.01). Conclusion:Second-stage Masquelet technique with 3D printed quantitative bone implants for the treatment of long-segment bone defect following Gustilo type IIIB and IIIC tibial fractures is associated with shortened bone harvesting time, attenuated pain, reduced complications, accelerated bone healing and improved function.

Objective:To analyze the clinical phenotypes and pathogenic gene of a Han Chinese family with enhanced S-cone syndrome (ESCS).Methods:The method of pedigree investigation was adopted.A suspected ESCS Han Chinese family including 8 members of 3 generations was recruited in Henan Eye Hospital from June to September 2021.There was one patient in the family.A thorough ophthalmic examination of the proband was carried out to evaluate the phenotypes, including visual acuity, degree of strabismus, anterior segment and fundus, autofluorescence imaging, fluorescein fundus angiography, full-field electroretinogram (ERG), multifocal ERG, optical coherence tomography.DNA was extracted from peripheral blood samples from the proband and family members.The pathogenic gene and variation were screened by whole exome sequencing (WES).The variation and co-segregation were verified by Sanger sequencing.The deleteriousness of the variation was analyzed by SIFT, Polyphen2 and MutationTaster.The pathogenicity of the variation was evaluated in accordance with the American College of Medical Genetics and Genomics (ACMG) Standards and Guidelines.The analysis of amino acid sequence conservation was performed by SIFT.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Henan Eye Hospital (No.HNEECKY-2017[6]).Written informed consent was obtained from each subject.Results:This pedigree was consistent with autosomal recessive inheritance.The proband had clinical features such as night blindness, hyperopia, accommodative esotropia, peripheral retinal pigmentation, retinoschisis, and photopic ERG responses dominated by large-amplitude waves.Variations including a compound heterozygous variation, c.671C>T: p.S224L on exon 5 and c. 955G>A: p.E319K on exon 6 of NR2E3 were identified by WES.The variations were confirmed to be consistent with co-segregation.The both loci were missense variations, the variation frequency of which was 0 in the East Asian population via the gnomAD database.The variations were predicted to be deleterious by SIFT, Polyphen2 and MutationTaster.The c.671C>T variation was recorded with unknown significance in ClinVar database, and the c.955G>A variation was an unreported new locus.According to the ACMG Standards and Guidelines, the both variations were labeled as with uncertain clinical significance, and the corresponding amino acid sequences were highly conservative across multiple species. Conclusions:This family has the clinical characteristics of ESCS and meets the genetic diagnosis criteria.Two novel variations in NR2E3 gene, c.671C>T: p.S224L and 955G>A: p.E319K, are found.

Objective:To explore the advantages, disadvantages, and precautions of clinically applying four types of non-trunk vessels as recipient vessels in the free flap transplantation for repairing peri-knee wounds.Methods:A retrospective analysis of the clinical data was conducted of 23 patients (12 males and 11 females) with peri-knee skin and soft tissue defects who underwent free anterolateral thigh flaps or free latissimus dorsi flaps. The patients were admitted to the Department of Traumatic Orthopedics, Wuxi 9th People’s Hospital, from January 2015 to December 2019. The patients were aged 20-72 years (mean 41.9). The wound size with vital tissue exposure was 18.0 cm×5.0 cm-42.0 cm×9.0 cm. Preoperative color Doppler and computerized tomography angiography techniques were used to assist in positioning and to measure the recipient vessels (including the descending genicular vessel, descending branch of the lateral femoral circumflex vessel, the medial sural vessel, and the medial inferior genicular vessel) and blood vessels in the donor area (including descending branches of lateral femoral circumflex vessels and thoracic dorsal vessels). The caliber was measured and verified by a soft ruler with a scale intraoperative. The measured values of the caliber were recorded in the preoperative and intraoperative. The color and texture of the two flaps and the healing of the donor sites were observed postoperatively. The conformance ratio of preoperative and intraoperative measured values of vessels in the donor and recipient areas (except for the only case of the medial inferior genicular vessel) was compared. SPSS 26.0 software was used for data analysis. The measurement data were showed as Mean±SD, and the comparison results were analyzed by paired t-test. P<0.05 was considered statistically significant. Results:A total of 23 patients with skin and soft tissue defects around the knee were enrolled. The size of the tissue flap was 18.0 cm×5.0 cm-46.0 cm×9.0 cm. Twelve of 14 anterolateral thigh flaps anastomosed to the descending genicular vessel survived. The remaining two cases had a length of 6 cm and 4 cm necrosis at the distal flap, which was finally given skin-grafting and healed. One of the 12 survived flaps failed in limb salvage in Phase Ⅰ and was thus given flap reconstruction in Phase Ⅱ. Another case was given amputation due to serious infection of limbs, with incomplete ends of the survived flaps rotated and covered. In four cases anastomosed to the descending branch of the lateral circumflex femoral vessel, three anterolateral thigh flaps survived completely, and one distal latissimus dorsi flap had a length of 12 cm necrosis which was given debridement and Ilizarov bone transport for heal. Two anterolateral thigh flaps and two latissimus dorsi flaps anastomosed to the medial sural vessel survived completely, of which one anterolateral thigh flap had vein crisis which was later solved. The anterolateral thigh flap of 1 case anastomosed to medial inferior genicular vessels completely survived. The postoperative follow-up lasted 3-30 months with an average of 13.6 months. All the flaps have good color and textures with good incision heal at the donor site in Phase Ⅰ. There was no statistically significant difference in the preoperative and intraoperative measurement values of blood vessel caliber in the donor and recipient areas ( P>0.05). Conclusions:Four non-trunk peri-knee blood vessels can serve as recipient vessels of the free tissue flaps, and proper selection of the vessels can effectively improve the survival rate of the tissue flaps. The descending genicular vessel can serve as the recipient vessel for a priority, as with a superficial position, fixed dissection, simple positioning, and convenient intraoperative dissection.

Objective:To explore the advantages, disadvantages, and precautions of clinically applying four types of non-trunk vessels as recipient vessels in the free flap transplantation for repairing peri-knee wounds.Methods:A retrospective analysis of the clinical data was conducted of 23 patients (12 males and 11 females) with peri-knee skin and soft tissue defects who underwent free anterolateral thigh flaps or free latissimus dorsi flaps. The patients were admitted to the Department of Traumatic Orthopedics, Wuxi 9th People’s Hospital, from January 2015 to December 2019. The patients were aged 20-72 years (mean 41.9). The wound size with vital tissue exposure was 18.0 cm×5.0 cm-42.0 cm×9.0 cm. Preoperative color Doppler and computerized tomography angiography techniques were used to assist in positioning and to measure the recipient vessels (including the descending genicular vessel, descending branch of the lateral femoral circumflex vessel, the medial sural vessel, and the medial inferior genicular vessel) and blood vessels in the donor area (including descending branches of lateral femoral circumflex vessels and thoracic dorsal vessels). The caliber was measured and verified by a soft ruler with a scale intraoperative. The measured values of the caliber were recorded in the preoperative and intraoperative. The color and texture of the two flaps and the healing of the donor sites were observed postoperatively. The conformance ratio of preoperative and intraoperative measured values of vessels in the donor and recipient areas (except for the only case of the medial inferior genicular vessel) was compared. SPSS 26.0 software was used for data analysis. The measurement data were showed as Mean±SD, and the comparison results were analyzed by paired t-test. P<0.05 was considered statistically significant. Results:A total of 23 patients with skin and soft tissue defects around the knee were enrolled. The size of the tissue flap was 18.0 cm×5.0 cm-46.0 cm×9.0 cm. Twelve of 14 anterolateral thigh flaps anastomosed to the descending genicular vessel survived. The remaining two cases had a length of 6 cm and 4 cm necrosis at the distal flap, which was finally given skin-grafting and healed. One of the 12 survived flaps failed in limb salvage in Phase Ⅰ and was thus given flap reconstruction in Phase Ⅱ. Another case was given amputation due to serious infection of limbs, with incomplete ends of the survived flaps rotated and covered. In four cases anastomosed to the descending branch of the lateral circumflex femoral vessel, three anterolateral thigh flaps survived completely, and one distal latissimus dorsi flap had a length of 12 cm necrosis which was given debridement and Ilizarov bone transport for heal. Two anterolateral thigh flaps and two latissimus dorsi flaps anastomosed to the medial sural vessel survived completely, of which one anterolateral thigh flap had vein crisis which was later solved. The anterolateral thigh flap of 1 case anastomosed to medial inferior genicular vessels completely survived. The postoperative follow-up lasted 3-30 months with an average of 13.6 months. All the flaps have good color and textures with good incision heal at the donor site in Phase Ⅰ. There was no statistically significant difference in the preoperative and intraoperative measurement values of blood vessel caliber in the donor and recipient areas ( P>0.05). Conclusions:Four non-trunk peri-knee blood vessels can serve as recipient vessels of the free tissue flaps, and proper selection of the vessels can effectively improve the survival rate of the tissue flaps. The descending genicular vessel can serve as the recipient vessel for a priority, as with a superficial position, fixed dissection, simple positioning, and convenient intraoperative dissection.