[Use of the ETV6/RUNX1 probe to verify the performance of the fluorescence in situ hybridization probe before clinical detection].

Objective: To explore the standardized performance of a FISH probe before clinical detection. Methods: The probe sensitivity and specificity of ETV6/RUNX1 were analyzed via interphase and metaphase FISH in 20 discarded healthy bone marrow samples. The threshold system of the probe was established using an inverse beta distribution, and an interpretation standard was established. Finally, a parallel-controlled polymerase chain reaction detection study was conducted on 286 bone marrow samples from patients at our hospital. The clinical sensitivity, specificity, and diagnostic coincidence rate of ETV6/RUNX1 FISH detection were analyzed, and the diagnostic consistency of the two methods was analyzed by the kappa test. Results: The probe sensitivity and specificity of the ETV6/RUNX1 probe were 98.47% and 100%, respectively. When 50, 100, and 200 cells were counted, the typical positive signal pattern cutoffs were 5.81%, 2.95%, and 1.49%, respectively, and the atypical positive signal pattern cutoffs were 13.98%, 9.75%, and 6.26%, respectively. The clinical sensitivity of FISH was 96.1%, clinical specificity was 99.6%, diagnostic coincidence rate was 99.00%, diagnostic consistency test kappa value was 0.964, and P value was <0.001. Conclusion: For FISH probes without a national medical device registration certificate, standardized performance verification and methodology performance verification can be performed using laboratory developed test verification standards to ensure a reliable and accurate reference basis for clinical diagnosis and treatment.

[Splicing abnormalities caused by a novel mutation in the PHKA2 gene in children with glycogen storage disease type IX].

Objective: Glycogen storage disease type IX (GSD-IX) is a rare primary glucose metabolism abnormality caused by phosphorylase kinase deficiency and a series of pathogenic gene mutations. The clinical characteristics, gene analysis, and functional verification of a mutation in a child with hepatomegaly are summarized here to clarify the pathogenic cause of the disease. Methods: The clinical data of a child with GSD-IX was collected. Peripheral blood from the child and his parents was collected for genomic DNA extraction. The patient's gene diagnosis was performed by second-generation sequencing. The suspected mutations were verified by Sanger sequencing and bioinformatics analysis. The suspected splicing mutations were verified in vivo by RT-PCR and first-generation sequencing. Results: Hepatomegaly, transaminitis, and hypertriglyceridemia were present in children. Liver biopsy pathological examination results indicated glycogen storage disease. Gene sequencing revealed that the child had a c.285 + 2_285 + 5delTAGG hemizygous mutation in the PHKA2 gene. Sanger sequencing verification showed that the mother of the child was heterozygous and the father of the child was of the wild type. Software such as HSF3.1 and ESEfinder predicted that the gene mutation affected splicing. RT-PCR of peripheral blood from children and his mother confirmed that the mutation had caused the skipping of exon 3 during the constitutive splicing of the PHKA2 gene. Conclusion: The hemizygous mutation in the PHKA2 gene (c.285 + 2_285 + 5delTAGG) is the pathogenic cause of the patient's disease. The detection of the novel mutation site enriches the mutation spectrum of the PHKA2 gene and serves as a basis for the family's genetic counseling.

[Surgical treatment of the complex bicondylar tibial plateau fracture using a midline longitudinal incision].

Objective: To explore the outcomes after treatment of the complex bicondylar tibial plateau fracture through a midline longitudinal approach. Methods: A review of fifteen patients with an average age of (51.3±12.3) years old (range17-65yers;7 males, 8 females) surgically treated from October 2013 to Febuary 2018 were included. Sahatker Ⅴ in 10, Sahatker Ⅵ in 5; fractures of medial and lateral columns in 9, fractures of three columns in 6. All the patients were adopt a midline longitudinal approach combined with the posterior approach and bone grafting were conducted. Results: All cases were followed-up for (14.4±3.8) month, with an average of 12-24 month. All patients gained bone union during 12-16 weeks after operation, with an average of (15.2±1.3) weeks. There were significant differeces in both tibial plateau angle and posterior slope angle on radiography between preoperation and postoperation (P<0.05), there were no significant differeces in either tibial plateau angle or posterior slope angle on radiography between immediate postoperation and 12 months postoperation (P>0.05). At final follow-up,both the Lachman test and the Pivot-shift test were negative. All patients had complete knee extension, knee flexion angle 100°-135°, with an average of 117.7°±11.3°. The HSS (the Hospital for Special Surgery) score were 66-98, with an average of 85.1±9.3, six cases were excellent and seven cases were good, two cases was fair, the excellent and good rate was 86.7%. The Rasmussen radiological evaluationre were 9-18, with an average of 15.1±2.5, three cases were excellent and eleven cases were good, one cases was fair, the excellent and good rate was 93.3%. 1 patient had fat liquefactionof in antero incision, and got good outcomes after debridement dressing. Conclusion: The treatment of the complex bicondylar tibial plateau fracture through a midline longitudinal approach combined with the posterior approach can result in good exposure and satisfying knee function in short-term.

The appropriate hybrid surgical strategy in three-level cervical degenerative disc disease: a finite element analysis.

OBJECTIVE: The purpose of this FE study was to analyze the biomechanical characteristics of different HS strategies used in the treatment of three-level CDDD (one-level CDA and two-level ACDF). METHODS: We validated the FE model of an intact cervical spine established by transferring the data, collected by 3D CT scan, to the FE software ABAQUS and comparing these data with the data from published studies. Then, the FE model of hybrid surgery was reconstructed to analyze the range of motion (ROM), facet joint force, and stress distribution on an ultrahigh molecular weight polyethylene (UHMWPE) core. RESULTS: The current cervical FE model was able to measure the biomechanical changes in a follow-up hybrid surgery simulation. The total ROM of the cervical HS models was substantially decreased compared with the total ROM of the intact group, and the M2 (C3/4 ACDF, C4/5 CDA, and C5/6 ACDF) model had the closest total ROM to the intact group, but the facet joint force adjacent to the treatment levels showed very little difference among them. The stress distribution showed noticeable similarity: two flanks were observed in the center core, but the inlay of M2 was more vulnerable. CONCLUSIONS: Through the comparison of ROM, the facet joint force after CDA, and the stress distribution of the prosthesis, we find that M2 model has a better theoretical outcome, especially in preserving the maximum total ROM.

[Cytogenetic test and clinical study on cryptic acute promyelocytic leukemia with ins (15; 17)].

Objective: To investigate the genetic screening methods for cryptic acute promyelocytic leukemia (APL) to further explore its clinical prognosis. Methods: From June 2016 to November 2018, we collected 373 newly diagnosed APL cases. The patients were retrospected by the results of PML-RARα detections both by RT-PCR and i-FISH, those who harbored positive PML-RARα detection by RT-PCR and negative by i-FISH were chosen. Metaphase FISH and Sanger sequencing were further performed to verify these results. Results: A total of 7 cryptic APL cases were discovered. These cases had tiny fragment of RARα inserted into PML in chromosome 15, formed ins (15;17) . The 7 cryptic APL cases had no PML-RARα gene subtype specificity, involving 5 cases in L subtype, 1 case in S subtype and 1 case in V subtype respectively. After the treatment of retinoic acid and arsenic or anthracyclines, 6 cases achieved complete remission, 1 case died of intracranial hemorrhage on the 6th day of therapy. Conclusion: The size and covering position of PML-RARα probe should be taken into account when PML-RARα was performed by FISH on APL patients. Furthermore, combination with Metaphase FISH could improve the recognition of cryptic APL. There were no differences between the cryptic and common APL patients in terms of clinical features and treatment choices. Cryptic APL patients also had a good response to the therapy of retinoic acid and arsenic or anthracyclines.

[Cardiovascular magnetic resonance imaging characteristics and influence factors of aortic insufficiency patients with myocardial fibrosis].

Objective: To investigate the cardiovascular magnetic resonance (CMR) imaging characteristics and influence factors of aortic insufficiency (AI) patients with myocardial fibrosis. Method: This retrospective study included 59 AI patients who received CMR and transthoracic echocardiography (TTE) examinations from June 2011 to February 2015. AI patients were divided into 2 groups: bicuspid aortic valve (BAV) group (n=30) and non-BAV group (n=29). Patients were also divided into late gadolinium enhancement (LGE) group (n=27) and non-LGE group (n=32). The baseline clinical characteristics were collected through electronic medical records. Hemodynamic parameters such as grade of AI, cardiac functional parameters and LGE mass fraction (LGE%) were measured by CMR post-processing analysis. Kappa test was used to assess the consistency of AI severity between CMR and TTE, and the multivariate logistic regression analysis was performed to evaluate influence factors of myocardial fibrosis in AI patients. Results: (1) 56 (94.9%) patients were male, and the mean age was (44.2±11.0) years old. There was no significant difference in age and gender, hypertension, hyperlipidemia, alcoholic consumption between BAV and non-BAV group (all P>0.05). There were a higher proportion of smoking history (P=0.008), a lower body mass index (BMI) (P=0.020) in the LGE group than in the non-LGE group. (2) The accuracy of CMR in diagnosis of BAV was 96.7% (29/30) compared to the gold standard. In the BAV group, 20 patients (66.7%) were with fusion of left and right cusp (L-R), 5 patients (16.7%) were with fusion of right and noncoronary cusp (R-N), 5 patients (16.7%) were with fusion of left and noncoronary cusp (L-N); patients with BAV had larger left ventricular end diastolic volume index (LVEDVi), left ventricular end systolic volume index (LVESVi), higher proportion of LGE and lower left ventricular ejection fraction (LVEF) than those in non-BAV group (all P<0.05). There were 19 patients with BAV in the LGE group, the cases of L-R, R-N, L-N were 10 (52.6%), 5 (26.3%), and 4 (21.1%), respectively. In the non-LGE group, patients with BAV of L-R, R-N, L-N were 10 (90.9%), 0, and 1 (9.1%), respectively. Patients with LGE had lower body surface area (BSA), LVEF and larger LVEDVi, LVESVi, left ventricular mass index (LVMi) and higher proportion of BAV compared patients without LGE. In addition, the proportion of moderate and severe AI patients was significantly higher in BAV group than in non-BAV group (P=0.009). (3) The consistency of CMR and TTE in evaluating the severity of AI patients: the agreement between TTE and CMR regarding AI severity was satisfactory (kappa value was 0.624, 95%CI 0.402-0.831, P<0.001). (4) The linear regression analysis demonstrated a negative correlation between LVEF and LGE% in BAV and non-BAV group (P<0.001). The multivariate logistic regression analysis showed that the presence of BAV was an independent risk factor of left ventricucar fibrosis (OR=5.050, 95%CI 1.220-20.908, P=0.025) after adjustment for LVEF, hypertension, LVEDVi and LVMi. Conclusion: Multi-parametric CMR provides a satisfactory noninvasive tool for estimation of myocardial fibrosis and ventricular remodeling in patients with AI, and BAV is an independent risk factor for myocardial fibrosis in patients with AI.

[The necessity of artificial kidney seeper in the ultrasonography-guided percutaneous nephrolithotomy: a randomized controlled study].

Objective: To investigate the necessity of artificial kidney seeper which made through inserting a ureteral tube in the ureter to the ultrasonography-guided percutaneous nephrolithotomy (PCNL). Methods: This was a randomized prospective study. Patients who conformed to the inclusion and exclusion criteria were enrolled at Department of Urology, Frist Affiliated Hospital of Wenzhou Medical University from January 2016 to May 2017. Totally 291 patients were included in the study. Patients were randomly assigned into two groups (artificial kidney seeper group and non-artificial kidney seeper group) in different kidney seeper level (5 to <10 mm, 10 to 20 mm) respectively. The artificial kidney seeper group was inserted by a ureteral cathedral, then were underwent the ultrasonography-guided PCNL in prone position. The non-artificial kidney seeper group were underwent the ultrasonography-guided PCNL in prone position directly. The t test, χ(2) test, repeated measure analysis was used to data measurement, respectively. Results: Four patients who diagnosed pyonephrosis were excluded. On the 5 to <10 mm level, fever rate (14.6% vs. 4.8%, χ(2)=5.07, P=0.03), operation time ((65.7±9.9)min vs. (50.3±7.4)min, t=11.47, P=0.00), cost ((18 327±903) yuan vs. (14 583±784) yuan, t=24.50, P=0.00) about artificial kidney seeper group and non-artificial kidney seeper group had statistical differences. And on the 10 to 20 mm level, fever rate (14.5% vs. 3.39%, χ(2)=4.53, P=0.03), operation time ((66.0±9.9)min vs. (52.4±8.9)min, t=8.30, P=0.00), cost ((16 548±537) yuan vs. (13 102±629) yuan, t=32.10, P=0.00) about artificial kidney seeper group and non-artificial kidney seeper group had statistical differences. And there were no statistical differences in the success rate of puncturing, the failures of the treatment to the stone pieces falling into the ureter and clearance rate of the stone and so on. In this study, 2 cases recovered after received transfusion and digital subtraction angiography artery embolization treatments; D-J tube was indwelled into 4 cases guiding by super smooth thread; 4 cases were finished the surgeries with the help of ureteroscopy, because the stone pieces fell into the ureter during the surgeries. And there was no patients developing septic shock, adjacent viscera injury or other serious complications. Conclusion: For seasoned doctors, there is no necessity of regularly artificial kidney seeper for PCNL when the pre-operation seeper ranging from 5 to 20 mm.

Activated natural killer cells accelerate liver damage in patients with chronic hepatitis B virus infection.

Emerging evidence indicates that natural killer (NK) cells may contribute to liver injury in patients with hepatitis B virus (HBV) infection. Because HBV infection progresses through various disease phases, the cytolytic profiles of peripheral and intrahepatic NK cells in HBV-infected patients remain to be defined. In this study, we comprehensively characterized intrahepatic and peripheral NK cells in a cohort of HBV-infected individuals, and investigated their impact on liver pathogenesis during chronic HBV infection. The study population included 34 immune-clearance (IC) patients, 36 immune-tolerant (IT) carriers and 10 healthy subjects. We found that the activity of peripheral NK cells from IC patients was functionally elevated compared to IT carriers and controls, and NK cell activation was indicated by an increased expression of CD69, CD107a, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Further analysis showed that the increased activity of both peripheral and hepatic NK cells was correlated positively with liver injury, which was assessed by serum alanine aminotransferase levels (ALT) and the liver histological activity index (HAI). Interestingly, the frequency of peripheral NK cells was reduced in IC patients (especially those with higher HAI scores of 3-4), but there was a concomitant increase in hepatic NK cells. The functionally activated NK cells are enriched preferentially in the livers of IC patients and skew towards cytolytic activity that accelerates liver injury in chronic hepatitis B (CHB) patients.

Structure and expression of the nicotinic acetylcholine receptor beta subunit of Xenopus laevis.

A cDNA encoding the beta subunit of the Xenopus muscle nicotinic acetylcholine receptor (AChR) was cloned from an embryonic Xenopus cDNA library. The predicted mature polypeptide has 469 amino acids and four membrane spanning regions corresponding to the M1-M4 regions identified in other AChR subunit clones. The polypeptide bears greater homology to beta subunits of Torpedo and mouse than to alpha, gamma or delta subunits of Xenopus. The earliest beta subunit transcripts were detected by RNase protection assays at the neural plate stage of development (stage 14) and the level of transcripts, as a fraction of total RNA, continued to increase through the age of hatching (stages 34-36). Co-injection of Xenopus alpha, beta, gamma and delta cRNAs into Xenopus oocytes led to expression of functional AChRs. Micromolar concentrations of ACh activated depolarizing AChR currents which reversed at -5 mV and were blocked by alpha bungarotoxin. Injection of alpha, gamma and delta subunits alone did not yield detectable ACh responses. With the cloning of the Xenopus beta subunit, structure/function relations of AChRs can now be studied using receptors composed entirely of Xenopus subunits.

A study of molecular epidemiology of adenovirus of types 3 and 7 on infant pneumonia in northern China.

This paper analysed 102 strains of adenovirus types 3(Ad3) and 7(Ad7) causing infant pneumonia from 1976 to 1988 in northern China. Two genotypes of Ad7, 7b and 7d, were identified by using restriction endonucleases, BamHI, BcLI, BgLI, SmaI, XbaI and HindIII. 3 genotypes of Ad3, 3I, 3II and 3III, were identified by using BgLII and BamHI. Of 56 Ad7 strains, 34 were 7b (76.8%) spread over last 10 years; 13 7d(23.2%) occurred from 1982, together with 7b. Of 46 Ad3 strains, 42 3I(91.3%) spread over the past 12 years. 3II and 3III scattered all over these years. Ad3I and Ad7b were the dominant genotypes. The results indicated that Ad7d tended to increase with time from 1982. It is possible that Ad7d will become dominant genotype and replace Ad7b.