BACKGROUND:A unified standard for cervical pedicle screw placement does not currently exist;therefore, it is difficult to quantitatively evaluate the clinical effects of the technique. Digital navigation can provide a reference for accurate and safe location, orientation, and placement of cervical pedicle screws. OBJECTIVE:To investigate whether digital navigation can greatly increase the accuracy and safety of cervical pedicle screw placement. METHODS:This was a prospective, single-center, randomized control ed, open-label trial. Seventy-six patients with cervical spine fracture scheduled to receive treatment in the Department of Orthopedics, Affiliated Hospital of Nantong University, China were randomly divided into three groups to undergo cervical pedicle screw internal fixation. Patients in the cervical lamina partial excision group (n=26, 160 screws) underwent partial cervical lamina excision and cervical pedicle screw internal fixation;those in the pipeline-dredge discharge group (n=27, 156 screws) underwent pipeline-dredge discharge and cervical pedicle screw internal fixation;and those in the digital navigation group (n=23, 162 screws) underwent digital navigation-assisted cervical pedicle placement. Al patients were evaluated at 12 and 36 months. The primary outcome was the percentage of screws graded I when evaluating the penetration degree of the cervical pedicle screws, which evaluates the accuracy of screw placement, 12 months after internal fixation. Secondary outcomes included:(1) the percentage of screws graded I when evaluating the penetration degree of cervical pedicle screws 36 months after internal fixation;(2) bony fusion rate of the atlantoaxial joint, used to evaluate fracture healing, 12 and 36 months after internal fixation;(3) Visual Analogue Scale spine score, used to evaluate cervical neck pain, prior to and 12 and 36 months after internal fixation;(4) American Spinal Injury Association Classification, used to evaluate improvement in neurological function, prior to and 12 and 36 months after internal fixation;and (5) adverse events, used to evaluate the safety of each pedicle screw implantation method, 12 and 36 months after internal fixation. This trial protocol was approved by Medical Ethics Committee, Affiliated Hospital of Nantong University, China, and was performed in accordance with the guidelines of the Declaration of Helsinki, formulated by the World Medical Association. Signed informed consent regarding trial procedure and treatment was obtained from each patient. DISCUSSION:This trial protocol compared the effects of three cervical pedicle screw internal fixation methods for the treatment of cervical spine fracture, and investigated and compared the accuracy and safety of digital navigation-assisted cervical pedicle screw placement with partial cervical lamina excision and pipeline-dredge discharge. We hoped to provide quantitative evidence for the clinical use of digital navigation in orthopedics, especial y in cervical pedicle screw placement. TRIAL REGISTRATION:This trial was registered at ClinicalTrials.gov identifier:NCT02880839 on 19 August 2016.

Objective To analyze the prognosis of T1-T2 stage breast cancer with 1 -3 positive axillary nodes after mastectomy, and to explore a subgroup of patients who could benefit from adjuvant radiotherapy. Methods In the retrospective study of 412 eligible patients, survival analysis was performed using the Kaplan-Meier method. Univariate and multivariate analyses were performed using the Log-rank method and Cox regression analysis, respectively. Results The follow-up rate was 98. 7%. 215 and 41patients were followed up for 5 and 10 years,respectively. The 5-and 10-year overall survival (OS) rate was 90. 0% and 81.3%, respectively. The 5-and 10-year locoregional recurrence (LRR) rate was 10. 7% and 18. 6%, respectively. In univariate analysis, T2 statging, more than one positive node, hormone receptornegative ( ER&PR-negative), ratio of positive lymph nodes (LNR) ＞ 25%, Her-2 positive, no hormonal therapy were associated with a significantly higher rate of LRR. T2 staging, more than one positive node,hormone receptor-negative were the risk factors for LRR with statistical significance in the multivariate analysis. Basing on these 3 risk factors, the high-risk group (with 2 -3 factors) had a 10-year LRR rate of 36. 9% compared with 3.9% in the low-risk group ( with 0 - 1 factors;x2 =20. 64,P =0. 000). The 5-year and 10-year distant metastasis (DM) rate was 12.9% and 24. 5%, respectively. LRR, and LNR ＞25%were statistically significant predictors of DM in the multivariate analysis. The 5-year DM rate for patients with LRR was 36. 6% compared with 9. 7% without LRR (x2 = 16. 34,P =0. 000). The 5-year OS rate for patients with LRR was 69. 9% compared with 92. 9% without LRR ( x2 = 20. 79, P = 0. 000). LRR was associated with a higher risk of distant metastasis and worse survival. Conclusions LRR after mastectomy has a significant impact on the outcome of patients with T1 -T2 breast cancer and 1 - 3 positive axillary nodes.Patients who have 2 -3 risk factors might benefit from radiotherapy.

Objective To evaluate the treatment of current status and prognosis in childhood APL with APL2008 ,which was administrated since 2008 in our center .Methods A total of 43 children with newly diagnosed APL between 2008 to 2014 were studied retrospectively .Treatment options and current status were summarized from 28 patients who received APL2008 therapy . Results Studied 43 patients were at median age of 8 years and 4 months ,with 28 boys and 15 girls .The main clinical manifestations were infection ,anemia ,bleeding ,fever ,hepatomegaly ,splenomegaly and lymphadenopathy .The proportions of low ,intermediate and high risk groups were 27 .9% ,48 .8% and 23 .3% ,respectively .Eleven cases could be diagnosed as DIC .Bone marrow morphology showed abnormal elevation of promyelocyte .37 patients had distinctive immunophenotype such as frequent expression of CD33 , CD117 and MPO .PML/RARαfusion gene positive rate was 100% in 43 children and cytogenetic analysis were positive in 37 cases , of which specific genetic lesion in APL cells with t (15 ;17)(q22 ;q12) was found in 28 cases ,and karyotypes was found in 9 cases as infrequent chromosomal abnormalities .In 43 patients ,4 cases were early dead from intracranial hemorrhage at early stage ,and 11 cases were given up early .There were only 2 cases dead ,2 cases relapsed and 1 case lost among 28 APL children ,which enabled ef‐ficacy analysis possible .96 .4% of these 28 cases achieved HCR .The 2 year Kaplan Meier estimates of OS and EFS were 85 .9% ± 7 .6% and 80 .4% ± 8 .8% .But OS and EFS would be 94 .7% ± 5 .1% and 88 .9% ± 7 .4% if 3 patients who had non standard treat‐ment were excluded .Conclusion Childhood APL were characterized by anemia ,bleeding ,fever and infiltration .APL′s coincidence rate between PML/RARa fusion gene and morphology ,immunology and cytogenetics were 95 .3% ,90 .2% and 86 .5% ,respective‐ly .APL2008 significantly improved the prognosis of APL .

Objective To investigate the differentiation of neuroendocrine component (NEC) in colorectal adenocarcinoma in relation to its significance by comparing the outcome between patients with or without NEC.Methods The paraffin sections from patients with pathologically confirmed colorectal adenocarcinoma were retrospectively collected and screened for those with NEC by morphological examination and immunohistochemistry with neuroendocrine markers.Control patients (n=54) without NEC were selected from colorectal cancer database and 2: 1 matched on the basis of clinical features with NEC positive patients (n=27).Relative analysis was performed between two groups.Results With a median follow-up of 72 months,the 5-year disease free survival was 58.0% (16/27) in NEC positive group and 79.1% (43/54) in control group (P=0.036).Similarly,the 5-year cancer-specific overall survival was significantly lower in NEC positive group than in control group (58.3% versus 81.1%,P=0.037).Cox regression showed that the 5-year cumulative risks of disease recurrence and cancer-caused death in NEC positive patients were 2.38 and 2.41 times higher than those in control patients,respectively.Conclusions NEC appears to bear a poor prognosis in patients with colorectal adenocarcinoma.

Objective To investigate the role of radiotherapy (RT) and prognostic factors in the combined modality treatment (CMT) of patients with stage ⅠE-ⅡE extranodal nasal type NK/T-cell lym-phoma. Methods From Dec. 1990 to Dec. 2006,177 patients who were diagnosed and treated in our hos-pital were retrospectively analyzed,induding 37 received chemotherapy (CT) alone ( median 4 cycles), 128 received CT (median 3 cycles) followed by RT (median 52 Gy) ,6 received RT alone (median 58 Gy) and 6 received RT ( median 54 Gy) followed by CT ( median 5 cycles). Results The overall response ( CR + PR) rate after initial CT was 60.8% compared with 83.8% after RT ( x2 = 28.63, P < 0.01 ). The 5-year overall survival (OS) and progress-free survival (PFS) rates were 46.2% and 36.8% ,respectively. The lo-cal control rates were 80.9% for RT ( alone or with CMT) and 50.0% for CT alone (x2 = 14.39, P < 0.01 ), and corresponding 5-year OS and PFS were 53.4% vs. 18.3 % ( x2 = 23.38, P < 0.01 ) and 45.0% vs. 10.9% (x2 =23.46,P <0.01 ),respectively. Compared with CT alone,the following definitive RT for patients who achieved response or not after initial CT significantly improved the local control [83.5%, 76.2% vs. 50.0% (x2 = 14.13,P <0.01;x2 =5.78,P <0.01)] and 5-year OS[56.2%,48.6% vs. 18.3%(x2 =28.87,P <0. 05;x2 =4.80,P <0.05)]. Concinsions Compared with CT alone, RT a-chieves better tumor response, local control and survival of patients not only with tumor response but also with local progression after CT. Definitive RT should be the reasonable choice of treatment for early stage extran-odal nasal type NK/T-cell lymphoma.

OBJECTIVETo establish a simple method to detect four ATP7B gene mutations in Wilson disease using allele-specific PCR(AS-PCR) with whole blood polymerase chain reaction.

METHODSFour allele-specific PCR primers specific for the mutations(G2333T, C2850T, G2855A, G2975T) were designed, and PCR was optimized to screen the whole blood samples. The amplified gene products with mutation were separated with agarose gel electrophoresis to detect the pattern of point mutation and allele types. Exons 8, 12 and 13 of the ATP7B gene were amplified with PCR, and the amplification products were sequenced to confirm the mutation.

RESULTSThe detection of four ATP7B gene mutations by AS-PCR with whole blood was accomplished with 100% accuracy. In the 27 healthy subjects, the mutation rate of G2855A was 51.8%. No mutation was detected for G2333T, C2850T and G2975T. Among the 22 patients, 11 were mutated for G2333T, C2850T or G2975T. The mutation rate was therefore 50%.

CONCLUSIONOur experiment has established an AS-PCR based method for detecting four ATP7B gene mutations using whole blood samples, which has provided a simple and effective means for the early diagnosis of Wilson disease. This method is rapid, convenient, accurate and economical for detecting point mutations of the ATP7B gene.

Adenosine Triphosphatases ; genetics ; Adolescent ; Adult ; Alleles ; Cation Transport Proteins ; genetics ; Child ; Copper-transporting ATPases ; Female ; Hepatolenticular Degeneration ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; methods

Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53 x 10(-4), 1.64 x 10(-4), 4.47 x 10(-3), respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10(-3) and 10(-2).
Bacillus subtilis ; classification ; genetics ; DNA Shuffling ; Genetic Techniques ; Genome, Bacterial ; genetics ; Protein Engineering ; Transformation, Bacterial

Purpose: To evaluated the efficacy and safety of gelatinized Maca (Lepidium meyenii) for eugonadal patients with late onset hypogonadism symptoms (LOH). Materials and Methods: Participants were instructed to receive 1,000 mg of Maca or placebo, two pills at a time, three times per day for 12 weeks before food intake. To evaluate the efficacy of the drug, Aging Males’ Symptoms scale (AMS), Androgen Deficiency in the Aging Males (ADAM), International Prostate Symptom Score (IPSS), and International Index of Erectile Function (IIEF) questionnaires, serologic tests (total testosterone and free testosterone, total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglyceride), body weight, and waist circumference were assessed at 4 and 12 weeks after treatment. Results: A total of 80 participants were enrolled and randomly assigned to Maca treated group (n=41) or the placebo group (n=39). AMS, IIEF, and IPSS were significantly (p<0.05) improved in Maca treated group than in the placebo group. ADAM positive rate was also significantly (p<0.0001) decreased in Maca treated group. Conclusions Maca may be considered an effective and safe treatment for eugonadal patients with late onset hypogonadism symptoms.

Objective: To study the effect of growth differentiation factor (GDF) 11-silenced bone marrow stromal cells (BMSCs) on bone regeneration in early steroid-induced osteonecrosis of the femoral head in rats. Methods: After GDF11 expression in BMSCs was inhibited by siRNA, the knockdown efficiency and transfection cytotoxicity were detected. The further experiments both in vitro (n=3) and in vivo (n=8) were divided into 4 groups respectively: blank control group (without any intervention), model group (glucocorticoid treatment), experimental group (siRNA transfection and glucocorticoid treatment) and negative control group (negative control transfection and glucocorticoid treatment). The BMSCs were induced into osteogenic differentiation. Alkaline phosphatase (ALP) staining and alizarin red staining were applied to evaluate the osteogenic differentiation ability of the cells while reverse transcription polymerase chain reaction (qRT-PCR) was employed to detect the relative expression levels of osteogenic markers. The osteogenesis in the necrotic femoral head was evaluated by microCT, H&E staining, immunohistochemistry staining and biomechanical test. Results: No transfection cytotoxicity was found (P>0.05). The ALP staining and alizarin red staining showed that the osteogenic differentiation of BMSCs in the experimental group was better than that in the model group. At the level of mRNA, the relative expression of ALP, runt-related transcription factor (Runx) 2, osteocalcin (OCN) and type Ⅰ collagen (α1) (COL1A1) in the blank control group (1.00±0.09, 1.02±0.23, 1.03±0.30 and 1.02±0.25, respectively) were significantly higher than those in the model group (0.46±0.11, 0.50±0.11, 0.35±0.01 and 0.57±0.02, respectively) but significantly lower than those in the experimental group (1.97±0.30, 0.94±0.19, 1.50±0.18 and 1.28±0.37) (all P<0.05). MicroCT images and quantitative analysis showed that the bone mass in the experimental group was significantly increased compared with the model group (P<0.05). Histological examination showed better bone regeneration and higher expression of Runx2 and COL1 in the necrotic femoral head in the experimental group than in the model group. Improved biomechanical properties were shown in the experimental group compared with the model group (P<0.05). Conclusions Silence of GDF11 expression may alleviate the inhibitory effect of glucocorticoid on osteogenic differentiation of BMSCs. Early transplantation of GDF11-silenced BMSCs may promote osteogenesis in the necrotic femoral head in rats.

Glioma-associated microglial cells, a key component of the tumor microenvironment, play an important role in glioma progression. In this study, the mouse glioma cell line GL261 and the mouse microglia cell line BV2 were chosen. First, circadian gene expression in glioma cells co-cultured with either M1 or M2 microglia was assessed and the exosomes of M2-polarized and unpolarized BV-2 microglia were extracted. Subsequently, we labeled the exosomes with PKH67 and treated GL261 cells with them to investigate the exosome distribution. GL261 cell phenotypes and related protein expression were used to explore the role of M2 microglial exosomes in gliomas. Then a specific miR-7239-3p inhibitor was added to verify miR-7239-3p functions. Finally, the mouse subcutaneous tumorigenic model was used to verify the tumorigenic effect of M2 microglial exosomes in vivo. Our results showed that in gliomas co-cultured with M2 microglia, the expression of the BMAL1 protein was decreased (P < 0.01), while the expression of the CLOCK protein was increased (P < 0.05); opposite results were obtained in gliomas co-cultured with M1 microglia. After treatment with M2 microglial exosomes, the apoptosis of GL261 cells decreased (P < 0.001), while the viability, proliferation, and migration of GL261 cells increased. Increased expression of N-cadherin and Vimentin, and decreased E-cadherin expression occurred upon treatment with M2 microglial exosomes. Addition of an miR-7239-3p inhibitor to M2 microglial exosomes reversed these results. In summary, we found that miR-7239-3p in the glioma microenvironment is recruited to glioma cells by exosomes and inhibits Bmal1 expression. M2 microglial exosomes promote the proliferation and migration of gliomas by regulating tumor-related protein expression and reducing apoptosis.