Direct genotyping from whole blood using alkaline polyethylene glycol.

In order to simplify biological sample preparation to meet the demand of rapid genotyping, we improve alkaline polyethylene glycol (APEG) based on Chomczynski's procedure for the PCR-based lateral flow genotyping system, which enables the rapid and efficient direct genotyping from whole blood without DNA purification. The improved APEG has a high tolerance for extreme storage conditions. Testing whole blood with an abnormal hematological index indicates that APEG efficiency would not be influenced by pathological factors. Compared with sequencing, the accuracy of this genotyping system was 100% on testing with 200 clinical samples.

Multiple SNPs Detection Based on Lateral Flow Assay for Phenylketonuria Diagnostic.

Single nucleotide polymorphisms (SNPs) are closely related to genetic diseases, but current SNP detection methods, such as DNA microarrays that include tedious procedures and expensive, sophisticated instruments, are unable to perform rapid SNPs detection in clinical practice, especially for those multiple SNPs related to genetic diseases. In this study, we report a sensitive, low cost, and easy-to-use point-of-care testing (POCT) system formed by combining amplification refractory mutation system (ARMS) polymerase chain reaction with gold magnetic nanoparticles (GMNPs) and lateral flow assay (LFA) noted as the ARMS-LFA system, which allow us to use a uniform condition for multiple SNPs detection simultaneously. The genotyping results can be explained by a magnetic reader automatically or through visual interpretation according to the captured GMNPs probes on the test and control lines of the LFA device. The high sensitivity (the detection limit of 0.04 pg/µL with plasmid) and specificity of this testing system were found through genotyping seven pathogenic SNPs in phenylalanine hydroxylase gene ( PAH, the etiological factor of phenylketonuria). This system can also be applied in DNA quantification with a linear range from 0.02 to 2 pg/µL of plasmid. Furthermore, this ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families including 69 individuals. The concordance rate of the genotyping results detected by the ARMS-LFA system was up to 97.8% compared with the DNA sequencing results. This method is a very promising POCT in the detection of multiple SNPs caused by genetic diseases.

[The value of 3 dimensional-fat suppression-spoiled gradient-recalled acquisition sequence on single compartment osteoarthritis for unicompartmental arthroplasty preoperative assessment].

OBJECTIVE: To analyze the 3 dimensional-fat suppression-spoiled gradient-recalled acquisition (3D-FS-SPGR) sequence in the diagnosis of knee articular cartilage injury. METHODS: A total of 56 knee osteoarthritis patients (26 males, 30 females, ages 52-73 years, mean 61.8 years) treated in Department of Orthopedics, Chinese People's Liberation Army General Hospital between June 2013 and May 2014 were involved in this study. All patients underwent knee MRI, plus 3D-FS-SPGR sequence, arthroscopic exploration, and in contrast to the results of MRI results analysis, evaluation 3D-FS-SPGR and conventional sequence of cartilage damage consistent with the arthroscopic accuracy. RESULTS: Divided 56 knee joints into 336 cartilage articular surface, included 55.1% normal articular surface, 21.4% early osteoarthritis and 23.5% advanced osteoarthritis. The accordance of 3D-FS-SPGR sequence grading and arthroscopic was 90.2%. The sensitivity of 3D-FS-SPGR sequence was 93.1%, specificity was 98.3%, and Kappa value was 0.849. The sensitivity of T2WI sequence was 84.4%, specificity was 96.9%, and the Kappa value was 0.671. CONCLUSION: For unicompartment osteoarthritis , MRI 3D-FS-SPGR sequence is effective in sensitivity and specificity of cartilage damage.

[Experimental study of tendon graft fixation in anterior cruciate ligament reconstruction with cortical press-fit bolt in rabbits].

OBJECTIVE: To explore the histological outcomes of tendon-bone healing in anterior cruciate ligament (ACL) reconstruction with cortical press-fit bolt (CPB). METHODS: Twenty-four healthy female or male New Zealand White rabbits (2-3 months old) underwent bilateral ACL reconstruction with extensor digitorum longus tendon. A random method was used to decide one knee would receive the routine ACL reconstruction (control group) and another cortical press-fit bolt fixation (experimental group). After general anesthesia, extensor digitorum longus tendon was harvested and ACL reconstruction performed. All animals were sacrificed at 4, 8 and 12 weeks postoperation. Radiological and histological examinations were made at each timepoint. The specimens were stained with different methods to observe the pathological changes of tendon graft, bone tunnel and cortical press-fit bolt. RESULTS: More revascularization and massive new bone were found in tendon-bone junction of experimental group at 4, 8 and 12 weeks postoperation. The circum-graft new vessel proportion of the experimental and control groups were 0.48 ± 0.12 and 0.26 ± 0.05 respectively (P < 0.05). In the experimental group, more cartilage cells were present in tendon-bone junction at 12 weeks and the circum-graft new bone areas in two groups were 0.41 ± 0.11 and 0.21 ± 0.10 mm(2) respectively (P < 0.05). CONCLUSION: Cortical press-fit blot may improve tendon-bone healing after ACL reconstruction in rabbits. The application prospects of this procedure are promising.

Evaluation of the effects of 20 nonsynonymous single nucleotide polymorphisms of CYP2C19 on S-mephenytoin 4'-hydroxylation and omeprazole 5'-hydroxylation.

CYP2C19 is a highly polymorphic enzyme that affects the metabolism of a wide range of therapeutic drugs. Almost all the identified alleles of CYP2C19 are derived from nonsynonymous single nucleotide polymorphisms (nsSNPs). The objective of this study was to functionally characterize 20 nsSNPs of CYP2C19, distributed throughout the entire coding region, most of which have not been thoroughly characterized. cDNAs of these variants were constructed and expressed in yeast cells. All variants had similar levels of apoprotein and holoprotein expression, except for CYP2C19.16 and D360N, which had significantly lower holoprotein levels than the wild-type (WT) CYP2C19 enzyme, and CYP2C19.5B, which showed only apoprotein. The activity of the CYP2C19 variants was investigated using two substrates, S-mephenytoin and omeprazole, and six different kinetic parameters were measured. CYP2C19.5B, CYP2C19.6, and CYP2C19.8 were found to be catalytically inactive. The entire dataset of the remaining 17 variants, together with the WT, was analyzed by multivariate analysis. This analysis indicated that CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.18, CYP2C19.19, A161P, W212C, and D360N were substantially altered in catalytic properties in comparison with the WT, with each of these variants exhibiting either dramatically decreased catalytic activities or higher K(m) values. These results not only generally confirmed the function of previously reported variants but also identified additional reduced-function variants. These findings will greatly extend our understanding of CYP2C19 genetic polymorphisms in humans as well as facilitate the structure-function study of the CYP2C19 protein.

Evaluation of the effects of 18 non-synonymous single-nucleotide polymorphisms of CYP450 2C19 on in vitro drug inhibition potential by a fluorescence-based high-throughput assay.

To comprehensively understand the effects of CYP2C19 genetic polymorphisms on inhibition-based drug-drug interactions (DDIs), 18 human CYP2C19 non-synonymous single-nucleotide polymorphic variants and the wild-type isoform (CYP2C19.1A) were expressed in yeast cells. Using a fluorescence-based high-throughput method, the kinetic constants of these variants, as well as the inhibition constants for 10 drugs, were determined. CYP2C19.5B and CYP2C19.6 showed no activity towards CEC (3-cyano-7-ethoxycoumarin) O-deethylation. CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.19, E122A and A161P* (an allele containing both A161P and I331V) exhibited significantly reduced catalytic activities compared with CYP2C19.1A. The inhibition assay showed that the CYP2C19 genotype significantly affected the in vitro drug inhibition potential. Although the effect on drug inhibition potential is genotype- and inhibitor-dependent, there was an obvious trend: drugs tended to exhibit higher IC50 values (i.e. decreased inhibition potential) towards variants with reduced activity compared with variants with normal activity. This indicated that patients with reduced-function alleles may be less susceptible to CYP2C19-related DDIs. In this study, we provided the first in vitro evidence of CYP2C19 genotype-dependent effects on drug inhibition potential. This work greatly extends our understanding of the functional consequences of CYP2C19 genetic polymorphisms, and thus should prove valuable for CYP2C19 genotype-based therapy.

Genome-Wide Identification and Co-Expression Analysis of ARF and IAA Family Genes in Euscaphis konishii: Potential Regulators of Triterpenoids and Anthocyanin Biosynthesis.

Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.

[Biomechanical study on different high-strength sutures and suture site for repairing posterior root tear of the medial meniscus].

OBJECTIVE: To compare biomechanical characteristic of different high-strength sutures and suture sites for repairing posterior root tear of the medial meniscus with modified Mason-Allen technique. METHODS: Forty-eight specimen of medial meniscus of knee joint from fresh porcine (female, aged from 5 to 9 months with an average of 7 months) were chosen and established experimental model. The samples were divided into red zone fixation group and red-white zone fixation group according to suture sites, 24 in each group; and then were randomly divided into 3 subgroups which 8 in each group, and fixed with Ethibond suture, Ultrabraid suture and FiberWire suture, respectively. Biomechanical tests were performedon universal electromagnetic and mechanical testing machine. Each specimen was underwent 1 000 cyclic tests on the first time, then pull out test until failure. The maximum failure load, yield load, stiffness and displacement were analyzed. RESULTS: All specimen were successfully completed biomechanical tests. The failure mode of Ethibond group was caused by suture fracture; 6 cases of Ultrabraid suture group was caused by suture fracture which belong to red zone fixation group, 10 cases were caused by suture pull out, which 2 cases belong to red zone fixation group, 8 cases belong to red-white zone fixation group;8 cases of FiberWire group was caused by suture pull-out. Biomechanical test showed that:(1)In terms of suture strength, comparison of the maximum failure load, yield load and stiffness showed that Ethibond suture group Ultrabraid suture group >FiberWire suture group, and had statistical differences among groups (P<0.05);the results showed the suture strength in Ethibond was the best, Ultrabraid suture took the second place, and FiberWire suture was the worst. (2)As for different suture sites, comparison of the maximum failure load, yield load and stiffness showed that red zone fixation group >red-white region fixation group, and had statistical difference between two groups(P< 0.05);comparison of cyclic displacement at 100, 500 and 1 000 cycles showed that red zone fixation group

[Case-control study on remnant-preserving strategy for preservation and reconstruction of anterior cruciate ligament].

OBJECTIVE: To investigate and compare the clinical efficacies of remnant-preserving and remnant-non-preserving, remnant-non-preserving remnant segment preserving and remnant root preserving with anterior cruciate ligament reconstruction. METHODS: From March 2014 to December 2017, 204 patients with anterior cruciate ligament (ACL) injuries were treated by single-bundle ACL reconstruction with hamstring tendon autograft. According to the different methods of remnant preservation, the procedures were divided into remnant segment preserving group (A), remnant root preserving group (B), and remnant-non-preserving group (C). There were 37 males and 39 femalesin group A aged from 16 to 43 years old with an average of (28.80±5.41) years old. The time from injury to operation ranged from 2 to 11 weeks with an average of (3.68±1.04) weeks. In group B, there were 39 males and 25 females aged from 18 to 41 years old with an average of (28.42±5.60) years old. The time from injury to operation ranged from 2 to 10 weeks with an average of (3.36±1.68) weeks. In group C, there were 37 males and 27 females aged from 18 to 43 years old with an average of (29.10±6.11) years old. The time from injury to operation ranged from 3 to 11 weeks with an average of (3.54±1.46) weeks. The range of motion (ROM) of the knee was used to assess the range of extension and flextion of the knee at pre-operation and 24 months after operation. Lysholm score and the international knee documentation committee (IKDC) score were used to assess the knee function. The differences among three procedures were judged by comparing among the three groups at 6, 12 and 24 months postoperatively. RESULTS: All incisions got a one stage healing, and no complications, such as vascular injury, nerve damage and articular infect or the like, occurred. All the patients were followed up, and the follow-up duration of group A ranged from 24.00 to 45.96 months with a mean of (35.52±14.40) months;the follow up duration of group B ranged from 27.96 to 48.00 months with a mean of (37.56±10.68) month;and the follow up duration of group C ranged from 24.00 to 66.00 months with a mean of (37.08±13.44) month. There were no significant differences in follow up time among three groups (P>0.05). Six months after operation, Lysholm score 80.74±3.14 and IKDC score 79.92±3.44 in group A were higher than those in group B 80.74±3.14 and 78.21±4.63, and higher than those in group C 79.22±3.63 and 76.63±3.80 (P<0.05);12 months after operation, Lysholm score 89.84± 5.13 and IKDC score 87.90±3.93 in group A were higher than those in group B 85.74±6.04 and 83.62±5.64, and higher than those in group C 82.83±3.43 and 79.21±4.04(P<0.05). CONCLUSION: Compared with remnant-non-preserving group, the residual tissue of anterior cruciate ligament is preserved, which is conducive to promote the healing and remodeling of tendon graft and accelerate the recovery of joint function. Proper fixation of residual tissue and restoration of its tension are the key factors affecting the postoperative efficacy.

Metabolism of tanshinol borneol ester in rat and human liver microsomes.

Tanshinol borneol ester (DBZ) is an experimental drug that exhibits efficacious anti-ischemic activity in rats. Although the specific metabolic properties of DBZ are still unknown, previous studies in rats have strongly suggested that DBZ is extensively metabolized after administration and thus probably acts as a prodrug. Because the enzymes involved in drug metabolism differ between humans and rats in isoform composition, expression, and catalytic activity, the pharmacokinetics of the same drug in the two species may also differ. Establishing the differences between DBZ metabolism in human and rat liver microsomes can help to predict DBZ pharmacokinetics in humans and aid in the assessment of its potential efficacy, toxicity, and mechanism of action. In this work, the microsomal stabilities and metabolic kinetics of DBZ in rat and human liver microsomes were compared, and the DBZ metabolites generated in human liver microsomes (HLMs) were identified. The results suggested that DBZ is more stable in HLMs than in rat liver microsomes (RLMs). The intrinsic clearance of DBZ in HLMs was 10- to 17-fold lower than that in RLMs, which indicates lower DBZ clearance in humans. Metabolite analysis suggested that DBZ is hydroxylated by liver microsomal enzymes, resulting in the production of five metabolites. Although the kinetics of metabolite formation in HLMs and RLMs were different, the same metabolites were generated, indicating that the same metabolic pathway is present in both species. The results obtained from this work suggest the potential for DBZ to act as a prodrug with anti-ischemic activity in humans.

Evaluation of the effects of cytochrome P450 nonsynonymous single-nucleotide polymorphisms on tanshinol borneol ester metabolism and inhibition potential.

Nonsynonymous single-nucleotide polymorphisms (nsSNPs) in cytochrome P450 (P450) genes may affect drug metabolism and drug-drug interactions (DDIs), potentially leading to adverse drug reactions. Functional characterization of the nsSNPs in P450 genes is important to help us understand the impact of genetic factors on P450-mediated drug metabolism and DDIs. To evaluate the effects of P450 nsSNPs on the metabolism and inhibition potential of a candidate drug, tanshinol borneol ester (DBZ), we obtained and experimentally validated eight yeast-expressed human P450 isoforms and their nsSNP variants and tested DBZ using these recombinant P450 enzymes. The results suggested that CYP2C8 is the major enzyme responsible for DBZ metabolism. In addition, compared with prototypic CYP2C8, the allelic variant, CYP2C8.3, produced a 54% decrease in the intrinsic clearance of DBZ. The inhibitory potency of DBZ toward CYP3A4 was greater than that toward other P450 isoforms, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. Moreover, the inhibitory potency toward three CYP3A4 allelic variants, CYP3A4.2, CYP3A4.12, and CYP3A4.16, was reduced 2- to 10-fold relative to prototype CYP3A4. These results provide useful information for understanding the influence of P450 genetic polymorphisms on DBZ metabolism and may help to design future clinical trials of DBZ. Our results suggest applications for in vitro P450 assays both for basic research in pharmacogenomics and for drug development.

Functional characterization of five CYP2C8 variants and prediction of CYP2C8 genotype-dependent effects on in vitro and in vivo drug-drug interactions.

1. To analyze the polymorphic activities of CYP2C8 and evaluate their impact on drug inhibitory potential, three CYP2C8 allelic variants (CYP2C8.2, CYP2C8.3, and CYP2C8.4), two non-synonymous single nucleotide polymorphic variants (R139K and K399R, carried by CYP2C8.3), and wild-type CYP2C8 (CYP2C8.1) were heterologously expressed in yeast, and their enzymatic activities were characterized. CYP2C8 inhibition-based in vitro and in vivo drug-drug interactions (DDIs) in wild-type and variant CYP2C8s were then predicted. 2. Functional characterization of five CYP2C8 variants revealed similar enzymatic activity in R139K and low activity in CYP2C8.2, CYP2C8.3, CYP2C8.4, and K399R compared with CYP2C8.1. The systematic analysis of these CYP2C8 variants can provide more homogeneous data for predicting CYP2C8 phenotypes and could be applied to personalized drug therapy. 3. Prediction of DDIs indicated that CYP2C8.4, R139K, and K399R dramatically alter the IC(50) values of nifedipine, troglitazone, and raloxifene, and R139K qualitatively and quantitatively reduces the risk of in vivo paclitaxel-raloxifene and paclitaxel-troglitazone interactions. The results provide the first evidence that CYP2C8 inhibition-based DDIs may be influenced by CYP2C8 genetic polymorphisms. These inhibition data can be used by pharmacologists in the design of in vivo studies to further assess and address the potential role of CYP2C8 genotype-dependent inhibition in clinical DDIs.

[Effect of arthroscopic debridement for adolescent ankylosing spondylitis with early hip-joint disease].

OBJECTIVE: To study the effect of arthroscopy debridement for adolescent ankylosing spondylitis (AS) with early hip-joint disease. METHODS: A total of 22 cases (26 hips) of adolescent AS were recruited. There were 15 males and 7 females. The distribution was as follows: left side (n = 10), right side (n = 8) and bilateral (n = 4). The average age was 16 (14 - 19) years old. The procedures included adhesion relief of hip joint, removal of hyperplasic synovial membrane, degenerated cartilage debris and repairing exfoliated debris or exposed subchondral bone on hip joint. RESULTS: All patients were followed up for an average of 26 (6 - 84) months. Most patients experienced pain relief, restored function and the range of motion. According to Harris hip joint score and VAS pain score evaluation system, the outcomes were excellent (n = 6) fair (n = 14) and poor (n = 2). There was no case of total hip replacement. CONCLUSION: Arthroscopic debridement for adolescent ankylosing spondylitis with early hip joint disease is effective to improve joint motion and relieve pain.

[Classification and arthroscopic surgery of chronic achilles tendinitis].

OBJECTIVE: To investigate the clinical classification of chronic achilles tendinitis and analyze the surgical technique and efficacy of arthroscopic surgery. METHODS: Twenty-two patients (16 males, 6 females) with chronic achilles tendinitis were recruited. The average age was 33.5 years old (range: 17 - 53). Sixteen cases were caused by sport injury while 6 cases had no definite etiological factor. The Achilles tendinopathy was divided into three types according to clinical characteristics and the results of X ray, CT scan and MRI examination of ankle: Type 1, hypertrophy (n = 10); Type 2, calcified tubercle (n = 5); Type 3, fiber tear (n = 7). All cases were treated with endoscopic debridement of ventral neovascularized area, peritendineum and Achilles tendon by shaver and radiofrequency (RF) probe. RESULTS: The patients were followed-up for a mean of 14 months (range: 9 - 15). Evaluated by our criteria and visual analogue scale, the post-operative efficacy was excellent in 12 cases, good in 8 and fair in 2. No postoperative complications, such as neurovascular injury, infection and rupture of Achilles tendon, was recorded. CONCLUSION: This scheme of classifying is helpful to the diagnosis and effective treatment of chronic Achilles tendonitis. With a high safety and a satisfactory efficacy, arthroscopic surgery has the advantages of minimally invasiveness.

[Arthroscopic repair of Bankart lesion with bioabsorbable suture anchors].

OBJECTIVE: To evaluate early clinical effects of bioabsorbable suture anchors for the treatment of Bankart lesion. METHODS: Total 23 patients with the Bankart lesion were treated with arthroscopic repair using bioabsorbable suture anchors from January 2010 to June 2017. There were 20 males and 3 females, with an average age of (23.4±3.9) years old (ranged, 19 to 34 years old). Fourteen patients had injuries on the right shoulder joint and 9 patients had the injuries on the left side. The mechanism of primary dislocation included 17 cases of training, 5 cases of sports injury and 1 case of falling down. The mean interval time from injury to surgery was(10.9±5.8) months (ranged, 3 to 36 months). The Bankart lesion was repaired by bio-cortical suture anchors. The Rowes rating system for Bankart repair was used to evaluate therapeutic effects. RESULTS: All 23 patients were followed up, with a mean duration of(24.5±3.7) months(ranged, 18 to 39 months). At the latest follow up, there was no recurrent dislocation occurred, and all patients had returned to sports and work. The Rowes rating system for Bankart repair was 53.91±11.67 pre-operationally and 91.74±12.30 post operationally, respectively (P<0.01). According to the Rowes rating system, there were 0 case of excellent, 0 case of fine, 9 cases of good and 14 cases of bad pre-operationally;16 cases of excellent, 4 case of fine, 3 cases of good and 0 cases of bad post operationally;the difference was statistically significant (P<0.01). CONCLUSION: Applying bio-cortical bone suture anchors for the Bankart lesion is a reliable, efficient and cost effective treatment, which is also suitable for the revision of the Bankart lesion.

Tetra-primer ARMS-PCR combined with GoldMag lateral flow assay for genotyping: simultaneous visual detection of both alleles.

Rapid and simple detection of single nucleotide polymorphism (SNP) is vital for individualized diagnosis and eventual treatment in the current clinical setting. In this study, we developed a tetra-primer ARMS-PCR combined lateral flow assay (T-ARMS-PCR-LFA) method for simultaneous visual detection of two alleles. By using four primers labeled with digoxin, biotin and Cy5 separately in one PCR reaction, the amplified allele-specific products could be captured by streptavidin and the anti-Cy5 antibody on two separated test lines of a LFA strip, which allows the presentation of both alleles within the single LFA strip. Both DNA and whole blood can be used as templates in this genotyping method in which the whole detection process is completed within 75 minutes. The performance assay of T-ARMS-PCR-LFA demonstrates the accuracy, specificity and sensitivity of this method. One hundred human whole blood samples were used for MTHFR C677T genotyping in T-ARMS-PCR-LFA. The concordance rate of the results detected was up to 100% when compared with that of the sequencing results. Collectively, this newly developed method is highly applicable for SNP screening in clinical practices.

[RIGIDfix tibial and femur cross pin system used for hamstring grafted anterior cruciate ligament reconstruction].

OBJECTIVE: To evaluate the curative effect of RIGIDfix tibial and femur cross pin system used for hamstring grafted reconstruction of anterior cruciate ligament (ACL) in arthroscopy. METHODS: Thirty two cases with ACL ruptures were reconstructed arthroscopically with hamstring grafts and the grafts fixed with RIGIDfix cross-pin on both femoral and tibial sides. They included 18 males and 14 females with a mean age of 28 (20-45) years old; the mean time from injury to operation was 3.6 (2.5-6) months. And each knee was checked by MRI and X-ray preoperatively. Through arthroscopy, we found 19 ACL ruptured from femoral attachment point, 13 from tibial point, 3 cases combined with medial meniscus injury and 4 cases with articular cartilage injury of medial femoral condyle. After semitendinosus or gracilis harvesting, the hamstring grafts were pre-tensioned and woven, the diameter of 4 or 5 strands grafts was 7-8 mm. To position and drill tibial tunnel on ACL stump of tibial crest, and to drill femoral tunnel at 10:00 to 11 o'clock of femoral intercondylar fossa transtibial with knee flexed to 90 degree, the depth of femoral tunnel was 30 mm. The RIGIDfix guide was inserted through tibial tunnel up into the femoral tunnel to drill the sleeve and interlocking Trocar across the lateral femur and keep the two sleeves fixed to the lateral femur. Insert RIGIDfix guide into the tibial tunnel, keeping the top of guide 2-3 mm beneath the endostoma of tibial tunnel, drill the sleeve and interlocking Trocar across the lateral tibia and keep the two sleeve fixed to the lateral tibia. The graft was pulled into the tunnel (the 30 mm mark on the graft should be at the edge of femoral hole) to insert the RIGIDfix cross pins from femoral tunnel to tibial tunnel, while inserting the second, third, fourth cross pin, the graft should be kept under tension. Then a knot was tied through tibial bony bridge using the Enthibond thread switched to the end of tendon grafts. We finally observed the tension of tendon grafts and the impingement of fossa intercondylic under arthroscopy. RESULTS: With a 16-month follow-up evaluation, all of the patients' injured knees were stable and the average Lysholm knee score increased from 62.5 to 94.5. Rulermetr device values were less than 2 mm of sagittal displacement in 28 patients and 4 mm in 4. Postoperative Lachman was negative in 30 patients and weakly positive in 2. According to the IKDC scores, 30 patients reported normal function, 2 reported nearly normal function and none reported abnormal or severely abnormal function. CONCLUSION: The grafting method of fixing both femur and tibia sides with absorbable cross pins is feasible. In this way, the graft is stabilized to allow for reconstruction. A surgeon should refrain from dissecting the tendon and enlarging the tunnel so as to promote the healing of tendon and bone.

[Arthroscopically assisted radiofrequency probe to treat achilles tendinitis].

OBJECTIVE: To evaluate the effectiveness of micro-tenotomy using a radiofrequency (RF) probe to treat chronicity achilles tendinitis. METHODS: Seventeen cases of chronicity achilles tendinitis were treated by RF probe. Eleven cases were male, and 6 female. The average age of the patients was 25 (17-48) years. Nine were athlete, 4 sports activity and 4 students. Seven were in left side, and 10 right side. Before receiving the RF therapy all patients had achilles tendinitis symptoms. The patients were treated with local anesthesia under arthroscopy by Arthrocare 2000 and TOPAZ. The operation were performed through a artificial lacuna under the subcutaneous tissue of achilles tend, insert arthroscope. The pathology test found the achilles tendon surface fibrous tissue hyperplasia and tear. The probe of RF to perforate just as meshwork, the space was 3-5 mm. RESULTS: Patients reported significantly reduced pain from 7 to 14 d postoperatively. The symptoms of pain was completely disappeared in 15, obviously relieve in 2. The functional outcome was assessed using the VAS score evaluation, perioperative 8.7 and postoperative 1.6. There were no perioperative and postoperative complications related to the procedure, as rupture of achilles tendon, blood vessel and nerve injury. No infection and recur was found in the cases. CONCLUSIONS: RF therapy for chronicity achilles tendinitis under the arthroscopy with minimum invasion is less pain and easy for early rehabilitation. The result is satisfactory.

[Arthroscope monitored solution of adult intramuscular injection associated gluteal muscle contracture by radiofrequency].

OBJECTIVES: To evaluate the result of releasing adult intramuscular injection associated gluteal muscle contracture under the monitor of arthroscope by radiofrequency probe. METHODS: From June 2001 to June 2005, 108 cases of bilateral gluteal muscle contracture were treated with radiofrequency colation under the arthroscope and solution with an average age of 24 years (from 18 to 40 years). There were 57 males and 51 females. Preoperatively, the course of the outline of the femur greater trochanter the sciatic nerve in buttocks and the area of gluteal muscle contracture were marked. With the patients firmly anchored in the straight lateral position, normal saline (which contains Adnephrin) was injected between the surface of contracted gluteus and subcutaneous fat to reduce bleeding in operation. The ports for the motorized shaver and radiofrequency probe were located at the edge of gluteal muscle contracture and were 5 mm superior to the greater trochanter. The 6 mm diameter port for the arthroscope was 3 cm inferior to the greater trochanter. Space was made between contracture bands and overlying subcutaneous tissue with a periosteal elevator by blunt dissection. After the anterior and posterior edge of the contracture bands were fully revealed, normal saline were filled in the space. With the monitor of arthroscope, the procedures were: removing fatty tissue from the surface of the contracture bands with motorized shaver, then cutting off the contracture bands curve and carefully probing and cutting off contracture bands which were mixed in gluteus maximus with radiofrequency probe, finally hemostasis by radiofrequency probe. In the operation flexion, adduction, internal rotation and straightening hip joint were repeated, until it got normal range of motion without snap and bleeding. Results One hundred and one patients were followed up with an average of 19 months. According to a comprehensive evaluating system, 91 cases were excellent, 7 were good, and 3 were fair. No infection, recurrence and neurovascular injury occurred. CONCLUSIONS: Gluteal muscle contracture could be effectively released with radiofrequency vaporization and solution. The technique has the advantage of easy to manipulate, minimally invasive, painless, safety and reliable curative effect, and is good for early functional exercises.

Genotyping of Multiple Clinical Samples with a Combined Direct PCR and Magnetic Lateral Flow Assay.

Developing a sensitive, low-cost, and easy-to-use point-of-care testing system for genotyping is important for informing treatment decisions and predicting the risk of underlying diseases. Conventional methods normally require complex operational procedures as well as expensive and sophisticated instruments. Here, we report a general approach that enables us to detect the genotype of multiple sample types directly without DNA purification. Moreover, the PCR results can be further quantitatively analyzed based on a magnetic lateral flow assay (MLFA) system, which avoids multiple steps needed for conventional nucleic acid biosensors. As a demonstration, we show that three genotypes of aldehyde dehydrogenase 2 (ALDH2) can be identified using a small volume of sample with an accuracy of 100% and a sensitivity of 1.0 × 102 cells/µL, which are better than those of the gold standard methods. We believe that the direct PCR-MLFA system represents a significant advance toward the development of portable, sensitive biomedical platforms.