Knee extension deficit during gait and knee extensor weakness persisting after saucerization and repair of discoid lateral meniscus tears.

BACKGROUND: Physical function and knee kinematics recovery after discoid lateral meniscus (DLM) tear surgery are essential for a better prognosis. However, these alterations remain unclear. Therefore, this study aimed to investigate changes in physical function and knee kinematics following saucerization and DLM tear repair. METHODS: We enrolled 16 patients who underwent saucerization and DLM tear repair. Postoperative changes in knee kinematics during gait, and physical function, were evaluated at 3, 6, and 12 months. RESULTS: The peak flexion angle of the operated limb during weight acceptance was significantly higher than that of the contralateral limb at 3 (operated limb: 34.6 ± 8.9°, contralateral limb: 23.7 ± 8.3°; P < 0.01) and 6 months (operated limb: 32.1 ± 9.7°, contralateral limb: 24.6 ± 8.2°; P = 0.03) postoperatively, but not at 12 months (operated limb: 27.1 ± 7.1°, contralateral limb: 23.1 ± 9.5°; P = 0.22) postoperatively. The knee extensor strength of the operated limb was significantly lower than that of the contralateral limb at 3 (operated limb: 1.00 ± 0.59 Nm/kg, contralateral limb: 1.37 ± 0.59 Nm/kg; P = 0.01), 6 (operated limb: 1.22 ± 0.55 Nm/kg, contralateral limb: 1.48 ± 0.60 Nm/kg; P < 0.01), and 12 months (operated limb: 1.39 ± 0.57 Nm/kg, contralateral limb: 1.55 ± 0.64 Nm/kg; P = 0.04) postoperatively. CONCLUSION: Knee extension deficits and extensor weakness persisted at 6 months after saucerization and repair of DLM tears. Postoperative rehabilitation should be focused on knee extension function.

Effect of Slightly Acidic Electrolyzed Water Immersion at Different Frequencies on Quality of Raw Chicken Legs.

Slightly acidic electrolyzed water (SAEW) is used as a disinfectant for raw chicken meat. Because its volume for a single immersion exceeds 10 times the weight of meat, a large amount of wastewater is generated. Importantly, a higher frequency of immersion is believed to reduce microbial contamination. The objective of this study was to investigate the effect of SAEW immersion at different frequencies on the disinfection and quality of raw chicken legs, thereby possibly limiting the usage of SAEW. Immersion for 1, 3, and 5 times, with a 7:1 SAEW:meat ratio, and duration of 15 min was tested. Meat quality was evaluated based on total aerobic bacteria, Enterobactericeae, total volatile basic nitrogen, thiobarbituric acid reactive substances, and color. A higher immersion frequency lowered the numbers of total aerobic bacteria and Enterobacteriaceae. Moreover, two immersions with a SAEW:meat ratio of 4:1 and a total immersion time of 6 min reduced the bacterial load as effectively as a single 15-min immersion with a SAEW:meat ratio of 7:1. Higher frequencies of SAEW immersion also resulted in lower total volatile basic nitrogen and lipid oxidation after 0 or 3 days of storage. They did, however, magnify the change in color, resulting in brighter meat. Overall, SAEW treatments with two to five immersions can improve the quality of raw chicken legs and reduce wastewater generation.

Change in cerebral circulation during the induction of anesthesia with remimazolam.

PURPOSE: Remimazolam is a new ultra-short-acting benzodiazepine with unknown effects on cerebral circulation. We measured total cerebral hemoglobin concentrations, which reflect cerebral blood volume (CBV), and cerebral oxygen saturation, using time-domain near-infrared spectroscopy, which can measure the absolute values of cerebral hemoglobin concentrations. We also measured cerebral blood flow velocity (CBFV) in the middle cerebral artery using transcranial Doppler as an indicator of cerebral blood flow (CBF). We did so to examine the effect of remimazolam on cerebral circulation in humans, as assessed CBV, CBF, and cerebral oxygen saturation. METHODS: This was a prospective, observational study. Fifteen patients without serious complications scheduled for general anesthesia were recruited. We measured total cerebral hemoglobin concentrations, CBFV, and cerebral oxygen saturation throughout the anesthetic induction course with remimazolam. RESULTS: Total cerebral hemoglobin concentrations did not change during the process (p = 0.51). In contrast, the mean CBFV was reduced by 11% (significant, p = 0.04). The drop in mean blood pressure following the induction of anesthesia was 17%; however, it was within the range of cerebrovascular autoregulation. Moreover, cerebral oxygen saturation increased by 4% (statistically significant, p < 0.01). CONCLUSIONS: We found that anesthetic induction with remimazolam did not alter CBV and reduced CBF in uncomplicated patients.

Clinical features and significance of leukopenia occurring immediately after endovascular surgery.

PURPOSE: Inflammation after stent graft surgery is known as postimplantation syndrome (PIS) and it causes leukocytosis. However, we have experienced leukopenia in the very early postoperative phase of endovascular surgery at our institution. We investigated leukopenia, an under-recognized phenomenon that occurred after transcatheter aortic valve implantation (TAVI), endovascular aortic repair (EVAR), and thoracic endovascular aortic repair (TEVAR). METHODS: Records of patients who underwent TAVI, EVAR, and TEVAR between March 2018 and February 2019 were retrospectively reviewed. Primary outcomes were the decline rate of white blood cell count (DR-WBC) in the immediate postoperative period and its differences among surgical procedures. The secondary endpoint was the relationship between DR-WBC and infectious complications. Furthermore, the incidence of PIS and its differences among the procedures and associations with DR-WBC were evaluated. RESULTS: A total of 108 patients (TAVI 41, EVAR 37, TEVAR 30) were included. DR-WBC immediately after surgery was higher in the TAVI group when compared with other groups (TAVI, 43.1 ± 22.6%; EVAR, 27.6 ± 17.3%; TEVAR, 25.4 ± 27.4%; P < 0.01). DR-WBC was not significantly different regardless of postoperative infection (P = 0.45) or PIS (P = 0.62). The incidence rate of PIS was higher in the EVAR group compared with the TAVI group, and was not associated with DR-WBC. CONCLUSIONS: Leukopenia was a common phenomenon immediately after endovascular surgery, especially TAVI. It resolved a day after surgery and was not associated with PIS or infectious complications. Therefore, it seems to be a transient abnormal hematological finding and a self-limiting condition.

Exclusive use of digital PCR allows an absolute assay of heat-killed Lactobacilli in foods targeting multiple copies of 16S rDNA.

The real-time PCR (qPCR) and digital PCR (dPCR) to amplify a single-copy of house-keeping genes (i.e., hsp60, pheS or tuf) are used for the assay of limited microbial species. In general, with a single-copy gene, there are obviously varied DNA sequences for even the same microbial species, which could cause difficulties with design of primers and probes for PCR when targeting various single copy genes. In general, for identification by dPCR (as a representative case: Lactobacillus paracasei), accumulated DNA sequence information of 16S rDNA, which is much more frequently used, should be targeted. In contrast, next-generation sequencing revealed that there are five copies of 16S rDNA in a live L. paracasei MCC1849. Therefore, we aimed to reveal, if heat-killed L. paracasei supplemented in nutritional foods that aid the host immune system have the relevant five copies per chromosomal DNA, and if the relevant copies remain unchanged on the same chromosomal DNA or remain to be different chromosomal DNA fragments. So, we revealed the actual distribution of the potential original five copies of 16S rDNA using our innovative dPCR, in which both 16S rDNA and hsp60 genes were simultaneously elongated. The molecular ratios of 16S rDNA/hsp60 dispersed in the dPCR chip were then estimated. The 16S rDNA/hsp60 molecular ratios of the heat-killed L. paracasei in foods, resultantly ranged from 5.0 to 7.2, being the same or higher than that of the five copies determined by next-generation sequencing. The 16S rDNA copy number/ratio indicated the chromosomal DNA molecular number and the associated cell number. As significance, different nutritional foods could potentially cause the loss of chromosomal DNA of supplemented beneficial microbes to a much greater degree. Our absolute dPCR does not require standard correlative samples for the estimation of final products. The estimation principle of the ratio of 16S rDNA/a house-keeping single-copy gene by our absolute dPCR could lead to a useful and accurate assay for various nutritional foods.

Innovative Use of Palladium Compounds To Selectively Detect Live Enterobacteriaceae in Milk by PCR.

Ethidium monoazide and propidium monoazide (EMA and PMA) have been used in combination with PCR for more than a decade to facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving lower limits of detection and quantification of targeted live cells, fewer laborious procedures, lower costs, and potentially higher-throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds. Palladium compounds can penetrate dead (compromised) but not live bacteria and can be chelated primarily by chromosomal DNA and cell wall transmembrane proteins, with small amounts of DNA-binding proteins in vivo The new mechanism for palladium compounds is obviously different from that of platinum compounds, which primarily target DNA. Combining palladium compounds with PCR (Pd-PCR) in water resulted in discrimination between live and dead Enterobacteriaceae bacteria that was much clearer than that seen with the PMA method. Pd-PCR correlated with reference plating or with the currently used PMA-PCR method for pasteurized milk, based on EN ISO 16140:2003 validation. Pd-PCR enabled us to specifically detect and assay viable Enterobacteriaceae cells at concentrations of 5 to 10 CFU/ml in milk while following U.S./EU regulations after a 4.5-h process in a typical laboratory exposed to natural or electric light, as specified by U.S./EU regulations.IMPORTANCE Ethidium monoazide and propidium monoazide (EMA and PMA) facilitate the discrimination of live and dead bacteria (LD discrimination). These methods, however, require many laborious procedures, including the use of a darkroom. Here, we demonstrate an innovative use of palladium compounds involving fewer laborious procedures, lower costs, and potentially higher-throughput analysis than the use of EMA and PMA. We have also recently reported platinum compounds for LD discrimination, but platinum compounds carry costs that are 3 times higher because of the requirement for much larger amounts for LD discrimination than palladium compounds, which have also a novel reaction mechanism different from that of platinum compounds. In view of testing cost, palladium compounds are also very useful here compared with platinum compounds. Ultimately, the innovative Pd-PCR method may be also substituted for the currently used reference plating methods.

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

An All-Inside Repair for Full Radial Posterior Lateral Meniscus Tears.

There is controversy about the treatment for unstable full radial posterior lateral meniscus tears, particularly that involving the posterior root. Some surgeons have advocated repairing these types of meniscus tears using various techniques, but their methods are somewhat technical. We developed the technique for an all-inside repair for full radial posterior lateral meniscus tears using the Meniscal Viper (Arthrex, Naples, FL). A doubled thread is passed through 1 edge of the radial tear by the Meniscal Viper and is kept in place without tying the knot. The Meniscal Viper is used again to set a new thread, repeating the same procedure to another edge of the tear. At this step, 2 doubled threads are passed through each stump of the tear, and both a loop end and 2 free ends of each thread are located outside of the joint. Then, 2 doubled threads pass the third thread into its own loop, pulling it out. Finally, the third thread becomes the mattress suture over the radial tear site and is fastened by sliding knot techniques. This procedure makes it easy to strictly, smoothly, and less invasively shorten the gap by drawing each stump of the meniscus in the direction of the circumference.

Anatomic Oblong Double Bundle Anterior Cruciate Ligament Reconstruction.

Double-bundle anterior cruciate ligament (ACL) reconstruction using hamstring tendon grafts is a standard procedure for ACL injury. However, its clinical effectiveness is not always satisfactory. One cause of this was problems with the graft-tunnel healing of the posterolateral bundle (PLB) on the femur. To solve this problem, we devised a new anatomic ACL reconstruction technique to improve the graft-tunnel healing of the femoral PLB by using a single-bundle with one bone tunnel on the femoral side and a double-bundle on the tibial side. We have performed 40 procedures with excellent results and no cases of intra- or postoperative complication. This procedure can help improve the graft-tunnel healing around the femoral bone tunnel aperture for the PLB.

Innovative use of platinum compounds to selectively detect live microorganisms by polymerase chain reaction.

PCR cannot distinguish live microorganisms from dead ones. To circumvent this disadvantage, ethidium/propidium-monoazide (EMA/PMA) and psoralen to discriminate live from dead bacteria have been used for 2 decades. These methods require the use of numerous laborious procedures. We introduce an innovative method that uses platinum compounds, which are primarily used as catalysts in organic chemistry and partly used as anti-cancer drugs. Microorganisms are briefly exposed to platinum compounds in vivo, and these compounds penetrate dead (compromised) microorganisms but not live ones and are chelated by chromosomal DNA. The use of platinum compounds permits clear discrimination between live and dead microorganisms in water and milk (including Cronobacter sakazakii and Escherichia coli) via PCR compared with typically used PMA. This platinum-PCR method could enable the specific detection of viable coliforms in milk at a concentration of 5-10 CFU mL(-1) specified by EU/USA regulations after a 4-h process. For sample components, environmental water contains lower levels of PCR inhibitors than milk does, and milk is similar to infant formula, skim milk and blood; thus, the use of the platinum-PCR method could also prevent food poisoning due to the presence of C. sakazakii in dairy products. This method could provide outstanding rapidity for use in environmental/food/clinical tests. Platinum-PCR could also be a substitute for the typical culture-based methods currently used.

Tendon-to-bone healing using autologous bone marrow-derived mesenchymal stem cells in ACL reconstruction without a tibial bone tunnel-A histological study-.

BACKGROUND: after anterior cruciate ligament (ACL) reconstruction, it is necessary to integrate free tendon graft biologically to the bone. In the present study, to verify whether a structure identical to the normal ligament-bone insertion could be regenerated at the tendon-bone interface without bone tunnel, we designed ACL reconstruction model without a tibial bone tunnel. Moreover, to enhance the integration process in this model, bone marrow-derived mesenchymal stem cells (bMSCs) were transplanted, and histological changes investigated. Our first hypothesis was that the grafted tendon would be anchored at part of the tendon-bone interface even if a bone tunnel was not created. Second hypothesis was that application of bMSCs at the tendon-bone interface would yield results histologically superior to those in controls. METHODS: bilateral ACL reconstruction using our originally designed method was performed. Autologous bMSCs with the carrier were transplanted between the bottom of the grafted tendon and the bone pit of the tibia in the experimental limb, whereas the control limb received the carrier only. At 4 and 8 weeks after the operation, histological comparison between bMSCs and the control group was carried out. RESULTS/CONCLUSIONS: even in our present ACL reconstruction model without a tibial bone tunnel, integration via chondroid tissue was seen at part of the tendon-bone interface. However, there were no appreciable differences between the groups. In ACL reconstruction, to enhance the tendon-bone integration without a bone tunnel would lead to save the graft length and prevent from bone tunnel complications (ex. Bone-tunnel enlargement after surgery).

Regeneration of ring-shaped lateral meniscus after partial resection of discoid meniscus with anterior cruciate ligament reconstruction.

INTRODUCTION: The ring-shaped lateral meniscus is very rare. Although it is essentially known as a congenital anomaly, a central tear in an incomplete discoid meniscus or an old bucket-handle tear in a meniscus may be easily mistaken for a ring-shaped meniscus. We experienced a ring-shaped lateral meniscus that regenerated after partial resection of a discoid meniscus together with anterior cruciate ligament (ACL) reconstruction. PRESENTATION OF CASE: A 37-year-old female patient still experienced unrelenting knee pain 6 months after ACL reconstruction and partial meniscectomy of a discoid lateral meniscus. A repeat arthroscopy was performed. The lateral tibial plateau was covered in the form of a ring by meniscus-like tissue. The meniscus-like tissue appeared to have regenerated inward toward the center from the stump after the partial meniscectomy and was connected from the anterior to posterior horn, forming an interhorn bridge. Partial meniscectomy was repeated. Histologically, the regenerated tissue was not meniscal, but comprised mature fibrocartilage; macroscopically; however, it was very similar to meniscal tissue. Two years after the initial operation, the patient had no complaints and experienced full return of function. DISCUSSION: The reason for such regeneration is unknown, but may have been attributed to the specific intra-articular environment that developed after the ACL reconstruction. CONCLUSION: This is the first report of regenerative development of a ring-shaped lateral meniscus. When a ring-shaped lateral meniscus is diagnosed, we must accurately determine whether it is a true congenital anomaly in consideration of the present case.

Proline/arginine-rich end leucine-rich repeat protein converts stem cells to ligament tissue and Zn(II) influences its nuclear expression.

Our objective was to facilitate ligament tissue reconstruction by characterizing the mechanism of expression of ligament tissue. To accomplish this, we searched for proteins specific to the tissue and introduced them into mesenchymal stem cells. In the two-dimensional phosphorescent gel electrophoresis, the spots in common with the normal human ligament tissue were selected after removing the spots of the normal bone tissue from those of the ossified tissue in the spinal ligament. Proline/arginine-rich end leucine-rich repeat protein (PRELP) was identified in ligament-specific locations by liquid chromatography-tandem mass spectrometry. Transfection of PRELP into mouse mesenchymal stem cells yielded ligament-like connective tissue comprised of parallel fibers. Thus, expression of the PRELP protein could reconstruct the ligament tissue. Since zinc-related proteins were found with high incidence as a result of an array analysis of PRELP's ProtoArray, it was considered that there is a relationship to the zinc metabolism. Tissue induction was mediated by the tumor necrosis factor (TNF)-α via the zinc pathway. PRELP may be a useful gene in syndesmoplasty, provided zinc is present for tissue reconstruction. Chromosome division becomes active with the addition of zinc, and rapid tissue induction takes place in the presence of zinc and TNF-α. Currently, the reconstruction of a ruptured ligament tissue is difficult, but we expect that the PRELP protein expression may facilitate this process. This study describes the discovery of the gene responsible for the differentiation of stem cells into ligament tissue. This important finding may lead to treatments for gonarthrosis, cruciate ligament, and periodontal ligament ruptures, and ossification of the posterior longitudinal ligament.

Direct real-time PCR with ethidium monoazide: a method for the rapid detection of viable Cronobacter sakazakii in powdered infant formula.

The goal of this study was to establish a rapid assay for the specific detection of viable Cronobacter sakazakii in powdered infant formula (PIF). Samples were subjected to treatment multiple times with ethidium monoazide with a concentration gradient (gEMA) prior to PCR to discriminate viable from dead C. sakazakii cells. To improve the current detection limits, we developed a new buffer for direct quantitative real-time PCR (DqPCR) without DNA isolation. Using 17 PIF samples, our rapid assay was compared with the new U.S. Food and Drug Administration (FDA) method published in the Bacteriological Analytical Manual in 2012. Although both the new FDA method and our rapid assay, which consists of DqPCR combined with gEMA (gEMA-DqPCR), produced negative results for all 17 PIF samples, 5 of the 17 PIFs were positive by DqPCR when they were not treated with EMA. Furthermore, for PIF samples artificially contaminated with viable C. sakazakii, gEMA-DqPCR successfully detected between 1 and 9 CFU of viable C. sakazakii in 300 g of PIF within 9 h, including a 6-h preincubation. Our results indicate that multiple EMA treatments are required to avoid false-positive results due to the contamination of commercial PIF with dead or injured C. sakazakii cells. Our rapid assay may also improve the sensitivity of the screening portion required by the new FDA method published in the Bacteriological Analytical Manual in 2012.

The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility.

BACKGROUND: Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007). METHODS: We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria. RESULTS: For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics. CONCLUSIONS: The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture. GENERAL SIGNIFICANCE: This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.

An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk.

Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451 bp) following EMA treatment to completely suppress the amplification of DNA of up to 7 logs (10(7) cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25 U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1 colony forming unit (CFU)/2.22 ml within 7.5 h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1 CFU/2.22 ml).

Inducement of semitendinosus tendon regeneration to the pes anserinus after its harvest for anterior cruciate ligament reconstruction-A new inducer grafting technique.

PURPOSE: To investigate the usefulness of the "inducer grafting" technique for regeneration of the semitendinosus (ST) tendon after its harvest for anterior cruciate ligament (ACL) reconstruction. METHODS: Twenty knees of 20 patients (mean age at the time of surgery, 23.1 years) underwent ACL reconstruction with a double bundle autograft using the ST tendon (7 patients) and the ST + the gracilis (G) tendons (13 patients)."Inducer grafting" techniqueAfter harvesting the ST tendon, a passing pin with a loop thread is inserted along with the tendon stripper. The passing pin is pulled out from the medial thigh and the loop thread retained. As an inducer graft, the ST tendon branch is used. After the ACL graft has been secured, the inducer graft is sutured to the pes anserinus and the proximal end passed through by pulling the thread out. Then the inducer graft is placed within the tendon canal. The mean follow-up period was 15 months. The presence and morphology of the regenerated ST tendon were examined by MRI. And the isometric hamstring strength was examined at 45°, 90° and 120° of knee flexion. RESULTS: One month after the operation in all the patients, MRI demonstrated a low-intensity structure at the anatomical location of the ST, at the level of the superior pole of the patella and the joint line, apparently representing the regenerated ST tendon. Four months after the operation, the distal portion of the regenerated ST tendon had reached the pes anserinus in all patients. Twelve months after the operation, the regenerated ST tendon was hypertrophic in 19 of the 20 patients (95%). The isometric knee flexion torque of the ACL-reconstructed limb was significantly lower at 90° and 120° compared with the contralateral limb. CONCLUSION: These results suggest that the "inducer grafting" technique is able to improve the regeneration rate of the harvested ST tendon and promote hypertrophy of the regenerated ST tendon, extending all the way to the pes anserinus. However, this technique couldn't improve the deficits in knee flexion torque after ACL reconstruction.

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae.

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.

Rapid detection of viable bacteria by nested polymerase chain reaction via long DNA amplification after ethidium monoazide treatment.

In assays to determine whether viable cells of Enterobacteriaceae are present in pasteurized milk, the typical ethidium monoazide (EMA) polymerase chain reaction (PCR) targets a short stretch of DNA. This process often triggers false-positive results owing to the high level of dead cells of Enterobacteriaceae that had initially contaminated the sample. We have developed a novel, direct, real-time PCR that does not require DNA isolation (DQ-PCR) to detect low levels of cells of Enterobacteriaceae regardless of live and dead cells first. We confirmed that the DQ-PCR targeting a long DNA (the 16S ribosomal RNA [rRNA] gene, amplified length of 1514 bp) following EMA treatment is a promising tool to detect live bacteria of all genera owing to the complete suppression of background signal from high levels of dead bacteria in pasteurized milk. However, when identifying viable bacteria in pasteurized milk, commercial PCR primers designed for detecting long stretches of DNA are generally not available. Thus, we treated samples with EMA and then carried out an initial round of PCR of a long stretch of DNA (16S gene, 1514 bp). We then performed another round of PCR, a novel nested PCR to generate short products using commercial primers. This procedure resulted in the rapid detection of low levels of viable cells of Enterobacteriaceae.

"Hybrid exercise" prevents muscle atrophy in association with a distinct gene expression pattern.

"Hybrid exercise" utilizing combined electrical stimulation and voluntary muscle contraction has been developed as a muscle exercise method. Although our previous studies have confirmed the effectiveness of the procedure, the mechanisms of its efficacy still remain unclear. In the present study, we identified genes that are specifically expressed in disused muscles, using the semitendinosus muscle from patients who underwent anterior cruciate ligament (ACL) reconstruction. Preoperative exercise was performed by four ACL-injured patients, who were subjected either to hybrid exercise (n=2), electrical stimulation (n=1), or no electrical stimulation (n=1), in addition to standard weight training for 4 weeks. Cross-sectional area (CSA) of the semitendinosus muscle was measured before and after the exercise by magnetic resonance imaging (MRI). A piece of the semitendinosus muscle was isolated during the surgery, and comprehensive analysis of the gene expression in this sample was performed using DNA microarray analysis. CSA increased in size by 4.2 and 14.7%, respectively, after hybrid exercise, and by 1.4% after electrical stimulation. However it shrunk by 7.7% without electrical stimulation. DNA microarray analysis revealed that hybrid exercise was more effective at stimulating the expression of signal transduction-, transcription- and cytoskeleton-related genes in semitendinosus muscles than electrical stimulation alone. In particular, gene ontology analysis revealed that hybrid exercise induced significantly higher expression of eukaryotic translation initiation factor 5A (EIFSA), peroxisomal biogenesis factor 6 (PEX6) and histone cluster 1 H4 (HIST1H4), compared with electrical stimulation alone. The expression of signal transduction-, transcription- and cytoskeleton-related genes may play an important role in muscle bulk increasing mechanisms in hybrid exercise.