Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized milk.

Pasteurized milk is a complex food and contains numerous PCR inhibitors and can often contain high levels of dead Enterobacteriaceae cells, depending on the condition of food sanitation. Usually, propidium monoazide (PMA) or ethidium monoazide PCR techniques decrease the number of dead bacteria by up to 3.5 log to the associated dead bacteria with no treatment. However, this difference could be insufficient to completely inhibit DNA amplification in the PCR from 10(6) cells of dead Enterobacteriaceae bacteria/mL, potentially contaminated in pasteurized milk. Actually, such potentially high levels of dead Enterobacteriaceae cells in milk has prevented milk researchers from applying PMA- or ethidium monoazide PCR to the assay of viable Enterobacteriaceae cells in milk. We, therefore, developed a rapid PMA real-time PCR whose minimum levels of detection were 1.5 log cfu/PCR for Cronobacter muytjensii and Escherichia coli, and 2.5 log cfu/PCR for Salmonella enteritidis without DNA purification in milk matrices. The PMA real-time PCR allowed us to specifically detect viable Enterobacteriaceae cells (5-10 cfu/mL) in pasteurized milk (20 mL) within 7.5h of total testing time, following the hygienic guidelines for pasteurized milk in the United States and European Union. The long DNA amplification (mainly 2,451 bp) of the 16S-23S rRNA gene was completely suppressed in highly contaminated dead Enterobacteriaceae cells (7.5 log cfu of Cronobacter muytjensii) in 20 mL of pasteurized milk by 23-µM PMA treatment. Although the contamination of the PCR reaction with 5% milk usually causes great inhibition, our method led to the successful elongation of PCR from viable Enterobacteriaceae cells still in the pasteurized milk matrices finally corresponding to 2 to 4 mL of milk PCR inhibitors without a DNA purification step. To comply with current customer demands for chilled pasteurized milk at the most excellent possible quality, our new technique could enable laboratory persons in a factory to conduct rapid milk coliform testing before shipping from a factory.

New approach to use ethidium bromide monoazide as an analytical tool.

AIMS: Ethidium bromide monoazide (EMA) has been determined to cause delay in DNA amplification from dead bacteria at real-time PCR. However, there is concern that the increasing EMA concentration to suppress amplification from high number of dead bacteria also affects live bacteria. The aim is to disclose a novel application of EMA for food hygienic test. METHODS AND RESULTS: We performed a low-dose double EMA treatment. Live or heat-dead Enterobacter sakazakii (reclassified as Cronobacter spp.) in 10% powdered infant formula (PIF) solution was subjected to a treatment with 20 µg ml(-1) of EMA followed by a treatment with 10 µg ml(-1) of EMA without washing, and direct real-time PCR. We observed that DNA amplification from 10(7) cells ml(-1) of dead Ent. sakazakii was completely suppressed within 50 cycles of PCR, whereas 10(2) -10(3) cells ml(-1) of viable cells could be detected. When a 3-h enrichment step in liquid medium was included after the first EMA treatment, live Ent. sakazakii could be detected at initial levels of 10(0) -10(2) cells ml(-1) . We compared the low-dose double-treated EMA-PCR with the culture method using 80 samples of PIF, and completely correlative results were obtained for both methods. CONCLUSIONS: We concluded that the newly developed low-dose double-treated EMA-PCR is a very effective tool for live Ent. sakazakii detection in PIF. SIGNIFICANCE AND IMPACT OF THE STUDY: We focused on the specific nature of photoreactive compound that residual EMA is cancelled by irradiation. We were successful in treating bacteria with EMA in gradient concentration to increase live and dead distinction ability.

Risk evaluation for staphylococcal food poisoning in processed milk produced with skim milk powder.

The growth of S. aureus and the production of staphylococcal enterotoxin A (SEA) in skim milk concentrates stored at inappropriate temperatures in a recovery milk tank (tank for excess concentrated skim milk) used in the manufacture of skimmed milk powder were investigated. Also, it was estimated if a possible outbreak of food poisoning would occur if the contaminated skimmed milk powder was used in the manufacture of processed milk. Skim milk concentrates with milk solid content of 15, 25, and 35% were inoculated with S. aureus at 1-2 log CFU/ml and incubated at 15, 25, or 35 degrees C for 0 to 24 h with or without shaking. Bacterial growth and the level of SEA production were measured. At 35 degrees C with shaking, there was a significant difference (p<0.05) in one way layout analysis of variance, and it was demonstrated that the growth of S. aureus and SEA production could be milk solid content-dependent. Shaking accelerated the growth of S. aureus and SEA production at 35 degrees C. Generally, skim milk powder is produced by mixing a set percentage of skim milk concentrates (recovery milk) from the recovery milk tank into raw milk. If recovery milk contaminated with S. aureus at levels of 1-2 log CFU/ml is kept at 15 to 35 degrees C due to a power failure, it was estimated that processed milk consumption of 670-1200 ml, 420-1500 ml and 18-83 ml would trigger the onset of food poisoning symptoms when skim milk concentrates (recovery milk) are stored at 25 degrees C for 24 h, 35 degrees C for 10 h, and 35 degrees C for 24 h, respectively, during the production of the skim milk powder. Based on these consumption levels, it was concluded that, if recovery milk cannot be refrigerated and is stored at room temperature (25 to 35 degrees C), it must be used within 8 h and preferably within 6 h.

An immunohistological study of the integration at the bone-tendon interface after reconstruction of the anterior cruciate ligament in rabbits.

We studied bone-tendon healing using immunohistochemical methods in a rabbit model. Reconstruction of the anterior cruciate ligament was undertaken using semitendinosus tendon in 20 rabbits. Immunohistochemical evaluations were performed at one, two, four and eight weeks after the operation. The expression of CD31, RAM-11, VEGF, b-FGF, S-100 protein and collagen I, II and III in the bone-tendon interface was very similar to that in the endochondral ossification. Some of the type-III collagen in the outer layer of the graft, which was deposited at a very early phase after the operation, was believed to have matured into Sharpey-like fibres. However, remodelling of the tendon grafted into the bone tunnel was significantly delayed when compared with this ossification process. To promote healing, we believe that it is necessary to accelerate remodelling of the tendon, simultaneously with the augmentation of the ossification.

Inducement of tissue regeneration of harvested hamstring tendons in a rabbit model.

OBJECTIVES: The objective of this study was to determine if the use of fascia lata as a tendon regeneration guide (placed into the tendon canal following harvesting the semitendinosus tendon) would improve the incidence of tissue regeneration and prevent fatty degeneration of the semitendinosus muscle. MATERIALS AND METHODS: Bilateral semitendinosus tendons were harvested from rabbits using a tendon stripper. On the inducing graft (IG) side, the tendon canal and semitendinosus tibial attachment site were connected by the fascia lata, which was harvested at the same width as the semitendinosus tendon. On the control side, no special procedures were performed. Two groups of six rabbits were killed at post-operative weeks 4 and 8, respectively. In addition, three healthy rabbits were killed to obtain normal tissue. We evaluated the incidence of tendon tissue regeneration, cross-sectional area of the regenerated tendon tissue and proportion of fatty tissue in the semitendinosus muscle. RESULTS: At post-operative week 8, the distal end of the regenerated tissue reached the vicinity of the tibial insertion on the control side in two of six specimens. On the IG side, the regenerated tissue maintained continuity with the tibial insertion in all specimens. The cross-sectional area of the IG side was significantly greater than that of the control side. The proportion of fatty tissue in the semitendinosus muscle on the IG side was comparable with that of the control side, but was significantly greater than that of the normal muscle. CONCLUSIONS: Tendon tissue regenerated with the fascia lata graft was thicker than naturally occurring regenerated tissue. However, the proportion of fatty tissue in the semitendinosus muscle was greater than that of normal muscle.Cite this article: K. Tabuchi, T. Soejima, H. Murakami, K. Noguchi, N. Shiba, K. Nagata. Inducement of tissue regeneration of harvested hamstring tendons in a rabbit model. Bone Joint Res 2016;5:247-252. DOI: 10.1302/2046-3758.56.2000585.

Clinical significance of serum pulmonary surfactant proteins a and d for the early detection of radiation pneumonitis.

PURPOSE: Radiation pneumonitis (RP) is one of the most serious complications for patients who receive thoracic irradiation. To avoid this, early diagnosis of radiation pneumonitis is extremely important. The purpose of the present study is to investigate whether serum pulmonary surfactant proteins A and D (SP-A and SP-D, respectively) could be useful markers for RP. METHODS AND MATERIALS: Eighty-six patients (lung cancer: 42 [primary: 39, metastatic: 3], breast cancer: 23, esophageal cancer: 21) who underwent radiation therapy were prospectively studied. Radiation doses ranged from 30-76 Gy (median, 58 Gy). Serum SP-A and SP-D levels were evaluated sequentially by a sandwich enzyme-linked immunosorbent assay (ELISA) method before, during, and throughout the follow-up period until the development of symptomatic RP or until one year after completion of radiotherapy. Specificity of the ELISA results was confirmed by Western blot analysis. Patients symptomatic for RP were graded according to the Common Toxicity Criteria. RESULTS: RP occurred in 19 patients. Serum SP-D levels of patients with RP were sequentially higher than those in patients without RP. In the monitoring, serum SP-D levels at 50-60 Gy showed greater sensitivity and positive predictive values for RP detection (74% and 68%, respectively) than SP-A (26% and 21%, respectively). Western blot analysis showed that the development of RP was due to overproduction, but not proteolysis of surfactant proteins. CONCLUSION: We confirm that serum SP-A and SP-D monitoring is a practical and useful diagnostic method for the early detection of RP.

Endoscopic findings of radiation esophagitis in concurrent chemoradiotherapy for intrathoracic malignancies.

BACKGROUND AND PURPOSE: The incidence and extent of radiation esophagitis were assessed endoscopically in patients treated with concurrent chemoradiotherapy. PATIENTS AND METHODS: Eighty-two patients who received thoracic radiotherapy for lung, thymic, or esophageal cancer were investigated endoscopically from July 1991 to the end of 1997. Among them, 23 esophageal cancer patients were treated with radiation alone, and the others were treated with concurrent chemoradiotherapy. Esophageal endoscopy was performed during or just after radiotherapy. The presence of radiation esophagitis was assessed and assigned an endoscopic score (i.e. grade 0 for normal, 1 for erythema, 2 for erosion or sloughing, 3 for ulcer, hemorrhage, or stricture). The symptomatic grade was assessed using the RTOG (Radiation Therapy Oncology Group) acute radiation morbidity score. RESULTS: A correlation was seen between endoscopic and RTOG scores. However, even some patients with RTOG grade 0 to 1 had endoscopic grade 3 esophagitis. Endoscopic grade 3 was observed in 16 (27.1%) patients in the concurrent chemoradiotherapy group, whereas it did not occur in any patient in the radiation alone group (P=0.004). CONCLUSIONS: Our results suggest that (1) RTOG score correlates closely to esophageal mucosal damage, and (2) more severe esophagitis occurs in those undergoing concurrent chemoradiotherapy than those undergoing radiotherapy alone [corrected].

Radiotherapy after hyperbaric oxygenation for malignant gliomas: a pilot study.

The results of radiotherapy combined with hyperbaric oxygen in 9 patients with malignant glioma were compared with those of radiotherapy without hyperbaric O2 in 12 patients. This is the first report of a pilot study of irradiation immediately after exposure to hyperbaric O2 in humans. All patients receiving this treatment showed more than 50% regression of the tumor, and in 4 of them, the tumors disappeared completely. Only 4 out of 12 patients without hyperbaric O2 showed decreases in tumor size, and all 12 patients died within 36 months. So far, this new regimen seems to be a useful form of radiotherapy for malignant gliomas.

Prognostic factors and value of adjunctive nephrectomy in patients with stage IV renal cell carcinoma.

We reviewed 57 cases of Stage IV renal cell carcinoma to clarify the factors influencing prognosis and to evaluate the value of nephrectomy. Cumulative survival from the initial diagnosis was analyzed with respect to the patients' age, sex, side of primary tumor, initial performance status (PS), site of metastasis, and nephrectomy. Overall survival for the patients was 51 percent at one year, 22 percent at three years, and 11 percent at five years. Age, sex, and side of primary tumor had no influence on survival. Improved survival was correlated with good PS, metastases limited to single organ, and removal of the primary tumor. With regard to histopathologic features in nephrectomized patients, low grade and stage were correlated with longer survival. These factors should be considered in the analysis of results of future clinical trials of metastatic renal cell carcinoma.

Target cells of apoptosis in the adult murine dentate gyrus and O4 immunoreactivity after ionizing radiation.

The occurrence of radiation-induced apoptosis and the determination of target cells were investigated by using the TdT-mediated dUTP-biotin nick end labeling assay and immunohistochemical analyses. The O4 immunoreactivity, an oligodendrocytes surface antigen, was also evaluated by using western blotting analysis. C57BL/6J adult female mice were subjected to single dose irradiation of 10 Gy. Eight hours after irradiation, the most significant increase of apoptotic cells was detected in the subgranular zone and the hilus of the dentate gyrus. The target cells of radiation-induced apoptosis are the subgranular progenitor cells and the oligodendrocytes in the hilus. The amount of the O4 immunoreactivity, a marker for premature oligodendrocytes, was unchanged until 8 h but enhanced after 12 h of irradiation. These results are the first to show the increase of the O4 immunoreactivity after irradiation and may be associated with the pathogenesis of radiation injury.

An integrated solar and artificial light system for internal illumination of photobioreactors.

Exploitation of photosynthetic cells for the production of useful metabolites requires efficient photobioreactors. Many laboratory scale photobioreactors have been reported but most of them are extremely difficult to scale up. Furthermore, the use of open ponds and outdoor tubular photobioreactors is limited by the requirement for large spaces and the difficulty in maintaining sterile conditions. In view of this, we have designed and constructed an internally illuminated stirred tank photobioreactor. The photobioreactor is simple, heat sterilizable and mechanically agitated like the conventional stirred tank bioreactors. Furthermore, it can easily be scaled up while maintaining the light supply coefficient and thus the productivity constant. A device was installed for collecting solar light and distributing it inside the reactor through optical fibers. It was equipped with a light tracking sensor so that the lenses rotate with the position of the sun. This makes it possible to use solar light for photosynthetic cell cultivation in indoor photobioreactors. As a solution to the problems of night biomass loss and low productivity on cloudy days, an artificial light source was coupled with the solar light collecting device. A light intensity sensor monitors the solar light intensity and the artificial light is automatically switched on or off, depending on the solar light intensity. In this way, continuous light supply to the reactor is achieved by using solar light during sunny period, and artificial light at night and on cloudy days.

Pachymeningoencephalitis: case report.

Hypertrophic cranial pachymeningitis is uncommon. We report the first case of pachymeningitis extending to the cerebral parenchyma (pachymeningoencephalitis) and involving the bone and extracranial soft tissue. The clinical features and pathogenesis are discussed.

Marked growth of an angiographically occult arteriovenous malformation: case report.

The authors report a case of an angiographically occult arteriovenous malformation (AVM) that showed a marked increase in size over a period of 7 years. The importance of accurate diagnosis, follow-up computed tomography and repeated angiography, if needed, is stressed in cases where enlargement of an occult AVM is suspected.

Comparison between ultrafiltration and trichloroacetic acid precipitation method for concentration of Staphylococcus aureus enterotoxin in dairy samples.

Staphylococcus aureus enterotoxin that may be contained at low concentrations in milk and dairy products can cause food poisoning. To detect this enterotoxin at low concentrations, samples should be concentrated. We evaluated the performance of centrifugal ultrafiltration method (UF) in comparison with trichloroacetic acid precipitation method (TCA) for the concentration of S. aureus enterotoxin in milk and dairy products. S. aureus enterotoxin A (SEA) were added at various concentrations to ultra high-temperature heating process (UHT) milk, UHT concentrated skim milk, UHT skim milk powder, low heat-treated (LH) skim milk powder, and raw milk. SEA was concentrated by TCA and UF once a day on a total of 3 days by different researchers to prepare test solutions. The fluorescence value (TV) of test solutions was determined using an immunofluorescence autoanalyzer (miniVIDAS), and the linearity and slope of the regression line, relative standard deviation (RSD(RW)) at each added concentration, detection limit (DL), quantification limit (QL), and the recovery rate by each concentration method were obtained according to the guidelines of the International Conference on Harmonization (ICH). The slope of the regression line obtained by UF was steeper than that by TCA for all dairy samples excluding LH (74 degrees C, 20 s) skim milk powder. RSDRW, DL, and QL obtained by UF were comparable to or more excellent than those obtained by TCA. The procedure of UF was simpler than that of TCA. The recovery rate and rapidity were similar between the two methods. The DL and QL of enterotoxins other than SEA in dairy products by UF or TCA were estimated based on the DL and QL of SEA. In this estimation, consideration was given to reactions between each enterotoxin and its antibody, and also to the immunoactivity maintenance rate of each enterotoxin after addition of trichloroacetic acid in TCA. The estimated values were similar to those obtained by experiments using enterotoxin C1 (SEC1). UF using a centrifugal ultrafiltration membrane can be more readily performed and similar to or more reliable than TCA. UF combined with a miniVIDAS can be used for quantitative routine analysis.

Multivariate analysis of single unit cells in electron crystallography.

High-resolution electron cryomicroscopy of two-dimensional protein crystals is associated with extremely noisy raw data in which even the crystal lattice often cannot be discerned. Correlation averaging procedures, aimed at calculating the total average of all unit cells of crystals in order to reduce noise, are now used routinely in electron crystallography. Multivariate statistical analysis (MSA) may be used for finding not only the average structure but also for quantifying the systematic departures from that average within the population of individual unit cells. We show that the MSA approach is applicable to single unit-cell images in the low-dose (< 10 electrons/A2), high-resolution (< 5 A) realm using 400 keV electron spot-scan images of ice-embedded gp32*I protein crystals. Our feasibility study opens a pathway toward exploiting these naturally occurring variations on the unit-cell theme in order to achieve higher-resolution three-dimensional reconstruction results, or to better understand the dynamic behaviour of molecules within two-dimensional crystals. We explain how single unit-cell images can be processed and classified into homogeneous groups, and we review how the results of such discriminate averaging may subsequently be exploited within the context of conventional "h, k"-space electron crystallographic approaches. Variations among the individual unit cells may thus be one of the most significant resolution-limiting factors currently experienced in electron crystallography. The quantitative assessment and exploitation of such variations may lead to an increased performance of electron crystallographic procedures.

Tachykinin-induced responses via neurokinin-1 and -3 receptors in hamster submandibular ganglion neurones.

Both substance P and neurokinin A are known as neurotransmitters of the submandibular ganglion cell. In this study, the effects of neurokinin (NK) receptor-subtype agonists on hamster submandibular ganglion cells were investigated using the whole-cell patch-clamp technique. Membrane currents evoked by a ramp pulse from +50 to -100 mV (-150 mV/1000 msec) were compared in both the absence and presence of NK receptor agonist. The NK-1 receptor agonist [Sar9, Met (O2)11]-substance P, the NK-2 receptor agonist [Ala5, beta-Ala8]-alpha-neurokinin fragment 4-10, and the NK-3 receptor agonist senktide were used. The three agonists dose-dependently increased the amplitude of the inward current with a reversal potential near 0 mV. Their rank order was NK-1 = NK-3 > NK-2. Even when the external solution was replaced with Cs+ or N-methyl-D-glucamine+ instead of Na+, the NK receptor agonists also increased the amplitude of the inward current. Thus, NK-1 and NK-3 receptors are apparently coupled with non-selective cation channels in submandibular ganglion cells.

Sinus Pericranii.

Two cases of sinus pericranii are reported. X-ray films, taken with the direct injection of contrast medium into the extracranial blood cavity, made visualization of the intracranial venous systems possible in both cases. The pericranial blood sinuses were totally removed and communications through the osseous holes were closed with bone wax. The pericranial blood sinuses, surrounded by connective tissues, were located mainly in the subgaleal and supraperiosteal space. The literature is reviewed.

Mixed colloid cyst-xanthogranuloma of the third ventricle. A light and electron microscopic study.

A mixed colloid cyst-xanthogranuloma of the third ventricle is described. The xanthogranuloma associated with the cyst completely occupied the third ventricle. On light microscopy, a single epithelial layer with a basal lamina was seen lining the cyst wall. Electron microscopic study revealed three different kinds of cells in the epithelial layer: ciliated, nonciliated, and basal cells. Many lipid droplets were present in the superficial epithelial cells, macrophages, pericytes, and endothelia. The pathogeneses of the cyst and of the xanthogranuloma are discussed.

Analysis of strain distribution in the medial collateral ligament using a photoelastic coating method.

The strain distribution over the entire medial collateral ligament (MCL) was measured using a photoelastic coating method. This new approach utilized a polyurethane monomer as a photoelastic coating film. The initial experiments investigating MCL strain measurement showed that this film had a high sensitivity for strain and good adhesion to the ligament. It was confirmed that strain distribution could be obtained qualitatively over the entire ligament using this method. The mechanism of MCL injury was studied by applying this polyurethane coating film to the entire MCL in a femur-MCL-tibia complex. When simple tension was applied to the complex, strain concentrations were centred at the tibial insertion site, and all the specimens ruptured at the MCL tibial insertion site. With application of a valgus bending moment, increased strain was seen in the MCL from the medial femoral condyle to the medial epicondyle. Histological analysis demonstrated midsubstance ligament ruptures in this same region. For both tests, rupture sites and increased strain concentration sites correlated. In addition, an impingement phenomenon of the MCL on the medial femoral condyle can be seen during application of valgus force, and this phenomenon may explain the higher incidence of MCL injuries on the femoral side seen in the clinical setting. This polyurethane coating method allows for direct and visual measurements, and can qualitatively measure the strain behaviour over the entire MCL surface. This new technique represents a significant improvement over previous point-by-point strain measurement methods.

Development of a water-soluble preparation of emamectin benzoate and its preventative effect against the wilting of pot-grown pine trees inoculated with the pine wood nematode, Bursaphelenchus xylophilus.

Water-soluble preparations have been investigated to develop a trunk injection agent based on the poorly water-soluble anti-nematode emamectin benzoate. Following tests on the phytotoxicity of some solvents and solubilizers and demonstration of the ability of some solubilizers to dissolve emamectin benzoate in water, acetone + methanol was selected as the solvent and Polysorbate 80 as the solubilizer. This water-soluble preparation of emamectin benzoate prevented the wilting of pot-grown 4-year-old trees of the Japanese black pine, Pinus thunbergii, artificially inoculated with the pine wood nematode, Bursaphelenchus xylophilus, at a dose of 20 g emamectin benzoate per cubic metre of pine tree.