Brachial plexus injury and upper rib fracture after median sternotomy in cardiac surgery.

OBJECTIVES: Sternal retractors utilized during open-heart surgeries through median sternotomy can cause upper rib fractures which sometimes further leads to brachial plexus injury. We aimed to investigate the incidence of brachial plexus injury and upper rib fractures in open-heart surgeries and how these injuries are associated with each other. METHODS: We investigated 1050 cases during the past five years. The incidence of brachial plexus injury and upper rib fractures after median sternotomy was assessed in all patients and the patients who sustained were evaluated for the affected side, the level of paralysis. RESULTS: Ten cases (0.95%) exhibited brachial plexus injury after median sternotomy. Nine cases developed paralysis on left upper extremity. In all ten cases, sensory and motor nerve impairment were exhibited in the lower plexus. Rib fractures were observed in 35.0% of cases after median sternotomy and the usage of asymmetric sternal retractors to harvest left internal thoracic artery (LITA) significantly affected the side of fracture. CONCLUSION: Sternal retractors utilized during open-heart surgeries through median sternotomy may cause rib fractures and brachial plexus injury, so operators should be aware of these complications.

Development of a Novel Hygiene Monitoring System Based on the Detection of Total Adenylate (ATP+ADP+AMP).

ATP is the universal energy molecule found in animals, plants, and microorganisms. ATP rapid hygiene monitoring tests have been employed in the food industry to ensure that adequate cleanliness is being maintained. However, because ATP is hydrolyzed to ADP and AMP by metabolic processes, by heat treatment, or under acidic or alkaline conditions, total adenylate (ATP+ADP+AMP [A3]) could be a more reliable sanitation indicator of food residues that may cause biofilm formation and allergen contamination. Therefore, a novel hygiene monitoring system to measure A3 was developed based on the luciferin-luciferase assay with the combination of two enzymes, pyruvate kinase and pyruvate phosphate dikinase, that can convert ADP into ATP and recycle AMP into ATP, respectively. The newly developed A3 assay system afforded stable bioluminescence signals and equivalent linear calibration curves between relative light units (RLU) and the amounts of ATP, ADP, and AMP, respectively. To verify the significance of the A3 method, the ratios of ATP, ADP, and AMP in various food samples were determined; large amounts of ADP and AMP were found in a variety of foods, such as meat, seafood, dairy, nuts, fruits, vegetables, and fermented foods. Sanitation monitoring of stainless steel exposed to raw meat was also examined, and the A3 method achieved a 200-RLU level, the typical benchmark value, after complete washing with detergent and rinsing. In contrast, a conventional ATP method showed less than 200 RLU after only a light cold and hot water rinse. In conclusion, the A3 assay appeared to be suitable for detection of adenylates from food residues that are not detected by the conventional ATP assay.

Assembly of zinc finger motif-fused enzymes on a dsDNA scaffold for catalyzing consecutive reactions with a proximity effect.

The feasibility of assembling enzymes, catalyzing consecutive reactions, on to a double-stranded DNA (dsDNA) scaffold utilizing zinc finger motifs is described. The catalytic activities of two zinc finger motif-fused enzymes catalyzing a bioluminescence reaction with energy recycling, namely pyruvate phosphate dikinase and firefly luciferase, have been evaluated. Bioluminescence measurements with dsDNA scaffolds coding a different distance between the binding sites for each zinc finger motif-fused enzyme confirmed the effect of the distance, proving the proximity effect of ATP recycling presumed to be the result of efficient intermediate diffusion. Thus, fusion to zinc finger motifs offers a promising option for the assembly of bi-enzymes, catalyzing a consecutive reaction, onto a dsDNA scaffold with a proximity effect.

Evaluation of an improved bioluminescence assay for the detection of bacteria in soy milk.

Because soy milk is nutrient rich and nearly neutral in pH, it favors the growth of microbial contaminants. To ensure that soy milk meets food-safety standards, it must be pasteurized and have its sterility confirmed. ATP bioluminescence assay has become a widely accepted means of detecting food microorganisms. However, the high background bioluminescence intensity of soy milk has rendered it unsuitable for ATP analysis. Here, we tested the efficacy of an improved pre-treated bioluminescence assay on soy milk. By comparing background bioluminescence intensities obtained by the conventional and improved methods, we demonstrated that our method significantly reduces soy milk background bioluminescence. The dose-response curve of the assay was tested with serial dilutions of Bacillus sp. culture. An extremely strong log-linear relation between the bioluminescence intensity relative light units and colony formation units CFU/ml emerged for the tested strain. The detection limit of the assay was estimated as 5.2×10(3) CFU/ml from the dose-response curve and an imposed signal limit was three times the background level. The results showed that contaminated samples could be easily detected within 24 h using our improved bioluminescence assay.

A model to evaluate toxic factors influencing islets during collagenase digestion: the role of serine protease inhibitor in the protection of islets.

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.

Adenine nucleotide levels in a closed enzymatic digestion system for porcine islet isolation.

Obtaining viable islets is a crucial step for successful islet transplantation. Adenosine triphosphate (ATP) is a marker of cell viability. However, little is known about any changes in the energy status of the tissues that are being digested during the digestion phase. We herein examined whether the ATP content in serially digested pancreatic tissue samples could be specific objective parameters that signal the optimal point to stop the digestion process. We obtained partial pancreata (body to tail) from 4- to 5-year-old pigs from a slaughterhouse. The tissue samples were preserved in M-Kyoto solution for less than 3 h. They were digested using an automated enzymatic and mechanical dissociation system at 37°C for 90 min following intraductal injection of Liberase HI. Samples were collected from the digestive circuit every 5 or 10 min to determine the ATP level, total adenine nucleotide (TAN) level, islet count (count/g), and yield of islet equivalent (IEQ) in the serial digestive fluids. The ATP and TAN levels, IEQ and islet count were increased and then decreased during digestion process. The profile of these parameters differed from case to case. However, when ATP changing ratio (respective value/precedent value) was compared with IEQ changing ratio, a greater than threefold increase in the ATP changing ratio followed by an increase in the islet count changing ratio within 5 min was consistently observed, indicating the optimal time to stop the digestion. The ATP levels of the handpicked islets in the digested samples were lower in the overdigested phase in comparison to those in the earlier digested phase. These results indicate that the ATP level in digested fluid could be an effective indicator to estimate the viability of cells as well as determine the optimal time to terminate the digestion process in order to obtain viable islets.

Preservation of pancreatic islets in cold UW solution before transplantation.

Culture of islets prior to transplantation needs to be revisited for maintaining functional islet capacity. This study was conducted to compare cold UW (University of Wisconsin) preservation with conventional culture based on insulin secretory capacity in vitro and in vivo. Islets isolated from Wistar rats were either cultured for 24 h at 37°C in RPMI1640 medium or DMEM containing various concentrations of glucose or preserved for the same period in UW solution or in DMEM solution at 4°C. The islet yield in UW group, but not in other groups, was maintained as comparable with that of fresh islets. Insulin secretory capacity in response to glucose was maintained only in the islets of UW group, but not in other groups. SCID mice given 300 IEQ islets of UW group showed gradual restoration of normoglycemia as found in the mice given freshly isolated islets. Meanwhile, those mice given cultured islets for 24 h at 37°C in RPMI1640 medium showed rapid decrease of blood glucose levels on day 1 followed by relatively elevated levels on day 2, suggesting unstable insulin secretory capacity of islets.   Morphological staining with anti-HMGB1 (high mobility group B1) antibody revealed central damage of islets in all culture groups regardless of glucose concentration and in islets of cold DMEM group, whereas those in the UW group were quite intact. These results suggest that cold preservation in UW solution is simple and beneficial in protecting islets morphologically and functionally before transplantation.

Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis.

A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 µm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.

Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay.

Aberrant expression of miRNAs often correlates with various human diseases. Therefore, miRNAs have been focused as disease biomarkers. Here, a novel application of a bioluminescence (BL) assay for small RNA quantification is described. The assay is based on detecting pyrophosphate (PPi) molecules released during branched rolling circle amplification (BRCA) with a second primer in the presence of target RNA molecules. The number of released PPi molecules is correlated with the target RNA copy number. This assay was capable of detecting at least 20 amol of target RNA molecules, and the dynamic range extended over at least three orders of magnitude. Appropriate use of a second primer allowed for sensitive detection of RNA molecules with a high S/N ratio in less time. Moreover, the assay could specifically detect as low as 0.1 fmol of a target small RNA within a total RNA extract with high reproducibility. These data suggest that our assay has the potential to become a simple, rapid, and highly sensitive method to detect miRNA. Furthermore, this method combined with a BL assay, which utilizes a widely used inexpensive luminometer, could be used for a wider, versatile range of applications.

Nano-sized bacterial magnetic particles displaying pyruvate phosphate dikinase for pyrosequencing.

There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost.

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis.

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3' end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either alpha-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.

Enzyme system for improving the detection limit in pyrosequencing.

Highly sensitive real-time pyrosequencing seems promising for constructing an inexpensive and small DNA sequencer with a low running cost. A DNA sample of a picomole level is usually used in the conventional pyrosequencing based on a luciferase assay coupled with an APS-ATP surfurylase reaction for producing ATP from pyrophosphate (PPi). Although the luminescence intensity could be increased by increasing the amount of luciferase, it was impossible to reduce the target DNA amount because of a large background luminescence due to the luciferase-APS reaction. In this report, a novel approach using a new conversion reaction of PPi to ATP is proposed. This method has a very low background and can produce high signals in the presence of a large amount of luciferase; thus, the sample amount required for sequencing is significantly reduced. The ATP production from PPi is catalyzed with pyruvate orthophosphate dikinase (PPDK) using AMP and phosphoenolpyruvate as the substrates, which are inactive for the luciferase-catalyzed reaction. All of the components in the AMP-PPDK-based pyrosequencing system are suitable for highly sensitive DNA sequencing in one tube. Real-time DNA sequencing with a readable length up to 70 bases was successfully demonstrated by using this system. By increasing the amount of luciferase, as low as 2.5 fmol of DNA templates was accurately sequenced by the proposed method with a novel simple and inexpensive DNA sequencer having a photodiode array as a sensor instead of a PMT or CCD camera. A sample amount as low as 2 orders of magnitude smaller than that used in the conventional pyrosequencer can be used.

Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reactions and a novel bioluminescent pyrophosphate assay.

We developed a novel bioluminescent assay for detection of pyrophosphate in polymerase chain reaction (PCR) product. The principle of this method is as follows: pyrophosphate released by PCR is converted to adenosine 5'-triphosphate (ATP) by pyruvate phosphate dikinase in the presence of the substrate pyruvate phosphate and the coenzyme adenosine 5'-monophosphate; subsequently, ATP concentration is determined by firefly luciferase reaction. The detection limit of pyrophosphate is 1.56 x 10(-15)mol/assay. Additionally, luminescent intensity reached a maximum at approximately 100 s and remained elevated beyond 10 min. This approach is applicable to the detection of cariogenic bacteria in dental plaque. Thus, the allele-specific PCR products of Streptococcus mutans and Streptococcus sobrinus developed in this study were measured via the proposed bioluminescent assay. This protocol, which does not require expensive equipment, can be utilized to rapidly monitor cariogenic bacteria in dental plaque.