Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number.

Recently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators' concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

Novel Bioprinting Application for the Production of Reference Material Containing a Defined Copy Number of Target DNA.

Nucleic acid amplification methods, such as polymerase chain reaction (PCR), are extensively used in many applications to detect target DNA because of their high sensitivity, good reproducibility, and wide dynamic range of quantification. However, analytical quality control when detecting low copy number target DNA is often missing because of a lack of appropriate reference materials. Recent advances in analytical sciences require a method to accurately quantify DNA at the single molecule level. Herein, we have developed a novel method to produce reference material containing a defined copy number of target DNA (referred to as "cell number-based DNA reference material"). In this method, a suspension of cells carrying a single target DNA sequence was ejected by an inkjet head, and the number of cells in each droplet was counted using highly sensitive cameras. The resulting solutions contained a defined copy number of target DNA and could be used as reference materials. The use of the newly developed reference material was compared with that of diluted solutions of target DNA to evaluate the performance of qualitative real-time PCR in terms of the limit of detection (LOD). Our results demonstrated that cell number-based DNA reference material provides more accurate information regarding performance quality. The reference material produced by this method is a promising tool to evaluate assay performance.

Right coronary artery stenosis associated with tricuspid valve ring annuloplasty.

A 70-year-old man with severe symptomatic functional mitral valve regurgitation underwent successful mitral valve repair combined with tricuspid valve ring annuloplasty. Pre-operative coronary angiography (CAG) showed no significant stenosis. One-and-a-half years later, the patient presented with an acute exacerbation of heart failure. Repeat CAG demonstrated tight stenosis in the right coronary artery (RCA) with arterial kinking that corresponded to the same point as the stenosis where the RCA is the closest to the tricuspid valve ring. The new lesion probably occurred as a consequence of the tricuspid valve ring annuloplasty.

A successful treatment for concomitant injury of the coronary artery and tricuspid valve after blunt chest trauma.

A 63-year-old woman involved in an automobile accident was brought to our hospital with thoracic injury sustained by the impact of her vehicle's steering wheel. Cardiac auscultation revealed a grade III/VI systolic murmur and the electrocardiogram showed ST elevation in leads 2, 3 and aVF. A 2D echocardiogram revealed severe tricuspid regurgitation and a hypokinetic right ventricle. Coronary angiography revealed dissection of the proximal right coronary artery (RCA) with 90 % stenosis. Urgent CABG for the RCA and tricuspid valvuloplasty were performed, as the anterior leaflet of the tricuspid valve had prolapsed as a result of chordal rupture. Blunt thoracic trauma causing both tricuspid insufficiency and coronary artery dissection is a very rare and life-threatening situation. Prompt diagnosis and timely surgery enabled us to save this patient's life.

Cardiac myxosarcoma with thoracic spinal metastasis.

Echocardiography revealed a left atrial tumor in a 59-year-old man with back pain that concurrently worsened with left foot drop and loss of the left ankle reflex soon after admission to our hospital. Magnetic resonance imaging of the spine revealed an epidural tumor extending from Th5 with spinal cord compression. The patient was immediately treated by emergency Th4-5 laminectomy and epidural decompression. One month later, a cardiac tumor excised via the left atrial approach was histopathologically diagnosed as myxosarcoma, and the Th5 tumor was consistent with this finding. This is the first report to describe spinal metastasis of cardiac myxosarcoma.

A case of rectal obstruction caused by bilateral internal iliac artery aneurysms.

In this article, we report a rare case of rectal obstruction caused by bilateral internal iliac artery aneurysms that required open surgical repair. A 73-year-old man was admitted to our hospital complaining of abdominal pain and persistent constipation for >1 month. Computed tomography demonstrated bilateral internal iliac artery aneurysms, 5.0 and 7.0 cm each in diameter, which occupied the intrapelvic space. An urgent surgery was performed to reduce the volume of the aneurysms and release the obstructed rectum. The postoperative course was uneventful, in which he had good evacuation. Aneurysms in the iliac region can be a good indication for the use of newly developed endovascular devices; however, open surgery should be considered without delay to avoid ileus or subileus symptoms when the aneurysms cause space-occupying complications.

Awake partial sternotomy pacemaker implantation under thoracic epidural anesthesia.

An 82-year-old man with symptomatic bradycardia had infective endocarditis with tricuspid vegetation. The patient also had severe obstructive pulmonary disease, and endotracheal general anesthesia was contraindicated. Under satisfactory epidural anesthesia with catheterization at the T5/6 level, lower partial sternotomy was performed, and dual pacing electrodes were placed on the heart. Throughout the surgery, spontaneous breathing was maintained with a sufficient level of oxygenation. The postoperative course was uneventful. Although awake cardiac surgery under thoracic epidural anesthesia is challenging, this less invasive technique was useful for epicardial pacemaker implantation in this patient with severe pulmonary dysfunction.

Successful surgical treatment for infective endocarditis during pregnancy.

We herein describe the case of a 31-year-old woman. In the 27th week of pregnancy, the patient was hospitalized because of fever and a lumbar backache. In the 29th week of pregnancy, she developed embolic symptom in her left lower limb. A cardiac murmur was detected, and a significant regurgitation of the mitral valve, along with a mobile vegetation at the posterior leaflet of the mitral valve, was detected by cardiac ultrasound examination, thus resulting in a diagnosis of infective endocarditis. At the time of diagnosis, the fetus was estimated to weigh 1400 g, and it was delivered by cesarian section, with a mitral valve repair being performed 4 days later. The mother did well and was discharged from the hospital after remission on the 30th hospital day. The infant was admitted to the NICU and was discharged from the hospital with good development and no complications at the age of 59 days, weighing 3066 g. Cardiac surgery under extracorporeal circulation in pregnant women is rare, and it is believed to have a high mortality rate for both the mothers and fetuses. On the other hand, the survival rate of low birth weight infants has improved as a consequence of progress in neonatal care. We herein report a case of mitral valve repair in the second trimester with a good outcome for both the mother and the infant.

Endovascular stent-grafting for recurrent aneurysm in Behcet's disease.

Arterial aneurysms represent a severe complication of Behcet's disease. A 42-year-old woman with Behcet's disease had a recurrence of an aneurysm after two surgical repair attempts using grafts. A covered stent-graft was implanted in her iliac external artery to occlude the neck of the aneurysm at the anastomosis of the bypass graft to her external iliac artery. The procedure reduced the size of the aneurysm by allowing the formation of a thrombus within its cavity. The implantation of an endovascular stent-graft may be a sound alternative to surgical repair for aneurysms associated with Behcet's disease.

Inverted L-shape sternotomy as a minimally invasive approach: re-do cardiac surgery for papillary muscle rupture.

We report a successful treatment of the complete papillary muscle rupture occurring 16 months after coronary artery bypass grafting (CABG). A 57-year-old man was admitted for the sudden onset of chest pain and cardiogenic shock. Emergency cardiac catheterization revealed severe mitral regurgitation and total occlusion in the right coronary artery, which was successfully revascularized by percutaneous coronary intervention under intra-aortic balloon pumping. The right internal thoracic artery grafted to the left anterior descending artery in the previous CABG was functioning well. An echocardiogram distinctly indicated the ruptured head of the papillary muscle. Since an emergency operation revealed complete rupture of the posterior papillary muscle, mitral valve replacement was carried out through an inverted L-shape sternotomy with T-shape left atriotomy. Our case indicates that the inverted L-shape sternotomy was a useful approach to preserve the function of grafts, and that T-shape left atriotomy offered a good exposure of the mitral valve in the limited surgical field.

Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography.

Two flavonoid glycosides, kaempferol 3-O-(6"-acetyl)-beta-glucopyranoside (KAG) and quercetin 3-O-(6"-acetyl)-beta-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol alpha, delta, and epsilon), but not other pol beta, eta, kappa, and lambda activities. KAG was a stronger inhibitor and more selective to pol alpha than QAG. The IC(50) values of KAG for pol alpha, delta, and epsilon were 41, 164, and 127 microM, respectively. The pol alpha inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol alpha, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol alpha was selectively adsorbed and eluted.

Effects of glycolipids from spinach on mammalian DNA polymerases.

We purified the major glycolipids in the class of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) from a green vegetable, spinach (Spinacia oleracea L.). MGDG was an inhibitor of the growth of NUGC-3 human gastric cancer cells, but DGDG and SQDG had no such cytotoxic effect. Therefore, we studied MGDG and its monoacyglycerol-form, monogalactosyl monoacylglycerol (MGMG), in detail. MGMG with one fatty acid molecule was obtained from MGDG with two fatty acid molecules by hydrolyzing with a pancreatic lipase. MGMG was also found to prevent the cancer cell growth. MGDG was a potent inhibitor of replicative DNA polymerases such as alpha, delta and epsilon. MGMG inhibited the activities of all mammalian DNA polymerases including repair-related DNA polymerase beta with IC(50) values of 8.5-36 microg/mL, and the inhibition by MGMG was stronger than that by MGDG. Both MGDG and MGMG could halt the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The relationship between the DNA polymerase inhibition and the cell growth effect by these glycolipids is discussed.

Rupture of a smoldering mycotic aneurysm of the thoracic aorta into the lung.

A 65-year-old man was a diagnosed with meningitis and bacteremia, as Streptococcus pneumoniae was isolated from spinal fluid and blood cultures. After three weeks of antibiotic therapy, computed tomography revealed a ruptured aneurysm of the descending thoracic aorta. The aneurysm had appeared during the first episode of meningitis but had remained silent for two years. The patient underwent in situ Dacron graft replacement and his postoperative course was uneventful with no infectious complication.