Identification of Genetic and Genomic Influences on Progressive Ethanol Consumption in Diversity Outbred Mice.

Genetic factors play a significant role in the risk for development of alcohol use disorder (AUD). Using 3-bottle choice intermittent access ethanol (IEA), we have employed the Diversity Outbred (DO) mouse panel as a model of alcohol use disorder in a genetically diverse population. Through use of gene expression network analysis techniques, in combination with expression quantitative trait loci (eQTL) mapping, we have completed an extensive analysis of the influence of genetic background on gene expression changes in the prefrontal cortex (PFC). This approach revealed that, in DO mice, genes whose expression was significantly disrupted by intermittent ethanol in the PFC also tended to be those whose expression correlated to intake. This finding is in contrast to previous studies of both mice and nonhuman primates. Importantly, these analyses identified genes involved in myelination in the PFC as significantly disrupted by IEA, correlated to ethanol intake, and having significant eQTLs. Genes that code for canonical components of the myelin sheath, such as Mbp, also emerged as key drivers of the gene expression response to intermittent ethanol drinking. Several regulators of myelination were also key drivers of gene expression, and had significant QTLs, indicating that genetic background may play an important role in regulation of brain myelination. These findings underscore the importance of disruption of normal myelination in the PFC in response to prolonged ethanol exposure, that genetic variation plays an important role in this response, and that this interaction between genetics and myelin disruption in the presence of ethanol may underlie previously observed behavioral changes under intermittent access ethanol drinking such as escalation of consumption.

Hospital-acquired Clostridium difficile infection: an institutional costing analysis.

BACKGROUND: Healthcare-acquired Clostridium difficile infection (HA-CDI) is a common infection and a financial burden on the healthcare system. AIM: To estimate the hospital-based financial costs of HA-CDI by comparing time-fixed statistical models that attribute cost to the entire hospital stay to time-varying statistical models that adjust for the time between admission, diagnosis of HA-CDI, and discharge and that only attribute HA-CDI costs post diagnosis. METHODS: A retrospective cohort study was conducted (April 2008 to March 2011) using clinical and administrative costing data of inpatients (≥15 years) who were admitted to The Ottawa Hospital with stays >72 h. Two time-fixed analyses, ordinary least square regression and generalized linear regression, were contrasted with two time-dependent approaches using Kaplan-Meier survival curve. FINDINGS: A total of 49,888 admissions were included and 366 (0.73%) patients developed HA-CDI. Estimated total costs (Canadian dollars) from time-fixed models were as high as $74,928 per patient compared to $28,089 using a time-varying model, and these were 1.47-fold higher compared to a patient without HA-CDI (incremental cost $8,997 per patient). The overall annual institutional cost at The Ottawa Hospital associated with HA-CDI was as high as $10.07 million using time-fixed models and $1.62 million using time-varying models. CONCLUSION: When calculating costs associated with HA-CDI, accounting for the time between admission, diagnosis, and discharge can substantially reduce the estimated institutional costs associated with HA-CDI.

Regeneration of hyaline cartilage by cell-mediated gene therapy using transforming growth factor beta 1-producing fibroblasts.

Transforming growth factor beta (TGF-beta) has been considered as a candidate for gene therapy of orthopedic diseases. The possible application of cell-mediated TGF-beta gene therapy as a new treatment regimen for degenerative arthritis was investigated. In this study, fibroblasts expressing active TGF-beta 1 were injected into the knee joints of rabbits with artificially made cartilage defects to evaluate the feasibility of this therapy for orthopedic diseases. Two to 3 weeks after the injection there was evidence of cartilage regeneration, and at 4 to 6 weeks the cartilage defect was completely filled with newly grown hyaline cartilage. Histological analyses of the regenerated cartilage suggested that it was well integrated with the adjacent normal cartilage at the sides of the defect and that the newly formed tissue was indeed hyaline cartilage. Our findings suggest that cell-mediated TGF-beta 1 gene therapy may be a novel treatment for orthopedic diseases in which hyaline cartilage damage has occurred.

Transthoracic echocardiographic demonstration of massive pulmonary thrombus caused by protein C deficiency.

Few cases of pulmonary embolism detected by transthoracic echocardiography (TTE) have been reported. We present a case of a patient affected by pulmonary embolism caused by protein C deficiency. Transthoracic echocardiography showed a thrombus in transit (ie, visualization of a thrombus within the pulmonary artery). A hypercoagulable state caused by deficiency of protein C is a rare cause of pulmonary thromboembolism. Our experience demonstrates a massive pulmonary thrombus resulting from such a deficiency. Transthoracic echocardiography should be considered as the first diagnostic method for patients with suspected pulmonary embolism.

Purification and characterization of coclaurine N-methyltransferase from cultured Coptis japonica cells.

S-Adenosyl-L-methionine (SAM): coclaurine N-methyltransferase (CNMT), which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. was purified 340-fold from Coptis japonica cells in 1% yield to give an almost homogeneous protein. The purified enzyme, which occurred as a homotetramer with a native Mr of 160 kDa (gel-filtration chromatography) and a subunit Mr of 45 kDa (SDS-polyacrylamide gel electrophoresis), had an optimum pH of 7.0 and a pI of 4.2. Whereas (R)-coclaurine was the best substrate for enzyme activity, Coptis CNMT had broad substrate specificity and no stereospecificity CNMT methylated norlaudanosoline, 6,7-dimethoxyl-1,2,3,4-tetrahydroisoquinoline and 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. The enzyme did not require any metal ion. p-Chloromercuribenzoate and iodoacetamide did not inhibit CNMT activity, but the addition of Co2+, Cu2+ or Mn2+ at 5 mM severely inhibited such activity by 75, 47 and 57%, respectively. The substrate-saturation kinetics of CNMT for norreticuline and SAM were of the typical Michaelis-Menten-type with respective Km values of 0.38 and 0.65 mM.

Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor.

A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12-15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100-500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture.

Prevention of primary nonfunction after canine liver allotransplantation: the effect of gadolinium chloride.

PURPOSE: Effective suppression of Kupffer cell function is believed to contribute to the prevention of preservation/reperfusion injury. In this study, effect of gadolinium, a synthetic Kupffer cell suppressant, on the reperfusion injury was examined using a canine partial liver transplantation model. METHODS: About a 70% partial liver segment was harvested and reimplanted in a mongrel recipient dog weighing 20 to 25 kg. Gadolinium chloride (10 mg/kg) was infused via the cephalic vein 24 hours before harvest of the partial liver (gadolinium group, n = 5). Serum aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and morphologic grading of graft were compared with those of a control group (n = 5). Statistical analysis was done with an independent t-test. RESULTS: Average total ischemic time was 4 hours and 27 minutes. At 1 hour after reperfusion, there were no significant differences in AST, ALP, or LDH levels, or pathologic scores. At 48 hours after reperfusion, AST (P = .03) and LDH (P = .05) levels were significantly lower in the gadolinium group. CONCLUSION: Kupffer cell blockade using gadolinium chloride may be effective to reduce ischemia reperfusion injury, but the effect is not evident at an early stage of reperfusion.

Multijet and multistage aerosol concentrator: design and performance analysis.

A multijet and multistage aerosol concentrator was designed and fabricated with two virtual impactors in a series. Collection efficiency, internal loss, and concentration factors were calculated at ambient conditions for each stage. The total inlet flow rate of the aerosol concentrator was set at 1000 L/min(-1), while the minor flow rate for the first stage was at 6.0% of the total inlet flow and the minor flow rate of the second stage was at 6.7% of the first stage minor flow. The aerosol concentrator was calibrated using polystyrene latex particles in aerodynamic sizes ranging from 0.5 to 10 microm. Several configurations of the multijet acceleration nozzles and multitube receptors were designed in this study. The effects of the different designs were subsequently evaluated through experimentation. It was found that a properly designed multijet and multistage aerosol concentrator can significantly improve aerosol concentration performance. Results showed that the concentration factor increases from 1 to 240 over the particle size range studied. Applications of the multijet and multistage aerosol concentrator with high-volume flow rate can vary widely, from detection of biological aerosols at low concentration, laboratory aerosol sampling, clean room monitoring, and ambient aerosol measurements.

Metabolic engineering of plant alkaloid biosynthesis.

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

Lipopolysaccharide mediates endothelial apoptosis by a FADD-dependent pathway.

Endothelial cells play a pivotal role in the inflammatory process by coordinating the recruitment of inflammatory cells to sites of tissue injury. Lipopolysaccharide (LPS) activates many of the proinflammatory and procoagulant responses of endothelial cells, and endothelial injury is thought to play a crucial role in the pathogenesis of septic shock due to Gram-negative bacteria. The receptor used by LPS to signal endothelial responses has not been identified. It is also not known how LPS induces endothelial injury/death. In this study, we demonstrate that LPS mediates endothelial apoptosis by a FADD-dependent pathway. FADD is a death domain-containing protein that binds to certain members of the tumor necrosis factor receptor family, namely TNFR1, Fas, and DR3. However, none of these receptors appear to be involved in LPS-mediated death, suggesting that LPS may utilize a novel death domain-containing protein to transduce a death signal.

Relationship of peritoneal membrane transport characteristics to the nutritional status in CAPD patients.

BACKGROUND: The study was carried out to evaluate the role of individual peritoneal membrane transport characteristics in the nutritional status expressed as the composite nutritional index (CNI) METHODS: Cross-sectional analyses of the overall nutritional status of 147 continuous ambulatory peritoneal dialysis (CAPD) patients were performed using the CNI. CNIs based on a scoring system of 10 nutritional indices including subjective global assessment, biochemical parameters and anthropometry were compared according to the results of a standard peritoneal equilibration test (PET) RESULTS: Patients were classified as low (n = 16, 10.9%), low average (n=59, 40.2%), high average (n=54, 36.7%) and high (n=18, 12.2%) transporters based on the D/P(Cr) after 4 h dwells. The mean 4 h D/P(Cr) was 0.65 +/- 0.12 (0.34-0.95), and there was no significant correlation between D/P(Cr) and other demographic parameters such as age, duration of peritoneal dialysis and body surface area. D/P(Cr) was correlated with dialytic albumin loss (r=0.47, P<0.001), serum albumin (r=-0.46, P<0.001), serum creatinine (r= -0.38, P<0.001), serum TGF-1 (r=-0.37, P<0.01) and LBM(Cr) (r= -0.26, P<0.05). In high transporters, the serum albumin was significantly lower while dialysate protein and albumin losses were significantly greater compared with low transporters. Serum creatinine and IGF-1 concentrations as well as LBM(Cr) were also decreased in higher transporters. The mean CNI score was 8.1 +/- 4.9, with a range of 0-24. CNI was positively correlated with age, duration of peritoneal dialysis, incidence of peritonitis, CRP and dialytic protein loss, whereas it was inversely correlated with ultrafiltration volume, haemoglobin and NPNA. The CNI score was significantly higher in high transporters compared with low transporters (11.7 +/- 4.3 vs. 5.9 +/- 1.6, P < 0.01). There was also a significant correlation between D/P(Cr) and CNI (r = 0.29, P < 0.05). Multiple regression analysis revealed that the incidence of peritonitis, duration of CAPD, CRP and D/P(Cr) were the independent factors affecting the CNI. CONCLUSION: Peritoneal membrane transport characteristics correlate with the overall nutritional status of peritoneal dialysis patients assessed by the scoring system of the CNI, although it is associated with a different impact on the individual nutritional indices. The results of this cross-sectional study also suggest that a high permeability state is a risk factor for malnutrition in CAPD patients. Prospective studies evaluating the changes in nutritional parameters among patients with different membrane transport rates are needed to understand better the relationship of peritoneal membrane characteristics to the nutritional status of CAPD patients.

Effects of extremely low frequency magnetic fields on pain thresholds in mice: roles of melatonin and opioids.

1. We studied the effects of extremely low frequency (ELF, 60 Hz) magnetic fields (MFs) on pain thresholds using the hot plate test. The implication of opioid and benzodiazepine system in the MFs-induced alteration of pain thresholds was also studied. 2. There was an increase at night time and a decrease at daytime of pain thresholds in normal mice. Exposure of MFs (24 h, 20 gauss (G)) inhibited the increase of pain thresholds at night time and even produced hyperalgesia at daytime. 3. The increase of pain thresholds induced by melatonin at daytime was inhibited by exposure to MFs (24 h, 20 G) or opioid antagonist naloxone. The MFs and naloxone synergically inhibited hypoalgesia produced by melatonin. The hyperalgesia at daytime after MFs exposure was potentiated by the benzodiazepine agonist, diazepam, and inhibited by the benzodiazepine antagonist, flumazenil. There was no significant difference in all rotarod performance we tested. 4. From these results, it is suggested that exposure to MFs inhibits the increase of pain thresholds at night time and produces hyperalgesia at daytime with the involvement of opioid and benzodiazepine systems.

A case of spontaneous pneumomediastinum and pneumopericardium in a young adult.

Spontaneous medialstinal emphysema (pneumomediastinum) and pneumopericardium may be defined as the presence of free air or gas in the mediastinal structures and in the pericardial sac without an apparent precipitating cause. It most frequently occurs in young healthy adults without serious underlying pulmonary disease. Although pneumomediastinum and pneumopericardium is often asymptomatic, it may cause pain in the neck and chest, dysphonia and shortness of breath. Treatment is supportive unless the patient has a history of trauma from foreign body aspiration. The course of spontaneous pneumomediastinum and pneumopericardium is usually benign and self-limited. A case of spontaneous pneumomediastinum, pneumopericardium and subcutaneous emphysema in a 20-year-old male is reported in this paper.

Recurrent asystoles associated with vasovagal reaction during venipuncture.

A 17-year-old high school student presented with a history of habitual faintings. On 24-hour Holter monitoring, cardiac asystoles were recorded, the longest lasting approximately 7 or 8 seconds during venipuncture procedures. The asystole associated with venipuncture demonstrated the cardioinhibitory effects of vasovagal reaction with blood-injury phobia. He also had a positive response during head-up tilt test showing hypotension and relative bradycardia after intravenous isoproterenol injection. After administration of oral beta blocker, he did not show further or recurrent cardiac asystole during blood injury procedure on electrocardiographic examination. Venipuncture is the most common invasive medical procedure performed in hospital settings. While venipuncture is considered to be reasonably safe, serious complication may occur even when only a small volume of blood is withdrawn. Therefore, medical personnel should be prepared to provide appropriate care.

A clinical consideration of systemic embolism complicated to infective endocarditis in Korea.

OBJECTIVES: Infective endocarditis is still one of the important fatal diseases in Korea, especially when systemic embolisms are supervene. So, identification of patients who are in the high risk of embolism and who can be helped by early surgical intervention is very important. Considering these, we tried to elaborate the risk factors for the systemic embolism in patients with an infective endocarditis and the influence of systemic embolism on the mortality and morbidity in patients with an infective endocarditis. METHODS: We retrospectively reviewed the clinical records of 97 patients who were admitted with the infective endocarditis between January 1983 and October 1993. RESULTS: Among 97 patients, 80 patients met our diagnostic criteria. The mean age of patients was 38 years old. There were 43 males and 37 females. The mean duration of fever since the fever developed by history was 38 days. Valvular heart disease was the most frequent underlying heart disease. Mitral regurgitation and aortic regurgitation were the most common among valvular heart diseases. Pneumonia and acute pharyngotonsillitis were the most frequent predisposing factors of infective endocarditis. Blood cultures were positive in 51 patients (63.8%). Streptococcus viridans was the organism isolated most frequently, and Staphylococcus aureus was the second most frequently isolated one. Vegetations were detected in 58 patients (76.3%) by an echocardiography. Mitral valve and aortic valve were the most frequently involved incidence rate of embolism was 50% and the most frequent embolism site was the central nervous system and extremities were the next. Embolism occurred at the mean of 37 days after onset of fever. Overall in-hospital mortality rate was 26.3% and a cardiac-origin was the major cause of death. The only statistically significant risk factor for mortality was systemic embolism. The analysis of the relation between an incidence of embolism and the multivariables (age, presence of vegetation, location of vegetation, size of vegetation, causative organisms) showed that only the growth of Staphylococcus aureus had a significant trend toward a risk of subsequent systemic embolism. CONCLUSION: This study suggests that systemic emboli increases the mortality rate in patients with infective endocarditis. Age of patients, presence of vegetation, size and location of vegetation are not the risk factors for embolism, while certain organism, especially Staphylococcus aureus, could be a risk factor for the systemic embolism.

Transesophageal echocardiographic detection of thoracic aortic plaque could noninvasively predict significant obstructive coronary artery disease.

OBJECTIVE: Previous pathologic and roentgenographic studies have suggested a relation between aortic plaque and coronary artery disease but have lacked clinical utility. The study was undertaken to elucidate whether atherosclerotic aortic plaque detected by transesophageal echocardiography can be a clinically useful marker for significant obstructive coronary artery disease. METHODS: Clinical and angiographic features and intraoperative transesophageal echocardiographic findings were prospectively analyzed in 131 consecutive patients (58 women and 73 men, aged 17 to 75 years [mean 54 +/- 12]) undergoing open heart surgery. Significant obstructive coronary artery disease was defined as > or = 50% stenosis of > or = 1 major branch. RESULTS: Seventy-six (58%) of 131 patients were found to have obstructive coronary artery disease. In 76 patients with significant coronary artery disease, 71 had thoracic aortic plaque. In contrast, aortic plaque existed in only 10 of the remaining 55 patients with normal or minimally abnormal coronary arteries. The presence of aortic plaque on transesophageal echocardiographic studies had a sensitivity of 93%, a specificity of 82% and positive and negative predictive values of 88% and 90%, respectively, for significant coronary artery disease. There was a significant relationship between the degree of aortic intimal changes and the severity of coronary artery disease (r = 0.74, P < 0.0001). Multivariate logistic regression analysis of patient age, sex, risk factors of cardiovascular disease and transesophageal, echocardiographic findings revealed that atherosclerotic aortic plaque was the most significant independent predictor of coronary artery disease. CONCLUSION: This study indicates that transesophageal echocardiographic detection of atherosclerotic plaque in the thoracic aorta is useful in the noninvasive prediction of the presence and severity of coronary artery disease.