Pilot study of arbitrarily primed PCR-single stranded DNA conformation polymorphism analysis for screening genetic polymorphisms related to specific phenotypes

BACKGROUND: To investigate relationships between phenotypes and genotypes is not simple. We propose a phenotype-to-genotype screening strategy and pooled DNA system. As a pilot study of this strategy, we used arbitrarily primed polymerase chain reaction (AP-PCR) in combination with single-stranded DNA conformation polymorphism (SSCP) to screen for genetic polymorphisms associated with longevity. METHODS: Study subjects were separated into 3 age groups, individuals aged >100 years, 90-99 years and 60-69 years. Genomic DNAs were prepared from each individual, pooled to represent the 5 study groups, and then the pooled genomic DNAs were subjected to AP-PCR-SSCP analysis. RESULTS: We found 1 SNP more frequently in senior citizens with longevity. The genotype frequency of the 82133G>A polymorphism of human chromosome 3 clone RP11-61K12 (AC011199) differed significantly (P=0.0189, Fisher's exact test) between older subjects (>90 years) and younger subjects (<70 years). It is noteworthy that the strategy we describe herein was useful for identifying an SNP that showed statistically significant differences in its distribution across the subject groups. CONCLUSIONS: The pooled DNA strategy and quantitative genotype discrimination can also be applied to screening for the relationship between phenotype and genotype more effectively.     

Detection of Legionella pneumophila serogroup 7 strain from bathwater samples in a Japanese hospital

Hospital-acquired legionellosis is one of the serious problems in nosocomial infection. For risk assessment of nosocomial Legionella infection, we surveyed samples from bathrooms for public use in three hospitals and two nursing homes to determine whether Legionella pneumophila was present. A total of 70 hot bathwater samples and samples wiped from bathtubs were collected at 1-h intervals. Fifteen shower-water and 15 inner-head samples were obtained at the start of a bath. Water samples were cultured using the Legionella spp. selective medium, and discrimination between L. pneumophila and other Legionella spp. was performed by PCR analysis. L. pneumophila serogroup 7 was detected in 1 bathwater and 1 wiped sample, both of which were collected 1 h after daily use from the same bathtub in a hospital. However, L. pneumophila SG7 was not detected in any other samples. Furthermore, the concentrations of free residual chlorine in most bath- and shower-water samples were lower than 0.1 mg/l. These results suggest that L. pneumophila has become a potential pathogen for nosocomial infections in public-type hospital baths. From the point of view of an infection-control program, it might be advisable to hold the concentration of free residual chlorine at 0.2–0.4 mg/l, which is generally required for public baths in Japan.     

Devising a technique for distal anastomosis during total arch replacement using the frozen elephant trunk technique: reverse stepwise anastomosis

Frozen elephant technique is an effective procedure used to repair complex thoracic aortic disease involving the aortic arch and the descending aorta, or to treat aortic dissection. However, the technique often requires some creativity to properly place the stent graft and anastomose the graft to the aorta. We recently used the J Graft FROZENIX® (Japan Lifeline Co., Ltd., Tokyo, Japan) in total arch replacement, and devise a technique for distal anastomosis (called “reverse stepwise anastomosis”) that was simple and easy and caused minimal bleeding.     

Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi

Using the polymerase chain reaction (PCR) assay, we developed a rapid diagnosis method for Salmonella typhi infection in blood specimens from patients with typhoid fever. Primers were designed from the flagellin gene sequence, which would give an amplification product of 367 base pairs. In this study, the specificity of the assay, with no amplification, was seen for the other Salmonella strains with the flagellin gene, and not for non-Salmonella bacteria. For the sensitivity test, the protocol described allowed the detection of two to three copies of the Salmonella typhi genome, as determined by serial dilution of genomic DNA from Salmonella typhi. With the PCR technique, genomic DNA of Salmonella typhi was detected in 46 of 73 blood samples collected from patients with clinically suspected typhoid fever who had fever within 3 days of admission to the General Hospital, Makassar, South Sulawesi, Indonesia, and who had had no prior antibiotic treatment. The PCR results (63% positive cases) were compared with those of blood culture (13.7% positive cases) and the Widal test (35.6% positive cases), using the same samples from each of the 73 patients admitted to the General Hospital in Makassar. The time taken for PCR analysis of each sample was less than 12h, compared with 3 to 5 days for blood or clot culture. The PCR with one pair of primers can be used as a novel, rapid diagnotic method for typhoid fever, particularly when results of standard culture assays are negative.     

PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.     

Removing the effects of task-related motion using independent-component analysis

Task-related motion is a major source of noise in functional magnetic-resonance imaging (fMRI) time series. The motion effect usually persists even after perfect spatial realignment is achieved. Here, we propose a new method to remove a certain type of task-related motion effect that persists after realignment. The procedure consists of the following: the decomposition of the realigned time-series data into spatially-independent components using independent-component analysis (ICA); the automatic classification and rejection of the ICs of the task-related residual motion effects; and finally, a reconstruction without them. To classify the ICs, we utilized the associated task-related changes in signal intensity and variance. The effectiveness of the method was verified using an fMRI experiment that explicitly included head motion as a main effect. The results indicate that our ICA-based method removed the task-related motion effects more effectively than the conventional voxel-wise regression-based method.     

Deficiency in systemic interleukin-1 receptor antagonist production as an operative risk factor in malnourished elderly patients with colorectal carcinoma

OBJECTIVE: Aging and tumor-related malnutrition are associated with increased inflammatory cytokine levels. However, it is unclear whether this influences the outcomes of surgery. We explored the relationships between cytokines and surgical complications among patients undergoing surgery for colorectal cancer. DESIGN: Laboratory experiment. SETTING: Surgery department of school of medicine. PATIENTS: One hundred patients with colorectal cancer. INTERVENTIONS: The perioperative circulating levels of interleukin (IL)-1beta, IL-6, and IL-1 receptor antagonist (Ra) were determined, and the numbers of circulating lymphocytes and neutrophils were counted. MEASUREMENTS AND MAIN RESULTS: Trends toward increasing postoperative infection were observed among patients who were older and had lower body mass index. Preoperative IL-1Ra and intraoperative blood loss, however, remained the only two independent predictors of postoperative infection. Clinically, patients with low preoperative IL-1Ra most frequently were the elderly with low body mass index. Postoperatively, elderly patients with low body mass index showed an exaggerated IL-6 response, followed by an exaggerated postoperative inflammatory response and increased postoperative loss of body weight. In contrast, normal immunoreactivity was preserved in well-nourished elderly patients. CONCLUSIONS: In colorectal cancer patients undergoing surgery, low preoperative IL-1Ra is associated with postoperative infection. In our patient population, lower IL-1Ra level is commonly observed in the elderly with low body mass index. These findings suggest that postoperative infection, frequently seen in the nutritionally deficient elderly, may be the result of defective immunoinflammatory adaptation system.