A novel high-throughput method for molecular serotyping and serotype-specific quantification of Streptococcus pneumoniae using a nanofluidic real-time PCR system

Serotype-specific quantification data are essential for elucidating the complex epidemiology of Streptococcus pneumoniae and evaluating pneumococcal vaccine efficacy. Various PCR-based assays have been developed to circumvent the drawback of labour-intensive and time-consuming culture-based procedures for serotype determination and quantification of pneumococcus. Here, we applied a nanofluidic real-time PCR system to establish a novel assay. Twenty-nine primer pairs, 13 of which were newly designed, were selected for the assay to cover 50 serotypes including all currently available conjugate and polysaccharide vaccine serotypes. All primer pairs were evaluated for their sensitivity, specificity, efficiency, repeatability, accuracy and reproducibility on the Fluidigm Biomark HD System, a nanofluidic real-time PCR system, by drawing standard curves with a serial dilution of purified DNA. We applied the assay to 52 nasopharyngeal swab samples from patients with pneumonia confirmed by chest X-ray to validate its accuracy. Minimum detection levels of this novel assay using the nanofluidic real-time PCR system were comparable to the conventional PCR-based assays (between 30 and 300 copies per reaction). They were specific to their targets with good repeatability (SD of copy number of 0.1), accuracy (within ±0.1 fold difference in log10 copy number) and reproducibility (SD of copy number of 0.1). When artificially mixed DNA samples consisting of multiple serotypes in various ratios were tested, all the serotypes were detected proportionally, including a minor serotype of one in 1000 copies. In the nasopharyngeal samples, the PCR system detected all the culturepositive samples and 22 out of 23 serotypes identified by the conventional method were matched with PCR results. We conclude that this novel assay, which is able to differentially quantify 29 pneumococcus groups for 45 test samples in a single run, is applicable to the large-scale epidemiological study of pneumococcus. We believe that this assay will facilitate our understanding of the roles of serotype-specific bacterial loads and implications of multiple serotype detections in pneumococcal diseases

Enhanced Auditory Brainstem Response and Parental Bonding Style in Children with Gastrointestinal Symptoms

The electrophysiological properties of the brain and influence of parental bonding in childhood irritable bowel syndrome (IBS) are unclear. We hypothesized that children with chronic gastrointestinal (GI) symptoms like IBS may show exaggerated brainstem auditory evoked potential (BAEP) responses and receive more inadequate parental bonding. = 0.024). Multiple regression analysis in females also supported these findings.It is suggested that children with chronic GI symptoms have exaggerated brainstem responses to environmental stimuli and inadequate parental behaviors aggravate these symptoms

Impact of the Tohoku earthquake and tsunami on pneumonia hospitalisations and mortality among adults in northern Miyagi, Japan: a multicentre observational study

Background: On 11 March 2011, the Tohoku earthquake and tsunami struck off the coast of northeastern Japan. Within 3 weeks, an increased number of pneumonia admissions and deaths occurred in local hospitals. Methods: A multicentre survey was conducted at three hospitals in Kesennuma City (population 74 000), northern Miyagi Prefecture. All adults aged ≥18 years hospitalised between March 2010 and June 2011 with community-acquired pneumonia were identified using hospital databases and medical records. Segmented regression analyses were used to quantify changes in the incidence of pneumonia. Results: A total of 550 pneumonia hospitalisations were identified, including 325 during the pre-disaster period and 225 cases during the post-disaster period. The majority (90%) of the post-disaster pneumonia patients were aged ≥65 years, and only eight cases (3.6%) were associated with near-drowning in the tsunami waters. The clinical pattern and causative pathogens were almost identical among the pre-disaster and post-disaster pneumonia patients. A marked increase in the incidence of pneumonia was observed during the 3-month period following the disaster; the weekly incidence rates of pneumonia hospitalisations and pneumonia-associated deaths increased by 5.7 times (95% CI 3.9 to 8.4) and 8.9 times (95% CI 4.4 to 17.8), respectively. The increases were largest among residents in nursing homes followed by those in evacuation shelters. Conclusions: A substantial increase in the pneumonia burden was observed among adults after the Tohoku earthquake and tsunami. Although the exact cause remains unresolved, multiple factors including population aging and stressful living conditions likely contributed to this pneumonia outbreak

Myosin-Va Regulates Exocytosis through the Submicromolar Ca(2)+-dependent Binding of Syntaxin-1A

Myosin-Va is an actin-based processive motor that conveys intracellular cargoes. Synaptic vesicles are one of the most important cargoes for myosin-Va, but the role of mammalian myosin-Va in secretion is less clear than for its yeast homologue, Myo2p. In the current studies, we show that myosin-Va on synaptic vesicles interacts with syntaxin-1A, a t-SNARE involved in exocytosis, at or above 0.3 μM Ca(2+). Interference with formation of the syntaxin-1A–myosin–Va complex reduces the exocytotic frequency in chromaffin cells. Surprisingly, the syntaxin-1A-binding site was not in the tail of myosin-Va but rather in the neck, a region that contains calmodulin-binding IQ-motifs. Furthermore, we found that syntaxin-1A binding by myosin-Va in the presence of Ca(2+) depends on the release of calmodulin from the myosin-Va neck, allowing syntaxin-1A to occupy the vacant IQ-motif. Using an anti-myosin-Va neck antibody, which blocks this binding, we demonstrated that the step most important for the antibody's inhibitory activity is the late sustained phase, which is involved in supplying readily releasable vesicles. Our results demonstrate that the interaction between myosin-Va and syntaxin-1A is involved in exocytosis and suggest that the myosin-Va neck contributes not only to the large step size but also to the regulation of exocytosis by Ca(2+)