mRNA detection of individual cells with the single cell nanoprobe method compared with in situ hybridization

<p>Abstract</p> <p>Background</p> <p>The localization of specific mRNA generates cell polarity by controlling the translation sites of specific proteins. Although most of these events depend on differences in gene expression, no method is available to examine time dependent gene expression of individual living cells. <it>In situ </it>hybridization (ISH) is a powerful and useful method for detecting the localization of mRNAs, but it does not allow a time dependent analysis of mRNA expression in single living cells because the cells have to be fixed for mRNA detection. To overcome these issues, the extraction of biomolecules such as mRNAs, proteins, and lipids from living cells should be performed without severe damage to the cells. In previous studies, we have reported a single cell nanoprobe (SCN) method to examine gene expression of individual living cells using atomic force microscopy (AFM) without killing the cells.</p> <p>Results</p> <p>In order to evaluate the SCN method, we compared the SCN method with <it>in situ </it>hybridization (ISH). First, we examined spatial β-actin mRNA expression in single living cells with the SCN method, and then the same cells were subjected to ISH for β-actin mRNA. In the SCN method, quantity of β-actin mRNA were analysed by quantitative PCR, and in ISH we used intensity of ISH as a parameter of concentration of β-actin mRNA. We showed that intensity of ISH is higher; quantity of β-actin mRNA detected by the SCN method increased more.</p> <p>Conclusion</p> <p>In this study, we compare the SCN method with the ISH. We examined β-actin mRNA expression in single cells using both methods. We picked up β-actin mRNA from several loci of a single living cell using an AFM nanoprobe, and identical cells were subjected to ISH. The results showed a good correlation between the SCN method and ISH. The SCN method is suitable and reliable to examine mRNAs at medium or higher expression level.</p

The Relationship between H. pylori

Background and Objective. H. pylori infection causes a chronic inflammation in the gastric mucosa. However, this local inflammation may result in extra-digestive conditions. Our aim is to investigate the relationship between H. pylori infection and osteoporosis in Japan. Methods. This cross-sectional study was conducted among outpatients at the Juntendo University Hospital between 2008 and 2014. Participants for patient profile, H. pylori infection status, comorbidity, internal medical therapies, lumbar dual-energy X-ray absorptiometry (DXA), and bone turnover marker were collected and upper gastrointestinal endoscopy for reflux esophagitis, hiatal hernia, peptic ulcer disease (PUD), and endoscopic gastric mucosal atrophy (EGA) was performed. The diagnosis of osteoporosis was performed in accordance with the Japanese criteria. We investigated risk factors of osteoporosis. Results. Of the eligible 200 study subjects, 41 cases were of osteoporosis. Bivariate analysis showed that age, being female, BMI, alcohol, smoking, H. pylori, bone-specific ALP, PUD, and EGA were related to osteoporosis. Multivariate analysis showed that age (OR 1.13; 95%CI 1.07–1.20), being female (OR 4.77; 95%CI 1.78–12.77), BMI (OR 0.79; 95%CI 0.68–0.92), H. pylori (OR 5.33; 95%CI 1.73–16.42), and PUD (OR 4.98; 95%CI 1.51–16.45) were related to osteoporosis. Conclusions. H. pylori infection may be a risk factor of osteoporosis in Japan

Identification of a high incidence region for retroviral vector integration near exon 1 of the LMO2 locus

Therapeutic retroviral vector integration near the oncogene LMO2 is thought to be a cause of leukemia in X-SCID gene therapy trials. However, no published studies have evaluated the frequency of vector integrations near exon 1 of the LMO2 locus. We identified a high incidence region (HIR) of vector integration using PCR techniques in the upstream region close to the LMO2 transcription start site in the TPA-Mat T cell line. The integration frequency of the HIR was one per 4.46 × 10(4 )cells. This HIR was also found in Jurkat T cells but was absent from HeLa cells. Furthermore, using human cord blood-derived CD34(+ )cells we identified a HIR in a similar region as the TPA-Mat T cell line. One of the X-linked severe combined immunodeficiency (X-SCID) patients that developed leukemia after gene therapy had a vector integration site in this HIR. Therefore, the descriptions of the location and the integration frequency of the HIR presented here may help us to better understand vector-induced leukemogenesis

Radial Shortening Osteotomy Using Volar Locking Plate for Kienb&ouml;ck\u27s Disease

Purpose:The purpose of this study was to report the preliminary results for radial shortening osteotomyfollowed by volar locking plate fixation without post-operative immobilization for the treatment of Kienb&ouml;ck\u27sdisease.Methods:Ten consecutive patients with Kienb&ouml;ck\u27s disease of stages III were treated by radial shorteningosteotomy at the metaphysis using a volar locking plate system. Radial shortening osteotomy was performed for patients with negative or neutral ulnar variance, and combined shortening of radius and ulna for those with positive ulnar variance. The active motion of the digits, wrist, and forearm was encouraged immediately after surgery, and no splints were used.Results:The average follow-up was 26 months. In all the patients, the osteotomized bone united after an average of 11 weeks. Follow-up radiographs showed no further progression of the disease in carpal height,St&aring;hl\u27s index, or Lichtman\u27s stage classification. Moderate pain reported by all the patients preoperativelysignificantly improved by the final follow-up. Wrist extension, flexion, grip strength, and the Mayo wrist score were significantly improved compared with preoperative values.Conclusions:Volar locking plate fixation without immobilization is a safe and effective procedure for radial shortening osteotomy of Kienb&ouml;ck\u27s disease

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Differences in mediolateral dynamic stability during gait initiation according to whether the non-paretic or paretic leg is used as the leading limb.

We investigated mediolateral dynamic stability at first foot off and first initial contact during gait initiation according to whether the paretic or non-paretic leg was used as the leading limb. Thirty-eight individuals with stroke initiated gait with the paretic and non-paretic legs as the leading limb, and their movements were measured using a 3D motion analysis system. Margin of stability (i.e., the length between the extrapolated center of mass and lateral border of the stance foot) was used as an index of dynamic stability, with a large value indicating dynamic stability in the lateral direction. However, an excessively large margin of stability value (i.e., when the extrapolated center of mass is outside the medial border of the stance foot) indicates dynamic instability in the medial direction. Differences in the margin of stability between tasks were compared using the Wilcoxon signed-rank test. The minimum margin of stability was observed just before first foot off. When the non-paretic leg was used as the leading limb, the margin of stability tended to be excessively large at first foot off compared with when the paretic leg was used (p < 0.001). In other words, the extrapolated center of mass was outside the medial border of the paretic stance foot. In conclusion, lateral stability was achieved when using the non-paretic leading limb because the extrapolated center of mass was located outside the medial border of the stance foot. However, medial dynamic stability was lower for the non-paretic leading limb compared with the paretic leading limb

Differential functioning of retrieval/comparison processing in detection of the presence and absence of change.

BACKGROUND: The phenomenon of change blindness may reflect the failure to detect the presence of change or the absence of change. Although performing the latter is considered more difficult than the former, the differential functioning of retrieval/comparison processing that leads to differences between the detection of the presence and the absence of change has not been clarified. This study aimed to fill this research gap by comparing performance in the detection of the presence and the absence of a change in one item among a set of items. METHODOLOGY/PRINCIPAL FINDINGS: Twenty subjects performed two types of change detection tasks, the first task was detection of one changed item among a set of unchanged items (detection of the presence of a change) and the other was the detection of one unchanged item among a set of changed items (detection of the absence of a change). The ANOVA results for the percentage of correct responses and signal detection measurement of A' values regarding change detection and the pattern of the results indicate that the subjects found (1) detection of the presence of change less difficult than detection of the absence of change (2), rejection of the presence of change less difficult than acceptance of the presence of change, and (3) rejection of the absence of change as difficult as acceptance of the absence of change. CONCLUSIONS/SIGNIFICANCE: Retrieval/comparison processing for the detection of the presence of change differs from that for the absence of change, likely because the retrieval/comparison process appears aimed at determining whether an item has changed but not whether an item appears the same as it had previously. This conclusion suggests the existence of an identification process that recognizes each item as the same as that observed previously that exists apart from the mechanism underlying retrieval/comparison processing