A high-density cDNA microarray with colorimetry detection system to simultaneously monitor the expression of many genes on nylon membrane is described and characterized. To quantify the expression of ...genes and to isolate differentially expressed genes, the southern hybridization process on filter membranes was employed. The levels of gene expression were represented by color intensities generated by colorimetric reactions in place of hazardous radioisotopes or costly laser-induced fluorescence detection. The gene expression patterns on nylon membranes were digitized by devices such as an economical flatbed scanner or a digital camera. The quantitative information of gene expression was retrieved by image analysis software. Quantitative comparison of the northern dot-blotting method with the microarray system is described. Applications employing single-color detection as well as dual-color detection to isolate differentially expressed genes among thousands of genes are demonstrated.
Recent products of piperacillin/tazobactam (PTZ) from the original manufacturer, previously considered a major cause of galactomannan (GM) false-positivity, are reported not to be related to it. ...However, data regarding generic PTZ are limited and controversial. To evaluate the effect of generic PTZ on GM false-positivity in Korea, we performed a case–control study in adult patients with cancer. A case–control study was designed. Electronic medical records of cancer patients who were admitted and tested for serum GM between March and June 2014 at a tertiary care university hospital were reviewed. During the study period, a single generic PTZ (C manufacturer, Korea) was used. Patients who received PTZ within 24 h prior to serum GM testing were enrolled. Age- and GM test date-matched non-PTZ patients were selected as controls. A total of 110 patients received PTZ within 24 h prior to serum GM testing during the study period. The GM optical density index (ODI) of the PTZ group did not vary significantly from that of the control group (
p
= 0.251). The percentage of false-positive patients in the PTZ group was also similar to that of the control group (
p
= 0.538). There was no statistical relationship between GM ODI titer and time interval from PTZ administration (
p
= 0.095) or cumulative PTZ dose (
p
= 0.416). In a case–control study that evaluated 220 patients, a generic PTZ in Korea was not related to GM false-positivity.
Genetic variation in the cholinergic muscarinic-2 (M(2)) receptor gene (CHRM2) has been associated with the risk for developing depression. We previously reported that M(2)-receptor distribution ...volume (V(T)) was reduced in depressed subjects with bipolar disorder (BD) relative to depressed subjects with major depressive disorder (MDD) and healthy controls (HCs). In this study, we investigated the effects of six single-nucleotide polymorphisms (SNPs) for CHRM2 on M(2)-receptor binding to test the hypotheses that genetic variation in CHRM2 influences M(2)-receptor binding and that a CHRM2 polymorphism underlies the deficits in M(2)-receptor V(T) observed in BD. The M(2)-receptor V(T) was measured using positron emission tomography and (18)FFP-TZTP in unmedicated, depressed subjects with BD (n=16) or MDD (n=24) and HCs (n=25), and the effect of genotype on V(T) was assessed. In the controls, one SNP (with identifier rs324650, in which the ancestral allele adenine (A) is replaced with one or two copies of thymine (T), showed a significant allelic effect on V(T) in the pregenual and subgenual anterior cingulate cortices in the direction AA<AT<TT. In contrast, in BD subjects with the TT genotype, V(T) was significantly lower than in BD subjects with the AT genotype in these regions. The BD subjects homozygous for the T -allele also showed markedly lower V(T) (by 27 to 37% across regions) than HCs of the same genotype. Post hoc analyses suggested that T homozygosity was associated with a more severe illness course, as manifested by lower socioeconomic function, poorer spatial recognition memory and a greater likelihood of having attempted suicide. These data represent novel preliminary evidence that reduced M(2)-receptor V(T) in BD is associated with genetic variation within CHRM2. The differential impact of the M(2)-receptor polymorphism at rs324650 in the BD and HC samples suggests interactive effects with an unidentified vulnerability factor for BD.
Metastasis is a complicated multistep process that involves interactions between cancer cells and their surrounding microenvironments. Previously, we have established a series of lung adenocarcinoma ...cell lines with varying degrees of invasiveness. Tracheal graft assay confirmed that cell lines with higher in vitro invasiveness had greater in vivo invasive potential. In this study, we used these model cell lines to identify invasion-associated genes using cDNA microarray with colorimetric detection. A more invasive subline, CL 1-5-F 4, derived from metastatic lung tumor of severe combined immunodeficient mice inoculated with CL 1-5 cells, was combined with CL 1-0, CL 1-1, and CL 1-5 in cDNA microarray screening. cDNA microarray membranes, each containing 9600 nonredundant expressed sequence tag clones, were used to identify differentially expressed genes in these cell lines. For statistical analysis, self-organizing map algorithm was performed to identify the expression patterns. Positive correlation between gene expression levels and cell line invasiveness was found in 2.9% of the 9600 putative genes. On the other hand, negative correlation was found in 3.3% of the genes. The trends of expression of some of the genes were also confirmed by Northern hybridization and flow cytometry. Our data demonstrated that genes related to cell adhesion, motility, angiogenesis, signal transduction, and some other expressed sequence tag genes may play significant roles in the metastasis process. These results substantiate the model system with which one can identify invasion-associated genes by using cDNA microarray and cancer cell lines of different invasiveness. This technique may allow us to explore complex interactions between multiple genes that orchestrate the process of cancer metastasis.
This Letter reports new results from the MINOS experiment based on a two-year exposure to muon neutrinos from the Fermilab NuMI beam. Our data are consistent with quantum-mechanical oscillations of ...neutrino flavor with mass splitting |Deltam2| = (2.43+/-0.13) x 10(-3) eV2 (68% C.L.) and mixing angle sin2(2theta) > 0.90 (90% C.L.). Our data disfavor two alternative explanations for the disappearance of neutrinos in flight: namely, neutrino decays into lighter particles and quantum decoherence of neutrinos, at the 3.7 and 5.7 standard-deviation levels, respectively.
Background Studies in animals suggest that enteric neurons decrease in density or number with increasing age. Neurons containing nitric oxide (NO), vasoactive intestinal peptide (VIP) and Substance ...P (SP) have been implicated. In human large intestine, NO‐utilizing neurons decrease during childhood or early adulthood but it is not known if the innervation of the muscle changes. This study examined the density of nerve fibres containing these transmitters in sigmoid colon circular muscle from children and adults.
Methods Fluorescence immunohistochemistry using antibodies to neuronal NO synthase (nNOS), VIP and SP was performed on sigmoid colon from 18 adults with colorectal cancer, two children with familial adenomatous polyposis, and normal colon from nine children with Hirschsprung’s disease. The percentage area of immunoreactive (IR) nerve fibres containing each transmitter in circular muscle was quantified in confocal images.
Key Results In the adult sigmoid colon circular muscle, the percentage area of nerve fibres containing nNOS>VIP>SP (6 : 2 : 1). Paediatric groups had significantly higher percentage area of nerve fibres containing nNOS, VIP or SP‐IR than adults, with the decrease in nerve fibre density occurring from birth to 30 years. Circular muscle thickness increased between 12 and 30 years. Total nerve fibre area remained constant, while the muscle increased in thickness.
Conclusions & Inferences In human sigmoid colon circular muscle, there are reductions in nNOS‐, VIP‐ and SP‐IR nerve fibre density with growth from newborn to late adolescence but little further change with aging. The reduction in nerve density is due to an increase in circular muscle thickness rather than a loss of nerve fibres.
Detection and quantitation of circulating cancer cells in peripheral blood may improve cancer staging and monitoring. This study explored the feasibility of using circulating cancer cell detection in ...peripheral blood for the rapid assessment of chemotherapeutic response. Cytokeratin 19 mRNA was amplified by nested reverse transcriptase-PCR in the peripheral blood of 29 healthy volunteers, 33 pneumonia patients, and 86 lung cancer patients. Circulating cancer cells in the peripheral blood were semiquantitatively determined by taking the ratio of cytokeratin 19 band intensity from the second round of nested PCR to the glyceraldehyde-3-phosphate dehydrogenase band intensity from the first round of PCR amplification. The detection limit of the method was 1 cancer cell in 107 peripheral blood mononuclear cells. The positive detection rate was 40% for lung adenocarcinoma patients of all stages, 41% for squamous carcinoma patients of all stages, and 27% for small cell lung cancer patients. Only one control sample from a pneumonia patient showed a positive result (1.6%). The quantitative method reliably and sensitively estimated cancer cell numbers in the peripheral blood of lung cancer patients. Serial measurement of the relative number of circulating cancer cells correlated with the tumor burden and treatment response of patients. This method may help rapidly assess the efficacy of anticancer treatment, redefine cancer staging, and facilitate the design of better therapeutic strategies for the treatment of cancer patients.
Introduction Due to the Covid-19 pandemic and associated travel restrictions, the physical presence of international external examiners was a challenge when assessing the exit level outcomes of the ...MSc (Dent) in Paediatric Dentistry at the University of the Western Cape.External moderation of final examinations ensure an acceptable standard, coverage of content as specified by the programme outcomes and eliminates bias during assessment. Internationalization of the moderation and examination process allows countries to compare and maintain international standards and graduate attributes expected for professional qualifications. Qualifications requiring assessment of skills often rely on Objective Structured Clinical Examinations, Objective Structured Practical Examinations and simulated cases in combination with an oral examination, which requires the presence of all examiners to assess the student.Aims and objectives This paper describes how the final examination in this MSc (Dent) degree was adapted and conducted in order to overcome the challenges of the Covid-19 pandemic, to maintain the academic integrity and rigour of the programme.Design and Methods A narrative essay-style approach was adopted, which reflects on the challenges and opportunities created by Covid-19.Conclusion The adapted assessment method proved to be an effective alternative to the more traditional assessment approaches employed pre-Covid.
The phosphatase and tensin homology deleted on chromosome 10 (PTEN) is a tumor suppressor gene with sequence homology to tyrosine phosphatases and the cytoskeletal proteins tensin and auxilin. PTEN ...has recently been shown to inhibit cell migration and the spreading and formation of focal adhesions. This study investigated the role of PTEN in carcinoma invasion in a lung-cancer cell line and examined the downstream genes regulated by PTEN. We have previously established a cell-line model in human lung adenocarcinoma with different invasive abilities and metastatic potentials. Examining PTEN gene expression in these cell lines, we found that a homozygous deletion in exon 5 is associated with high invasive ability. We then constructed stable constitutive and inducible wild-type PTEN-overexpressed transfectants in the highly invasive cell line CL(1-5). We found that an overexpression of PTEN can inhibit invasion in lung cancer cells. To further explore the downstream genes regulated by PTEN, a high-density complementary DNA (cDNA) microarray technique was used to profile gene changes after PTEN overexpression. Our results indicate a panel of genes that can be modulated by PTEN. PTEN overexpression downregulated genes, including integrin alpha(6), laminin beta(3), heparin-binding epidermal growth factor-like growth factor, urokinase-type plasminogen activator, myb protein B, Akt2, and some expressed sequence tag (EST) clones. In contrast, PTEN overexpression upregulated protein phosphatase 2A1B, ubiquitin protease (unph), secreted phosphoprotein 1, leukocyte elastase inhibitor, nuclear factor-kappaB, cyclic adenosine monophosphate response element binding protein, DNA ligase 1, heat shock protein 90, and some EST genes. Northern hybridization and flow cytometry analysis also confirmed that PTEN overexpression results in the reduced expression of the integrin alpha(6) subunit. The results of this study indicate that PTEN overexpression may inhibit lung cancer invasion by downregulation of a panel of genes including integrin alpha(6). The cDNA microarray technique may be an effective tool to study the downstream function of a tumor suppressor gene.
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