A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors

There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-kappa B (NF-κB) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-κB or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-κB or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors

Ca2+ monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor

Calcium (Ca2+)-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (300?nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening.We are grateful to Japanese Red Cross Blood Society for providing human RBC and plasma. We also thank Tanaka R, Ogoshi (Sakura) M and Matsumoto N for technical assistance and Templeton TJ for critical reading. This study was conducted at the Joint Usage / Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University, Japan. KP was a Tokyo Biochemical Research Foundation (TBRF, http://www.tokyobrf.or.jp) post-doctoral fellow and PEF was a Japanese Society of Promotion Sciences (JSPS) post-doctoral fellow. This work was supported in part by the TBRF (K.P.), JSPS (P.E.F.), Takeda Science Foundation (K.Y.), Grants-in-Aids for Scientific Research 24590509 (K.Y.), 22390079 (O.K.), and for Scientific Research on Innovative Areas 23117008 (O.K.), MEXT, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Electrocardiogram analysis in Anderson-Fabry disease: a valuable tool for progressive phenotypic expression tracking

BackgroundElectrocardiogram (ECG) has proven to be useful for early detection of cardiac involvement in Anderson-Fabry disease (AFD); however, little evidence is available on the association between ECG alterations and the progression of the disease.Aim and MethodsTo perform a cross sectional comparison of ECG abnormalities throughout different left ventricular hypertrophy (LVH) severity subgroups, providing ECG patterns specific of the progressive AFD stages. 189 AFD patients from a multicenter cohort underwent comprehensive ECG analysis, echocardiography, and clinical evaluation.ResultsThe study cohort (39% males, median age 47 years, 68% classical AFD) was divided into 4 groups according to different degree of left ventricular (LV) thickness: group A & LE; 9 mm (n = 52, 28%); group B 10-14 mm (n = 76, 40%); group C 15-19 mm (n = 46, 24%); group D & GE; 20 mm (n = 15, 8%). The most frequent conduction delay was right bundle branch block (RBBB), incomplete in groups B and C (20%,22%) and complete RBBB in group D (54%, p < 0.001); none of the patients had left bundle branch block (LBBB). Left anterior fascicular block, LVH criteria, negative T waves, ST depression were more common in the advanced stages of the disease (p < 0.001). Summarizing our results, we suggested ECG patterns representative of the different AFD stages as assessed by the increases in LV thickness over time (Central Figure). Patients from group A showed mostly a normal ECG (77%) or minor anomalies like LVH criteria (8%) and delta wave/slurred QR onset + borderline PR (8%). Differently, patients from groups B and C exhibited more heterogeneous ECG patterns: LVH (17%; 7% respectively); LVH + LV strain (9%; 17%); incomplete RBBB + repolarization abnormalities (8%; 9%), more frequently associated with LVH criteria in group C than B (8%; 15%). Finally, patients from group D showed very peculiar ECG patterns, represented by complete RBBB + LVH and repolarization abnormalities (40%), sometimes associated with QRS fragmentation (13%).ConclusionsECG is a sensitive tool for early identification and long-term monitoring of cardiac involvement in patients with AFD, providing "instantaneous pictures" along the natural history of AFD. Whether ECG changes may be associated with clinical events remains to be determined

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts

Age adjustment in ecological studies: using a study on arsenic ingestion and bladder cancer as an example

<p>Abstract</p> <p>Background</p> <p>Despite its limitations, ecological study design is widely applied in epidemiology. In most cases, adjustment for age is necessary, but different methods may lead to different conclusions. To compare three methods of age adjustment, a study on the associations between arsenic in drinking water and incidence of bladder cancer in 243 townships in Taiwan was used as an example.</p> <p>Methods</p> <p>A total of 3068 cases of bladder cancer, including 2276 men and 792 women, were identified during a ten-year study period in the study townships. Three methods were applied to analyze the same data set on the ten-year study period. The first (Direct Method) applied direct standardization to obtain standardized incidence rate and then used it as the dependent variable in the regression analysis. The second (Indirect Method) applied indirect standardization to obtain standardized incidence ratio and then used it as the dependent variable in the regression analysis instead. The third (Variable Method) used proportions of residents in different age groups as a part of the independent variables in the multiple regression models.</p> <p>Results</p> <p>All three methods showed a statistically significant positive association between arsenic exposure above 0.64 mg/L and incidence of bladder cancer in men and women, but different results were observed for the other exposure categories. In addition, the risk estimates obtained by different methods for the same exposure category were all different.</p> <p>Conclusions</p> <p>Using an empirical example, the current study confirmed the argument made by other researchers previously that whereas the three different methods of age adjustment may lead to different conclusions, only the third approach can obtain unbiased estimates of the risks. The third method can also generate estimates of the risk associated with each age group, but the other two are unable to evaluate the effects of age directly.</p

Association of residential dampness and mold with respiratory tract infections and bronchitis: a meta-analysis

<p>Abstract</p> <p>Background</p> <p>Dampness and mold have been shown in qualitative reviews to be associated with a variety of adverse respiratory health effects, including respiratory tract infections. Several published meta-analyses have provided quantitative summaries for some of these associations, but not for respiratory infections. Demonstrating a causal relationship between dampness-related agents, which are preventable exposures, and respiratory tract infections would suggest important new public health strategies. We report the results of quantitative meta-analyses of published studies that examined the association of dampness or mold in homes with respiratory infections and bronchitis.</p> <p>Methods</p> <p>For primary studies meeting eligibility criteria, we transformed reported odds ratios (ORs) and confidence intervals (CIs) to the log scale. Both fixed and random effects models were applied to the log ORs and their variances. Most studies contained multiple estimated ORs. Models accounted for the correlation between multiple results within the studies analyzed. One set of analyses was performed with all eligible studies, and another set restricted to studies that controlled for age, gender, smoking, and socioeconomic status. Subgroups of studies were assessed to explore heterogeneity. Funnel plots were used to assess publication bias.</p> <p>Results</p> <p>The resulting summary estimates of ORs from random effects models based on all studies ranged from 1.38 to 1.50, with 95% CIs excluding the null in all cases. Use of different analysis models and restricting analyses based on control of multiple confounding variables changed findings only slightly. ORs (95% CIs) from random effects models using studies adjusting for major confounding variables were, for bronchitis, 1.45 (1.32-1.59); for respiratory infections, 1.44 (1.31-1.59); for respiratory infections excluding nonspecific upper respiratory infections, 1.50 (1.32-1.70), and for respiratory infections in children or infants, 1.48 (1.33-1.65). Little effect of publication bias was evident. Estimated attributable risk proportions ranged from 8% to 20%.</p> <p>Conclusions</p> <p>Residential dampness and mold are associated with substantial and statistically significant increases in both respiratory infections and bronchitis. If these associations were confirmed as causal, effective control of dampness and mold in buildings would prevent a substantial proportion of respiratory infections.</p

Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins

Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP® Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner

The Physics of the B Factories

This work is on the Physics of the B Factories. Part A of this book contains a brief description of the SLAC and KEK B Factories as well as their detectors, BaBar and Belle, and data taking related issues. Part B discusses tools and methods used by the experiments in order to obtain results. The results themselves can be found in Part C