8 research outputs found
Multiple MeCP2 immunoreactive bands in neural cell lines.
<p>(<b>A</b>) Diagram of the hMeCP2e1 protein illustrating the position of the MeCP2 antibodies. (<b>B</b>) Phase-contrast photomicrographs (PhC) of proliferating neural cell lines. Scale bar = 100μm. (<b>C</b>) Western-blot analysis of proliferating neural cell lines with antibodies against the N-terminal (AAH11612, a.a.93-182) and C-terminal region (H300, a.a.198-496) of MeCP2 protein. Blots were stained with Ponceau solution as a loading control. Protein size markers (in kilodaltons) are indicated on the side of each panel.</p
MeCP2 antibodies available commercially against different epitopes of MeCP2 protein detected multiple bands by western-blot analysis.
<p>MeCP2 antibodies available commercially against different epitopes of MeCP2 protein detected multiple bands by western-blot analysis.</p
p.T158M MeCP2e1-RFP mutant expressing neural cell line.
<p>(<b>A</b>) Sequencing chromatogram of hMeCP2e1-RFP expression vector. Red box indicated codon ACG (threonine). (<b>B</b>) Single nucleotide mutation converting T158 to methionine (T158M). Sequencing chromatogram of mutant hMeCP2e1-RFP expression vector. Red box indicated codon ATG (methionine). (<b>C</b>) Photomicrographs show phase-contrast (PhC) and fluorescence images of wild-type and p.T158M hMeCP2e1-RFP<sup>+</sup> mutant expressing neural cell line. Scale bar = 100μm.</p
Multiple MeCP2 and RFP immunoreactive bands in hMeCP2e1-RFP expressing neural cell lines.
<p>(<b>A</b>) Diagram of the hMeCP2e1-RFP protein illustrating the position of the MeCP2 and RFP antibodies. (<b>B-D</b>) Western-blot analysis of hMeCP2e1-RFP<sup>+</sup> HEK293 cell line with antibodies against the N- and C-terminal region of MeCP2. (<b>E</b>) RFP immunoreactive bands in transfected HEK293 cell line. (<b>F-H)</b> Western-blot analysis of hMeCP2e1-RFP<sup>+</sup> PC12 cell line with antibodies against the N- and C-terminal region of MeCP2. <b>(I)</b> RFP immunoreactive bands in transfected PC12 cell line. <b>(J-L)</b> Western-blot analysis of hMeCP2e1-RFP<sup>+</sup> N2A cell line with antibodies against the N- and C-terminal region of MeCP2. (<b>M</b>) RFP immunoreactive bands in transfected N2A cell line. (<b>N-P</b>) Western-blot analysis of hMeCP2e1-RFP<sup>+</sup> SHSY5Y cell line with antibodies against the N- and C-terminal region of MeCP2. (<b>Q</b>) RFP immunoreactive bands in transfected SHSY5Y cell line. Protein size markers (in kilodaltons) are indicated on the right of each panel.</p
Immunoblot analysis with MeCP2 antibody revealed multiple MeCP2 immunoreactive bands at the same position as the fluorescent signals detected via SDS-PAGE and in gel fluorescence scanning.
<p>(<b>A</b>) Fluorescence pattern of total cell lysate from hMeCP2e1-RFP<sup>+</sup> N2A neural cell line. (<b>B,C,D</b>) Fluorescence pattern, Ponceau staining and MeCP2 immunoblot of total cell lysate from hMeCP2e1-RFP<sup>+</sup> SHSY5Y neural cell line. (<b>E,F,G</b>) Fluorescence pattern, Ponceau staining and MeCP2 immunoblot of total cell lysate from HEK293 and hMeCP2e1-RFP<sup>+</sup> HEK293 cell lines. Protein ladder (M) and protein size markers (in kilodaltons) are indicated on the right of each panel.</p
Correct localization of hMeCP2e1-RFP fusion protein in stable transfected neural cell lines.
<p>(<b>A</b>). Photomicrographs show phase-contrast (PhC) and fluorescence images of hMeCP2e1-RFP<sup>+</sup> expressing neural cell lines. Scale bar = 100μm. (<b>B</b>) Nuclear localization of hMeCP2e1-RFP in mouse and human interphase nuclei. Scale bar = 100μm. (<b>C</b>) hMeCP2e1-RFP fusion protein localized to metaphase chromosomes in mitotic nuclei. Scale bar = 50μm.</p
Multiple MeCP2 and RFP immunoreactive bands in p.T158M MeCP2e1-RFP mutant expressing neural cell line.
<p>(<b>A</b>) Diagram of the hMeCP2e1-RFP protein illustrating the position of the MeCP2 and RFP antibodies. (<b>B</b>) RFP immunoreactive bands in wild-type and p.T158M hMeCP2e1-RFP<sup>+</sup> mutant expressing neural cell lines. Blots were also double-stained for β-actin, as a loading control. The asterisks marks β-actin bands. (<b>C</b>) Higher denaturing conditions did not affect the recognition of the RFP immunoreactive bands. (<b>D-G</b>) RFP and MeCP2 immunoreactive bands in wild-type and p.T158M hMeCP2e1-RFP<sup>+</sup> mutant expressing neural cell lines. (<b>H-K</b>) Only one RFP immunoreactive band around 70kDa (faster migration band) was visible in hMeCP2e1-RFP<sup>+</sup> HEK293 and N2A cell lines in the absence of phosphatase inhibitor. Blots were stained with Ponceau solution as a loading control. Protein size markers (in kilodaltons) are indicated on the right of each panel.</p
Diabetes mortality and trends before 25 years of age: an analysis of the Global Burden of Disease Study 2019
Background: Diabetes, particularly type 1 diabetes, at younger ages can be a largely preventable cause of death with the correct health care and services. We aimed to evaluate diabetes mortality and trends at ages younger than 25 years globally using data from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019. Methods: We used estimates of GBD 2019 to calculate international diabetes mortality at ages younger than 25 years in 1990 and 2019. Data sources for causes of death were obtained from vital registration systems, verbal autopsies, and other surveillance systems for 1990–2019. We estimated death rates for each location using the GBD Cause of Death Ensemble model. We analysed the association of age-standardised death rates per 100 000 population with the Socio-demographic Index (SDI) and a measure of universal health coverage (UHC) and described the variability within SDI quintiles. We present estimates with their 95% uncertainty intervals. Findings: In 2019, 16 300 (95% uncertainty interval 14 200 to 18 900) global deaths due to diabetes (type 1 and 2 combined) occurred in people younger than 25 years and 73·7% (68·3 to 77·4) were classified as due to type 1 diabetes. The age-standardised death rate was 0·50 (0·44 to 0·58) per 100 000 population, and 15 900 (97·5%) of these deaths occurred in low to high-middle SDI countries. The rate was 0·13 (0·12 to 0·14) per 100 000 population in the high SDI quintile, 0·60 (0·51 to 0·70) per 100 000 population in the low-middle SDI quintile, and 0·71 (0·60 to 0·86) per 100 000 population in the low SDI quintile. Within SDI quintiles, we observed large variability in rates across countries, in part explained by the extent of UHC (r2=0·62). From 1990 to 2019, age-standardised death rates decreased globally by 17·0% (−28·4 to −2·9) for all diabetes, and by 21·0% (–33·0 to −5·9) when considering only type 1 diabetes. However, the low SDI quintile had the lowest decline for both all diabetes (−13·6% [–28·4 to 3·4]) and for type 1 diabetes (−13·6% [–29·3 to 8·9]). Interpretation: Decreasing diabetes mortality at ages younger than 25 years remains an important challenge, especially in low and low-middle SDI countries. Inadequate diagnosis and treatment of diabetes is likely to be major contributor to these early deaths, highlighting the urgent need to provide better access to insulin and basic diabetes education and care. This mortality metric, derived from readily available and frequently updated GBD data, can help to monitor preventable diabetes-related deaths over time globally, aligned with the UN's Sustainable Development Targets, and serve as an indicator of the adequacy of basic diabetes care for type 1 and type 2 diabetes across nations. Funding: Bill & Melinda Gates Foundation