ALMS1-Deficient Fibroblasts Over-Express Extra-Cellular Matrix Components, Display Cell Cycle Delay and Are Resistant to Apoptosis

Alström Syndrome (ALMS) is a rare genetic disorder (483 living cases), characterized by many clinical manifestations, including blindness, obesity, type 2 diabetes and cardiomyopathy. ALMS is caused by mutations in the ALMS1 gene, encoding for a large protein with implicated roles in ciliary function, cellular quiescence and intracellular transport. Patients with ALMS have extensive fibrosis in nearly all tissues resulting in a progressive organ failure which is often the ultimate cause of death. To focus on the role of ALMS1 mutations in the generation and maintenance of this pathological fibrosis, we performed gene expression analysis, ultrastructural characterization and functional assays in 4 dermal fibroblast cultures from ALMS patients. Using a genome-wide gene expression analysis we found alterations in genes belonging to specific categories (cell cycle, extracellular matrix (ECM) and fibrosis, cellular architecture/motility and apoptosis). ALMS fibroblasts display cytoskeleton abnormalities and migration impairment, up-regulate the expression and production of collagens and despite the increase in the cell cycle length are more resistant to apoptosis. Therefore ALMS1-deficient fibroblasts showed a constitutively activated myofibroblast phenotype even if they do not derive from a fibrotic lesion. Our results support a genetic basis for the fibrosis observed in ALMS and show that both an excessive ECM production and a failure to eliminate myofibroblasts are key mechanisms. Furthermore, our findings suggest new roles for ALMS1 in both intra- and extra-cellular events which are essential not only for the normal cellular function but also for cell-cell and ECM-cell interactions

Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes

Copyright: © 2010 Stimpson et al.Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extrachromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.This work was supported by the Tumorzentrum Heidelberg/Mannheim grant (D.10026941)and by March of Dimes Research Foundation grant #1-FY06-377 and NIH R01 GM069514

Prevalence and etiology of false normal aEEG recordings in neonatal hypoxic-ischaemic encephalopathy.

BACKGROUND: Amplitude-integrated electroencephalography (aEEG) is a useful tool to determine the severity of neonatal hypoxic-ischemic encephalopathy (HIE). Our aim was to assess the prevalence and study the origin of false normal aEEG recordings based on 85 aEEG recordings registered before six hours of age. METHODS: Raw EEG recordings were reevaluated retrospectively with Fourier analysis to identify and describe the frequency patterns of the raw EEG signal, in cases with inconsistent aEEG recordings and clinical symptoms. Power spectral density curves, power (P) and median frequency (MF) were determined using the raw EEG. In 7 patients non-depolarizing muscle relaxant (NDMR) exposure was found. The EEG sections were analyzed and compared before and after NDMR administration. RESULTS: The reevaluation found that the aEEG was truly normal in 4 neonates. In 3 neonates, high voltage electrocardiographic (ECG) artifacts were found with flat trace on raw EEG. High frequency component (HFC) was found as a cause of normal appearing aEEG in 10 neonates. HFC disappeared while P and MF decreased significantly upon NDMR administration in each observed case. CONCLUSION: Occurrence of false normal aEEG background pattern is relatively high in neonates with HIE and hypothermia. High frequency EEG artifacts suggestive of shivering were found to be the most common cause of false normal aEEG in hypothermic neonates while high voltage ECG artifacts are less common

Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients

Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human beta-globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34(+) cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for a future clinical translation

Fluticasone/formoterol combination therapy is as effective as fluticasone/salmeterol in the treatment of asthma, but has a more rapid onset of action: an open-label, randomized study

<p>Abstract</p> <p>Background</p> <p>The inhaled corticosteroid (ICS) fluticasone propionate (fluticasone) and the long-acting β₂-agonist (LABA) formoterol fumarate (formoterol) are being made available as a combination product (fluticasone/formoterol, <b><it>flutiform</it></b>^®) in a single aerosol inhaler. This 12-week, open-label, randomized, active-controlled, parallel-group, multicentre, phase 3 study compared the efficacy and safety of fluticasone/formoterol with the commercially available combination product fluticasone/salmeterol.</p> <p>Methods</p> <p>Patients aged ≥ 18 years (N = 202) with mild-to-moderate–severe, persistent asthma for ≥ 6 months prior to screening were included in the study. After a screening phase (4–10 days), eligible patients were randomized 1:1 to receive fluticasone/formoterol or fluticasone/salmeterol during the 12-week treatment period. The primary objective was to demonstrate non-inferiority of fluticasone/formoterol versus fluticasone/salmeterol, measured by pre-dose forced expiratory volume in the first second (FEV₁), at week 12.</p> <p>Results</p> <p>Fluticasone/formoterol was comparable to fluticasone/salmeterol for the primary efficacy endpoint, mean pre-dose FEV₁at week 12. The new combination was also comparable to fluticasone/salmeterol for change from baseline to week 12 in pre-dose FEV₁, change from pre-dose FEV₁at baseline to 2-hour post-dose FEV₁at week 12 and discontinuations due to lack of efficacy. Importantly, fluticasone/formoterol was superior to fluticasone/salmeterol in time to onset of action throughout the duration of the study. The two treatments demonstrated similar results for various other secondary efficacy parameters, including other lung function tests, patient-reported outcomes, rescue medication use, asthma exacerbations and Asthma Quality of Life Questionnaire scores. Fluticasone/formoterol was well tolerated and had a good safety profile that was similar to fluticasone/salmeterol.</p> <p>Conclusions</p> <p>The results of this study indicate that fluticasone/formoterol is as effective as fluticasone/salmeterol, and has a more rapid onset of action, reflecting the faster bronchodilatory effects of formoterol compared with those of salmeterol. If patients perceive the benefits of therapy with fluticasone/formoterol more rapidly than with fluticasone/salmeterol, this could have a positive impact on preference and adherence.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT00476073">NCT00476073</a></p

Use of Oral Cholera Vaccines in an Outbreak in Vietnam: A Case Control Study

Simple measures such as adequate sanitation and clean water stops the spread of cholera; however, in areas where these are not available, cholera spreads quickly and may lead to death in a few hours if treatment is not initiated immediately. The use of life-saving rehydration therapy is the mainstay in cholera control, however, the rapidity of the disease and the limited access to appropriate healthcare units in far-flung areas together result in an unacceptable number of deaths. The WHO has recommended the use of oral cholera vaccines as a preventive measure against cholera outbreaks since 2001, but this was recently updated so that vaccine use may also be considered once a cholera outbreak has begun. The findings from this study suggest that reactive use of killed oral cholera vaccines provides protection against the disease and may be a potential tool in times of outbreaks. Further studies must be conducted to confirm these findings

Does green tea affect postprandial glucose, insulin and satiety in healthy subjects: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Results of epidemiological studies have suggested that consumption of green tea could lower the risk of type 2 diabetes. Intervention studies show that green tea may decrease blood glucose levels, and also increase satiety. This study was conducted to examine the postprandial effects of green tea on glucose levels, glycemic index, insulin levels and satiety in healthy individuals after the consumption of a meal including green tea.</p> <p>Methods</p> <p>The study was conducted on 14 healthy volunteers, with a crossover design. Participants were randomized to either 300 ml of green tea or water. This was consumed together with a breakfast consisting of white bread and sliced turkey. Blood samples were drawn at 0, 15, 30, 45, 60, 90, and 120 minutes. Participants completed several different satiety score scales at the same times.</p> <p>Results</p> <p>Plasma glucose levels were higher 120 min after ingestion of the meal with green tea than after the ingestion of the meal with water. No significant differences were found in serum insulin levels, or the area under the curve for glucose or insulin. Subjects reported significantly higher satiety, having a less strong desire to eat their favorite food and finding it less pleasant to eat another mouthful of the same food after drinking green tea compared to water.</p> <p>Conclusions</p> <p>Green tea showed no glucose or insulin-lowering effect. However, increased satiety and fullness were reported by the participants after the consumption of green tea.</p> <p>Trial registration number</p> <p>NCT01086189</p

Identifying factors relevant in the assessment of return-to-work efforts in employees on long-term sickness absence due to chronic low back pain: a focus group study

ABSTRACT: BACKGROUND: Efforts undertaken during the return to work (RTW) process need to be sufficient to prevent unnecessary applications for disability benefits. The purpose of this study was to identify factors relevant to RTW Effort Sufficiency (RTW-ES) in cases of sick-listed employees with chronic low back pain (CLBP). METHODS: Using focus groups consisting of Labor Experts (LE's) working at the Dutch Social Insurance Institute, arguments and underlying grounds relevant to the assessment of RTW-ES were investigated. Factors were collected and categorized using the International Classification of Functioning, Disability and Health (ICF model). RESULTS: Two focus groups yielded 19 factors, of which 12 are categorized in the ICF model under activities (e.g. functional capacity) and in the personal (e.g. age, tenure) and environmental domain (e.g. employer-employee relationship). The remaining 7 factors are categorized under intervention, job accommodation and measures. CONCLUSIONS: This focus group study shows that 19 factors may be relevant to RTW-ES in sick-listed employees with CLBP. Providing these results to professionals assessing RTW-ES might contribute to a more transparent and systematic approach. Considering the importance of the quality of the RTW process, optimizing the RTW-ES assessment is essential

Self-reported colorectal cancer screening of Medicare beneficiaries in family medicine vs. internal medicine practices in the United States: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>The benefit of screening for decreasing the risk of death from colorectal cancer (CRC) has been shown, yet many patients in primary care are still not undergoing screening according to guidelines. There are known variations in delivery of preventive health care services among primary care physicians. This study compared self-reported CRC screening rates and patient awareness of the need for CRC screening of patients receiving care from family medicine (FPs) vs. internal medicine (internists) physicians.</p> <p>Methods</p> <p>Nationally representative sample of non-institutionalized beneficiaries who received medical care from FPs or internists in 2006 (using Medicare Current Beneficiary Survey). The main outcome was the percentage of patients screened in 2007. We also examined the percentage of patients offered screening.</p> <p>Results</p> <p>Patients of FPs, compared to those of internists, were less likely to have received an FOBT kit or undergone home FOBT, even after accounting for patients' characteristics. Compared to internists, FPs' patients were more likely to have heard of colonoscopy, but were less likely to receive a screening colonoscopy recommendation (18% vs. 27%), or undergo a colonoscopy (43% vs. 46%, adjusted odds ratios [AOR], 95% confidence interval [CI]-- 0.65, 0.51-0.81) or any CRC screening (52% vs. 60%, AOR, CI--0.80, 0.68-0.94). Among subgroups examined, higher income beneficiaries receiving care from internists had the highest screening rate (68%), while disabled beneficiaries receiving care from FPs had the lowest screening rate (34%).</p> <p>Conclusion</p> <p>Patients cared for by FPs had a lower rate of screening compared to those cared for by internists, despite equal or higher levels of awareness; a difference that remained statistically significant after accounting for socioeconomic status and access to healthcare. Both groups of patients remained below the national goal of 70 percent.</p

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry for Probing the Microenvironment of Histidine Residues in Dihydrofolate Reductase

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins