Dual role of Bmi1 loss in the preservation of photoreceptor layers in the Rd1 mouse

Purpose: In the Rd1 and Rd10 mouse models of retinitis pigmentosa, a mutation in the Pde6ß gene leads to the rapid loss of photoreceptors. As in several neurodegenerative diseases, Rd1 and Rd10 photoreceptors re-express cell cycle proteins prior to death. Bmi1 regulates cell cycle progression through inhibition of CDK inhibitors, and its deletion efficiently rescues the Rd1 retinal degeneration. The present study evaluates the effects of Bmi1 loss in photoreceptors and Müller glia, since in lower vertebrates, these cells respond to retinal injury through dedifferentiation and regeneration of retinal cells. Methods: Cell death and Müller cell activation were analyzed by immunostaining of wild-type, Rd1 and Rd1;Bmi1-/- eye sections during retinal degeneration, between P10 and P20. Lineage tracing experiments use the GFAP-Cre mouse (JAX) to target Müller cells. Results: In Rd1 retinal explants, inhibition of CDKs reduces the amount of dying cells. In vivo, Bmi1 deletion reduces CDK4 expression and cell death in the P15 Rd1;Bmi1-/- retina, although cGMP accumulation and TUNEL staining are detected at the onset of retinal degeneration (P12). This suggests that another process acts in parallel to overcome the initial loss of Rd1;Bmi1-/- photoreceptors. We demonstrate here that Bmi1 loss in the Rd1 retina enhances the activation of Müller glia by downregulation of p27Kip1, that these cells migrate toward the ONL, and that some cells express the retinal progenitor marker Pax6 at the inner part of the ONL. These events are also observed, but to a lesser extent, in Rd1 and Rd10 retinas. At P12, EdU incorporation shows proliferating cells with atypical elongated nuclei at the inner border of the Rd1;Bmi1-/- ONL. Lineage tracing targeting Müller cells is in process and will determine the implication of this cell population in the maintenance of the Rd1;Bmi1-/- ONL thickness and whether downregulation of Bmi1 in Rd10 Müller cells equally stimulates their activation. Conclusions: Our results show a dual role of Bmi1 deletion in the rescue of photoreceptors in the Rd1;Bmi1-/- retina. Indeed, the loss of Bmi1 reduces Rd1 retinal degeneration, and as well, enhances the Müller glia activation. In addition, the emergence of cells expressing a retinal progenitor marker in the ONL suggests Bmi1 as a blockade to the regeneration of retinal cells in mammals.

Interventions to achieve long-term weight loss in obese older people

Purpose: the prevalence of obesity is rapidly increasing in older adults. Information is required about what interventions are effective in reducing obesity and influencing health outcomes in this age group. Design: systematic review and meta-analysis. Data sources: thirteen databases were searched, earliest date 1966 to December 2008, including Medline, CINAHL, PsycINFO, the Cochrane database and EMBASE. Study selection: we included studies with participants�۪ mean age 60 years and mean body mass index 30 kg/m2, with outcomes at a minimum of 1 year. Data were independently extracted by two reviewers and differences resolved by consensus. Data extraction: nine eligible trials were included. Study interventions targeted diet, physical activity and mixed approaches. Populations included patients with coronary artery disease, diabetes mellitus and osteoarthritis. Results: meta-analysis (seven studies) demonstrated a modest but significant weight loss of 3.0 kg [95% confidence interval (CI) 5.1���0.9] at 1 year. Total cholesterol (four studies) did not show a significant change: ���0.36 mmol/l (95% CI ���0.75 to 0.04). There was no significant change in high-density lipoprotein, low-density lipoprotein or triglycerides. In one study, recurrence of hypertension or cardiovascular events was significantly reduced (hazard ratio 0.65, 95% CI 0.50���0.85). Six-minute walk test did not significantly change in one study. Health-related quality of life significantly improved in one study but did not improve in a second study. Conclusions: although modest weight reductions were observed, there is a lack of high-quality evidence to support the efficacy of weight loss programmes in older people. ��Keywords: obesity, older, weight loss, meta-analysis, elderly, systematic review��

A New Large Deletion in the DFNB1 Locus Causes Nonsyndromic Hearing Loss.

Mutations in the GJB2 gene encoding the gap junction protein connexin 26 are responsible for up to 30% of all cases of autosomal recessive nonsyndromic hearing impairment (HI) with prelingual onset in most populations. The corresponding locus DFNB1, located on chromosome 13q11-q12, is also affected by three distinct deletions. These deletions extended distally to GJB2, which remains intact. We report a novel large deletion in DFNB1 observed in a patient presenting profound prelingual HI. This deletion was observed in trans to a GJB2 mutated allele carrying the p.Val84Met (V84M) mutation and was shown to be associated with hearing loss. The deletion caused a false homozygosity of V84M in the proband. Quantification of alleles by quantitative fluorescent multiplex PCR (QFM-PCR) enabled us to study the breakpoints of the deletion. The deleted segment extended through at least 920kb and removed the three connexin genes GJA3, GJB2 and GJB6. The distal breakpoint inside intron 2 of CRYL1 gene differed from the breakpoints of the known DFNB1 deletions. This case highlights the importance of screening for large deletions in molecular studies of GJB2.

Antibiotic-dependent correlation between drug-induced killing and loss of luminescence in Streptococcus gordonii expressing luciferase.

Measuring antibiotic-induced killing relies on time-consuming biological tests. The firefly luciferase gene (luc) was successfully used as a reporter gene to assess antibiotic efficacy rapidly in slow-growing Mycobacterium tuberculosis. We tested whether luc expression could also provide a rapid evaluation of bactericidal drugs in Streptococcus gordonii. The suicide vectors pFW5luc and a modified version of pJDC9 carrying a promoterless luc gene were used to construct transcriptional-fusion mutants. One mutant susceptible to penicillin-induced killing (LMI2) and three penicillin-tolerant derivatives (LMI103, LMI104, and LMI105) producing luciferase under independent streptococcal promoters were tested. The correlation between antibiotic-induced killing and luminescence was determined with mechanistically unrelated drugs. Chloramphenicol (20 times the MIC) inhibited bacterial growth. In parallel, luciferase stopped increasing and remained stable, as determined by luminescence and Western blots. Ciprofloxacin (200 times the MIC) rapidly killed 1.5 log10 CFU/ml in 2-4 hr. Luminescence decreased simultaneously by 10-fold. In contrast, penicillin (200 times the MIC) gave discordant results. Although killing was slow (< or = 0.5 log10 CFU/ml in 2 hr), luminescence dropped abruptly by 50-100-times in the same time. Inactivating penicillin with penicillinase restored luminescence, irrespective of viable counts. This was not due to altered luciferase expression or stability, suggesting some kind of post-translational modification. Luciferase shares homology with aminoacyl-tRNA synthetase and acyl-CoA ligase, which might be regulated by macromolecule synthesis and hence affected in penicillin-inhibited cells. Because of resemblance, luciferase might be down-regulated simultaneously. Luminescence cannot be universally used to predict antibiotic-induced killing. Thus, introducing reporter enzymes sharing mechanistic similarities with normal metabolic reactions might reveal other effects than those expected.

Heterogeneous secretion of individual B cells in response to D-glucose and to nonglucidic nutrient secretagogues.

We used a hemolytic plaque assay for insulin to determine whether the same pancreatic B cells respond to D-glucose, 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and the association of this nonmetabolized analogue of L-leucine with either the monomethyl ester of succinic acid (SME) or the dimethyl ester of L-glutamic acid (GME). During a 30-min incubation in the absence of D-glucose, BCH alone (5 mM) had no effect on insulin release. In contrast, the combination of BCH with either SME (10 mM) or GME (3 mM) stimulated insulin release to the same extent observed in the sole presence of 16.7 mM D-glucose. The effects of BCH plus SME and BCH plus GME on both percentage of secreting B cells and total insulin output were little affected in the presence of D-glucose concentrations ranging from 0 to 16.7 mM. Varying the concentration of SME from 2 to 10 mM also did not influence these effects. In other experiments, the very same B cells were first exposed 45 min to 16.7 mM D-glucose, then incubated 45 min in the presence of only BCH and SME. Under these conditions, most (80.3 +/- 2.5%) of the cells contributing to insulin release did so during both incubation periods. Furthermore, virtually all cells responding to BCH and SME during the second incubation corresponded to cells also responsive to D-glucose during the first incubation. Similar observations were made when the sequence of the two incubations was reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

Does metabolic compensation explain the majority of less-than-expected weight loss in obese adults during a short-term severe diet and exercise intervention?

Objective:We investigated to what extent changes in metabolic rate and composition of weight loss explained the less-than-expected weight loss in obese men and women during a diet-plus-exercise intervention.Design:In all, 16 obese men and women (41±9 years; body mass index (BMI) 39±6 kg m(-2)) were investigated in energy balance before, after and twice during a 12-week very-low-energy diet(565-650 kcal per day) plus exercise (aerobic plus resistance training) intervention. The relative energy deficit (EDef) from baseline requirements was severe (74%-87%). Body composition was measured by deuterium dilution and dual energy X-ray absorptiometry, and resting metabolic rate (RMR) was measured by indirect calorimetry. Fat mass (FM) and fat-free mass (FFM) were converted into energy equivalents using constants 9.45 kcal per g FM and 1.13 kcal per g FFM. Predicted weight loss was calculated from the EDef using the '7700 kcal kg(-1) rule'.Results:Changes in weight (-18.6±5.0 kg), FM (-15.5±4.3 kg) and FFM (-3.1±1.9 kg) did not differ between genders. Measured weight loss was on average 67% of the predicted value, but ranged from 39% to 94%. Relative EDef was correlated with the decrease in RMR (R=0.70, P<0.01), and the decrease in RMR correlated with the difference between actual and expected weight loss (R=0.51, P<0.01). Changes in metabolic rate explained on average 67% of the less-than-expected weight loss, and variability in the proportion of weight lost as FM accounted for a further 5%. On average, after adjustment for changes in metabolic rate and body composition of weight lost, actual weight loss reached 90% of the predicted values.Conclusion:Although weight loss was 33% lower than predicted at baseline from standard energy equivalents, the majority of this differential was explained by physiological variables. Although lower-than-expected weight loss is often attributed to incomplete adherence to prescribed interventions, the influence of baseline calculation errors and metabolic downregulation should not be discounted.

Functional Variants in NOS1 and NOS2A Are Not Associated with Progressive Hearing Loss in Ménière's Disease in a European Caucasian Population

Hearing loss in Meniere's disease (MD) is associated with loss of spiral ganglion neurons and hair cells. In a guinea pig model of endolymphatic hydrops, nitric oxide synthases (NOS) and oxidative stress mediate loss of spiral ganglion neurons. To test the hypothesis that functional variants of NOS1 and NOS2A are associated with MD, wed genotyped three functional variants of NOS1 (rs41279104,rs2682826, and a cytosine-adenosine microsatellite repeat in exon 1f) and the CCTTT repeat in the promoter of NOS2A gene (rs3833912) in two independent MD sets(273 patients in total) and 550 controls. A third cohort of American patients was genotyped as replication cohort for the CCTTT repeat. Neither allele nor genotype frequencies of rs41279104 and rs2682826 were associated with MD, although longer alleles of the cytosine-adenosine microsatellite repeat were marginally significant (corrected p = 0.05) in the Mediterranean cohort but not in a second Galicia cohort. Shorter numbers of the CCTTT repeat in NOS2A were significantly more frequent in Galicia controls (OR = 0.37 [CI, 0.18-0.76], corrected p =0.04), but this finding could not be replicated in Mediterranean or American case-control populations. Meta-analysis did not support an association between CCTTT repeats and risk for MD. Severe hearing loss (>75 dB) was also not associated with any functional variants studied. Functional variants of NOS1 and and NOS2A do not confer susceptibility for MD.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Genome-wide analysis of SREBP1 activity around the clock reveals its combined dependency on nutrient and circadian signals.

In mammals, the circadian clock allows them to anticipate and adapt physiology around the 24 hours. Conversely, metabolism and food consumption regulate the internal clock, pointing the existence of an intricate relationship between nutrient state and circadian homeostasis that is far from being understood. The Sterol Regulatory Element Binding Protein 1 (SREBP1) is a key regulator of lipid homeostasis. Hepatic SREBP1 function is influenced by the nutrient-response cycle, but also by the circadian machinery. To systematically understand how the interplay of circadian clock and nutrient-driven rhythm regulates SREBP1 activity, we evaluated the genome-wide binding of SREBP1 to its targets throughout the day in C57BL/6 mice. The recruitment of SREBP1 to the DNA showed a highly circadian behaviour, with a maximum during the fed status. However, the temporal expression of SREBP1 targets was not always synchronized with its binding pattern. In particular, different expression phases were observed for SREBP1 target genes depending on their function, suggesting the involvement of other transcription factors in their regulation. Binding sites for Hepatocyte Nuclear Factor 4 (HNF4) were specifically enriched in the close proximity of SREBP1 peaks of genes, whose expression was shifted by about 8 hours with respect to SREBP1 binding. Thus, the cross-talk between hepatic HNF4 and SREBP1 may underlie the expression timing of this subgroup of SREBP1 targets. Interestingly, the proper temporal expression profile of these genes was dramatically changed in Bmal1-/- mice upon time-restricted feeding, for which a rhythmic, but slightly delayed, binding of SREBP1 was maintained. Collectively, our results show that besides the nutrient-driven regulation of SREBP1 nuclear translocation, a second layer of modulation of SREBP1 transcriptional activity, strongly dependent from the circadian clock, exists. This system allows us to fine tune the expression timing of SREBP1 target genes, thus helping to temporally separate the different physiological processes in which these genes are involved.

Changes in gene expression profile in human subcutaneous adipose tissue during significant weight loss.

Objective: To analyze the expression of peroxisome proliferator-activated receptor-γ1 and 2 (PPARγ1 and 2), 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), and leptin in adipose tissue (AT) of obese women during weight loss following Roux-en-Y gastric bypass (RYGB) and to compare these levels with those obtained in AT of nonobese subjects. Methods: Gene expression was determined by real-time RT-PCR prior to surgery and at 3, 6, and 12 months after RYGB. Results: All obese patients lost weight, reaching a mean BMI of 29.3 ± 1.0 kg/m(2) at 1 year after surgery (-33.9 ± 1.5% of their initial body weight). In obese subjects leptin and 11βHSD1 were over-expressed, whereas PPARγ1 was expressed at lower levels compared to controls. After surgery, leptin and 11βHSD1 gene expression decreased, whereas PPARγ1 expression increased. At 12 months after RYGB, these 3 genes had reached levels similar to the controls. In contrast, PPARγ2 gene expression was not different between groups and types of tissue and remained unchanged during weight loss. We found a positive correlation between BMI and levels of gene expression of leptin and 11βHSD1. Conclusion: Gene expression of leptin, PPARγ1, and 11βHSD1 in AT is modified in human obesity. This default is completely corrected by RYGB. Copyright © 2012 S. Karger GmbH, Freiburg.

Loss of ovarian hormones increases salt-sensitivity in normotensive women

Salt sensitivity (SS) is associated with an elevated risk of developing hypertension(HTN) and is an independent risk factor for cardiovascular (CV) morbidity and mortality. Cross-sectional studies have suggested that postmenopausal women are more salt sensitive than premenopausal women. The purpose of the present study was to investigate prospectively the prevalence of SS among healthy premenopausal women and determine whether the loss of ovarian hormones increases SS.

ALDH1A3 loss of function causes bilateral anophthalmia/microphthalmia and hypoplasia of the optic nerve and optic chiasm.

The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.

Compliance with dietary and nutrient recommendations in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Granada cohort at recruitment

BACKGROUND: The overall intake of energy and nutrients in the Granada EPIC-cohort (European Prospective Investigation into Cancer and Nutrition) is examined in order to assess compliance with the Spanish Nutritional Objectives (NO) and the Recommended Intakes (RI). METHODS: During recruitment (1992-1996), 7,789 participants, aged 35-69, were asked about diet through a validated diet history questionnaire. Nutrient intake is compared to the NO and RI that were valid at that time. Risk of inadequate intake is estimated as the percentage of the sample with intakes: ≤ 1/3 RI (high risk), ≤ 2/3 RI- > 1/3 RI (moderate risk), ≤ RI- > 2/3 RI, > RI. Differences in intakes have been analyzed by sex and age, and by smoking status and BMI. RESULTS: The daily intake of nutrients did not meet the NO as the total contribution of energy from proteins and fats exceeded these guidelines. Whilst intake of most nutrients was above the RI, the amount of iron, magnesium and vitamins D and E provided by the diet was not enough to meet the RI: in women aged 20-49 years, about 55% were at moderate risk for iron inadequacy, and a 20% of women for magnesium. Both sexes were at high risk of inadequacy for vitamin D, although sunlight exposure may supply adequate amounts. Never smokers showed a higher compliance to the NO. CONCLUSION: At recruitment, the nutrient profile of the diet was unbalanced. The observed nutrient inadequacy for iron, magnesium and vitamin E might be attributed to inappropriate dietary habits, and may have implications for future disease risk.

TNF-alpha-dependent loss of IKKbeta-deficient myeloid progenitors triggers a cytokine loop culminating in granulocytosis.

Loss of IκB kinase (IKK) β-dependent NF-κB signaling in hematopoietic cells is associated with increased granulopoiesis. Here we identify a regulatory cytokine loop that causes neutrophilia in Ikkβ-deficient mice. TNF-α-dependent apoptosis of myeloid progenitor cells leads to the release of IL-1β, which promotes Th17 polarization of peripheral CD4(+) T cells. Although the elevation of IL-17 and the consecutive induction of granulocyte colony-stimulating factor compensate for the loss of myeloid progenitor cells, the facilitated induction of Th17 cells renders Ikkβ-deficient animals more susceptible to the development of experimental autoimmune encephalitis. These results unravel so far unanticipated direct and indirect functions for IKKβ in myeloid progenitor survival and maintenance of innate and Th17 immunity and raise concerns about long-term IKKβ inhibition in IL-17-mediated diseases.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.