Impaired Lysosome Reformation in Chloroquine-Treated Retinal Pigment Epithelial Cells.

Purpose: To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild chloroquine treatment on lysosome function and turnover. Methods: Effects of low-dose chloroquine treatment on lysosomal function and accessibility to newly endocytosed cargo were investigated in primary and embryonic stem cell-derived RPE cells and ARPE19 cells using fluorescence and electron microscopy of fluorescent and gold-labeled probes. Lysosomal protein expression and accumulation were measured by quantitative PCR and Western blotting. Results: Initial chloroquine-induced lysosome neutralization was followed by partial recovery, lysosomal expansion, and accumulation of undegraded endocytic, phagocytic, and autophagic cargo and inhibition of cathepsin D processing. Accumulation of enlarged lysosomes was accompanied by a gradual loss of accessibility of these structures to the endocytic pathway, implying impaired lysosome reformation. Chloroquine-induced accumulation of pro-cathepsin D, as well as the lysosomal membrane protein, LAMP1, was reproduced by treatment with protease inhibitors and preceded changes in lysosomal gene expression. Conclusions: Low-dose chloroquine treatment inhibits lysosome reformation, causing a gradual depletion of lysosomes able to interact with cargo-carrying vacuoles and degrade their content. The resulting accumulation of newly synthesized pro-cathepsin D and LAMP1 reflects inhibition of normal turnover of lysosomal constituents and possibly lysosomes themselves. A better understanding of the mechanisms underlying lysosome reformation may reveal new targets for the treatment of chloroquine-induced retinopathy.

Visceral Obesity Is Associated with Shorter Progression-Free Survival in Well-Differentiated Gastro-Entero-Pancreatic Neuroendocrine Neoplasia.

The association of well-differentiated gastro-entero-pancreatic neuroendocrine neoplasia (WD GEP-NEN) with metabolic syndrome (MetS), abdominal obesity, and fasting glucose abnormalities was recently described. However, whether obesity and metabolic syndrome risk factors are associated with GEP-NEN adverse outcomes and the poorer prognosis was unknown. The present study aimed to evaluate whether the presence of MetS or any of its individual components at WD GEP-NEN diagnosis influenced disease outcomes. A cohort of patients with non-localized WD GEP-NETs (n = 81), was classified according to the primary tumor site (gastrointestinal or pancreatic), pathological grading (G1 (Ki67 ≤ 2%) and G2 (3% ≤ Ki67 ≤ 20%) (WHO 2010)), disease extension (loco-regional or metastatic disease), presence of hormonal secretion syndrome (functioning or non-functioning), and evaluated for the presence of MetS criteria at diagnosis. MetS was present in 48 (59.3%) patients. During a median follow-up of 95.0 months (16.8-262.5), 18 patients died of the disease (10 with MetS vs. 8 without MetS). Overall survival (OS) at 5 years was 87.1% (95% CI: 73.6-94.0) for MetS and 90.9% (95% CI: 74.4-97.0) for non-Mets group, while OS at 10 years was 72.5% (95% CI: 55.3-84.0) for MetS, and 76.4% (95% CI: 53.6-89.0) for non-MetS group. Progression-Free Survival (PFS) at 5 years was 45.9% (95% CI: 30.8-59.8) for MetS and 40.0% (95% CI: 21.3-58.1) for non-MetS group, and PFS at 10 years was 18.1% (95% CI: 7.0-33.5) for MetS and 24.4% (95% CI: 9.0-43.7) for non-MetS group. Waist circumference (WC), a surrogate measure for visceral obesity, was associated with significantly shorter PFS (HR = 1.03; 95% CI: 1.01-1.06), although did not influence OS (HR = 1.01; 95% CI: 0.97-1.06). The findings of this study reinforce a potential link between visceral obesity and GEP-NEN and further suggest that obesity could influence disease prognosis.

Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction.

Purpose: We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers. Methods: We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing. Results: AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation. Conclusions: We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

Disseminated Well-Differentiated Gastro-Entero-Pancreatic Tumors Are Associated with Metabolic Syndrome.

The association of well-differentiated gastro-entero-pancreatic neuroendocrine tumors (WD GEP-NETs) with metabolic syndrome (MetS), abdominal obesity, and fasting glucose abnormalities was recently described. The aim of this study was to evaluate whether the presence of MetS or any MetS individual component was also influenced by GEP-NET characteristics at diagnosis. A cohort of patients with WD GEP-NETs (n = 134), classified according to primary tumor location (gastrointestinal or pancreatic), pathological grading (G1 (Ki67 ≤ 2%) and G2 (>3 ≤ 20%) (WHO 2010), disease extension (localized, loco-regional, and metastatic), and presence of hormonal secretion syndrome (functioning/non-functioning), was evaluated for the presence of MetS criteria. After adjustment for age and gender, the odds of having MetS was significantly higher for patients with WD GEP-NET grade G1 (OR 4.35 95%CI 1.30-14.53) and disseminated disease (OR 4.52 95%CI 1.44-14.15). GEP-NET primary tumor location or secretory syndrome did not influence the risk for MetS. None of the tumor characteristics evaluated were associated with body mass index, fasting plasma glucose category, or any of the individual MetS components. Patients with GEP-NET and MetS depicted a higher risk of presenting a lower tumor grade and disseminated disease. The positive association between MetS and GEP-NET characteristics further highlights the potential link between the two conditions.

Increase in ghrelin levels after weight loss in obese Zucker rats is prevented by gastric banding.

BACKGROUND: Gastric banding is thought to decrease appetite in addition to the mechanical effects of food restriction, although this has been difficult to demonstrate in human studies. Our aim was to investigate the changes in orexigenic signals in the obese Zucker rat after gastric banding. METHODS: Obese Zucker rats (fa/fa) were submitted to gastric banding (GBP), sham gastric banding fed ad libitum (sham), or sham operation with food restriction, pair-fed to the gastric banding group (sham-PF). Lean Zucker rats (fa/+) were used as additional controls. Body weight and food intake were daily recorded for 21 days after surgery when epididymal fat was weighed and fasting ghrelin and hypothalamic NPY mRNA expression were measured. RESULTS: Gastric banding in obese Zucker rats resulted in a significant decrease of cumulative body weight gain and food intake. Furthermore, gastric banded rats were leaner than Sham-PF, as expressed by a significantly lower epididymal fat weight. Ghrelin levels of gastric banded rats were not increased when compared to sham-operated animals fed ad libitum and were significantly lower than the levels of weight matched sham-PF rats (1116.9 +/- 103.3 g GBP vs 963.2 +/- 54.3 g sham, 3,079.5 +/- 221.6 sham-PF and 2,969.9 +/- 150.9 g lean rats, p < 0.001); hypothalamic NPY mRNA expression was not increased in GBP when compared to sham-operated rats. CONCLUSION: In obese Zucker rats, GBP prevents the increase in orexigenic signals that occur during caloric deprivation. Our data support the hypothesis that sustained weight loss observed after gastric banding does not depend solely on food restriction.

Decolourisation of a synthetic textile effluent using a bacterial consortium.

In the present study we examined the performance of a thermoalkalophilic bacterial consortium, where the predominant strain was Bacillus sp. SF, in the degradation of Reactive Black 5 (RB5). We used a reactor working in continuous mode and investigated the effects of pH, hydraulic retention time (HRT) and several added salts on colour and chemical oxygen demand (COD) reductions. For the chosen operational conditions (pH 9, 55 degrees C and HRT of 12 h) the efficiencies achieved were 91.2 +/- 0.8 % for colour removal and 81.2% for COD removal. The system tolerated, with no significant decrease in colour removal efficiency, 30 g/L Na(2)SO(4), Na(2)CO(3) or NaCl. The latter two salts, however, led to a reduction in COD removal of 30% and 50%, respectively. The system proved to be very effective in the decolourisation of C.I. RB5 under alkaline conditions and at a comparatively high temperature.

Rats submitted to gastric banding are leaner and show distinctive feeding patterns.

BACKGROUND: Bariatric surgery is expanding to meet the global epidemic of morbid obesity, because this surgery is successful in achieving sustained weight loss. After having recently established a rat model of gastric banding, our aim now was to investigate the relative fat mass content and the feeding patterns of gastric banded rats. METHODS: Two groups of Wistar rats, submitted either to gastric banding or to sham surgery, were followed-up for 26 days regarding weight, daily food intake and feeding patterns both under resting conditions and when refed after fasting. Weight of the epididymal fat pad was used as a measure to evaluate changes in white adipose tissue in the rats. RESULTS: 10 days after surgery and thereafter, rats submitted to gastric banding showed the same daily food intake that was observed in sham-operated rats. Nevertheless, gastric banded rats kept lower body weights and were leaner than controls. These differences were associated with distinctive feeding patterns, both under resting conditions and when refed after fasting, suggesting that gastric banded rats present a significant increase in feeding frequency when compared with controls. CONCLUSION: This data is the first experimental evidence that an increase in feeding frequency is associated with weight loss after gastric banding, even if there is no decrease in total energy intake. Thus, medical advice on the advantages of fractionating daily caloric intake into multiple meals is further supported by the herein new information obtained in an animal model of gastric banding.

A rat model of restrictive bariatric surgery with gastric banding.

BACKGROUND: Gastric banding is a well established weight reduction operation that is effective in the treatment of severe obesity. Its metabolic and endocrine mechanisms of action, however, remain unclear. The aim of this study was to establish a rat model of gastric banding that would replicate the procedure performed in human obese patients. METHODS: Male Wistar rats were submitted either to gastric banding (n=5) or sham gastric banding (n=4), and were followed for 21 days. Detailed description on how to perform gastric banding in rats are herein described. RESULTS: The Wistar rats submitted to gastric banding showed a decrease in weight gain and food intake when compared to sham-operated rats. The cumulative weight gain during the 21 days after the surgical procedure was 143+/-2.58 g for the gastric banded rats and 162+/-2.48 g for the sham-operated animals (P=0.001). The cumulative food intake was 329+/-0.53 g for the gastric banded rats and 380+/-15.22 g for the sham-operated animals, also statistically significant (P=0.025). CONCLUSION: A rat model to study gastric banding is described. This model can now be used for experimental investigation of biochemical and molecular mechanisms of weight loss resulting from this type of surgery.

Azo reductase activity of intact saccharomyces cerevisiae cells is dependent on the Fre1p component of plasma membrane ferric reductase.

Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae Deltafre1 and Deltafre1 Deltafre2 mutant strains, but not the Deltafre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae Deltafre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.

Characterization of azo reduction activity in a novel ascomycete yeast strain.

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.