Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis

The glomerulus is a highly specialized capillary tuft, which under pressure filters large amounts of water and small solutes into the urinary space, while retaining albumin and large proteins. The glomerular filtration barrier (GFB) is a highly specialized filtration interface between blood and urine that is highly permeable to small and midsized solutes in plasma but relatively impermeable to macromolecules such as albumin. The integrity of the GFB is maintained by molecular interplay between its 3 layers: the glomerular endothelium, the glomerular basement membrane and podocytes, which are highly specialized postmitotic pericytes forming the outer part of the GFB. Abnormalities of glomerular ultrafiltration lead to the loss of proteins in urine and progressive renal insufficiency, underlining the importance of the GFB. Indeed, albuminuria is strongly predictive of the course of chronic nephropathies especially that of diabetic nephropathy (DN), a leading cause of renal insufficiency. We found that high glucose concentrations promote autophagy flux in podocyte cultures and that the abundance of LC3B II in podocytes is high in diabetic mice. Deletion of Atg5 specifically in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky GFB and glomerulosclerosis. Strikingly, genetic alteration of autophagy on the other side of the GFB involving the endothelial-specific deletion of Atg5 also resulted in capillary rarefaction and accelerated DN. Thus autophagy is a key protective mechanism on both cellular layers of the GFB suggesting autophagy as a promising new therapeutic strategy for DN.     

Convex-Envelope Based Automated Quantitative Approach to Multi-Voxel 1H-MRS Applied to Brain Tumor Analysis

Background

Magnetic Resonance Spectroscopy (MRS) can measure in vivo brain tissue metabolism that exhibits unique biochemical characteristics in brain tumors. For clinical application, an efficient and versatile quantification method of MRS would be an important tool for medical research, particularly for exploring the scientific problem of tumor monitoring. The objective of our study is to propose an automated MRS quantitative approach and assess the feasibility of this approach for glioma grading, prognosis and boundary detection.

Methods

An automated quantitative approach based on a convex envelope (AQoCE) is proposed in this paper, including preprocessing, convex-envelope based baseline fitting, bias correction, sectional baseline removal, and peak detection, in a total of 5 steps. Some metabolic ratios acquired by this quantification are selected for statistical analysis. An independent sample t-test and the Kruskal-Wallis test are used for distinguishing low-grade gliomas (LGG) and high-grade gliomas (HGG) and for detecting the tumor, peritumoral and contralateral areas, respectively. Seventy-eight cases of pre-operative brain gliomas with pathological reports are included in this study.

Results

Cho/NAA, Cho/Cr and Lip-Lac/Cr (LL/Cr) calculated by AQoCE in the tumor area differ significantly between LGG and HGG, with p≤0.005. Using logistic regression combining Cho/NAA, Cho/Cr and LL/Cr to generate a ROC curve, AQoCE achieves a sensitivity of 92.9%, a specificity of 72.2%, and an area under ROC curve (AUC) of 0.860. Moreover, both Cho/NAA and Cho/Cr in the AQoCE approach show a significant difference (p≤0.019) between tumoral, peritumoral, and contralateral areas. The comparison between the results of AQoCE and Siemens MRS processing software are also discussed in this paper.

Conclusions

The AQoCE approach is an automated method of residual water removal and metabolite quantification. It can be applied to multi-voxel ¹H-MRS for evaluating brain glioma grading and demonstrating characteristics of brain glioma metabolism. It can also detect infiltration in the peritumoral area. Under the limited clinical data used, AQoCE is significantly more versatile and efficient compared to the reference approach of Siemens.     

Biodistribution of 137Cs in a mouse model of chronic contamination by ingestion and effects on the hematopoietic system

The aim of this work was to define the possible occurrence of hematological changes during the course of a chronic ingestion of ¹³⁷Cs. A mouse model was used, with ingestion through drinking water with a cesium concentration of 20 kBq l⁻¹. Ingestion started in parent animals before mating, and ¹³⁷Cs intake and its effect on the hematopoietic system was studied in offspring at various ages between birth and 20 weeks. ¹³⁷Cs content was measured in various organs, indicating that ¹³⁷Cs was distributed throughout the organism including lympho-hematopoietic organs, i.e., femurs, spleen and thymus. However, we did not observe any effect on the hematopoietic system, whatever the parameter used. In fact, blood cell counts, mononuclear cell counts and progenitor frequency in bone marrow and spleen, and Flt3-ligand, Erythropoietin, G-CSF and SDF-1 concentration in plasma remained unchanged when compared to control animals. Moreover, phenotypic analysis did not show any change in the proportions of bone marrow cell populations. These results indicate that, although ¹³⁷Cs was found in all organs implicated in the hematopoietic system, this did not induce any changes in bone marrow function.     

The Cytosolic Tail Dipeptide Ile-Met of the Pea Receptor BP80 Is Required for Recycling from the Prevacuole and for Endocytosis

Pea (Pisum sativum) BP80 is a vacuolar sorting receptor for soluble proteins and has a cytosolic domain essential for its intracellular trafficking between the trans-Golgi network and the prevacuole. Based on mammalian knowledge, we introduced point mutations in the cytosolic region of the receptor and produced chimeras of green fluorescent protein fused to the transmembrane domain of pea BP80 along with the modified cytosolic tails. By analyzing the subcellular location of these chimera, we found that mutating Glu-604, Asp-616, or Glu-620 had mild effects, whereas mutating the Tyr motif partially redistributed the chimera to the plasma membrane. Replacing both Ile-608 and Met-609 by Ala (IMAA) led to a massive redistribution of fluorescence to the vacuole, indicating that recycling is impaired. When the chimera uses the alternative route, the IMAA mutation led to a massive accumulation at the plasma membrane. Using Arabidopsis thaliana plants expressing a fluorescent reporter with the full-length sequence of At VSR4, we demonstrated that the receptor undergoes brefeldin A–sensitive endocytosis. We conclude that the receptors use two pathways, one leading directly to the lytic vacuole and the other going via the plasma membrane, and that the Ileu-608 Met-609 motif has a role in the retrieval step in both pathways.     

Evidence that yeast frataxin is not an iron storage protein in vivo

Yeast cells deficient in the yeast frataxin homolog (Yfh1p) accumulate iron in their mitochondria. Whether this iron is toxic, however, remains unclear. We showed that large excesses of iron in the growth medium did not inhibit growth and did not decrease cell viability. Increasing the ratio of mitochondrial iron-to-Yfh1p by decreasing the steady-state level of Yfh1p to less than 100 molecules per cell had very few deleterious effects on cell physiology, even though the mitochondrial iron concentration greatly exceeded the iron-binding capacity of Yfh1p in these conditions. Mössbauer spectroscopy and FPLC analyses of whole mitochondria or of isolated mitochondrial matrices showed that the chemical and biochemical forms of the accumulated iron in mitochondria of mutant yeast strains (Δyfh1, Δggc1 and Δssq1) displayed a nearly identical distribution. This was also the case for Δggc1 cells, in which Yfh1p was overproduced. In these mitochondria, most of the iron was insoluble, and the ratio of soluble-to-insoluble iron did not change when the amount of Yfh1p was increased up to 4500 molecules per cell. Our results do not privilege the hypothesis of Yfh1p being an iron storage protein in vivo.     

S3EPY: a Sparky extension for determination of small scalar couplings from spin-state-selective excitation NMR experiments

S3EPY is a Python extension to the program Sparky written to facilitate the assessment of coupling constants from in-phase/antiphase and spin-state-selective excitation (S³E) experiments. It enables the routine use of small scalar couplings by automating the coupling evaluation procedure. S3EPY provides an integrated graphical user interface to programs which outputs graphs and the table of determined couplings.     

Gender affects liver desaturase expression in a rat model of n−3 fatty acid repletion

Dietary n?3 polyunsaturated fatty acids (PUFA) are major components of cell membranes and have beneficial effects on human health. Docosahexaenoic acid (DHA; 22:6n?3) is the most biologically important n?3 PUFA and can be synthesized from its dietary essential precursor, α-linolenic acid (ALA; 18:3n?3). Gender differences in the efficiency of DHA bioconversion have been reported, but underlying molecular mechanisms are unknown. We compared the capacity for DHA synthesis from ALA and the expression of related enzymes in the liver and cerebral cortex between male and female rats. Wistar rats, born with a low-DHA status, were supplied with a suboptimal amount of ALA from weaning to 8 weeks of age. Fatty acid composition was determined by gas chromatography, the mRNA expression of different genes involved in PUFA metabolism was determined by RT-PCR (low-density array) and the expression of proteins was determined by Western blot analysis. At 8 weeks, DHA content was higher (+20 to +40%) in each phospholipid class of female livers compared to male livers. The “Δ4,” Δ5 and Δ6 desaturation indexes were 1.2–3 times higher in females than in males. The mRNA expression of Δ5- and Δ6-desaturase genes was 3.8 and 2.5 times greater, respectively, and the Δ5-desaturase protein was higher in female livers (+50%). No gender difference was observed in the cerebral cortex. We conclude that female rats replete their DHA status more readily than males, probably due to a higher expression of liver desaturases. Our results support the hypothesis on hormonal regulation of PUFA metabolism, which should be taken into account for specific nutritional recommendations.