Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Osteopontin and systemic lupus erythematosus association: a probable gene-gender interaction

Osteopontin (SPP1) is an important bone matrix mediator found to have key roles in inflammation and immunity. SPP1 genetic polymorphisms and increased osteopontin protein levels have been reported to be associated with SLE in small patient collections. The present study evaluates association between SPP1 polymorphisms and SLE in a large cohort of 1141 unrelated SLE patients [707 European-American (EA) and 434 African-American (AA)], and 2009 unrelated controls (1309 EA and 700 AA). Population-based case-control association analyses were performed. To control for potential population stratification, admixture adjusted logistic regression, genomic control (GC), structured association (STRAT), and principal components analysis (PCA) were applied. Combined analysis of 2 ethnic groups, showed the minor allele of 2 SNPs (rs1126616T and rs9138C) significantly associated with higher risk of SLE in males (P = 0.0005, OR = 1.73, 95% CI = 1.28-2.33), but not in females. Indeed, significant gene-gender interactions in the 2 SNPs, rs1126772 and rs9138, were detected (P = 0.001 and P = 0.0006, respectively). Further, haplotype analysis identified rs1126616T-rs1126772A-rs9138C which demonstrated significant association with SLE in general (P = 0.02, OR = 1.30, 95%CI 1.08-1.57), especially in males (P = 0.0003, OR = 2.42, 95%CI 1.51-3.89). Subgroup analysis with single SNPs and haplotypes also identified a similar pattern of gender-specific association in AA and EA. GC, STRAT, and PCA results within each group showed consistent associations. Our data suggest SPP1 is associated with SLE, and this association is especially stronger in males. To our knowledge, this report serves as the first association of a specific autosomal gene with human male lupus.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Capsaicin Modulates Proliferation,Migration, and Activation of Hepatic Stellate Cells

Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro.     

Mitotracker Green Is a P-Glycoprotein Substrate

P-glycoprotein has a widespread expression on normal tissues. The protein has also been strongly associated with the multidrug resistance phenotype (MDR) on tumor cells. The employment of flow cytometry and confocal microscopy has contributed to the discovery and application of new particular fluorescent dyes. Nevertheless, several studies are being performed in different cellular types neglecting the expression/activity of MDR proteins. Because many fluorochromes have been reported as P-glycoprotein substrates, an especial attention must be given to the properties of new dyes in the presence of MDR proteins. Flow cytometric analyzes of Mitotracker Green (MTG) fluorescence profile were performed in a human erythroleukemic cell line and its resistant counterpart. In this report we demonstrated that MTG, a probe used to evaluate the mitochondrial mass, is a P-glycoprotein substrate and its staining profile is dependent on the activity of this protein. In vitro studies on a human erythroleukemic cell line and its resistant counterpart revealed that MDR modulators (Cyclosporin A, Verapamil, and Trifluoperazine) alter the MTG fluorescence pattern on a resistant cell line. The findings suggest that attention should be given to the expression of P-glycoprotein when performing an evaluation of mitochondria properties with MTG.     

What shapes the altitudinal range of a high mountain Mediterranean plant? Recruitment probabilities from ovule to seedling stage

Recruitment is a complex process consisting of sequential stages affected by biotic interactions and abiotic factors. Assessment of these sequential stages and corresponding subprocesses may be useful in identifying the most critical stages. Accordingly, to assess the factors that may determine the altitudinal range limits of the high mountain Mediterranean plant Silene ciliata, a set of demographic stages, from flower production to establishment of 2‐yr‐old plants, and their influence on recruitment probability were examined using a step‐by‐step approach. We integrated florivory, pollination and pre‐dispersal seed predation as pre‐dispersal factors, and seedling emergence and survival as post‐dispersal determinants of recruitment. Three populations were monitored at the southernmost margin of the species along its local altitudinal range. Previous studies suggest that seediness is strongly limited by summer drought especially at the lower boundary of the species, a situation that may worsen under current global warming. Our results showed that recruitment was mainly limited by low seed production in the pre‐dispersal stage and low seedling emergence and survival in the post‐dispersal stage, probably due to environmental harshness in summer. By contrast, biotic factors responsible for propagule loss, such as flower and fruit predation, had a minor effect on the probability of plant recruitment. Although the relative importance of transition probabilities was similar among populations along the altitudinal range, comparatively lower flower production significantly reduced the number of recruited plants at the lowest altitude population. This demographic bottleneck, together with increased competition with other species favoured by climate warming, might collapse population growth and limit persistence at the lower altitudinal range of the species, raising its low local altitudinal edge.     

Agroecological basis for the design of biotechnological traps based on Isaria fumosorosea for the biological control of Bemisia tabaci in strawberry crops

Isaria fumosorosea is an entomopathogenic fungus that is used as a control alternative for nymphs and adults of Bemisia tabaci. Currently there are some commercial products, however, in greenhouse or field, these do not reach the levels of control as in the laboratory because the viability of the spores decreases as a result of the conditions of application of these products in situ. The objective of this work is to implement, through agroecological data, a system of biotechnological traps based on I. fumosorosea to increase the control efficiency mainly of adults of B. tabaci in strawberry greenhouses. One way to quantify the degree of infestation of a crop is the use of yellow traps, likewise to determine the spatial distribution of adults. The Taylor method [(1984). Assessing and interpreting the spatial distributions of insect populations. Annual Reviews of Entomology, 29, 321–357) was used in five different strawberry cultivation models, finding aggregate and regular distributions. Finally, once the crop model with the highest degree of infestation was selected, the designed traps were tested and mortalities were obtained between 50% and 90% in both the laboratory and the greenhouse. The biotechnological traps based on I. fumosorosea both in the laboratory and in the greenhouse had statistically the same effect as those used under the traditional method used in the field that is aspersion; therefore, this alternative method of application can be a tool important for the biological control of this pest.