The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear ...translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKβ and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.
The old Greek saying “Panta Rhei” (“everything flows”) is true for all life and all living things in general. It also becomes nicely evident when looking closely into cells. There, material from the ...extracellular space is taken up by endocytic processes and transported to endosomes where it is sorted either for recycling or degradation. Cargo is also packaged for export through exocytosis involving the Golgi network, lysosomes and other organelles. Everything in this system is in constant motion and many proteins are necessary to coordinate transport along the different intracellular pathways to avoid chaos. Among these proteins are ion channels., in particular TRPML channels (mucolipins) and two-pore channels (TPCs) which reside on endosomal and lysosomal membranes to speed up movement between organelles, e.g. by regulating fusion and fission; they help readjust pH and osmolarity changes due to such processes, or they promote exocytosis of export material. Pathophysiologically, these channels are involved in neurodegenerative, metabolic, retinal and infectious diseases, cancer, pigmentation defects, and immune cell function, and thus have been proposed as novel pharmacological targets, e.g. for the treatment of lysosomal storage disorders, Duchenne muscular dystrophy, or different types of cancer. Here, we discuss the similarities but also differences of TPCs and TRPMLs in regulating phagocytosis, autophagy and lysosomal exocytosis, and we address the contradictions and open questions in the field relating to the roles TPCs and TRPMLs play in these different processes.
The growing worldwide demand for more efficient and less polluting forms of energy production has led to a renewed interest in the use of micro-cogeneration technologies in the residential. Among the ...others technologies, internal combustion engine-based micro-cogeneration devices are a market-ready technology gaining an increasing appeal thanks to their high efficiency, fuel flexibility, low emissions, low noise and vibration.
In order to explore and assess the feasibility of using internal combustion engine-based cogeneration systems in the residential sector, an accurate and practical simulation model that can be used to conduct sensitivity and what-if analyses is needed. A residential cogeneration device model has been developed within IEA/ECBCS Annex 42 and implemented into a number of building simulation programs. This model is potentially able to accurately predict the thermal and electrical outputs of the residential cogeneration devices, but it relies almost entirely on empirical data because the model specification uses experimental measurements contained within a performance map to represent the device specific performance characteristics coupled with thermally massive elements to characterize the device's dynamic thermal performance.
At the Built Environment Control Laboratory of Seconda Università degli studi di Napoli, an AISIN SEIKI micro-cogeneration device based on natural gas fuelled reciprocating internal combustion engine is available. This unit has been intensively tested in order to calibrate and validate the Annex 42 model. This paper shows in detail the series of experiments conducted for the calibration activity and examines the validity of this model by contrasting simulation predictions to measurements derived by operating the system in electric load following control strategy. The statistical comparison was made both for the whole database and the segregated data by system mode operation.
The good agreement found in the predictions of net electric power production, useful thermal output and primary power consumption allowed to conclude that the Annex 42 model can be used to carry out a detailed performance assessment in order to examine the applicability of the AISIN SEIKI unit for supplying building electrical and thermal energy requirements according to different load profiles during annual or multi-year operation.
► MCHP systems can reduce primary energy consumption and greenhouse gas emissions. ► A simulation model can be used to assess the feasibility of using MCHP systems. ► A MCHP simulation model has been experimentally calibrated and validated.
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•Hydrogels from glycol chitosan were obtained in physiological conditions.•PEG diglycidyl ether was used as the crosslinker.•A purification step is not required after synthesis.•The ...obtained GCS-PEG scaffolds have antibacterial activity.•The GCS-PEG scaffolds stimulate angiogenesis like the positive control VEGF.
We aimed at producing a hydrogel from a chitosan (CS) derivative soluble in physiological conditions to avoid any purification step thus allowing to use the materials also as an in-situ forming material. So, we crosslinked glycol chitosan (GCS) with poly(ethylene glycol) diglycidyl ether (PEGDE) in water at 37 °C. The scaffolds, referred as GCS-PEG, were specifically designed to be used as wound dressing materials as such (after crosslinking) or as in-situ forming materials.
Different amounts of PEGDE were tested. The obtained scaffolds showed macroscopic pores and a tailorable swelling in water by controlling the crosslinking degree. Moreover, GCS-PEG scaffolds displayed a significant antimicrobial activity against Staphylococcus aureus. In-vivo study using the chick embryo choriallantoic membrane resulted in a highly pronounced pro-angiogenic activity suggesting important tissue regeneration properties. Moreover, the employed materials are commercially available, no organic solvents are required and the scaling up is quite predictable.
Menkes disease Bertini, I.; Rosato, A.
Cellular and molecular life sciences : CMLS,
01/2008, Letnik:
65, Številka:
1
Journal Article
Recenzirano
.
Menkes disease is caused by mutations in the copper-transporting P
1B
-type ATPase ATP7A. ATP7A has a dual function: it serves to incorporate copper into copper-dependent enzymes, and it maintains ...intracellular copper levels by removing excess copper from the cytosol. To accomplish both functions, the protein traffics between different cellular locations depending on copper levels.The mechanism for sensing the concentration of copper, for trafficking, as well as the details of the mechanism of copper translocation across the membrane are unknown.
Structure determination of complex molecular machines requires a combination of an increasing number of experimental methods with highly specialized software geared toward each data source to ...properly handle the gathered data. Recently, we introduced the two software packages PowerFit and DisVis. These combine high-resolution structures of atomic subunits with density maps from cryo-electron microscopy or distance restraints, typically acquired by chemical cross-linking coupled with mass spectrometry, respectively. Here, we report on recent advances in both GPGPU-accelerated software packages: PowerFit is a tool for rigid body fitting of atomic structures in cryo-electron density maps and has been updated to also output reliability indicators for the success of fitting, through the use of the Fisher z-transformation and associated confidence intervals; DisVis aims at quantifying the information content of distance restraints and identifying false-positive restraints. We extended its analysis capabilities to include an analysis of putative interface residues and to output an average shape representing the putative location of the ligand. To facilitate their use by a broad community, they have been implemented as web portals harvesting both local CPU resources and GPGPU-accelerated EGI grid resources. They offer user-friendly interfaces, while minimizing computational requirements, and provide a first interactive view of the results. The portals can be accessed freely after registration via http://milou.science.uu.nl/services/DISVIS and http://milou.science.uu.nl/services/POWERFIT.
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•Core-weighted local cross-correlation and edge-enhancing Laplace filter in PowerFit•New occupancy analysis and statistics for interacting residues in DisVis•Grid-based, GPGPU-enabled modular web portals•Web portals with advanced post-processing for online visualization of the results
Abstract Muscle tissue engineering can provide support to large congenital skeletal muscle defects using scaffolds able to allow cell migration, proliferation and differentiation. Acellular ...extracellular matrix (ECM) scaffold can generate a positive inflammatory response through the activation of anti-inflammatory T-cell populations and M2 polarized macrophages that together lead to a local pro-regenerative environment. This immunoregulatory effect is maintained when acellular matrices are transplanted in a xenogeneic setting, but it remains unclear whether it can be therapeutic in a model of muscle diseases. We demonstrated here for the first time that orthotopic transplantation of a decellularized diaphragmatic muscle from wild animals promoted tissue functional recovery in an established atrophic mouse model. In particular, ECM supported a local immunoresponse activating a pro-regenerative environment and stimulating host muscle progenitor cell activation and migration. These results indicate that acellular scaffolds may represent a suitable regenerative medicine option for improving performance of diseased muscles.
Microbial transglutaminase (mTGase) is an enzyme that catalyzes site-specific protein derivatization at specific glutamines. mTGase-mediated conjugation with PEG-NH
2
to granulocyte colony ...stimulating factor (G-CSF) yields a site selective mono-derivative conjugate involving Gln135. The same enzymatic reaction of mTGase,
i.e.
the transfer of the Gln acyl group to an amino donor, was investigated by reversing the substrates. A specific acyl donor PEG derivative was synthesized by coupling the Z-QG mTGase substrate to PEG. The mTGase-mediated conjugation of this PEG-ZQG in the presence of G-CSF generated a high-yield PEG-G-CSF conjugate in which the polymer was selectively coupled to Lys41 of the protein. The PEG-K41-G-CSF conjugate was compared with the PEG-Q135-G-CSF one obtained through mTGase conjugation of PEG-NH
2
to Gln135. Biophysical characterization showed that the two positional isomers have similar behaviors, and pharmacokinetic studies in rats demonstrated that they have comparable half-life extensions. Overall, the study demonstrates that mTGase protein derivatization is linked to inherent advantages because it carries with it the possibility of targeting lysines or glutamines, in both cases with a high site-selective specificity.
Microbial transglutaminase (mTGase) is an enzyme that catalyzes site-specific protein derivatization at specific glutamines and lysines.
Summary
Although iron under anaerobic conditions is more accessible and highly reactive because of its reduced form, iron‐dependent regulation is not well known in anaerobic bacteria. Here, we ...investigated iron‐ and hemin‐dependent gene regulation in Porphyromonas gingivalis, an established periodontopathogen that primarily inhabits anaerobic pockets. Whole‐genome microarrays of P. gingivalis genes were used to compare the levels of gene expression under iron‐replete and iron‐depleted conditions as well as under hemin‐replete and hemin‐depleted conditions. Under iron‐depleted conditions, the expression of genes encoding proteins that participate in iron uptake and adhesion/invasion of host cells was increased, while that of genes encoding proteins involved in iron storage, energy metabolism, and electron transport was decreased. Interestingly, many of the genes with altered expression had no known function. Limiting the amount of hemin also resulted in a reduced expression of the genes encoding proteins involved in energy metabolism and electron transport. However, hemin also had a significant effect on many other biological processes such as oxidative stress protection and lipopolysaccharide synthesis. Overall, comparison of the data from iron‐depleted conditions to those from hemin‐depleted ones showed that although some regulation is through the iron derived from hemin, there also is significant distinct regulation through hemin only. Furthermore, our data showed that the molecular mechanisms of iron‐dependent regulation are novel as the deletion of the putative Fur protein had no effect on the expression of iron‐regulated genes. Finally, our functional studies demonstrated greater survivability of host cells in the presence of the iron‐stressed bacterium than the iron‐replete P. gingivalis cells. The major iron‐regulated proteins encoded by PG1019–20 may play a role in this process as deletion of these sequences also resulted in reduced survival of the bacterium when grown with eukaryotic cells. Taken together, the results of this study demonstrated the utility of whole‐genome microarray analysis for the identification of genes with altered expression profiles during varying growth conditions and provided a framework for the detailed analysis of the molecular mechanisms of iron and hemin acquisition, metabolism and virulence of P. gingivalis.
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