Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, ...cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis.
Abstract
Megalin/LRP2 is a major receptor supporting apical endocytosis in kidney proximal tubular cells. We have previously reported that kidney-specific perinatal ablation of the megalin gene in ...cystinotic mice, a model of nephropathic cystinosis, essentially blocks renal cystine accumulation and partially preserves kidney tissue integrity. Here, we examined whether inhibition of the megalin pathway in adult cystinotic mice by dietary supplementation (5x-fold
vs
control regular diet) with the dibasic amino-acids (dAAs), lysine or arginine, both of which are used to treat patients with other rare metabolic disorders, could also decrease renal cystine accumulation and protect cystinotic kidneys. Using surface plasmon resonance, we first showed that both dAAs compete for protein ligand binding to immobilized megalin in a concentration-dependent manner, with identical inhibition curves by
l
- and
d
-stereoisomers. In cystinotic mice, 2-month diets with 5x-
l
-lysine and 5x-
l
-arginine were overall well tolerated, while 5x-
d
-lysine induced strong polyuria but no weight loss. All diets induced a marked increase of dAA urinary excretion, most prominent under 5x-
d
-lysine, without sign of kidney insufficiency. Renal cystine accumulation was slowed down approx. twofold by L-dAAs, and totally suppressed by
d
-lysine. We conclude that prolonged dietary manipulation of the megalin pathway in kidneys is feasible, tolerable and can be effective in vivo.
Electrohydrodynamic jetting is emerging as a successful technique for printing inks with resolutions well beyond those offered by conventional inkjet printers. However, the variety of printable inks ...is still limited to those with relatively low viscosities (typically <20 mPa s) due to nozzle clogging problems. Here, we show the possibility of printing ordered microdots of high viscous inks such as poly(lactic-co-glycolic acid) (PLGA) by exploiting the spontaneous breakup of a thin fiber generated through nozzle-free pyro-electrospinning. The PLGA fiber is deposited onto a partially wetting surface, and the breakup is achieved simply by applying an appropriate thermal stimulation, which is able to induce polymer melting and hence a mechanism of surface area minimization due to the Plateau–Rayleigh instability. The results show that this technique is a good candidate for extending the printability at the microscale to high viscous inks, thus extending their applicability to additional applications, such as cell behavior under controlled morphological constraints.
Remarkable advances have been made during the last few decades in defining the organizational principles of the secretory pathway. The Golgi complex in particular has attracted special attention due ...to its central position in the pathway, as well as for its fascinating and complex structure. Analytical studies of this organelle have produced significant advances in our understanding of its function, although some aspects still seem to elude our comprehension. In more recent years a level of complexity surrounding this organelle has emerged with the discovery that the Golgi complex is involved in cellular processes other than the 'classical' trafficking and biosynthetic pathways. The resulting picture is that the Golgi complex can be considered as a cellular headquarters where cargo sorting/processing, basic metabolism, signalling and cell-fate decisional processes converge.
Spiral shapes occur frequently in nature as in the case of snail shells or the cochlea - the auditory portion of the inner ear. They also inspire many technological devices that take advantage of ...this geometry. Here we show that μ-pyro-electrospinning is able to control whipping instabilities in order to form spiralling fibres (down to 300 nm thick) directly on a support with true microscale regularity. The results show that polymer concentration plays a key role in producing reliable and long spirals. We investigate the cell response to these spiral templates that, thanks to their true regularity, would be useful for developing innovative cochlea regeneration scaffolds.
Nod-like Receptor Pyrin domain containing proteins (NLRPs) expressed by resident renal cells may contribute to the pathogenesis of multiple renal diseases. Cystinosis is a genetic disorder that ...affects kidney and particularly proximal tubular epithelial cells (PTEC). Here, we investigated the expression of NLRP family members in human control and cystinotic conditionally immortalized PTEC. Among all the NLRPs tested, we found that NLRP2 is highly expressed in cystinostic PTEC, but not in PTEC from healthy subjects. The NLRP2 overexpression was confirmed in primary PTEC and in kidney biopsies from cystinotic patients. In order to elucidate the role of NLRP2 in PTEC, we stably transfected control PTEC with an NLRP2-containing plasmid. We showed that NLRP2 markedly increases the production of several NF-κB regulated cytokines and chemokines. Accordingly, we demonstrated that NLRP2 interacts with IKKa and positively regulates the DNA-binding activity of p50 and p65 NF-κB, by modulating the p65 NF-κB phosphorylation status in Serine 536. Transcriptome analysis revealed that NLRP2 also upregulates the expression of profibrotic mediators and reduces that of several interferon-inducible genes. Finally, NLRP2 overexpression decreased the apoptotic cell rate. Consistently, silencing of NLRP2 by small-interfering RNA in cystinotic PTEC resulted in a significant decrease in cytokine and chemokine production as well as in an increase in the apoptosis rate. Altogether, our data reveals a previously unrecognized role for NLRP2 in regulating proinflammatory, profibrotic and antiapoptotic responses in PTEC, through NF-κB activation. Moreover, our findings unveil a novel potential mechanism involving NLRP2 overexpression in the pathogenesis of cystinosis.
The unique deformability and the compliance ability of thin sheets on soft substrates attract much interest for studying the phenomena related to elastic instabilities as well as for sensing very ...weak forces such as those generated by live cells in vitro. However, the techniques used currently for producing such platforms are affected by a high degree of complexity and poor repeatability. Moreover, their deformability is usually used as a passive response to the action of an external force. Herein we propose a novel concept for a reliable and dynamic skin-over-liquid system made of a periodic array of highly compliant microbumps actuated through electrode-free electrohydrodynamic (EHD) pressure. We demonstrate that these structures are highly repeatable and capable of swelling and deflating easily under a simple thermal stimulation driven by the pyroelectric effect, thus providing a challenging platform that can be actively controlled at the microscale. Furthermore, we show the proof of principle by swelling these microbumps for mechanically stimulating live cells in vitro, thus opening the route to more reliable and easy to accomplish assays in the field of mechanobiology.Biomechanics: Giving artificial skin the goosebumpsResearchers curious about how mechanical stress impacts cell growth can now turn to silicone plastics that grow microbumps on demand. Simonetta Grilli from Italy’s Institute of Applied Sciences and Intelligent Systems in Pozzuoli and colleagues created a controllable ‘skin-over-liquid’ system by coating a silicone polymer solution onto lithium niobate, a crystal that contains patterned regions of electric charge. An initial plasma treatment produced a flat stiff film on top of the liquid silicone. Thermal heating caused the skin to swell and form dimpled, air mattress-like patterns, corresponding to the underlying lithium niobate domains. Removal of heat restored the sheet to its initial state. The chemically inert skin supported live fibroblast cells and was used to determine reactions to mechanical stress. Significant variations in nuclei and cytoskeletons were observed between cells grown under flat or swollen-skin conditions.
Biofilms are detrimental to human life and industrial processes due to potential infections, contaminations, and deterioration. Therefore, the evaluation of microbial capability to form biofilms is ...of fundamental importance for assessing how different environmental factors may affect their vitality. Nowadays, the approaches used for biofilm evaluation are still poor in reliability and rapidity and often provide contradictory results. Here, we present what we call biofilm electrostatic test (BET) as a simple, rapid, and highly reproducible tool for evaluating in vitro the ability of bacteria to form biofilms through electrostatic interaction with a pyroelectrified carrier. The results show how the BET is able to produce viable biofilms with a density 6-fold higher than that on the control, after just 2 h incubation. The BET could pave the way to a rapid standardization of the evaluation of bacterial resistance among biofilm-producing microorganisms. In fact, due to its simplicity and cost-effectiveness, it is well suited for a rapid and easy implementation in a microbiology laboratory.
•We presented a deep investigation of the pyroelectric effect induced into ferroelectric LN by micro-heater structures integrated directly onto the surface of the crystals. Four configurations were ...considered in order to fully understand the distribution of the electric field under different stimulation conditions.•The effect of the micro-heater shape on the temperature distribution and consequently on the pyroelectric effect was evaluated using COMSOL™ Multiphysics. Simulations were validated by different experimental measurements.•In particular, the pyroelectric effect activated by the micro-heater was investigated analysing the current impulses detected using a micrometric metallic probe connected to an oscilloscope.•Finally, the high repeatability of the pyroelectric effect induced by the integrated micro-heater was demonstrated reporting the occurrence of the impulses.•The reported results let us to claim that by regulating the rate of heating or cooling of the different design of micro-heaters, the occurrence of the pyroelectric effect from the –Z surface of the LiNbO3 can be manipulated and/or controlled is an easy and efficient manner compared with traditional ways of thermalizing the crystal.
We present a deep investigation of pyroelectric fields generated by lithium niobate crystals through integrated microheater structures. The microheaters are made of highly compact titanium microcircuits able to dissipate heat through a low-power consuming Joule effect. Microheaters with diverse geometries were designed and fabricated on the +Z face of lithium niobate crystals, in order to characterize pyroelectric fields with different distributions. The pyroelectric effect was studied under ambient conditions analysing the current impulses detected using a metallic probe connected to an oscilloscope. The current impulses were related to the air breakdown induced by the electric field arising between the −Z face of the crystal and the metallic tip. We show that the fabrication technique is relatively easy to accomplish and we analyse the thermal behaviour of the microheaters both theoretically and experimentally. The results show how such microheaters are able to control the intensity and the spatial distribution of the pyroelectric fields at microscale.
During the Renaissance, Leonardo Da Vinci was the first person to successfully detail the anatomy of the aortic root and its adjacent structures. Ever since, novel insights into morphology, function, ...and their interplay have accumulated, resulting in advanced knowledge on the complex functional characteristics of the aortic valve (AV) and root. This has shifted our vision from the AV as being a static structure towards that of a dynamic interconnected apparatus within the aortic root as a functional unit, exhibiting a complex interplay with adjacent structures via both humoral and mechanical stimuli. This paradigm shift has stimulated surgical treatment strategies of valvular disease that seek to recapitulate healthy AV function, whereby AV disease can no longer be seen as an isolated morphological pathology which needs to be replaced. As prostheses still cannot reproduce the complexity of human nature, treatment of diseased AVs, whether stenotic or insufficient, has tremendously evolved, with a similar shift towards treatments options that are more hemodynamically centered, such as the Ross procedure and valve-conserving surgery. Native AV and root components allow for an efficient Venturi effect over the valve to allow for optimal opening during the cardiac cycle, while also alleviating the left ventricle. Next to that, several receptors are present on native AV leaflets, enabling messenger pathways based on their interaction with blood and other shear-stress-related stimuli. Many of these physiological and hemodynamical processes are under-acknowledged but may hold important clues for innovative treatment strategies, or as potential novel targets for therapeutic agents that halt or reverse the process of valve degeneration. A structured overview of these pathways and their implications for cardiothoracic surgeons and cardiologists is lacking. As such, we provide an overview on embryology, hemodynamics, and messenger pathways of the healthy and diseased AV and its implications for clinical practice, by relating this knowledge to current treatment alternatives and clinical decision making.
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