How the Electrical Conductivity of Water Fluids Affects Micro-EDM in the Short-Pulse Regime.

This work investigates micro-electro discharge machining (EDM) performance involving deionized and tap water. The chosen machining regime was semi-finishing, where open voltage (from 100 to 130 V) and current values (5-10 A) were applied using a 0.5 µs pulse-on time and a frequency of 150 kHz, i.e., a duty cycle of 25%. First, numerical analyses were performed via COMSOL Multiphysics and used to estimate the plasma channel distribution and melted material, varying the current, sparking gap, electrical conductivity, and permittivity of the two fluids. Then, experimentally, the micro-EDM of holes and channels in hardened thin steel plates were replicated three times for each considered fluid. The material removal rate (MRR), tool wear ratio (TWR), radius overcut, and surface roughness were plotted as a function of open voltage and electrical conductivity. The study proves that as voltage and current increase, the MRR and TWR decrease with electrical conductivity. Nonetheless, for higher electrical conductivity (tap water), the process did not proceed for lower open voltages and currents, and the radius overcut was reduced, contrary to what is commonly acknowledged. Finally, the crater morphology and size were evaluated using a confocal microscope and compared to simulated outcomes.

Rapid Prototyping of Bio-Inspired Dielectric Resonator Antennas for Sub-6 GHz Applications.

Bio-inspired Dielectric Resonator Antennas (DRAs) are engaging more and more attention from the scientific community due to their exceptional wideband characteristic, which is especially desirable for the implementation of 5G communications. Nonetheless, since these antennas exhibit peculiar geometries in their micro-features, high dimensional accuracy must be accomplished via the selection of the most suitable fabrication process. In this study, the challenges to the manufacturing process presented by the wideband Spiral shell Dielectric Resonator Antenna (SsDRA), based on the Gielis superformula, are addressed. Three prototypes, made of three different photopolymer resins, were manufactured by bottom-up micro-Stereolithography (SLA). This process allows to cope with SsDRA's fabrication criticalities, especially concerning the wavy features characterizing the thin spiral surface and the micro-features located in close proximity to the spiral origin. The assembly of the SsDRAs with a ground plane and feed probe was also accurately managed in order to guarantee reliable and repeatable measurements. The scattering parameter S11 trends were then measured by means of a Vector Network Analyzer, while the realized gains and 3D radiation diagrams were measured in the anechoic chamber. The experimental results show that all SsDRAs display relevant wideband behavior of 2 GHz at -10 dB in the sub-6 GHz range.

A small molecule UPR modulator for diabetes identified by high throughput screening.

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and in vivo disease models. Using asialoglycoprotein receptor 1 (ASGR) fused with Cypridina luciferase (CLuc) as reporter assay for folding capacity, we have screened a million small molecule library and identified APC655 as a potent activator of protein folding, that appears to act by promoting chaperone expression. Furthermore, APC655 improved pancreatic ß cell viability and insulin secretion under ER stress conditions induced by thapsigargin or cytokines. APC655 was also effective in preserving ß cell function and decreasing lipid accumulation in the liver of the leptin-deficient (ob/ob) mouse model. These results demonstrate a successful uHTS campaign that identified a modulator of UPR, which can provide a novel candidate for potential therapeutic development for a host of metabolic diseases.

Pulse-Type Influence on the Micro-EDM Milling Machinability of Si3N4-TiN Workpieces.

In this paper, the effect of the micro-electro discharge machining (EDM) milling machinability of Si3N4-TiN workpieces was investigated. The material removal rate (MRR) and tool wear rate (TWR) were analyzed in relation to discharge pulse types in order to evaluate how the different pulse shapes impact on such micro-EDM performance indicators. Voltage and current pulse waveforms were acquired during micro-EDM trials, scheduled according to a Design of Experiment (DOE); then, a pulse discrimination algorithm was used to post-process the data off-line and discriminate the pulse types as short, arc, delayed, or normal. The analysis showed that, for the considered process parameter combinations, MRR was sensitive only to normal pulses, while the other pulse types had no remarkable effect on it. On the contrary, TWR was affected by normal pulses, but the occurrence of arcs and delayed pulses induced unexpected improvements in tool wear. Those results suggest that micro-EDM manufacturing of Si3N4-TiN workpiece is relevantly different from the micro-EDM process performed on metal workpieces such as steel. Additionally, the inspection of the Si3N4-TiN micro-EDM surface, performed by SEM and EDS analyses, showed the presence of re-solidified droplets and micro-cracks, which modified the chemical composition and the consequent surface quality of the machined micro-features.

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems.

PKC and PKN in heart disease.

The protein kinase C (PKC) and closely related protein kinase N (PKN) families of serine/threonine protein kinases play crucial cellular roles. Both kinases belong to the AGC subfamily of protein kinases that also include the cAMP dependent protein kinase (PKA), protein kinase B (PKB/AKT), protein kinase G (PKG) and the ribosomal protein S6 kinase (S6K). Involvement of PKC family members in heart disease has been well documented over the years, as their activity and levels are mis-regulated in several pathological heart conditions, such as ischemia, diabetic cardiomyopathy, as well as hypertrophic or dilated cardiomyopathy. This review focuses on the regulation of PKCs and PKNs in different pathological heart conditions and on the influences that PKC/PKN activation has on several physiological processes. In addition, we discuss mechanisms by which PKCs and the closely related PKNs are activated and turned-off in hearts, how they regulate cardiac specific downstream targets and pathways, and how their inhibition by small molecules is explored as new therapeutic target to treat cardiomyopathies and heart failure.

Targeting early PKCθ-dependent T-cell infiltration of dystrophic muscle reduces disease severity in a mouse model of muscular dystrophy.

Chronic muscle inflammation is a critical feature of Duchenne muscular dystrophy and contributes to muscle fibre injury and disease progression. Although previous studies have implicated T cells in the development of muscle fibrosis, little is known about their role during the early stages of muscular dystrophy. Here, we show that T cells are among the first cells to infiltrate mdx mouse dystrophic muscle, prior to the onset of necrosis, suggesting an important role in early disease pathogenesis. Based on our comprehensive analysis of the kinetics of the immune response, we further identify the early pre-necrotic stage of muscular dystrophy as the relevant time frame for T-cell-based interventions. We focused on protein kinase C θ (PKCθ, encoded by Prkcq), a critical regulator of effector T-cell activation, as a potential target to inhibit T-cell activity in dystrophic muscle. Lack of PKCθ not only reduced the frequency and number of infiltrating T cells but also led to quantitative and qualitative changes in the innate immune cell infiltrate in mdx/Prkcq-/- muscle. These changes were due to the inhibition of T cells, since PKCθ was necessary for T-cell but not for myeloid cell infiltration of acutely injured muscle. Targeting T cells with a PKCθ inhibitor early in the disease process markedly diminished the size of the inflammatory cell infiltrate and resulted in reduced muscle damage. Moreover, diaphragm necrosis and fibrosis were also reduced following treatment. Overall, our findings identify the early T-cell infiltrate as a therapeutic target and highlight the potential of PKCθ inhibition as a therapeutic approach to muscular dystrophy. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functions via epigenetic modulation of Sca-1 and Pw1 promoters.

Understanding the regulation of the stem cell fate is fundamental for designing novel regenerative medicine strategies. Previous studies have suggested that pharmacological treatments with small molecules provide a robust and reversible regulation of the stem cell program. Previously, we showed that treatment with a vanadium compound influences muscle cell fatein vitro In this study, we demonstrate that treatment with the phosphotyrosine phosphatase inhibitor bisperoxovanadium (BpV) drives primary muscle cells to a poised stem cell stage, with enhanced function in muscle regenerationin vivofollowing transplantation into injured muscles. Importantly, BpV-treated cells displayed increased self-renewal potentialin vivoand replenished the niche in both satellite and interstitial cell compartments. Moreover, we found that BpV treatment induces specific activating chromatin modifications at the promoter regions of genes associated with stem cell fate, includingSca-1andPw1 Thus, our findings indicate that BpV resets the cell fate program by specific epigenetic regulations, such that the committed myogenic cell fate is redirected to an earlier progenitor cell fate stage, which leads to an enhanced regenerative stem cell potential.-Smeriglio, P., Alonso-Martin, S., Masciarelli, S., Madaro, L., Iosue, I., Marrocco, V., Relaix, F., Fazi, F., Marazzi, G., Sassoon, D. A., Bouché, M. Phosphotyrosine phosphatase inhibitor bisperoxovanadium endows myogenic cells with enhanced muscle stem cell functionsviaepigenetic modulation of Sca-1 and Pw1 promoters.

Targeting PKCθ in skeletal muscle and muscle diseases: good or bad?

Protein kinase Cθ (PKCθ) is a member of the novel calcium-independent PKC family, with a relatively selective tissue distribution. Most studies have focused on its unique role in T-lymphocyte activation and suggest that inhibition of PKCθ could represent a novel therapeutic approach in the treatment of chronic inflammation, autoimmunity and allograft rejection. However, considering that PKCθ is also expressed in other cell types, including skeletal muscle cells, it is important to understand its function in different tissues before proposing it as a molecular target for the treatment of immune-mediated diseases. A number of studies have highlighted the role of PKCθ in mediating several intracellular pathways, regulating muscle cell development, homoeostasis and remodelling, although a comprehensive picture is still lacking. Moreover, we recently showed that lack of PKCθ in a mouse model of Duchenne muscular dystrophy (DMD) ameliorates the progression of the disease. In the present article, we review new developments in our understanding of the involvement of PKCθ in intracellular mechanisms regulating skeletal muscle development, growth and maintenance under physiological conditions and recent advances showing a hitherto unrecognized role of PKCθ in promoting muscular dystrophy.

Gold strip gratings with binary supercell.

In this Letter, the study of a periodic structure composed of gold strips arranged in double-period unit cells, in a symmetric and asymmetric environment, is reported. The spectral maps show that the formation of the plasmonic bandgap and the extraordinary optical transmission are subjected to the proportion between the strip widths. Moreover, when the asymmetric environment is considered, high-transmittance and high-absorbance states arise. Hence, by controlling the geometrical parameters of the binary-periodic structure, it is possible to tailor the spectral response of the grating enhancing the desired features and exploiting them for different applications.

Intracellular signaling in ER stress-induced autophagy in skeletal muscle cells.

Skeletal muscle remodeling in response to muscle disuse and unloading is known to be associated with so-called ER stress, which, in turn, activates autophagy and contributes to muscle atrophy. Different molecules are involved in ER stress-induced autophagy, among which PKCθ has recently been described. In this study, we dissected both in vitro and in vivo ER stress-induced autophagy pathways in muscle. Using C2C12 muscle cells in culture, we demonstrated that PKC activation induced autophagy in the absence of ER stress. We further demonstrated that PKCθ was strongly activated in cultured myoblasts and myotubes during ER stress induced by different stimuli, such as TG or TN treatment, and that it localized into Lc3-positive autophagic dots upon TG treatment. Neither Akt dephosphorylation nor Foxo or GSK3ß activation was observed in these conditions. Moreover, PKCθ inhibition in myoblasts and myotubes prevented ER stress-induced Lc3 activation and autophagic dot formation, but not ER stress. In vivo, lack of PKCθ prevented both food deprivation- and immobilization-induced autophagy and muscle atrophy, irrespective of Akt pathway inhibition. Taken together, these results demonstrate that PKCθ functions as an ER stress sensor in skeletal muscle, required for ER-stress-dependent autophagy activation, and can be proposed as a novel molecular target to maintain muscle homeostasis in response to external stimuli, such as disuse and unloading, still allowing intracellular clearance.

PKC theta ablation improves healing in a mouse model of muscular dystrophy.

Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, limiting muscle regeneration and resulting in fibrotic and fatty tissue replacement of muscle, which exacerbates the wasting process in dystrophic muscles. Limiting immune response is thus one of the therapeutic options to improve healing, as well as to improve the efficacy of gene- or cell-mediated strategies to restore dystrophin expression. Protein kinase C θ (PKCθ) is a member of the PKCs family highly expressed in both immune cells and skeletal muscle; given its crucial role in adaptive, but also innate, immunity, it is being proposed as a valuable pharmacological target for immune disorders. In our study we asked whether targeting PKCθ could represent a valuable approach to efficiently prevent inflammatory response and disease progression in a mouse model of muscular dystrophy. We generated the bi-genetic mouse model mdx/θ(-/-), where PKCθ expression is lacking in mdx mice, the mouse model of Duchenne muscular dystrophy. We found that muscle wasting in mdx/θ(-/-) mice was greatly prevented, while muscle regeneration, maintenance and performance was significantly improved, as compared to mdx mice. This phenotype was associated to reduction in inflammatory infiltrate, pro-inflammatory gene expression and pro-fibrotic markers activity, as compared to mdx mice. Moreover, BM transplantation experiments demonstrated that the phenotype observed was primarily dependent on lack of PKCθ expression in hematopoietic cells.These results demonstrate a hitherto unrecognized role of immune-cell intrinsic PKCθ activity in the development of DMD. Although the immune cell population(s) involved remain unidentified, our findings reveal that PKCθ can be proposed as a new pharmacological target to counteract the disease, as well as to improve the efficacy of gene- or cell- therapy approaches.

Experimental demonstration of a novel bio-sensing platform via plasmonic band gap formation in gold nano-patch arrays.

In this paper we discuss the possibility of implementing a novel bio-sensing platform based on the observation of the shift of the leaky surface plasmon mode that occurs at the edge of the plasmonic band gap of metal gratings, when an analyte is deposited on top of the metallic structure. We report numerical calculations, fabrication and experimental measurements to prove the sensing capability of a two-dimensional array of gold nano-patches in the detection of a small quantity of Isopropyl Alcohol (IPA) deposited on top of sensor surface. The calculated sensitivity of our device approaches a value of 1000 nm/RIU with a corresponding Figure of Merit (FOM) of 222 RIU(-1). The presence of IPA can also be visually estimated by observing a color variation in the diffracted field. We show that color brightness and intensity variations can be ascribed to a change in the aperture size, keeping the periodicity constant, and to different types of analyte deposited on the sample, respectively. Moreover, we demonstrate that unavoidable fabrication imperfections revealed by the presence of rounded corners and surface roughness do not significantly affect device performance.

Plasmonic bandgap formation in two-dimensional periodic arrangements of gold patches with subwavelength gaps.

We report on the formation of plasmonic bandgaps in two-dimensional periodic arrangements of gold patches. Orthogonal arrays of subwavelength slits with different periodicities have been studied by means of a three-dimensional finite-difference time-domain (FDTD) code, changing incident polarization and geometrical parameters. Spectral response of gold patches having different a form factor and surrounded by different media have been also investigated and compared in order to give a full description of bandgap shifts paving the way for the design of polarization-sensitive devices.

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and ß1D integrin upstream focal adhesion kinase.

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and ß1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.