Aberrant mitochondrial dynamics contributes to diaphragmatic weakness induced by mechanical ventilation.

In critical care patients, the ""temporary inactivity of the diaphragm caused by mechanical ventilation (MV) triggers a series of events leading to diaphragmatic dysfunction and atrophy, commonly known as ventilator-induced diaphragm dysfunction (VIDD). While mitochondrial dysfunction related to oxidative stress is recognized as a crucial factor in VIDD, the exact molecular mechanism remains poorly understood. In this study, we observe that 6 h of MV triggers aberrant mitochondrial dynamics, resulting in a reduction in mitochondrial size and interaction, associated with increased expression of dynamin-related protein 1 (DRP1). This effect can be prevented by P110, a molecule that inhibits the recruitment of DRP1 to the mitochondrial membrane. Furthermore, isolated mitochondria from the diaphragms of ventilated patients exhibited increased production of reactive oxygen species (ROS). These mitochondrial changes were associated with the rapid oxidation of type 1 ryanodine receptor (RyR1) and a decrease in the stabilizing subunit calstabin 1. Subsequently, we observed that the sarcoplasmic reticulum (SR) in the ventilated diaphragms showed increased calcium leakage and reduced contractile function. Importantly, the mitochondrial fission inhibitor P110 effectively prevented all of these alterations. Taken together, the results of our study illustrate that MV leads, in the diaphragm, to both mitochondrial fragmentation and dysfunction, linked to the up-/down-regulation of 320 proteins, as assessed through global comprehensive quantitative proteomics analysis, primarily associated with mitochondrial function. These outcomes underscore the significance of developing compounds aimed at modulating the balance between mitochondrial fission and fusion as potential interventions to mitigate VIDD in human patients.

Mitochondria in cell senescence: A Friend or Foe?

Cell senescence denotes cell growth arrest in response to continuous replication or stresses damaging DNA or mitochondria. Mounting research suggests that cell senescence attributes to aging-associated failing organ function and diseases. Conversely, it participates in embryonic tissue maturation, wound healing, tissue regeneration, and tumor suppression. The acute or chronic properties and microenvironment may explain the double faces of senescence. Senescent cells display unique characteristics. In particular, its mitochondria become elongated with altered metabolomes and dynamics. Accordingly, mitochondria reform their function to produce more reactive oxygen species at the cost of low ATP production. Meanwhile, destructed mitochondrial unfolded protein responses further break the delicate proteostasis fostering mitochondrial dysfunction. Additionally, the release of mitochondrial damage-associated molecular patterns, mitochondrial Ca2+ overload, and altered NAD+ level intertwine other cellular organelle strengthening senescence. These findings further intrigue researchers to develop anti-senescence interventions. Applying mitochondrial-targeted antioxidants reduces cell senescence and mitigates aging by restoring mitochondrial function and attenuating oxidative stress. Metformin and caloric restriction also manifest senescent rescuing effects by increasing mitochondria efficiency and alleviating oxidative damage. On the other hand, Bcl2 family protein inhibitors eradicate senescent cells by inducing apoptosis to facilitate cancer chemotherapy. This review describes the different aspects of mitochondrial changes in senescence and highlights the recent progress of some anti-senescence strategies.

Mitochondrial targeted antioxidants, mitoquinone and SKQ1, not vitamin C, mitigate doxorubicin-induced damage in H9c2 myoblast: pretreatment vs. co-treatment.

BACKGROUND: Preconditioning of the heart ameliorates doxorubicin (Dox)-induced cardiotoxicity. We tested whether pretreating cardiomyocytes by mitochondrial-targeted antioxidants, mitoquinone (MitoQ) or SKQ1, would provide better protection against Dox than co-treatment. METHODS: We investigated the dose-response relationship of MitoQ, SKQ1, and vitamin C on Dox-induced damage on H9c2 cardiomyoblasts when drugs were given concurrently with Dox (e.g., co-treatment) or 24 h prior to Dox (e.g., pretreatment). Moreover, their effects on intracellular and mitochondrial oxidative stress were evaluated by 2,7-dichlorofluorescin diacetate and MitoSOX, respectively. RESULTS: Dox (0.5-50 µM, n = 6) dose-dependently reduced cell viability. By contrast, co-treatment of MitoQ (0.05-10 µM, n = 6) and SKQ1 (0.05-10 µM, n = 6), but not vitamin C (1-2000 µM, n = 3), significantly improved cell viability only at intermediate doses (0.5-1 µM). MitoQ (1 µM) and SKQ1 (1 µM) significantly increased cell viability to 1.79 ± 0.12 and 1.59 ± 0.08 relative to Dox alone, respectively (both p < 0.05). Interestingly, when given as pretreatment, only higher doses of MitoQ (2.5 µM, n = 9) and SKQ1 (5 µM, n = 7) showed maximal protection and improved cell viability to 2.19 ± 0.13 and 1.65 ± 0.07 relative to Dox alone, respectively (both p < 0.01), which was better than that of co-treatment. Moreover, the protective effects were attributed to the significant reduction in Dox-induced intracellular and mitochondrial oxidative stress. CONCLUSION: The data suggest that MitoQ and SKQ1, but not vitamin C, mitigated DOX-induced damage. Moreover, MitoQ pretreatment showed significantly higher cardioprotection than its co-treatment and SKQ1, which may be due to its better antioxidant effects.

Ultrasonic Welding of Novel Carbon/ Elium® Thermoplastic Composites with Flat and Integrated Energy Directors: Lap Shear Characterisation and Fractographic Investigation.

The current research work presents a first attempt to investigate the welding attributes of Elium® thermoplastic resin and the fusion bonding using ultrafast ultrasonic welding technique. The integrated energy director (ED) polymer-matrix composites (PMCs) panel manufacturing was carried out using the Resin Transfer Moulding (RTM) technique and the scheme is deduced to manufacture a bubble-free panel. Integrated ED configurations and flat specimens with Elium® film of different thickness at the interface were investigated for ultrasonic welding optimization. Optimised weld time for integrated ED and flat Elium® panels with film (0.5 mm thick) configuration was found to be 1 s and 5.5 s, respectively. The ED integrated configuration showed the best welding results with a lap shear strength of 18.68 MPa. The morphological assessment has shown significant plastic deformation of Elium® resin and the shear cusps formation, which enhances the welding strength. This research has the potential to open up an excellent and automated way of joining Elium® composite parts in automotive, wind turbines, sports, and many other industrial applications.

Investigation on Ultrasonic Welding Attributes of Novel Carbon/Elium® Composites.

Joining large and complex polymer-matrix composite structures is becoming increasingly important in industries such as automobiles, aerospace, sports, wind turbines, and others. Ultrasonic welding is an ultra-fast joining process and also provides excellent joint quality as a cost-effective alternative to other joining processes. This research aims at investigating the welding characteristics of novel methyl methacrylate Elium®, a liquid thermoplastic resin. Elium® is the first of its kind of thermoplastic resin, which is curable at room temperature and is suitable for mass production processes. The welding characteristics of Elium® composites were investigated by optimizing the welding parameters with specially designed integrated energy directors (ED) and manufactured using the Resin transfer molding process. The results showed a 23% higher lap shear strength for ultrasonically welded composite joints when compared to the adhesively bonded joints. The optimized welding time for the ultrasonic welded joint was found to be 1.5 s whereas it was 10 min for the adhesively bonded joint. Fractographic analysis showed the significant plastic deformation and shear cusps formation on the fractured surface, which are typical characteristics for strong interfacial bonding.

Mitochondrial oxidative stress induces leaky ryanodine receptor during mechanical ventilation.

RATIONALE: Ventilator-induced diaphragm dysfunction (VIDD) increases morbidity and mortality in critical care patients. Although VIDD has been associated with mitochondrial oxidative stress and calcium homeostasis impairment, the underling mechanisms are still unknown. We hypothesized that diaphragmatic mitochondrial oxidative stress causes remodeling of the ryanodine receptor (RyR1)/calcium release channel, contributing to sarcoplasmic reticulum (SR) Ca2+ leak, proteolysis and VIDD. METHOD: In mice diaphragms mechanically ventilated for short (6 h) and long (12 h) period, we assessed mitochondrial ROS production, mitochondrial aconitase activity as a marker of mitochondrial oxidative stress, RyR1 remodeling and function, Ca2+ dependent proteolysis, TGFß1 and STAT3 pathway, muscle fibers cross-sectional area, and diaphragm specific force production, with or without the mitochondrial targeted anti-oxidant peptide d-Arg-2', 6'-dimethyltyrosine-Lys-Phe-NH2 (SS31). MEASUREMENTS AND MAIN RESULTS: 6 h of mechanical ventilation (MV) resulted in increased mitochondrial ROS production, reduction of mitochondrial aconitase activity, increased oxidation, S-nitrosylation, S-glutathionylation and Ser-2844 phosphorylation of RyR1, depletion of stabilizing subunit calstabin1 from RyR1, increased SR Ca2+ leak. Preventing mROS production by SS31 treatment does not affect the TGFß1 and STAT3 activation, which suggests that mitochondrial oxidative stress is a downstream pathway to TGFß1 and STAT3, early involved in VIDD. This is further supported by the fact that SS-31 rescue all the other described cellular events and diaphragm contractile dysfunction induced by MV, while SS20, an analog of SS31 lacking antioxidant properties, failed to prevent these cellular events and the contractile dysfunction. Similar results were found in ventilated for 12 h. Moreover, SS31 treatment prevented calpain1 activity and diaphragm atrophy observed after 12 h of MV. This study emphasizes that mitochondrial oxidative stress during 6 h-MV contributes to SR Ca2+ leak via RyR1 remodeling, and diaphragm weakness, while longer periods of MV (12 h) were also associated with increased Ca2+-dependent proteolysis and diaphragm atrophy.

Collagen fibrils and proteoglycans of peripheral and central stroma of the keratoconus cornea - Ultrastructure and 3D transmission electron tomography.

Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet's membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Ultrastructural study of peripheral and central stroma of keratoconus cornea.

PURPOSE: Assess the lamellar organisation of the peripheral and central stroma of the keratoconus (KC) and normal cornea. METHODS: Five normal and three KC corneas were fixed in 2.5% glutaraldehyde and processed for electron microscopy. The ultrathin sections were observed under JEOL 1400 TEM, and digital images were taken with a bottom-mounted 11-megapixel Quamisa camera, using the iTEM software. Measurements of the lamellae were carried out using the iTEM software. Statistical analysis was performed using the SPSS software. RESULTS: The lamellar organisation at the centre and periphery of the KC cornea was disrupted by the presence of multiple undulations, which were more aggressive at the posterior stroma. Among the KC cornea, the mean lamellar thickness of the peripheral middle (1030.32±86.25 nm) and posterior (615.68±30.94 nm) stroma was also significantly (p<0.05) thinner than their corresponding areas of the central KC cornea (1151.1±48 nm; 783.57±31.10 nm). At the periphery of KC cornea, just above the Descemet's membrane (DM), small undulations appeared to emerge out from the DM. Furthermore, the anterior stroma of the peripheral cornea contained several lamellar sutures. The mean lamellar thickness of the peripheral and central KC cornea was significantly (p<0.0001) thinner than the corresponding areas of the normal cornea. CONCLUSION: The present study reveals the involvement of lamellae in the peripheral stroma in the pathogenicity of the KC cornea. The emergence of small undulations in the DM suggests that the formation of undulation might be starting from the DM.

Unaffected contractility of diaphragm muscle fibers in humans on mechanical ventilation.

Several studies have indicated that diaphragm dysfunction develops in patients on mechanical ventilation (MV). Here, we tested the hypothesis that the contractility of sarcomeres, i.e., the smallest contractile unit in muscle, is affected in humans on MV. To this end, we compared diaphragm muscle fibers of nine brain-dead organ donors (cases) that had been on MV for 26 ± 5 h with diaphragm muscle fibers from nine patients (controls) undergoing surgery for lung cancer that had been on MV for less than 2 h. In each diaphragm specimen we determined 1) muscle fiber cross-sectional area in cryosections by immunohistochemical methods and 2) the contractile performance of permeabilized single muscle fibers by means of maximum specific force, kinetics of cross-bridge cycling by rate of tension redevelopment, myosin heavy chain content and concentration, and calcium sensitivity of force of slow-twitch and fast-twitch muscle fibers. In case subjects, we noted no statistically significant decrease in outcomes compared with controls in slow-twitch or fast-twitch muscle fibers. These observations indicate that 26 h of MV of humans is not invariably associated with changes in the contractile performance of sarcomeres in the diaphragm.

Increased proteolysis, myosin depletion, and atrophic AKT-FOXO signaling in human diaphragm disuse.

RATIONALE: Patients on mechanical ventilation who exhibit diaphragm inactivity for a prolonged time (case subjects) develop decreases in diaphragm force-generating capacity accompanied by diaphragm myofiber atrophy. OBJECTIVES: Our objectives were to test the hypotheses that increased proteolysis by the ubiquitin-proteasome pathway, decreases in myosin heavy chain (MyHC) levels, and atrophic AKT-FOXO signaling play major roles in eliciting these pathological changes associated with diaphragm disuse. METHODS: Biopsy specimens were obtained from the costal diaphragms of 18 case subjects before harvest (cases) and compared with intraoperative specimens from the diaphragms of 11 patients undergoing surgery for benign lesions or localized lung cancer (control subjects). Case subjects had diaphragm inactivity and underwent mechanical ventilation for 18 to 72 hours, whereas this state in controls was limited to 2 to 4 hours. MEASUREMENTS AND MAIN RESULTS: With respect to proteolysis in cytoplasm fractions, case diaphragms exhibited greater levels of ubiquitinated-protein conjugates, increased activity of the 26S proteasome, and decreased levels of MyHCs and α-actin. With respect to atrophic signaling in nuclear fractions, case diaphragms exhibited decreases in phosphorylated AKT, phosphorylated FOXO1, increased binding to consensus DNA sequence for Atrogin-1 and MuRF-1, and increased supershift of DNA-FOXO1 complexes with specific antibodies against FOXO1, as well as increased Atrogin-1 and MuRF-1 transcripts in whole myofiber lysates. CONCLUSIONS: Our findings suggest that increased activity of the ubiquitin-proteasome pathway, marked decreases in MyHCs, and atrophic AKT-FOXO signaling play important roles in eliciting the myofiber atrophy and decreases in diaphragm force generation associated with prolonged human diaphragm disuse.

Assembly of metal nanoparticles into nanogaps.

The directed assembly of nanoparticles and nanoscale materials onto specific locations of a surface is one of the major challenges in nanotechnology. Here we present a simple and scalable method and model for the assembly of nanoparticles in between electrical leads. Gold nanoparticles, 20 nm in diameter, were assembled inside electrical gaps ranging from 15 to 150 nm with the use of positive ac dielectrophoresis. In this method, an alternating current is used to create a gradient of electrical field that attracts particles in between the two leads used to create the potential. Assembly is achieved when dielectrophoretic forces exceed thermal and electrostatic forces; the use of anchoring molecules, present in the gap, improves the final assembly stability. We demonstrate with both experiment and theory that nanoparticle assembly inside the gap is controlled by the applied voltage and the gap size. Experimental evidence and modeling suggest that a gap-size-dependent threshold voltage must be overcome before particle assembly is realized. Assembly results as a function of frequency and time are also presented. Assembly of fewer than 10 isolated particles in a gap is demonstrated. Preliminary electrical characterization reveals that stable conductance of the assembled particles can be achieved.

Tuning caged calcium: photolabile analogues of EGTA with improved optical and chelation properties.

The physico-chemical properties of several Ca(2+)-selective, photolabile chelators are described. These molecules have been developed as part of an effort to produce a caged Ca(2+) that improved upon the Ca(2+) chelation properties and light absorption capability of nitrophenyl-EGTA (NP-EGTA). Four dimethoxy-ortho-nitrophenyl derivatives of EGTA (called DMNPE-1 through -4), and one analogue of EGTA (DMNPE-5) have been characterized, each of which is bisected upon irradiation. One of these cages has a higher affinity than NP-EGTA: DMNPE-4 has a K(d) for Ca(2+) of 48 nm at pH 7.2 (19 nM at pH 7.4). Furthermore, this cage has a large extinction coefficient of 5120 M(-1)cm(-1) at 350 nm (cf. 975 M(-1)cm(-1) for NP-EGTA). The other physico-chemical properties of DMNPE-4 are: quantum yield of photolysis of 0.09; bipasic Ca(2+) release kinetics (70% released with a rate of about 48,000 s(-1) and 30% at 1.5s(-1)) and photoproducts that bind Ca(2+) with very low affinity (K(d) in the range of 2mM, pH 7.2), hence most of the bound Ca(2+) is released rapidly and efficiently upon photolysis. Thus, DMNPE-4 has a unique combination of properties that make it an extremely effective Ca(2+) cage.

Kinetics of cardiac thin-filament activation probed by fluorescence polarization of rhodamine-labeled troponin C in skinned guinea pig trabeculae.

A genetically engineered cardiac TnC mutant labeled at Cys-84 with tetramethylrhodamine-5-iodoacetamide dihydroiodide was passively exchanged for the endogenous form in skinned guinea pig trabeculae. The extent of exchange averaged nearly 70%, quantified by protein microarray of individual trabeculae. The uniformity of its distribution was verified by confocal microscopy. Fluorescence polarization, giving probe angle and its dispersion relative to the fiber long axis, was monitored simultaneously with isometric tension. Probe angle reflects underlying cTnC orientation. In steady-state experiments, rigor cross-bridges and Ca2+ with vanadate to inhibit cross-bridge formation produce a similar change in probe orientation as that observed with cycling cross-bridges (no Vi). Changes in probe angle were found at [Ca2+] well below those required to generate tension. Cross-bridges increased the Ca2+ dependence of angle change (cooperativity). Strong cross-bridge formation enhanced Ca2+ sensitivity and was required for full change in probe position. At submaximal [Ca2+], the thin filament regulatory system may act in a coordinated fashion, with the probe orientation of Ca2+-bound cTnC significantly affected by Ca2+ binding at neighboring regulatory units. The time course of the probe angle change and tension after photolytic release [Ca2+] by laser photolysis of NP-EGTA was Ca2+ sensitive and biphasic: a rapid component approximately 10 times faster than that of tension and a slower rate similar to that of tension. The fast component likely represents steps closely associated with Ca2+ binding to site II of cTnC, whereas the slow component may arise from cross-bridge feedback. These results suggest that the thin filament activation rate does not limit the tension time course in cardiac muscle.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells.

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

Activation kinetics of skinned cardiac muscle by laser photolysis of nitrophenyl-EGTA.

The kinetics of Ca(2+)-induced contractions of chemically skinned guinea pig trabeculae was studied using laser photolysis of NP-EGTA. The amount of free Ca(2+) released was altered by varying the output from a frequency-doubled ruby laser focused on the trabeculae, while maintaining constant total [NP-EGTA] and [Ca(2+)]. The time courses of the rise in stiffness and tension were biexponential at 23 degrees C, pH 7.1, and 200 mM ionic strength. At full activation (pCa < 5.0), the rates of the rapid phase of the stiffness and tension rise were 56 +/- 7 s(-1) (n = 7) and 48 +/- 6 s(-1) (n = 11) while the amplitudes were 21 +/- 2 and 23 +/- 3%, respectively. These rates had similar dependencies on final [Ca(2+)] achieved by photolysis: 43 and 50 s(-1) per pCa unit, respectively, over a range of [Ca(2+)] producing from 15% to 90% of maximal isometric tension. At all [Ca(2+)], the rise in stiffness initially was faster than that of tension. The maximal rates for the slower components of the rise in stiffness and tension were 4.1 +/- 0.8 and 6.2 +/- 1.0 s(-1). The rate of this slower phase exhibited significantly less Ca(2+) sensitivity, 1 and 4 s(-1) per pCa unit for stiffness and tension, respectively. These data, along with previous studies indicating that the force-generating step in the cross-bridge cycle of cardiac muscle is marginally sensitive to [Ca(2+)], suggest a mechanism of regulation in which Ca(2+) controls the attachment step in the cross-bridge cycle via a rapid equilibrium with the thin filament activation state. Myosin kinetics sets the time course for the rise in stiffness and force generation with the biexponential nature of the mechanical responses to steps in [Ca(2+)] arising from a shift to slower cross-bridge kinetics as the number of strongly bound cross-bridges increases.

Morphology control in self-assembled monolayers written by dip pen nanolithography.

Here, we describe the effect of writing speed in dip pen nanolithography on the morphology (height and density) of self-assembled monolayers of alkanethiols on gold surfaces. The analysis of atomic force microscopy images of written monolayers shows that molecules assemble according to a nucleation and growth mechanism. Slow writing speeds lead to dense monolayers that can be used either to direct the self-assembly of metal nanoparticles or as masks for selective etching of conductive gold nanowires.

Stretch-induced calcium release in smooth muscle.

Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues.