Bioreactor development for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain: proof of concept.

Skeletal muscles overcome terrestrial, gravitational loading by producing tensile forces that produce movement through joint rotation. Conversely, the microgravity of spaceflight reduces tensile loads in working skeletal muscles, causing an adaptive muscle atrophy. Unfortunately, the design of stable, physiological bioreactors to model skeletal muscle tensile loading during spaceflight experiments remains challenging. Here, we tested a bioreactor that uses initiation and cessation of cyclic, tensile strain to induce hypertrophy and atrophy, respectively, in murine lineage (C2C12) skeletal muscle myotubes. Uniaxial cyclic stretch of myotubes was conducted using a StrexCell® (STB-1400) stepper motor system (0.75 Hz, 12% strain, 60 min day^-1). Myotube groups were assigned as follows: (a) quiescent over 2- or (b) 5-day (no stretch), (c) experienced 2-days (2dHY) or (d) 5-days (5dHY) of cyclic stretch, or (e) 2-days of cyclic stretch followed by a 3-day cessation of stretch (3dAT). Using ß-sarcoglycan as a sarcolemmal marker, mean myotube diameter increased significantly following 2dAT (51%) and 5dAT (94%) vs. matched controls. The hypertrophic, anabolic markers talin and Akt phosphorylation (Thr308) were elevated with 2dHY but not in 3dAT myotubes. Inflammatory, catabolic markers IL-1ß, IL6, and NF-kappaB p65 subunit were significantly higher in the 3dAT group vs. all other groups. The ratio of phosphorylated FoxO3a/total FoxO3a was significantly lower in 3dAT than in the 2dHY group, consistent with elevated catabolic signaling during unloading. In summary, we demonstrated proof-of-concept for a spaceflight research bioreactor, using uniaxial cyclic stretch to produce myotube hypertrophy with increased tensile loading, and myotube atrophy with subsequent cessation of stretch.

A Second Space Age Spanning Omics, Platforms, and Medicine Across Orbits.

The recent acceleration of commercial, private, and multi-national spaceflight has created an unprecedented level of activity in low Earth orbit (LEO), concomitant with the highest-ever number of crewed missions entering space and preparations for exploration-class (>1 year) missions. Such rapid advancement into space from many new companies, countries, and space-related entities has enabled a"Second Space Age." This new era is also poised to leverage, for the first time, modern tools and methods of molecular biology and precision medicine, thus enabling precision aerospace medicine for the crews. The applications of these biomedical technologies and algorithms are diverse, encompassing multi-omic, single-cell, and spatial biology tools to investigate human and microbial responses to spaceflight. Additionally, they extend to the development of new imaging techniques, real-time cognitive assessments, physiological monitoring, and personalized risk profiles tailored for astronauts. Furthermore, these technologies enable advancements in pharmacogenomics (PGx), as well as the identification of novel spaceflight biomarkers and the development of corresponding countermeasures. In this review, we highlight some of the recent biomedical research from the National Aeronautics and Space Administration (NASA), Japan Aerospace Exploration Agency (JAXA), European Space Agency (ESA), and other space agencies, and also detail the commercial spaceflight sector's (e.g. SpaceX, Blue Origin, Axiom, Sierra Space) entrance into aerospace medicine and space biology, the first aerospace medicine biobank, and the myriad upcoming missions that will utilize these tools to ensure a permanent human presence beyond LEO, venturing out to other planets and moons.

Machine Learning Identifies Key Proteins in Primary Sclerosing Cholangitis Progression and Links High CCL24 to Cirrhosis.

Primary sclerosing cholangitis (PSC) is a rare, progressive disease, characterized by inflammation and fibrosis of the bile ducts, lacking reliable prognostic biomarkers for disease activity. Machine learning applied to broad proteomic profiling of sera allowed for the discovery of markers of disease presence, severity, and cirrhosis and the exploration of the involvement of CCL24, a chemokine with fibro-inflammatory activity. Sera from 30 healthy controls and 45 PSC patients were profiled with proximity extension assay, quantifying the expression of 2870 proteins, and used to train an elastic net model. Proteins that contributed most to the model were tested for correlation to enhanced liver fibrosis (ELF) score and used to perform pathway analysis. Statistical modeling for the presence of cirrhosis was performed with principal component analysis (PCA), and receiver operating characteristics (ROC) curves were used to assess the useability of potential biomarkers. The model successfully predicted the presence of PSC, where the top-ranked proteins were associated with cell adhesion, immune response, and inflammation, and each had an area under receiver operator characteristic (AUROC) curve greater than 0.9 for disease presence and greater than 0.8 for ELF score. Pathway analysis showed enrichment for functions associated with PSC, overlapping with pathways enriched in patients with high levels of CCL24. Patients with cirrhosis showed higher levels of CCL24. This data-driven approach to characterize PSC and its severity highlights potential serum protein biomarkers and the importance of CCL24 in the disease, implying its therapeutic potential in PSC.

The Role of CCL24 in Primary Sclerosing Cholangitis: Bridging Patient Serum Proteomics to Preclinical Data.

Primary sclerosing cholangitis (PSC) is an inflammatory and fibrotic biliary disease lacking approved treatment. We studied CCL24, a chemokine shown to be overexpressed in damaged bile ducts, and its involvement in key disease-related mechanisms. Serum proteomics of PSC patients and healthy controls (HC) were analyzed using the Olink® proximity extension assay and compared based on disease presence, fibrosis severity, and CCL24 levels. Disease-related canonical pathways, upstream regulators, and toxicity functions were elevated in PSC patients compared to HC and further elevated in patients with high CCL24 levels. In vitro, a protein signature in CCL24-treated hepatic stellate cells (HSCs) differentiated patients by disease severity. In mice, CCL24 intraperitoneal injection selectively recruited neutrophils and monocytes. Treatment with CM-101, a CCL24-neutralizing antibody, in an α-naphthylisothiocyanate (ANIT)-induced cholestasis mouse model effectively inhibited accumulation of peribiliary neutrophils and macrophages while reducing biliary hyperplasia and fibrosis. Furthermore, in PSC patients, CCL24 levels were correlated with upregulation of monocyte and neutrophil chemotaxis pathways. Collectively, these findings highlight the distinct role of CCL24 in PSC, influencing disease-related mechanisms, affecting immune cells trafficking and HSC activation. Its blockade with CM-101 reduces inflammation and fibrosis and positions CCL24 as a promising therapeutic target in PSC.

The Impact of SRT2104 on Skeletal Muscle Mitochondrial Function, Redox Biology, and Loss of Muscle Mass in Hindlimb Unloaded Rats.

Mechanical unloading during microgravity causes skeletal muscle atrophy and impairs mitochondrial energetics. The elevated production of reactive oxygen species (ROS) by mitochondria and Nox2, coupled with impairment of stress protection (e.g., SIRT1, antioxidant enzymes), contribute to atrophy. We tested the hypothesis that the SIRT1 activator, SRT2104 would rescue unloading-induced mitochondrial dysfunction. Mitochondrial function in rat gastrocnemius and soleus muscles were evaluated under three conditions (10 days): ambulatory control (CON), hindlimb unloaded (HU), and hindlimb-unloaded-treated with SRT2104 (SIRT). Oxidative phosphorylation, electron transfer capacities, H2O2 production, and oxidative and antioxidant enzymes were quantified using high-resolution respirometry and colorimetry. In the gastrocnemius, (1) integrative (per mg tissue) proton LEAK was lesser in SIRT than in HU or CON; (2) intrinsic (relative to citrate synthase) maximal noncoupled electron transfer capacity (ECI+II) was lesser, while complex I-supported oxidative phosphorylation to ECI+II was greater in HU than CON; (3) the contribution of LEAK to ECI+II was greatest, but cytochrome c oxidase activity was lowest in HU. In both muscles, H2O2 production and concentration was greatest in SIRT, as was gastrocnemius superoxide dismutase activity. In the soleus, H2O2 concentration was greater in HU compared to CON. These results indicate that SRT2104 preserves mitochondrial function in unloaded skeletal muscle, suggesting its potential to support healthy muscle cells in microgravity by promoting necessary energy production in mitochondria.

Combined effects of heavy ion exposure and simulated Lunar gravity on skeletal muscle.

BACKGROUND: The limitations to prolonged spaceflight include unloading-induced atrophy of the musculoskeletal system which may be enhanced by exposure to the space radiation environment. Previous results have concluded that partial gravity, comparable to the Lunar surface, may have detrimental effects on skeletal muscle. However, little is known if these outcomes are exacerbated by exposure to low-dose rate, high-energy radiation common to the space environment. Therefore, the present study sought to determine the impact of highly charge, high-energy (HZE) radiation on skeletal muscle when combined with partial weightbearing to simulate Lunar gravity. We hypothesized that partial unloading would compromise skeletal muscle and these effects would be exacerbated by radiation exposure. METHODS: For month old female BALB/cByJ mice were -assigned to one of 2 groups; either full weight bearing (Cage Controls, CC) or partial weight bearing equal to 1/6th bodyweight (G/6). Both groups were then divided to receive either a single whole body absorbed dose of 0.5 Gy of 300 MeV 28Si ions (RAD) or a sham treatment (SHAM). Radiation exposure experiments were performed at the NASA Space Radiation Laboratory (NSRL) located at Brookhaven National Laboratory on Day 0, followed by 21 d of CC or G/6 loading. Muscles of the hind limb were used to measure protein synthesis and other histological measures. RESULTS: Twenty-one days of Lunar gravity (G/6) resulted in lower soleus, plantaris, and gastrocnemius muscle mass. Radiation exposure did not further impact muscle mass. 28Si exposure in normal ambulatory animals (RAD+CC) did not impact gastrocnemius muscle mass when compared to SHAM+CC (p>0.05), but did affect the soleus, where mass was higher following radiation compared to SHAM (p<0.05). Mixed gastrocnemius muscle protein synthesis was lower in both unloading groups. Fiber type composition transitioned towards a faster isoform with partial unloading and was not further impacted by radiation. The combined effects of partial loading and radiation partially mitigated fiber cross-sectional area when compared to partial loading alone. Radiation and G/6 reduced the total number of myonuclei per fiber while leading to elevated BrdU content of skeletal muscle. Similarly, unloading and radiation resulted in higher collagen content of muscle when compared to controls, but the effects of combined exposure were not additive. CONCLUSIONS: The results of this study confirm that partial weightbearing causes muscle atrophy, in part due to reductions of muscle protein synthesis in the soleus and gastrocnemius as well as reduced peripheral nuclei per fiber. Additionally, we present novel data illustrating 28Si exposure reduced nuclei in muscle fibers despite higher satellite cell fusion, but did not exacerbate muscle atrophy, CSA changes, or collagen content. In conclusion, both partial loading and HZE radiation can negatively impact muscle morphology.

Cellular and Molecular Signaling Meet the Space Environment.

During space missions that travel beyond the cocoon of the Earth's magnetosphere, astronauts are subjected to the microgravity and radiation stressors of outer space [...].

Correction: Single-conformation spectroscopy of cold, protonated DPG-containing peptides: switching ß-turn types and formation of a sequential type II/II' double ß-turn.

Correction for 'Single-conformation spectroscopy of cold, protonated DPG-containing peptides: switching ß-turn types and formation of a sequential type II/II' double ß-turn' by John T. Lawler et al., Phys. Chem. Chem. Phys., 2022, 24, 2095-2109, https://doi.org/10.1039/D1CP04852J.

Single-conformation spectroscopy of cold, protonated DPG-containing peptides: switching ß-turn types and formation of a sequential type II/II' double ß-turn.

D-Proline (DPro, DP) is widely utilized to form ß-hairpin loops in engineered peptides that would otherwise be unstructured, most often as part of a DPG sub-unit that forms a ß-turn. To observe whether DPG facilitated this effect in short protonated peptides, conformation specific IR-UV double resonance photofragment spectra of the cold (∼10 K) protonated DP and LP diastereomers of the pentapeptide YAPGA was carried out in the hydride stretch (2800-3700 cm-1) and amide I/II (1400-1800 cm-1) regions. A model localized Hamiltonian was developed to better describe the 1600-1800 cm-1 region commonly associated with the amide I vibrations. The CO stretch fundamentals experience extensive mixing with the N-H bending fundamentals of the NH3+ group in these protonated peptides. The model Hamiltonian accounts for experiment in quantitative detail. In the DP diastereomer, all the population is funneled into a single conformer which presented as a type II ß-turn with A and DP in the i + 1 and i + 2 positions, respectively. This structure was not the anticipated type II' ß-turn across DPG that we had hypothesized based on solution-phase propensities. Analysis of the conformational energy landscape shows that both steric and charge-induced effects play a role in the preferred formation of the type II ß-turn. In contrast, the LP isomer forms three conformations with very different structures, none of which were type II/II' ß-turns, confirming that LPG is not a ß-turn former. Finally, single-conformation spectroscopy was also carried out on the extended peptide [YAADPGAAA + H]+ to determine whether moving the protonated N-terminus further from DPG would lead to ß-hairpin formation. Despite funneling its entire population into a single peptide backbone structure, the assigned structure is not a ß-hairpin, but a concatenated type II/type II' double ß-turn that displaces the peptide backbone laterally by about 7.5 Å, but leaves the backbone oriented in its original direction.

Nox2 Inhibition Regulates Stress Response and Mitigates Skeletal Muscle Fiber Atrophy during Simulated Microgravity.

Insufficient stress response and elevated oxidative stress can contribute to skeletal muscle atrophy during mechanical unloading (e.g., spaceflight and bedrest). Perturbations in heat shock proteins (e.g., HSP70), antioxidant enzymes, and sarcolemmal neuronal nitric oxidase synthase (nNOS) have been linked to unloading-induced atrophy. We recently discovered that the sarcolemmal NADPH oxidase-2 complex (Nox2) is elevated during unloading, downstream of angiotensin II receptor 1, and concomitant with atrophy. Here, we hypothesized that peptidyl inhibition of Nox2 would attenuate disruption of HSP70, MnSOD, and sarcolemmal nNOS during unloading, and thus muscle fiber atrophy. F344 rats were divided into control (CON), hindlimb unloaded (HU), and hindlimb unloaded +7.5 mg/kg/day gp91ds-tat (HUG) groups. Unloading-induced elevation of the Nox2 subunit p67phox-positive staining was mitigated by gp91ds-tat. HSP70 protein abundance was significantly lower in HU muscles, but not HUG. MnSOD decreased with unloading; however, MnSOD was not rescued by gp91ds-tat. In contrast, Nox2 inhibition protected against unloading suppression of the antioxidant transcription factor Nrf2. nNOS bioactivity was reduced by HU, an effect abrogated by Nox2 inhibition. Unloading-induced soleus fiber atrophy was significantly attenuated by gp91ds-tat. These data establish a causal role for Nox2 in unloading-induced muscle atrophy, linked to preservation of HSP70, Nrf2, and sarcolemmal nNOS.

Nox2 signaling and muscle fiber remodeling are attenuated by losartan administration during skeletal muscle unloading.

Reduced mechanical loading results in atrophy of skeletal muscle fibers. Increased reactive oxygen species (ROS) are causal in sarcolemmal dislocation of nNOS and FoxO3a activation. The Nox2 isoform of NADPH oxidase and mitochondria release ROS during disuse in skeletal muscle. Activation of the angiotensin II type 1 receptor (AT1R) can elicit Nox2 complex formation. The AT1R blocker losartan was used to test the hypothesis that AT1R activation drives Nox2 assembly, nNOS dislocation, FoxO3a activation, and thus alterations in morphology in the unloaded rat soleus. Male Fischer 344 rats were divided into four groups: ambulatory control (CON), ambulatory + losartan (40 mg kg-1  day-1 ) (CONL), 7 days of tail-traction hindlimb unloading (HU), and HU + losartan (HUL). Losartan attenuated unloading-induced loss of muscle fiber cross-sectional area (CSA) and fiber-type shift. Losartan mitigated unloading-induced elevation of ROS levels and upregulation of Nox2. Furthermore, AT1R blockade abrogated nNOS dislocation away from the sarcolemma and elevation of nuclear FoxO3a. We conclude that AT1R blockade attenuates disuse remodeling by inhibiting Nox2, thereby lessening nNOS dislocation and activation of FoxO3a.

Protective Effects of Ghrelin on Fasting-Induced Muscle Atrophy in Aging Mice.

Sarcopenia is the aging-associated progressive loss of skeletal muscle; however, the pathogenic mechanism of sarcopenia is not clear. The orexigenic hormone ghrelin stimulates growth hormone secretion, increases food intake, and promotes adiposity. Here we showed that fasting-induced muscle loss was exacerbated in old ghrelin-null (Ghrl-/-) mice, exhibiting decreased expression of myogenic regulator MyoD and increased expression of protein degradation marker MuRF1, as well as altered mitochondrial function. Moreover, acylated ghrelin and unacylated ghrelin treatments significantly increased mitochondrial respiration capacity in muscle C2C12 cells. Consistently, acylated ghrelin and unacylated ghrelin treatments effectively increased myogenic genes and decreased degradation genes in the muscle in fasted old Ghrl-/- mice, possibly by stimulating insulin and adenosine monophosphate-activated protein kinase pathways. Furthermore, Ghrl-/- mice showed a profile of pro-inflammatory gut microbiota, exhibiting reduced butyrate-producing bacteria Roseburia and ClostridiumXIVb. Collectively, our results showed that ghrelin has a major role in the maintenance of aging muscle via both muscle-intrinsic and -extrinsic mechanisms. Acylated ghrelin and unacylated ghrelin enhanced muscle anabolism and exerted protective effects for muscle atrophy. Because unacylated ghrelin is devoid of the obesogenic side effect seen with acylated ghrelin, it represents an attractive therapeutic option for sarcopenia.

Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction.

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH2CONH2. The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua, where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.

Muscular apoptosis but not oxidative stress increases with old age in a long-lived diver, the Weddell seal.

Seals experience repeated bouts of ischemia-reperfusion while diving, potentially exposing their tissues to increased oxidant generation and thus oxidative damage and accelerated aging. We contrasted markers of oxidative damage with antioxidant profiles across age and sex for propulsive (longissismus dorsi) and maneuvering (pectoralis) muscles of Weddell seals to determine whether previously observed morphological senescence is associated with oxidative stress. In longissismus dorsi, old (age 17-26 years) seals exhibited a nearly 2-fold increase in apoptosis over young (age 9-16 years) seals. There was no evidence of age-associated changes in lipid peroxidation or enzymatic antioxidant profiles. In pectoralis, 4-hydroxynonenal-Lys (4-HNE-Lys) levels increased 1.5-fold in old versus young seals, but lipid hydroperoxide levels and apoptotic index did not vary with age. Glutathione peroxidase activity was 1.5-fold higher in pectoralis of old versus young animals, but no other antioxidants changed with age in this muscle. With respect to sex, no differences in lipid hydroperoxides or apoptosis were observed in either muscle. Males had higher HSP70 expression (1.4-fold) and glutathione peroxidase activity (1.3-fold) than females in longissismus dorsi, although glutathione reductase activity was 1.4-fold higher in females. No antioxidants varied with sex in pectoralis. These results show that apoptosis is not associated with oxidative stress in aged Weddell seal muscles. Additionally, the data suggest that adult seals utilize sex-specific antioxidant strategies in longissismus dorsi but not pectoralis to protect skeletal muscles from oxidative damage.

Gold(I) Cationization Promotes Ring Opening in Lysine-Containing Cyclic Peptides.

A strategy to sequence lysine-containing cyclic peptides by MSn is presented. Doubly protonated cyclic peptides ions are transformed into gold (I) cationized peptide ions via cation switching ion/ion reaction. Gold(I) cationization facilitates the oxidation of neutral lysine residues in the gas phase, weakening the adjacent amide bond. Upon activation, facile cleavage N-terminal to the oxidized lysine residue provides a site-specific ring opening pathway that converts cyclic peptides into acyclic analogs. The ensuing ion contains a cyclic imine as the new N-terminus and an oxazolone, or structural equivalent, as the new C-terminus. Product ions are formed from subsequent fragmentation events of the linearized peptide ion. Such an approach simplifies MS/MS data interpretation as a series of fragment ions with common N- and C-termini are generated. Results are presented for two cyclic peptides, sunflower trypsin inhibitor and the model cyclic peptide, ß-Loop. The power of this strategy lies in the ability to generate the oxidized peptide, which is easily identified via the loss of HAuNH3 from [M + Au]+. While some competitive processes are observed, the site of ring opening can be pinpointed to the lysine residue upon MS4 enabling the unambiguous sequencing of cyclic peptides.

Effect of combined fish oil & Curcumin on murine skeletal muscle morphology and stress response proteins during mechanical unloading.

Skeletal muscle is a highly adaptable tissue capable of remodeling when dynamic stress is altered, including changes in mechanical loading and stretch. When muscle is subjected to an unloaded state (e.g., bedrest, immobilization, spaceflight) the resulting loss of muscle cross sectional area (CSA) impairs force production. In addition, muscle fiber-type shifts from slow to fast-twitch fibers. Unloading also results in a downregulation of heat shock proteins (e.g., HSP70) and anabolic signaling, which further exacerbate these morphological changes. Our lab recently showed reactive oxygen species (ROS) are causal in unloading-induced alterations in Akt and FoxO3a phosphorylation, muscle fiber atrophy, and fiber-type shift. Nutritional supplements such as fish oil and curcumin enhance anabolic signaling, glutathione levels, and heat shock proteins. We hypothesized that fish oil, rich in omega-3-fatty acids, combined with the polyphenol curcumin would enhance stress protective proteins and anabolic signaling in the rat soleus muscle, concomitant with synergistic protection of morphology. C57BL/6 mice were assigned to 3 groups (n = 6/group): ambulatory controls (CON), hindlimb unloading (HU), and hindlimb unloading with 5% fish oil, 1% curcumin in diet (FOC). FOC treatments began 10 days prior to HU and tissues were harvested following 7 days of HU. FOC mitigated the unloading induced decrease in CSA. FOC also enhanced abundance of HSP70 and anabolic signaling (Akt phosphorylation, p70S6K phosphorylation), while reducing Nox2, a source of oxidative stress. Therefore, we concluded that the combination of fish oil and curcumin prevents skeletal muscle atrophy due to a boost of heat shock proteins and anabolic signaling in an unloaded state.

Infrared Population Transfer Spectroscopy of Cryo-Cooled Ions: Quantitative Tests of the Effects of Collisional Cooling on the Room Temperature Conformer Populations.

The single-conformation spectroscopy and infrared-induced conformational isomerization of a model protonated pentapeptide [YGPAA + H]+ is studied under cryo-cooled conditions in the gas phase. Building on recent results ( DeBlase , A. F. ; J. Am. Chem. Soc. 2017 , 139 , 5481 - 5493 ), firm assignments are established for the presence of two conformer families with distinct infrared and ultraviolet spectra, using IR-UV depletion spectroscopy. Families (A and B) share a similar structure near the N-terminus but differ in the way that the C-terminal COOH group configures itself (cis versus trans) in forming H-bonds with the peptide backbone. Infrared population transfer (IR-PT) spectroscopy is used to study the IR-induced conformational isomerization following single-conformer infrared excitation. IR-induced isomerization is accomplished in both directions (A → B and B → A) in the hydride stretch region and is used to determine fractional abundances for the two conformer families (FA = 0.65 ± 0.04, FB = 0.35 ± 0.04, 2σ error bars). The time scale for collisional cooling of the room-temperature ions to Tvib = 10 K by cold helium in the octupole trap is established as 1.0 ms. Key stationary points on the isomerization potential energy surface are calculated at the DFT B3LYP/6-31+G(d) G3DBJ level of theory. Using RRKM theory, the energy-dependent isomerization rates and populations are calculated as a function of energy. According to the model, the observed population distribution after collisional cooling is close to that of the 298 K Boltzmann distribution and is in near-quantitative agreement with experiment. On the basis of this success, inferences are drawn for the circumstances that govern the population distribution in the trap, concluding that, in ions the size of [YGPAA + H]+ and larger, the observed distributions will be near those at 298 K.

Near-UV Water Splitting by Cu, Ni, and Co Complexes in the Gas Phase.

(2,2'-Bipyridine)M═O+ ions (M = Cu, Ni, Co) were generated by collision-induced dissociation and near-UV photodissociation of readily available [(2,2'-bipyridine)MII(NO3)]+ ions in the gas phase, and their structure was confirmed by ion-molecule reactions combined with isotope labeling. Upon storage in a quadrupole ion trap, the (2,2'-bipyridine)M═O+ ions spontaneously added water, and the formed [(2,2'-bipyridine)M═O + H2O]+ complexes eliminated OH upon further near-UV photodissociation. This reaction sequence can be accomplished at a single laser wavelength in the range of 260-340 nm to achieve stoichiometric homolytic cleavage of gaseous water. Structures, spin states, and electronic excitations of the metal complexes were characterized by ion-molecule reactions using 2H and 18O labeling, photodissociation action spectroscopy, and density functional theory calculations.

Effect of Eukarion-134 on Akt-mTOR signalling in the rat soleus during 7 days of mechanical unloading.

NEW FINDINGS: What is the central question of this study? Translocation of nNOSµ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading-induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion-134) also mitigate suppression of the Akt-mTOR pathway? What is the main finding and its importance? Eukarion-134 rescued Akt-mTOR signalling and sarcolemmal nNOSµ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre-type shift from slow to fast twitch. The loss of nNOSµ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved. ABSTRACT: Mechanical unloading stimulates rapid changes in skeletal muscle morphology, characterized by atrophy of muscle fibre cross-sectional area and a partial fibre-type shift from slow to fast twitch. Recent studies revealed that oxidative stress contributes to activation of forkhead box O3a (FoxO3a), proteolytic signalling and unloading-induced muscle atrophy via translocation of the µ-splice variant of neuronal nitric oxide synthase (nNOSµ) and activation of FoxO3a. There is limited understanding of the role of reactive oxygen species in the Akt-mammalian target of rapamycin (mTOR) pathway signalling during unloading. We hypothesized that Eukarion-134 (EUK-134), a mimetic of the antioxidant enzymes superoxide dismutase and catalase, would protect Akt-mTOR signalling in the unloaded rat soleus. Male Fischer 344 rats were separated into the following three study groups: ambulatory control (n = 11); 7 days of hindlimb unloading + saline injections (HU, n = 11); or 7 days of HU + EUK-134; (HU + EUK-134, n = 9). EUK-134 mitigated unloading-induced dephosphorylation of Akt, as well as FoxO3a, in the soleus. Phosphorylation of mTOR in the EUK-treated HU rats was not different from that in control animals. However, EUK-134 did not significantly rescue p70S6K phosphorylation. EUK-134 attenuated translocation of nNOSµ from the membrane to the cytosol, reduced nitration of tyrosine residues and suppressed upregulation of caveolin-3 and dysferlin. EUK-134 ameliorated HU-induced remodelling, atrophy of muscle fibres and the 12% increase in type II myosin heavy chain-positive fibres. Attenuation of the unloaded muscle phenotype was associated with decreased reactive oxygen species, as assessed by ethidium-positive nuclei. We conclude that oxidative stress affects Akt-mTOR signalling in unloaded skeletal muscle. Direct linkage of abrogation of nNOSµ translocation with Akt-mTOR signalling during unloading is the subject of future investigation.

Clinical relevance of the pharmacokinetic characteristics of an abuse-deterrent, extended-release, injection-molded morphine tablet.

OBJECTIVE: To characterize the pharmacokinetics (PK) and in vitro alcohol dissolution characteristics of extended-release (ER), injection-molded (IM) morphine tablets with abuse-deterrent (AD) features (morphine-ADER-IMT). DESIGN: In vivo, in vitro, and in silico studies were conducted. A randomized, two-cohort study evaluated the bioequivalence of morphine-ADER-IMT (60 mg) to morphine ER (60 mg; MS Contin®; Purdue Pharma LP, Stamford, CT) and characterized the effect of food on the PK profile of morphine-ADER-IMT. A three-treatment, three-period crossover study assessed the bioequivalence of morphine-ADER-IMT (30 and 2 ȕ 15 mg) to morphine ER (30 mg). Bioequivalence studies were performed in healthy male and female subjects (18-55 y of age) in the presence of naltrexone blockade. PK modeling was performed to assess steady-state bioequivalence for morphine-ADER-IMT 60 mg compared with morphine ER 60 mg. In vitro alcohol dissolution studies were performed with morphine-ADER-IMT (15 and 60 mg). RESULTS: Fifty-nine and 56 subjects completed the 60-mg bioequivalence/food effect study and 30-mg bioequivalence study, respectively. Bioequivalence of morphine-ADER-IMT 60, 30, and 2 ȕ 15 mg and morphine ER was demonstrated to comparable doses of morphine ER. No clinically significant food effect was observed with morphine-ADER-IMT. Treatment-emergent adverse events were similar among all treatment groups. Steady-state modeling indicated bioequivalence between morphine-ADER-IMT 60 mg and morphine ER 60 mg when administered every 8 or 12 hours. No evidence of alcohol dose-dumping was observed with morphine-ADER-IMT. CONCLUSIONS: Morphine-ADER-IMT, an ER morphine formulation with robust AD features, has a clinical PK profile that is well suited for patients with chronic pain.