Tetrakis(N-heterocyclic Carbene)-Diboron(0): Double Single-Electron-Transfer Reactivity.

The use of 1,3,4,5-tetramethylimidazol-2-ylidene (IMe) to coordinate with diatomic B2 species afforded a tetrakis(N-heterocyclic carbene)-diboron(0) [(IMe)2B-B(IMe)2] (2). The singly bonded B2 moiety therein possesses a valence electronic configuration 1σg21πu21πg*2 with four vacant molecular orbitals (1σu*, 2σg, 1πu', 1πg'*) coordinated with IMe. Its unprecedented electronic structure is analogous to the energetically unfavorable planar hydrazine with a D2h symmetry. The two highly reactive πg* antibonding electrons enable double single-electron-transfer (SET) reactivity in small-molecule activation. Compound 2 underwent a double SET reduction with CO2 to form two carbon dioxide radical anions CO2•-, which then reduced pyridine to yield a carboxylated pyridine reductive coupling dianion [O2CNC5(H)5-C5(H)5NCO2]2- and converted compound 2 to the tetrakis(N-heterocyclic carbene)-diborene dication [(IMe)2B═B(IMe)2]2+ (32+). This is a remarkable transition-metal-free SET reduction of CO2 without ultraviolet/visible (UV/vis) light conditions.

Non-Fullerene Electron Acceptors Based on Hybridisation of Corannulene and Thiophene-S,S-Dioxide Motifs.

Herein we show that hybridisation of buckybowl corannulene and thiophene-S,S-dioxide motifs is a general approach for the preparation of high electron affinity molecular materials. The devised synthesis is modular and relies on thienannulation of corannnulene-based phenylacetylene scaffolds. The final compounds are highly soluble in common organic solvents. These compounds also exhibit interesting optical properties such as absorption and emission in the blue/green regions of the electromagnetic spectrum. Importantly, a bis-S,S-dioxide derivative exhibits three reversible reductions similar in their strength to the prevalent fullerene-based electron acceptor phenyl-C61 -butyric acid methyl ester (PC61 BM).

Evolution of the Electronic Structure of Ultrathin MnBi2Te4 Films.

Ultrathin films of intrinsic magnetic topological insulator MnBi2Te4 exhibit fascinating quantum properties such as the quantum anomalous Hall effect and the axion insulator state. In this work, we systematically investigate the evolution of the electronic structure of MnBi2Te4 thin films. With increasing film thickness, the electronic structure changes from an insulator type with a large energy gap to one with in-gap topological surface states, which is, however, still in drastic contrast to the bulk material. By surface doping of alkali-metal atoms, a Rashba split band gradually emerges and hybridizes with topological surface states, which not only reconciles the puzzling difference between the electronic structures of the bulk and thin-film MnBi2Te4 but also provides an interesting platform to establish Rashba ferromagnet that is attractive for (quantum) anomalous Hall effect. Our results provide important insights into the understanding and engineering of the intriguing quantum properties of MnBi2Te4 thin films.

Nickel-Catalyzed Enantioselective Reductive Conjugate Arylation and Heteroarylation via an Elementary Mechanism of 1,4-Addition.

A nickel complex of isoquinox promoted enantioselective conjugate arylation and heteroarylation of enones using aryl and heteroaryl halides directly. The reaction was successfully applied to stereoselective syntheses of ar-turmerone, chiral fragments of (+)-tolterodine and AZD5672. Mechanistically, experiments and calculations supported that an arylnickel(I) complex inserted to enones via an elementary 1,4-addition.

TRIC-A Channel Maintains Store Calcium Handling by Interacting With Type 2 Ryanodine Receptor in Cardiac Muscle.

RATIONALE: Trimeric intracellular cation (TRIC)-A and B are distributed to endoplasmic reticulum/sarcoplasmic reticulum intracellular Ca2+ stores. The crystal structure of TRIC has been determined, confirming the homotrimeric structure of a potassium channel. While the pore architectures of TRIC-A and TRIC-B are conserved, the carboxyl-terminal tail (CTT) domains of TRIC-A and TRIC-B are different from each other. Aside from its recognized role as a counterion channel that participates in excitation-contraction coupling of striated muscles, the physiological function of TRIC-A in heart physiology and disease has remained largely unexplored. OBJECTIVE: In cardiomyocytes, spontaneous Ca2+ waves, triggered by store overload-induced Ca2+ release mediated by the RyR2 (type 2 ryanodine receptor), develop extrasystolic contractions often associated with arrhythmic events. Here, we test the hypothesis that TRIC-A is a physiological component of RyR2-mediated Ca2+ release machinery that directly modulates store overload-induced Ca2+ release activity via CTT. METHODS AND RESULTS: We show that cardiomyocytes derived from the TRIC-A-/- (TRIC-A knockout) mice display dysregulated Ca2+ movement across sarcoplasmic reticulum. Biochemical studies demonstrate a direct interaction between CTT-A and RyR2. Modeling and docking studies reveal potential sites on RyR2 that show differential interactions with CTT-A and CTT-B. In HEK293 (human embryonic kidney) cells with stable expression of RyR2, transient expression of TRIC-A, but not TRIC-B, leads to apparent suppression of spontaneous Ca2+ oscillations. Ca2+ measurements using the cytosolic indicator Fura-2 and the endoplasmic reticulum luminal store indicator D1ER suggest that TRIC-A enhances Ca2+ leak across the endoplasmic reticulum by directly targeting RyR2 to modulate store overload-induced Ca2+ release. Moreover, synthetic CTT-A peptide facilitates RyR2 activity in lipid bilayer reconstitution system, enhances Ca2+ sparks in permeabilized TRIC-A-/- cardiomyocytes, and induces intracellular Ca2+ release after microinjection into isolated cardiomyocytes, whereas such effects were not observed with the CTT-B peptide. In response to isoproterenol stimulation, the TRIC-A-/- mice display irregular ECG and develop more fibrosis than the WT (wild type) littermates. CONCLUSIONS: In addition to the ion-conducting function, TRIC-A functions as an accessory protein of RyR2 to modulate sarcoplasmic reticulum Ca2+ handling in cardiac muscle.

TRIC-A regulates intracellular Ca2+ homeostasis in cardiomyocytes.

Trimeric intracellular cation (TRIC) channels have been identified as monovalent cation channels that are located in the ER/SR membrane. Two isoforms discovered in mammals are TRIC-A (TMEM38a) and TRIC-B (TMEM38b). TRIC-B ubiquitously expresses in all tissues, and TRIC-B-/- mice is lethal at the neonatal stage. TRIC-A mainly expresses in excitable cells. TRIC-A-/- mice survive normally but show abnormal SR Ca2+ handling in both skeletal and cardiac muscle cells. Importantly, TRIC-A mutations have been identified in human patients with stress-induced arrhythmia. In the past decade, important discoveries have been made to understand the structure and function of TRIC channels, especially its role in regulating intracellular Ca2+ homeostasis. In this review article, we focus on the potential roles of TRIC-A in regulating cardiac function, particularly its effects on intracellular Ca2+ signaling of cardiomyocytes and discuss the current knowledge gaps.

Butyrate Feeding Reverses CypD-Related Mitoflash Phenotypes in Mouse Myofibers.

Mitoflashes are spontaneous transients of the biosensor mt-cpYFP. In cardiomyocytes, mitoflashes are associated with the cyclophilin D (CypD) mediated opening of mitochondrial permeability transition pore (mPTP), while in skeletal muscle they are considered hallmarks of mitochondrial respiration burst under physiological conditions. Here, we evaluated the potential association between mitoflashes and the mPTP opening at different CypD levels and phosphorylation status by generating three CypD derived fusion constructs with a red shifted, pH stable Ca2+ sensor jRCaMP1b. We observed perinuclear mitochondrial Ca2+ efflux accompanying mitoflashes in CypD and CypDS42A (a phosphor-resistant mutation at Serine 42) overexpressed myofibers but not the control myofibers expressing the mitochondria-targeting sequence of CypD (CypDN30). Assisted by a newly developed analysis program, we identified shorter, more frequent mitoflash activities occurring over larger areas in CypD and CypDS42A overexpressed myofibers than the control CypDN30 myofibers. These observations provide an association between the elevated CypD expression and increased mitoflash activities in hindlimb muscles in an amyotrophic lateral sclerosis (ALS) mouse model previously observed. More importantly, feeding the mice with sodium butyrate reversed the CypD-associated mitoflash phenotypes and protected against ectopic upregulation of CypD, unveiling a novel molecular mechanism underlying butyrate mediated alleviation of ALS progression in the mouse model.

Lineage-associated underrepresented permutations (LAUPs) of mammalian genomic sequences based on a Jellyfish-based LAUPs analysis application (JBLA).

Motivation: This study addresses several important questions related to naturally underrepresented sequences: (i) are there permutations of real genomic DNA sequences in a defined length (k-mer) and a given lineage that do not actually exist or underrepresented? (ii) If there are such sequences, what are their characteristics in terms of k-mer length and base composition? (iii) Are they related to CpG or TpA underrepresentation known for human sequences? We propose that the answers to these questions are of great significance for the study of sequence-associated regulatory mechanisms, such cytosine methylation and chromosomal structures in physiological or pathological conditions such as cancer. Results: We empirically defined sequences that were not included in any well-known public databases as lineage-associated underrepresented permutations (LAUPs). Then, we developed a Jellyfish-based LAUPs analysis application (JBLA) to investigate LAUPs for 24 representative species. The present discoveries include: (i) lengths for the shortest LAUPs, ranging from 10 to 14, which collectively constitute a low proportion of the genome. (ii) Common LAUPs showing higher CG content over the analysed mammalian genome and possessing distinct CG*CG motifs. (iii) Neither CpG-containing LAUPs nor CpG island sequences are randomly structured and distributed over the genomes; some LAUPs and most CpG-containing sequences exhibit an opposite trend within the same k and n variants. In addition, we demonstrate that the JBLA algorithm is more efficient than the original Jellyfish for computing LAUPs. Availability and implementation: We developed a Jellyfish-based LAUP analysis (JBLA) application by integrating Jellyfish (Marçais and Kingsford, 2011), MEME (Bailey, et al., 2009) and the NCBI genome database (Pruitt, et al., 2007) applications, which are listed as Supplementary Material. Supplementary information: Supplementary data are available at Bioinformatics online.

Dysregulated mitochondrial Ca2+ and ROS signaling in skeletal muscle of ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disease characterized by motor neuron loss and prominent skeletal muscle wasting. Despite more than one hundred years of research efforts, the pathogenic mechanisms underlying neuromuscular degeneration in ALS remain elusive. While the death of motor neuron is a defining hallmark of ALS, accumulated evidences suggested that in addition to being a victim of motor neuron axonal withdrawal, the intrinsic skeletal muscle degeneration may also actively contribute to ALS disease pathogenesis and progression. Examination of spinal cord and muscle autopsy/biopsy samples of ALS patients revealed similar mitochondrial abnormalities in morphology, quantity and disposition, which are accompanied by defective mitochondrial respiratory chain complex and elevated oxidative stress. Detailing the molecular/cellular mechanisms and the role of mitochondrial dysfunction in ALS relies on ALS animal model studies. This review article discusses the dysregulated mitochondrial Ca2+ and reactive oxygen species (ROS) signaling revealed in live skeletal muscle derived from ALS mouse models, and a potential role of the vicious cycle formed between the dysregulated mitochondrial Ca2+ signaling and excessive ROS production in promoting muscle wasting during ALS progression.

[Finite element analysis on biomechanical properties of medial collateral ligament of elbow joint under different flexion angles].

Three-dimensional finite element model of elbow was established to study the effect of medial collateral ligament (MCL) in maintaining the stability of elbow joint. In the present study a three-dimensional geometric model of elbow joint was established by reverse engineering method based on the computed tomography (CT) image of healthy human elbow. In the finite element pre-processing software, the ligament and articular cartilage were constructed according to the anatomical structure, and the materials and contacts properties were given to the model. In the neutral forearm rotation position and 0° flexion angle, by comparing the simulation data of the elbow joint with the experimental data, the validity of the model is verified. The stress value and stress distribution of medial collateral ligaments were calculated at the flexion angles of elbow position in 15°, 30°, 45°, 60°, 75°, 90°, 105°, 120°, 135°, respectively. The result shows that when the elbow joint loaded at different flexion angles, the anterior bundle has the largest stress, followed by the posterior bundle, transverse bundle has the least, and the stress value of transverse bundle is trending to 0. Therefore, the anterior bundle plays leading role in maintaining the stability of the elbow, the posterior bundle plays supplementary role, and the transverse bundle does little. Furthermore, the present study will provide theoretical basis for clinical recognizing and therapy of elbow instability caused by medial collateral ligament injury.

ROS-related mitochondrial dysfunction in skeletal muscle of an ALS mouse model during the disease progression.

In amyotrophic lateral sclerosis (ALS), mitochondrial dysfunction and oxidative stress form a vicious cycle that promotes neurodegeneration and muscle wasting. To quantify the disease-stage-dependent changes of mitochondrial function and their relationship to the generation of reactive oxygen species (ROS), we generated double transgenic mice (G93A/cpYFP) that carry human ALS mutation SOD1G93A and mt-cpYFP transgenes, in which mt-cpYFP detects dynamic changes of ROS-related mitoflash events at individual mitochondria level. Compared with wild type mice, mitoflash activity in the SOD1G93A (G93A) mouse muscle showed an increased flashing frequency prior to the onset of ALS symptom (at the age of 2 months), whereas the onset of ALS symptoms (at the age of 4 months) is associated with drastic changes in the kinetics property of mitoflash signal with prolonged full duration at half maximum (FDHM). Elevated levels of cytosolic ROS in skeletal muscle derived from the SOD1G93A mice were confirmed with fluorescent probes, MitoSOX™ Red and ROS Brite™570. Immunoblotting analysis of subcellular mitochondrial fractionation of G93A muscle revealed an increased expression level of cyclophilin D (CypD), a regulatory component of the mitochondrial permeability transition pore (mPTP), at the age of 4 months but not at the age of 2 months. Transient overexpressing of SOD1G93A in skeletal muscle of wild type mice directly promoted mitochondrial ROS production with an enhanced mitoflash activity in the absence of motor neuron axonal withdrawal. Remarkably, the SOD1G93A-induced mitoflash activity was attenuated by the application of cyclosporine A (CsA), an inhibitor of CypD. Similar to the observation with the SOD1G93A transgenic mice, an increased expression level of CypD was also detected in skeletal muscle following transient overexpression of SOD1G93A. Overall, this study reveals a disease-stage-dependent change in mitochondrial function that is associated with CypD-dependent mPTP opening; and the ALS mutation SOD1G93A directly contributes to mitochondrial dysfunction in the absence of motor neuron axonal withdrawal.

Irisin Protects Heart Against Ischemia-Reperfusion Injury Through a SOD2-Dependent Mitochondria Mechanism.

Irisin, a muscle-origin protein derived from the extracellular domain of the fibronectin domain-containing 5 protein (FNDC5), has been shown to modulate mitochondria welfare through paracrine action. Here, we test the hypothesis that irisin contributes to cardioprotection after myocardial infarction by preserving mitochondrial function in cardiomyocytes. Animal model studies show that intravenous administration of exogenous irisin produces dose-dependent protection against ischemia/reperfusion (I/R)-induced injury to the heart as reflected by the improvement of left ventricular ejection fraction and the reduction in serum level of cTnI (n = 15, P < 0.05). I/R-induced apoptosis of cardiomyocytes is reduced after irisin treatment. The irisin-mediated protection has, at least in part, an effect on mitochondrial function because administration of irisin increases irisin staining in the mitochondria of the infarct area. Irisin also reduces I/R-induced oxidative stress as determined by mitochondrial membrane potential evaluation and superoxide FLASH event recording (n = 4, P < 0.05). The interaction between irisin and superoxide dismutase2 (SOD2) plays a key role in the protective process because irisin treatment increases SOD activity (n = 10, P < 0.05) and restores the mitochondria localization of SOD2 in cardiomyocytes (n = 5, P < 0.05). These results demonstrate that irisin plays a protective role against I/R injury to the heart. Targeting the action of irisin in mitochondria presents a novel therapeutic intervention for myocardial infarction.

[Finite element analysis of male lower urinary tract based on the collodion slice images].

Males typically have high rates of morbidity of primary bladder neck obstruction, while the existing urodynamic examination is invasive and more likely to cause false diagnosis. To build a non-invasive biomechanical detecting system for the male lower urinary tract, a finite element model for male lower urinary tract based on the collodion slice images of normal male lower urinary tract was constructed, and the fluid-structure interaction of the lower urinary tract was simulated based on the real urination environment. The finite element model of the lower urinary tract was validated by comparing the clinical experiment data with the simulation result. The stress, flow rate and deformation of the lower urinary tract were analyzed, and the results showed that the Von Mises stress and the wall shear stress at the membrane sphincter in the normal male lower urinary tract model reached a peak, and there was nearly 1 s delay than in the bladder pressure, which helped to validate the model. This paper lays a foundation for further research on the urodynamic response mechanism of the bladder pressure and flow rate of the lower urinary tract obstruction model, which can provide a theoretical basis for the research of non-invasive biomechanical detecting system.

Generation and Screening of T-DNA Insertion Mutants Mediated by Agrobacterium tumefaciens in the Garden Asparagus Stem Blight Pathogen Phomopsis asparagi.

The garden asparagus stem blight caused by filamentous fungus Phomopsis asparagi exposes a serious threat on asparagus production globally. However, to present, we understand poorly about the molecular mechanisms of fungal pathogenicity. To facilitate functional genomics research of P. asparagi, here we developed a highly efficient and stable Agrobacterium tumefaciens-mediated transformation approach which yielded 150-200 transformants per 1 × 106 conidia. Our results indicated that 25 °C, acetosyringone concentration of 150 µmol/L, and 72 h were recommended as optimal co-cultivation conditions for the transformation. Using this transformation approach, we constructed a T-DNA insertion mutant library containing 1253 strains. Twenty randomly selected T-DNA insertion mutants were able to grow on 0.2 × PDA selective media after five successive subcultures without selective pressure, indicating that the exogenous T-DNA was stably integrated into the P. asparagi genome. We confirmed several randomly selected mutants using PCR with primers specific to the hph gene. Southern blots suggested that three out of the five selected mutants have a single T-DNA insertion. Interestingly, multiple mutant candidates with growth defects were obtained from the growth assay. Moreover, several mutants were selected for further analysis on the T-DNA flanking sequences through TAIL-PCR analysis. A sequence comparison of total junction fragments implied that the insertion of T-DNA within P. asparagi genome appeared to be a random event. The transformation technology and genetic resources developed here will facilitate studies of pathogenic mechanisms in this devastating filamentous fungal pathogen of garden asparagus.

Impaired bone homeostasis in amyotrophic lateral sclerosis mice with muscle atrophy.

There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and ß-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.

MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor ß/δ-dependent manner.

MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showed that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA-induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor ß/δ (PPARß/δ) was a downstream molecule of Sirt1, and its knockout by PPARß/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARß/δ-dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic differentiation through Sirt1 in a PPARß/δ-dependent manner, indicating that miR-132 and Sirt1-PPARß/δ may act as potential therapeutic targets for T2DM-induced osteoporosis.

Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 reduces pancreatic ß-cells apoptosis in glucotoxicity through activation of autophagy.

Chronic elevated glucose is harmful to pancreatic ß-cells, resulting in pancreatic ß-cells dysfunction and apoptosis. Understanding the molecular mechanisms associated with ß-cells survival is pivotal for the prevention of ß-cells injury caused by glucotoxicity. The role of Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) in the fate of pancreatic ß-cells constantly exposed to high glucose was studied. Sustained high glucose increased PINK1 protein expression both in rat pancreatic ß-cells and INS-1 ß-cells, and that this increase can be inhibited by PINK1 knockdown and further enhanced by PINK1 over-expression. PINK1 deficiency aggravated glucotoxicity-induced pancreatic ß-cells apoptosis and inhibition of autophagy whereas PINK1 could reverse these adverse effects. This study provides fundamental data supporting the potential protective role of PINK1 as a new therapeutic target necessary to preserve ß-cells survival under non-physiological hyperglycemia conditions.

Muscle-Bone Crosstalk in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease, is a fatal neuromuscular disorder characterized by degeneration of motor neurons and by skeletal muscle atrophy. Although the death of motor neurons is a pathological hallmark of ALS, the potential role of other organs in disease progression remains to be elucidated. Skeletal muscle and bone are the two largest organs in the human body. They are responsible not only for locomotion but also for maintaining whole body normal metabolism and homeostasis. Patients with ALS display severe muscle atrophy, which may reflect intrinsic defects in mitochondrial respiratory function and calcium (Ca) signaling in muscle fibers, in addition to the role of axonal withdrawal associated with ALS progression. Incidence of fractures is high in ALS patients, indicating there are potential bone defects in individuals with this condition. There is a lifelong interaction between skeletal muscle and bone. The severe muscle degeneration that occurs during ALS progression may potentially have a significant impact on bone function, and the defective bone may also contribute significantly to neuromuscular degeneration in the course of the disease. Due to the nature of the rapid and severe neuromuscular symptoms, a majority of studies on ALS have focused on neurodegeneration. Just a few studies have explored the possible contribution of muscle defects, even fewer on bone defects, and fewer still on possible muscle-bone crosstalk in ALS. This review article discusses current studies on bone defects and potential defects in muscle-bone crosstalk in ALS.

Isolation of Mitochondria from Murine Skeletal Muscle.

Skeletal muscle is one of the largest tissues in human body. Besides enabling voluntary movements and maintaining body's metabolic homeostasis, skeletal muscle is also a target of many pathological conditions. Mitochondria occupy 10-15% volume of a muscle myofiber and regulate many cellular processes, which often determine the fate of the cell. Isolation of mitochondria from skeletal muscle provides opportunities for various multi-omics studies with a focus on mitochondria in biomedical research field. Here we describe a protocol to efficiently isolate mitochondria with high quality and purity from skeletal muscle of mice using Nycodenz density gradient ultracentrifugation.

Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7 + satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12 , along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro . Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible "response biomarkers" in pre-clinical and clinical studies.