From amino-acid to disease: the effects of oxidation on actin-myosin interactions in muscle.

Actin-myosin interactions form the basis of the force-producing contraction cycle within the sarcomere, serving as the primary mechanism for muscle contraction. Post-translational modifications, such as oxidation, have a considerable impact on the mechanics of these interactions. Considering their widespread occurrence, the explicit contributions of these modifications to muscle function remain an active field of research. In this review, we aim to provide a comprehensive overview of the basic mechanics of the actin-myosin complex and elucidate the extent to which oxidation influences the contractile cycle and various mechanical characteristics of this complex at the single-molecule, myofibrillar and whole-muscle levels. We place particular focus on amino acids shown to be vulnerable to oxidation in actin, myosin, and some of their binding partners. Additionally, we highlight the differences between in vitro environments, where oxidation is controlled and limited to actin and myosin and myofibrillar or whole muscle environments, to foster a better understanding of oxidative modification in muscle. Thus, this review seeks to encompass a broad range of studies, aiming to lay out the multi layered effects of oxidation in in vitro and in vivo environments, with brief mention of clinical muscular disorders associated with oxidative stress.

Oxidation alters myosin-actin interaction and force generation in skeletal muscle filaments.

The interaction between actin and myosin is the basis of contraction and force production in muscle fibers. Studies have shown that actin and myosin oxidation cause myofibrillar weakness in healthy and diseased muscles. The degree to which oxidation of each of these proteins contributes to an attenuated force in myofibrils is unclear. In this study, we show that exposure of actin and myosin to the chemical 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride (SIN-1), an NO and O2•- donor, affected actin-myosin interactions, as shown by a decreased myosin-propelled actin velocity in the in vitro motility assay. We also observed that oxidation of actin and myosin resulted in a decrease in force generated by myosin and actin filaments, as determined by a system of microfabricated cantilevers. Although myosin is more sensitive to oxidative modifications than actin, as indicated by a steeper decrease in velocity and force by the filaments, modifications on actin are sufficient to affect force and velocity and also contribute to a decrease in contractile activity in muscles.

Millisecond Conformational Dynamics of Skeletal Myosin II Power Stroke Studied by High-Speed Atomic Force Microscopy.

Myosin-based molecular motors are responsible for a variety of functions in the cells. Myosin II is ultimately responsible for muscle contraction and can be affected by multiple mutations, that may lead to myopathies. Therefore, it is essential to understand the nanomechanical properties of myosin II. Due to the lack of technical capabilities to visualize rapid changes in nonprocessive molecular motors, there are several mechanistic details in the force-generating steps produced by myosin II that are poorly understood. In this study, high-speed atomic force microscopy was used to visualize the actin-myosin complex at high temporal and spatial resolutions, providing further details about the myosin mechanism of force generation. A two-step motion of the double-headed heavy meromyosin (HMM) lever arm, coupled to an 8.4 nm working stroke was observed in the presence of ATP. HMM heads attached to an actin filament worked independently, exhibiting different lever arm configurations in given time during experiments. A lever arm rotation was associated with several non-stereospecific long-lived and stereospecific short-lived (∼1 ms) HMM conformations. The presence of free Pi increased the short-lived stereospecific binding events in which the power stroke occurred, followed by release of Pi after the power stroke.

The load dependence and the force-velocity relation in intact myosin filaments from skeletal and smooth muscles.

In the present study we evaluated the load dependence of force produced by isolated muscle myosin filaments interacting with fluorescently labeled actin filaments, using for the first time whole native myosin filaments. We used a newly developed approach that allowed the use of physiological levels of ATP. Single filaments composed of either skeletal or smooth muscle myosin and single filaments of actin were attached between pairs of nano-fabricated cantilevers of known stiffness. The filaments were brought into contact to produce force, which caused sliding of the actin filaments over the myosin filaments. We applied load to the system by either pushing or pulling the filaments during interactions and observed that increasing the load increased the force produced by myosin and decreasing the load decreased the force. We also performed additional experiments in which we clamped the filaments at predetermined levels of force, which caused the filaments to slide to adjust the different loads, allowing us to measure the velocity of length changes to construct a force-velocity relation. Force values were in the range observed previously with myosin filaments and molecules. The force-velocity curves for skeletal and smooth muscle myosins resembled the relations observed for muscle fibers. The technique can be used to investigate many issues of interest and debate in the field of muscle biophysics.

Deep Learning in Automated Region Proposal and Diagnosis of Chronic Otitis Media Based on Computed Tomography.

OBJECTIVES: The purpose of this study was to develop a deep-learning framework for the diagnosis of chronic otitis media (COM) based on temporal bone computed tomography (CT) scans. DESIGN: A total of 562 COM patients with 672 temporal bone CT scans of both ears were included. The final dataset consisted of 1147 ears, and each of them was assigned with a ground truth label from one of the 3 conditions: normal, chronic suppurative otitis media, and cholesteatoma. A random selection of 85% dataset (n = 975) was used for training and validation. The framework contained two deep-learning networks with distinct functions: a region proposal network for extracting regions of interest from 2-dimensional CT slices; and a classification network for diagnosis of COM based on the extracted regions. The performance of this framework was evaluated on the remaining 15% dataset (n = 172) and compared with that of 6 clinical experts who read the same CT images only. The panel included 2 otologists, 3 otolaryngologists, and 1 radiologist. RESULTS: The area under the receiver operating characteristic curve of the artificial intelligence model in classifying COM versus normal was 0.92, with sensitivity (83.3%) and specificity (91.4%) exceeding the averages of clinical experts (81.1% and 88.8%, respectively). In a 3-class classification task, this network had higher overall accuracy (76.7% versus 73.8%), higher recall rates in identifying chronic suppurative otitis media (75% versus 70%) and cholesteatoma (76% versus 53%) cases, and superior consistency in duplicated cases (100% versus 81%) compared with clinical experts. CONCLUSIONS: This article presented a deep-learning framework that automatically extracted the region of interest from two-dimensional temporal bone CT slices and made diagnosis of COM. The performance of this model was comparable and, in some cases, superior to that of clinical experts. These results implied a promising prospect for clinical application of artificial intelligence in the diagnosis of COM based on CT images.

High-speed AFM reveals subsecond dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding.

High-speed atomic force microscopy (HS-AFM) can be used to study dynamic processes with real-time imaging of molecules within 1- to 5-nm spatial resolution. In the current study, we evaluated the 3-state model of activation of cardiac thin filaments (cTFs) isolated as a complex and deposited on a mica-supported lipid bilayer. We studied this complex for dynamic conformational changes 1) at low and high [Ca2+] (pCa 9.0 and 4.5), and 2) upon myosin binding to the cTF in the nucleotide-free state or in the presence of ATP. HS-AFM was used to directly visualize the tropomyosin-troponin complex and Ca2+-induced tropomyosin movements accompanied by structural transitions of actin monomers within cTFs. Our data show that cTFs at relaxing or activating conditions are not ultimately in a blocked or activated state, respectively, but rather the combination of states with a prevalence that is dependent on the [Ca2+] and the presence of weakly or strongly bound myosin. The weakly and strongly bound myosin induce similar changes in the structure of cTFs as confirmed by the local dynamical displacement of individual tropomyosin strands in the center of a regulatory unit of cTF at the relaxed and activation conditions. The displacement of tropomyosin at the relaxed conditions had never been visualized directly and explains the ability of myosin binding to TF at the relaxed conditions. Based on the ratios of nonactivated and activated segments within cTFs, we proposed a mechanism of tropomyosin switching from different states that includes both weakly and strongly bound myosin.

Cleavage of loops 1 and 2 in skeletal muscle heavy meromyosin (HMM) leads to a decreased function.

BACKGROUND: The mechanical work and the actin-activated ATP kinetics in skeletal muscles are closely associated with two surface loops that are present in the myosin molecule: loop 1 and loop 2. They are located close to the ATP-loop (loop 1), and the actin binding domain (loop 2). In this study we investigated the roles of loops 1 and 2 in the regulation of the load-dependent velocity of actin sliding and ATPase activity. METHODS: Heavy meromyosin (HMM) from rabbit skeletal muscle was subjected to limited tryptic proteolysis to obtain fragments containing different amounts of loops 1 and 2. The amino-acid sequences of these fragments were confirmed with quantitative mass-spectrometry. The velocity of actin motility propelled by the HMM fragments was measured using in-vitro motility assays, with varying loads induced by the addition of different concentrations of α-actinin. RESULTS: The load-dependent velocity of the myosin-propelled actin motility, and the fraction of actin filaments motility, were decreased in close association with the depletion of loop 1 in the HMM. The ATPase activity was decreased in close association with depletion of loops 1 and 2. CONCLUSIONS: Loop 1 is responsible for regulating the load-dependent velocity of actin motility. GENERAL SIGNIFICANCE: Myosin-actin interaction is closely regulated by two flexible loops in the structure of myosin. The results of this study are important for the understanding of the molecular mechanisms of contraction, and therefore the most basic functions of life, such as locomotion, heart beating, and breathing.

Long-term Brain Tissue Monitoring after Semi-brain Irradiation in Rats Using Proton Magnetic Resonance Spectroscopy: A Preliminary Study In vivo.

BACKGROUND: In head and neck neoplasm survivors treated with brain irradiation, metabolic alterations would occur in the radiation-induced injury area. The mechanism of these metabolic alterations has not been fully understood, while the alternations could be sensitively detected by proton (1H) nuclear magnetic resonance spectroscopy (MRS). In this study, we investigated the metabolic characteristics of radiation-induced brain injury through a long-term follow-up after radiation treatment using MRS in vivo. METHODS: A total of 12 adult Sprague-Dawley rats received a single dose of 30 Gy radiation treatment to semi-brain (field size: 1.0 cm × 2.0 cm; anterior limit: binocular posterior inner canthus connection; posterior limit: external acoustic meatus connection; internal limit: sagittal suture). Conventional magnetic resonance imaging and single-voxel 1H-MRS were performed at different time points (in month 0 before irradiation as well as in the 1st, 3rd, 5th, 7th, and 9th months after irradiation) to investigate the alternations in irradiation field. N-acetylaspartate/choline (NAA/Cho), NAA/creatinine (Cr), and Cho/Cr ratios were measured in the bilateral hippocampus and quantitatively analyzed with a repeated-measures mixed-effects model and multiple comparison test. RESULTS: Significant changes in the ratios of NAA/Cho (F = 57.37, P_{g < 0.001), NAA/Cr (F = 54.49, Pg < 0.001), and Cho/Cr (F = 9.78, Pg = 0.005) between the hippocampus region of the irradiated semi-brain and the contralateral semi-brain were observed. There were significant differences in NAA/Cho (F = 9.17, Pt < 0.001) and NAA/Cr (F = 13.04, Pt < 0.001) ratios over time. The tendency of NAA/Cr to change with time showed no significant difference between the irradiated and contralateral sides. Nevertheless, there were significant differences in the Cho/Cr ratio between these two sides. CONCLUSIONS: MRS can sensitively detect metabolic alternations. Significant changes of metabolites ratio in the first few months after radiation treatment reflect the metabolic disturbance in the acute and early-delayed stages of radiation-induced brain injuries.}

Association Between Subtotal Gastrectomy with Billroth II Anastomosis and Coronary Heart Disease.

BACKGROUND: We assessed the risk of coronary heart disease (CHD) after subtotal gastrectomy with Billroth II anastomosis (SGBIIA) for peptic ulcer disease (PUD). METHODS: The Taiwan National Health Insurance Research Database was used, and 6160 patients undergoing SGBIIA for PUD were identified as the surgical cohort. A total of 24,540 patients from the PUD population not undergoing surgery selected by frequency-matching were identified as the non-surgical cohort. All patients were followed until the end of 2011 to measure the incidence of CHD. RESULTS: The cumulative incidence of CHD was lower in patients with SGBIIA than in those without surgery (16.9 vs 22.9 per 1000 person-year, adjusted hazard ratio [aHR] = 0.79, 95% confidence interval [CI] = 0.71-0.88). The risk of CHD, either acute coronary syndrome (ACS) (aHR = 0.83, 95% CI = 0.75-0.91) or other non-ACS CHD (aHR = 0.78, 95% CI = 0.68-0.88), was lower for the SGBIIA cohort than for the non-surgery cohort (aHR = 0.79, 95% CI = 0.71-0.88) after adjusting for age and the comorbidities of hypertension, diabetes mellitus, hyperlipidemia, stroke, congestive heart failure, chronic kidney disease, and chronic obstructive pulmonary disease. CONCLUSIONS: We found SGBIIA is associated with a reduced risk of CHD for PUD patients.

Reduced passive force in skeletal muscles lacking protein arginylation.

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.

Elastic proteins in the flight muscle of Manduca sexta.

The flight muscles (DLM1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spike for each contraction. Stress/strain curves of skinned DLM1 showed hysteresis indicating the presence of titin-like elastic proteins. Projectin and kettin are titin-like proteins previously identified in Lethocerus and Drosophila flight muscles. Analysis of Manduca muscles with 1% SDS-agarose gels and western blots showed two bands near 1 MDa that cross-reacted with antibodies to Drosophila projectin. Antibodies to Drosophila kettin cross-reacted to bands at ∼500 and ∼700 kDa, but also to bands at ∼1.6 and ∼2.1 MDa, that had not been previously observed in insect flight muscles. Mass spectrometry identified the 2.1 MDa protein as a product of the Sallimus (sls) gene. Analysis of the gene sequence showed that all 4 putative Sallimus and kettin isoforms could be explained as products of alternative splicing of the single sls gene. Both projectin and sallimus isoforms were expressed to higher levels in ventrally located DLM1 subunits, primarily responsible for active work production, as compared to dorsally located subunits, which may act as damped springs. The different expression levels of the 2 projectin isoforms and 4 sallimus/kettin isoforms may be adaptations to the specific requirements of individual muscle subunits.

Arginylation of myosin heavy chain regulates skeletal muscle strength.

Protein arginylation is a posttranslational modification with an emerging global role in the regulation of actin cytoskeleton. To test the role of arginylation in the skeletal muscle, we generated a mouse model with Ate1 deletion driven by the skeletal muscle-specific creatine kinase (Ckmm) promoter. Ckmm-Ate1 mice were viable and outwardly normal; however, their skeletal muscle strength was significantly reduced in comparison to controls. Mass spectrometry of isolated skeletal myofibrils showed a limited set of proteins, including myosin heavy chain, arginylated on specific sites. Atomic force microscopy measurements of contractile strength in individual myofibrils and isolated myosin filaments from these mice showed a significant reduction of contractile forces, which, in the case of myosin filaments, could be fully rescued by rearginylation with purified Ate1. Our results demonstrate that arginylation regulates force production in muscle and exerts a direct effect on muscle strength through arginylation of myosin.

Sparing sentinel node biopsy through axillary lymph node fine needle aspiration in primary breast cancers.

BACKGROUND: Axillary lymph node status is an important staging and prognostic factor in breast cancer. This study aimed to evaluate the efficacy of axilla fine needle aspiration cytology (FNAC) in primary breast cancer without a palpable node and even without image characteristics of a metastatic node. METHODS: From June 2008 to January 2012, 77 patients met the inclusion criteria of having received a FNAC procedure during the diagnostic protocol of primary breast cancer with the characteristic of impalpable axilla nodes, and of having received axillary surgery after that, according to the guidelines. The patients' characteristics, clinical-pathological features, pre-operative axillary lymph node FNAC findings, surgical lymph node report, and definite pathologic staging were reviewed. RESULTS: The FNAC procedures had a reported sensitivity of 58.82%, specificity of 100%, positive predictive value of 100%, negative predictive value of 72.55%, and accuracy of 80.28%. There were no false positives on FNAC; therefore, the positive likelihood ratio approached infinity. The negative likelihood ratio was 41.18%. Axillary lymph node FNAC is feasible in newly diagnosed breast cancer patients to evaluate metastatic lymph nodes even in those without clinical or ultrasonic evidence of lymphadenopathy. CONCLUSIONS: FNAC can be a routine evaluation for most primary breast cancer patients with benefits in expediting treatment. For those patients with positive findings of the axilla, sentinel node biopsy can be avoided.

Labyrinthine sequestrum: four case studies.

Labyrinthine sequestrum, a rare form of labyrinthitis, is highly distinct from the more commonly encountered labyrinthitis ossificans based on its unique clinical, radiologic, and histologic characteristics. The study included 4 such patients who had undergone clinical and laboratory investigations, computed tomography (CT), and magnetic resonance imaging (MRI) assessments followed by surgical procedures and pathological evaluation. Their major symptoms were otorrhea, otalgia, tinnitus, and profound hearing loss. Imaging studies showed an osteolytic soft mass with calcified debris in the inner ear, and the bony labyrinth was eroded partly or completely by granulation mass, with loss of bony morphology. Further pathological examination was coincident with inflammatory granulation tissue with some calcification or osseous tissue. The disease process is attributed to chronic osteomyelitis due to the presence of osteonecrosis. Prompt CT and MRI examinations and optimal therapeutic management facilitate definitive diagnosis and protect against fatal complications.

[Neuroendocrine carcinoma of the breast: mammographic features correlated with sonography and histopathological findings].

OBJECTIVE: The purpose of this study was to describe the X-ray features of the neuroendocrine carcinoma (NEC) of breast to raise the awareness for the disease. METHODS: The mammography, sonography and clinicopathologic features in a total of 16 cases of pathologically proven breast neuroendocrine carcinoma in Fudan University Cancer Hospital were analyzed retrospectively. RESULTS: All the 16 patients were women with a mean age of 67.3 years old (ranged from 45 to 75 years old). Twelve patients had palpable masses and the other four patients had outflow from the nipple. Pathological diagnosis included endocrine ductal carcinoma in situ (E-DCIS) in 4 cases, E-DCIS with microinvasion in 5, and invasive solid neuroendocrine carcinoma in 7. On mammography, 9 of 16 cases exhibited round or slightly lobulated masses. Five of the 16 cases exhibited irregular or asymmetric opacities. Two of the 16 cases had negative findings. The borders of the 14 masses detected on mammography were vague in 7, partly unclear in 3 and clear-cut in 4 cases. Malignant microcalcification was not found in all cases. The accuracy rates of preoperative qualitative diagnosis for NEC with mammography and sonography were 68.8% (11/16) and 81.3% (13/16), respectively. Given the application of combined mammography and sonography, the accuracy rates could be improved to 87.5% (14/16). CONCLUSIONS: A round mass and irregular or asymmetric opacities without malignant calcification are the most frequent appearances of mammography in neuroendocrine carcinoma of the breast. Combination of mammography and sonography may be useful to improve the accuracy of diagnosis and early detection of neuroendocrine carcinoma of the breast.