Hypoglycemic and antioxidative activity evaluation of phenolic compounds derived from walnut diaphragm produced in Xinjiang.

Walnut diaphragm is defined as a dry wood septum located between the walnut shell and kernel. In this work, seven phenolic compounds from walnut diaphragm were purified and characterized, and their antioxidant activities and mechanisms of hypoglycemia were investigated. Compounds 1-7 were tested for DPPH, ABTS scavenging ability, and FRAP assay to evaluate the antioxidant activity. α-Amylase inhibition assay was introduced to assess the hypoglycemic activity, and the mechanism was investigated by kinetic analysis, CD spectrum, and molecular docking. Compound 6 showed the strongest antioxidant ability, while compound 1 exhibited the strongest inhibition of α-amylase by changing the secondary structure of α-amylase in a mixed competitive inhibition mode. Molecular docking test predicted that the tetrahydropyran part in compound 1 may contribute to its hypoglycemic effect. This study furnishes a new theoretical reference for the utilization and development of walnut diaphragm into a health food with antioxidant and hypoglycemic properties. PRACTICAL APPLICATIONS: The finding of this research may serve as a basis for the subsequent development of walnut diaphragm into instant tea-based health food or added to other food carriers to achieve auxiliary antioxidant and hypoglycemic effects. This study revealed that polyphenolic components were the material basis for the antioxidant and hypoglycemic effects of walnut diaphragm, which could be identified as landmark chemical components for controlling quality standards in the development of walnut diaphragm, thus accelerating the research process of quality standards for walnut diaphragm-related products. Furthermore, the studies on the mechanism of hypoglycemic activity supply more credible data to support the development of walnut diaphragm into a safe and consumer-friendly health food. With abundant resources and clear efficacy, walnut diaphragm has great development prospect and application value.

Simultaneous removal of NOx and SO2 from simulated marine ship flue gas in a novel wet scrubbing system based on divided diaphragm seawater electrolysis technology: efficiency optimization and economic assessment.

This work constructed a divided diaphragm seawater electrolysis system with two tandem packed towers for the synergistic removal of NOx and SO2. The first tower was mainly used to oxidize NO and SO2 by AC (active chlorine), and the second tower was used to further absorb NOx. The factors affecting on NO removal, including ACC (active chlorine concentration), pH value, initial NO concentration and temperature in the oxidation tower were investigated. Moreover, the effect of different inlet gas concentrations and current values were explored. The results showed that with the increase of ACC, the NO and NOx removal efficiency increased rapidly, but when the ACC was higher than 500 mg/L [Cl2], the removal efficiency did not increase further in the oxidation tower. Low pH values in the oxidation tower were favorable for NO removal. NO removal efficiency reached a maximum at 40 °C. Higher NO and SO2 concentrations were favorable for NO removal. The decline of pH in the anode cell was not conducive to the storage of AC in the continuous electrolysis removal process. NOx and SO2 were almost completely removed after being scrubbed in the oxidation and absorption towers. The relationship between current and removal efficiency of NO and SO2 in the oxidation tower was also analyzed. Finally, the removal mechanism and the application prospects were discussed.

Noninvasive ultrasound imaging for assessment of intact microstructure of extracellular matrix in tissue engineering.

Development of artificial tissues or organs is one of the actual tasks in regenerative medicine that requires observation and evaluation of intact volume microstructure of tissue engineering products at all stages of their formation, from native donor tissues and decellularized scaffolds to recipient cell migration in the matrix. Unfortunately in practice, methods of vital noninvasive imaging of volume microstructure in matrixes are absent. In this work, we propose a new approach based on high-frequency acoustic microscopy for noninvasive evaluation and visualization of volume microstructure in tissue engineering products. The results present the ultrasound characterization of native rat diaphragms and lungs and their decellularized scaffolds. Verification of the method for visualization of tissue formation in the matrix volume was described in the model samples of diaphragm scaffolds with stepwise collagenization. Results demonstrate acoustic microscopic sensitivity to cell content concentration, variation in local density, and orientation of protein fibers in the volume, micron air inclusions, and other inhomogeneities of matrixes.

Estimation of Decellularization and Recellularization Quality of Tissue-Engineered Constructions by the Chemiluminescence Method.

It was found that the chemiluminescence intensity in native and recellularized tissues of rat muscular organs as well as in their decellularized scaffolds can serve as an express criterion that, along with ultrastructural analysis, makes it possible to perform quantitative assessment of the viability of cellular structures in biological samples of the diaphragm.

Identification and Quantification of Bioactive Compounds in Diaphragma juglandis Fructus by UHPLC-Q-Orbitrap HRMS and UHPLC-MS/MS.

Diaphragma juglandis fructus is the dry wooden diaphragm inside walnuts and a byproduct in food processing of walnut kernels. The purpose of our research is to enrich the information on compounds in Diaphragma juglandis fructus to further discover and exploit its potential nutritional value. In this study, new quali-quantitative analytical approaches were developed to identify and determine bioactive compounds in Diaphragma juglandis fructus. Two-hundred compounds, including hydrolyzable tannins, flavonoids, phenolic acids, and quinones, were identified by UHPLC-Q-Orbitrap HRMS, more than 150 of which were first discovered in Diaphragma juglandis fructus. Among them, 21 major dietary polyphenols with health-promoting effects were successfully quantified using UHPLC-MS/MS, with total contents of 2.88-6.18 mg/g. This successful characterization and quantification of bioactive compounds in Diaphragma juglandis fructus gives a better understanding of its potential nutritional value and supports efficiently developing and reusing it instead of discarding it as agrofood waste.

Mitochondrial accumulation of doxorubicin in cardiac and diaphragm muscle following exercise preconditioning.

Doxorubicin (DOX) is a highly effective anthracycline antibiotic. Unfortunately, the clinical use of DOX is limited by the risk of deleterious effects to cardiac and respiratory (i.e. diaphragm) muscle, resulting from mitochondrial reactive oxygen species (ROS) production. In this regard, exercise is demonstrated to protect against DOX-induced myotoxicity and prevent mitochondrial dysfunction. However, the protective mechanisms are currently unclear. We hypothesized that exercise may induce protection by increasing the expression of mitochondria-specific ATP-binding cassette (ABC) transporters and reducing mitochondrial DOX accumulation. Our results confirm this finding and demonstrate that two weeks of exercise preconditioning is sufficient to prevent cardiorespiratory dysfunction.

Decellularized Diaphragmatic Muscle Drives a Constructive Angiogenic Response In Vivo.

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31⁺, αSMA⁺, and vWF⁺ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF⁺ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration.

Palynological Investigation of Mummified Human Remains.

Pollen analysis was applied to a mummified homicide victim in Nebraska, U.S.A., to determine the location of death. A control sample showed the normal ambient pollen in the garage crime scene. Ambient windborne types, common in the air of the region, dominated the control. Internal samples were analyzed from the sacrum, intestine, and diaphragm. Microfossils were recovered from the rehydrated intestine lumen. The intestinal sample was dominated by Brassica (broccoli). The sacrum sample was high in dietary types but with a showing of ambient types. The pollen from the diaphragm was dominated by ambient pollen similar to the control samples. The discovery of diverse pollen spectra from within a single mummy was unexpected. They show that ingested and inhaled pollen mixed in the corpse. The data linked the decedent to a specific crime scene in her Nebraska home in the southern tier of eastern counties on the border with Kansas.

How to Wire the Diaphragm: Wholemount Staining Methods to Analyze Mammalian Respiratory Innervation.

Direct or indirect impairment of breathing in humans by diseases or environmental factors can either cause long-term disability and pain, or can ultimately result in death. Automatic respiratory centers in the brainstem control the highly structured process of breathing and signal to a specialized group of motor neurons in the cervical spinal cord that constitute the phrenic nerves. In mammals, the thoracic diaphragm separates the thorax from the abdomen and adopts the function of the primary respiratory musculature. Faithful innervation by the phrenic nerves is a prerequisite for correct functionality of this highly specialized musculature and thus, ultimately, the viability of the entire organism.To analyze the effects of diseases and genetic defects responsible for deleterious or lethal respiratory phenotypes, accurate imaging of respiratory innervation during embryonic development, e.g., in genetically modified mouse models enables the characterization of specific marker genes and pathways that underlie appropriate wiring of the diaphragm. Among the different available immunostaining techniques, wholemount staining methods provide the advantage of clear and faithful three-dimensional information about the location of the antigens of interest. In comparison to routine histological techniques, however, the researcher has to deal with technical challenges, such as antibody penetration, the stability and availability of the antigen, and clearing of the relevant tissue, and the need to be equipped with state-of-the-art microscope equipment.In this methodological chapter, we explain and share our expertise concerning wholemount processing of mouse embryos and thoracic diaphragms for the analysis of mammalian respiratory innervation.

EPR spectroscopy solutions for assessment of decellularization of intrathoracic organs and tissues.

Using EPR spectroscopy it was established that the determination of the concentration of paramagnetic centers in lyophilized tissues allows indirect evaluation of the quality of decellularization of intrathoracic organs (diaphragm, heart, and lungs), since the content of paramagnetic particles in them can serve as a criterion of cell viability and points to the necessity to repeat decellularization. Experiments in rats showed that the EPR spectra of the native thoracic organs contained paramagnetic centers with g-factor values ranging from 2.007 to 2.011 at a concentration of 10(-8) to 6.62 × 10(-7) mol/g of lyophilized tissue, whereas in all decellularized tissues of the same organs paramagnetic particles were not detected.

WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.

BACKGROUND: Intrapleural injection of CTB-Alexa 488, a retrograde tracer, provides an alternative labeling technique to the surgically invasive laparotomy required for intradiaphragmatic injection. However, CTB-Alexa 488 is incapable of crossing synapses restricting the tracer to the phrenic nuclei and the intercostal motor nuclei in the spinal cord. NEW METHOD: Intrapleural injection of WGA-Alexa 488, a transsynaptic tracer, provides a method to label the respiratory motor pathway in both the spinal cord and medulla. Intradiaphragmatic injection of WGA-Alexa 594 and vagal nerve injections of True blue were used to confirm the phrenic nuclei and to differentiate between the rVRG and the NA in the medulla. RESULTS: Following intrapleural injection, WGA-Alexa 488 was retrogradely transported to the phrenic nuclei and to the intercostal motor nuclei. Subsequently WGA-Alexa 488 was transsynaptically transported from the phrenic motoneurons to the pre-motor neurons in the rVRG that provide the descending drive to the phrenic neurons during inspiration. In addition WGA-Alexa 488 was identified in select cells of the NA confirmed by a dual label of both WGA-Alexa 488 and True blue. COMPARISON WITH EXISTING METHOD: WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling. CONCLUSIONS: Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells.

Intrinsic transient tracheal occlusion training and myogenic remodeling of rodent parasternal intercostal fibers.

It is recognized that diaphragm muscle plasticity occurs with mechanical overloads, yet less is known about synergistic parasternal intercostal muscle fiber remodeling. We conducted overload training with intrinsic transient tracheal occlusion (ITTO) exercises in conscious animals. We hypothesized that ITTO would yield significant fiber hypertrophy and myogenic activation that would parallel diaphragm fiber remodeling. Sprague-Dawley rats underwent placement of a tracheal cuff and were randomly assigned to receive daily 10 min sessions of conscious ITTO or observation (sham) over 2 wk. After training, fiber morphology, myosin heavy chain (MHC) isoform composition, cross-sectional area, proportion of Pax7-positive nuclei, and presence of embryonic MHC (eMHC) were quantified. Type IIx/b fibers were 20% larger after ITTO training than with sham training (ITTO: 4,431 +/­ 676 µm2, sham: 3,689 +/­ 400 µm2, p < 0.05), and type I fibers were more prevalent after ITTO (p < 0.01). Expression of Pax7 was increased in ITTO parasternals and diaphragm (p < 0.05). In contrast, the proportion of eMHC-positive fibers was increased only in ITTO parasternals (1.2% [3.4%­0.6%], sham: 0% [0.6%­0%], p < 0.05). Although diaphragm and parasternal type II fibers hypertrophy to a similar degree, myogenic remodeling appears to differ between the two muscles.

Giant solitary fibrous tumor of the diaphragm: a case report and review of literature.

A young gentleman presented with difficulty in breathing. Computed tomography (CT) scan showed a huge mass located between the heart and stomach, which might have rooted in the diaphragm. Magnetic resonance imaging (MRI) with enhanced three dimensional construction showed a lobulated, heterogeneous soft tissue mass with short T1 weighted imaging signal and flake long T2-weighted imaging (T2WI). Tumor-enhanced scanning demonstrated heterogeneous contrast enhancement. The preliminary diagnosis was intra-abdominal huge mass and considering sarcoma. Resection was conducted where the base of the tumor was located in the diaphragm oppressing the left liver lobe and heart. The base of the tumor, together with partial surrounding of the diaphragm, pericardium base, and the left lateral hepatic segment, was resected. The defect in the diaphragm and pericardium was repaired by patching, and thoracic close drainage and abdominal drainage were placed following the surgical operation. The pathological report showed giant solitary fibrous tumor (SFT). This case report may provide a reference resource for the diagnosis and treatment of SFT located in the diaphragm.

Cavernous hemangioma arising from the diaphragm.

A 51-year-old man was referred to our hospital with an abnormal nodule in the right lung field. Computed tomography revealed a homogeneous nodule adjacent to the diaphragm, which appeared to be an extrapulmonary lesion. No hilar or mediastinal lymph node swelling was detected, and positron-emission tomography showed no significant uptake. At surgery, 2 red papillary tumors were found, originating from the right diaphragm, and tumor extirpation was performed. The pathological diagnosis was cavernous hemangioma.

[Changes of rat respiratory and locomotory muscles during aerobic exercise training in continuous and interval regimens].

Male Wistar rats were treadmill-trained for 8 weeks using one of the two regimens: with the constant running speed or with alternating high-speed and low-speed intervals. Both training regimens led to an increase of rat aerobic capacities and to a higher citrate synthase activity in the medial head of gastrocnemius muscle. No differences between the effects of two training regimens were observed. However, in contrast to constant-speed training the interval one resulted in myocardium hypertrophy and also in less pronounced changes in diaphragm muscle, such as slow-direction shift of myosin phenotype and reduction of muscle fiber cross-sectional area. Neither of the training regimens had an effect on corticosterone and thyroid hormones levels in rat blood, whereas the interval training resulted in a higher level of testosterone. Anabolic influence of testosterone during interval aerobic training may be favorable for heart hemodynamic capacity and force characteristics of the diaphragm.

IgG anti-Galnac-GD1a antibodies bind to neuromuscular junctions of rat hemidiaphragm.

INTRODUCTION: We investigated the localization of a ganglioside, N-acetylgalactosaminyl GD1a (GalNAc-GD1a), in peripheral nerves with an IgG anti-GalNAc-GD1a antibody, which was produced in rabbits immunized with GalNAc-GD1a. METHODS: Teased fibers from ventral and dorsal roots and hemidiaphragm sections of rats were assessed using fluorescent double- and triple-labeling methods. RESULTS: The nodal and paranodal regions of teased fibers from ventral roots were immunostained with IgG anti-GalNAc-GD1a antibodies. After collagenase treatment, no staining was seen with IgG anti-GalNAc-GD1a or anti-NF200 antibodies, whereas α-bungarotoxin selectively stained nerve terminals. In cross-sectional and longitudinal sections of rat hemidiaphragm, IgG anti-GalNAc-GD1a antibodies overlapped with α-BuTx and anti-NF200 antibodies, indicating that GalNAc-GD1a is localized to the nerve terminal. IgG anti-GalNAc-GD1a antibody staining also overlapped with that of AChR clusters and syntaxin-positive presynaptic nerve terminals. CONCLUSION: GalNAc-GD1 is localized in both pre- and postsynaptic nerve terminals of neuromuscular junctions.

[Reduction of muscle mass mediated by myostatin in an experimental model of pulmonary emphysema]. / Reducción de la masa muscular mediada por miostatina en un modelo experimental de enfisema pulmonar.

INTRODUCTION: Among the extrapulmonary manifestations of COPD, dysfunction and loss of muscle mass/weight are those that have the greatest impact on the quality of life of patients. Our objective was to evaluate the molecular mechanisms that are potentially implicated in the limited development of muscle mass in the diaphragm and gastrocnemius of mice with experimentally-induced emphysema. METHODS: An experimental model in mice, in which emphysema was induced by means of the local instillation of elastase (n=6), while saline was administered to the controls (n=7). We determined the levels of oxidative stress, proteolytic systems, signaling pathways, growth factors and cell differentiation (western-blot) in the diaphragm and gastrocnemius of all the mice after 34 weeks. RESULTS: Upon comparing the mice with emphysema with the controls, the following findings were observed: (1) lower total body weight and lower weight of the diaphragm and gastrocnemius; (2) in the diaphragm, the levels of protein oxidation were increased, the mitochondrial antioxidant systems reduced, the levels of myostatin and of the ERK1/2 and FoxO1 signaling pathways were higher, and the myosin content was lower (67%); and (3) in the gastrocnemius of the emphysematous mice, the cytosolic antioxidants were decreased and the levels of myostatin and of the JNK and NF-kB signaling pathways were increased. CONCLUSIONS: The reduction of the myosin content observed in the diaphragm of mice with emphysema could explain their smaller size. Oxidative stress, myostatin and FoxO could be implicated in the loss of this structural protein.

Effect of type of muscle and Cu supplementation on trace element concentrations in cattle meat.

Considering that meat is an important source of metals exposure to humans it is important to explore trace element concentrations in different types of muscles. Because of the demonstrated effect of Cu-supplementation on mineral status, the influence of Cu-supplementation was also evaluated. Samples of four different muscles (diaphragm, cardiac, semitendinous and pectoral, n=120) from beef calves receiving typical commercial diets Cu-supplemented (15 mg Cu(2)SO(4)/kg DM) and non-supplemented were taken and acid digested. The levels of non-essential (As, Cd, Hg, Pb and Sn) and essential (Co, Cr, Fe, Mn, Mo, Ni, Se and Zn) elements were analyzed by ICP-MS. The statistical analyzes included two way Anova, post hoc DHS Tukey and Spearman correlations. The most active and less fat containing muscles showed in general the highest essential and the lowest non-essential trace element accumulation. As and Hg muscular residues are indicative of animal exposure, however, in situations of an adequate mineral status, essential trace element concentrations in muscle are irrespective of the mineral status of the animal and could be possibly related to their own particular muscular metabolism. Cu-supplementation significantly reduced As but caused a significant decrease of Se, which could have significance for the animal's health.

N-Acetylcysteine protects the rat diaphragm from the decreased contractility associated with controlled mechanical ventilation.

OBJECTIVE: Controlled mechanical ventilation results in diaphragmatic dysfunction, and oxidative stress has been shown to be an important contributor to ventilator-induced diaphragm dysfunction. We hypothesized that the administration of an antioxidant, N-acetylcysteine, would restore the redox balance in the diaphragm and prevent against the deleterious effects of controlled mechanical ventilation. DESIGN: Randomized, controlled experiment. SETTINGS: Basic science animal laboratory. SUBJECTS: Male Wistar rats, 14 wks old. INTERVENTIONS: Anesthetized rats were submitted for 24 hrs to either spontaneous breathing receiving 150 mg/kg N-acetylcysteine (SBNAC) or saline (SBSAL) or to controlled mechanical ventilation receiving 150 mg/kg N-acetylcysteine (MVNAC) or saline (MVSAL). MEASUREMENTS AND MAIN RESULTS: After 24 hrs of controlled mechanical ventilation, diaphragmatic force production was significantly lower in MVSAL compared with all groups. Importantly, administration of N-acetylcysteine completely abolished this controlled mechanical ventilation-induced diaphragmatic contractile dysfunction. Diaphragmatic protein oxidation was significantly increased after 24 hrs of controlled mechanical ventilation (+53%, p < .01) in MVSAL animals, whereas administration of N-acetylcysteine prevented this controlled mechanical ventilation-induced oxidative stress. Diaphragmatic 20S proteasome activity was increased in MVSAL (+62%, p < .05). Further, compared with SBSAL, diaphragm caspase-3 activity was significantly increased in MVSAL (+279%, p < .001), and N-acetylcysteine treatment provided partial protection against caspase-3 activation. Diaphragmatic calpain activity was significantly increased after controlled mechanical ventilation (+137%, p < .001) in MVSAL animals, but N-acetylcysteine treatment protected against this event. Finally, significant negative correlations existed between calpain activity and diaphragm force production (r from -0.56 to -0.49, p < .05). CONCLUSIONS: These data show that the administration of N-acetylcysteine protects the diaphragm from the deleterious effects of controlled mechanical ventilation. Specifically, N-acetylcysteine prevents against controlled mechanical ventilation-induced diaphragmatic oxidative stress and proteolysis and abolishes controlled mechanical ventilation-induced diaphragmatic contractile dysfunction.

Rapidly progressive diaphragmatic weakness and injury during mechanical ventilation in humans.

RATIONALE: Diaphragmatic function is a major determinant of the ability to successfully wean patients from mechanical ventilation (MV). Paradoxically, MV itself results in a rapid loss of diaphragmatic strength in animals. However, very little is known about the time course or mechanistic basis for such a phenomenon in humans. OBJECTIVES: To determine in a prospective fashion the time course for development of diaphragmatic weakness during MV; and the relationship between MV duration and diaphragmatic injury or atrophy, and the status of candidate cellular pathways implicated in these phenomena. METHODS: Airway occlusion pressure (TwPtr) generated by the diaphragm during phrenic nerve stimulation was measured in short-term (0.5 h; n = 6) and long-term (>5 d; n = 6) MV groups. Diaphragmatic biopsies obtained during thoracic surgery (MV for 2-3 h; n = 10) and from brain-dead organ donors (MV for 24-249 h; n = 15) were analyzed for ultrastructural injury, atrophy, and expression of proteolysis-related proteins (ubiquitin, nuclear factor-κB, and calpains). MEASUREMENTS AND MAIN RESULTS: TwPtr decreased progressively during MV, with a mean reduction of 32 ± 6% after 6 days. Longer periods of MV were associated with significantly greater ultrastructural fiber injury (26.2 ± 4.8 vs. 4.7 ± 0.6% area), decreased cross-sectional area of muscle fibers (1,904 ± 220 vs. 3,100 ± 329 µm²), an increase of ubiquitinated proteins (+19%), higher expression of p65 nuclear factor-κB (+77%), and greater levels of the calcium-activated proteases calpain-1, -2, and -3 (+104%, +432%, and +266%, respectively) in the diaphragm. CONCLUSIONS: Diaphragmatic weakness, injury, and atrophy occur rapidly in critically ill patients during MV, and are significantly correlated with the duration of ventilator support.