The structural and functional characteristics of soluble dietary fibers modified from tomato pomace with increased content of lycopene.

The tomato pomace, a by-product of tomato processing, was rich in nutrients such as lycopene (Lyc), vitamins, phenols and soluble dietary fibers (SDF). Homogenization combined with enzymatic hydrolysis (HE) was firstly applied to obtain HE-pomace. The yield of Lyc was raised by 57.2% after HE treatment by the optimal condition. The extraction rate of HE-SDF was increased by 73.4%. In order to clarify the relationship between the SDF and the release of Lyc, SDFs were characterized by structural analysis and morphological determination. The results suggested that HE-SDF possessed smaller molecular weight and loose microstructure with shorter chains. It implied that the degradation of dietary fiber led to the release of Lyc molecules. Besides, HE-SDF exhibited stronger capacity of water-holding, glucose adsorption and bile acid binding. In conclusion, HE treatment possessed the potential to be applied as an excellent modification method, which improved the nutritional and economic value of tomato pomace.

Inhibition Mechanism of L-Cysteine on Maillard Reaction by Trapping 5-Hydroxymethylfurfural.

The Maillard reaction (MR) can affect the color, flavor, organoleptic properties, and nutritional value of food. Sometimes, MR is undesirable due to lowering the nutrient utilization, producing harmful neo-formed compounds, etc. In this case, it is necessary to control MR. Some chemical substances, such as phenolic acid, vitamins, aminoguanidine, and thiols extracted from garlic or onion, can effectively prevent MR. In this study, L-cysteine (L-cys) was found to inhibit MR after screening 10 sulfhydryl compounds by comparing their ability to mitigate browning. The inhibition mechanism was speculated to be related to the removal of 5-hydroxymethylfurfural (HMF), a key mid-product of MR. The reaction product of HMF and L-cys was identified and named as 1-dicysteinethioacetal-5-hydroxymethylfurfural (DCH) according to the mass spectrum and nuclear magnetic resonance spectrum of the main product. Furthermore, DCH was detected in the glutamic-fructose mixture after L-cys was added. In addition, the production of DCH also increased with the addition of L-cys. It also was worth noting that DCH showed no cell toxicity to RAW 264.7 cells. Moreover, the in vitro assays indicated that DCH had anti-inflammatory and antioxidant activities. In conclusion, L-cys inhibits MR by converting HMF into another adduct DCH with higher safety and health benefits. L-cys has the potential to be applied as an inhibitor to prevent MR during food processing and storage.

Sudden cardiac death of Duchenne muscular dystrophy with NT-proBNP in pericardial fluid as a useful biomarker for diagnosis of the cause of death: a case report.

Duchenne muscular dystrophy (DMD) is one of the most common and severest muscular dystrophies. Although it can be a cause of death when associated with cardiac muscle and/or respiratory muscles, no cases of sudden deaths in the setting of undiagnosed DMD with cardiac involvement have been reported in the literatures. Previous studies showed that N-terminal-proBNP (NT-proBNP) was a robust laboratory biomarker to diagnose and monitor cardiac failure in clinical situations, suggesting that it may be used as an auxiliary indicator for diagnosis on left ventricular dysfunction in sudden cardiac deaths in forensic settings. Here, we reported a case of 29-year-old man who died suddenly. Autopsy revealed that muscles of the body were almost replaced by fatty and fibrotic tissues. The heart was enlarged with disarray and degeneration of cardiomyocytes in cardiac muscle. Total absence of dystrophin was detected by immunohistochemical staining, which confirmed DMD. Postmortem biochemical test of pericardial fluid revealed a high level of NT-proBNP, indicating dysfunction of the left ventricle before death. The cause of death was certified as an early dilated cardiomyopathy (DCM)/dysfunction of the left ventricle secondary to DMD, suggesting that sudden cardiac death with cardiac dysfunction could be identified by immunohistochemical method in combination with pericardial fluid NT-proBNP determination after systemic autopsy.

Complete Genome Sequence of Kosakonia radicincitans GXGL-4A, a Nitrogen-Fixing Bacterium with Capability to Degrade TEX.

Kosakonia radicincitans GXGL-4A, a free-living nitrogen-fixing (NF) bacterial strain isolated from maize (Zea mays L.) roots was found to have ability to degrade aromatic hydrocarbons. In this study, we describe the main morphological characteristics of bacterium, aromatic hydrocarbon-degrading capability, and the complete genome of K. radicincitans GXGL-4A. The genome is consisted of only one 5,687,681 bp linear chromosome with a G + C content of 53.96%. The strain has two genetically distinct nitrogenase systems, one based on molybdenum (Mo) similar to nitrogenase isolated from a wide range of nitrogen-fixing organisms, and the other contains iron (Fe). The differences in transcriptional level of several important nitrogen fixation (nif) genes between LB (nitrogen-rich, NR) and A15 nitrogen-free (nitrogen-limited, NL) culture conditions were detected using Real-time Quantitative Reverse Transcription PCR (qRT-PCR). The bacterial cells of GXGL-4A can grow well in LB liquid medium containing 1% toluene, ethylbenzene or xylene, suggesting a good resistance to the tested aromatic hydrocarbons. The results of GC-MS analysis showed that K. radicincitans GXGL-4A has a good capability to degrade toluene, ethylbenzene, and xylene (TEX). Completion of the genome sequencing will no doubt contribute to the deep exploration and comprehensive utilization of this NF bacterium in sustainable agriculture and bioremediation of aromatic pollutants.

Impact of short-term application of seaweed fertilizer on bacterial diversity and community structure, soil nitrogen contents, and plant growth in maize rhizosphere soil.

The effects of the short-term application of Ascophyllum nodosum-fermented seaweed fertilizer on the bacterial community, soil nitrogen contents, and plant growth in maize rhizosphere soil were evaluated. The changes in the bacterial community composition and nitrogen contents including those of total nitrogen (TN), nitrate nitrogen (NO3--N) and ammonium nitrogen (NH4+-N) in rhizosphere soils in response to treatment with seaweed fertilizer were determined. Furthermore, soil enzymatic activity and crop biomass were analyzed. The relative abundance of the dominant phyla varied regularly with fertilization, and bacterial α-diversity was apparently influenced by seaweed fertilizer amendment. The TN contents of all soil samples decreased gradually, and the NO3--N and NH4+-N contents of the soils treated with seaweed fertilizer were much higher than those of the control soils. Similarly, the enzymatic activities of dehydrogenase, nitrite reductase, urease, and cellulase in the soil were significantly increased on day 3, day 8, and day 13 after the application of seaweed fertilizer to the maize rhizosphere soil. However, there was no difference in the activity of soil sucrase between the treatment group and the control group. In this study, the growth of maize seedlings was confirmed to be greatly promoted by the utilization of seaweed fertilizer. These results deepen our understanding of plant-microbe interactions in agroecosystems and should benefit the wide use of seaweed fertilizer in sustainable agricultural production.

Cannabinoid 2 receptor attenuates inflammation during skin wound healing by inhibiting M1 macrophages rather than activating M2 macrophages.

BACKGROUND: The anti-inflammatory properties of the cannabinoid 2 receptor (CB2R) in injury and inflammatory diseases have been widely substantiated. Specifically, the anti-inflammatory effect of CB2R may be achieved by regulating macrophage polarisation. Several research findings suggested that the activation of CB2R could attenuate inflammation by reducing pro-inflammatory M1 macrophage polarisation and promoting anti-inflammatory M2 polarisation. However, considering CB2R inhibits fibrosis and M2 promotes fibrosis, that the activation of CB2R may lead to an increase in M2 macrophages seems contradictory. Therefore, we hypothesised that the activation of CB2R to attenuate inflammation is not achieved by up-regulating M2 macrophages. METHODS: We established an incised wound model using mouse skin and used this to evaluate the effect of CB2R agonists (JWH133 or GP1a) and an antagonist (AM630) on wound healing. At various post-injury intervals, we used western blot analysis, immunofluorescence staining, enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction assays to determine CB2R protein expression, M1/M2 macrophage infiltration, and the protein and gene expression of M1/M2-associated markers and cytokines in skin lesions. RESULTS: Activation of CB2R significantly reduced M1 macrophage infiltration and slightly increased M2 macrophage infiltration. Similarly, gene expression and protein levels of M1-associated markers and cytokines (interleukin [IL]-6, IL-12, CD86 and inducible nitric oxide synthase) were significantly down-regulated after CB2R agonist administration; in contrast, markers and cytokines were increased in the CB2R antagonist-treated group. Conversely, the administration of agonists slightly increased gene expression and protein levels of M2-associated markers and cytokines (IL-4, IL-10, CD206 and arginase-1 [Arg-1]); however, a statistical significance at most time points post-injury was not noted. CONCLUSION: In summary, our findings suggested that during incised skin wound healing in mice, increased levels of CB2R may affect inflammation by regulating M1 rather than M2 macrophage subtype polarisation. These results offer a novel understanding of the molecular mechanisms involved in the inhibition of inflammation by CBR2 that may lead to new treatments for cutaneous inflammation.

α7-nAChR Activation Has an Opposite Effect on Healing of Covered and Uncovered Wounds.

The α7 nicotinic acetylcholine receptor (α7-nAChR) is associated with inflammation, re-epithelialization, and angiogenesis in wound healing process. A recent study demonstrated that PNU-282987, a selective agonist of α7-nAChR, accelerates the repair of diabetic excisional wounds. Whether α7-nAChR activation promotes non-diabetic wounds healing is unknown. The aim of this study was to evaluate the effects of α7-nAChR activation on non-diabetic wound healing. The effects were evaluated in two wound models. In the first model, the wound was covered with a semi-permeable transparent dressing. In the second model, the wound was left uncovered. In both models, the mice were randomly assigned to two treatment groups: saline or PNU282987 (25 mice in each group). In covered wounds, we found that α7-nAChR activation inhibited re-epithelialization, angiogenesis, and epithelial cells proliferation, promoted neo-epithelial detachment, and suppressed neutrophil infiltration and the expression of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). However, in uncovered wounds, we observed that α7-nAChR activation promoted re-epithelialization and angiogenesis, inhibited neutrophil infiltration and the expression of high mobility group box (HMGB)-1, epidermal growth factor (EGF), and VEGF. In conclusion, this data demonstrated that α7-nAChR activation inhibited wound healing in covered wounds but played an opposite role in uncovered wounds. The opposite effect might be primarily due to inhibition of inflammation.

Pharmacological activation of cannabinoid 2 receptor attenuates inflammation, fibrogenesis, and promotes re-epithelialization during skin wound healing.

Previous studies showed that cannabinoid 2 (CB2) receptor is expressed in multiple effector cells during skin wound healing. Meanwhile, its functional involvement in inflammation, fibrosis, and cell proliferation in other organs and skin diseases implied CB2 receptor might also regulate skin wound healing. To verify this hypothesis, mice excisional wounds were created and treated with highly selective CB2 receptor agonist GP1a (1-(2,4-dichlorophenyl)-6-methyl- N-piperidin-1-yl-4H-indeno[1,2-c]pyrazole-3-carboxamide) and antagonist AM630 ([6-iodo-2- methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone) respectively. The inflammatory infiltration, cytokine expression, fibrogenesis, and wound re-epithelialization were analyzed. After CB2 receptor activation, neutrophil and macrophage infiltrations were reduced, and expressions of monocyte chemotactic protein (MCP)-1, stromal cell-derived factor (SDF)-1, Interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1 and vascular endothelial growth factor (VEGF)-A were decreased. Keratinocyte proliferation and migration were enhanced. Wound re-epithelialization was accelerated. Fibroblast accumulation and fibroblast-to-myofibroblast transformation were attenuated, and expression of pro-collagen I was decreased. Furthermore, HaCaT cells in vitro were treated with GP1a or AM630, which revealed that CB2 receptor activation promoted keratinocyte migration by inducing the epithelial to mesenchymal transition. These results, taken together, indicate that activating CB2 receptor could ameliorate wound healing by reducing inflammation, accelerating re-epithelialization, and attenuating scar formation. Thus, CB2 receptor agonist might be a novel perspective for skin wound therapy.

Cannabinoid CB2 receptors are involved in the regulation of fibrogenesis during skin wound repair in mice.

Studies have shown that cannabinoid CB2 receptors are involved in wound repair, however, its physiological roles in fibrogenesis remain to be elucidated. In the present study, the capacity of cannabinoid CB2 receptors in the regulation of skin fibrogenesis during skin wound healing was investigated. To assess the function of cannabinoid CB2 receptors, skin excisional BALB/c mice were treated with either the cannabinoid CB2 receptor selective agonist, GP1a, or antagonist, AM630. Skin fibrosis was assessed by histological analysis and profibrotic cytokines were determined by immunohistochemistry, immunofluorescence staining, reverse transcription­quantitative polymerase chain reaction and immunoblotting in these animals. GP1a decreased collagen deposition, reduced the levels of transforming growth factor (TGF)­ß1, TGF­ß receptor I (TßRI) and phosphorylated small mothers against decapentaplegic homolog 3 (P­Smad3), but elevated the expression of its inhibitor, Smad7. By contrast, AM630 increased collagen deposition and the expression levels of TGF­ß1, TßRI and P­Smad3. These results indicated that cannabinoid CB2 receptors modulate fibrogenesis and the TGF­ß/Smad profibrotic signaling pathway during skin wound repair in the mouse.

A fundamental study on the dynamics of multiple biomarkers in mouse excisional wounds for wound age estimation.

Wound age estimation is a classic but still modern theme in forensic practice. More experiments on different types of wound are needed to further improve its accuracy. In this study, mouse skin excisional wounds were created to simulate dermal defective injury. The neutrophil and macrophage infiltration, fibroblast and fibrocyte accumulation as well as their myofibroblastic transformation were examined. In addition, some wound healing-related molecules, including IL-1ß, IL-6, TNF-α, IFN-γ, MCP-1, CXCL12, VEGF-A, EGF, KGF, pro-col Iα2 and pro-col IIIα1, were quantified by Western blotting and real-time quantitative PCR. Neutrophils and macrophages profoundly infiltrated in the wound at 12 h-1 d and 3 d-10 d respectively. Fibroblasts and fibrocytes accumulated in the wound from 3 d, and transformed into contractile myofibroblasts from 5 d post injury. The transformation ratios of fibroblasts and fibrocytes were highest at 7 d-10 d and 10 d respectively (over 50%). MCP-1 and CXCL12 increased from 12 h to 5 d, and IL-1ß, TNF-α and pro-col IIIα1 up to 7 d. IL-6 and VEGF-A increased from 12 h to 1 d-10 d. Pro-col Iα2 increased from 7 d to 21 d. IFN-γ decreased from 12 h to 10 d. By comprehensive analysis of these factors from the perspective of morphometrics, protein and gene expressions, this study provided us with fundamental information for wound age estimation, especially in the wounds with full-thickness defection.

Detection of satellite cells during skeletal muscle wound healing in rats: time-dependent expressions of Pax7 and MyoD in relation to wound age.

The study was focused on time-dependent expressions of paired-box transcription factor 7 (Pax7) and myoblast determination protein (MyoD) during skeletal muscle wound healing. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17, and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. By morphometric analysis, the data based on the number of Pax7(+)/MyoD(-), Pax7(+)/MyoD(+), and Pax7(-)/MyoD(+) cells were highly correlated with the wound age. Pax7 and MyoD expressions were upregulated after injury by Western blot and quantitative real-time PCR assays. The relative quantity of Pax7 protein peaked at 5 days after injury, which was >1.13, and decreased thereafter. Similarly, the relative quantity of MyoD mRNA expression peaked at 3 days after injury, which was >2.59. The relative quantity of Pax7 protein >0.73 or mRNA expression >2.38 or the relative quantity of MyoD protein >1.33 suggested a wound age of 3 to 7 days. The relative quantity of MyoD mRNA expression >2.02 suggested a wound age of 1 to 7 days post-injury. In conclusion, the expressions of Pax7 and MyoD are upregulated in a time-dependent manner during skeletal muscle wound healing, suggesting that Pax7 and MyoD may be potential markers for wound age estimation in skeletal muscle.

α7nAChR is expressed in satellite cells at different myogenic status during skeletal muscle wound healing in rats.

Recent study has reported that α7 nicotine acetylcholine receptor (α7nAChR) is expressed in regenerated multinucleated myotubes. But the distribution of α7nAChR in satellite cells in different myogenic status is unknown. A preliminary study on the dynamic distribution of α7nAChR in satellite cells was performed by double indirect immunofluorescent procedures during skeletal muscle wound healing in rats. An animal model of skeletal muscle contusion was established in 40 Sprague-Dawley male rats. Samples were taken at 1, 3, 5, 7, 9, 13, 17 and 21 days after injury, respectively (five rats in each posttraumatic interval). Five rats were employed as control. In normal muscle specimens, weak immunoreactivity for α7nAChR was detected in a few satellite cells (considered as quiescent). α7nAChR-positive signals were observed in proliferated and differentiated satellite cells and regenerated multinucleated myotubes in the wounded areas. By morphometric analysis, the average number of α7nAChR+/Pax7+ and α7nAChR+/MyoD+ cells climaxed at 5 days post-injury. The average number of α7nAChR+/myogenin+ cells was significantly increased from 3 to 9 days post-injury as compared with other posttraumatic intervals. The protein level of α7nAChR maximized at 9 days post-injury, which implies that α7nAChR was associated with the satellite cells status. Our observations on expression of α7nAChR in satellite cells from quiescence to myotube formation suggest that α7nAChR may be involved in muscle regeneration by regulating satellite cell status.

CB2R orchestrates fibrogenesis through regulation of inflammatory response during the repair of skeletal muscle contusion.

Skeletal muscle injuries repair typically is an overlapping event between inflammation and tissue repair. Our previous study has demonstrated that activation of cannabinoid receptor type 2 (CB2R) by JWH-133 alleviates fibrosis in the repair of rat skeletal muscle contusion. Meanwhile, accumulated data show that CB2R stimulation exerts anti-inflammatory property in sepsis and cystitis. However, the effects of CB2R on inflammatory cytokines in response to the repair of skeletal muscle contusion are still unknown. In this study, we used selective agonist or antagonist of CB2R to observe the role of CB2R on inflammation and fibrogenesis during the repair of contused skeletal muscles in rats. Our results revealed that treatment with Gp1a, a selective CB2R agonist, significantly decreased the infiltration of neutrophils and macrophages, the expression of pro-inflammatory cytokines MCP-1, TNF-α, IL-1ß and IL-6, the expression of pro-fibrotic cytokines IL-4, IL-13, TGF-ß and P-Smad3 while increased anti-fibrotic cytokine IL-10 production as compared with Vehicle. The opposite results were observed in the CB2R inhibition group with AM630. Our study demonstrated that CB2R orchestrates fibrogenesis through regulation of inflammatory response during the repair of skeletal muscle contusion.

The monoacylglycerol lipase inhibitor JZL184 decreases inflammatory response in skeletal muscle contusion in rats.

Muscle wound healing process is a typical inflammation-evoked event. The monoacylglycerol lipase (MAGL) inhibitor (4-nitrophenyl)4-[bis(1,3-benzodioxol -5-yl)-hydroxymethyl]piperidine-1-carboxylate (JZL184) has been previously reported to reduce inflammation in colitis and acute lung injury in mice, which provide a new strategy for primary care of skeletal muscle injury. We investigated the effect of JZL184 on inflammation in rat muscle contusion model, and found decreased neutrophil and macrophage infiltration and pro-inflammatory cytokine expression. With extension of post-traumatic interval, myofiber regeneration was significantly hindered with increased collagen types I and ІІІ mRNAfibroblast infiltration as well as promoted fibrosis. Furthermore, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide (AM281, a selective cannabinoid CB1 receptor antagonist) and [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone (AM630, a selective cannabinoid CB2 receptor antagonist) treatment alleviated the anti-inflammatory effect of JZL184. Our findings demonstrate that JZL184 is able to inhibit the inflammatory response and interfere with contused muscle healing, in which the anti-inflammatory action may be mediated through cannabinoid CB1 and CB2 receptors.

[The number of circulating fibrocytes of skeletal muscle in rats after contusion].

OBJECTION: To investigate the time-dependent appearance of circulating fibrocytes of skeletal muscle in rats after contusion. METHODS: The model of skeletal muscle wound was established in rat. The circulating fibrocytes in contused skeletal muscle were detected by CD45 and procollagen I double immunofluorescence staining method. RESULTS: In the control group, CD45- and procollagen I-positive cells were not detected in skeletal muscle. A few CD45 cells were observed aged from 6 h to 1 d after contusion. A few CD45- and procollagen I-positive cells (fibrocytes) initially gathered in injury area 3d after injury. The ratio of positive fibrocytes significantly increased 5 d after injury. The ratio of fibrocytes was highest at 7 d after contusion and then decreased. The volume of fibrocytes showed bigger with injury time increase compared with 3 d group. The expression of procollagen I and CD45 were weakened at 14d after injury. CONCLUSION: The circulating fibrocytes are detected in contused skeletal muscle in time-dependent pattern. Circulating fibrocytes may be a marker in the wound age determination for contused skeletal muscle.