Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice

Chronic inflammation and excessive protease activity have a major role in the persistence of non-healing wounds. Granzyme B (GzmB) is a serine protease expressed during chronic inflammation that, in conjunction with perforin, has a well-established role in initiating apoptotic cell death. GzmB is also capable of acting extracellularly, independent of perforin and can degrade several extracellular matrix (ECM) proteins that are critical during wound healing. We used apolipoprotein E (ApoE) knockout (AKO) mice as a novel model of chronic inflammation and impaired wound healing to investigate the role of GzmB in chronic wounds. Wild-type and AKO mice were grown to 7 weeks (young) or 37 weeks (old) of age on a regular chow or high-fat diet (HFD), given a 1-cm diameter full thickness wound on their mid dorsum and allowed to heal for 16 days. Old AKO mice fed a HFD exhibited reduced wound closure, delayed contraction, chronic inflammation and altered ECM remodeling. Conversely, GzmB/ApoE double knockout mice displayed improved wound closure and contraction rates. In addition, murine GzmB was found to degrade both fibronectin and vitronectin derived from healthy mouse granulation tissue. In addition, GzmB-mediated degradation of fibronectin generated a fragment similar in size to that observed in non-healing mouse wounds. These results provide the first direct evidence that GzmB contributes to chronic wound healing in part through degradation of ECM.     

Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection

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Low-dose photodynamic therapy effect on closure of scratch wounds of normal and diabetic fibroblast cells: An in vitro study

Chronic wounds such as diabetic ulcers are a serious public health problem. Extensive research is needed to find new alternatives for wound treatment. Photodynamic therapy (PDT) is a non-invasive method, which has been studied for several decades to treat cancer, infections, and other diseases. PDT involves the administration of a photosensitizer compound followed by irradiation with using light at specific wavelength to produce reactive oxygen species (ROS) using molecular oxygen. It is possible that low dose photodynamic therapy (LDPDT) could improve wound healing and stimulates the cell repair process. This study we explored the effect of LDPDT on wound healing in vitro using normal and diabetic cellular wound models. The effects of different concentrations of 5-ALA and different energy densities (dark or light) on the cell viability of human fibroblast cells were studied using the MTT assay. After ascertaining the optimum parameters, a scratch wound assay was performed on both normal and diabetic cells and then cells treated with 1 and 5 μg/mL of 5-ALA at 1 J/cm² energy density. ROS production and morphological alteration of the cells were studied. The mortality of normal fibroblast cells increased with increasing 5-ALA concentration and also increasing energy density (up to 3 J/cm²). However, in diabetic cells, the mortality rate did not decrease. Diabetic cells showed increased migration and closure of the scratch compared to normal cells under similar conditions. A low concentration of 5-ALA (5 μg/mL) and low energy density of 1 J/cm² in both normal and diabetic cells gave a small increase in ROS levels compared to controls. This may explain the positive effects of LDPDT on wound healing. The findings of this study suggest that LDPDT may have a potential effect on the wound healing of diabetic wounds.     

Synthesis of a novel nanocomposite containing chitosan as a three-dimensional printed wound dressing technique: Emphasis on gene expression

In this study, a highly porous three-dimensional (3D)-printed wound healing core/shell scaffold fabricated using poly-lactic acid (PLA). The core of scaffold was composed of hyaluronic acid (HA), copper carbon dots (Cu-CDs), rosmarinic acid, and chitosan hydrogel. Cu-CDs were synthesized using ammonium hydrogen citrate under hydrothermal conditions. Formulation containing 1 mg ml⁻¹concentration of Cu-CDs showed an excellent antibacterial activity against gram bacteria. At 0.25 mg ml⁻¹ of Cu-CDs concentration, scaffold had a good biocompatibility as confirmed by cytotoxicity assay on L929 fibroblast stem cells. in vivo wound healing experiments on groups of rats revealed that after 15 days of treatment, the optimal formulation of composite scaffold significantly improves the wound healing process compared to the PLA scaffold. This finding was confirmed by histological analysis and the relative expression of PDGF, TGF-β, and MMP-1 genes. The biocompatible antibacterial CU-CDS/PLA/HA/chitosan/rosmarinic acid nanocomposite is a promising wound healing scaffold which highly accelerates the process of skin regeneration.     

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1beta (IL-1beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alpha/beta (CINC-2alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process.     

Injury Activates a Dynamic Cytoprotective Network to Confer Stress Resilience and Drive Repair

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PINK1 as a molecular checkpoint in the maintenance of mitochondrial function and integrity

Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder, is characterized by an age-dependent selective loss of dopaminergic (DA) neurons. Although most PD cases are sporadic, more than 20 responsible genes in familial cases were identified recently. Genetic studies using Drosophila models demonstrate that PINK1, a mitochondrial kinase encoded by a PD-linked gene PINK1, is critical for maintaining mitochondrial function and integrity. This suggests that mitochondrial dysfunction is the main cause of PD pathogenesis. Further genetic and cell biological studies revealed that PINK1 recruits Parkin, an E3 ubiquitin ligase encoded by another PD-linked gene parkin, to mitochondria and regulates the mitochondrial remodeling process via the Parkin-mediated ubiquitination of various mitochondrial proteins. PINK1 also directly phosphorylates the mitochondrial proteins Miro and TRAP1, subsequently inhibiting mitochondrial transport and mitochondrial oxidative damage, respectively. Moreover, recent Drosophila genetic analyses demonstrate that the neuroprotective molecules Sir2 and FOXO specifically complement mitochondrial dysfunction and DA neuron loss in PINK1 null mutants, suggesting that Sir2 and FOXO protect mitochondria and DA neurons downstream of PINK1. Collectively, these recent results suggest that PINK1 plays multiple roles in mitochondrial quality control by regulating its mitochondrial, cytosolic, and nuclear targets.     

Thrombin as a Regulator of Inflammation and Reparative Processes in Tissues

Activation of blood coagulation and thrombin formation accompany inflammation, wound healing, atherogenesis, and other processes induced by endothelial injury. Systems of hemostasis and inflammation play an important role in the pathogenesis of acute coronary syndromes. This paper reviews thrombin functions involved in its interaction with PAR family receptors, activation of platelets, endothelial cells, leukocytes, smooth muscle cells, and mast cells. Mechanisms of regulatory effects of thrombin on mast cells associated with nitric oxide release are discussed.     

The influence of a synthetic growth hormone-releasing hormone analogue G11 and opioid peptide biphalin on selected fibroblasts parameters relevant to wound healing

The treatment of hard-to-heal chronic wounds is still a major medical problem and an economic and social burden. In this work, we examine the proregenerative potential of two peptides, G11 (a trypsin-resistant analogue of growth hormone-releasing hormone [GHRH]) and biphalin (opioid peptide), and their combination in vitro on human fibroblasts (BJ). G11, biphalin and their combination exhibited no toxicity against BJ cells. On the contrary, these treatments significantly stimulated proliferation and migration of fibroblasts. Under inflammatory conditions (LPS-induced BJ cells), we noticed that the tested peptides decreased the levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and interleukin 1β (IL-1β). This was correlated with diminished phosphorylation levels of p38 kinase, but not those of ERK1/2. We found also that G11, biphalin and their combination activated the ERK1/2 signalling pathway, which has been previously implicated in promigratory activity of some regeneration enhancers, including opioids or GHRH analogues. Potential application of their combination requires further work, in particular in vivo experiments, in which the organism-level relevance of the discussed cell-level effects would be proven and, additionally, analgesic action of the opioid ingredient could be quantified.     

The role of oxidised regenerated cellulose/collagen in wound repair: effects in vitro on fibroblast biology and in vivo in a model of compromised healing

Irrespective of underlying chronic wound pathology, delayed wound healing is normally characterised by impaired new tissue formation at the site of injury. It is thought that this impairment reflects both a reduced capacity to synthesize new tissue and the antagonistic activities of high levels of proteinases within the chronic wound environment. Historically, wound dressings have largely been passive devices that offer the wound interim barrier function and establish a moist healing environment. A new generation of devices, designed to interact with the wound and promote new tissue formation, is currently being developed and tested. This study considers one such device, oxidised regenerated cellulose (ORC) /collagen, in terms of its ability to promote fibroblast migration and proliferation in vitro and to accelerate wound repair in the diabetic mouse, a model of delayed wound healing. ORC/collagen was found to promote both human dermal fibroblasts proliferation and cell migration. In vivo studies considered the closure and histological characteristics of diabetic wounds treated with ORC/collagen compared to those of wounds given standard treatment on both diabetic and non-diabetic mice. ORC/collagen was found to significantly accelerate diabetic wound closure and result in a measurable improvement in the histological appearance of wound tissues. As the diabetic mouse is a recognised model of impaired healing, which may share some characteristics of human chronic wounds, the results of this in vivo study, taken together with those relating the positive effects of ORC/collagen in vitro, may predict the beneficial use of this device in the clinical setting.     

Jwa participates the maintenance of intestinal epithelial homeostasis via ERK/FBXW7-mediated NOTCH1/PPARγ/STAT5 axis and acts as a novel putative aging related gene

The intestinal epithelium is a rapid self-renewal and regenerated tissue of which the structural integrity is beneficial for maintaining health. The integrity of intestinal epithelium depends on the balance of cell proliferation, differentiation, migration, and the function of intestinal stem cells, which declines due to genetic defect or aging. Jwa participates in multiple cellular processes; it also responds to oxidative stress and repairs DNA damage. However, whether Jwa plays a role in maintaining the homeostasis of intestinal renewal and regeneration is not clear. In the present study, we firstly described that the deletion of Jwa disturbed the homeostasis of intestinal epithelial renewal and regeneration. Jwa deficiency promoted NOTCH1 degradation in the ERK/FBXW7-mediated ubiquitin-proteasome pathway, thus disturbing the PPARγ/STAT5 axis. These mechanisms might partially contribute to the reduction of intestinal stem cell function and alteration of intestinal epithelial cell lineage distribution, finally suppressing the renewal and regeneration of intestinal epithelium. Moreover, our results also revealed that Jwa was a novel putative aging related gene.     

抑菌生的研究总结报告

本文报告一种由枯草杆菌制成的生态制剂,并命名为抑菌生(Subtilobiogen)。该制剂对创、烧伤感染有治疗作用。经过安全试验、急性毒性试验、Ames试验和微核试验证明,该制剂是一种无害、无毒和有致突变作用的活菌制剂。抑菌生有膏剂、乳剂及粉剂3种剂型。抑菌生在试管和体内对金黄色葡萄球菌、绿脓杆菌和大肠杆菌均具有抑菌作用。临床观察证明,将抑菌生喷洒在创面上,对浅Ⅱ°度、深Ⅱ°度及混合型烧伤感染均具有明显疗效。实验组(181例)与对照组(174例)相比较,在统计学上具有显著性差异。抑菌生的作用机制,经过初步试验证明,与营养争夺和占位性保护有关,因为枯草杆菌的生长速度超过金黄色葡萄球菌、绿脓杆菌和大肠杆菌的生长速度。     

A DNA methylation based measure outperforms circulating CRP as a marker of chronic inflammation and partly reflects the monocytic response to long-term inflammatory exposure: A Canadian Longitudinal Study on Aging analysis

A key hallmark in the age-related dysfunction of physiological systems is disruption related to the regulation of inflammation, often resulting in a chronic, low-grade inflammatory state (i.e., inflammaging). In order to understand the causes of overall system decline, methods to quantify the life-long exposure or damage related to chronic inflammation are critical. Here, we characterize a comprehensive epigenetic inflammation score (EIS) based on DNA methylation loci (CpGs) that are associated with circulating levels of C-reactive protein (CRP). In a cohort of 1446 older adults, we show that associations to age and health-related traits such as smoking history, chronic conditions, and established measures of accelerated aging were stronger for EIS than CRP, while the risk of longitudinal outcomes such as outpatient or inpatient visits and increased frailty were relatively similar. To determine whether variation in EIS actually reflects the cellular response to chronic inflammation we exposed THP1 myelo-monocytic cells to low levels of inflammatory mediators for 14 days, finding that EIS increased in response to both CRP (p = 0.011) and TNF (p = 0.068). Interestingly, a refined version of EIS based only on those CpGs that changed in vitro was more strongly associated with many of the aforementioned traits as compared to EIS. In conclusion, our study demonstrates that EIS outperforms circulating CRP with regard to its association to health-traits that are synonymous with chronic inflammation and accelerated aging, and substantiates its potential role as a clinically relevant tool for stratifying patient risk of adverse outcomes prior to treatment or following illness.     

Evolution on a volcanic conveyor belt: using phylogeographic reconstructions and K–Ar-based ages of the Hawaiian Islands to estimate molecular evolutionary rates

The Hawaiian Islands form as the Pacific Plate moves over a 'hot spot' in the earth's mantle where magma extrudes through the crust to build huge shield volcanos. The islands subside and erode as the plate carries them to the north-west, eventually to become coral atolls and seamounts. Thus islands are ordered linearly by age, with the oldest islands in the north-west (e.g. Kauai at 5.1 Ma) and the youngest in the south-east (e.g. Hawaii at 0.43 Ma). K–Ar estimates of the date of an island's formation provide a maximum age for the taxa inhabiting the island. These ages can be used to calibrate rates of molecular change under the following assumptions: (i) K–Ar dates are accurate; (ii) tree topologies show that derivation of taxa parallels the timing of island formation; (iii) populations do not colonize long after island emergence; (iv) the coalescent point for sister taxa does not greatly predate the formation of the colonized younger island; (v) saturation effects and (vi) among-lineage rate variation are minimal or correctable; and (vii) unbiased standard errors of distances and regressions can be estimated from multiple pairwise comparisons. We use the approach to obtain overall corrected rate calibrations for: (i) part of the mitochondrial cytochrome b gene in Hawaiian drepanidines (0.016 sequence divergence/Myr); (ii) the Yp1 gene in Hawaiian Drosophila (0.019/Myr Kambysellis et al. 1995 ); and (iii) parts of the mitochondrial 12S and 16S rRNA and tRNA^val in Laupala crickets (0.024–0.102/Myr, Shaw 1996 ). We discuss the reliability of the estimates given the assumptions (i–vii) above and contrast the results with previous calibrations of Adh in Hawaiian Drosophila and chloroplast DNA in lobeliods.     

Assessment on the binding characteristics of dasatinib,a tyrosine kinase inhibitor to calf thymus DNA: insights from multi-spectroscopic methodologies and molecular docking as well as DFT calculation

Abstract

The binding characteristics of calf thymus DNA (ct-DNA) with dasatinib (DSTN), a tyrosine kinase inhibitor was assessed through multi-spectroscopic methodologies and viscosity measurement combined with molecular docking as well as DFT calculation to understand the binding mechanism, affinity of DSTN onto ct-DNA, effect of DSTN on ct-DNA conformation, and among others. The results confirmed DSTN bound onto ct-DNA, leading to forming the DSTN–ct-DNA complex with the binding constant of 4.82?×?10³ M^?1 at 310?K. DSTN preferentially inserted to the minor groove of ct-DNA with rich A-T region, that was the binding mode of DSTN onto ct-DNA was groove binding. The enthalpic change (ΔH⁰) and entropic change (ΔS⁰) during the binding process of DSTN with ct-DNA were 128.9?kJ mol^?1 and 489.2?J mol^?1 K^?1, respectively, confirming clearly that the association of DSTN with ct-DNA was an endothermic process and the dominative driven-force was hydrophobic interaction. Meanwhile, the results also indicated that there was a certain extent of electrostatic force and hydrogen bonding, but they maybe play an auxiliary role. The CD measurement results confirmed the alteration in the helical configuration of ct-DNA but almost no change in the base stacking after binding DSTN. The results revealed that there was the obvious change in the conformation, the dipole moment, and the atomic charge distribution of DSTN in the B-DNA complexes, compared with free DSTN, to satisfy the conformational adaptation. From the obtained fronitier molecular orbitals of DSTN, it can be inferred that the nature of DSTN alters with the change of the environment around DSTN.

Communicated by Ramaswamy H. Sarma     

Molecular functions of the iron-regulated metastasis suppressor,NDRG1, and its potential as a molecular target for cancer therapy

N-myc down-regulated gene 1 (NDRG1) is a known metastasis suppressor in multiple cancers, being also involved in embryogenesis and development, cell growth and differentiation, lipid biosynthesis and myelination, stress responses and immunity. In addition to its primary role as a metastasis suppressor, NDRG1 can also influence other stages of carcinogenesis, namely angiogenesis and primary tumour growth. NDRG1 is regulated by multiple effectors in normal and neoplastic cells, including N-myc, histone acetylation, hypoxia, cellular iron levels and intracellular calcium. Further, studies have found that NDRG1 is up-regulated in neoplastic cells after treatment with novel iron chelators, which are a promising therapy for effective cancer management. Although the pathways by which NDRG1 exerts its functions in cancers have been documented, the relationship between the molecular structure of this protein and its functions remains unclear. In fact, recent studies suggest that, in certain cancers, NDRG1 is post-translationally modified, possibly by the activity of endogenous trypsins, leading to a subsequent alteration in its metastasis suppressor activity. This review describes the role of this important metastasis suppressor and discusses interesting unresolved issues regarding this protein.