Α1 Chain Research Articles

Vedolizumab: Potential Mechanisms of Action for Reducing Pathological Inflammation in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD), encompassing ulcerative colitis (UC), and Crohn’s disease (CD), are a group of disorders characterized by chronic, relapsing, and remitting, or progressive inflammation along the gastrointestinal tract. IBD is accompanied by massive infiltration of circulating leukocytes into the intestinal mucosa. Leukocytes such as neutrophils, monocytes, and T-cells are recruited to the affected site, exacerbating inflammation and causing tissue damage. Current treatments used to block inflammation in IBD include aminosalicylates, corticosteroids, immunosuppressants, and biologics. The first successful biologic, which revolutionized IBD treatment, targeted the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFα). Infliximab, adalimumab, and other anti-TNF antibodies neutralize TNFα, preventing interactions with its receptors and reducing the inflammatory response. However, up to 40% of people with IBD become unresponsive to anti-TNFα therapy. Thus, more recent biologics have been designed to block leukocyte trafficking to the inflamed intestine by targeting integrins and adhesins. For example, natalizumab targets the α4 chain of integrin heterodimers, α4β1 and α4β7, on leukocytes. However, binding of α4β1 is associated with increased risk for developing progressive multifocal leukoencephalopathy, an often-fatal disease, and thus, it is not used to treat IBD. To target leukocyte infiltration without this life-threatening complication, vedolizumab was developed. Vedolizumab specifically targets the α4β7 integrin and was approved to treat IBD based on the presumption that it would block T-cell recruitment to the intestine. Though vedolizumab is an effective treatment for IBD, some studies suggest that it may not block T-cell recruitment to the intestine and its mechanism(s) of action remain unclear. Vedolizumab may reduce inflammation by blocking recruitment of T-cells, or pro-inflammatory monocytes and dendritic cells to the intestine, and/or vedolizumab may lead to changes in the programming of innate and acquired immune cells dampening down inflammation.

Analysis of hydroxylation and O-glycosylation on lysine sites in deer-hide gelatin

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

Diagnostic Value of Type IV Collagen Expression in Renal Glomeruli at Alport’s Syndrome

The Alport’s syndrome is the hereditary multisystem disease characterized by the development of the progressive nephropathy. The early diagnosis and subsequent prescription of nephroprotective therapy improves significantly the nephrological prognosis. Purpose of the Study. Determine the value of the immunohistochemical method for the Alport’s syndrome diagnosis. Material and methods. The clinical, laboratory and morphological data of 35 patients with suspected Alport’s syndrome (13 years of age [11; 16]; 18 boys and 17 girls) examined in the Nephrology Department in 2013–2019 were summarized. The study of the renal tissue included the light, immunofluorescence, electron microscopy of the kidney biopsy sample, determination of the expression of α1, α3 and α5 chains of type IV collagen in the renal glomeruli using the immunohistochemical method; the genetic testing was carried out for 26 patients. The children were divided into groups depending on the glomerular expression of α5 chain of type IV collagen: normal (group 1, n=18), decreased (group 2, n=4), negative (group 3, n=13). Results are as the following: The disorder of the expression of α5 chain was detected in ¾ (q = 0.78) patients with genetically confirmed Alport’s syndrome and in almost all children with the X-linked variant of the disease (q = 0.94). Results. Based on the genetic testing, the Alport’s syndrome was confirmed in ¼ of the children of the 1st group (the children with the heterozygous variants of COL4A3, COL4A5 genes) and in all children of the 2nd and 3rd groups (COL4A5 variants). The sensitivity/ specificity of the immunohistochemical study for the Alport’s syndrome diagnosis was 78% /100%, that of the electron microscopy – 93% /87%. The predictive value of the positive/negative result of the immunohistochemical study was 100% /66%, that of the electron microscopy – 95% / 88% compared with 100% / 88% with the combine use of two methods. Conclusion. The determination of the expression of α5 chain of type IV collagen in the renal glomeruli has the independent diagnostic value, but it is inferior to the electron microscopy in the heterozygous variants of the Alport’s syndrome. The high specificity of the immunohistochemical method makes it possible to confirm the Alport’s syndrome in the case of the change in the expression of α5 chain of type IV collagen in the renal glomeruli.

Discovering pathways in benign prostate hyperplasia: A functional genomics pilot study.

Benign prostate hyperplasia (BPH) is one of the well-known urological neoplasms common in males with an increasing number of associated deaths in aging males. It causes uncomfortable urinary symptoms, including urine flow blockage, and may cause bladder, urinary tract or kidney problems. The histopathological and clinical knowledge regarding BPH is limited. In the present study, an in silico approach was applied that uses genome-scale microarray expression data to discover a wide range of protein-protein interactions in addition to focusing on specific genes responsible for BPH to develop prognostic biomarkers. Various genes that were differentially expressed in BPH were identified. Gene and functional annotation clusters were determined and an interaction analysis with disease phenotypes of BPH was performed, as well as an RNA tissue specificity analysis. Furthermore, a molecular docking study of certain short-listed gene biomarkers, namely anterior gradient 2 (AGR2; PDB ID: 2LNT), steroid 5α-reductase 2 (PDB ID: 6OQX), zinc finger protein 3 (PDB ID: 5T00) and collagen type XII α1 chain (PDB ID: 1U5M), was performed in order to identify alternative Chinese herbal agents for the treatment of BPH. Data from the present study revealed that AGR2 receptor (PDB ID: 2LNT) and berberine (Huang Bo) form the most stable complex and therefore may be assessed in further pharmacological studies for the treatment of BPH.

Preventive Effect of Canstatin against Ventricular Arrhythmia Induced by Ischemia/Reperfusion Injury: A Pilot Study.

Ventricular arrhythmia induced by ischemia/reperfusion (I/R) injury is a clinical problem in reperfusion therapies for acute myocardial infarction. Ca2+ overload through reactive oxygen species (ROS) production is a major cause for I/R-induced arrhythmia. We previously demonstrated that canstatin, a C-terminal fragment of type IV collagen α2 chain, regulated Ca2+ handling in rat heart. In this study, we aimed to clarify the effects of canstatin on I/R-induced ventricular arrhythmia in rats. Male Wistar rats were subjected to I/R injury by ligating the left anterior descending artery followed by reperfusion. Ventricular arrhythmia (ventricular tachycardia and ventricular fibrillation) was recorded by electrocardiogram. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and ROS production in neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen glucose deprivation/reperfusion (OGD/R) were measured by lucigenin assay and 2′,7′-dichlorodihydrofluorescein diacetate staining, respectively. The H2O2-induced intracellular Ca2+ ([Ca2+]i) rise in NRCMs was measured by a fluorescent Ca2+ indicator. Canstatin (20 µg/kg) inhibited I/R-induced ventricular arrhythmia in rats. Canstatin (250 ng/mL) inhibited OGD/R-induced NOX activation and ROS production and suppressed the H2O2-induced [Ca2+]i rise in NRCMs. We for the first time demonstrated that canstatin exerts a preventive effect against I/R-induced ventricular arrhythmia, perhaps in part through the suppression of ROS production and the subsequent [Ca2+]i rise.

Retraction Note: Inhibition of Tumor Angiogenesis by Tumstatin: Insights into Signaling Mechanisms and Implications in Cancer Regression

Growing tumors develop additional new blood vessels to meet the demand for adequate nutrients and oxygen, a process called angiogenesis. Cancer is a highly complex disease promoted by excess angiogenesis; interfering with this process poses for an attractive approach for controlling tumor growth. This hypothesis led to the identification of endogenous angiogenesis inhibitors generated from type IV collagen, a major component of vascular basement membrane (VBM). Type IV collagen and the angiogenesis inhibitors derived from it are involved in complex roles, than just the molecular construction of basement membranes. Protease degradation of collagens in VBM occurs in various physiological and pathological conditions and produces several peptides. Some of these peptides are occupied in the regulation of functions conflicting from those of their original integral molecules. Tumstatin (α3(IV)NC1), a proteolytic C-terminal non-collagenous (NC1) domain from type IV collagen α3 chain has been highlighted recently because of its potential role in anti-angiogenesis, however its biological actions are not limited to these processes. α3(IV)NC1 inhibits proliferation by promoting endothelial cell apoptosis and suppresses diverse tumor angiogenesis, thus making it a potential candidate for future cancer therapy. The present review surveys the physiological functions of type IV collagen and discovery of α3(IV)NC1 as an antiangiogenic protein with a comprehensive overview of the knowledge gained by us towards understanding its signaling mechanisms.

Development of basic research toward clinical application of cleaved fragment of type IV collagen

Basement membrane is a dense sheet-like extracellular matrix (ECM), which separates cells from surrounding interstitium. Type IV collagen is a major component of basement membrane and three of six α chains (namely α1-α6 chains) form a triple-helix structure. Recently, endogenous bioactive factors called "matricryptins" or "matrikines", which are produced by degrading and cleaving C-terminal domain of type IV collagen, attract attentions as a novel therapeutic target or a candidate for biomarkers. In all type IV collagens, matricryptins called arresten (α1 chain), canstatin (α2), tumstatin (α3), tetrastatin (α4), pentastatin (α5), and hexastatin (α6), have been identified. The type IV collagen-derived matricryptins have been previously studied as new therapeutic targets for neoplastic diseases since they exert anti-angiogenic and/or anti-tumor effects. On the other hand, we have recently demonstrated the cardioprotective effects of matricryptins in addition to the altered expression levels in cardiac diseases. In this review, we introduce the results of fundamental studies for the type IV collagen-derived matricryptins in various diseases, such as neoplastic diseases and cardiac diseases, and discuss the potential clinical application as novel therapeutic agents and biomarkers.

Anti-glomerular basement membrane disease

Background Anti-glomerular basement membrane (GBM) disease is an autoimmune disorder defined by the presence of autoantibody directed against α3 chain of type IV collagen. It is clinically characterized by rapidly progressive glomerulonephritis and lung hemorrhage. Patients and methods A retrospective study of 25 patients with anti-GBM disease, who were diagnosed and followed up at our institute from January 2011 to December 2017, was undertaken. Most patients presented with hypertension (76%), oliguria (80%), and dialysis-dependent renal failure (96%). Hemoptysis was seen in only one-third (28%) of patients. Diffuse glomerulosclerosis with crescents was the most common renal histopathology (60%) seen. Results Of all patients who were able to get adequate treatment in the form of plasmapheresis (56%) and immunosuppression (100%), only one (4%) patient recovered. Conclusion The majority of our patients with anti-GBM presented late in their clinical course of the disease, with advanced renal failure and irreversible lesion on kidney biopsy. High index of suspicion, early diagnosis, and aggressive management may improve the outcome of these patients.

Feasibility of canstatin, a cleaved product of type IV collagen α2 chain, as a novel biomarker and therapeutic target in cardiac diseases

Feasibility of canstatin, a cleaved product of type IV collagen α2 chain, as a novel biomarker and therapeutic target in cardiac diseases

Arresten, a cleaved fragment of type IV collagen α1 chain, regulates functions of cardiac fibroblasts and left atria

Arresten, a cleaved fragment of type IV collagen α1 chain, regulates functions of cardiac fibroblasts and left atria

Effects of laminin-111 peptide coatings on rat neural stem/progenitor cell culture

Neurons require adhesive scaffolds for their growth and differentiation. Laminins are a major cell adhesive component of basement membranes and have various biological activities in the peripheral and central nervous systems. Here, we evaluated the biological activities of 5 peptides derived from laminin-111 as a scaffold for mouse neuroblastoma Neuro2a cells and rat neural stem/progenitor cells (NPCs). The 5 peptides showed Neuro2a cell attachment activity similar to that of poly-d-lysine. However, when NPCs were cultured on the peptides, 2 syndecan-binding peptides, AG73 (RKRLQVQLSIRT, mouse laminin α1 chain 2719-2730) and C16 (KAFDITYVRLKF, laminin γ1 chain 139–150), demonstrated significantly higher cell attachment and neurite extension activities than other peptides including integrin-binding ones. Long-term cell culture experiments showed that both AG73 and C16 supported the growth of neurons and astrocytes that had differentiated from NPCs. Furthermore, C16 markedly promoted the expression of neuronal markers such as synaptosomal-associated protein-25 and syntaxin 1A. These results indicate that AG73 and C16 are useful for NPC cultures and that C16 can be applied to specialized research on synapses in differentiated neurons. These peptides have the potential for use as valuable biomaterials for NPC research.

A2 Chain: A Blockchain-Based Decentralized Authentication Scheme for 5G-Enabled IoT

The fifth-generation mobile communication technology (5G) provides high-bandwidth and low-latency data channels for massive IoT terminals to access the core business network. At the same time, it also brings higher security threats and challenges. Terminal identity authentication is an important security mechanism to ensure the core business network; however, most of the existing solutions adopt a centralized authentication model. Once the number of authentication requests exceeds the processing capacity of the authentication center service, it will cause authentication request congestion or deadlock. The decentralized authentication model can effectively solve the above problems. This article proposes a decentralized IoT authentication scheme called A2 Chain. First, A2 Chain uses edge computing to decentralize the processing of authentication requests and eliminate the burden on authentication services and the network. Second, to implement cross-domain identity verification of IoT devices, A2 Chain uses blockchain, and sidechain technologies are used to securely share the identity verification information of IoT devices. Additionally, A2 Chain replaces public key infrastructure (PKI) algorithm with identity-based cryptography (IBC) algorithm to eliminate the management overhead caused by centralized authentication model.

Effect of demineralized bone matrix modified by laminin α4 chain functional peptide on H-type angiogenesis and osteogenesis to promote bone defect repair

Based on the cell-extracellular matrix adhesion theory in selective cell retention (SCR) technology, demineralized bone matrix (DBM) modified by simplified polypeptide surface was designed to promote both bone regeneration and angiogenesis. Functional peptide of α4 chains of laminin protein (LNα4), cyclic RGDfK (cRGD), and collagen-binding domain (CBD) peptides were selected. CBD-LNα4-cRGD peptide was synthesized in solid phase and modified on DBM to construct DBM/CBD-LNα4-cRGD scaffold (DBM/LN). Firstly, scanning electron microscope and laser scanning confocal microscope were used to examine the characteristics and stability of the modified scaffold. Then, the adhesion, proliferation, and tube formation properties of CBD-LNα4-cRGD peptide on endothelial progenitor cells (EPCs) were detected, respectively. Western blot method was used to verify the molecular mechanism affecting EPCs. Finally, 24 10-week-old male C57 mice were used to establish a 2-mm-length defect of femoral bone model. DBM/LN and DBM scaffolds after SCR treatment were used to repair bone defects in DBM/LN group ( n=12) and DBM group ( n=12), respectively. At 8 weeks after operation, the angiogenesis and bone regeneration ability of DBM/LN scaffolds were evaluated by X-ray film, Micro-CT, angiography, histology, and immunofluorescence staining [CD31, endomucin (Emcn), Ki67]. Material related tests showed that the surface of DBM/LN scaffold was rougher than DBM scaffold, but the pore diameter did not change significantly ( t=0.218, P=0.835). After SCR treatment, DBM/LN scaffold was still stable and effective. Compared with DBM scaffold, DBM/LN scaffold could adhere to more EPCs after the surface modification of CBD-LNα4-cRGD ( P<0.05), and the proliferation rate and tube formation ability increased. Western blot analysis showed that the relative expressions of VEGF, phosphorylated FAK (p-FAK), and phosphorylated ERK1/2 (p-ERK1/2) proteins were higher in DBM/LN than in DBM ( P<0.05). In the femoral bone defect model of mice, it was found that mice implanted with DBM/LN scaffold had stronger angiogenesis and bone regeneration capacity ( P<0.05), and the number of CD31 hiEmcn hi cells increased significantly ( P<0.05). DBM/LN scaffold can promote the adhesion of EPCs. Importantly, it can significantly promote the generation of H-type vessels and realize the effective coupling between angiogenesis and bone regeneration in bone defect repair.

Efficient homing of antibody-secreting cells to the bone marrow requires RNA-binding protein ZFP36L1.

Cell migration relies on coordinated activity of chemotactic and guidance receptors. Here, we report a specific role for the RNA-binding protein ZFP36L1 in limiting the abundance of molecules involved in the homing of antibody-secreting cells (ASCs) to the bone marrow (BM). In the absence of ZFP36L1, ASCs build up in the spleen and the liver and show diminished accumulation in the BM. ZFP36L1 facilitates migration by directly regulating G protein-coupled receptor kinase 2 (GRK2) and the integrin chains α4 and β1 in splenic ASCs. Expression of CXCR4 and of the integrins α4 and β1 is differentially regulated on ASCs produced at the early and late stages of the immune response. Consequently, deletion of the Zfp36l1 gene has a stronger effect on BM accumulation of high-affinity ASCs formed late in the response. Thus, ZFP36L1 is an integral part of the regulatory network controlling gene expression during ASC homing.

Role of Zip1 in the regulation of NPY expression by MLT to promote fracture healing in rats.

Our previous study documented that melatonin (MLT) induced the osteogenic differentiation of mesenchymal stem cells (MSCs) and promoted the healing of femoral fractures in rats via the neuropeptide Y (NPY)/neuropeptide Y1 receptor (NPY1R) signaling pathway. MLT treatment upregulated the expression of the zinc uptake transporter zinc transporter 1 (Zip1) in nerve cells. Prior research demonstrated that oral zinc upregulated NPY expression. MSCs were isolated from rat bone marrow and identified using flow cytometry in our study. The results showed that MLT treatment upregulated NPY and NPY1R levels in MSCs with osteogenic differentiation, which was accompanied by upregulated Zip1 expression. However, the MLT-induced osteogenic differentiation of MSCs was reversed after interference of Zip1 expression. It was confirmed by the decreased alkaline phosphatase (ALP) level; downregulated activities of type I collagen α1 chain (COL1A1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2) and ALP; and reduced mineralized nodule formation. MLT promoted fracture healing in rats with femoral fracture, which was accompanied by increased expression of NPY and NPY1R and significantly increased expression of Zip1. In contrast, the silencing of Zip1 expression reversed MLT-mediated fracture healing. In summary, Zip1 participated in the regulation of the NPY/NPY1R signaling pathway via MLT to promote the osteogenic differentiation of MSCs and fracture healing.

Characterization of Collagen from Sakhalin Taimen Skin as Useful Biomass.

SUMMARYResearch backgroundAnimal collagen has been widely utilized in foods, cosmetics and biomedical fields. The non-edible portion, such as fish skin and bones, are obtained during cooking. Most of them are currently discarded as wastes, although the nutritional value of the skin and bones is high. The non-edible portion needs to be reused in order to reduce environmental impact, as it is one of the sources of environmental pollution.Experimental approachCollagen was prepared by cold acetone treatment from Sakhalin taimen skin as a waste produced during cooking. Next, the colour, SDS-polyacrylamide gel electrophoresis, ultraviolet absorption, subunit composition, amino acid composition, denaturation temperature and attenuated total reflectance-Fourier transform infrared spectroscopy analyses were conducted to explore the properties of the collagen. Lastly, we attempted to improve the functional properties of the collagen for future applications using chemical modification technique (succinylation).Results and conclusionsCold acetone treatment easily removed the fats and pigments from the skin. The odourless and pure white collagen was obtained with high yield. The α3 chain did not exist in the collagen. Sakhalin taimen skin collagen had rich α-helix and low β-sheet structures. Succinylation caused the secondary structural changes of the collagen molecule. Moreover, it made it possible not only to increase the viscosity of the collagen solution but also to improve the solubility of the collagen under the physiological conditions around pH=6.Novelty and scientific contributionThis finding is the first report on the absence of the α3 chain from salmonid fish skin collagens. The succinylated collagen from Sakhalin taimen skins as useful biomass has potential to utilize in foods, cosmetics and related industries.

Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood. To study the effect that loss of the laminin subunit alpha-3 (α3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment. We examined the role of the basement membrane proteins laminin subunits α3, β3 and γ2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples. Analysis of SCC tumours and cell lines using antibodies specific to laminin chains α3, β3 and γ2 identified a link between poorly differentiated SCC and reduced expression of laminin α3 but not the other laminin subunits investigated. Knockdown of laminin α3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin α3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin α3 knockdown cells. Knockdown of laminin subunits α3 and γ2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin α3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples. These data provide evidence that loss of the laminin α3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment.

Genetic background, recent advances in molecular biology, and development of novel therapy in Alport syndrome

Alport syndrome (AS) is a progressive inherited kidney disease characterized by hearing loss and ocular abnormalities. There are three forms of AS depending on inheritance mode: X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, which encodes type IV collagen α5 chain, while ADAS and ARAS are caused by variants in COL4A3 or COL4A4, which encode type IV collagen α3 or α4 chain, respectively. In male XLAS or ARAS cases, end-stage kidney disease (ESKD) develops around a median age of 20 to 30 years old, while female XLAS or ADAS cases develop ESKD around a median age of 60 to 70 years old. The diagnosis of AS is dependent on either genetic or pathological findings. However, determining the pathogenicity of the variants detected by gene tests can be difficult. Recently, we applied the following molecular investigation tools to determine pathogenicity: 1) in silico and in vitro trimer formation assay of α345 chains to assess triple helix formation ability, 2) kidney organoids constructed from patients’ induced pluripotent stem cells to identify α5 chain expression on the glomerular basement membrane, and 3) in vitro splicing assay to detect aberrant splicing to determine the pathogenicity of variants. In this review article, we discuss the genetic background and novel assays for determining the pathogenicity of variants. We also discuss the current treatment approaches and introduce exon skipping therapy as one potential treatment option.

Post-translational modifications in collagen type I of bone in a mouse model of aging.

The fracture resistance of cortical bone and matrix hydration are known to decline with advanced aging. However, the underlying mechanisms remain poorly understood, and so we investigated levels of matrix proteins and post-translational modifications (PTM) of collagen I in extracts from the tibia of 6-mo. and 20-mo. old BALB/c mice (female and male analysis done separately). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the levels of collagen I deamidation at specific asparagine (Asn) and glutamine (Gln) residues significantly increased with age. Other non-enzymatic PTMs such as carboxymethylation of lysine (CML) were detected as well, but the relative abundance did not vary with age. No significant age-related differences in the abundance of hydroxylysine glycosylation sites were found, but hydroxylation levels at a few of the numerous lysine and proline hydroxylation sites significantly changed by a small amount with age. We performed molecular modeling and dynamics (MD) simulations for three triple helical fragments representing collagen I regions with prominent age-dependent increases in deamidation as identified by LC-MS/MS of male extracts. These 3 fragments included deamidated Asn and Gln residues as follows: 1) an Asn428 site of the α2(I) chain in which deamidation levels increased from 4.4% at 6-mo. to 8.1% at 20-mo., 2) an Asn983 site of the α2(I) chain with a deamidation increase from 18.3% to 36.8% with age and an Asn1052 site of the α1(I) chain with consistent deamidation levels of ~60% across the age groups, and 3) a Gln410 site of the α1(I) chain that went from no detectable deamidation at 6-mo. to 2.7% at 20-mo. and a neighboring Asn421 site of the same chain with an age-related deamidation increase from 3.6% to 16.3%. The deamidation levels at these sites inversely correlated with an estimate of toughness determined from three-point bending tests of the femur mid-diaphysis. MD revealed that the sidechains become more negatively charged at deamidated sites and that deamidation alters hydrogen bonding with water along the collagen backbone while increasing water interactions with the aspartic and glutamic acid sidechains. Our findings suggest a new mechanism of the age-dependent reduction in the fracture resistance of cortical bone whereby deamidation of Asn and Glu residues redistributes bound water within collagen I triple helix.

Identification of adhesion-associated DNA methylation patterns in the peripheral nervous system.

Schwann cells are unique glial cells in the peripheral nervous system. These cells provide a range of cytokines and nutritional factors to maintain axons and support axonal regeneration. However, little is known concerning adhesion-associated epigenetic changes that occur in Schwann cells after peripheral nerve injury (PNI). In the present study, adhesion-associated DNA methylation biomarkers were assessed between normal and injury peripheral nerve. Specifically, normal Schwann cells (NSCs) and activated Schwann cells (ASCs) were obtained from adult Wistar rats. After the Schwann cells were identified, proliferation and adhesion assays were used to assess differences between NSCs and ASCs. Methylated DNA immunoprecipitation-sequencing and bioinformatics analysis were used to identify and analyze the differentially methylated genes. Reverse transcription-quantitative PCR was performed to assess the expression levels of adhesion-associated genes. In the present study, the proliferation and adhesion assays demonstrated that ASCs had a more robust proliferative activity and adhesion compared with NSCs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to identify methylation-associated biological processes and signaling pathways. Protein-protein interaction network analysis revealed that Fyn, Efna1, Jak2, Vav3, Flt4, Epha7, Crk, Kitlg, Ctnnb1 and Ptpn11 were potential markers for Schwann cell adhesion. The expression levels of several adhesion-associated genes, such as vinculin, BCAR1 scaffold protein, collagen type XVIII α1 chain and integrin subunit β6, in ASCs were altered compared with those in NSCs. The current study analyzed adhesion-associated DNA methylation patterns of Schwann cells and identified candidate genes that may potentially regulate Schwann cell adhesion in Wistar rats before and after PNI.