The role of peroxidasin in solid cancer progression.

Peroxidasin is a heme-containing peroxidase enzyme that plays a vital role in the cross-linking of collagen IV molecules in basement membranes. Collagen IV cross-links are essential for providing structure and mechanical stability throughout tissue development, homeostasis, and wound healing. During cancer progression, the basement membrane is degraded, and proteins typically found in the basement membrane, including peroxidasin and collagen IV, can be found spread throughout the tumour microenvironment where they interact with cancer cells and alter cell behaviour. Whilst peroxidasin is reported to be up-regulated in a number of different cancers, the role that it plays in disease progression and metastasis has only recently begun to be studied. This review highlights the current literature exploring the known roles of peroxidasin in normal tissues and cancer progression, regulators of peroxidasin expression, and the reported relationships between peroxidasin expression and patient outcome in cancer.

A recombinant technique for mapping functional sites of heterotrimeric collagen helices: Collagen IV CB3 fragment as a prototype for integrin binding.

Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α1ß1 and α2ß1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.

Regulation of the collagen IV network by the basement membrane protein perlecan is crucial for squamous epithelial cell morphogenesis and organ architecture.

All epithelia have their basal side in contact with a specialized extracellular matrix, the basement membrane (BM). During development, the BM contributes to the shaping of epithelial organs via its mechanical properties. These properties rely on two core components of the BM, collagen type IV and perlecan/HSPG2, which both interact with another core component, laminin, the initiator of BM assembly. While collagen type IV supplies the BM with rigidity to constrain the tissue, perlecan antagonizes this effect. Nevertheless, the number of organs that has been studied is still scarce, and given that epithelial tissues exhibit a wide array of shapes, their forms are bound to be regulated by distinct mechanisms. This is underscored by mounting evidence that BM composition and assembly/biogenesis is tissue-specific. Moreover, previous reports have essentially focused on the mechanical role of the BM in morphogenesis at the tissue scale, but not the cell scale. Here, we took advantage of the robust conservation of core BM proteins and the limited genetic redundancy of the Drosophila model system to address how this matrix shapes the wing imaginal disc, a complex organ comprising a squamous, a cuboidal and a columnar epithelium. With the use of a hypomorphic allele, we show that the depletion of Trol (Drosophila perlecan) affects the morphogenesis of the three epithelia, but particularly that of the squamous one. The planar surface of the squamous epithelium (SE) becomes extremely narrow, due to a function for Trol in the control of the squamous shape of its cells. Furthermore, we find that the lack of Trol impairs the biogenesis of the BM of the SE by modifying the structure of the collagen type IV lattice. Through atomic force microscopy and laser surgery, we demonstrate that Trol provides elasticity to the SE's BM, thereby regulating the mechanical properties of the SE. Moreover, we show that Trol acts via collagen type IV, since the global reduction in the trol mutant context of collagen type IV or the enzyme that cross-links its 7S -but not the enzyme that cross-links its NC1- domain substantially restores the morphogenesis of the SE. In addition, a stronger decrease in collagen type IV achieved by the overexpression of the matrix metalloprotease 2 exclusively in the BM of the SE, significantly rescues the organization of the two other epithelia. Our data thus sustain a model in which Trol counters the rigidity conveyed by collagen type IV to the BM of the SE, via the regulation of the NC1-dependant assembly of its scaffold, allowing the spreading of the squamous cells, spreading which is compulsory for the architecture of the whole organ.

Non-triple helical form of type IV collagen alpha1 chain suppresses vascular endothelial-cadherin mediated cell-to-cell junctions.

Non-triple helical collagen polypeptide α1(IV) (NTH α1(IV)) is a gene product of COL4A1 and is secreted as a polypeptide chain without the triple helix structure under physiological conditions. Studies have shown that NTH α1(IV) is up-regulated in and around vascular endothelial cells during neovascularization and vascular-like networks of in vitro angiogenesis models, suggesting its involvement in angiogenesis. In the present study, we examined the effect of NTH α1(IV) on endothelial cell-to-cell junctions, and we found that NTH α1(IV) suppressed VE-cadherin (vascular endothelial cadherin) mediated junctions and promoted cellular migration in human umbilical vein endothelial cell cultures. NTH α1(IV) is potentially a factor that induces VE-cadherin endocytosis and promotes neovascular sprouting and elongation. The possible mechanism entails endocytosis of NTH α1(IV) by its cellular receptor(s), Endo180 and/or other proteins, which results in the clearance of the cellular receptor(s) from the cell surface, thus inducing the endocytosis of VE-cadherin. Because the NC1 domain of the α1 chain of type IV collagen, called arresten, is considered an endogenous inhibitor of angiogenesis, it seems that the single polypeptide chain of NTH α1(IV) has conflicting functions.

Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine.

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO- as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1-33 and 2.1-2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1ß1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.

A pH-sensitive T7 peptide-decorated liposome system for HER2 inhibitor extracellular delivery: an application for the efficient suppression of HER2+ breast cancer.

HER2+ breast cancer is highly aggressive and proliferative even after multiple chemotherapy regimens. At present, the available clinical treatment duration of chemotherapeutic agents is limited by severe toxicity to noncancerous tissues, which are attributed to insufficient targeting. Here, we designed an active-targeted and pH-responsive liposome to improve the treatment. The ideas were as follows: (1) using liposome as a nano-delivery system for HER2 inhibitor (lapatinib; LAP) to reduce the toxicity; (2) modifying the capsule with T7 peptide for specific targeted delivery to the tumor cells, and (3) enabling the capsule with the pH-sensitive ability and triggering sustained drug release at extracellular weakly acidic microenvironment to emerge toxicity in tumors and to improve curative effects. It was found that T7 peptide-modified pH-sensitive liposome (T7-LP) was more effective and safer than free drug and unmodified liposome, and reduced drug-induced side effects and noncancerous toxicity. These results support the application potential of T7-LP in improving the efficacy of LAP in HER2+ breast cancer treatment. It might be a novel LAP formulation as a clinical agent.

Targeted delivery of protein arginine deiminase-4 inhibitors to limit arterial intimal NETosis and preserve endothelial integrity.

AIMS: Recent evidence suggests that 'vulnerable plaques', which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. METHODS AND RESULTS: We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. CONCLUSIONS: NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

A novel heterozygous variant of the COL4A4 gene in a Chinese family with hematuria and proteinuria leads to focal segmental glomerulosclerosis and chronic kidney disease.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS), as the frequent primary glomerular diseases in adults, accounts for symptomless proteinuria or nephrotic syndrome with or without renal insufficiency. As the crucial lesion of chronic kidney disease (CKD), accumulating evidence from recent studies show that mutations in Collagen-related genes may be responsible for FSGS. The aim of this study was to identify the genetic lesion of a Chinese family with FSGS and CKD. METHODS: In this study, we recruited a Han-Chinese family with unexplained high serum creatinine, hematuria, and proteinuria. Further renal biopsy and renal pathology indicated the diagnosis of FSGS in the proband. Whole-exome sequencing and Sanger sequencing were employed to explore the pathogenic mutation of this family. RESULTS: A novel heterozygous mutation (NM_000092 c.2030G>A, p.G677D) of the collagen type IV alpha-4 gene (COL4A4) was detected. Co-segregation analysis revealed that the novel mutation was carried by all the five affected individuals and absent in other healthy members as well as in our 200 local control cohorts. Bioinformatics predication indicated that this novel mutation was pathogenic and may disrupt the structure and function of type IV collagen. Simultaneously, this variant is located in an evolutionarily conserved site of COL4A4 protein. CONCLUSION: Here, we identified a novel mutation of COL4A4 in a family with FSGS and CKD. Our study expanded the variants spectrum of the COL4A4 gene and contributed to the genetic counseling and prenatal genetic diagnosis of the family. In addition, we also recommended the new classification of collagen IV nephropathies, which may be a benefit to the diagnosis, target drug treatment, and management of patients with COL4A3/COL4A4 mutations.

Enhanced Anti-Brain Metastasis from Non-Small Cell Lung Cancer of Osimertinib and Doxorubicin Co-Delivery Targeted Nanocarrier.

PURPOSE: Currently, the treatment of brain metastases from non-small cell lung cancer (NSCLC) is rather difficult in the clinic. A combination of small molecule-targeted drug and chemo-drug is a promising therapeutic strategy for the treatment of NSCLC brain metastases. But the efficacy of this combination therapy is not satisfactory due to the blood-brain barrier (BBB). Therefore, it is urgent to develop a drug delivery system to enhance the synergistic therapeutic effects of small molecule-targeted drug and chemo-drug for the treatment of NSCLC brain metastases. METHODS: T7 peptide installed and osimertinib (AZD9291) loaded intracellular glutathione (GSH) responsive doxorubicin prodrug self-assembly nanocarriers (T7-DSNPs/9291) have been developed as a targeted co-delivery system to enhance the combined therapeutic effect on brain metastases from NSCLC. In vitro cell experiments, including intracellular uptake assay, in vitro BBB transportation, and MTT assay were used to demonstrate the efficacy of T7-DSNPs/9291 in NSCLC brain metastasis in vitro. Real-time fluorescence imaging analysis, magnetic resonance imaging analysis, and Kaplan-Meier survival curves were used to study the effect of T7-DSNPs/9291 on an animal model in vivo. RESULTS: T7-DSNPs/9291 could significantly enhance BBB penetration of AZD9291 and doxorubicin via transferrin receptor-mediated transcytosis. Moreover, T7-DSNPs/9291 showed significant anti-NSCLC brain metastasis effect and prolonged median survival of an intracranial NSCLC brain metastasis animal model. CONCLUSION: T7-DSNPs/9291 is a potential drug delivery system for the combined therapy of brain metastasis from NSCLC.

Transferrin receptors targeting peptide (T7 peptide) surface-modified sorafenib nanoliposomes enhance the anti-tumor effect in colorectal cancer.

In this work, T7 modified nanoliposome loaded SNF (T7-SNF-NLPs) was developed. The physicochemical properties and characteristics of T7-SNF-NLPs, including morphology, particle size, zeta potential, stability, and in vitro release, were determined. In vitro toxicity and cellular uptake were evaluated in RKO cells. Antitumor efficiency was examined in RKO cells-bearing Kunming mice to assess their potential applications in the development of nanoliposomes therapeutics. The average particle size of T7-SNF-NLPs was observed to be 131.6 ± 1.7 nm and the polydispersity index represented a uniform mono-dispersion with PDI = 0.19. SNF was sustainably released from T7-SNF-NLPs at a release rate of 65% at 48 h in pH 7.4 PBS. The release rate of SNF was over 72% from T7-SNF-NLPs in pH 6.5 PBS, faster than that in pH 7.4, which indicated that the release rate of SNF was enhanced under the acidic environment. In vitro study clearly showed that T7 modified NLPs was more effective in inducing uptake and apoptosis in cancer cells than nonmodified NLPs. The IC50 values of T7-SNF-NLPs treated RKO cells was 9.54 µg/mL, 9.23 µg/mL for SNF-NLPs and 16.85 µg/mL for free SNF. T7-SNF-NLPs was highly efficient in suppressing the tumor growth in xenograft tumor model. The proportion of Ki67 in T7-SNF-NLPs group was significantly lower than that of either free drug or nonmodified NLPs.

3D-printed chitosan scaffolds modified with D-(+) raffinose and enriched with type IV collagen to improve epithelial cell colonization.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

Compound Mutations of the COL4A3 including a Novel Allele Identified in a Patient with Alport Syndrome.

Alport syndrome (AS) is a hereditary nephropathy which is characterized by molecular abnormalities in collagen IV. Here, we report compound mutations of the COL4A3 gene including a novel allele identified in a patient with Alport syndrome. The patient was a 25-year-old Chinese woman. She has a history of proteinuria and hematuria with cleft lip and palate. The pathologic results were consistent with Alport syndrome. The patient received ACEI treatment but did not respond well to the treatment. Sequencing results revealed that the patient carried two heterozygous mutations in the COL4A3 gene, including a known mutation (c.4243G>C, p.G1415R), which was inherited from her father, and a previously undescribed allele (c.4216G>A, p.G1406R) inherited from her mother. To date, at least 294 different variants of COL4A3 have been reported according to the Human Gene Mutation Database (HGMD). Identification of c.4216G>A as a new AS-related mutation may contribute to both genetic diagnosis of AS and further functional study of COL4A3.

Basement membrane-like structures containing NTH α1(IV) are formed around the endothelial cell network in a novel in vitro angiogenesis model.

Angiogenesis is a process through which new blood vessels are formed by sprouting and elongating from existing blood vessels. Several methods have been used to replicate angiogenesis in vitro, including culturing vascular endothelial cells on Matrigel and coculturing with endothelial cells and fibroblasts. However, the angiogenesis elongation process has not been completely clarified in these models. We therefore propose a new in vitro model of angiogenesis, suitable for observing vascular elongation, by seeding a spheroid cocultured from endothelial cells and fibroblasts into a culture dish. In this model, endothelial cells formed tubular networks elongated from the spheroid with a lumen structure and were connected with tight junctions. A basement membrane (BM)-like structure was observed around the tubular network, similarly to blood vessels in vivo. These results suggested that blood vessel-like structure could be reconstituted in our model. Laminin and type IV collagen, main BM components, were highly localized around the network, along with nontriple helical form of type IV collagen α1-chain [NTH α1(IV)]. In an ascorbic acid-depleted condition, laminin and NTH α1(IV) were observed around the network but not the triple-helical form of type IV collagen and the network was unstable. These results suggest that laminin and NTH α1(IV) are involved in the formation of tubular network and type IV collagen is necessary to stabilize the network.

A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.

Protective effect of T3 peptide, an active fragment of tumstatin, against ischemia/reperfusion injury in rat heart.

Ischemia/reperfusion (I/R)-induced oxidative stress is a serious clinical problem in the reperfusion therapy for ischemic diseases. Tumstatin is an endogenous bioactive peptide cleaved from type IV collagen α3 chain. We previously reported that T3 peptide, an active subfragment of tumstatin, exerts cytoprotective effects on H2O2-induced apoptosis through the inhibition of intracellular reactive oxygen species (ROS) production in H9c2 cardiomyoblasts. In this study, we investigated whether T3 peptide has cardioprotective effects against I/R injury by using in vitro and ex vivo experimental models. H9c2 cardiomyoblasts were stimulated with oxygen and glucose deprivation (OGD) for 12 h followed by reoxygenation for 1-8 h (OGD/R; in vitro model). The cells were treated with T3 peptide (30-1000 ng/ml) during OGD. Ten minutes after the pre-perfusion of T3 peptide (300 ng/ml), Langendorff perfused rat hearts were exposed to ischemia for 30 min followed by reperfusion for 1 h (ex vivo model). T3 peptide inhibited OGD/R-induced apoptosis through the inhibition of mitochondrial ROS production and dysfunction in H9c2 cardiomyoblasts. T3 peptide also prevented I/R-induced cardiac dysfunction, arrhythmia and myocardial infarction in the perfused rat heart. In conclusion, we for the first time demonstrated that T3 peptide exerts cardioprotective effects against I/R injury.

Poly-l/dl-lactic acid films functionalized with collagen IV as carrier substrata for corneal epithelial stem cells.

Limbal epithelial stem cells (LESCs) are responsible for the renewal of corneal epithelium. Cultivated limbal epithelial transplantation is the current treatment of choice for restoring the loss or dysfunction of LESCs. To perform this procedure, a substratum is necessary for in vitro culturing of limbal epithelial cells and their subsequent transplantation onto the ocular surface. In this work, we evaluated poly-L/DL-lactic acid 70:30 (PLA) films functionalized with type IV collagen (col IV) as potential in vitro carrier substrata for LESCs. We first demonstrated that PLA-col IV films were biocompatible and suitable for the proliferation of human corneal epithelial cells. Subsequently, limbal epithelial cell suspensions, isolated from human limbal rings, were cultivated using culture medium that did not contain animal components. The cells adhered significantly faster to PLA-col IV films than to tissue culture plastic (TCP). The mRNA expression levels for the LESC specific markers, K15, P63α and ABCG2 were similar or greater (significantly in the case of K15) in limbal epithelial cells cultured on PLA-col IV films than limbal epithelial cells cultured on TCP. The percentage of cells expressing the corneal (K3, K12) and the LESC (P63α, ABCG2) specific markers was similar for both substrata. These results suggest that the PLA-col IV films promoted LESC attachment and helped to maintain their undifferentiated stem cell phenotype. Consequently, these substrata offer an alternative for the transplantation of limbal cells onto the ocular surface.

Engineering oncolytic vaccinia virus with functional peptides through mild and universal strategy.

Oncolytic virotherapy is one of promising tumor therapy modalities. However, its therapeutic efficacy is still limited due to the immunogenicity and poor tumor-targeting capability. In this report, an engineered oncolytic vaccinia virus (OVV) was constructed by site-specifically introducing azide groups to the envelope of OVV during the in situ assembling process of virions. Subsequently, dibenzocyclooctynes (DBCO) derivate T7 peptide and DBCO derivate self-peptide were simultaneously conjugated to the azide-modified OVV (azide-OVV) via copper-free click chemistry. The infectivity of peptide-conjugated virus was well kept. Meanwhile, both of the targeting capacity to transferrin receptor (TfR)-overexpressed tumor cells and the in vivo blood circulation time increased. Therefore, the growth of TfR-positive tumor could be significantly inhibited after intravenously injecting the engineered OVV, while no noticeable side effects. This construction strategy can be popularized to other enveloped oncolytic virus (OV), thus a universal engineering platform can be provided for OV cancer therapy. Graphical Abstract An engineered oncolytic vaccinia virus (OVV) was constructed by bioconjugating DBCO derivate T7 peptide and DBCO derivate self-peptide with azide-modified OVV via copper-free click chemistry. As a result, the tumor inhibit effect was significantly enhanced attributed to the prolonged in vivo circulation time and improved targeting recognition capability.

Identification of a common epitope in the sequences of COL4A1 and COL6A1 recognized by monoclonal antibody #141.

Identification of a type IV collagen α1 polypeptide in non-triple helical form [NTH α1(IV)], possibly involved in angiogenesis, introduces the further possibility of the existence of non-triple helical forms of other collagen chains. We previously reported that an anti-NTH α1(IV) monoclonal antibody #141 recognizes not only NTH α1(IV) but also a novel non-triple helical collagen polypeptide NTH α1(VI) encoded by COL6A1. In this study, we identified the recognition sequence in order to better understand the properties of antibody #141 and provide clues regarding the biological function of the two non-triple helical molecules. Additionally, we determined the common epitope between COL4A1 and COL6A1 as PXXGXPGLRG, with surface plasmon resonance analyses revealing KD values for the COL4A1 epitope as 5.56±1.81×10-9 M and for the COL6A1 epitope as 7.15±0.44×10-10 M. The specific recognition of NTH α1(IV) and NTH α1(VI) by antibody #141 can be explained by the common epitope sequence. Moreover, epitope localization supports previous finding that NTH α1(IV) and NTH α1(VI) differ in conformation from the α1 chains in triple-helical type IV and type VI collagen. These findings suggest that antibody #141 might be useful for diagnosis of type VI collagen myopathies.

Collagen type-IV Langmuir and Langmuir-Schäfer layers as model biointerfaces to direct stem cell adhesion.

In biomaterial development, the design of material surfaces that mimic the extra-cellular matrix (ECM) in order to achieve favorable cellular instruction is rather challenging. Collagen-type IV (Col-IV), the major scaffolding component of Basement Membranes (BM), a specialized ECM with multiple biological functions, has the propensity to form networks by self-assembly and supports adhesion of cells such as endothelial cells or stem cells. The preparation of biomimetic Col-IV network-like layers to direct cell responses is difficult. We hypothesize that the morphology of the layer, and especially the density of the available adhesion sites, regulates the cellular adhesion to the layer. The Langmuir monolayer technique allows for preparation of thin layers with precisely controlled packing density at the air-water (A-W) interface. Transferring these layers onto cell culture substrates using the Langmuir-Schäfer (LS) technique should therefore provide a pathway for preparation of BM mimicking layers with controlled cell adherence properties. In situ characterization using ellipsometry and polarization modulation-infrared reflection absorption spectroscopy of Col-IV layer during compression at the A-W interface reveal that there is linear increase of surface molecule concentration with negligible orientational changes up to a surface pressure of 25 mN m-1. Smooth and homogeneous Col-IV network-like layers are successfully transferred by LS method at 15 mN m-1 onto poly(ethylene terephthalate) (PET), which is a common substrate for cell culture. In contrast, the organization of Col-IV on PET prepared by the traditionally employed solution deposition method results in rather inhomogeneous layers with the appearance of aggregates and multilayers. Progressive increase in the number of early adherent mesenchymal stem cells (MSCs) after 24 h by controlling the areal Col-IV density by LS transfer at 10, 15 and 20 mN m-1 on PET is shown. The LS method offers the possibility to control protein characteristics on biomaterial surfaces such as molecular density and thereby, modulate cell responses.

D-T7 Peptide-Modified PEGylated Bilirubin Nanoparticles Loaded with Cediranib and Paclitaxel for Antiangiogenesis and Chemotherapy of Glioma.

The blood-brain tumor barrier (BTB) and blood-brain barrier (BBB) have always been the major barriers in glioma therapy. In this report, we proposed D-T7 peptide-modified nanoparticles actively targeted glioma by overcoming the BBB and BTB to improve the antiglioma efficacy. Glioma-targeting experiments showed that the penetration effect of the D-T7 peptide-modified nanoparticles was 7.89-fold higher than that of unmodified nanoparticles. Furthermore, cediranib (CD) and paclitaxel (PTX) were used for the combination of the antiangiogenesis and chemotherapy for glioma. PEGylated bilirubin nanoparticles (BRNPs) were selected as a suitable drug delivery system (CD&PTX@TBRBPs) owing to the antioxidant, anti-inflammatory, and reactive oxygen species-responsive ability. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assays showed that CD&PTX@TBRBPs had the highest cytotoxicity and the median survival time of the CD&PTX@TBRNP group was 3.31-fold and 1.23-fold longer than that of the saline and CD&PTX@BRNP groups, respectively. All the results showed that we constructed a novel and accessible peptide-modified dual drug carrier with an enhanced antiglioma effect.