Laminin B1 Research Articles

Bottlenose dolphin (Tursiops truncatus) immortalized fibroblasts on novel 3D in vitro collagen-free scaffolds.

Dolphins, as apex predators, can be considered relevant sentinels of the health of marine ecosystems. The creation of 3D cell models to assess in vitro cell-to-cell and cell-to-matrix interactions in environmental-mimicking conditions, is of considerable interest. However, to date the establishment of cetacean 3D culture systems has not yet been accomplished. Thus, in this study, different 3D systems of bottlenose dolphin (Tursiops truncatus) skin fibroblasts have been analyzed. Particularly, novel scaffolds based on hyaluronic acid and ionic-complementary self-assembling peptides such as RGD-EAbuK and EAbuK-IKVAV have been compared to Matrigel. Histological and fluorescent staining, electron microscopy (TEM) analyses and viability assays have been performed and RT-PCR has been used to detect extracellular matrix (ECM) components produced by cells. Results showed that Matrigel induced cells to form aggregates with lower viability and no ECM production compared to the novel scaffolds. Moreover, scaffolds allowed dispersed cells to produce a collagenous ECM containing collagen1a1, laminin B1 and elastin. The HA-EAbuK-IKVAV scaffold resulted in the most suitable 3D model in terms of cell quantity and viability. The development of this innovative approach is the first step towards the possibility to create 3D in vitro models for this protected species.

Protein retention in the endoplasmic reticulum rescues Aβ toxicity in Drosophila

Amyloid β (Aβ) accumulation is a hallmark of Alzheimer’s disease. In adult Drosophila brains, human Aβ overexpression harms climbing and lifespan. It’s uncertain whether Aβ is intrinsically toxic or activates downstream neurodegeneration pathways. Our study uncovers a novel protective role against Aβ toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high Aβ, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential Aβ resistance mechanism. Other laminin subunits and collagen IV also alleviate Aβ toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering Aβ secretion. LanB1’s rescue function operates independently of the IRE1α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates Aβ toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal Aβ toxicity, offering a new therapeutic avenue for Alzheimer’s disease.

Autoimmunity to synovial extracellular matrix proteins in patients with postinfectious Lyme arthritis.

BACKGROUNDAutoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive.METHODSUsing immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-mimic (Bb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins.RESULTSOf 24 postinfectious LA patients, 58% had CD4+ T cell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen Vα1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Bb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive CD4+ T cells in synovial fluid were T-bet-expressing Th1 cells, a small percentage were RoRγt-expressing Th17 cells, and a minimal percentage were FoxP3-expressing Tregs.CONCLUSIONAutoreactive, proinflammatory CD4+ T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Rather than the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity.FUNDINGSupported by National Institute of Allergy and Infectious Diseases R01-AI101175, R01-AI144365, and F32-AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01-AR062098 and T32-AR007258; NIH grants P41-GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital.

Abstract 071: Hypertension Rewrites The Kidney Transcriptome And Drives Nephropathy

Hypertensive nephropathy is a commonly diagnosed form of chronic kidney disease (CKD), but the biological processes underlying disease initiation are poorly understood. Here, we explore if non-malignant hypertension (HTN) is sufficient to initiate kidney damage and seek to identify pathways that drive disease development. We created a genetically defined hypertensive mouse by introducing activating mutations in the Ste20p-related proline alanine-rich kinase (SPAK) that are solely expressed in distal convoluted tubule cells. These constitutively active SPAK mice (CA-SPAK) develop salt-sensitive HTN due to hyperactivation of the thiazide-sensitive sodium chloride cotransporter (NCC). For these studies, CA-SPAK and control mice were fed either control salt (0.74% NaCl, CSD) or high salt diets (2% NaCl, HSD) for 24 weeks, and renal function and blood pressure were evaluated. RNASeq was performed to assess changes in the kidney transcriptional profile, the results of which were used for pathway enrichment analysis. CA-SPAK mice fed CSD develop HTN and show declining renal function starting at 26 weeks old and progresses over the next 10 weeks until they develop azotemia (BUN 30.8 vs 21.5 mg/dl; PCr - 0.42 vs 0.25 mg/dl, n=8) and albuminuria (5.2 vs 1.8 mg/day; n=8). HSD exacerbates HTN in CA-SPAK and initiates the same decline of renal function after only 8 weeks (20 weeks old). To identify changes in renal gene expression associated with the onset of function decline, we performed RNASeq on cortical tissue from both genotypes fed HSD for 8 weeks. We identified 3568 deferentially expressed genes in CA-SPAK. Pathway enrichment analysis found 3 biological processes associated with the early decline in CA-SPAK renal function that includes alterations of the filtration barrier (e.g. decreased nephrin, podocalyxin, synaptopodin, CD2AP, collagen 4a3, laminin B2), albuminuria associated genes (e.g. decreased cubilin, megalin, mucin 1), and immune system activation (e.g. increased MHC class II, T and B lymphocytes markers). Our findings from CA-SPAK mice demonstrate that non-malignant HTN can induce altered renal gene expression, the consequence of which is sufficient to disrupt kidney function resulting in the onset of hypertensive nephropathy.

Breast cancer cells grown on hyaluronic acid-based scaffolds as 3D in vitro model for electroporation

Nowadays, electroporation (EP) represents a promising method for the intracellular delivery of anticancer drugs. To setting up the process, the EP efficiency is usually evaluated by using cell suspension and adherent cell cultures that are not representative of the in vivo conditions. Indeed, cells are surrounded by extracellular matrix (ECM) whose composition and physical characteristics are different for each tissue. So, various three-dimensional (3D) in vitro models, such as spheroids and hydrogel-based cultures, have been proposed to mimic the tumour microenvironment.Herein, a 3D breast cancer in vitro model has been proposed. HCC1954 cells were seeded on crosslinked and lyophilized matrices composed of hyaluronic acid (HA) and ionic complementary self-assembling peptides (SAPs) already known to provide a fibrous structure mimicking collagen network. Herein, SAPs were functionalized with laminin derived IKVAV adhesion motif. Cultures were characterized by spheroids surrounded by ECM produced by cancer cells as demonstrated by collagen1a1 and laminin B1 transcripts. EP was carried out on both 2D and 3D cultures: a sequence of 8 voltage pulses at 5 kHz with different amplitude was applied using a plate electrode. Cell sensitivity to EP seemed to be modulated by the presence of ECM and the different cell organization. Indeed, cells cultured on HA-IKVAV were more sensitive than those treated in 2D and HA cultures, in terms of both cell membrane permeabilization and viability. Collectively, our results suggest that HA-IKVAV cultures may represent an interesting model for EP studies. Further studies will be needed to elucidate the influence of ECM composition on EP efficiency.

Serpent/dGATAb regulates Laminin B1 and Laminin B2 expression during Drosophila embryogenesis

Transcriptional regulation of Laminin expression during embryogenesis is a key step required for proper ECM assembly. We show, that in Drosophila the Laminin B1 and Laminin B2 genes share expression patterns in mesodermal cells as well as in endodermal and ectodermal gut primordia, yolk and amnioserosa. In the absence of the GATA transcription factor Serpent, the spatial extend of Laminin reporter gene expression was strongly limited, indicating that Laminin expression in many tissues depends on Serpent activity. We demonstrate a direct binding of Serpent to the intronic enhancers of Laminin B1 and Laminin B2. In addition, ectopically expressed Serpent activated enhancer elements of Laminin B1 and Laminin B2. Our results reveal Serpent as an important regulator of Laminin expression across tissues.

Expression of Laminin gene family in porcine pluripotent stem cells

Laminin (LN) proteins are important components of extracellular matrix. These proteins regulate cell proliferation, differentiation, migration, and tissue repair. The LN family has 12 genes that encode 5 α, 4 β, and 3 γ proteins. LamininA5 (LAMA5) as an important gene can support pluripotent cell growth and have been widely studied. However, porcine LAMA5 is absent in all tested porcine genomic databases so far. In this study, we confirmed for the first time the existence of porcine LAMA5 through bioinformatics analysis, and verified this result by cDNA cloning and sequencing. To reveal the expression pattern of Laminin gene family, we detected the expression of Laminin genes in porcine tissues, somatic cells, and porcine induced pluripotent stem cells (piPSCs). The results showed that an alternative splicing variant of Laminin B1 (LAMB1-a) was found exclusively in all tested piPSCs. The expression of this alternative splicing variant is positively correlated with the pluripotent state of piPSCs. The above findings provide evidences and foundations for the father use of LN as extracellular matrix to facilitate the derivation and culture of porcine pluripotent stem cells.

Nephrology Crossword: Genetics and glomerulopathies

ACROSS 3. Monoclonal antibody used in the treatment of atypical hemolytic uremic syndrome, a disorder caused by mutations in regulatory components of complement system. 5. Protein originally identified in lymphocytes but also critical in slit diaphragm structure and assembly; mutations in encoding gene result in proteinuric renal disease and foot process effacement (abbv). 7. Syndrome of isolated glomerular hematuria caused by mutations in type IV collagen (abbv). 9. First ion channel to be implicated in hereditary nephrotic syndrome and focal segmental glomerulosclerosis (FSGS); mutation results in increased calcium influx, which disrupts podocyte structure and function (abbv). 10. Gene critical for normal embryonic development of the urogenital system and kidneys; mutation results in nephrotic syndrome, urogenital anomalies, and predisposition to urogenital tumors (abbv). 11. Transmembrane protein critical to slit diaphragm structure and podocyte function; mutations in encoding gene cause congenital nephrotic syndrome of Finnish type. 15. Distinct clinical syndrome of hematuria, proteinuria, sensorineural deafness, and ocular abnormality caused by mutations in type 4 collagen family. 16. Gene encoding glomerular basement membrane (GBM) protein laminin b2; mutation results in clinical syndrome of ocular defects (microcoria), neurological abnormalities, and mesangial sclerosis with onset shortly after birth (abbv). 17. Gene identified to confer additional risk for FSGS and glomerulosclerosis from secondary causes in African Americans (abbv). DOWN 1. Mutation in encoded protein __ results in dysregulation of cross-linking of actin filaments in podocyte foot processes and presents as FSGS. 2. Mutation in the encoding gene results in __ enzyme deficiency and accumulation of glycol-sphingolipids, presents as renal failure in the third to sixth decade. 4. Gene critical in dorsoventral patterning of the vertebrae limb; mutations cause nail patella syndrome (abbv). 6. Recently identified mutation that results in hemolytic uremic syndrome without complement activation, suggesting alternate pathophysiological mechanisms1 (abbv). 8. __ deficiency causes abnormalities of lipid metabolism and proteinuric kidney disease with foamy appearance of GBM secondary to lipid deposition (abbv). 12. Hairpin-shaped protein of podocytes; mutation results in malfunction of slit diaphragm and steroid-resistant nephrotic syndrome in children. 13. Mutation in this gene results in defective podocyte development, diffuse mesangial sclerosis, and infantile nephrotic syndrome (abbv). 14. Mutation in this formin gene causes mislocalization of encoded protein, resulting in FSGS in early adolescence to adulthood (abbv). http://www.kidney-international.org m a k e y o u r d i a g n o s i s

Gene expression profiles of patients with cerebral hematoma following spontaneous intracerebral hemorrhage.

The present study aimed to investigate the gene functions and expression profiles in perihematomal (PH) brain regions following spontaneous intracerebral hemorrhage. The gene expression profiles were downloaded from the Gene Expression Omnibus database under accession number GSE24265, which includes 11 brain samples from different regions, including four samples from PH areas, four from contralateral grey matter (CG) and three from contralateral white matter (CW). The gene expression profiles were pre-processed and the differentially expressed genes (DEGs) between PH and CG tissue, and PH and CW tissue were identified using R packages. The expression of genes in different tissues was analyzed by hierarchical clustering. Then, the interaction network between the DEGs was constructed using String software. Finally, Gene Ontology was performed and pathway analysis was conducted using FuncAssociate and Expression Analysis Systematic Explorer to identify the gene function. As a result, 399 DEGs were obtained between PH and CG, and 756 DEGs were identified between PH and CW. There were 35 common DEGs between the two groups. These DEGs may be involved in PH edema by regulating the calcium signaling pathway [calcium channel, voltage-dependent, T-type, α1I subunit, Ca2+/calmodulin-dependent protein kinase II α (CAMK2A), ryanodine receptor 2 (RYR2) and inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1)], cell proliferation (sphingosine kinase 1), neuron differentiation (Ephrin-A5) or extracellular matrix-receptor interaction [collagen, type I, α 2, laminin B1 (LAMB1), syndecan 2, fibronectin 1 and integrin α5 (ITGA5)]. A number of genes may cooperate to participate in the same pathway, such as ITPR1-RYR2, CAMK2A-RYR2 and ITGA5-LAMB1 interaction pairs. The present study provides several potential targets to decrease hematoma expansion and alleviate neuronal cell death following spontaneous intracerebral hemorrhage.

Age and SPARC dependent cardiac collagen changes (1120.7)

Secreted protein acidic and rich in cysteine (SPARC), a collagen‐binding matricellular protein, has been implicated in procollagen processing. The aim of this study was to investigate age and SPARC dependent changes in cardiac extracellular matrix (ECM). We studied 6 groups of mice (n=4/group): young (4‐5 month old), middle‐aged (11‐12 m.o.), and old (18‐23 m.o.) C57BL/6J wild type and SPARC‐null mice. The left ventricle was decellularized in 1% SDS with protease inhibitors in PBS to enrich for ECM proteins. Protein extracts (10 μg) were separated by SDS‐PAGE, digested in‐gel and analyzed by HPLC‐ESI‐MS/MS. Relative quantification was by spectral counting (Scaffold). We identified 321 proteins, of which 27 proteins were ECM or ECM‐related proteins. Of these proteins, 16 showed age‐dependent changes. Collagens I, III, IV and VI, dystrophin, and laminin b2 increased with age, which may explain previously reported age‐associated increases in cardiac stiffness. SPARC deletion blunted the age changes observed in collagens I and III and laminin b2. Of interest, fibrillin and elastin showed a significant increase with age in the Null mice, suggestive of increased microfibril deposition in the absence of SPARC. In summary, our data suggest SPARC is a possible therapeutic target for aging induced cardiac dysfunction.Grant Funding Source: Supported by HHSN 268201000036C (N01‐HV‐00244), R01 HL075360, HL051971, 1SC2 HL101430, 5I01BX000505

Recombinant silk fibroin incorporated cell-adhesive sequences produced by transgenic silkworm as a possible candidate for use in vascular graft.

Interest in vascular grafts has recently grown because more patients are undergoing procedures that involve these grafts. However, smaller grafts with diameters <6 mm made from conventional biomaterials are associated with a high incidence of thrombosis, and therefore the development of improved materials suitable for small vascular grafts is highly desirable. In this paper, four kinds of recombinant Bombyx mori silk fibroins were prepared using transgenic techniques for use as silk vascular graft with a diameter of <6 mm. The peptide sequence TS(CDPGYIGSRAS)8 derived from the laminin B1 chain or the combination of two kinds of sequences, TS(CDPGYIGSRAS)8 and (TGRGDSPAS)8 derived from fibronectin, was incorporated into the light (L)-chain or the heavy (H)-chain of the silk fibroin. The fractions of the incorporated peptide sequences range from 0.8% to 7.2% by weight in the recombinant silk fibroins. This incorporation causes a very small increase in the random coil fraction of silk fibroin and a decrease in the tensile strength. Compared with native silk fibroin, the adhesive activities of mouse endothelial and smooth muscle cells increase significantly with the recombinant silk fibroin films incorporating only the TS(CDPGYIGSRAS)8 sequence independent of the L- or H-chains. A similar tendency was observed for the high migration activities of the endothelial cells in vitro and also the longer migration distance of the endothelial cell from the anastomotic part of rat abdominal aorta in vivo when this recombinant silk fibroin was used as a coating material for the silk graft. In view of the results, the recombinant silk fibroin incorporating the laminin peptide sequence can be potentially used as a vascular graft material.

Comprehensive Genetic Dissection of the Hemocyte Immune Response in the Malaria Mosquito Anopheles gambiae

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.

A Rab10-Dependent Mechanism for Polarized Basement Membrane Secretion during Organ Morphogenesis

Basement membranes (BMs) are specialized extracellular matrices that are essential for epithelial structure and morphogenesis. However, little is known about how BM proteins are delivered to the basal cell surface or how this process is regulated during development. Here, we identify a mechanism for polarized BM secretion in the Drosophila follicle cells. BM proteins are synthesized in a basal endoplasmic reticulum (ER) compartment from localized mRNAs and are then exported through Tango1-positive ER exit sites to basal Golgi clusters. Next, Crag targets Rab10 to structures in the basal cytoplasm, where it restricts protein delivery to the basal surface. These events occur during egg chamber elongation, a morphogenetic process that depends on follicle cell planar polarity and BM remodeling. Significantly, Tango1 and Rab10 are also planar polarized at the basal epithelial surface. We propose that the spatial control of BM production along two tissue axes promotes exocytic efficiency, BM remodeling, and organ morphogenesis.

PDGF enhances IRES-mediated translation of Laminin B1 by cytoplasmic accumulation of La during epithelial to mesenchymal transition

The extracellular matrix protein Laminin B1 (LamB1) regulates tumor cell migration and invasion. Carcinoma cells acquire invasive properties by epithelial to mesenchymal transition (EMT), which is a fundamental step in dissemination of metastatic cells from the primary tumor. Recently, we showed that enhanced translation of LamB1 upon EMT of malignant hepatocytes is mediated by an internal ribosome entry site (IRES). We demonstrated that the IRES transacting factor La binds the minimal IRES motif and positively modulates IRES activity of LamB1. Here, we show that platelet-derived growth factor (PDGF) enhances IRES activity of LamB1 by the increasing cytoplasmic localization of La during EMT. Accordingly, cells expressing dominant negative PDGF receptor display reduced cytoplasmic accumulation of La and show no elevation of IRES activity or endogenous LamB1 levels after stimulation with PDGF. Furthermore, La-mediated regulation of LamB1 IRES activity predominantly depends on MAPK/ERK signaling downstream of PDGF. Notably, LamB1 expression is not significantly downregulated by the impairment of the translation initiation factor eIF4E. In vivo, knockdown of La associated with decreased LamB1 expression and reduced tumor growth. Together, these data suggest that PDGF is required for the cytoplasmic accumulation of La that triggers IRES-dependent translation of LamB1 during EMT.

La enhances IRES-mediated translation of laminin B1 during malignant epithelial to mesenchymal transition

The majority of transcripts that harbor an internal ribosome entry site (IRES) are involved in cancer development via corresponding proteins. A crucial event in tumor progression referred to as epithelial to mesenchymal transition (EMT) allows carcinoma cells to acquire invasive properties. The translational activation of the extracellular matrix component laminin B1 (LamB1) during EMT has been recently reported suggesting an IRES-mediated mechanism. In this study, the IRES activity of LamB1 was determined by independent bicistronic reporter assays. Strong evidences exclude an impact of cryptic promoter or splice sites on IRES-driven translation of LamB1. Furthermore, no other LamB1 mRNA species arising from alternative transcription start sites or polyadenylation signals were detected that account for its translational control. Mapping of the LamB1 5′-untranslated region (UTR) revealed the minimal LamB1 IRES motif between −293 and −1 upstream of the start codon. Notably, RNA affinity purification showed that the La protein interacts with the LamB1 IRES. This interaction and its regulation during EMT were confirmed by ribonucleoprotein immunoprecipitation. In addition, La was able to positively modulate LamB1 IRES translation. In summary, these data indicate that the LamB1 IRES is activated by binding to La which leads to translational upregulation during hepatocellular EMT.

Laminin B1 expression during gonadogenesis in rat using immunohistochemical technique

Laminin B1 expression during gonadogenesis in rat using immunohistochemical technique

Peripheral Nerve Defect Repair With Epineural Tubes Supported With Bone Marrow Stromal Cells

This study was performed to assess the effects of nerve gap repair with isogenic epineural tubes filled with isogenic bone marrow stromal cells (BMSC) as an alternative to autograft repair. A total of 24 epineural tubes were transplanted as a conduit to bridge 20 mm nerve gap defects in 2 experimental groups. In group 1, the tube was filled with saline, whereas in group 2 with isogenic BMSC prestained with PKH-dye. In all, 12 autograft sciatic nerve repairs served as a control. Sensory and motor recovery was evaluated by gastrocnemius muscle index; pinprick, toe-spread tests; and somatosensory-evoked potentials at 6 and 12 weeks postrepair. Histomorphometry and immunostaining were also performed. Evidence of successful nerve regeneration was present in all animals at 6 and 12 weeks. There were no significant differences between experimental groups in functional recovery--toe-spread and pinprick tests; however, epineural conduit groups trended toward better toe-spread scores compared with autograft repair at 6 weeks. All animals had full sensory recovery as evaluated by pinprick at 12 weeks. Saline group had significantly higher gastrocnemius muscle index compared with other groups at 6 weeks; however, no differences were noted at 12 weeks. Histomorphometrical assessment did not reveal superiority of any group at 6 weeks postrepair. However, at 12 weeks, the BMSC group had significantly increased myelin thickness compared with other groups and larger diameter nerve fibers than autograft. In group 2, PKH-positive cells and expression of nerve growth factor, Laminin B2, glial fibrillary acidic protein, and vascular endothelial growth factor were confirmed. nerve growth factor- and glial fibrillary acidic protein-positive BMSC were also found inside the tube. The epineural tube is a viable, naturally occurring biologic conduit for nerve repair. Cotransplantation of BMSC-enhanced nerve regeneration by means of increased myelinization and expression of neurotrophic factors. Overall, obtained results with epineural tube/BMSC construct were comparable to autograft repair.

Proteomic Analysis of Human Small Cell Lung Cancer Tissues: Up-Regulation of Coactosin-Like Protein-1

Small cell lung cancer (SCLC) is the leading cause of cancer death, with a high propensity for aggressiveness and metastasis even in an early stage. Thus, identification of biomarkers as early diagnostics and treatment is needed. In this study, we investigated differentially regulated proteins between human SCLC tissues and normal bronchial epithelium by proteomic analysis using two-dimensional electrophoresis (2-DE) and MALDI-TOF mass spectrometry. Seven proteins and protein isoforms, including, γ-actin, tubulin α-1B, laminin B1, coactosin-like protein-1 (COTL-1), ubiquitin carboxyl-terminal esterase L1, ubiquitin-conjugating enzyme E2-25K, and carbonic anhydrase 1, were up-regulated more than 2 fold in SCLC tissues. In particular, up-regulated COTL-1 expression was validated by Western blot analysis, immunohistochemistry, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Moreover, most SCLC tissues (93%; 28/30) were COTL-1-positive in immunohistochemistry, whereas only 16% (10/64) of nonsmall cell lung cancer (NSLC) tissues were. Taken together, this SCLC proteomic data may help in establishing a human SCLC proteome database. COTL-1 may be a biomarker or a therapeutic target in SCLC patients.

Genetics of ulcerative colitis

Ulcerative colitis (UC) and Crohn's disease (CD) are related polygenic inflammatory bowel diseases (IBDs), with distinct and overlapping susceptibility loci. Recently, hypothesis-free genome-wide association (GWA) studies have revolutionized the field of complex disease genetics. Substantial advances have been achieved in defining the genetic architecture of IBD. To date, over 60 published IBD susceptibility loci have been discovered and replicated, of which approximately a third are associated with both UC and CD, although 21 are specific to UC and 23 to CD. In CD, the breakthrough identification of NOD2 as a susceptibility gene was followed by a rapid phase of gene discovery from GWA studies between 2006 and 2008. Progress in UC was slower; however, by initially testing hits for CD in UC, and later scanning larger UC cohorts, significant new loci for UC have been discovered, with exciting novel insights into disease pathogenesis. Notably, genes implicated in mucosal barrier function (ECM1, CDH1, HNF4α, and laminin B1) confer risk of UC; furthermore, E-cadherin is the first genetic correlation between colorectal cancer and UC. Impaired IL10 signaling has reemerged as a key pathway in intestinal inflammation, and is perhaps the most amenable to therapeutic intervention in UC. Collaborative international efforts with large meta-analyses of GWA studies and replication will yield many new UC genes. Furthermore, a large effort is required to characterize the loci found. Fine-mapping, deep resequencing, and functional studies will be critical to translating these gene discoveries into pathogenic insights, and ultimately into clinical insights and novel therapeutics.

Effect of a novel botanical agent Drynol Cibotin on human osteoblast cells and implications for osteoporosis: promotion of cell growth, calcium uptake and collagen production

Osteoporosis is a widespread problem afflicting millions of people. Drynol Cibotinis is a newly developed proprietary botanical combination of eight botanicals including Angelica sinensis, Glycine max, Wild yam, Ligustrum lucidum, Astragalus membranaceus, Cuscuta chinensis, Psoraleae corylifoliae, and Drynaria fortune. Each of the botanicals has been used in traditional Chinese medicine to treat osteoporosis. The effect of Drynol Cibotinis, with the specific combination of these anti-osteoporosis botanicals for promoting bone growth, was examined in this study. The effects of Drynol Cibotin on cell growth, apoptosis, cell spreading, calcium uptake and production of bone matrix proteins Collagen I and Laminin B2 on human osteoblast cells were assessed by BrdU incorporation, TUNEL assay, cell staining, intracellular Ca2+ measurement and Western blot analysis. The results showed that Drynol Cibotin significantly increased cell proliferation and inhibited apoptosis in osteoblasts (P < 0.01). In addition, Drynol Cibotin was found to promote cell spreading and greatly increase calcium uptake both instantaneously and in the long term (P < 0.01). Furthermore, Drynol Cibotin significantly increased production of two key extracellular matrix proteins in bone cells: Collagen I and Laminin B2. These results indicate that Drynol Cibotin alone or in combination with amino acids and vitamins may have prophylactic potentials in osteoporosis.