Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The use of trehalose-treated freeze-dried amniotic membrane for ocular surface reconstruction

The aim of this study was to evaluate the efficacy and safety of trehalose-treated freeze-dried amniotic membrane (TT-FDAM) for ocular surface reconstruction. Human AM deprived of amniotic epithelial cells was first incubated with 10% trehalose solution, and then freeze-dried, vacuum-packed, and sterilized with gamma-irradiation. The resultant newly developed TT-FDAM was characterized for its physical, biological, and morphological properties by comprehensive physical assays, immunohistochemistry, electron microscopy, cell adhesion assay, 3D cell culture, and an in vivo biocompatibility test. The adaptability of TT-FDAM was markedly improved as compared to FDAM. Immunohistochemistry for several extracellular matrix molecules revealed that the process of freeze-drying and irradiation apparently did not affect its biological properties, however, electron microscopy revealed that the detailed morphological appearance of TT-FDAM is more similar to that of native AM than to FDAM, Intracorneal and scleral-surface transplantation of TT-FDAM showed excellent biocompatibility with ocular surface tissues. Thus, TT-FDAM retained most of the physical, biological, and morphological characteristics of native AM, consequently it is a useful biomaterial for ocular surface reconstruction. (C) 2008 Published by Elsevier Ltd

Novel sutureless transplantation of bioadhesive-coated, freeze-dried amniotic membrane for ocular surface reconstruction. Invest Ophthalmol Vis Sci 48

PURPOSE. To evaluate the efficacy and safety of a novel sutureless transplantation of bioadhesive-coated, sterilized, freezedried amniotic membrane (FD-AM) for ocular surface reconstruction. METHODS. A bioadhesive-coated, freeze-dried amniotic membrane was made by freeze drying the denuded AM in a vacuum, applying the minimum amount of fibrin glue (mixture of fibrinogen and thrombin) necessary to retain adhesion on the chorionic side, and sterilizing it by ␥-radiation. The resultant AM was characterized for its biological and morphologic properties by immunohistochemical and electron microscopic examination. In addition, fibrin glue-coated, freeze-dried (FCFD) AM was transplanted onto a rabbit scleral surface without sutures, to examine its biocompatibility. RESULTS. Immunohistochemistry of the FCFD-AM revealed that fibrinogen existed on its chorionic side, and the process of applying fibrin glue did not affect its biological and morphologic properties. Moreover, electron microscopic examination of the chorionic side of the FCFD-AM revealed tiny microfibrils (which are probably fibrinogen protofibrils), and showed that the epithelial surface of FCFD-AM consisted of intact basal lamina similar to that of FD-AM. FCFD-AM transplantation was very easily performed, and the graft adhered to the bare sclera immediately. Though the fibrinogen naturally biodegraded within 2 weeks, the FCFD-AM remained for at least 12 weeks after transplantation. Epithelialization on the FCFD-AM was achieved within 2 weeks, as was the case with FD-AM transplantation. The conjunctival epithelium on the FCFD-AM was well stratified and not keratinized, suggesting that FCFD-AM supports normal cell differentiation. CONCLUSIONS. The FCFD-AM retained most of the biological characteristics of FD-AM. Consequently, this sutureless method of transplantation of FCFD-AM is safe, simple, and useful for ocular surface reconstruction. (Invest Ophthalmol Vis Sci

Novel sutureless transplantation freeze-dried amniotic membrane surface reconstruction

PURPOSE. To evaluate the efficacy and safety of a novel sutureless transplantation of bioadhesive-coated, sterilized, freeze-dried amniotic membrane (FD-AM) for ocular surface reconstruction. METHODS. A bioadhesive-coated, freeze-dried amniotic membrane was made by freeze drying the denuded AM in a vacuum, applying the minimum amount of fibrin glue (mixture of fibrinogen and thrombin) necessary to retain adhesion on the chorionic side, and sterilizing it by gamma-radiation. The resultant AM was characterized for its biological and morphologic properties by immunohistochemical and electron microscopic examination. In addition, fibrin glue-coated, freeze-dried (FCFD) AM was transplanted onto a rabbit scleral surface without sutures, to examine its biocompatibility. RESULTS. Immunohistochemistry of the FCFD-AM revealed that fibrinogen existed on its chorionic side, and the process of applying fibrin glue did not affect its biological and morphologic properties. Moreover, electron microscopic examination of the chorionic side of the FCFD-AM revealed tiny microfibrils (which are probably fibrinogen protofibrils), and showed that the epithelial surface of FCFD-AM consisted of intact basal lamina similar to that of FDAM. FCFD-AM transplantation was very easily performed, and the graft adhered to the bare sclera immediately. Though the fibrinogen naturally biodegraded within 2 weeks, the FCFD-AM remained for at least 12 weeks after transplantation. Epithelialization on the FCFD-AM was achieved within 2 weeks, as was the case with FD-AM transplantation. The conjunctival epithelium on the FCFD-AM was well stratified and not keratinized, suggesting that FCFD-AM supports normal cell differentiation. CONCLUSIONS. The FCFD-AM retained most of the biological characteristics of FDAM. Consequently, this sutureless method of transplantation of FCFD-AM is safe, simple, and useful for ocular surface reconstruction

Sutureless amniotic membrane transplantation for ocular surface reconstruction with a chemically defined bioadhesive

The purpose of this study was to evaluate the efficiency and safety of a sutureless amniotic membrane transplantation (AMT) for ocular surface reconstruction with a chemically defined bioadhesive (CDB). The CDB was synthesized from aldehyded polysaccharides and epsilon-poly(L-lysine), two kinds of medical and food additives, as starting materials. Biocompatibility assay indicated that the CDB showed excellent biocompatibility with in vitro and in vivo ocular surface tissues and most of the CDB was histologically degraded within 4 weeks. Sutureless AMT using the CDB was safely and successfully performed onto a rabbit scleral surface. Transplanted amniotic membrane (AM) evaluated by histological, electron microscopic- and immunohistochemical examination indicated that the CDB did not affect normal differentiation of the cells or the integrity of the surrounding tissue. Thus, this newly developed CDB was found to be very useful for sutureless AMT for ocular surface reconstruction, without considering the risk of infection. It has the ability to fix AM to the ocular surface for a long time-period without additional inflammation, scarring, or damage to the surrounding tissues. (C) 2008 Elsevier Ltd. All rights reserved

Novel Sutureless Keratoplasty with a Chemically Defined Bioadhesive

PURPOSE. The purpose of this study was to evaluate sutureless keratoplasty using a chemically-defined bioadhesive (CDB) made from food or medical additives. METHODS. Sutureless automated lamellar therapeutic keratoplasty (ALTK) using a CDB was performed on three rabbit eyes. Allogenic lamellar graft was transplanted onto the recipient bed using either suture fixation or a sutureless technique using the CDB. Slit-lamp examination was performed at selected intervals to evaluate the grade of epithelialization and the corneal clarity. The rabbits were killed at 90 days after operation and the eyes processed for histology, electron microscopic examination, and immunohistochemistry for cytokeratins and cell junction-related proteins. RESULTS. Sutureless keratoplasty was successfully performed with appropriate handling time before the CDB gelatinized. All the glued grafts were rapidly epithelialized within 7 days, and thereafter remained clear and attached for 90 days. Histologic and ultrastructural findings on the sutureless group showed the normal feature of stromal and epithelial cells and the grafts to be closely adhered with no inflammatory or scarring changes on the interface. Immunohistochemistry of the epithelial cells on the sutureless group revealed a similar expression pattern to the control group. CONCLUSIONS. These results demonstrate that sutureless keratoplasty using the CDB is easy to perform, with reliable attachment and no fear of toxic effects or disease transmissions. The authors expect the CDB to become a major choice for corneal treatment, especially in lamellar keratoplasty, posterior keratoplasty, and amniotic membrane transplantation on corneas. (Invest Ophthalmol Vis Sci. 2009; 50: 2679-2685) DOI:10.1167/iovs.08-294

LRIG1 inhibits STAT3-dependent inflammation to maintain corneal homeostasis.

Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis

Tumor-Associated Calcium Signal Transducer 2 Is Required for the Proper Subcellular Localization of Claudin 1 and 7 : Implications in the Pathogenesis of Gelatinous Drop-Like Corneal Dystrophy

Gelatinous drop-like dystrophy (GDLD) is a rare autosomal recessive form of corneal dystrophy characterized by subepithelial amyloid depositions on the cornea. Previous clinical and laboratory observations have strongly suggested that epithelial barrier function is significantly decreased in GDLD. Despite the decade-old identification of the tumor-associated calcium signal transducer 2 (TACSTD2) gene as a causative gene for GDLD, the mechanism by which the loss of function of this causative gene leads to the pathological consequence of this disease remains unknown. In this study, we investigated the functional relationship between the TACSTD2 gene and epithelial barrier function. Through the use of immunoprecipitation and a proximity ligation assay, we obtained evidence that the TACSTD2 protein directly binds to claudin 1 and 7 proteins. In addition, the loss of function of the TACSTD2 gene leads to decreased expression and change in the subcellular localization of tight junction-related proteins, including claudin 1, 4, 7, and ZO1 and occludin, both in diseased cornea and cultured corneal epithelial cells. These results indicate that loss of function of the TACSTD2 gene impairs epithelial barrier function through decreased expression and altered subcellular localization of tight junction-related proteins in GDLD corneas