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

Erratum: Corrigendum: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

International Chicken Genome Sequencing Consortium. The Original Article was published on 09 December 2004. Nature432, 695–716 (2004). In Table 5 of this Article, the last four values listed in the ‘Copy number’ column were incorrect. These should be: LTR elements, 30,000; DNA transposons, 20,000; simple repeats, 140,000; and satellites, 4,000. These errors do not affect any of the conclusions in our paper. Additional information. The online version of the original article can be found at 10.1038/nature0315

Tissue Engineering Strategies for the Treatment of Peripheral Vascular Diseases

Peripheral vascular diseases such as peripheral artery disease (PAD) and critical limb ischemia (CLI) are growing at an ever-increasing rate in the Western world due to an aging population and the incidence of type II diabetes. A growing economic burden continues because these diseases are common indicators of future heart attack or stroke. Common therapies are generally limited to pharmacologic agents or endovascular therapies which have had mixed results still ending in necrosis or limb loss. Therapeutic angiogenic strategies have become welcome options for patients suffering from PAD due to the restoration of blood flow in the extremities. Capillary sprouting and a return to normoxic tissue states are also demonstrated by the use of angiogenic cytokines in conjunction with bone marrow cell populations. To this point, it has been determined that spatial and temporal controlled release of growth factors from vehicles provides a greater therapeutic and angiogenic effect than growth factors delivered intramuscularly, intravenously, or intraarterialy due to rapid metabolization of the cytokine, and non-targeted release. Furthermore, bone marrow cells have been implicated to enhance angiogenesis in numerous ischemic diseases due to their ability to secrete angiogenic cytokines and their numerous cell fractions present which are implicated to promote mature vessel formation. Use of angiogenic peptides, in conjunction with bone marrow cells, has been hypothesized in EPC mobilization from the periphery and marrow tissues to facilitate neovessel formation. For this purpose, controlled release of angiogenic peptides basic fibroblast growth factor (FGF-2) and granulocyte-colony stimulating factor (G-CSF) was performed using tunable ionic gelatin hydrogels or fibrin scaffolds with ionic albumin microspheres. The proliferation of endothelial cell culture was determined to have an enhanced effect based on altering concentrations of growth factors and method of release: co-delivery versus sequential. Scaffolds with these angiogenic peptides were implanted in young balb/c mice that underwent unilateral hindlimb ischemia by ligation and excision of the femoral artery. Endpoints for hindlimb reperfusion and angiogenesis were determined by Laser Doppler Perfusion Imaging and immunohistochemical staining for capillaries (CD-31) and smooth muscle cells (alpha-SMA). In addition to controlled release of angiogenic peptides, further studies combined the use of a fibrin co-delivery scaffold with FGF-2 and G-CSF with bone marrow stem cell transplantation to enhance vessel formation following CLI. Endpoints also included lipophilic vascular painting to evaluate the extent of angiogenesis and arteriogenesis in an ischemic hindlimb. Tissue engineering strategies utilizing bone marrow cells and angiogenic peptides demonstrate improved hindlimb blood flow compared to BM cells or cytokines alone, as well as enhanced angiogenesis based on immunohistochemical staining and vessel densities.</p

HoxA5 is expressed in EC in TRE-HoxA5/TIE2-tTA mice.

<p>(<b>A</b>) Schematic of the TRE-HoxA5-TIE2-tTA system for inducible and restricted expression of Hox A5 in ECs in FvBn mice. When the double transgenic mice are maintained on a Dox diet the activator cannot bind the TRE promoter. However in the absence of Dox, tTA binds the TRE activating transcription and HoxA5 expression. Expression of the transgene is restricted to EC by driving tTA using the TIE-2 promoter enhancer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121720#pone.0121720.ref012" target="_blank">12</a>]. (<b>B</b>) Real Time PCR analysis of HoxA5 transgene mRNA expression in mouse liver at various times following withdrawal of Dox from the diet in control TIE-2-tTA (tTA) and TRE-HoxA5-TIE2-tTA (HoxA5-tTA) mice. Results are expressed relative to the housekeeping gene GUSB (n = 3). (<b>C</b>) HoxA5 expression levels in ECs isolated from lungs of tTA and HoxA5-tTA mice. Histogram shows HoxA5 mRNA levels measured by real time PCR in the presence of Dox (1μg/ml) or 48 hours following removal of Dox in the cell culture media. Insert shows corresponding Western blot of protein lysates extracted from lung EC isolated from tTA and HoxA5-TA mice and detected via polyclonal antibodies against HoxA5. (<b>D</b>) Real time PCR analysis of relative mRNA expression levels of thrombospndin-2 (TSP-2) and VEGF-A levels one month after removal of Dox from the diet of HoxA5-tTA mice. Results are expressed relative to mRNA levels in age-matched tTA control mice lacking the HoxA5 transgene or Dox in the diet. (<b>E</b>) Vascular permeability in tTA or HoxA5-tTA mice. Measurement of extravasated Evans Blue dye 30 minutes following topical application of mineral oil (control, left panel) or Mustard oil to induce an acute leakage (right panel; n = 4).</p

<i>De novo</i> neoplastic progression is delayed with sustained expression of HoxA5 in EC.

<p>(<b>A</b>) Real time PCR analysis of Thrombospondin-2 (TSP-2) and VEGF-A mRNA levels in ear tissue harvested from 5 month old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice (n = 5). All mice were given Dox (+Dox) for 3 weeks during gestation, and subsequently removed from Dox (-Dox) for the remainder of the study. (<b>B</b>) Vascular density analysis of CD31+ vessels following staining of ear tissue harvested from 5 month old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice (p<0.05; n = 5). (<b>C</b>) Photomicrographs of confocal images of ear tissues harvested from HPV16 or HPV16/HoxA5 mice. Mice were perfused with endothelial binding FITC-labeled <i>lycospersicon esculentum</i> (FITC lectin) and tissues stained for anti-smooth <i>actin</i> (SM actin). Upper panel shows images of SM actin coverage of vessels and lower panels show merged images of FITC lectin in ECs and associated SM coverage. (<b>D</b>) Vascular permeability in HPV16 or HPV16/HoxA5 mice. Measurement of extravasated Evans blue dye performed 30 minutes following treatment with mineral oil (p<0.05; n = 4). (<b>E</b>) Quantitation of mast cell infiltration assessed by toluidine blue staining in ear tissue harvested from 5-month-old control tTA (-LM), K14-HPV16 (HPV16) or K14-HPV16/HoxA5-tTA (HPV16/HoxA5) mice. (<b>F</b>) Micrographs showing H&E staining of 5 μm-paraffin sections of 5 month old HPV16 (upper panels) and HPV16/HoxA5 (lower panels) mice. Mice exhibited mild hyperplasia, moderate hyperplasia and dysplasia as measured by epidermal thickness (hyperpasia) or invasion of granulation tissue or immature cell types (dysplasia) in different proportions according to the genotype. (<b>G</b>) Quantitative analysis of mild hyperplasia (white), hyperplasia (grey) and dysplasia (black) in 5-month-old HPV16 and HPV16/HoxA5 mice. HPV16/HoxA5 mice exhibited a significantly (p<0.01) higher proportion of mild hyperplasia, and a significantly (** p<0.05) reduced incidence of dysplasia as compared to age-matched HPV16 mice (n = 8). (<b>H</b>) Quantitation of Ki-67 positive cells in ear tissue harvested from 5-month-old HPV16 or HPV16/HoxA5 mice where the transgene had been constitutively active since one month of age (n = 3; ** p<0.05).</p

HoxA5 expression in EC inhibits angiogenesis and growth of allograph mammary tumors.

<p>(<b>A</b>) Tumor volume in tTA (square) and HoxA5-tTA (triangle) 8 week old mice (3 weeks + Dox, 5 weeks—Dox), 32 days following subcutaneous injection of MMTV-PyMT tumor cells into syngeneic female FVB/n mice. The analysis revealed a significant reduction (p<0.01) in volume of tumors grown in HoxA5-tTA mice on days 25 through 32 (n = 5). (<b>B</b>) Micrograph showing representative tumors obtained 32 days after implantation of tumor cells into tTA (left) and HoxA5-tTA (right) mice. (<b>C</b>) Quantitative analysis of tumor weight 32 days after implantation into tTA or HoxA5-tTA mice. HoxA5-tTA mice showed a significant reduction (p<0.05) in tumor weight as compared to tTA mice (n = 5). (<b>D</b>) Immunofluoresence analysis of vascular density in tumors in tTA (left panel) or HoxA5-tTA (right panel). Vascular density was assessed by CD31+ staining of frozen OCT-embedded tissue sections of peri-tumor tissue from each animal. (<b>E</b>) Quantitative analysis of CD31+ vessels in tumors isolated at day 32 from HoxA5-tTA mice compared to those from tTA mice. HoxA5-tTA mice showed a significant reduction (P<0.01) in the number of vessels (n = 5).</p

Synergistic Angiogenic Effect of Codelivering Fibroblast Growth Factor 2 and Granulocyte-Colony Stimulating Factor from Fibrin Scaffolds and Bone Marrow Transplantation in Critical Limb Ischemia

Increasing evidence suggests that therapeutic angiogenesis strategies utilizing cytokines and stem cells are necessary to treat traumatic vascular events such as critical limb ischemia and peripheral artery disease. In this study, basic fibroblast growth factor 2 (FGF-2) and granulocyte-colony stimulating factor (G-CSF) were immobilized in fibrin matrices and codelivered in combination with unfractionated bone marrow cells. Hindlimb ischemia was induced on young (6–7 weeks) Balb/C mice, and fibrin gels containing 100 ng/mL of FGF-2 and G-CSF were implanted adjacent to the ligation points. In addition, 1 × 10 6 bone marrow (BM) cells were injected into five locations in the ischemic muscle immediately after ligation and artery excision. Hindlimb reperfusion was determined by Laser Doppler Perfusion Imaging and immunohistochemistry for CD31+ and smooth muscle actin-positive cells at 2, 4, and 8 weeks postsurgery to identify capillary formation and maturation. A fluorescent vessel painting technique was also utilized to determine the extent of angiogenesis and arteriogenesis in the hindlimb at 8 weeks postsurgery. The codelivery of FGF-2 and G-CSF in combination with BM cells led to enhanced therapeutic recovery in critical limb ischemia Balb/C mice after 8 weeks of treatment with 87.2% blood flow recovery and a significant increase ( p  < 0.05) in capillary formation in comparison to growth factor delivery or BM cell administration alone

Enhanced angiogenic efficacy through controlled and sustained delivery of FGF-2 and G-CSF from fibrin hydrogels containing ionic-albumin microspheres

Neo-vessel formation in ischemic tissues relies on numerous growth factors and cell fractions for the formation of mature, stable, functional vasculature. However, the efforts to regenerate tissues typically rely on the administration of a single growth factor or cells alone. Conversely, polymeric matrices have been investigated extensively to deliver multiple growth factors at pre-determined rates to form stable blood vessels in ischemic tissues. We report on a novel sequential delivery system of a fibrin hydrogel containing ionic-albumin microspheres that allows for the controlled release of two growth factors. The use of this system was investigated in the context of therapeutic angiogenesis. Material properties were determined based on degree of swelling measurements and degradation characteristics. Release kinetics of model angiogenic polypeptides FGF-2 and G-CSF were determined using ELISA and the bioactivity of released protein was evaluated in human endothelial cell cultures. The release of growth factors from ionic-albumin microspheres was significantly delayed compared to the growth factor released from fibrin matrices in the absence of spheres. The scaffolds were implanted in a murine critical limb ischemia model at two concentrations, 40 ng (low) and 400 ng (high), restoring 92% of the blood flow in a normally perfused limb using a fibrin hydrogel releasing FGF-2 containing albumin-PLL microspheres releasing G-CSF (measured by LDPI at the high concentration), a 3.2-fold increase compared to untreated limbs. The extent of neo-vessel formation was delineated by immunohistochemical staining for capillary density (CD-31+) and mature vessel formation (α-SMA+). In conclusion, our study demonstrated that the release kinetics from our scaffold have distinct kinetics previously unpublished and the delivery of these factors resulted in hindlimb reperfusion, and robust capillary and mature vessel formation after 8 weeks compared to either growth factor alone or bolus administration of growth factor