Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

The trishanku gene and terminal morphogenesis in Dictyostelium discoideum

P>Multicellular development in the social amoeba Dictyostelium discoideum is triggered by starvation. It involves a series of morphogenetic movements, among them being the rising of the spore mass to the tip of the stalk. The process requires precise coordination between two distinct cell types-presumptive (pre-) spore cells and presumptive (pre-) stalk cells. Trishanku (triA) is a gene expressed in prespore cells that is required for normal morphogenesis. The triA- mutant shows pleiotropic effects that include an inability of the spore mass to go all the way to the top. We have examined the cellular behavior required for the normal ascent of the spore mass. Grafting and mixing experiments carried out with tissue fragments and cells show that the upper cup, a tissue that derives from prestalk cells and anterior-like cells (ALCs), does not develop properly in a triA- background. A mutant upper cup is unable to lift the spore mass to the top of the fruiting body, likely due to defective intercellular adhesion. If wild-type upper cup function is provided by prestalk and ALCs, trishanku spores ascend all the way. Conversely, Ax2 spores fail to do so in chimeras in which the upper cup is largely made up of mutant cells. Besides proving that under these conditions the wild-type phenotype of the upper cup is necessary and sufficient for terminal morphogenesis in D. discoideum, this study provides novel insights into developmental and evolutionary aspects of morphogenesis in general. Genes that are active exclusively in one cell type can elicit behavior in a second cell type that enhances the reproductive fitness of the first cell type, thereby showing that morphogenesis is a cooperative process.

Elevated circulating sclerostin concentrations in individuals with high bone mass, with and without LRP5 mutations

CONTEXT: The role and importance of circulating sclerostin is poorly understood. High bone mass (HBM) caused by activating LRP5 mutations has been reported to be associated with increased plasma sclerostin concentrations; whether the same applies to HBM due to other causes is unknown. OBJECTIVE: Our objective was to determine circulating sclerostin concentrations in HBM. DESIGN AND PARTICIPANTS: In this case-control study, 406 HBM index cases were identified by screening dual-energy x-ray absorptiometry (DXA) databases from 4 United Kingdom centers (n = 219 088), excluding significant osteoarthritis/artifact. Controls comprised unaffected relatives and spouses. MAIN MEASURES: Plasma sclerostin; lumbar spine L1, total hip, and total body DXA; and radial and tibial peripheral quantitative computed tomography (subgroup only) were evaluated. RESULTS: Sclerostin concentrations were significantly higher in both LRP5 HBM and non-LRP5 HBM cases compared with controls: mean (SD) 130.1 (61.7) and 88.0 (39.3) vs 66.4 (32.3) pmol/L (both P < .001, which persisted after adjustment for a priori confounders). In combined adjusted analyses of cases and controls, sclerostin concentrations were positively related to all bone parameters found to be increased in HBM cases (ie, L1, total hip, and total body DXA bone mineral density and radial/tibial cortical area, cortical bone mineral density, and trabecular density). Although these relationships were broadly equivalent in HBM cases and controls, there was some evidence that associations between sclerostin and trabecular phenotypes were stronger in HBM cases, particularly for radial trabecular density (interaction P < .01). CONCLUSIONS: Circulating plasma sclerostin concentrations are increased in both LRP5 and non-LRP5 HBM compared with controls. In addition to the general positive relationship between sclerostin and DXA/peripheral quantitative computed tomography parameters, genetic factors predisposing to HBM may contribute to increased sclerostin levels.

Multistage genome-wide association meta-analyses identified two new loci for bone mineral density

Aiming to identify novel genetic variants and to confirm previously identified genetic variants associated with bone mineral density (BMD), we conducted a three-stage genome-wide association (GWA) meta-analysis in 27 061 study subjects. Stage 1 meta-analyzed seven GWA samples and 11 140 subjects for BMDs at the lumbar spine, hip and femoral neck, followed by a Stage 2 in silico replication of 33 SNPs in 9258 subjects, and by a Stage 3 de novo validation of three SNPs in 6663 subjects. Combining evidence from all the stages, we have identified two novel loci that have not been reported previously at the genome-wide significance (GWS; 5.0 × 10-8) level: 14q24.2 (rs227425, P-value 3.98 × 10-13, SMOC1) in the combined sample of males and females and 21q22.13 (rs170183, P-value 4.15 × 10-9, CLDN14) in the female-specific sample. The two newly identified SNPs were also significant in the GEnetic Factors for OSteoporosis consortium (GEFOS, n 5 32 960) summary results. We have also independently confirmed 13 previously reported loci at the GWS level: 1p36.12 (ZBTB40), 1p31.3 (GPR177), 4p16.3 (FGFRL1), 4q22.1 (MEPE), 5q14.3 (MEF2C), 6q25.1 (C6orf97, ESR1), 7q21.3 (FLJ42280, SHFM1), 7q31.31 (FAM3C, WNT16), 8q24.12 (TNFRSF11B), 11p15.3 (SOX6), 11q13.4 (LRP5), 13q14.11 (AKAP11) and 16q24 (FOXL1). Gene expression analysis in osteogenic cells implied potential functional association of the two candidate genes (SMOC1 and CLDN14) in bone metabolism. Our findings independently confirm previously identified biological pathways underlying bone metabolism and contribute to the discovery of novel pathways, thus providing valuable insights into the intervention and treatment of osteoporosis. © The Author 2013. Published by Oxford University Press.

Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.

Sosiaalityön ammattilaiset refleksiivisinä toimijoina : tutkimus erikoistumiskoulutuksesta Margaret Archerin teorian valossa

In Finland, specialized studies in social work (professional licentiate education) were begun in the year 2000. The education is targeted at experienced social workers and leads to a licentiate degree (a degree between master s and doctorate). In this study, the experiences of members of the first study cohort, specializing in social work with children and young people, are examined. The study s theoretical frame of reference is based on the morphogenetic approach, developed by British sociologist Margaret Archer. In it, the potential powers of both an agent as well as social and cultural structures are considered important and worth taking into account. Archer sees reflexivity, a person s ability to analyze herself/himself, as an essential starting point for agency. Thanks to reflexivity, people are able to engage in internal conversations , discuss the concerns that are important to them and form agential projects . In Archer s theory, the social structures and traits of the cultural system are seen as having potential power in relation to people s agential projects; these powers can enable but also restrain the realization of the projects. On the other hand, individuals can try to review the factors affecting their agential projects and find ways of action that facilitate them. The research task is to study the self-understanding of social work professionals in the 21st century, the issues and goals professionally important for them, as well as the contexts framing the realization of these goals. The research questions are as follows: 1) What kind of internal conversations, concerns and agential projects related to their work did the professionals taking part in licentiate education bring to light? 2) What kind of enabling and restraining factors can be identified in their situations? And 3) What kind of social structures and traits related to the cultural system are connected to these factors? The research material was collected by interviewing the students in different phases of their education. In 2001 and 2004 all members of the study group (n = 25) were interviewed. In 2007, 13 students took part. The themes of the internal conversations brought to light in the interviews were divided into four broad thematic categories: professional development, the position of children in social work, multiprofessional work and structural social work. In relation to these themes the students formed different kinds of agential projects. In addition, the study reveals several cultural and social structures that have enabled but also restrained the realization of the agential projects. These structures are linked, for example, to the relations between employees and employers, students and teachers, children and adults as well as between the representatives of different professions. Working conditions which social workers often consider weak are discussed as a focal issue related to many themes. These working conditions become evident, for example, in the great imbalance which exists between the professional tasks and the amount of time that social workers have for them. Difficult situations arise when social workers feel they cannot reach the goals that are professionally important to them because of the strict external conditions of the work.

Molecular background of three lethal fetal syndromes

This study identified the molecular defects underlying three lethal fetal syndromes. Lethal Congenital Contracture Syndrome 1 (LCCS1, MIM 253310) and Lethal Arthrogryposis with Anterior Horn Cell Disease (LAAHD, MIM 611890) are fetal motor neuron diseases. They affect the nerve cells that control voluntary muscle movement, and eventually result in severe atrophy of spinal cord motor neurons and fetal immobility. Both LCCS1 and LAAHD are caused by mutations in the GLE1 gene, which encodes for a multifunctional protein involved in posttranscriptional mRNA processing. LCCS2 and LCCS3, two syndromes that are clinically similar to LCCS1, are caused by defective proteins involved in the synthesis of inositol hexakisphosphate (IP6), an essential cofactor of GLE1. This suggests a common mechanism behind these fetal motor neuron diseases, and along with accumulating evidence from genetic studies of more late-onset motor neuron diseases such as Spinal muscular atrophy (SMA) and Amyotrophic lateral sclerosis (ALS), implicates mRNA processing as a common mechanism in motor neuron disease pathogenesis. We also studied gle1-/- zebrafish in order to investigate whether they would be a good model for studying the pathogenesis of LCCS1 and LAAHD. Mutant zebrafish exhibit cell death in their central nervous system at two days post fertilization, and the distribution of mRNA within the cells of mutant zebrafish differs from controls, encouraging further studies. The third lethal fetal syndrome is described in this study for the first time. Cocoon syndrome (MIM 613630) was discovered in a Finnish family with two affected individuals. Its hallmarks are the encasement of the limbs under the skin, and severe craniofacial abnormalities, including the lack of skull bones. We showed that Cocoon syndrome is caused by a mutation in the gene encoding the conserved helix-loop-helix ubiquitous kinase CHUK, also known as IκB kinase α (IKKα). The mutation results in the complete lack of CHUK protein expression. CHUK is a subunit of the IκB kinase enzyme that inhibits NF-κB transcription factors, but in addition, it has an essential, independent role in controlling keratinocyte differentiation, as well as informing morphogenetic events such as limb and skeletal patterning. CHUK also acts as a tumor suppressor, and is frequently inactivated in cancer. This study has brought significant new information about the molecular background of these three lethal fetal syndromes, as well as provided knowledge about the prerequisites of normal human development.

Control of morphogenesis in tobacco protoplast cultures: organogenesis vs embryogenesis

The morphogenetic pathway leading to plant differentiation in tobacco mesophyll protoplasts could be regulated. The course of development via organogenesis or embryogenesis was controlled by manipulating nutrient media, culture conditions and hormone requirements. A lowering of molarity of medium after 5 weeks of protoplast culture, inclusion of GA3 (0.5 mg/l) in the medium for first 8 weeks of culture and exclusion of reduced nitrogen in the medium resulted in shoot organogenesis, while maintenance of higher molarity of the medium till 8 weeks, reduced nitrogen in the medium and removal of 2, 4-D after 5 weeks of culture induced embryogenesis. Regenerability of viable plants was obtained by both developmental pathways. The implications of tobacco embryogenesis system in plant molecular genetics were highlighted.

GATA Factors Regulate Inner Ear Development and Midbrain Neurogenesis

The zinc-finger transcription factors GATA2 and GATA3 in vertebrates belong to the six-member family that are essential regulators in the development of various organs. The aim of this study was to gain new information of the roles of GATA2 and GATA3 in inner ear morphogenesis and of the function of GATA2 in neuronal fate specification in the midbrain using genetically modified mouse and chicken embryos as models. A century ago the stepwise process of inner ear epithelial morphogenesis was described, but the molecular players regulating the cellular differentiation of the otic epithelium are still not fully resolved. This study provided novel data on GATA factor roles in several developmental processes during otic development. The expression analysis in chicken suggested that GATA2 and GATA3 possess redundant roles during otic cup and vesicle formation, but complementary cell-type specific functions during vestibular and cochlear morphogenesis. The comparative analysis between mouse and chicken Gata2 and Gata3 expression revealed many conserved aspects, especially during later stages of inner ear development, while the expression was more divergent at early stages. Namely, expression of both Gata genes was initiated earlier in chicken than mouse otic epithelium relative to the morphogenetic stages. Likewise, important differences concerning Gata3 expression in the otic cup epithelium were detected between mouse and chicken, suggesting that distinct molecular mechanisms regulate otic vesicle closure in different vertebrate species. Temporally distinct Gata2 and Gata3 expression was also found during otic ganglion formation in mouse and chicken. Targeted inactivation of Gata3 in mouse embryos caused aberrant morphology of the otic vesicle that in severe cases was disrupted into two parts, a dorsal and a ventral vesicle. Detailed analyses of Gata3 mutant embryos unveiled a crucial role for GATA3 in the initial inner ear morphogenetic event, the invagination of the otic placode. A large-scale comparative expression analysis suggested that GATA3 could control cell adhesion and motility in otic epithelium, which could be important for early morphogenesis. GATA3 was also identified as the first factor to directly regulate Fgf10 expression in the otic epithelium and could thus influence the development of the semicircular ducts. Despite the serious problems in the early inner ear development, the otic sensory fate establishment and some vestibular hair cell differentiation was observable in pharmacologically rescued Gata3-/- embryos. Cochlear sensory differentiation was, however, completely blocked so that no auditory hair cells were detected. In contrast to the early morphogenetic phenotype in Gata3-/- mutants, conditional inactivation of Gata2 in mouse embryos resulted in a relatively late growth defect of the three semicircular ducts. GATA2 was required for the proliferation of the vestibular nonsensory epithelium to support growing of the three ducts. Concurrently, with the role in epithelial semicircular ducts, GATA2 was also required for the mesenchymal cell clearance from the vestibular perilymphatic region between the membranous labyrinth and bony capsule. The gamma-aminobutyric acid-secreting (GABAergic) neurons in the midbrain are clinically relevant since they contribute to fear, anxiety, and addiction regulation. The molecular mechanisms regulating the GABAergic neuronal development, however, are largely unknown. Using tissue-specific mutagenesis in mice, GATA2 was characterized as a critical determinant of the GABAergic neuronal fate in the midbrain. In Gata2-deficient mouse midbrain, GABAergic neurons were not produced, instead the Gata2-mutant cells acquired a glutamatergic neuronal phenotype. Gain-of-function experiments in chicken also revealed that GATA2 was sufficient to induce GABAergic differentiation in the midbrain.

UDP-glucose 4, 6-dehydratase Activity Plays an Important Role in Maintaining Cell Wall Integrity and Virulence of Candida albicans

Candida albicans, a human fungal pathogen, undergoes morphogenetic changes that are associated with virulence. We report here that GAL102 in C. albicans encodes a homolog of dTDP-glucose 4,6-dehydratase, an enzyme that affects cell wall properties as well as virulence of many pathogenic bacteria. We found that GAL102 deletion leads to greater sensitivity to antifungal drugs and cell wall destabilizing agents like Calcofluor white and Congo red. The mutant also formed biofilms consisting mainly of hyphal cells that show less turgor. The NMR analysis of cell wall mannans of gal102 deletion strain revealed that a major constituent of mannan is missing and the phosphomannan component known to affect virulence is greatly reduced. We also observed that there was a substantial reduction in the expression of genes involved in biofilm formation but increase in the expression of genes encoding glycosylphosphatidylinositol-anchored proteins in the mutant. These, along with altered mannosylation of cell wall proteins together might be responsible for multiple phenotypes displayed by the mutant. Finally, the mutant was unable to grow in the presence of resident peritoneal macrophages and elicited a weak pro-inflammatory cytokine response in vitro. Similarly, this mutant elicited a poor serum pro-inflammatory cytokine response as judged by IFN gamma and TNF alpha levels and showed reduced virulence in a mouse model of systemic candidiasis. Importantly, an Ala substitution for a conserved Lys residue in the active site motif YXXXK, that abrogates the enzyme activity also showed reduced virulence and increased filamentation similar to the gal102 deletion strain. Since inactivating the enzyme encoded by GAL102 makes the cells sensitive to antifungal drugs and reduces its virulence, it can serve as a potential drug target in combination therapies for C. albicans and related pathogens.

Influência da superexpressão da claudina-3 na radiorresposta de células de câncer de cólon

O câncer colorretal (CCR) é o terceiro tipo de câncer mais incidente no mundo para o sexo masculino, o segundo para o sexo feminino e a radioterapia é um dos tratamentos de primeira linha no combate a este tipo de câncer. Durante a progressão do CCR as células sofrem alterações morfogenéticas, sendo a desorganização do complexo juncional apical (CJA) um dos eventos iniciais desse processo. As junções oclusivas (JTs) são um dos principais componentes da CJA e desempenham papel importante no controle do fluxo paracelular, na determinação da polaridade celular e na transdução de sinais relacionados com a progressão tumoral. As claudinas são proteínas transmembrana, constituintes das JTs e cumprem um importante papel no controle desses eventos. Alterações na expressão das claudinas são observadas em tumores de diferentes órgãos e têm sido relacionadas com a progressão tumoral. No entanto os mecanismos que regulam essas alterações e sua consequência na progressão do CCR são poucos conhecidos. Desta forma, o presente estudo teve como objetivo avaliar a influência da superexpressão da claudina-3 na radiorresposta de células CCR. Nossos resultados mostraram que a superexpressão de claudina-3 minimiza alterações morfológicas causadas pela radiação, causa diminuição da resistência elétrica transepitelial e não tem efeito na permeabilidade a macromoléculas após a irradiação. Além disso, observamos que a superexpressão de claudina-3 aumenta o potencial proliferativo das células e que esta característica torna as células mais sensíveis a radiação. Porém quando avaliamos eventos celulares relacionados a progressão tumoral observamos que apesar da radiação diminuir a capacidade migratória das progênies, as células que superexpressam claudina-3 apresentam migração mais elevada. Além disso, verificamos que a superexpressão de claudina-3 diminui a invasão e a capacidade de formação de colônias frente ao tratamento com a radiação. Em seguida fomos avaliar o efeito da inibição das vias de proliferação (MEK/ERK) e sobrevivência (PI3K-Akt) na resposta das células que superexpressam claudina-3 frente a radiação. Observamos que a inibição de MEK é capaz de sensibilizar as células que superexpressam claudina-3 à radiação no ensaio de proliferação celular, no entanto a inibição de MEK e PI3K antes da exposição à radiação é capaz aumentar a migração e a capacidade de formação de colônias de células que superexpressam claudina-3 contribuindo para o aumento do potencial maligno. Em conjunto nossos resultados mostram que a superexpressão de claudina-3 contribui para um fenótipo mais maligno, no entanto frente ao tratamento com a radiação é capaz de sensibilizar as células.

Evidence for Positive Selection on the Osteogenin (BMP3) Gene in Human Populations

Background: Human skeletal system has evolved rapidly since the dispersal of modern humans from Africa, potentially driven by selection and adaptation. Osteogenin (BMP3) plays an important role in skeletal development and bone osteogenesis as an antagonist of the osteogenic bone morphogenetic proteins, and negatively regulates bone mineral density. Methodology/Principal Findings: Here, we resequenced the BMP3 gene from individuals in four geographically separated modern human populations. Features supportive of positive selection in the BMP3 gene were found including the presence of an excess of nonsynonymous mutations in modern humans, and a significantly lower genetic diversity that deviates from neutrality. The prevalent haplotypes of the first exon region in Europeans demonstrated features of long-range haplotype homogeneity. In contrast with findings in European, the derived allele SNP Arg192Gln shows higher extended haplotype homozygosity in East Asian. The worldwide allele frequency distribution of SNP shows not only a high-derived allele frequency in Asians, but also in Americans, which is suggestive of functional adaptation. Conclusions/Significance: In conclusion, we provide evidence for recent positive selection operating upon a crucial gene in skeletal development, which may provide new insight into the evolution of the skeletal system and bone development.

Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells

In the present study, five homologous feeder cell lines were developed for the culture and maintenance of rhesus monkey embryonic stem cells (rESCs). Monkey ear skin fibroblasts (MESFs), monkey oviductal fibroblasts (MOFs), monkey follicular granulosa fibroblast-like (MFG) cells, monkey follicular granulosa epithelium-like (MFGE) cells, and clonally derived fibroblasts from MESF (CMESFs) were established and compared with the ability of mouse embryonic fibroblasts (MEFs) to support rESC growth. MESF, MOF, MFG, and CMESF cells, but not MFGE cells, were as good as or better than MEFs in supporting undifferentiated growth while maintaining the differentiation potential of the rESCs. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. MOF, MESF, and MEF cells highly expressed leukemia inhibitory factor, ciliary neurotrophic factor, basic fibroblast growth factor, stem cell factor, transforming growth factor PI, bone morphogenetic protein 4, and WNT3A, whereas WNT2, WNT4, and WNT5A were downregulated, compared with MFGE cells. Additionally, all monkey feeder cell lines expressed Dkk1 and LRP6, antagonists of the WNT signaling pathway, but not WNT1, WNT8B, or Dkk2. rESCs grown on homologous feeders maintained normal karyotypes, displayed the characteristics of ESCs, including morphology, alkaline phosphatase, Oct4, the cell surface markers stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumor-related antigen (TRA)-1-60, and TRA-1-81, and formed cystic embryoid bodies in vitro that included differentiated cells representing the three major germ layers. These results indicate that the four homologous feeder cell lines can be used to support the undifferentiated growth and maintenance of pluripotency in rESCs.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

Characterization of eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.