The EWS-WT1 translocation product induces PDGFA in desmoplastic small round-cell tumour.

Chromosomal translocations resulting in chimaeric transcription factors underlie specific malignancies, but few authentic target genes regulated by these fusion proteins have been identified. Desmoplastic small round-cell tumour (DSRT) is a multiphenotypic primitive tumour characterized by massive reactive fibrosis surrounding nests of tumour cells. The t(11;22)(p13;q12) chromosomal translocation that defines DSRT produces a chimaeric protein containing the potential transactivation domain of the Ewing-sarcoma protein (EWS) fused to zinc fingers 2-4 of the Wilms tumour suppressor and transcriptional repressor WT1 (refs 2,3). By analogy with other EWS fusion products, the EWS-WT1 chimaera may encode a transcriptional activator whose target genes overlap with those repressed by WT1 (ref. 4). To characterize its functional properties, we generated osteosarcoma cell lines with tightly regulated inducible expression of EWS-WT1. Expression of EWS-WT1 induced the expression of endogenous platelet-derived growth factor-A (PDGFA), a potent secreted mitogen and chemoattractant whose promoter contains the many potential WT1-binding sites. Native PDGFA was not regulated by wild-type WT1, indicating a difference in target gene specificity between this tumour suppressor and its oncogenic derivative. PDGFA was expressed within tumour cells in primary DSRT specimens, but it was absent in Wilms tumours expressing WT1 and Ewing sarcomas with an EWS-Fli translocation. We conclude that the oncogenic fusion of EWS to WT1 in DSRT results in the induction of PDGFA, a potent fibroblast growth factor that contributes to the characteristic reactive fibrosis associated with this unique tumour.

The role of self-control strength in the development of state anxiety in test situations.

Self-control strength may affect state anxiety because emotion regulation is impaired in individuals whose self-control strength has been temporarily depleted. Increases in state anxiety were expected to be larger for participants with depleted compared to nondepleted self-control strength, and trait test anxiety should predict increases in state anxiety more strongly if self-control strength is depleted. In a sample of 76 university students, trait test anxiety was assessed, self-control strength experimentally manipulated, and state anxiety measured before and after the announcement of a test. State anxiety increased after the announcement. Trait test anxiety predicted increases in state anxiety only in students with depleted self-control strength, suggesting that increased self-control strength may be useful for coping with anxiety.

Liver transplantation in a child with liver failure due to chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation from the same unrelated living donor.

We report a case of a six-yr-old boy who developed chronic GVHD of the liver, intestines, and skin following allogeneic hematopoietic SCT. The boy received an allogeneic hematopoietic stem cell transplant at the age of two yr because of early recurrence of ALL. Chimerism analysis showed complete chimerism. In the following year, he developed GVHD despite adequate immunosuppressive therapy. Liver biopsy showed liver GVHD resulting in liver cirrhosis by the age of five yr. LTx was performed with a left liver lobe from the unrelated donor from whom the stem cells had been taken. Immunosuppressive therapy consisted of low-dose steroids and low-dose cyclosporine. The postoperative course was uneventful. Graft function was excellent, and we performed protocol biopsies at seven days and three wk as well as three, six, and nine months after transplantation; none of these showed any signs of rejection or GVHD. Immunosuppressive therapy was discontinued nine months after LTx. Three yr after transplantation, the boy is in good condition with normal graft function. To our knowledge, this is the first report on LTx following allogeneic hematopoietic SCT from the same unrelated living donor.

Cranio-lenticulo-sutural dysplasia associated with defects in collagen secretion.

Cranio-lenticulo-sutural dysplasia (CLSD) is a rare autosomal recessive syndrome manifesting with large and late-closing fontanels and calvarial hypomineralization, Y-shaped cataracts, skeletal defects, and hypertelorism and other facial dysmorphisms. The CLSD locus was mapped to chromosome 14q13-q21 and a homozygous SEC23A F382L missense mutation was identified in the original family. Skin fibroblasts from these patients exhibit features of a secretion defect with marked distension of the endoplasmic reticulum (ER), consistent with SEC23A function in protein export from the ER. We report an unrelated family where a male proband presented with clinical features of CLSD. A heterozygous missense M702V mutation in a highly conserved residue of SEC23A was inherited from the clinically unaffected father, but no maternal SEC23A mutation was identified. Cultured skin fibroblasts from this new patient showed a severe secretion defect of collagen and enlarged ER, confirming aberrant protein export from the ER. Milder collagen secretion defects and ER distention were present in paternal fibroblasts, indicating that an additional mutation(s) is present in the proband. Our data suggest that defective ER export is the cause of CLSD and genetic element(s) besides SEC23A may influence its presentation.

Temperature Tides Across the Mid-Latitude Summer Turbopause Measured by a Sodium Lidar and MIGHTI/ICON.

Local full diurnal coverage of temperature variations across the turbopause (~90-115 km altitude) is achieved by combining the nocturnal observations of a Sodium (Na) Doppler lidar on the Utah State University (USU) campus (41.7°N, 248.2°E) and NASA Michelson interferometer for global high-resolution thermospheric imaging (MIGHTI)/Ionospheric connection explorer (ICON) daytime observations made in the same vicinity. In this study, utilizing this hybrid data set during summer 2020 between June 12th and July 15th, we retrieve the temperature signatures of diurnal and semidiurnal tides in this region. The tidal amplitudes of both components have similar vertical variation with increasing altitude: less than 5 K below ~98 km but increase considerably above, up to 19 K near 104 km. Both experience significant dissipation near turbopause altitudes, down to ~12 K up to 113 km for the diurnal tide and ~13 K for the semidiurnal tide near 110 km. In addition, while the semidiurnal tidal behavior is consistent with the theoretical predictions, the diurnal amplitude is considerably larger than what is expected in the turbopause region. The tidal phase profile shows a dominance of tidal components with a long vertical wavelength (longer than 40 km) for the semidiurnal tide. On the other hand, the diurnal tide demonstrates close to an evanescent wave behavior in the turbopause region, which is absent in the model results and Thermosphere ionosphere mesosphere energetics and dynamics (TIMED)/Sounding of the atmosphere using broadband radiometry (SABER) observations.

Synergistic effects of growth and differentiation factor-5 (GDF-5) and insulin on expanded chondrocytes in a 3-D environment.

OBJECTIVE: To investigate the effects of growth and differentiation factor-5 (GDF-5) alone or in combination with insulin on engineered cartilage from primary or expanded chondrocytes during 3-dimensional in vitro culture. DESIGN: Juvenile bovine chondrocytes were seeded either as primary or as expanded (passage 2) cells onto polyglycolic acid fiber meshes and cultured for 3 weeks in vitro. Additionally, adult human chondrocytes were grown in pellet culture after expansion (passage 2). The culture medium was supplemented either with GDF-5 in varying concentrations or insulin alone, or with combinations thereof. RESULTS: For primary chondrocytes, the combination of GDF-5 and insulin led to increased proliferation and construct weight, as compared to either factor alone, however, the production of glycosaminoglycans (GAG) and collagen per cell were not affected. With expanded bovine chondrocytes, the use of GDF-5 or insulin alone led to only very small constructs with no type II collagen detectable. However, the combination of GDF-5 (0.01 or 0.1 microg/ml) and insulin (2.5 microg/ml) yielded cartilaginous constructs and, in contrast to the primary cells, the observed redifferentiating effects were elicited on the cellular level independent of proliferation (increased production of GAG and collagen per cell, clear shift in collagen subtype expression with type II collagen observed throughout the construct). The synergistic redifferentiating effects of the GDF-5/insulin combination were confirmed with expanded adult human cells, also exhibiting a clear shift in collagen subtype expression on the mRNA and protein level. CONCLUSIONS: In combination with insulin, GDF-5 appears to enable the redifferentiation of expanded chondrocytes and the concurrent generation of cartilaginous constructs. The demonstration of these synergistic effects also for adult human chondrocytes supports the clinical relevance of the findings.

WT1-mediated growth suppression of Wilms tumor cells expressing a WT1 splicing variant.

A human Wilms tumor cell line (RM1) was developed to test the tumor suppressor activity of WT1, a zinc finger transcription factor that is expressed in the developing human kidney and is mutationally inactivated in a subset of Wilms tumors. Transfection of each of four wild-type WT1 isoforms suppressed the growth of RM1 cells. The endogenous WT1 transcript in these cells was devoid of exon 2 sequences, a splicing alteration that was also detected in varying amounts in all Wilms tumors tested but not in normal kidney. Production of this abnormal transcript, which encodes a functionally altered protein, may represent a distinct mechanism for inactivating WT1 in Wilms tumors.

WT1--more than a transcription factor?

The Wilms tumor-suppressor gene WT1 was originally identified through its involvement in the development of a pediatric kidney tumor. Recent genetic data show that mutations in the WT1 gene cause a variety of other diseases, and new biochemical evidence suggests that the WT1 protein is not only a transcription factor but might also act at the post-transcriptional level.

T-peel test for the analysis of articular cartilage integration.

A central aim of current research is to determine the molecular mechanisms of articular cartilage repair. One major issue of articular cartilage repair is the achievable mechanical strength which has been correlated with the collagen metabolism, deposition and collagen cross-linking. Current in vitro techniques, leading to cartilage integration used a shear test to failure. Another well established in vitro method to investigate articular cartilage integration is the insert-ring push out model which is mainly utilized investigating the integration of tissue engineered cartilage to native cartilage. Finite element modeling illustrates at least for the shear test to failure that the contact area is not homogeneously loaded. For the mechanical analysis of articular cartilage integration in regard to its inhomogeneous integration a higher mechanical resolution method is needed. Furthermore the shear test to failure as well as the ring-insert model lacks a comparison to in situ trauma situation, where ruptured or fractured articular cartilage surfaces are opposed after surgical reduction. Considering all these a T-peel test has been introduced in literature but never been experimentally performed. This project deals with the establishment of a T-peel test as a topographical sensitive tool in mechanical analysis of T-peel data and its potential to investigate articular cartilage in vitro integration in comparison to articular cartilage rupture strength.

The Ionospheric Connection Explorer Mission: Mission Goals and Design.

The Ionospheric Connection Explorer, or ICON, is a new NASA Explorer mission that will explore the boundary between Earth and space to understand the physical connection between our world and our space environment. This connection is made in the ionosphere, which has long been known to exhibit variability associated with the sun and solar wind. However, it has been recognized in the 21st century that equally significant changes in ionospheric conditions are apparently associated with energy and momentum propagating upward from our own atmosphere. ICON's goal is to weigh the competing impacts of these two drivers as they influence our space environment. Here we describe the specific science objectives that address this goal, as well as the means by which they will be achieved. The instruments selected, the overall performance requirements of the science payload and the operational requirements are also described. ICON's development began in 2013 and the mission is on track for launch in 2017. ICON is developed and managed by the Space Sciences Laboratory at the University of California, Berkeley, with key contributions from several partner institutions.

The Wilms tumor suppressor gene wt1 is required for development of the spleen.

The Wilms tumor suppressor gene WT1 (wt1 in mouse) is unique among tumor suppressors because, in addition to its involvement in cancer [1] [2] and various other diseases [3] [4] [5] [6], it has an essential role in the development of certain organs. This is revealed by the phenotype of mice with inactivated wt1 alleles [7]. These animals exhibit a complete failure of kidney and gonad development as well as abnormalities of the heart and mesothelial structures. On a C57BL/6 genetic background, wt1(-/-) animals die between day 13.5 (E13.5) and 15.5 (E15.5) of embryonic development [7]. We report here that crossing of the wt1 mutation onto different mouse backgrounds delayed embryonic lethality until birth. In wt1(-/-) mice on these different genetic backgrounds, we observed a dramatic failure of spleen development, in addition to the well characterized phenotypic abnormalities. The spleen anlage formed at around E12 to E13 and involuted by the E15 stage, before the invasion of hematopoietic cells. The absence of proper spleen development in these wt1(-/-) embryos correlated with enhanced apoptosis in the primordial spleen cells. The expression of hox11, a gene that also controls development of the spleen [8] [9], was not altered by the inactivation of wt1. In situ hybridization revealed that the two genes are regulated independently. These findings demonstrate that the penetrance of the wt1(-/-) phenotype depends on the existence of one or more modifier gene(s) and that wt1 plays a pivotal role in the development of the spleen, thereby extending its role in organogenesis.

A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1.

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.

Lipidic implants for controlled release of bioactive insulin: effects on cartilage engineered in vitro.

Controlled release systems for growth factors and morphogens are potentially powerful tools for the engineering or the treatment of living tissues. However, due to possible instabilities of the protein during manufacture, storage, and release, in the development of new release systems it is paramount to investigate into the maintenance of bioactivity of the protein. Within this study, recently developed protein releasing lipid matrix cylinders of 2 mm diameter and 2 mm height made from glycerol tripalmitate were manufactured in a compression process without further additives. Insulin in different concentrations (0.2%, 1%, and 2%) served as model protein. The bioactivity of the protein released from the matrices was investigated in a long-term cartilage engineering culture for up to four weeks; additionally, the release profiles were determined using ELISA. Insulin released from the matrices increased the wet weights of the cartilaginous cell-polymer constructs (up to 3.2-fold), the amount of GAG and collagen in the constructs (up to 2.4-fold and 3.2-fold, respectively) and the GAG and collagen content per cell (1.8-fold and 2.5-fold, respectively), compared to the control. The dose-dependent effects on tissue development correlated well with release profiles from the matrices with different insulin loading. In conclusion, the lipid matrices, preserving the bioactivity of incorporated and released protein, are suggested as a suitable carrier system for use in tissue engineering or for the localized treatment of tissues with highly potent protein drugs such as used in the therapy of brain cancer or neurodegenerative CNS diseases.

Biomechanical properties of articular cartilage as a standard for biologically integrated interfaces.

Articular cartilage integration has been described in in-vitro models, which compare mechanical to biochemical behaviour and histological analysis, respectively. The emphasis of these findings is mainly on the biochemical and histological analysis, rather than on the mechanical performance. The complex in vitro loading conditions and high deviations in the mechanical results due to the biological variance, make interpretations difficult. The aim of this study is to analyse and define the mechanical stress and strain distribution in a single lap configuration by means of an optical strain measurement system. Supportive finite element computation is performed to indicate the heterogeneous stress strain distribution in the integration area. The optical failure analysis of the experiment reveals crack propagation through the integration area comparable to plane shear in fracture mode two. Using the optical strain measurement set up a direct estimation of the shear modulus is achievable by analysing the relative displacement within the bonded joint before the onset of delamination in the adhesive layer. This result lead to a better interpretation of the mechanical behaviour of articular cartilage integration in vitro.

Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1.

Telomerase, an enzymatic activity responsible for the replication of chromosome end structures, is strongly upregulated in most human cancers. In contrast, most differentiated tissues are telomerase negative. The rate-limiting step for telomerase activity seems to be the expression of the catalytic subunit of the enzyme, encoded by the human telomerase reverse transcriptase (hTERT) gene. The precise mechanism of how hTERT is regulated has not been elucidated yet. We show here that the down-regulation of hTERT mRNA during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of human U937 cells is a consequence of a fast decrease in the rate of transcription rather than changes in its half-life. The only transcription factor that has so far been implicated in the regulation of hTERT expression is the c-Myc oncoprotein. Our analysis shows that another member of the myc/marx/mad network, mad1, encoding a transcriptional repressor that is significantly increased by 12-O-tetra-decanoylphorbol-13-acetate treatment, represses hTERT promoter-driven reporter gene activity in transient transfection assays. This effect is dependent on the NH2 terminal domain of Madl, which mediates the association with the transcriptional corepressor mSin3. Our findings suggest the involvement of an additional transcription factor in the regulation of hTERT expression and may provide a model for how hTERT activity is controlled during the differentiation process in human somatic tissues.

Induction of p21 by the Wilms' tumor suppressor gene WT1.

WT1 encodes a zinc finger transcription factor that is expressed in the developing kidney and the inactivation of which leads to Wilms' tumor, a pediatric kidney cancer. We have recently shown that inducible expression of WT1 in osteosarcoma cells triggers programmed cell death, an effect that is associated with transcriptional repression of the endogenous epidermal growth factor receptor. We now show that WT1-mediated apoptosis is preceded by induction of the cyclin-dependent kinase inhibitor p21, associated with G1 phase arrest. This effect is only demonstrated by WT1 isoforms with an intact DNA binding domain, and it is associated with increased expression of endogenous p21 mRNA. WT1-mediated induction of p21 is independent of p53, another tumor suppressor gene known to regulate p21 expression. In the kidney, p21 is expressed in differentiating glomerular podocytes along with WT1. We conclude that induction of p21 expression may contribute to WT1-dependent differentiation pathways in the kidney and potentially to the function of WT1 as a tumor suppressor gene.

Layered expression scanning: rapid molecular profiling of tumor samples.

Layered expression scanning is a new approach to comprehensive molecular analysis of tumor samples that uses a layered array of capture membranes coupled to antibodies or DNA sequences to perform multiplex protein or mRNA analysis. Cell or tissue samples are transferred through a series of individual capture layers, each linked to a separate antibody or DNA sequence. As the biomolecules traverse the membrane set, each targeted protein or mRNA is specifically captured by the layer containing its antibody or cDNA sequence. The two-dimensional relationship of the cell populations is maintained during the transfer process, thereby producing a molecular profile of each cell type present. Reduction-to-practice of the technique is demonstrated by analysis of prostate-specific antigen (PSA) protein, gelatinase A protein, and POV1 (PB39) cDNA. As layered expression scanning technology progresses, we envision a laboratory method that will have multiple applications for high-throughput molecular profiling of normal and tumor samples.

Analysis of WT1 target gene expression in stably transfected cell lines.

The Wilms' tumour suppressor gene WT1 encodes a zinc finger protein that is mutated in a subset of Wilms' tumours. Mutation screening and animal studies revealed essential roles during development and later function of the kidneys and the entire genitourinary system. Sequence similarity suggested a possible role for WT1 as a transcription factor. Indeed, sequence specific DNA binding and transcriptional activation or repression potential could be demonstrated in transient transfection assays with various reporter constructs. To identify endogenous WT1 target genes we established HEK293 cell lines expressing the different WT1 isoforms in a tetracycline dependent manner. Differential display PCR (ddPCR) was performed on RNA from stable WT1 transfected HEK293 cell lines and two other WT1 transfected lines (G401 and Saos-2). In an extended survey of several thousand ddPCR bands only few differences in intensity were seen and none of these could unambiguously be verified as being WT1 regulated by subsequent Northern blot analysis. In addition, almost none of the WT1 target genes identified to date in transient co-transfection assays could be confirmed by either ddPCR or Northern hybridization in the three stable transfected cell lines. Among the nine genes expressed, the only exceptions were CSF1 and to a lesser extent IGF1R being induced in Saos-2/G401 and HEK293 cells, respectively. At least two of the cell lines tested had previously shown clear biological effects though -- either WT1 dependent apoptosis (Saos-2) or greatly reduced tumorigenicity (G401). This suggests that WT1 may regulate only a very small set of genes that escape the detection methods used or it may not act as a transcription factor that influences steady state levels of mRNA.

Mutational analysis of the carboxy-terminal portion of p53 using both yeast and mammalian cell assays in vivo.

Increasing evidence indicates that p53 is a transcriptional trans-activator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the trans-activation ability mainly due to loss of its sequence-specific DNA binding. Using both yeast and mammalian cell assays, the effect of p53 mutations in the carboxy terminal portion was investigated in order to address how p53 mutations outside of the DNA binding domain affect p53 function. The p53 cDNA in the carboxy-terminus was randomly mutagenized by error-prone polymerase chain reactions and the amplified cDNA was screened for the ability to trans-activate using a yeast assay. Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomerization domain, were further analysed for trans-activation, cell cycle arrest and colony formation in a human osteosarcoma cell line, Saos-2. These functional properties of p53 were disrupted by the missense mutations. Surprisingly, one of the nonsense mutations disrupts the trans-activation function and the ability to G1 arrest but shows a strong inhibition of colony formation. These results confirm that mutations in the oligomerization domain can inactivate p53 function and also indicate that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.

E1B 55K sequesters WT1 along with p53 within a cytoplasmic body in adenovirus-transformed kidney cells.

WT1 encodes a tumor suppressor that is expressed in cells of the developing kidney and is inactivated in Wilms tumor, a pediatric kidney cancer. The adenovirus E1B 55K gene product contributes to the transformation of primary baby rat kidney (BRK) cells by binding and inactivating the product of the p53 tumor suppressor. We have previously demonstrated that WT1 and p53 are present within a protein complex in vivo. We now show that WT1 is physically associated with E1B 55K in adenovirus-transformed cells, an interaction that is mediated by the first two zinc fingers of WT1. Immunodepletion of p53 abrogates the coimmunoprecipitation of E1B 55K and WT1, consistent with the presence of a trimeric protein complex containing these three proteins. In the presence of E1B 55K, WT1 which is normally localized in the nucleus, is retained within a very high molecular weight complex and sequestered in the characteristic perinuclear cytoplasmic body that contains E1B 55K and p53. Expression of E1B 55K in osteosarcoma cells that undergo apoptosis following expression of WT1 inhibits WT1-mediated cell death. We conclude that E1B 55K may target WT1 along with p53, resulting in the functional inactivation of both tumor suppressor gene products by this viral oncoprotein.