Molecular diagnostics of gliomas: the clinical perspective.

Significant progress has been made in the molecular diagnostic subtyping of brain tumors, in particular gliomas. In contrast to the classical molecular markers in this field, p53 and epidermal growth factor receptor (EGFR) status, the clinical significance of which has remained controversial, at least three important molecular markers with clinical implications have now been identified: 1p/19q codeletion, O⁶-methylguanine methyltransferase (MGMT) promoter methylation and isocitrate dehydrogenase-1 (IDH1) mutations. All three are favorable prognostic markers. 1p/19q codeletion and IDH1 mutations are also useful to support and extend the histological classification of gliomas since they are strongly linked to oligodendroglial morphology and grade II/III gliomas, as opposed to glioblastoma, respectively. MGMT promoter methylation is the only potentially predictive marker, at least for alkylating agent chemotherapy in glioblastoma. Beyond these classical markers, the increasing repertoire of anti-angiogenic agents that are currently explored within registration trials for gliomas urgently calls for efforts to identify molecular markers that predict the benefit derived from these novel treatments, too.

Nuclear factor I-C regulates TGF-{beta}-dependent hair follicle cycling.

Skin appendages such as teeth and hair share several common signaling pathways. The nuclear factor I C (NFI-C) transcription factor has been implicated in tooth development, but a potential role in hair growth had not been assessed. In this study we found that NFI-C regulates the onset of the hair growth cycle. NFI-C(-/-) mice were delayed in the transition from the telogen to anagen phase of the hair follicle cycle after either experimental depilation or spontaneous hair loss. Lack of NFI-C resulted in delayed induction of the sonic hedgehog, Wnt5a, and Lef1 gene expression, which are key regulators of the hair follicle growth initiation. NFI-C(-/-) mice also showed elevated levels of transforming growth factor β1 (TGF-β1), an inhibitor of keratinocyte proliferation, and of the cell cycle inhibitor p21 at telogen. Reduced expression of Ki67, a marker of cell proliferation, was noted at the onset of anagen, indicating impaired activation of the hair progenitor cells. These findings implicate NFI-C in the repression of TGF-β1 signaling during telogen stage, resulting in the delay of progenitor cell proliferation and hair follicle regeneration in NFI-C-deficient mice. Taken together with prior observations, these findings also designate NFI-C as a regulator of adult progenitor cell proliferation and of postnatal tissue growth or regeneration.

A nuclear factor I-like activity and a liver-specific repressor govern estrogen-regulated in vitro transcription from the Xenopus laevis vitellogenin B1 promoter.

A hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to identify novel cis-acting elements within the vitellogenin gene B1 promoter region. In addition to the already well-documented estrogen-responsive element (ERE), two elements were found within the 140 base pairs upstream of the transcription initiation site. One of them, a negative regulatory element, is responsible for the lack of promoter activity in the absence of the hormone and, as demonstrated by DNA-binding assays, interacts with a liver-specific transcription factor. The second is required in association with the estrogen-responsive element to mediate hormonal induction and is recognized by the Xenopus liver homolog of nuclear factor I.

Development of Preliminary Load and Resistance Factor Design of Drilled Shafts in Iowa, RB03-012, 2014

The Federal Highway Administration (FHWA) mandated utilizing the Load and Resistance Factor Design (LRFD) approach for all new bridges initiated in the United States after October 1, 2007. To achieve part of this goal, a database for Drilled Shaft Foundation Testing (DSHAFT) was developed and reported on by Garder, Ng, Sritharan, and Roling in 2012. DSHAFT is aimed at assimilating high-quality drilled shaft test data from Iowa and the surrounding regions. DSHAFT is currently housed on a project website (http://srg.cce.iastate.edu/dshaft) and contains data for 41 drilled shaft tests. The objective of this research was to utilize the DSHAFT database and develop a regional LRFD procedure for drilled shafts in Iowa with preliminary resistance factors using a probability-based reliability theory. This was done by examining current design and construction practices used by the Iowa Department of Transportation (DOT) as well as recommendations given in the American Association of State Highway and Transportation Officials (AASHTO) LRFD Bridge Design Specifications and the FHWA drilled shaft guidelines. Various analytical methods were used to estimate side resistance and end bearing of drilled shafts in clay, sand, intermediate geomaterial (IGM), and rock. Since most of the load test results obtained from O-cell do not pass the 1-in. top displacement criterion used by the Iowa DOT and the 5% of shaft diameter for top displacement criterion recommended by AASHTO, three improved procedures are proposed to generate and extend equivalent top load-displacement curves that enable the quantification of measured resistances corresponding to the displacement criteria. Using the estimated and measured resistances, regional resistance factors were calibrated following the AASHTO LRFD framework and adjusted to resolve any anomalies observed among the factors. To illustrate the potential and successful use of drilled shafts in Iowa, the design procedures of drilled shaft foundations were demonstrated and the advantages of drilled shafts over driven piles were addressed in two case studies.

Stress intensity factor of wood from crack-tip displacement fields obtained from digital image processing

Abstract

Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1.

Families of clonally expressed major histocompatibility complex (MHC) class I-specific receptors provide specificity to and regulate the function of natural killer (NK) cells. One of these receptors, mouse Ly49A, is expressed by 20% of NK cells and inhibits the killing of H-2D(d) but not D(b)-expressing target cells. Here, we show that the trans-acting factor TCF-1 binds to two sites in the Ly49A promoter and regulates its activity. Moreover, we find that TCF-1 determines the size of the Ly49A NK cell subset in vivo in a dosage-dependent manner. We propose that clonal Ly49A acquisition during NK cell development is regulated by TCF-1.

Systematic assessment of factors influencing preferences of crohn's disease patients in selecting an anti-tumor necrosis factor agent (CHOOSE TNF TRIAL).

BACKGROUND: Infliximab (IFX), adalimumab (ADA), and certolizumab pegol (CZP) have similar efficacy in induction and maintenance of clinical remission in Crohn's disease (CD). Given the comparable nature of these drugs, patient preferences may influence the choice of the product. We aimed to identify factors that may contribute to CD patients' decision in selecting one anti-tumor necrosis factor (TNF) agent over the others. METHODS: A prospective survey was performed among anti-TNF-naïve CD patients. Prior to completion of a questionnaire, patients were provided with a written description of the three anti-TNF agents, focusing on indications, mode of administration, side effects, and scientific evidence of efficacy and safety for each drug. RESULTS: One hundred patients (47 females, mean age 45 ± 16 years, range 19-81) with an ileal, colonic, or ileocolonic (33%, 40%, and 27%, respectively) disease location completed the questionnaire. Based on the information provided, 36% of patients preferred ADA, 28% CZP, and 25% IFX, whereas 11% were undecided. The patients' decision in selecting a specific anti-TNF drug was influenced by the following factors: ease of use (69%), time required for therapy (34%), time interval between application of the drug (31%), scientific evidence for efficacy (19%), and fear of syringes (10%). CONCLUSIONS: The majority of patients preferred anti-TNF medications that were administered by subcutaneous injection rather than by intravenous infusion. Ease of use and time required for therapy were two major factors influencing the patients' selection of a specific anti-TNF drug. Patients' individual preferences should be taken into account when prescribing anti-TNF drugs. (Inflamm Bowel Dis 2012).

Correlation of CXCL10, tumor necrosis factor receptor type II, and galectin 9 with disease activity in juvenile dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is a systemic autoimmune disorder of unknown immunopathogenesis in which the immune system targets the microvasculature of skeletal muscles, skin, and other organs. The current mainstay of therapy is a steroid regimen in combination with other immunosuppressive treatments. To date, no validated markers for monitoring disease activity have been identified, which hampers personalized treatment. This study was undertaken to identify a panel of proteins specifically related to active disease in juvenile DM. METHODS: We performed a multiplex immunoassay for plasma levels of 45 proteins related to inflammation in 25 patients with juvenile DM in 4 clinically well-defined groups, as determined by clinical activity and treatment. We compared them to 14 age-matched healthy children and 8 age-matched children with nonautoimmune muscle disease. RESULTS: Cluster analysis of circulating proteins showed distinct profiles for juvenile DM patients and controls based on a group of 10 proteins. In addition to CXCL10, tumor necrosis factor receptor type II (TNFRII) and galectin 9 were significantly increased in active juvenile DM. The levels of these 3 proteins were tightly linked to active disease and correlated with clinical scores (as measured by the Childhood Myositis Assessment Scale and physician's global assessment of disease activity on a visual analog scale). CONCLUSION: Our findings indicate that CXCL10, TNFRII, and galectin 9 correspond to disease status in juvenile DM and thus could be helpful in monitoring disease activity and guiding treatment. Furthermore, they might provide new knowledge about the pathogenesis of this autoimmune disease.

Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA.

BACKGROUND: Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. METHODS: We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. RESULTS: Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. CONCLUSIONS: Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Formation of virus-like clusters is an intrinsic property of the tumor necrosis factor family member BAFF (B cell activating factor).

The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.

Triiodothyronine and nerve growth factor are required to induce cytoplasmic dynein expression in rat dorsal root ganglion cultures.

Beside the several growth factors which play a crucial role in the development and regeneration of the nervous system, thyroid hormones also contribute to the normal development of the central and peripheral nervous system. In our previous work, we demonstrated that triiodothyronine (T3) in physiological concentration enhances neurite outgrowth of primary sensory neurons in cultures. Neurite outgrowth requires microtubules and microtubule associated proteins (MAPs). Therefore the effects of exogenous T3 or/and nerve growth factors (NGF) were tested on the expression of cytoskeletal proteins in primary sensory neurons. Dorsal root ganglia (DRG) from 19 day old rat embryos were cultured under four conditions: (1) control cultures in which explants were grown in the absence of T3 and NGF, (2) cultures grown in the presence of NGF alone, (3) in the presence of T3 alone or (4) in the presence of NGF and T3 together. Analysis of proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of several proteins in the molecular weight region around 240 kDa. NGF and T3 together induced the expression of one protein, in particular, with a molecular weight above 240 kDa, which was identified by an antibody against MAP1c, a protein also known as cytoplasmic dynein. The immunocytochemical detection confirmed that this protein was expressed only in DRG explants grown in the presence of NGF and T3 together. Neither control explants nor explants treated with either NGF or T3 alone expressed dynein. In conclusion, a combination of nerve growth factor and thyroid hormone is necessary to regulate the expression of cytoplasmic dynein, a protein that is involved in retrograde axonal transport.

Decreased glucose-induced thermogenesis after weight loss in obese subjects: a predisposing factor for relapse of obesity?

Glucose-induced thermogenesis (GIT) after a 100-g oral glucose load was measured by continuous indirect calorimetry in 32 nondiabetic and diabetic obese subjects and compared to 17 young and 13 middle aged control subjects. The obese subjects were divided into three groups: A (n = 12) normal glucose tolerance, B (n = 13) impaired glucose tolerance, and C (n = 7) diabetics, and were studied before and after a body weight loss ranging from 9.6 to 33.5 kg consecutive to a 4 to 6 months hypocaloric diet. GIT, measured over 3 h and expressed as percentage of the energy content of the load, was significantly reduced in obese groups A and C (6.2 +/- 0.6, and 3.8 +/- 0.7%, respectively) when compared to their age-matched control groups: 8.6 +/- 0.7 (young) and 5.8 +/- 0.3% (middle aged). Obese group B had a GIT of 6.1 +/- 0.6% which was lower than that of the young control group but not different from the middle-aged control group. After weight loss, GIT in the obese was further reduced in groups A and B than before weight loss: ie, 3.4 +/- 0.6 (p less than 0.001), 3.7 +/- 0.5 (p less than 0.01) respectively, whereas in group C, weight loss induced no further diminution in GIT (3.8 +/- 0.6%). These results support the concept of a thermogenic defect after glucose ingestion in obese individuals which is not the consequence of their excess body weight but may be one of the factors favoring the relapse of obesity after weight loss.

Structure-function analysis of the human TFIIB-related factor II protein reveals an essential role for the C-terminal domain in RNA polymerase III transcription.

The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAP(c), a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.