Cell surface vimentin-positive circulating tumor cell-based relapse prediction in a long-term longitudinal study of postremission neuroblastoma patients.

Neuroblastoma (NB) is a deadly childhood disease that carries a 50% chance of relapse for anyone in remission and similar level of 5-year survival. We investigated the value of our proprietary approach-cell surface vimentin (CSV) positive circulating tumor cells (CTC) to monitor treatment response and predict relapse in NB patients under remission in a Phase II long-term preventative clinical trial. We longitudinally analyzed peripheral blood samples from 93 patients for 27 cycles (~25 months) and discovered that the presence of CSV+ CTCs in the first two sequential samples (baseline, cycle 4 [month 3-4]) was a significant indicator of earlier relapse. We observed strong correlation between relapse-free survival (RFS) and lack of CSV+ CTCs in first 4 cycles of therapy (95%). There was sensitivity reaching 100% in predicting RFS in patients who had neither CSV+ CTCs nor MycN amplification. Of note, the low number of CSV+ CTCs seems equivalent to low tumor load because the prevention therapy difluoromethylornithine yields faster reduction of relapse risk when none or only 1-2 CSV+ CTCs (every 6 mL) are present in the blood samples compared to >3 CSV+ CTCs. To the best of our knowledge, this is the first study that directly observes CTCs in under remission NB patients for relapse prediction and the first to gather sequential CSV+ CTC data in any study in a long-term longitudinal manner.

The molecular signature of murine T cell homeostatic proliferation reveals both inflammatory and immune inhibition patterns.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (lpr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient lpr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4-CD8-TCRαß+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.

Global analysis of the sporulation pathway of Clostridium difficile.

The Gram-positive, spore-forming pathogen Clostridium difficile is the leading definable cause of healthcare-associated diarrhea worldwide. C. difficile infections are difficult to treat because of their frequent recurrence, which can cause life-threatening complications such as pseudomembranous colitis. The spores of C. difficile are responsible for these high rates of recurrence, since they are the major transmissive form of the organism and resistant to antibiotics and many disinfectants. Despite the importance of spores to the pathogenesis of C. difficile, little is known about their composition or formation. Based on studies in Bacillus subtilis and other Clostridium spp., the sigma factors σ(F), σ(E), σ(G), and σ(K) are predicted to control the transcription of genes required for sporulation, although their specific functions vary depending on the organism. In order to determine the roles of σ(F), σ(E), σ(G), and σ(K) in regulating C. difficile sporulation, we generated loss-of-function mutations in genes encoding these sporulation sigma factors and performed RNA-Sequencing to identify specific sigma factor-dependent genes. This analysis identified 224 genes whose expression was collectively activated by sporulation sigma factors: 183 were σ(F)-dependent, 169 were σ(E)-dependent, 34 were σ(G)-dependent, and 31 were σ(K)-dependent. In contrast with B. subtilis, C. difficile σ(E) was dispensable for σ(G) activation, σ(G) was dispensable for σ(K) activation, and σ(F) was required for post-translationally activating σ(G). Collectively, these results provide the first genome-wide transcriptional analysis of genes induced by specific sporulation sigma factors in the Clostridia and highlight that diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes.

A simple method of measuring tibial tubercle to trochlear groove distance on MRI: description of a novel and reliable technique.

PURPOSE: Tibial tubercle-trochlear groove (TT-TG) distance is a variable that helps guide surgical decision-making in patients with patellar instability. The purpose of this study was to compare the accuracy and reliability of an MRI TT-TG measuring technique using a simple external alignment method to a previously validated gold standard technique that requires advanced software read by radiologists. METHODS: TT-TG was calculated by MRI on 59 knees with a clinical diagnosis of patellar instability in a blinded and randomized fashion by two musculoskeletal radiologists using advanced software and by two orthopaedists using the study technique which utilizes measurements taken on a simple electronic imaging platform. Interrater reliability between the two radiologists and the two orthopaedists and intermethods reliability between the two techniques were calculated using interclass correlation coefficients (ICC) and concordance correlation coefficients (CCC). ICC and CCC values greater than 0.75 were considered to represent excellent agreement. RESULTS: The mean TT-TG distance was 14.7 mm (Standard Deviation (SD) 4.87 mm) and 15.4 mm (SD 5.41) as measured by the radiologists and orthopaedists, respectively. Excellent interobserver agreement was noted between the radiologists (ICC 0.941; CCC 0.941), the orthopaedists (ICC 0.978; CCC 0.976), and the two techniques (ICC 0.941; CCC 0.933). CONCLUSION: The simple TT-TG distance measurement technique analysed in this study resulted in excellent agreement and reliability as compared to the gold standard technique. This method can predictably be performed by orthopaedic surgeons without advanced radiologic software. LEVEL OF EVIDENCE: II.

Identification of Orch3, a locus controlling dominant resistance to autoimmune orchitis, as kinesin family member 1C.

Experimental autoimmune orchitis (EAO), the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c) as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1c(D2) allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L(578) → P(578) and S(1027) → P(1027) polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases.

Milk yield responses to changes in milking frequency during early lactation are associated with coordinated and persistent changes in mammary gene expression.

BACKGROUND: The lactating mammary gland responds to changes in milking frequency by modulating milk production. This response is locally regulated and, in dairy cows, the udder is particularly sensitive during early lactation. Relative to cows milked twice-daily throughout lactation, those milked four-times-daily for just the first 3 weeks of lactation produce more milk throughout that lactation. We hypothesized that the milk yield response would be associated with increased mammary cell turnover and changes in gene expression during frequent milking and persisting thereafter. Cows were assigned to unilateral frequent milking (UFM; left udder halves milked twice-daily; right udder halves milked four-times daily) on days 1 to 21 of lactation, followed by twice-daily milking for the remainder of lactation. Relative to udder halves milked twice-daily, those milked four-times produced more milk during UFM; the difference in milk yield declined acutely upon cessation of UFM after day 21, but remained significantly elevated thereafter. We obtained mammary biopsies from both udder halves on days 21, 23, and 40 of lactation. RESULTS: Mammary cell proliferation and apoptosis were not affected by milking frequency. We identified 75 genes that were differentially expressed between paired udder halves on day 21 but exhibited a reversal of differential expression on day 23. Among those genes, we identified four clusters characterized by similar temporal patterns of differential expression. Two clusters (11 genes) were positively correlated with changes in milk yield and were differentially expressed on day 21 of lactation only, indicating involvement in the initial milk yield response. Two other clusters (64 genes) were negatively correlated with changes in milk yield. Twenty-nine of the 75 genes were also differentially expressed on day 40 of lactation. CONCLUSIONS: Changes in milking frequency during early lactation did not alter mammary cell population dynamics, but were associated with coordinated changes in mammary expression of at least 75 genes. Twenty-nine of those genes were differentially expressed 19 days after cessation of treatment, implicating them in the persistent milk yield response. We conclude that we have identified a novel transcriptional signature that may mediate the adaptive response to changes in milking frequency.

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo.

To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.

Mammary gland morphological and gene expression changes underlying pregnancy protection of breast cancer tumorigenesis.

A full-term pregnancy early in life reduces lifetime risk of developing breast cancer, and the effect can be mimicked in rodents by full-term pregnancy or short-term treatment with exogenous estrogen and progesterone. To gain insight into the protective mechanism, 15 3-mo-old postpubertal virgin Lewis rats were randomly assigned to three groups: control (C), pregnancy (P), or hormone (H). The P group animals underwent a full-term pregnancy, and H group animals were implanted subcutaneously with silastic capsules filled with ethynyl estradiol and megesterol acetate for 21 days. C and P animals were implanted with sham capsules. On day 21 capsules were removed, which was followed by a 49-day involution period, euthanasia, and mammary tissue collection. Global gene expression was measured using Rat Genome 230.2 Arrays. Histological analysis revealed that P and H treatments induced sustained morphological changes in the mammary gland with significantly increased percentages of mammary parenchyma and stromal tissues and higher ratio of stroma to parenchyma. Transcriptome analysis showed that P and H treatments induced sustained global changes in gene expression in the mammary gland. Analysis of commonly up- and downregulated genes in P and H relative to C treatment showed increased expression of three matrix metallopeptidases (Mmp3, 8, and 12), more differentiated mammary phenotype, enhanced innate and adaptive immunity, and reduced cell proliferation and angiogenic signatures. The sustained morphological and global gene expression changes in mammary tissue after pregnancy and hormone treatment may function together to provide the protective effect against breast cancer.

Dual energy computed tomography for quantification of tissue urate deposits in tophaceous gout: help from modern physics in the management of an ancient disease.

Gout has been recognized for centuries but is also a modern day scourge. It is the most common type of inflammatory arthritis in men and appears to be increasing in both incidence and prevalence (Arromdee et al. in J Rheumatol 29(11):2403-2406, 2002). Despite these facts, few advances have been made in the diagnosis and treatment of gout for over 50 years. Difficult cases of gout challenge available therapeutic options. It is only recently that the Food and Drug Administration has approved febuxostat as a treatment option for patients intolerant of allopurinol. We describe a difficult case of tophaceous gout notable for several reasons: utilization of rasburicase as uricolytic treatment to dramatically reduce tissue urate burden; treatment of gout flares with interleukin-1ß inhibition; and quantification of tissue urate with novel dual energy computed tomography technology before and after uricolytic therapy.

An app-enhanced cognitive fitness training program for athletes: The rationale and validation protocol.

The core dimensions of cognitive fitness, such as attention and cognitive control, are emerging through a transdisciplinary expert consensus on what has been termed the Cognitive Fitness Framework (CF2). These dimensions represent key drivers of cognitive performance under pressure across many occupations, from first responders to sport, performing arts and the military. The constructs forming the building blocks of CF2 come from the RDoC framework, an initiative of the US National Institute of Mental Health (NIMH) aimed at identifying the cognitive processes underlying normal and abnormal behavior. Similar to physical conditioning, cognitive fitness can be improved with deliberate practice. This paper reports the development of a prototype cognitive fitness training program for competitive athletes and the protocol for its evaluation. The program is focused on primary cognitive capacities and subtending skills for adjusting training rhythms and enhancing readiness for competition. The project is driven by the Australian Psychological Society's College of Sport & Exercise Psychology and includes the development of a Cognitive Gym program for a smartphone app-enhanced implementation. Its key building blocks are training protocols (drills) connected by a periodized training plan. A website with background supporting resources has also been developed as part of the project. National-level training squads will participate in a three-week pilot evaluation protocol, assessing the program's efficacy and usability through gamified cognitive assessment of participants' training gains and coaching staff evaluations, respectively. Both near and far transfer of training effects will be examined.

Advantages of CT Versus MRI for Preoperative Assessment of Osteochondritis Dissecans of the Capitellum.

BACKGROUND: Magnetic resonance imaging (MRI) is considered to be the gold standard for imaging of osteochondritis dissecans (OCD). PURPOSE/HYPOTHESIS: The purpose was to determine the additional value of a preoperative computed tomography (CT) scan in adolescent patients with capitellar OCD of the elbow. Consistent with the fact that OCD is a lesion involving the subchondral bone, the hypothesis was that CT would be superior to MRI for imaging OCD of the capitellum. STUDY DESIGN: Cohort study (diagnosis); Level of evidence, 3. METHODS: All patients being treated surgically for an OCD of the capitellum between 2006 and 2016 at one institution were reviewed for preoperative imaging. A total of 28 patients met the inclusion criteria. Corresponding MRI and CT scans were compared retrospectively. Multiple parameters were recorded, with special emphasis on OCD lesion size, fragmentation, and tilt as well as joint surface integrity, loose bodies, and osteophytes. RESULTS: The OCD lesions were best seen on CT scans, whereas MRI T1-weighted images overestimated and T2-weighted images underestimated the size of defects. A subchondral fracture nonunion was found on CT scans in 18 patients, whereas this was seen on MRI T1-weighted images in only 2 patients (P < .001) and MRI T2-weighted images in 4 patients (P < .001). Fragmentation of the OCD fragment was found on CT scans in 17 patients but on MRI scans in only 9 patients (P = .05). Osteophytes as a sign of secondary degenerative changes were seen on CT scans in 24 patients and were seen on MRI scans in 15 patients (P = .02). Altogether, only 51 of 89 secondary changes including loose bodies, effects on the radial head and ulnohumeral joint, and osteophytes that were seen on CT scans were also seen on MRI scans (P = .002). CONCLUSION: OCD fragmentation and secondary changes were more often diagnosed on CT. These factors indicate OCD instability or advanced OCD stages, which are indications for surgery. In an adolescent who is considered at risk for OCD (baseball, gymnastics, weightlifting, tennis) and who has lateral elbow joint pain with axial or valgus load bearing, CT is our imaging modality of choice for diagnosing and staging OCD of the capitellum.

Building a transdisciplinary expert consensus on the cognitive drivers of performance under pressure: An international multi-panel Delphi study.

Introduction: The ability to perform optimally under pressure is critical across many occupations, including the military, first responders, and competitive sport. Despite recognition that such performance depends on a range of cognitive factors, how common these factors are across performance domains remains unclear. The current study sought to integrate existing knowledge in the performance field in the form of a transdisciplinary expert consensus on the cognitive mechanisms that underlie performance under pressure. Methods: International experts were recruited from four performance domains [(i) Defense; (ii) Competitive Sport; (iii) Civilian High-stakes; and (iv) Performance Neuroscience]. Experts rated constructs from the Research Domain Criteria (RDoC) framework (and several expert-suggested constructs) across successive rounds, until all constructs reached consensus for inclusion or were eliminated. Finally, included constructs were ranked for their relative importance. Results: Sixty-eight experts completed the first Delphi round, with 94% of experts retained by the end of the Delphi process. The following 10 constructs reached consensus across all four panels (in order of overall ranking): (1) Attention; (2) Cognitive Control-Performance Monitoring; (3) Arousal and Regulatory Systems-Arousal; (4) Cognitive Control-Goal Selection, Updating, Representation, and Maintenance; (5) Cognitive Control-Response Selection and Inhibition/Suppression; (6) Working memory-Flexible Updating; (7) Working memory-Active Maintenance; (8) Perception and Understanding of Self-Self-knowledge; (9) Working memory-Interference Control, and (10) Expert-suggested-Shifting. Discussion: Our results identify a set of transdisciplinary neuroscience-informed constructs, validated through expert consensus. This expert consensus is critical to standardizing cognitive assessment and informing mechanism-targeted interventions in the broader field of human performance optimization.

Classifying variants of CDKN2A using computational and laboratory studies.

Variants in the CDKN2A tumor suppressor are associated with Familial Melanoma (FM), although for many variants the linkage is weak. The effects of missense variants on protein function and pathogenicity are often unclear. Multiple methods (e.g., laboratory, computational, epidemiological) have been developed to analyze whether a missense variant is pathogenic or not. It is not yet clear how to integrate these data types into a strategy for variant classification. We studied 51 CDKN2A missense variants using a cell cycle arrest assay. There was a continuum of results ranging from full wild-type effect through partial activity to complete loss of arrest. A reproducible decrease of 30% of cell cycle arrest activity correlated with FM association. We analyzed missense CDKN2A germline variants using a Bayesian method to combine multiple data types and derive a probability of pathogenicity. When equal to or more than two data types could be evaluated with this method, 22 of 25 FM-associated variants and 8 of 15 variants of uncertain significance were classified as likely pathogenic with >95% probability. The other 10 variants were classified as uncertain (probability 5-95%). For most variants, there were insufficient data to draw a conclusion. The Bayesian model appears to be a sound method of classifying missense variants in cancer susceptibility genes.

Genomic and Transcriptomic Analysis of Relapsed and Refractory Childhood Solid Tumors Reveals a Diverse Molecular Landscape and Mechanisms of Immune Evasion.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. SIGNIFICANCE: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

Genome-wide characterization of the SloR metalloregulome in Streptococcus mutans.

Streptococcus mutans is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the sloABCR metal ion transport operon in S. mutans. In the present study, we demonstrate that S. mutans coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the S. mutans chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional "class I" genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204 S. mutans genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of S. mutans virulence genes that are subject to transcriptional upregulation by SloR and noted that such "class II"-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating S. mutans-induced caries formation.

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases.

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from gamma-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k(obs) of over 2500min(-1).

Evaluation of intraneural ganglion cysts using three-dimensional fast spin echo-cube.

PURPOSE: To compare conventional two-dimensional fast spin echo (FSE) MRI sequences with a three-dimensional FSE extended echo train acquisition method, known as Cube, in the evaluation of intraneural ganglion cysts. Also, to demonstrate that Cube enables the consistent identification and thorough characterization of the cystic joint connection, and therefore improves patient care by superior preoperative planning. MATERIALS AND METHODS: Six patients with intraneural ganglia in the knee region (five involving the peroneal and one the tibial nerve) were evaluated using both conventional FSE MR sequences and the Cube sequence. Studies were interpreted by the consensus of three board certified musculoskeletal radiologists and one peripheral nerve neurosurgeon. Surgical correlation was available in five of the six cases. RESULTS: Both imaging methods demonstrated the cysts and at least part of their joint connections after variable amount of postprocessing. Cube proved superior to conventional imaging in its ability to acquire isotropic data that could easily be reconstructed in any plane and its ability to resolve fine anatomical details. CONCLUSION: Cube is a new MR pulse sequence that enables the consistent identification of the intraneural ganglion cyst joint connection. We believe that improved visualization and characterization of the entire cyst will improve patient outcomes by facilitating more accurate surgical intervention.

Methylation-controlled J protein promotes c-Jun degradation to prevent ABCB1 transporter expression.

Methylation-controlled J protein (MCJ) is a newly identified member of the DnaJ family of cochaperones. Hypermethylation-mediated transcriptional silencing of the MCJ gene has been associated with increased chemotherapeutic resistance in ovarian cancer. However, the biology and function of MCJ remain unknown. Here we show that MCJ is a type II transmembrane cochaperone localized in the Golgi network and present only in vertebrates. MCJ is expressed in drug-sensitive breast cancer cells but not in multidrug-resistant cells. The inhibition of MCJ expression increases resistance to specific drugs by inducing expression of the ABCB1 drug transporter that prevents intracellular drug accumulation. The induction of ABCB1 gene expression is mediated by increased levels of c-Jun due to an impaired degradation of this transcription factor in the absence of MCJ. Thus, MCJ is required in these cells to prevent c-Jun-mediated expression of ABCB1 and maintain drug response.

Utilization of gene profiling and proteomics to determine mineral pathogenicity in a human mesothelial cell line (LP9/TERT-1).

Identifying and understanding the early molecular events that underscore mineral pathogenicity using in vitro screening tests is imperative, especially given the large number of synthetic and natural fibers and particles being introduced into the environment. The purpose of the work described here was to examine the ability of gene profiling (Affymetrix microarrays) to predict the pathogenicity of various materials in a human mesothelial cell line (LP9/TERT-1) exposed to equal surface area concentrations (15 x 10(6) or 75 x 10(6) microm(2)/cm(2)) of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO(2)), or glass beads for 8 or 24 h. Since crocidolite asbestos caused the greatest number of alterations in gene expression, multiplex analysis (Bio-Plex) of proteins released from LP9/TERT-1 cells exposed to crocidolite asbestos was also assessed to reveal if this approach might also be explored in future assays comparing various mineral types. To verify that LP9/TERT-1 cells were more sensitive than other cell types to asbestos, human ovarian epithelial cells (IOSE) were also utilized in microarray studies. Upon assessing changes in gene expression via microarrays, principal component analysis (PCA) of these data was used to identify patterns of differential gene expression. PCA of microarray data confirmed that LP9/TERT-1 cells were more responsive than IOSE cells to crocidolite asbestos or nonfibrous talc, and that crocidolite asbestos elicited greater responses in both cell types when compared to nonfibrous talc, TiO(2), or glass beads. Bio-Plex analysis demonstrated that asbestos caused an increase in interleukin-13 (IL-13), basic fibroblast growth factor (bFGF), granulocyte colony-stimulating factor (G-CSF), and vascular endothelial growth factor (VEGF). These responses were generally dose-dependent (bFGF and G-CSF only) and tumor necrosis factor (TNF)-alpha independent (except for G-CSF). Thus, microarray and Bio-Plex analyses are valuable in determining early molecular responses to fibers/particles and may directly contribute to understanding the etiology of diseases caused by them. The number and magnitude of changes in gene expression or "profiles" of secreted proteins may serve as valuable metrics for determining the potential pathogenicity of various mineral types. Hence, alterations in gene expression and cytokine/chemokine changes induced by crocidolite asbestos in LP9/TERT-1 cells may be indicative of its increased potential to cause mesothelioma in comparison to the other nonfibrous materials examined.

The MDM2 inhibitor CGM097 combined with the BET inhibitor OTX015 induces cell death and inhibits tumor growth in models of neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Although less frequent, overexpression of MYC is similarly an indicator of poor prognosis. Most NB tumors initially respond to chemotherapy, however, most will relapse, resulting in chemoresistant disease. After relapse, there is growing evidence of p53 inactivation. MYC/MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. MDM2 inhibitors and Bromodomain and Extra-Terminal domain (BET) inhibitors have both shown promise in treating NB by increasing the expression of p53 and decreasing MYC/MYCN expression, respectively. Our study focuses on the combined treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) in neuroblastoma. METHODS: Two p53 wild-type and two p53 mutant established neuroblastoma cells lines were used to test this combination. Ray design assays were used to test whether this combination was synergistically cytotoxic to NB cells. Western blots were performed to check signaling pathways of interest after drug treatment. IncuCyte imaging and flow cytometry were utilized to quantify the apoptotic and cytostatic effects of these drugs on NB cells. In vivo studies were carried out to test the antitumor effect of this combination in a living host. RESULTS: The combination of CGM097 and OTX015 resulted in p53 activation, decreased expression of MYC family proteins and a subsequent synergistic increase in NB cell death. CONCLUSION: This study warrants further investigation into the combination of MDM2 inhibitors and BET inhibitors for the treatment in NB.