Analysis of primary Wilms tumors for WT1 RNA processing alterations

Wilms tumor (WT) is an embryonal renal tumor with a heterogeneous genetic etiology that serves as a valuable model for studying tumorigenesis. Biallelic inactivation of the tumor suppressor gene WT1, a zinc-finger transcriptional regulator located at 11p13, is critical for the development of some Wilms tumors. Interestingly, WT1 genomic analysis has demonstrated mutations in less than 20% of WT cases. This suggests either other genes play a more major role in Wilms tumorigenesis or WT1 is functionally altered by mechanisms other than DNA mutation. Previous observations in rat and in WT xenograft cell lines have suggested that abnormal WT1 RNA processing (exon 6 RNA editing and aberrant exon 2 splicing, respectively) is a potential mechanism of altering WT1 function in the absence of a WT1 DNA mutation. However, the role of this abnormal RNA processing has not previously been assessed in primary Wilms tumors. ^ To test the hypothesis that abnormal WT1 RNA processing is a mechanism of WT1alteration during tumor development, WT1 RNA from 85 primary tumors was analyzed using reverse transcription and polymerase chain reaction amplification (RT-PCR). Although no evidence for WT1 RNA editing was observed, variable levels (5% to 50%) of aberrant WT1 exon 2 splicing were detected for 11 tumors in the absence of a detectable WT1 DNA mutation. Also, alteration of normal WT1 alternative splicing, observed as RNA isoform loss, was detected in five tumors with no apparent WT1 genomic alteration, although no consistent pattern of RNA isoform loss was detected. This abnormal WT1 splicing, detected by either loss of exon 2 from some of the transcripts or loss of RNA isoforms, is statistically correlated with relapse (p = 0.005). These studies demonstrate that abnormal WT1 RNA processing is not a common mechanism of abrogating normal WT1 function in primary tumors. However, in those cases in which abnormal WTI splicing is present, these data indicate that it may serve as a useful prognostic marker for relapse in WT patients. ^

THE ISOLATION AND IDENTIFICATION OF O-ACETYLATED SIALIC ACIDS ON HUMAN CELLS (NEURAMINIC ACIDS, PAPER CHROMATOGRAPHY, HUMAN MALIGNANCY, BIOSYNTHETIC LABELING)

Unlike most carbohydrates, sialic acids have a restricted distribution in nature, being present in higher animals and in certain bacteriae. Unfortunately, most studies have not taken into account the fact that the parent sialic acid molecules, N-acetyl(or N-glycolyl)-neuraminic acid can be O-substituted at the 4, 7, 8 and 9 positions, generating many compounds and isomers. The approach and results of this research study demonstrates that proportions of non-, mono-, di-, and tri-O-acetylated sialic acids can be identified and quantitated on normal and malignant human cells. This was accomplished using a paper chromatographic technique to isolate and resolve individual species of non and O-substituted sialic acids. The chemical nature of these O-substituents, as an acetyl ester, was determined on the basis of chemical degradation, enzymatic and fast atom bombardment-mass spectrometry analysis.^ The working hypothesis of this study, that O-acetylated sialic acids are expressed in a restricted manner on normal and malignant cells, was confirmed using the above experimental approach; which identified mono-, di-, and tri-O-acetylated sialic acids on a variety of normal and malignant human cells. These O-acetylated sialic acids were expressed in restricted manner on subpopulations and subcellular fractions of PHL melanoma cells. Aberrant expression of O-acetylated sialic acids was associated with adenocarcinoma of the colon, leading to a nearly complete loss of di- and tri-O-acetylated sialic acids.^ Thus, the ability to isolate and identify biosynthetically radiolabeled O-acetylated sialic acids offers an efficient method of monitoring the expression of O-acetylated sialic acids in biochemical and cellular interactions. Furthermore, the ability to identify abnormal ratios of O-acetylated sialic acids in the human colon, represents a possible diagnostic tool to evaluate and identify patients who may be genetically or culturally predisposed to the development of adenocarcinoma of the colon. ^

Disorganization and timing of motor behavior : insight from gesture impairments and movement patterns in schizophrenia

Background: Motor symptoms are frequent phenomena across the entire course of schizophrenia1. Some have argued that disorganized behavior was associated with aberrant motor behavior. We have studied the association of motor disturbances and disorganization in two projects focusing on the timing of movements. Method: In two studies, we assessed motor behavior and psychopathology. The first study applied a validated test of upper limb apraxia in 30 schizophrenia patients2,3. We used standardized video assessments of hand gestures by a blinded rater. The second study tested the stability of movement patterns using time series analysis in actigraphy data of 100 schizophrenia patients4. Both stability of movement patterns and the overall amount of movement were calculated from data of two hours with high degrees of social interaction comparable across the 100 subjects. Results: In total, 67% of the patients had gesture performance deficits3. Most frequently, they made spatial, temporal and body-part-as-object errors. Gesture performance relied on frontal lobe function2. Poor gesture performance was associated with increased disorganization scores. In the second study, we found disorganization to be predicted only by more irregular movement patterns irrespective of the overall amount of movement4. Conclusion : Both studies provide evidence for a link between aberrant timing of motor behavior and disorganization. Disturbed movement control seems critical for disorganized behavior in schizophrenia.

Myoepithelioma of the cerebellopontine angle: a previously not documented benign salivary gland-type neoplasm within the cranium

Myoepithelioma is a dimorphic neoplasm with contractile-epithelial phenotype, originally interpreted as deriving from, but not actually restricted to the salivary glands. As a novel addition to the list of exquisitely rare intracranial salivary gland-type tumors and tumor-like lesions, we report on an example of myoepithelioma encountered in the left cerebellopontine angle of a 32-year-old male. Clinically presenting with ataxia and dizziness, this extraaxial mass of 4 × 3.5 × 3 cm was surgically resected, and the patient is alive 6 years postoperatively. Histologically, the tumor exhibited a continuum ranging from compact fascicles of spindle cells to epithelial nests and trabeculae partitioned by hyalinized septa, while lacking tubular differentiation. Regardless of architectural variations, there was robust immunoexpression of S100 protein, smooth muscle actin, GFAP, cytokeratin, and vimentin. Cytologic atypia tended to be modest throughout, and the MIB1 labeling index averaged less than 1%. Fluorescent in situ hybridization indicated no rearrangement of the EWSR1 locus. We interpret these results to suggest that myoepithelioma of the posterior fossa - along with related salivary epithelial tumors in this ostensibly incongruous locale - may possibly represent analogous neoplasms to their orthotopic counterparts, ones arising within aberrant salivary anlagen. The presence of the latter lends itself to being mechanistically accounted for by either postulating placodal remnants in the wake of branchial arch development, or linking them to exocrine glandular nests within endodermal cysts. Alternatively, myoepithelioma at this site could be regarded as a non tissue-specific lesion similar to its relatives ubiquitously occurring in the soft parts.

Right aortic arch and Kommerell's diverticulum associated with acute aortic dissection and pericardial tamponade

The right-sided aorta associated with an aberrant left subclavian artery is a rare anomaly of the aortic branches in the upper mediastinum. We present a 62-year-old patient suffering from an acute dissection of the ascending aorta associated with hemopericardium. In this case, there was also aneurysmal dilatation of the origin of the left subclavian artery, known as diverticulum of Kommerell.

The role of learning-related dopamine signals in addiction vulnerability

Dopaminergic signals play a mathematically precise role in reward-related learning, and variations in dopaminergic signaling have been implicated in vulnerability to addiction. Here, we provide a detailed overview of the relationship between theoretical, mathematical, and experimental accounts of phasic dopamine signaling, with implications for the role of learning-related dopamine signaling in addiction and related disorders. We describe the theoretical and behavioral characteristics of model-free learning based on errors in the prediction of reward, including step-by-step explanations of the underlying equations. We then use recent insights from an animal model that highlights individual variation in learning during a Pavlovian conditioning paradigm to describe overlapping aspects of incentive salience attribution and model-free learning. We argue that this provides a computationally coherent account of some features of addiction.

XIAP Restricts TNF- and RIP3-Dependent Cell Death and Inflammasome Activation

X-linked inhibitor of apoptosis protein (XIAP) has been identified as a potent regulator of innate immune responses, and loss-of-function mutations in XIAP cause the development of the X-linked lymphoproliferative syndrome type 2 (XLP-2) in humans. Using gene-targeted mice, we show that loss of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3 but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap−/− mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1β secretion in response to TNF, which might contribute to hyperinflammation in patients with XLP-2.

Mechanistic aspects of NMD in human cells

Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). To get more insight into the recruitment of the central NMD factor UPF1 to target mRNAs, we mapped transcriptome-wide UPF1-binding sites by individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) in human cells and found that UPF1 preferentially associated with 3′ UTRs in translationally active cells but underwent significant redistribution toward coding regions (CDS) upon translation inhibition. This indicates that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. Corroborated by RNA immunoprecipitation and by UPF1 cross-linking to long noncoding RNAs, our evidence for translation-independent UPF1-RNA interaction suggests that the triggering of NMD occurs after UPF1 binding to mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. Unlike in yeast, in mammalian cells NMD has been reported to be restricted to cap-binding complex (CBC)–bound mRNAs during the pioneer round of translation. However, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and show that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

Mechanistic aspects of mRNA targeting for nonsense-mediated mRNA decay in human cells

The nonsense-mediated mRNA decay (NMD) pathway is best known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with ORF-truncating premature termination codons (PTCs), but a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD substrates. We try to decipher the mechanism of mRNA targeting to the NMD pathway in human cells. Recruitment of the conserved RNA-binding helicase UPF1 to target mRNAs has been reported to occur through interaction with release factors at terminating ribosomes, but evidence for translation-independent interaction of UPF1 with the 3’ untranslated region (UTR) of mRNAs has also been reported. We have transcriptome-wide determined the UPF1 binding sites by individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) in human cells, untreated or after inhibiting translation. We detected a strongly enriched association of UPF1 with 3’ UTRs in undisturbed, translationally active cells. After translation inhibition, a significant increase in UPF1 binding to coding sequence (CDS) was observed, indicating that UPF1 binds RNA before translation and gets displaced from the CDS by translating ribosomes. This suggests that the decision to trigger NMD occurs after association of UPF1 with mRNA, presumably through activation of RNA-bound UPF1 by aberrant translation termination. In a second recent study, we re-visited the reported restriction of NMD in mammals to the ‘pioneer round of translation’, i.e. to cap-binding complex (CBC)-bound mRNAs. The limitation of mammalian NMD to early rounds of translation would indicate a – from an evolutionary perspective – unexpected mechanistic difference to NMD in yeast and plants, where PTC-containing mRNAs seem to be available to NMD at each round of translation. In contrast to previous reports, our comparison of decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells revealed that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.

eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E‐bound mRNPs are substrates for nonsense‐mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

eIF4E‐bound mRNPs are substrates for nonsense‐mediated mRNA decay in mammalian cells

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 would imply a similar basic mechanism of PTC recognition in all eukaryotes. However, unlike NMD in yeast, which targets PTC-containing mRNAs irrespectively of whether their 5' cap is bound by the cap-binding complex (CBC) or by the eukaryotic initiation factor 4E (eIF4E), mammalian NMD has been claimed to be restricted to CBC-bound mRNAs during the pioneer round of translation. In our recent study we compared decay kinetics of two NMD reporter systems in mRNA fractions bound to either CBC or eIF4E in human cells. Our findings reveal that NMD destabilizes eIF4E bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event. Additionally, our results indicate that the closed loop structure of mRNA forms only after the replacement of CBC with eIF4E at the 5' cap.

Brief Report: How Adolescents with ASD Process Social Information in Complex Scenes. Combining Evidence from Eye Movements and Verbal Descriptions

We investigated attention, encoding and processing of social aspects of complex photographic scenes. Twenty-four high-functioning adolescents (aged 11–16) with ASD and 24 typically developing matched control participants viewed and then described a series of scenes, each containing a person. Analyses of eye movements and verbal descriptions provided converging evidence that both groups displayed general interest in the person in each scene but the salience of the person was reduced for the ASD participants. Nevertheless, the verbal descriptions revealed that participants with ASD frequently processed the observed person’s emotion or mental state without prompting. They also often mentioned eye-gaze direction, and there was evidence from eye movements and verbal descriptions that gaze was followed accurately. The combination of evidence from eye movements and verbal descriptions provides a rich insight into the way stimuli are processed overall. The merits of using these methods within the same paradigm are discussed.

Eukaryotic Initiation Factor 4G Suppresses Nonsense-Mediated mRNA Decay by Two Genetically Separable Mechanisms

Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.