Minimal representations of possibility at age 3.

Young children do not always consider alternative possibilities when planning. Suppose a prize is hidden in a single occluded container and another prize is hidden in an occluded pair. If given a chance to choose one container and receive its contents, choosing the singleton maximizes expected reward because each member of the pair might be empty. Yet, 3-y-olds choose a member of the pair almost half the time. Why don't they maximize expected reward? Three studies provide evidence that 3-y-olds do not deploy possibility concepts like MIGHT, which would let them represent that each container in the pair might and might not contain a prize. Rather, they build an overly specific model of the situation that correctly specifies that the singleton holds a prize while inappropriately specifying which member of the pair holds a prize and which is empty. So, when asked to choose a container, they see two equally good options. This predicts approximately 50% choice of the singleton, observed in studies 1 and 3. But when asked to throw away a container so that they can receive the remaining contents (study 2), they mostly throw away a member of the pair. The full pattern of data is expected if children construct overly specific models. We discuss whether 3-year-olds lack possibility concepts or whether performance demands prevent deployment of them in our tasks.

Chronic lymphocytic leukaemia induces an exhausted T cell phenotype in the TCL1 transgenic mouse model.

Although chronic lymphocytic leukaemia (CLL) is a B cell malignancy, earlier studies have indicated a role of T cells in tumour growth and disease progression. In particular, the functional silencing of antigen-experienced T cells, called T cell exhaustion, has become implicated in immune evasion in CLL. In this study, we tested whether T cell exhaustion is recapitulated in the TCL1(tg) mouse model for CLL. We show that T cells express high levels of the inhibitory exhaustion markers programmed cell death 1 (PDCD1, also termed PD-1) and lymphocyte-activation gene 3 (LAG3), whereas CLL cells express high levels of CD274 (also termed PD-ligand 1). In addition, the fraction of exhausted T cells increases with CLL progression. Finally, we demonstrate that exhausted T cells are reinvigorated towards CLL cytotoxicity by inhibition of PDCD1/CD274 interaction in vivo. These results suggest that T cell exhaustion contributes to CLL pathogenesis and that interference with PDCD1/CD274 signalling holds high potential for therapeutic approaches.

Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia.

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5'-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy.

AID induces intraclonal diversity and genomic damage in CD86(+) chronic lymphocytic leukemia cells.

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sµ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.

The knowledge ("true belief") error in 4- to 6-year-old children: When are agents aware of what they have in view?

The standard view on explicit theory of mind development holds that children around the age of 4 years start to ascribe beliefs to themselves and others, typically tested with false belief (FB) tasks. The present study (N = 95, 53 female, 41 male, Austrian, 41 to 80 months) systematically investigated the puzzling phenomenon that FB achievers (FB+) fail knowledge (often subsumed under "true belief") tasks: Despite the story protagonist witnessing the displacement of an object these children predict that the protagonist will look for it in its original location. We replicate this result in Experiment 1. Interestingly, some of our children indicated uncertainty about the protagonist's awareness of the relevant event. Thus, in Experiment 2 a new active watching condition was designed to help children understand that the protagonist attended to the critical event. This practically eradicated the knowledge error. Experiment 3 successfully replicated these results. Implications for existing explanations, perceptual access reasoning (PAR, Fabricius, Boyer, Weimer, & Carroll, 2010) and pragmatic difficulties (Oktay-Gür & Rakoczy, 2017) are discussed.

HAX1 deficiency: impact on lymphopoiesis and B-cell development.

HAX1 was originally described as HS1-associated protein with a suggested function in receptor-mediated apoptotic and proliferative responses of lymphoid cells. Recent publications refer to a complex and multifunctional role of this protein. To investigate the in vivo function of HAX1 (HS1-associated protein X1) in B cells, we generated a Hax1-deficient mouse strain. Targeted deletion of Hax1 resulted in premature death around the age of 12 wk accompanied by a severe reduction of lymphocytes in spleen, thymus and bone marrow. In the bone marrow, all B-cell populations were lost comparably. In the spleen, B220(+) cells were reduced by almost 70%. However, as investigated by adoptive transfer experiments, this impairment is not exclusively B-cell intrinsic and we hypothesize that a HAX1-deficient environment cannot sufficiently provide the essential factors for proper lymphocyte development, trafficking and survival. Hax1(-/-) B cells show a significantly reduced expression of CXCR4, which might have an influence on the observed defects in B-cell development.

Why Do Children Who Solve False Belief Tasks Begin to Find True Belief Control Tasks Difficult? A Test of Pragmatic Performance Factors in Theory of Mind Tasks.

The litmus test for the development of a metarepresentational Theory of Mind is the false belief (FB) task in which children have to represent how another agent misrepresents the world. Children typically start mastering this task around age four. Recently, however, a puzzling finding has emerged: Once children master the FB task, they begin to fail true belief (TB) control tasks. Pragmatic accounts assume that the TB task is pragmatically confusing because it poses a trivial academic test question about a rational agent's perspective; and we do not normally engage in such discourse about subjective mental perspectives unless there is at least the possibility of error or deviance. The lack of such an obvious possibility in the TB task implicates that there might be some hidden perspective difference and thus makes the task confusing. In the present study, we test the pragmatic account by administering to 3- to 6-year-olds (N = 88) TB and FB tasks and structurally analogous true and false sign (TS/FS) tasks. The belief and sign tasks are matched in terms of representational and metarepresentational complexity; the crucial difference is that TS tasks do not implicate an alternative non-mental perspective and should thus be less pragmatically confusing than TB tasks. The results show parallel and correlated development in FB and FS tasks, replicate the puzzling performance pattern in TB tasks, but show no trace of this in TS tasks. Taken together, these results speak in favor of the pragmatic performance account.

AID Contributes to Accelerated Disease Progression in the TCL1 Mouse Transplant Model for CLL.

Adaptive somatic mutations conferring treatment resistance and accelerated disease progression is still a major problem in cancer therapy. Additionally in CLL, patients receiving novel, efficient drugs frequently become treatment refractory and eventually relapse. Activation-induced deaminase (AID) is a cytosine deaminase that catalyzes somatic hypermutation of genomic DNA at the immunoglobulin locus in activated B cells. As considerable off-target mutations by AID have been discerned in chronic lymphocytic leukemia, it is essential to investigate to which extent these mutations contribute to disease progression to estimate whether AID inhibition could counteract drug resistance mechanisms. In this study, we examined the TCL1 mouse model for CLL on an AID pro- and deficient background by comparing disease development and mutational landscapes. We provide evidence that AID contributes to the acquisition of somatic cancer-specific mutations also in the TCL1 model and accelerates CLL development particularly in the transplant setting. We conclude that AID is directly determining the fitness of the CLL clone, which prompts further studies to assess the effect of AID inhibition on the occurrence of drug resistance.

Mental files theory of mind: When do children consider agents acquainted with different object identities?

Mental files theory explains why children pass many perspective taking tasks like the false belief test around age 4 (Perner & Leahy, 2016). It also explains why older children struggle to understand that beliefs about an object depend on how one is acquainted with it (intensionality or aspectuality). If Heinz looks at an object that is both a die and an eraser, but cannot tell by looking that it is an eraser, he will not reach for it if he needs an eraser. Four- to 6-year olds find this difficult (Apperly & Robinson, 1998). We tested 129 35- to 86-month olds with a modified version of Apperly and Robinson's task. Each child faced four tasks resulting from two experimental factors, timing and mode of information. Timing: Children saw Heinz learn the die's location either before or after they learn that the die is an eraser. Mode of information: Heinz learns where the die is either perceptually or verbally. When Heinz' learning is verbal, he never perceives the die at all. We found that Apperly and Robinson's problem occurs only in the seen-after condition, where Heinz sees the die afterchildren had learnt that it was also an eraser. It vanishes when Heinz learns where the die is before children learn that it is also an eraser. The problem also vanishes when Heinz learns where the die is purely verbally (e.g., "The die is in the red box") and never sees it. This evidence lets us refine existing mental files theory, and eliminate several alternatives from the literature.

Multifocal motor neuropathy in Austria: a nationwide survey of clinical features and response to treatment.

BACKGROUND AND OBJECTIVES: Multifocal motor neuropathy (MMN) is a rare neuropathy and detailed descriptions of larger patient cohorts are scarce. The objective of this study was to evaluate epidemiological, clinical, and laboratory features of MMN patients and their response to treatment in Austria and to compare these data with those from the literature. METHODS: Anonymized demographic and clinical data about MMN patients until 31.12.2017 were collected from registered Austrian neurologists. Exploratory statistics on clinical and laboratory features as well as treatment regimens and responses were performed. RESULTS: 57 Patients with MMN were identified, resulting in a prevalence of 0.65/100.000. Mean age of onset was 44.1 ± 13.1 years, the diagnostic delay 5.5 ± 8.4 years. In 77% of patients, symptom onset was in the upper limbs, and in 92%, it occurred in distal muscles. Proximal onset was never observed in the lower limbs. At the final follow-up, the majority of patients had atrophy (88%) in affected regions. Definite motor conduction blocks (CB) were found in 54 patients. Anti-GM1-IgM antibodies were present in 43%. Treatment with intravenous immunoglobulins improved muscle strength and INCAT score initially, but at last follow-up, both scores deteriorated to values before treatment. DISCUSSION: The findings of the present study corroborate the previous findings in MMN. Onset typically occurs in the upper limbs and mostly distal, CBs are found in the majority of cases, while anti-GM1-IgM antibodies are detected in only approximately 40%. Our study underlines that the initial good response to treatment fades over time.

A novel sporadic mutation G14739A of the mitochondrial tRNA(Glu) in a girl with exercise intolerance.

We describe a 7-year-old girl who presented with loss of appetite, weakness and excercise intolerance. Enzyme investigation of the respiratory chain in muscle tissue revealed a combined complex I, III and IV deficiency. A novel heteroplasmic G-->A exchange at nucleotide position 14739 was found in the MTTE gene of the tRNA glutamic acid. The mutation load in muscle was 72%, urine sediment 38%, blood 31% and fibroblasts 29% and it correlated with COX-negative fibres. Our patient presented with a predominantly myopathic phenotype. The G14739A mutation is the third reported in the mitochondrial tRNA glutamic acid gene, and it occurred in a sporadic case.

Mental files and belief: A cognitive theory of how children represent belief and its intensionality.

We provide a cognitive analysis of how children represent belief using mental files. We explain why children who pass the false belief test are not aware of the intensionality of belief. Fifty-one 3½- to 7-year old children were familiarized with a dual object, e.g., a ball that rattles and is described as a rattle. They observed how a puppet agent witnessed the ball being put into box 1. In the agent's absence the ball was taken from box 1, the child was reminded of it being a rattle, and emphasising its being a rattle it was put back into box 1. Then the agent returned, the object was hidden in the experimenter's hands and removed from box 1, described as a "rattle," and transferred to box 2. Children who passed false belief had no problem saying where the puppet would look for the ball. However, in a different condition in which the agent was also shown that the ball was a rattle they erroneously said that the agent would look for the ball in box 1, ignoring the agent's knowledge of the identity of rattle and ball. Their problems cease with their mastery of second-order beliefs (she thinks she knows). Problems also vanish when the ball is described not as a rattle but as a thing that rattles. We describe how our theory can account for these data as well as all other relevant data in the literature.

AID/APOBEC deaminases and cancer.

Mutations are the basis for evolution and the development of genetic diseases. Especially in cancer, somatic mutations in oncogenes and tumor suppressor genes alongside the occurrence of passenger mutations have been observed by recent deep-sequencing approaches. While mutations have long been considered random events induced by DNA-replication errors or by DNA damaging agents, genome sequencing led to the discovery of non-random mutation signatures in many human cancer. Common non-random mutations comprise DNA strand-biased mutation showers and mutations restricted to certain DNA motifs, which recently have become attributed to the activity of the AID/APOBEC family of DNA deaminases. Hence, APOBEC enzymes, which have evolved as key players in natural and adaptive immunity, have been proposed to contribute to cancer development and clonal evolution of cancer by inducing collateral genomic damage due to their DNA deaminating activity. This review focuses on how mutagenic events through AID/APOBEC deaminases may contribute to cancer development.

Lysine residue at position 22 of the AID protein regulates its class switch activity.

BACKGROUND: Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells. CONCLUSIONS/SIGNIFICANCE: Our results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity.

Severe depletion of mitochondrial DNA in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder in childhood leading to a dramatic loss of muscle strength. Functional investigations with high-resolution polarography and enzyme measurements of the respiratory chain revealed lowered activities in muscle tissue of SMA patients. To gain a better understanding of this low energy supply we analyzed the amount of mitochondrial DNA (mtDNA) in skeletal muscle of 20 unrelated children with genetically proven SMA and 31 controls. Quantitative Southern blot analysis revealed a severe and homogeneous decrease in the content of muscle mtDNA in relation to nuclear DNA in SMA patients (90.3+/-7.8%), whereas by immunofluorescence no decrease in the number of mitochondria was detected. In addition, a two- to threefold reduction of the nuclear-encoded complex II (succinate dehydrogenase) activity was detected in SMA muscle tissue. Western blot analysis showed a significant reduction of both mitochondrial- and nuclear-encoded cytochrome c oxidase subunits. Our results indicate that mtDNA depletion in SMA is a consequence of severe atrophy, and has to be differentiated by measurement of complex II from an isolated reduction of mtDNA as found in patients with mitochondriocytopathies and the so-called mtDNA depletion syndrome.

Galanin and galanin receptors in human gliomas.

Galanin-like immunoreactivity (GAL-LI) and specific GAL binding sites have been shown to be widely distributed in the central nervous system (CNS) and in CNS tumors. GAL and its receptors have also been shown to be present in glial cells, but to date it is still unknown whether human gliomas produce GAL and express GAL receptors. In this study 20 brain tumors consisting of 15 glioblastomas, 4 meningiomas and 1 gliosarcoma were investigated for the presence of GAL-LI and GAL receptors. Immunofluorescence analysis revealed a dense network of GAL-LI positive cellular processes and cell bodies in 18 of the 20 tumors. In contrast, in vitro (125)I-labeled GAL receptor autoradiography showed substantial GAL binding in only 6 glioblastoma tissues. Reverse transcription-PCR analysis detected mRNA of all three known galanin receptors in the tumor tissues, with most tumors expressing multiple receptor subtypes. Pharmacological analysis of tumor membrane homogenates with GAL and the specific GAL receptor GalR2 agonist, AR-M1896, revealed that the GAL receptor GalR1 is most likely the receptor responsible for the observed GAL binding in the glioblastomas. No correlation could be found between GAL-LI, the level of GAL binding and proliferative activity as determined by immunostaining with the cell proliferation marker Ki-67.

Fluorescent in situ hybridization on isolated tumor cell nuclei: a sensitive method for 1p and 19q deletion analysis in paraffin-embedded oligodendroglial tumor specimens.

In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP-Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P =.001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.