Two Hydrolase Resistant Analogues of Diadenosine 5′,5″′-P,P-Triphosphate for Studies with FHIT,The Human Fragile Histidine Triad Protein¶

Abstract

The design and synthesis of analogues of diadenosine 5′,5″′-P,P-triphosphate that are resistant to pyrophosphate hydrolysis is described in relation to their rôle in signalling and tumorigenesis involving the Fhit protein, the human fragile histidine triad protein, which is a novel Ap3A binding/cleaving protein.

     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

Effects of Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.     

Reliable Neuronal Systems: The Importance of Heterogeneity

For every engineer it goes without saying: in order to build a reliable system we need components that consistently behave precisely as they should. It is also well known that neurons, the building blocks of brains, do not satisfy this constraint. Even neurons of the same type come with huge variances in their properties and these properties also vary over time. Synapses, the connections between neurons, are highly unreliable in forwarding signals. In this paper we argue that both these fact add variance to neuronal processes, and that this variance is not a handicap of neural systems, but that instead predictable and reliable functional behavior of neural systems depends crucially on this variability. In particular, we show that higher variance allows a recurrently connected neural population to react more sensitively to incoming signals, and processes them faster and more energy efficient. This, for example, challenges the general assumption that the intrinsic variability of neurons in the brain is a defect that has to be overcome by synaptic plasticity in the process of learning.     

TGFβs Modulate Permeability of the Blood-Epididymis Barrier in an In Vitro Model

The blood-epididymis barrier (BEB) is formed by epithelial tight junctions mediating selective permeability of the epididymal epithelium. Defective barrier function can disturb the balance of the epididymal milieu, which may result in infertility. The stroma of the epididymis contains high amounts of cytokines of the TGFβ family of unknown function. We screened possible effects of all three TGFβ isoforms on paracellular tightness in a BEB in vitro model based on the strongly polarized mouse epididymal epithelial MEPC5 cells in the transwell system. In this model we found a robust transepithelial electrical resistance (TER) of about 840 Ω x cm². Effects on the paracellular permeability were evaluated by two methods, TER and FITC-Dextran-based tracer diffusion assays. Both assays add up to corresponding results indicating a time-dependent disturbance of the BEB differentially for the three TGFβ isoforms (TGFβ3>TGFβ1>TGFβ2) in a TGFβ-recetor-1 kinase- and Smad-dependent manner. The tight junction protein claudin-1 was found to be reduced by the treatment with TGFβs, whereas occludin was not influenced. Epididymal epithelial cells are predominantly responsive to TGFβs from the basolateral side, suggesting that TGFβ may have an impact on the epididymal epithelium from the stroma in vivo. Our data show for the first time that TGFβs decrease paracellular tightness in epididymal epithelial cells, thus establishing a novel mechanism of regulation of BEB permeability, which is elementary for sperm maturation and male fertility.     

Emotion recognition in children and adolescents with attention-deficit/hyperactivity disorder (ADHD)

Children with attention-deficit/hyperactivity disorder (ADHD) are impaired in social adaptation and display deficits in social competence. Deficient emotion recognition has been discussed to underlie these social problems. However, comorbid conduct problems have not been considered in the majority of studies conducted so far, and the influence of medication on emotion recognition has rarely been studied. Here, emotion recognition performance was assessed in children with ADHD without medication compared with children with ADHD under stimulant medication and a matched control group. In order to rule out confounding by externalizing symptoms, children with comorbid conduct problems were excluded. Video clips with neutral faces developing a basic emotion (happiness, sadness, disgust, fear and anger) were presented in order to assess emotion recognition. Results indicated between-group differences neither concerning the number of correctly identified emotions nor concerning reaction times and their standard deviations. Thus, we suggest that ADHD per se is not associated with deficits in emotion recognition.     

Focal DNA Copy Number Changes in Neuroblastoma Target MYCN Regulated Genes

Neuroblastoma is an embryonic tumor arising from immature sympathetic nervous system cells. Recurrent genomic alterations include MYCN and ALK amplification as well as recurrent patterns of gains and losses of whole or large partial chromosome segments. A recent whole genome sequencing effort yielded no frequently recurring mutations in genes other than those affecting ALK. However, the study further stresses the importance of DNA copy number alterations in this disease, in particular for genes implicated in neuritogenesis. Here we provide additional evidence for the importance of focal DNA copy number gains and losses, which are predominantly observed in MYCN amplified tumors. A focal 5 kb gain encompassing the MYCN regulated miR-17∼92 cluster as sole gene was detected in a neuroblastoma cell line and further analyses of the array CGH data set demonstrated enrichment for other MYCN target genes in focal gains and amplifications. Next we applied an integrated genomics analysis to prioritize MYCN down regulated genes mediated by MYCN driven miRNAs within regions of focal heterozygous or homozygous deletion. We identified RGS5, a negative regulator of G-protein signaling implicated in vascular normalization, invasion and metastasis, targeted by a focal homozygous deletion, as a new MYCN target gene, down regulated through MYCN activated miRNAs. In addition, we expand the miR-17∼92 regulatory network controlling TGFß signaling in neuroblastoma with the ring finger protein 11 encoding gene RNF11, which was previously shown to be targeted by the miR-17∼92 member miR-19b. Taken together, our data indicate that focal DNA copy number imbalances in neuroblastoma (1) target genes that are implicated in MYCN signaling, possibly selected to reinforce MYCN oncogene addiction and (2) serve as a resource for identifying new molecular targets for treatment.     

Serum Protein Signatures Differentiating Autoimmune Pancreatitis versus Pancreatic Cancer

Autoimmune pancreatitis (AIP) is defined by characteristic lymphoplasmacytic infiltrate, ductal strictures and a pancreatic enlargement or mass that can mimic pancreatic cancer (PaCa). The distinction between this benign disease and pancreatic cancer can be challenging. However, an accurate diagnosis may pre-empt the misdiagnosis of cancer, allowing the appropriate medical treatment of AIP and, consequently, decreasing the number of unnecessary pancreatic resections.Mass spectrometry (MS) and two-dimensional differential gel electrophoresis (2D-DIGE) have been applied to analyse serum protein alterations associated with AIP and PaCa, and to identify protein signatures indicative of the diseases. Patients'' sera were immunodepleted from the 20 most prominent serum proteins prior to further 2D-DIGE and image analysis. The identity of the most-discriminatory proteins detected, was performed by MS and ELISAs were applied to confirm their expression. Serum profiling data analysis with 2D-DIGE revealed 39 protein peaks able to discriminate between AIP and PaCa. Proteins were purified and further analysed by MALDI-TOF-MS. Peptide mass fingerprinting led to identification of eleven proteins. Among them apolipoprotein A-I, apolipoprotein A-II, transthyretin, and tetranectin were identified and found as 3.0-, 3.5-, 2-, and 1.6-fold decreased in PaCa sera, respectively, whereas haptoglobin and apolipoprotein E were found to be 3.8- and 1.6-fold elevated in PaCa sera. With the exception of haptoglobin the ELISA results of the identified proteins confirmed the 2D-DIGE image analysis characteristics. Integration of the identified serum proteins as AIP markers may have considerable potential to provide additional information for the diagnosis of AIP to choose the appropriate treatment.