Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of pre‐mRNA splicing. Similar to other classes of nucleic acid‐based therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinyl‐tetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strain‐promoted azide‐alkyne cycloaddition conjugation with splice‐switching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest splice‐switching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2K‐mdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant splice‐switching at very low nanomolar concentrations. The presented aminoethylene‐lipopeptides are thus a promising platform for the generation of PMO‐therapeutics with a favorable activity/toxicity profile.     

Experimental and numerical analysis of the influence of cable tray arrangements on the resulting mass loss rate and fire spreading

In the context of industrial buildings and power plants, electrical installations and cable trays represent a main fuel load and a potential initial fire source due to possible short circuits or comparable malfunction. Furthermore, a fire can spread from one tray to additional trays mounted above and/or horizontally on one tray. Because of the high significance of cable fires, several research projects have been carried out, investigating the fire behaviour of cables from small‐scale tests, eg, the cone calorimeter, up to large‐scale tests, analysing complete cable tray constructions. The goal of the work presented in this paper is the extension of the knowledge regarding the influence of geometrical parameters like the packing density and tray distance on the burning behaviour and fire spread of cable tray installations. The results are considered, together with test results from the literature, to quantify the main physical parameters describing the burning behaviour. In a next step, the general applicability of these parameters as input data for the parametrization of the source term of numerical simulations is shown. The test results show that the burning behaviour and the fire spreading highly depend on the cable arrangement of the cables on the cable tray, in combination with other boundary conditions. By applying the results as input for a fire simulation, the mass loss rate is considered appropriately.     

Length of maternity leave and quality of mother-infant interactions

The aim of this study was to assess the association between the length of maternity leave and the quality of mother-infant interactions; 198 employed mothers of 4-month-old infants were interviewed and videotaped in their homes during a feeding time. Hierarchical multiple regression analyses indicated a direct association between shorter length of leave and more negative affect and behavior in maternal interactions with their infants. Infant and mother stressor/protective variables added significantly in predicting the quality of the mother-infant relationship. There were also significant interaction effects between the length of leave and these variables. Mothers who either reported more depressive symptoms or who perceived their infant as having a more difficult temperament and who had shorter leaves, compared with mothers who had longer leaves, were observed to express less positive affect, sensitivity, and responsiveness in interactions with their infants. The public policy implications of the relation between length of maternity leave, maternal and infant individual differences, and the quality of mother-infant interactions are discussed.     

Patient characteristics, MRI examination, and dopaminergic drug response in clinical possible Parkinson''s disease

As hypersensitivity to natural rubber latex (NRL) has become an increasingly recognized problem in children, identification of all groups at risk seems important. In this study hypersensitivity to NRL was evaluated in 337 children with potential risk factors. We identified by questionnaire children's underlying diseases, history of surgical procedures, evidence of atopy and patient's history of NRL-specific reactions. Hypersensitivity to NRL and other allergens relevant in the Düsseldorf area was evaluated by skin prick test and specific IgE. In 9.2% of all children hypersensitivity to NRL was observed. Significant risk factors for hypersensitivity to NRL were, among the underlying diseases, spina bifida (odds ratio 29.2), hydrocephalus internus (10.1), gastrointestinal malformation (5.2) and atopy (2.2). Surgical procedures with significant risk were the implantation of a ventriculo-peritoneal shunt (15.7) and surgery of the gastrointestinal tract (3.1). Frequency of surgical procedures correlated (p < 0.001) with risk of hypersensitivity. Frequent surgery and atopy were found to have an additional effect on the risk of hypersensitivity. Information about previous NRL-specific reactions obtained by questionnaire was of little predictive value when performing multivariate analysis. For children at high risk for hypersensitivity to NRL preoperative evaluation and, in case of hypersensitivity, preventive measures seem to be advised.     

Synthesis of Hexp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH2)7 CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex = 3-O-methyl-beta-D-Gal, 3-deoxy-beta-D-Gal, 3-deoxy-3-fluoro-beta-D-Gal, 3-amino-3-deoxy-beta-D-Gal, beta-D-Gul, alpha-L-Alt, or beta-L-Gal

Seven analogues of the trisaccharide beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3 have been synthesized as potential substrates for glycosyltransferases involved in the chain-termination of N-acetyllactosamine-type N-glycans. These compounds include: 3-O-methyl-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp -(1-->O) (CH2)7CH3, 3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1 -->O) (CH2)7CH3, 3-deoxy-3-fluoro-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-M anp- (1-->O)(CH2)7Ch3, 3-amino-3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Ma np- (1-->O)(CH2)7CH3, beta-D-Gulp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-- >O)(CH2)7CH3, beta-L-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3, and alpha-L-Altp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1- ->O) (CH2)7CH3. All trisaccharides were obtained by condensation of suitably modified glycosyl donors based on imidates or thioglycosides with the same disaccharide acceptor, octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)-alpha-D-mannopyranoside, followed by deprotection.     

Cerebrovascular effects of analgosedation

The in vitro drug sensitivity of three dihydrofolate reductase inhibitors (pyrimethamine, cycloguanil, trimethoprim) was determined against 29 strains and isolates of Plasmodium falciparum by an isotopic semi-microtest. Trimethoprim is less active than pyrimethamine or cycloguanil and its activity is correlated with that of two other inhibitors, suggesting cross-resistance in vitro among the dihydrofolate reductase inhibitors.     

Somatic deletion of the imprinted 11p15 region in sporadic persistent hyperinsulinemic hypoglycemia of infancy is specific of focal adenomatous hyperplasia and endorses partial pancreatectomy

Sporadic persistent hyperinsulinemic hypoglycemia of infancy (PHHI) or nesidioblastosis is a heterogeneous disorder characterized by profound hypoglycemia due to inappropriate hypersecretion of insulin. An important diagnostic goal is to distinguish patients with a focal hyperplasia of islet cells of the pancreas (FoPHHI) from those with a diffuse abnormality of islets (DiPHHI) because management strategies differ significantly. 16 infants with sporadic PHHI resistant to diazoxide and who underwent pancreatectomy were investigated. Selective pancreatic venous sampling coupled with peroperative surgical examination and analysis of extemporaneous frozen sections allowed us to identify 10 cases with FoPHHI and 6 cases with DiPHHI. We show here that in cases of FoPHHI, but not those of DiPHHI, there was specific loss of maternal alleles of the imprinted chromosome region 11p15 in cells of the hyperplastic area of the pancreas but not in normal pancreatic cells. This somatic event is consistent with a proliferative monoclonal lesion. It involves disruption of the balance between monoallelic expression of several maternally and paternally expressed genes. Thus, we provide the first molecular explanation of the heterogeneity of sporadic forms of PHHI such that it is possible to perform only partial pancreatectomy, limited to the focal somatic lesion, so as to avoid iatrogenic diabetes in patients with focal adenomatous hyperplasia.     

Biological evaluation of two anomeric glucose analogues iodinated in position 6

Two anomeric analogues of glucose labelled with 123 iodine in position 6, proposed as tracers of glucose transport in vivo, have been synthesized: alpha- and beta-methyl-6-deoxy-6-iodo-D-glucopyranoside (alpha MDIG and beta MDIG). The aim of this study was to determine whether these molecules interact with the glucose transporter and whether they could be used as tracers of glucose transport in vivo. The biodistribution of alpha MDIG and beta MDIG was studied in the mouse in vivo. To determine if these two anomers enter the cell via the glucose transporter, their uptake was measured in isolated perfused rat hearts, in human erythrocytes in suspension, and in cardiomyocytes of neonatal rat in culture. Both alpha MDIG and beta MDIG had similar repartitions in the mouse: myocardial uptake averaged 7% of the injected dose/g of organ at 2 min postinjection and alpha MDIG competed with D-glucose to enter the cells. Insulin produced a 123% increase of its uptake in isolated perfused rat hearts and a 100% increase in cardiomyocytes of neonatal rat in culture. alpha MDIG uptake was lowered in the presence of glucose transport inhibitors in each experimental model. An interaction between beta MDIG and glucose transporters was observed only in human erythrocytes in suspension. Only alpha MDIG interacts with the glucose transporter, and thus could be used to estimate glucose transport in vivo.     

Immunohistochemical localization of p21(WAF1/CIP1) in normal, hyperplastic, and neoplastic uterine tissues

Assuming that the chemical reactions used to synthesize a combinatorial library member are successful, then knowledge of the specific reaction sequence is equivalent to knowing the member's chemical identity. Because the determination of chemical identity is typically not automatable and requires a substantial amount of material, schemes that encode a member's reaction history onto the reaction platform are of value. The primary benefits of encoding are relational nomenclature (all methods) and automated handling (some methods). Encoding methods evaluated to date are spatial, graphical, chemical, spectrometric, electronic, and physical.     

Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus

Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1+ strain in a murine model of aspergillosis.