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.     

Microscopic simulation of electronic noise in semiconductormaterials and devices

We present a microscopic interpretation of electronic noise in semiconductor materials and two-terminal devices. The theory is based on Monte Carlo simulations of the carrier motion self-consistently coupled with a Poisson solver. Current and voltage noise operations are applied and their respective representations discussed. As application we consider the cases of homogeneous materials, resistors, n⁺nn ⁺ structures, and Schottky-barrier diodes. Phenomena associated with coupling between fluctuations in carrier velocity and self-consistent electric field are quantitatively investigated for the first time. At increasing applied fields hot-carrier effects are found to be of relevant importance in all the cases considered here. As a general result, noise spectroscopy is found to be a source of valuable information to investigate and characterize transport properties of semiconductor materials and devices     

Elevated temperature performance of pseudomorphic AlGaAs/InGaAsMODFETs

Parametric DC measurements on pseudomorphic AlGaAs/InGaAs modulation-doped field-effect transistors (MODFETs) were carried out over the 300-405 K temperature range. A gradual channel device model was developed to simulate the temperature dependent behavior and assist in the interpretation of the characteristics. The simulations are shown to provide good predictive ability and confirm the physical reasons why the zero temperature coefficient point of a MODFET occurs only for gate bias voltages below the threshold voltage     

Inhibition of 2-nitropropane-induced rat liver DNA and RNA damage by benzyl selenocyanate

We observed that pretreatment of male F344 rats with benzyl selenocyanate, a versatile organoselenium chemopreventive agent in several animal model systems, decreases the levels of DNA and RNA modifications produced in the liver by the hepatocarcinogen 2-nitropropane. To clarify the mechanisms involved, we pretreated male F344 rats with either benzyl selenocyanate, its sulfur analog benzyl thiocyanate, phenobarbital or cobalt protoporphyrin IX; the latter is a depletor of P450. We then determined (1) the ability of liver microsomes to denitrify 2-nitropropane, (2) effects on 2-nitropropane-induced liver DNA and RNA modifications and (3) amount of nitrate excreted in rat urine following administration of the carcinogen. Pretreatment with benzyl selenocyanate or phenobarbital increased the denitrification activity of liver microsomes by 217 and 765%, respectively, increased liver P4502B1 by 31- and 435-fold, respectively, decreased the levels of 2-nitropropane-induced modifications in liver DNA (29-70% and 17-30%, respectively) and RNA (67-85% and 30-50%, respectively), and increased the 24-h urinary excretion of nitrate by 157 and 209%, respectively. Pretreatment with benzyl thiocyanate had no significant effect on any of these parameters. Pretreatment with cobalt protoporphyrin IX decreased liver P4502B 1 by 87%, decreased the denitrification activity of liver microsomes by 76%, decreased the 24 h urinary excretion of nitrate by 88.5%, but increased the extent of 2-nitropropane-induced liver nucleic acid modifications by 17-67%. These results indicate that the metabolic sequence from 2-nitropropane to the reactive species causing DNA and RNA modifications does not involve the removal of the nitro group. Moreover, they suggest that benzyl selenocyanate inhibits 2-NP-induced liver nucleic acid modifications in part by increasing its detoxication through induction of denitrification, although it is evident that other mechanisms must also be involved.     

Colosplenic fistula in a patient treated with interleukin-2 for malignant melanoma

Interleukin-2 (IL-2) therapy often causes gastrointestinal side effects and at least 8 cases of bowel perforation have been reported. The patient reported here developed a colosplenic fistula, diagnosed by CT, with no neoplastic involvement of these organs. Awareness of these complications of IL-2 can help lead to earlier diagnosis.     

Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines

Certain bacterial immunostimulatory (i.s.) DNA sequences containing unmethylated CpG motifs stimulate antigen-presenting cells (APC) to express a full complement of costimulatory molecules and to produce cytokines including interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha. While IL-12 is key to their T helper cell (Th)1-promoting adjuvant activity, secretion of toxic levels of TNF-alpha is harmful in that it promotes toxic shock. Given the beneficial as well as harmful consequences of i.s. DNA, we investigated the possibility of identifying DNA sequences, i.e. CpG oligodeoxynucleotides (ODN) which differentially activate IL-12 versus TNF-alpha cytokine production in APC. Here, we describe an i.s. DNA sequence with these characteristics. While its potential to induce IL-12 is preserved, its ability to trigger TNF-alpha release is strongly curtailed both in vitro and in vivo. I.s. DNA could be segregated into lethal and non-lethal in a mouse toxic shock model. The non-toxic i.s. DNA was useful as an adjuvant, thus allowing cytotoxic T cell responses to the soluble protein ovalbumin and conferring a resistant Th 1 phenotype to BALB/c mice lethally infected with Leishmania major. This i.s. CpG motif may thus be prototypic for a useful immunostimulating DNA sequence that lacks harmful side effects.     

Mutagenic and antimutagenic activities of human whole blood, plasma and albumin

The seeds of Crepis capillaris were used to examine the mutagenic und antimutagenic properties of human whole blood, plasma, serum albumin and gamma-globulin by recording chromosomal and chromatid aberrations. The antimutagenic activity was determined by preliminary, simultaneous, and subsequent biosubstrate treatments of the seeds. The whole and twice-diluted blood, as well as plasma, induced aberrations exceeding the level of self-arbitrary mutagenesis by 3.7-5.3 and 2.6-3.1 times, respectively. When the blood was diluted to its 20% concentration, the antimutagenic efficiency of biological fluids was recorded. Human serum albumin and gamma-globulin were found to have an antimutagenic effect. In the dilutions having no antimutagenic effect, blood, plasma, and albumin showed their ability to be effectively decrease the level of induced aberrations.     

The effects of treatment with PIXY321 (GM-CSF/IL-3 fusion protein) on human polymorphonuclear leukocyte function

During a phase I trial of the genetically engineered hematopoietic growth factor PIXY321 (granulocyte-macrophage colony-stimulating factor/interleukin-3 [IL-3] fusion protein), we examined the effects of PIXY321 treatment on human polymorphonuclear leukocyte (PMN) locomotive, respiratory burst, and phagocytic responses. PIXY321 treatment was associated with transient suppression of both unstimulated locomotion and chemotaxis responses to multiple stimuli, as well as significant transient enhancement of formyl peptide-stimulated H2O2 production. No effects on opsonic phagocytosis of Staphylococcus aureus were observed. In vitro exposure of control PMN to PIXY321 resulted in suppression of unstimulated locomotion/chemotaxis and enhancement of formyl peptide-stimulated H2O2 production but had no effects on phagocytosis. When patient cells were exposed in vitro to PIXY321 during treatment, suppression of chemotaxis and enhancement of H2O2 production were observed before PIXY321 treatment, but these effects diminished during treatment. The in vivo and in vitro exposure effects of PIXY321 treatment on PMN function are similar to those of the parent molecule, granulocyte-macrophage colony-stimulating factor (GM-CSF).     

Detection of human papillomavirus in routinely processed biopsy specimens from laryngeal papillomas: evaluation of reproducibility of polymerase chain reaction and DNA in situ hybridization procedures

The frequency of human papillomavirus (HPV) in laryngeal papillomas varies largely among different studies. DNA in situ hybridization (ISH) has been the most widely used method for detection of HPV. The aim of this study was to compare the reproducibility and sensitivity of ISH with polymerase chain reaction (PCR) in 35 specimens of laryngeal papillomas routinely fixed in buffered or unbuffered formalin. Out of 12 specimens fixed in buffered formalin, 10 were positive for HPV 6/11 using ISH. The procedure was repeated three times and three specimens were positive only twice. Nine biopsies were positive for HPV using PCR with consensus primers (My 09/11) on dewaxed tissue without extracting DNA. In three repeated PCRs, the results were inconsistent in three samples. After DNA extraction, all 12 samples were positive with PCR. Of the 23 specimens fixed in unbuffered formalin, 14 were HPV-positive with ISH, while only one was positive with PCR. We concluded that PCR with My 09/11 consensus primers is a highly sensitive method for detection of HPV in laryngeal papillomas fixed in buffered formalin, but useless for samples fixed in unbuffered formalin. When DNA was extracted from the former type of fixed tissue, the results were highly reproducible. In contrast to PCR, ISH appeared to be less influenced by fixation procedure, but it was not as reproducible and sensitive as PCR. Negative results did not necessarily mean absence of HPV.