Recurrent Genomic Gains in Preinvasive Lesions as a Biomarker of Risk for Lung Cancer

Lung carcinoma development is accompanied by field changes that may have diagnostic significance. We have previously shown the importance of chromosomal aneusomy in lung cancer progression. Here, we tested whether genomic gains in six specific loci, TP63 on 3q28, EGFR on 7p12, MYC on 8q24, 5p15.2, and centromeric regions for chromosomes 3 (CEP3) and 6 (CEP6), may provide further value in the prediction of lung cancer. Bronchial biopsy specimens were obtained by LIFE bronchoscopy from 70 subjects (27 with prevalent lung cancers and 43 individuals without lung cancer). Twenty six biopsies were read as moderate dysplasia, 21 as severe dysplasia and 23 as carcinoma in situ (CIS). Four-micron paraffin sections were submitted to a 4-target FISH assay (LAVysion, Abbott Molecular) and reprobed for TP63 and CEP 3 sequences. Spot counts were obtained in 30–50 nuclei per specimen for each probe. Increased gene copy number in 4 of the 6 probes was associated with increased risk of being diagnosed with lung cancer both in unadjusted analyses (odds ratio = 11, p<0.05) and adjusted for histology grade (odds ratio = 17, p<0.05). The most informative 4 probes were TP63, MYC, CEP3 and CEP6. The combination of these 4 probes offered a sensitivity of 82% for lung cancer and a specificity of 58%. These results indicate that specific cytogenetic alterations present in preinvasive lung lesions are closely associated with the diagnosis of lung cancer and may therefore have value in assessing lung cancer risk.     

Oral administration of a tri-therapy for central pattern generator activation in paraplegic mice: Proof-of-concept of efficacy

Spinal cord injury (SCI) is a neurological condition, for which no cure exists, typically leading to an immediate and irreversible loss of sensory and voluntary motor functions accompanied by significant health problems. We conducted proof-of-concept experiments aimed at assessing efficacy upon oral administration of a novel combination therapy for central pattern generator (CPG) activation and corresponding locomotor movement generation in completely paraplegic animals. Co-administration orally (by gavage) of buspirone, levodopa and carbidopa was found to dose-dependently induce episodes of steady weight-bearing stepping in low-thoracic (Th9/10) spinal cord-transected (Tx) mice (with no other form of assistance or training). Robust hindlimb stepping with weight-bearing capabilities was induced with the tri-therapy but not with clinically relevant doses of these compounds administered separately. These results provide evidence suggesting that this drug combination may be ideally suited to constitute a first-in-class therapy (CPG activator) for locomotor activity induction in chronic SCI individuals, given that efficacy was shown using commercially available brain-permeable small molecules, already known as safe for the treatment of various neurological indications.     

Improving the pharmacokinetic properties of biologics by fusion to an anti-HSA shark VNAR domain

Advances in recombinant antibody technology and protein engineering have provided the opportunity to reduce antibodies to their smallest binding domain components and have concomitantly driven the requirement for devising strategies to increase serum half-life to optimise drug exposure, thereby increasing therapeutic efficacy. In this study, we adopted an immunization route to raise picomolar affinity shark immunoglobulin new antigen receptors (IgNARs) to target human serum albumin (HSA). From our model shark species, Squalus acanthias, a phage display library encompassing the variable binding domain of IgNAR (VNAR) was constructed, screened against target, and positive clones were characterized for affinity and specificity. N-terminal and C-terminal molecular fusions of our lead hit in complex with a naïve VNAR domain were expressed, purified and exhibited the retention of high affinity binding to HSA, but also cross-selectivity to mouse, rat and monkey serum albumin both in vitro and in vivo. Furthermore, the naïve VNAR had enhanced pharmacokinetic (PK) characteristics in both N- and C-terminal orientations and when tested as a three domain construct with naïve VNAR flanking the HSA binding domain at both the N and C termini. Molecules derived from this platform technology also demonstrated the potential for clinical utility by being available via the subcutaneous route of delivery. This study thus demonstrates the first in vivo functional efficacy of a VNAR binding domain with the ability to enhance PK properties and support delivery of multifunctional therapies.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

REVERSION FROM FLOWERING TO THE VEGETATIVE STATE IN XANTHIUM

Lam , S. L. and A. C. Leopold . (Purdue U., Lafayette, Indiana.) Reversion from flowering to the vegetative state in Xanthium. Amer. Jour. Bot. 47(4): 256—259. Illus. 1960.–Plants of Xanthium pensylvanicum Wallr. which had been induced with short photoperiods were caused to revert to the vegetative state either by decapitation or by pinching and subsequent partial de-budding. The ability of induced plants to revert varies with the intensity of the inductive stimulus, and with the extent to which the plant is cut back. Four successive decapitation treatments caused complete reversion of almost all plants which had been given 7 inductive cycles. Reversion can be obtained after 3 inductive cycles even when all of the induced leaves remain on the plant. It is suggested that under the conditions of these experiments the synthesis of the flowering stimulus is stopped or the stimulus is immobilized in the leaves and made ineffective.     

A genotype-to-phenotype map of in vitro selected RNA-cleaving DNAzymes: implications for accessing the target phenotype

Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. k_obs, maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.     

Strategy combining chiral chromatography with β-cyclodextrin and stereospecific enzyme reaction detection with hydroxysteroid dehydrogenases for resolving steroid epimers

Steroids are chiral molecules with multiple stereogenic centers. Studies of their intermediary metabolism often require analytical techniques to separate the isomers and determine their stereochemistry. Methods for resolving steroid stereoisomers by HPLC using β-cyclodextrin in the mobile phase are reported. Even with the improved selectivity of cyclodextrin chromatography, not all isomers within a steroid series can be resolved. Additional specificity is achieved by reaction detection using postcolumn reactors containing hydroxysteroid dehydrogenases stereospecific for the configuration of the hydroxy functions of steroids. The enzymes catalyze the oxidation of hydroxysteroids and reduction of the coenzyme NAD to NADH. NADH, which is highly fluorescent, is detected at the nanogram levels. Isomers not separated by chromatography were effectively resolved by reaction detection with stereospecific enzymes. © 1992 Wiley-Liss, Inc.     

Selective inhibition of integrin function by antibodies specific for ligand-occupied receptor conformers

We have hypothesized that ligand-induced binding sites (LIBS), i.e. sites expressed on cell surface receptors only after ligand binding causes the receptor to change shape, mediate subsequent biological events. To test this hypothesis, we have raised monoclonal antibodies that preferentially react with an integrin (platelet glycoprotein (GP) IIb-IIIa) after it bind Arg-Gly-Asp-containing ligands. The 13 anti-LIBS antibodies obtained define at least three distinct GPIIb-IIIa epitopes; one of these epitopes is also expressed following occupancy of another integrin, the vitronectin receptor. Certain of these LIBSs appear to mediate functions, since the antibodies that define them inhibit GPIIb-IIIa-mediated fibrin clot contraction or platelet adhesion to collagen. Nevertheless, none of the anti-LIBS antibodies inhibit binding of the primary ligand, fibrinogen. These data indicate that LIBS may mediate distinct consequences of receptor occupancy.