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.     

Engineering Plant-Microbe Symbiosis for Rhizoremediation of Heavy Metals

The use of plants for rehabilitation of heavy-metal-contaminated environments is an emerging area of interest because it provides an ecologically sound and safe method for restoration and remediation. Although a number of plant species are capable of hyperaccumulation of heavy metals, the technology is not applicable for remediating sites with multiple contaminants. A clever solution is to combine the advantages of microbe-plant symbiosis within the plant rhizosphere into an effective cleanup technology. We demonstrated that expression of a metal-binding peptide (EC20) in a rhizobacterium, Pseudomonas putida 06909, not only improved cadmium binding but also alleviated the cellular toxicity of cadmium. More importantly, inoculation of sunflower roots with the engineered rhizobacterium resulted in a marked decrease in cadmium phytotoxicity and a 40% increase in cadmium accumulation in the plant root. Owing to the significantly improved growth characteristics of both the rhizobacterium and plant, the use of EC20-expressing P. putida endowed with organic-degrading capabilities may be a promising strategy to remediate mixed organic-metal-contaminated sites.     

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.     

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 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.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.     

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.