A Quantitative Assessment of the Role of Amphetamine Metabolites in Acute Responses Evoked by d-Amphetamine1

ABSTRAC

Intensive Referral of Veterans to Mutual-Help Groups: A Mixed-Methods Implementation Evaluation

Community-based support group participation protects against substance use disorder (SUD) relapse, but referrals during treatment are inconsistently delivered and may not acknowledge barriers facing rural patients. This formative evaluation of a rural intensive referral intervention (RAIR) to community-based support groups for veterans seeking SUD treatment surveyed patients (N = 145) and surveyed and interviewed treatment staff (N = 28). Patients and staff did not differ significantly on quantitative ratings of the helpfulness of, or satisfaction with, seven RAIR components, but staff did not deliver the intervention consistently or as designed, citing two themes: lack of commitment and lack of resources

Intensive Referral to Mutual-Help Groups: A Field Trial of Adaptations for Rural Veterans

Objective: A multisite field trial testing whether improved outcomes associated with intensive referral to mutual help groups (MHGs) could be maintained after the intervention was adapted for the circumstances and needs of rural veterans in treatment for substance use disorder (SUD). Methods: In three Veterans Affairs treatment programs in the Midwest, patients (N = 195) received standard referral (SR) or rural-adapted intensive referral (RAIR) and were measured at baseline and 6-month follow-up. Results: Both groups reported significant improvement at 6 months but no significant differences between SR and RAIR groups in MHG participation, substance use, addiction severity, and posttraumatic stress symptoms. Inconsistent delivery of the intervention resulted in only one-third of the RAIR group receiving the full three sessions, but this group reported significantly greater 6-month abstinence from alcohol than those receiving no sessions. Conclusion: Further research should explore implementation problems and determine whether consistent delivery of the intervention enhances 12-step facilitation. Practice implications: The addition of rural-specific elements to the original intensive referral intervention has not been shown to increase its effectiveness among rural veterans

Epitope characterization of sero-specific monoclonal antibody to Clostridium botulinum neurotoxin type A.

Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (H(C)50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant H(C)50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of H(C)50/A (H(CN)25/A) comprising amino acid residues (985)WTLQDTQEIKQRVVF(999), an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis

Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

Background: Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components o

Genomic distance entrained clustering and regression modelling highlights interacting genomic regions contributing to proliferation in breast cancer

<p>Abstract</p> <p>Background</p> <p>Genomic copy number changes and regional alterations in epigenetic states have been linked to grade in breast cancer. However, the relative contribution of specific alterations to the pathology of different breast cancer subtypes remains unclear. The heterogeneity and interplay of genomic and epigenetic variations means that large datasets and statistical data mining methods are required to uncover recurrent patterns that are likely to be important in cancer progression.</p> <p>Results</p> <p>We employed ridge regression to model the relationship between regional changes in gene expression and proliferation. Regional features were extracted from tumour gene expression data using a novel clustering method, called genomic distance entrained agglomerative (GDEC) clustering. Using gene expression data in this way provides a simple means of integrating the phenotypic effects of both copy number aberrations and alterations in chromatin state. We show that regional metagenes derived from GDEC clustering are representative of recurrent regions of epigenetic regulation or copy number aberrations in breast cancer. Furthermore, detected patterns of genomic alterations are conserved across independent oestrogen receptor positive breast cancer datasets. Sequential competitive metagene selection was used to reveal the relative importance of genomic regions in predicting proliferation rate. The predictive model suggested additive interactions between the most informative regions such as 8p22-12 and 8q13-22.</p> <p>Conclusions</p> <p>Data-mining of large-scale microarray gene expression datasets can reveal regional clusters of co-ordinate gene expression, independent of cause. By correlating these clusters with tumour proliferation we have identified a number of genomic regions that act together to promote proliferation in ER+ breast cancer. Identification of such regions should enable prioritisation of genomic regions for combinatorial functional studies to pinpoint the key genes and interactions contributing to tumourigenicity.</p

UNC569, a Novel Small-Molecule Mer Inhibitor with Efficacy against Acute Lymphoblastic Leukemia In Vitro and In Vivo

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biological subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity, and delays development of leukaemia in vivo suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiation have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (ATRT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small molecule Mer inhibitor. This manuscript describes the biochemical and biological effects of UNC569 in ALL and ATRT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 µM for 2 weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced green fluorescent protein. UNC569 induced >50% reduction in tumor burden compared to vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and ATRT

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

BACKGROUND:Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. METHODOLOGY AND PRINCIPAL FINDINGS:Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. CONCLUSIONS:S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers