Andrew I. Brooks

Environmental and Occupational Health Sciences Institute, Edison, New Jersey, United States

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Publications (14)23.58 Total impact

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    ABSTRACT: A novel flow-through device for performing fast PCR thermal cycling is presented. The thermal gradient thermal cycling device is comprised of layers of highly thermally conducting material separated by insulating layers. Channels etched in the conducting and insulating layers create one continuous path through the device. When the device is held between platens at different temperatures and PCR sample mix is pumped through it, every fluid particle undergoes the time-temperature protocol necessary for PCR but with a temperature change rate not possible with conventional cyclers. Ultrafast thermal cycling makes it ideal for bio-defense applications, such as the instantaneous bio-aerosol agent identification system under development for the Department of Homeland Security. Its compact size and simplicity of use make it a natural choice for diagnostics, forensics, food and water testing and other DNA testing applications. Herein we describe the design and fabrication of the device developed for IBADS and the subsequent performance with various assays using plasmid and genomic template DNA. Performance under some circumstances was exceptional: Amplification rates of up to two decades per minute were recorded and total amplification of up to eight decades in 30 cycles was seen. We discuss how to optimize the performance of a device that pushes PCR to its fundamental limits and review a wide variety of performance data.
    IEEE Sensors Journal 06/2008; 8(5-8):476 - 487. DOI:10.1109/JSEN.2008.918248 · 1.85 Impact Factor
  • Sheryl J. Wildt, Andrew I. Brooks, Robert J. Russell
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    ABSTRACT: Laboratory mice (Mus musculus) and rats (Rattus norvegicus) are the most commonly used animals in biomedical and behavioral research. Today, there are many inbred, outbred, hybrid, congenic, and genetically modified mouse and rat models that have made essential contributions to advances in the diagnosis, prevention, and control of human disease. Routine genetic monitoring of these animals is essential to their successful use. Herein, we discuss the basic structure of the mouse and rat genome; common breeding protocols to preserve innate and exogenous genetic backgrounds; and the methods used for creating specific types of mutant model, research applications, and current genotyping methods.
    12/2007: pages 179-186;
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    ABSTRACT: Microarrays have revolutionized many areas of biology due to our technical ability to quantify tens of thousands of transcripts within a single experiment. However, there are still many areas that cannot benefit from this technology due to the amount of biological material needed for microarray analysis. In response to this demand, chemistries have been developed that boast the capability of generating targets from nanogram amounts of total RnA, reflecting minimal amounts of biological material, on the order of several hundred or thousand cells. Herein, we describe the evaluation of four chemistries for RnA amplification in terms of reproducibility, sensitivity, accuracy, and comparability to results from a single round of T7 amplification. No evidence for false-positive measurements of differential expression was observed. In contrast, clear differences between chemistries in sensitivity and accuracy were detected. PCR validation showed an interaction of probe sequence on the array and target labeling chemistry, resulting in a chemistry-dependent probe set sensitivity varying over an order of magnitude.
    Journal of biomolecular techniques: JBT 08/2007; 18(3):150-61.
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    ABSTRACT: Abstract Background The mouse C57BL/6 (C57) and DBA/2J (DBA) inbred strains differ substantially in many aspects of their response to drugs of abuse. The development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. In the current study, we carried out microarray analysis in C57 and DBA mice in the nucleus accumbens of drug-naïve and morphine-treated animals. Results We identified mRNAs with altered expression between the two strains. We validated the mRNA expression changes of several such mRNAs, including Gnb1, which has been observed to be regulated by several drugs of abuse. In addition, we validated alterations in the enzyme activity of one mRNA product, catechol-O-methyltransferase (Comt). Data mining of expression and behavioral data indicates that both Gnb1 and Comt expression correlate with aspects of drug response in C57/DBA recombinant inbred strains. Pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. These analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. Pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure. Conclusion mRNAs with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. One such mRNA is Comt and we hypothesize that altered expression of Comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. Similarly, a role for Gnb1 in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. Finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains.
    BMC Genomics 03/2007; 8. DOI:10.1186/1471-2164-8-76 · 4.04 Impact Factor
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    ABSTRACT: Over the past several years, microarray technology has evolved into a critical component of any discovery-based program. Since 1999, the Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) has conducted biennial surveys designed to generate a profile of microarray service laboratories and, more importantly, an overview of technology development and implementation. Survey questions addressed instrumentation, protocols, staffing, funding, and work flow in a microarray facility. Presented herein are the results of the MARG 2005 survey; where possible, trends in the field are discussed and compared to data collected from previous surveys.
    Journal of biomolecular techniques: JBT 05/2006; 17(2):176-86.
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    ABSTRACT: Western blots using an antibody which recognizes the orphanin FQ/nociceptin (OFQ/N) receptor reveals a band at approximately 69 kD in several cell lines, including the Raji human B cell lymphoma cell line. RT-PCR confirms the presence of this receptor in the Raji cells. Binding studies revealed a high affinity [125I][Tyr14]OFQ/N site in the Raji cells. The affinity of [125I][Tyr14]OFQ/N in the Raji cells (KD 68.4 pM) was similar to that in the transfected receptor (KD 36.7 pM). Its selectivity profile also was quite similar. OFQ/N competed binding quite potently (Ki 65 pM), as did [Tyr14]OFQ/N (Ki 33 pM). Traditional opioids displayed no appreciable affinity for the binding at any concentration examined, with the exception of naloxone benzoylhydrazone, which had only a very modest affinity. The receptors in the Raji cells were functionally active. OFQ/N inhibited forskolin-stimulated cyclase by 72% with an IC50 value of approximately 1 nM. Synapse 34:187–191, 1999. © 1999 Wiley-Liss, Inc.
    Synapse 12/1999; 34(3):187 - 191. DOI:10.1002/(SICI)1098-2396(19991201)34:3<187::AID-SYN3>3.0.CO;2-A · 2.43 Impact Factor
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    ABSTRACT: Quantitative structure activity relationships (QSAR) were determined for the Ki values measured for the interaction of 4′-substituted naloxone benzoylhydrazones with κ1, κ3, μ1, μ2, and δ receptors. Based on a comparison of the estimated and observed values of Ki for two groups which were not in the data sets used to obtain the initial QSAR the latter give very satisfactory estimates for all but the δ receptor. The validity of the QSAR are further supported by a comparison of the observed and calculated orders of Ki values for these two groups. The effects of the 4′ substituent on Ki are similar for the μ1 and μ2 receptors. No other similarities were observed. The results suggest that substituents of the type CH2Z and NHZ where Z has large values of the localized electrical effect parameter, σ1, should exhibit a ratio of Ki(κ3) to Ki(κ1) greater than 30. This is supported by the result for the acetylamino group. For all receptors but δ and possibly κ3 the 4′ substituent seems to be binding to a hydrophobic region of the molecular framework through Van der Waals interactions. The segmental parameterization seems to be a better choice for the representation of steric effects than a monoparametric parameterization. The intermolecular force model is a useful alternative to the Hansch-Fujita model. The QSAR are reliable for all but the δ receptor where the results are very uncertain.
    Quantitative Structure-Activity Relationships 12/1998; 17(02):109 - 121. DOI:10.1002/(SICI)1521-3838(199804)17:02<109::AID-QSAR109>3.0.CO;2-1
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    ABSTRACT: Naloxone benzoylhydrazone (NalBzoH) has proved a valuable tool in the investigation of opioid receptor subtypes. In the present study, we have examined a series of derivatives of NalBzoH in which substitutions have been made on the benzoyl ring. Overall, we see dramatic effects on the binding affinities of derivatives against the various opioid receptor subtypes. Although the range of affinities against the mu receptors is quite modest, ranges of the others vary almost 30-fold for kappa3, 50-fold for kappa1 and 100-fold for delta and kappa2 binding. Few substituted derivatives display greater affinity than NalBzoH for any of the receptors, except for delta sites where several derivatives have affinities almost tenfold greater than NalBzoH. Along with the wide variations in affinity, the compounds also appear to exhibit widely divergent activities in traditional biosasays. © 1996 Wiley-Liss, Inc.
    Synapse 10/1996; 24(2):193 - 201. DOI:10.1002/(SICI)1098-2396(199610)24:2<193::AID-SYN11>3.0.CO;2-# · 2.43 Impact Factor
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    ABSTRACT: To help characterize kappa3 receptors and establish their relationship to traditional mu and delta receptors, we have generated a kappa3-selective monoclonal antibody. Monoclonal antibodies were raised against BE(2)-C cells, a human neuroblastoma cell line containing mu, kappa3, and delta opioid receptors. Of the 5,000 hybridoma cell lines screened, approximately 2,000 hybridomas tested positive against BE(2)-C membranes by ELISA, but only 98 of these were negative against a different neuroblastoma cell line lacking opioid receptors. Supernatants from one hybridoma, 8D8, inhibited up to 90% of 3H-NalBzoH (kappa3) binding without affecting 3H-DAMGO (mu) or 3H-naltrindole (delta) binding in BE(2)-C membranes. The selectivity of the antibody was further demonstrated by its blockade of the inhibition of cAMP accumulation in BE(2)-C cells by the kappa3 agonist NalBzoH but not the mu agonist morphine. Monoclonal antibody 8D8 (mAb8D8) also recognizes kappa3 receptors from mouse, rat, and calf brain. Administered intracerebroventricularly, mAb8D8 blocked kappa3 but not morphine (mu) analgesia in vivo. On Western blots, mAb8D8 recognized a protein with a molecular mass of approximately 70 kilodaltons in BE(2)-C. These studies demonstrate the selectivity of mAb8D8 for kappa3 receptors and provide additional support for the existence of this unique opioid receptor subtype.
    Synapse 03/1996; 22(3):247-52. DOI:10.1002/(SICI)1098-2396(199603)22:3<247::AID-SYN7>3.0.CO;2-C · 2.43 Impact Factor
  • Synapse 01/1996; 22(3):247-252. DOI:10.1002/(SICI)1098-2396(199603)22:33.0.CO;2-C · 2.43 Impact Factor
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    ABSTRACT: We have identified a putative opioid receptor from mouse brain (KOR-3), belonging to the G protein-coupled receptor family, that is distinct from the previously cloned mu, delta, and kappa 1 receptors. Assignment of the clone to the opioid receptor family derives from both structural and functional studies. Its predicted amino acid sequence is highly homologous to that of the other opioid receptors, particularly in many of the transmembrane regions, where long stretches are identical to mu, delta, and kappa 1 receptors. Both cyclazocine and nalorphine inhibit cAMP accumulation in COS-7 cells stably expressing the clone. Northern analysis shows that the mRNA is present in brain but not in a number of other organs. Southern analysis suggests a single gene encoding the receptor. A highly selective monoclonal antibody directed against the native kappa 3 receptor recognizes, in Western analysis, the clone expressed in COS-7 cells. The in vitro translation product is also labeled by the antibody. Additional clones reveal the presence of several introns, including one in the second extracellular loop and another in the first transmembrane region. Antisense studies with an oligodeoxynucleotide directed against a region of the second extracellular loop reveal a selective blockade of kappa 3 analgesia in vivo that is not observed with a mismatch oligodeoxynucleotide based upon the antisense sequence. The mu, delta, and kappa 1 analgesia is unaffected by this antisense treatment. Antisense mapping of the clone downstream from the splice site in the first transmembrane region reveals that six different antisense oligodeoxynucleotides all block kappa 3 analgesia. In contrast, only one of an additional six different antisense oligodeoxynucleotides directed at regions upstream from this splice site is effective. This strong demarcation between the two regions raises the possibility of splice variants of the receptor. An additional clone reveals an insert in the 3' untranslated region. In conclusion, the antibody and antisense studies strongly associate KOR-3 with the kappa 3-opioid receptor, although it is not clear whether it is the kappa 3 receptor itself or a splice variant.
    Molecular Pharmacology 07/1995; 47(6):1180-8. · 4.12 Impact Factor
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    ABSTRACT: Total opioid binding in the human neuroblastoma cell line BE(2)-C has a density similar to that found in brain, with a Bmax value of 383 +/- 60 fmol/mg protein and a KD of 0.4 +/- 0.07 nM for the nonselective opioid antagonist 3H-diprenorphine. Selective assays reveal a binding distribution of mu (38%), delta (16%) and kappa 3 (43%) opioid receptors. There is no observable kappa 1 or kappa 2 binding. The sum of the Bmax values in the selective binding assays (370 +/- 39 fmol/mg protein) approximates closely that observed with 3H-diprenorphine, suggesting that mu, delta and kappa 3 sites account for most of the binding. The binding selectivities of various opiates and opioid peptides in the BE(2)-C cells are similar to those in rat brain. Delta and mu binding are defined easily by traditional selective ligands. The binding profiles also distinguish clearly mu from kappa 3 binding. The selective mu ligand DAMGO competes with mu binding over 35-fold more potently than kappa 3 binding, whereas morphine shows a 10-fold selectivity. Functionally, selective mu, delta and kappa 3 agonists inhibit forskolin-stimulated cAMP accumulation through distinct receptor mechanisms that are pertussis toxin-sensitive. In addition to demonstrating that BE(2)-C cells provide a useful model system for studying mu, kappa 3 and delta receptors, these studies confirm that kappa 3 receptors represent a pharmacologically distinct receptor class in this cell line.
    Journal of Pharmacology and Experimental Therapeutics 10/1994; 270(3):1246-55. · 3.86 Impact Factor
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    ABSTRACT: Opiate receptor expression in phylogenetically different species has played an important role in the study of opioid receptor pharmacology. Total opioid binding measured with the nonselective ligand 3H-diprenorphine reveals a Bmax of 21.7 +/- 1.37 fmol/mg tissue wet wt and a KD of 0.17 +/- 0.03 nM in Bufo marinus (giant toad), as well as a Bmax of 18.17 + 0.41 fmol/mg tissue wet wt and a KD of 0.47 +/- 0.18 nM in Carassius auratus (goldfish). Despite the similar levels of 3H-diprenorphine binding, the composition of binding subtypes in the two species differs. Approximately 30% of total binding corresponds to mu receptors in both species, whereas neither kappa 1 nor delta binding can be detected. However, the remaining 70% of binding differs between the toad and goldfish. In the toad, the non-mu binding corresponds to kappa 2 sites, whereas in the goldfish, the non-mu binding corresponds to kappa 3 sites. The sites can be distinguished biochemically, as well as pharmacologically. After affinity labeling the sites with 3H-NalBzoH, the retention times on an ion-exchange column differ for the peaks of kappa binding in the two species. Although Bufo marinus (giant toad) and Carassius auratus (goldfish) brains express kappa and mu opioid binding, the kappa subtypes in these two species differ.
    Receptor 02/1994; 4(1):55-62.
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