Chemical Biological Research Articles

Surface-Enhanced Raman Scattering (SERS) Evaluation Protocol for Nanometallic Surfaces

We present the results of a three-year collaboration between the U.S. Army Edgewood Chemical Biological Center and the U.S. Army Research Laboratory-Aldelphi Laboratory Center on the evaluation of selected nanometallic surfaces developed for the Defense Advanced Research Projects Agency Surface-Enhanced Raman Scattering (SERS) Science and Technology Fundamentals program. The primary role of the two Army labs was to develop the analytical and spectroscopic figures of merit to unambiguously compare the sensitivity and reproducibility of various SERS substrates submitted by the program participants. We present the design and implementation of an evaluation protocol for SERS active surfaces enabling an enhancement value calculation from which different substrates can be directly compared. This method was established to: (1) collect physical and spectral characterization data from the small number of substrates (performer supplied) typically encountered, and (2) account for the complex fabrication technique and varying nature of the substrate platforms encountered within this program.

Carcinogenic 4(5)-Methylimidazole Found in Beverages, Sauces, and Caramel Colors: Chemical Properties, Analysis, and Biological Activities

Since the National Toxicology Program (NTP) identified 4(5)-methylimidazole [4(5)-MI] as a cancer causing chemical in 2007 and the State of California added it to the Proposition 65 list of compounds as a carcinogen on January 7, 2011, many researchers and regulatory agencies have become focused on the presence of 4(5)-MI in foods and beverages. 4(5)-MI has been known to form in the Maillard reaction system consisting of a sugar and ammonia-a typical caramel-color preparation method for beverages. 4(5)-MI is identified in various beverages and sauces, which are colored with caramel, as well as in caramel color itself. Analysis of 4(5)-MI is extremely difficult due to its high water solubility, but the analytical method for 4(5)-MI has progressed from conventional paper chromatography, gas chromatography, and gas chromatography-mass spectrometry to the most advanced high-performance liquid chromatography-mass spectrometry. Various studies indicate that caramel colors and carbonated beverages contain 4(5)-MI in levels ranging from 0 to around 1000 ppm and from 0 to about 500 ppm, respectively. Reports of the toxicity of 4(5)-MI at relatively high levels suggest that it may cause some adverse effects on human consumers.

Genetically Marked, or “Barcoded” B. thuringiensis for Simulating Anthrax

By adding a genetic “barcode” to the genome of relatively harmless Bacillus thuringiensis subsp kurstaki (Btk), this bacterial species can be used much more effectively to simulate how Bacillus anthracis—a far more dangerous pathogen, whose spores might be used again by terrorists—disperses in various environments, according to Henry S. Gibbons of the Edgewood Chemical Biological Center at Aberdeen Proving Ground in Aberdeen, Md., and his collaborators. Without that special coding, it is virtually impossible to distinguish Btk spores that are deliberately released in simulatory experiments from those that are naturally occurring, they point out. Their findings appeared online September 21, 2012 in Applied and Environmental Microbiology (78:8601–8610).

Chemical Biology of Natural Products on the Basis of Identification of Target Proteins

Abstract Recent progress in chemical biology research has accelerated the target protein identification of biologically active small molecules, including naturally occurring ligands. It is widely recognized that a biologically active natural product functions as a “key” that fits a specific protein “lock.” However, recent studies have revealed multiligandable nature of natural products that works as “multiple keys” (or a “master key”) fitting multiple “locks” in a biological event. Biological experiments using natural products as ligands have often provided puzzling results, which can likely be attributed to their multiligandability. Thus, a novel research direction dealing with multiliganbale natural product ligand based on their target identification is strongly desired. This review focuses on the current status of natural products chemistry based on their target identification, and suggest the future direction, named chemical biological chemistry (Chem-Bio-Chem).

Rethinking Molecular Similarity: Comparing Compounds on the Basis of Biological Activity

Since the advent of high-throughput screening (HTS), there has been an urgent need for methods that facilitate the interrogation of large-scale chemical biology data to build a mode of action (MoA) hypothesis. This can be done either prior to the HTS by subset design of compounds with known MoA or post HTS by data annotation and mining. To enable this process, we developed a tool that compares compounds solely on the basis of their bioactivity: the chemical biological descriptor "high-throughput screening fingerprint" (HTS-FP). In the current embodiment, data are aggregated from 195 biochemical and cell-based assays developed at Novartis and can be used to identify bioactivity relationships among the in-house collection comprising ~1.5 million compounds. We demonstrate the value of the HTS-FP for virtual screening and in particular scaffold hopping. HTS-FP outperforms state of the art methods in several aspects, retrieving bioactive compounds with remarkable chemical dissimilarity to a probe structure. We also apply HTS-FP for the design of screening subsets in HTS. Using retrospective data, we show that a biodiverse selection of plates performs significantly better than a chemically diverse selection of plates, both in terms of number of hits and diversity of chemotypes retrieved. This is also true in the case of hit expansion predictions using HTS-FP similarity. Sets of compounds clustered with HTS-FP are biologically meaningful, in the sense that these clusters enrich for genes and gene ontology (GO) terms, showing that compounds that are bioactively similar also tend to target proteins that operate together in the cell. HTS-FP are valuable not only because of their predictive power but mainly because they relate compounds solely on the basis of bioactivity, harnessing the accumulated knowledge of a high-throughput screening facility toward the understanding of how compounds interact with the proteome.

Abstract SY02-03: Bromodomain inhibition in cancer

Abstract Cellular states are maintained by coordinated gene expression programs, attributable to master regulatory transcription factors. Toward the pharmacologic control of cellular states we have undertaken a chemical biological approach to inhibit chromatin-dependent transcriptional signaling. Transcriptional activation by master regulatory proteins is facilitated by chromatin remodeling at promoter/enhancer regions, in particular side-chain acetylation of lysine residues on nearby histone tails. Context-specific lysine acetylation prompts molecular recognition by transcriptional co-activator proteins possessing acetyl-lysine recognition modules, or bromodomains. Perhaps owing to perceptions regarding the feasibility of abrogating protein-protein interactions, research toward the direct inhibition of human bromodomains (or epigenetic reader proteins, in general) has received comparatively little attention. Based on the above rationale, we have developed chemical and biochemical platforms for the development and characterization of novel bromodomain inhibitors. Recently, we reported the first potent, small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional co-activators. Of broad relevance to cancer, we will present research demonstrating that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate down-regulation of c-Myc expression leading to suppression of a transcriptional program associated with proliferation, survival and metabolic adaptation. In translational models of Myc-dependent hematologic malignancies, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable, small molecules. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 150 academic, governmental and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer, inflammation and metabolic diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY02-03. doi:1538-7445.AM2012-SY02-03

Anti-inflammatory Effect of a Cell-Penetrating Peptide Targeting the Nrf2/Keap1 Interaction

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is increasingly recognized as a central regulator of multiple signaling pathways in inflammation and cancer, and the ability to use chemical biological tools to investigate its biological effects is very attractive. A peptide comprising a TAT-conjugated Nrf2 sequence is shown to activate Nrf2 and its downstream target gene heme-oxygenase-1 (HO-1) in a dose-dependent manner in intact human THP-1 monocytes. Levels of Nrf2 protein peak after 3 h, whereas HO-1 mRNA and protein peak after 6 and 12 h, respectively. The peptide is also shown to inhibit the production of the pro-inflammatory cytokine TNF. The TAT-14mer constitutes a useful chemical biology tool with potential therapeutic applications.

(P1-24) Future Weapons of Mass Destruction: Preparing For Emerging Threats

IntroductionCivilian Weapons of Mass Destruction (WMD) training involves preparing for threats well-known to the military since World War II. Present and future developments in Chemical-Biological (CB) research have resulted in new potential agents, modes of action, and methods of delivery. Chemical-Biological defense training should include these new agents and anticipate contact with previously unknown ones. The natural response to an unknown threat is fear and panic, out of proportion to the actual threat. Specific training in management of new pathogens and toxidromes should be incorporated into existing preparedness regimes. Leadership skills that address uncertainty and inspire constructive responses will increase resiliency.MethodsLiterature ReviewResultsRecent and Future CB Agents: (1) 4th Generation AchE inhibitors: Novichoks, Substance 33, etc.; (2) Genetically enhanced bacteria and engineered chimeric diseases; (3) Modified viral diseases: Variola, Influenza, filoviruses, flaviviruses, arenaviruses; (4) Bacteriophage induced diseases; (5) Agents targeting specific racial or genetic groups; (6) Mid-spectrum agents; (6) Bioregulators: Substance P, vasopressin, enkephalin, etc.; (7) Novel toxins: tetrodotoxin, SEB, saxitoxin, etc.; (8) Hallucinogens and incapacitants (LSD, DMT, carfentanyl, cis-fluoro-ohmefentanyl); (9) Prions and infectious nucleic acids Delivery: (1) New delivery methods: micro- and nano-robots modeled on insects (MEMS systems), microencapsulation; (2) Directed Energy Weapons Mitigation: (1) Development of systems for identifying and dealing with unknown agents and symptoms; (2) Hazard Identification, Risk Estimation, Risk Reduction Strategies, Residual Risk Evaluation and Monitoring, Mitigation and Recovery Leadership skills needed during uncertainty: Sense making, Visioning, Relating, Inventing.ConclusionsPreparing for events without training for new and novel CB agents leaves us unprepared. Incorporating modern science with leadership skills will lessen the impact of future CB release and improve organizational resiliency. The main mistake people make is that they fear current problems more than future ones. Carl von Clausewitz Chance favors the prepared mind. Louis Pasteur.

Book Reviews

Abstract Book reviewed in this article: PHARMACOLOGY, by J. H. Gaddum. CHEMICAL CONSTITUTION AND BIOLOGICAL ACTIVITY, by W. A. Sexton.

Homodimerization of the Death-Associated Protein Kinase Catalytic Domain: Development of a New Small Molecule Fluorescent Reporter

BackgroundDeath-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases.Methodology/Principal FindingsIn the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution.Conclusions/SignificanceThe use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.

The chemical biology of synapses and neuronal circuits

Excitatory synapses are located in confined chemical spaces called the dendritic spines. These are atypical femtoliter-order microdomains where the behavior of even single molecules may have important biological consequences. Powerful chemical biological techniques have now been developed to decipher the dynamic stability of the synapses and to further interrogate the complex properties of neuronal circuits.

Evaluating Suitability of a Military Duty Glove for Use in a Multi-Layered Chemical-Biological Protective Glove System

The Max Grip NT glove (Test Glove) has been approved for use as a duty glove for U.S. Army Combat Vehicle Crew and Aviation communities and is intended to either supplement or replace the current issue duty glove (Baseline) worn by these communities. The Baseline duty glove serves, however, as both a duty glove and as the outer glove component of a layered protective chemical biological (CB) glove system. In considering whether the Test Glove can fully replace the Baseline, it was necessary for the U.S. Army Program Manager Clothing and Individual Equipment (PM CIE) to determine whether the Test Glove could serve effectively in this second function as the outer layer of the CB glove system. The Ergonomics Team at U.S. Army Natick Soldier Research, Development, and Engineering Center was asked to conduct an evaluation to determine, from a human factors perspective, the suitability of the Test Glove to be worn in conjunction with the currently fielded CB protective inner glove layers, known as 7 mil and 14 mil butyls. The evaluation addressed, among other issues, these three questions: (1) Does the Test Glove duty size (i.e., worn over bare hands) fit effectively over the 7 mil and 14 mil butyl glove layers without upsizing? (2) If it is necessary to upsize, can the upsized Test Glove be effectively worn as a duty glove (i.e., over bare hands) to serve dual purpose? and, (3) When wearing the Test Glove as part of the CB protective glove system can the wearer perform manual dexterity tasks at least as well as in the Baseline CB protective glove system? Results showed that it was often necessary to upsize the Test Glove by one size to accommodate the CB inner layer but wearing this larger glove over the bare hands, as a duty glove, did not adversely impact manual dexterity, suggesting potential for a single-size dual purpose glove. In a comparison of manual dexterity performance between the Test Glove and Baseline CB glove configurations, minor differences, only, were observed. Practical implications including path forward for the Test Glove are discussed.

Quantitative Method To Determine Sporicidal Decontamination of Building Surfaces by Gaseous Fumigants, and Issues Related to Laboratory-Scale Studies

Chlorine dioxide gas and vaporous hydrogen peroxide sterilant have been used in the cleanup of building interiors contaminated with spores of Bacillus anthracis. A systematic study, in collaboration with the U.S. Environmental Protection Agency, was jointly undertaken by the U.S. Army-Edgewood Chemical Biological Center to determine the sporicidal efficacies of these two fumigants on six building structural materials: carpet, ceiling tile, unpainted cinder block, painted I-beam steel, painted wallboard, and unpainted pinewood. Critical issues related to high-throughput sample processing and spore recovery from porous and nonporous surfaces included (i) the extraction of spores from complex building materials, (ii) the effects of titer challenge levels on fumigant efficacy, and (iii) the impact of bioburden inclusion on spore recovery from surfaces and spore inactivation. Small pieces (1.3 by 1.3 cm of carpet, ceiling tile, wallboard, I-beam steel, and pinewood and 2.5 by 1.3 cm for cinder block) of the materials were inoculated with an aliquot of 50 microl containing the target number (1 x 10(6), 1 x 10(7), or 1 x 10(8)) of avirulent spores of B. anthracis NNR1Delta1. The aliquot was dried overnight in a biosafety cabinet, and the spores were extracted by a combination of a 10-min sonication and a 2-min vortexing using 0.5% buffered peptone water as the recovery medium. No statistically significant drop in the kill efficacies of the fumigants was observed when the spore challenge level was increased from 6 log units to 8 log units, even though a general trend toward inhibition of fumigant efficacy was evident. The organic burden (0 to 5%) in the spore inoculum resulted in a statistically significant drop in spore recovery (at the 2 or 5% level). The effect on spore killing was a function of the organic bioburden amount and the material type. In summary, a high-throughput quantitative method was developed for determining the efficacies of fumigants, and the spore recoveries from five porous materials and one nonporous material ranged between 20 and 80%.

Composição química e atividade biológica das folhas e frutos de Triphasia trifolia

CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF LEAVES AND FRUITS OF TRIPHASIA TRIFOLIA. The chemical composition of the essential oils from leaves and fruits of Triphasia trifolia was analyzed by GC-FID and GC-MS. The major constituents of oil obtained from leaves were sabinene (35.4%) and myrcene (34.1%), while the prevalent compounds in oil from fruits were sabinene (37.2%), β-pinene (23.95) and γ-terpinene (16.3%). Both oils showed moderate antimicrobial activity. The fruit decoction was also investigated leading to the isolation of the coumarins isopimpinelin, (R)-byakangelicin and (S)-mexoticin. From leaves were isolated the coumarins (R)-byakangelicin, aurapten, (S)-mexoticin, isosibiricin, isomerazin and coumurrayin and the flavonoid vitexin. All coumarins showed cholinesterase inhibition on TLC tests.

Chemical biology that controls DNA structure and function

All genetic information, which is necessary for life, is written in 3 billion base pairs of DNA. Sequence-dependent local DNA conformations are also thought to play an important role in gene expression. Many diseases, including cancer, and hereditary and viral diseases, can now be understood at the DNA sequence and structure level. Therefore, direct control of the expression level of a specific gene would provide a promising approach for knowledge-based therapy. In this presentation I focus on our long-term efforts in controlling of structure and function of DNA by chemical biological approach.

Online estimation of vapor path-integrated concentration and absorptivity using multiwavelength differential absorption lidar

Differential absorption lidar data processing traditionally assumes knowledge of the spectral dependence of the absorptivity coefficients. While this is sometimes a good assumption, it is often not in complicated collection environments where the material present is ambiguous. We present an alternative approach that estimates the vapor path-integrated concentration (CL) and absorptivity (rho) in parallel by a processor capable of online implementation. The algorithm is based on an extended Kalman filter (EKF) for CL and a sequential maximum likelihood estimator for rho. The state model parameters of the EKF are also estimated sequentially together with CL and rho. The approach is illustrated on simulated and real topographic backscatter lidar data collected by the Edgewood Chemical Biological Center.

Evaluating Tactility and Dexterity for Military Aviation Protective Gloves

To avoid penetration from chemical agents, U.S. military Aviators wear chemical-biological (CB) protective clothing, including gloves, while in CB threat environments. This protective requirement typically increases glove thickness which could interfere with tactility and dexterity needed to effectively operate aircraft. An applied laboratory evaluation was conducted to determine if current CB glove tactility and dexterity could be enhanced by the introduction of novel glove concepts. Aviator subjects performed objective tasks based upon standardized tests of tactility and dexterity. They also self-rated their ability to manipulate cockpit controls on aircraft simulator panels. A human factors questionnaire was administered following each test condition and at the conclusion of all conditions. Evidence from all data sources indicates that three novel glove concepts show promise for enhancing tactility and dexterity performance. The approach to combining multiple data sources, the methodological constraints, and lessons learned are discussed.

Photopolarimetric lidar duel-beam switching device and Mueller matrix standoff detection method

We present an optomechanical switching device (OSD) for a photopolarimetric lidar system with differential-absorption Mueller matrix spectroscopy standoff detection method. An output train of alternate continuous-wave CO2 laser beams [...L1:L2...] is directed onto suspect chemical-biological (CB) aerosol plume or the land mass it contaminates (S) vis-à-vis the OSD, where L1 [L2] is tuned on [detuned off] a resonant molecular absorption moiety of CB analyte. Moreover, both incident beams and their backscattered radiances, from S, are polarization-modulated synchronously so as to produce gated temporal voltage waveforms called scattergrams recorded on focus at the receiver end of polarization lidar sensor system. All 16 elements of the Mueller matrix (Mij) of S are measured via digital or analog filtration of constituent frequency components in these running scattergram data streams (phase-sensitive detection). A collective set of normalized differential elements {Mi,j} (ratioed to element M11) that are susceptible to the analyte, probed on-then-off its molecular absorption band, form a unique detection domain that is scrutinized. Any mapping onto this domain in Mueller-space, from incoming sensor scattergram data sets preprocessed by algorithm and forwarded through a trained neural network pattern recognition system, cues a standoff detection event.

Medical Preparedness Against Chemical and Biological Incidents for the NATO Summit in Istanbul and Lessons Learned

During the 2004 North Atlantic Treaty Organization (NATO) Summit, essential counter-measures, including medical preparedness, were taken to cope with any suspected terrorist case or events including the use of chemical or biological (CB) weapons. The Summit was held in Istanbul, a city that bridges two continents, and involved the participation of many Heads of State, Prime Ministers, and Defense Ministers from 26 NATO countries. First responders, including medical Chemical, Biological, Radiological, and Nuclear (CBRN) teams, received special training. Essential equipment, including drugs, antidotes, detectors, etc., was provided and stock-piled. Medical authorities augmented the capacity for identifying and controlling the injuries and any emerging CB incident through the set-up of decontamination units and the procurement of medical devices, antidotes, drugs, and personal protective suits. Additionally, a small part of the recently established NATO-CBRN battalion was welcomed to the Summit and was prepared to perform detection and identification of the agent found in suspicious appearing samples. Although no CB incident was reported during the Summit, extensive experience was gained with respect to medical preparedness against CB terrorism. Sampling, detection, and analysis of toxic materials were taken into account in the medical management. Much laboratory-related work was conducted in the following time period. The laboratory work involved the standardization of sampling and transportation procedures, development of both mobile and reference laboratories, and performing research activities aimed to make the CB analysis more efficient. Although the training of the medical staff was advanced, training should be continuous and supported with educational programs, conferences, meetings, and tabletop and hospital medical exercises throughout the country. Multidisciplinary cooperation, training, and preparedness should be provided to basic medical care units and centers as part of the medical planning aimed at perfect detection and surveillance, laboratory analysis, and emergency response.

RNA Interference mediated knockdown of genes in order to increase protein production using the baculovirus expression system

Address: 1Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742, USA, 2Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center, 3302 Gaston Ave., Dallas, TX 75246, USA, 3U. S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland 21010, USA and 4Department of Chemical and Biomolecular Engineering, University of Maryland College Park, College Park, MD 20742, USA * Corresponding author