Activity
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🌟 Speaker Spotlight: Raymond Colton🌟 Join us at the Extractables & Leachables Summit West 2024 as we welcome Raymond Colton, Director of E&L…
🌟 Speaker Spotlight: Raymond Colton🌟 Join us at the Extractables & Leachables Summit West 2024 as we welcome Raymond Colton, Director of E&L…
Liked by Mike Awada, PhD, MBA
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Looking forward to this workshop by FDA on Artificial Intelligence in Drug & Biological Product Development. #ArtificialIntelligence #pharmaceutical…
Looking forward to this workshop by FDA on Artificial Intelligence in Drug & Biological Product Development. #ArtificialIntelligence #pharmaceutical…
Liked by Mike Awada, PhD, MBA
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Here’s a fun segment I did for the SLCC Student Media Center a few months ago. I was impressed by what some of the students knew! Go Bruins! Salt…
Here’s a fun segment I did for the SLCC Student Media Center a few months ago. I was impressed by what some of the students knew! Go Bruins! Salt…
Liked by Mike Awada, PhD, MBA
Licenses & Certifications
Publications
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Application of BPOG standardized extractables protocol to container films: The impact of different sample conditions and analytical procedures on E&L profile.
ECI Conference, Leesburg, VA
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Practical Application of BPOG E&L Protocols for SU Films.
Merck Single-Use Symposium, Blue Bell, PA
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Measurement of Reverse Cholesterol Transport Pathways in Humans: In Vivo Rates of Free Cholesterol Efflux, Esterification and Excretion.
Journal of the American Heart Association
Reverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high‐density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and excretion of plasma‐derived FC.
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A Stable Isotope Method for the Simultaneous Measurement of Matrix Synthesis and Cell Proliferation in Articular Cartilage in Vivo.
Osteoarthritis and Cartilage
Measurements of cell proliferation and matrix synthesis in cartilage explants have identified regulatory factors [e.g., interleukin-1 (IL-1)] that contribute to osteoarthritis and anabolic mediators [e.g., bone morphogenic protein-7 (BMP-7)] that may have therapeutic potential. The objective of this study was to develop a robust method for measuring cell proliferation and glycosaminoglycan synthesis in articular cartilage that could be applied in vivo.
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Measurement of brain microglial proliferation rates in vivo in response to neuroinflammatory stimuli: application to drug discovery
J Neurosci Res .
Microglial activation is emerging as an important etiologic factor and therapeutic target in neurodegenerative and neuroinflammatory diseases. Techniques have been lacking, however, for measuring the different components of microglial activation independently in vivo. We describe a method for measuring microglial proliferation rates in vivo using heavy water (2H2O) labeling, and its application in screening for drugs that suppress neuro-inflammation. Brain microglia were isolated by flow…
Microglial activation is emerging as an important etiologic factor and therapeutic target in neurodegenerative and neuroinflammatory diseases. Techniques have been lacking, however, for measuring the different components of microglial activation independently in vivo. We describe a method for measuring microglial proliferation rates in vivo using heavy water (2H2O) labeling, and its application in screening for drugs that suppress neuro-inflammation. Brain microglia were isolated by flow cytometry as F4/80+, CD11b+, CD45(low) cells, and 2H enrichment in DNA was analyzed by gas chromatography/mass spectrometry. Basal proliferation rate was approximately 1%/week and systemic administration of bacterial lipopolysaccharide (LPS) markedly increased this rate in a dose-dependent manner. Induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice by MOG(35-55) peptide stimulated proliferation of CD45(low) microglia, which could be distinguished from the proliferation of CD45(high) infiltrating monocytes. Minocycline (45 mg/kg/day, i.p.) inhibited resident microglial proliferation in both the LPS and EAE models. Thirteen drugs were then screened for their ability to inhibit LPS-stimulated microglia proliferation. Female C57BL/6 mice were given LPS (1 mg/kg), and concomitant drug treatment while receiving 2H2O label for 7 days. Among the drugs screened, treatment with isotretinoin dose-dependently reduced LPS-induced microglial proliferation, representing an action of retinoids unknown previously. Follow-up studies in the EAE model confirmed that isotretinoin not only inhibited proliferation of microglia but also delayed the onset of clinical symptoms. In conclusion, 2H2O labeling represents a relatively high-throughput, quantitative, and highly reproducible technique for measuring microglial proliferation, and is useful for screening and discovering novel anti-neuroinflammatory drugs.
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Modulation of Hyperdynamic Microtubules Is Neuroprotective in ALS
Journal of Biological Chemistry
Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced…
Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders.
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Measurement of liver collagen synthesis by heavy water labeling: effects of profibrotic toxicants and antifibrotic interventions
Am J Physiol Gastrointest Liver Physiol .
Enhanced production of collagen is central to fibrotic disorders such as hepatic cirrhosis and pulmonary fibrosis. We describe a sensitive, quantitative, and high-throughput technique for measuring hepatic collagen synthesis in vivo through metabolic labeling with heavy water ((2)H(2)O). Rats and mice received (2)H(2)O in drinking water for up to 35 days. Deuterium incorporation into collagen-bound amino acids (AA) alanine and hydroxyproline (OHP) was measured by gas chromatography-mass…
Enhanced production of collagen is central to fibrotic disorders such as hepatic cirrhosis and pulmonary fibrosis. We describe a sensitive, quantitative, and high-throughput technique for measuring hepatic collagen synthesis in vivo through metabolic labeling with heavy water ((2)H(2)O). Rats and mice received (2)H(2)O in drinking water for up to 35 days. Deuterium incorporation into collagen-bound amino acids (AA) alanine and hydroxyproline (OHP) was measured by gas chromatography-mass spectrometry. A threefold stimulation of collagen fractional synthesis was observed under the maximum dosage of carbon tetrachloride (CCl(4); 1.67 ml/kg). Deuterium enrichment was systematically 20% higher in AA from monomeric collagen relative to dimeric collagen, consistent with slower turnover of the latter. Administration of 1% griseofulvin to mice resulted in a significant, threefold increase in liver collagen synthesis, observable within 12 days and consistent with predicted interstrain differences (C57/Bl6J > BALB/c). Deuterium enrichments of OHP from total liver proteins correlated well with alanine or OHP from isolated collagen. Fibrogenesis subsided after withdrawal of CCl(4) exposure and was reduced to various degrees by coadministration of interferon-gamma, rosiglitazone, atorvastatin, or enalapril. Changes in isotopically measured collagen synthesis correlated with, but were more sensitive and reproducible than, standard histological staining (trichrome) for fibrosis. In summary, liver collagen synthesis can be measured sensitively and with high precision over a short time period, without radioactivity, thereby providing a relatively high-throughput in vivo strategy for rapidly measuring profibrotic activities of suspected hepatotoxicants and antifibrotic activities of drug candidates.
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Measurement of cell proliferation by heavy water labeling
Nature Protocols
DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by…
DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.
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Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio-mass spectrometric analysis
Nature Protocols
DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be…
DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.
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Measurement of protein turnover rates by heavy water labeling of nonessential amino acids
Biochim Biophys Acta .
In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into…
In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.
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Whole-body glycolysis measured by the deuterated-glucose disposal test correlates highly with insulin resistance in vivo
Diabetes Care
The purpose of this study was to compare an in vivo test of whole-body glycolysis, the deuterated-glucose disposal test (2H-GDT), with insulin sensitivity measured by the euglycemic-hyperinsulinemic glucose clamp and the steady-state plasma glucose (SSPG) test.
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In vivo measurement of microtubule dynamics using stable isotope labeling with heavy water. Effect of taxanes
J Biol Chem .
Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures…
Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures microtubule dynamics as an exchange of tubulin dimers into microtubules in vivo. The incorporation of deuterium ((2)H(2)) from heavy water ((2)H(2)O) into tubulin dimers and polymers is measured by gas chromatography/mass spectrometry. In cultured human lung and breast cancer cell lines, or in tumors implanted into nude mice, tubulin dimers and polymerized microtubules exhibited nearly identical label incorporation rates, reflecting their rapid exchange. Administration of paclitaxel during 24 h of (2)H(2)O labeling in vivo reduced (2)H labeling in polymers while increasing (2)H in dimers, indicating diminished flux of dimers into polymers (i.e. inhibition of microtubule dynamic equilibrium). In vivo inhibition of microtubule dynamics was dose-dependent and correlated with inhibition of DNA replication, a stable isotopic measure of tumor cell growth. In contrast, microtubule polymers from sciatic nerve of untreated mice were not in dynamic equilibrium with tubulin dimers, and paclitaxel increased label incorporation into polymers. Our results directly demonstrate altered microtubule dynamics as an important action of MTPAs in vivo. This sensitive and quantitative in vivo assay of microtubule dynamics may prove useful for pre-clinical and clinical development of the next generation of MTPAs as anticancer drugs.
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Analysis of 3'-phosphoglycolaldehyde residues in oxidized DNA by gas chromatography/negative chemical ionization/mass spectrometry
Chem Res Toxicol .
Deoxyribose oxidation in DNA represents a biologically important facet of oxidative DNA damage that gives rise to protein-DNA cross-links and base adducts. Toward the goal of quantifying deoxyribose oxidation chemistry in cells, we report a method for the quantification of 3'-phosphoglycolaldehyde (PGA) residues, which likely arise from 3'-oxidation of deoxyribose in DNA. The method exploits the aldehyde moiety in PGA by derivatization as a stable oxime with pentafluorobenzylhydroxylamine…
Deoxyribose oxidation in DNA represents a biologically important facet of oxidative DNA damage that gives rise to protein-DNA cross-links and base adducts. Toward the goal of quantifying deoxyribose oxidation chemistry in cells, we report a method for the quantification of 3'-phosphoglycolaldehyde (PGA) residues, which likely arise from 3'-oxidation of deoxyribose in DNA. The method exploits the aldehyde moiety in PGA by derivatization as a stable oxime with pentafluorobenzylhydroxylamine, followed by solvent extraction and gas chromatography/negative chemical ionization/mass spectrometry. A stable isotopically labeled [(13)C(2)]PGA was synthesized and used as an internal standard. The assay showed a linear response over the range of 30 fmol to 300 pmol, and its precision was verified by analysis of a synthetic, PGA-containing oligodeoxynucleotide. The limit of detection in the presence of DNA was 30 fmol per sample, corresponding to two molecules of PGA in 10(6) nucleotides for 170 microg of DNA. Samples were exposed to 0-100 Gy of (60)Co gamma-radiation, which resulted in a linear dose-response of 1.5 PGA residues per 10(6) nucleotides per Gy and a radiation chemical yield (G-value) of 0.0016 micromol/J. When compared to the total quantity of deoxyribose oxidation occurring under the same conditions (141 oxidation events per 10(6) nucleotides per Gy; determined by plasmid topoisomer analysis), PGA formation occurs in 1% of deoxyribose oxidation events. This small fraction is consistent with current models of limited solvent accessibility of the 3'-position of deoxyribose, although partitioning of 3'-chemistry could lead to other damage products that would increase the fraction of oxidation at this site in deoxyribose.
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Formation of the 1,N2-glyoxal adduct of deoxyguanosine by phosphoglycolaldehyde, a product of 3'-deoxyribose oxidation in DNA
Chem Res Toxicol .
Oxidation of deoxyribose in DNA results in the formation of a variety of electrophilic products that have the potential to react with nucleobases to form adducts. We now report that 2-phosphoglycolaldehyde, a model for the 3'-phosphoglycolaldehyde residue generated by 3'-oxidation of deoxyribose in DNA, reacts with dG and DNA to form the diastereomeric 1,N2-glyoxal adducts of dG, 3-(2-deoxy-beta-D-erythro-pentofuransyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one. The glyoxal…
Oxidation of deoxyribose in DNA results in the formation of a variety of electrophilic products that have the potential to react with nucleobases to form adducts. We now report that 2-phosphoglycolaldehyde, a model for the 3'-phosphoglycolaldehyde residue generated by 3'-oxidation of deoxyribose in DNA, reacts with dG and DNA to form the diastereomeric 1,N2-glyoxal adducts of dG, 3-(2-deoxy-beta-D-erythro-pentofuransyl)-6,7-dihydro-6,7-dihydroxyimidazo[1,2-a]purine-9(3H)-one. The glyoxal adducts were the predominant species formed under biological conditions (pH 7.4 and 37 degrees C), with several minor fluorescent adducts, including 1,N6-ethenoadenine. The adducts were fully characterized by HPLC, mass spectrometry, and UV and NMR spectroscopy. The reaction of 2-phosphoglycolaldehyde with dG occurred with a rate constant of 10(-6) M(-1) s(-1) compared to the rate constants of 0.08 and approximately 10(-9) M(-1) s(-1) for the reactions of glyoxal and glycolaldehyde with dG, respectively. The kinetic results rule out contamination of 2-phosphoglycolaldehyde preparations with glyoxal as the basis for our observations. The rate constant for the formation of glyoxal from 2-phosphoglycolaldehyde (10(-8) s(-1)) is consistent with glyoxal generation being the rate-limiting step in the formation of dG adducts in reactions with 2-phosphoglycolaldehyde. Mechanistic studies were also undertaken to define the basis for the different oxidation states of glyoxal and 2-phosphoglycolaldehyde. Although 2-phosphoglycolaldehyde produced a weak ESR signal consistent with generation of hydroxyl radicals and it caused DNA strand breaks at high concentrations, the formation of the glyoxal adducts of dG was insensitive to radical quenchers (e.g., sorbitol) and independent of molecular oxygen.
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Analysis of oxidized DNA fragments by gel electrophoresis
Curr Protoc Nucleic Acid Chem .
Polyacrylamide gel electrophoresis is used to define and quantify products of deoxyribose oxidation in DNA, based on the unique electrophoretic mobility of DNA fragments possessing deoxyribose oxidation products on their termini. This approach allows initial estimation of the chemistry. Once the chemical identity of damage products has been confirmed, this technique allows sensitive quantitation of the various damage products.
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Electrodeposition of Metal Adlayers on Boron‐Doped Diamond Thin‐Film Electrodes
Journal of The Electrochemical Societ
A preliminary investigation of the electrochemical deposition of Pt, Pb, and Hg adlayers on conductive diamond thin‐film surfaces has been made using cyclic voltammetry and scanning electron microscopy. The diamond thin films employed were polycrystalline, grown on conductive Si substrates (1 cm2) to a thickness of ca. 14 μm, and doped with boron at a nominal atomic concentration ranging between 1019 and 1020 cm−3. The cyclic volammetric measurements were performed both in a conventional glass…
A preliminary investigation of the electrochemical deposition of Pt, Pb, and Hg adlayers on conductive diamond thin‐film surfaces has been made using cyclic voltammetry and scanning electron microscopy. The diamond thin films employed were polycrystalline, grown on conductive Si substrates (1 cm2) to a thickness of ca. 14 μm, and doped with boron at a nominal atomic concentration ranging between 1019 and 1020 cm−3. The cyclic volammetric measurements were performed both in a conventional glass electrochemical cell and in a thin‐layer flow cell. The results demonstrate that metallization of diamond film surfaces electrochemically is feasible, opening the door for the development of novel catalytic electrodes, sensors, and detectors using this advanced material.
More activity by Mike
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A bit late to post this but I am excited to announce that I completed my degree in Biological Engineering in May! I am super excited for what the…
A bit late to post this but I am excited to announce that I completed my degree in Biological Engineering in May! I am super excited for what the…
Liked by Mike Awada, PhD, MBA
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Momentum is celebrating #InventorsMonth recognizing our core team's contribution to the RapidFire high-throughput mass spectrometry platform, which…
Momentum is celebrating #InventorsMonth recognizing our core team's contribution to the RapidFire high-throughput mass spectrometry platform, which…
Liked by Mike Awada, PhD, MBA
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I'm looking forward to presenting on bacteriophage therapy with my colleague Emily Heffernan this June.
I'm looking forward to presenting on bacteriophage therapy with my colleague Emily Heffernan this June.
Liked by Mike Awada, PhD, MBA
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𝐇𝐢𝐤𝐦𝐚 𝐚𝐩𝐩𝐨𝐢𝐧𝐭𝐬 𝐃𝐫. 𝐇𝐚𝐟𝐫𝐮𝐧 𝐅𝐫𝐢𝐝𝐫𝐢𝐤𝐬𝐝𝐨𝐭𝐭𝐢𝐫 𝐚𝐬 𝐏𝐫𝐞𝐬𝐢𝐝𝐞𝐧𝐭 𝐨𝐟 𝐆𝐞𝐧𝐞𝐫𝐢𝐜𝐬 𝐛𝐮𝐬𝐢𝐧𝐞𝐬𝐬 Hikma…
𝐇𝐢𝐤𝐦𝐚 𝐚𝐩𝐩𝐨𝐢𝐧𝐭𝐬 𝐃𝐫. 𝐇𝐚𝐟𝐫𝐮𝐧 𝐅𝐫𝐢𝐝𝐫𝐢𝐤𝐬𝐝𝐨𝐭𝐭𝐢𝐫 𝐚𝐬 𝐏𝐫𝐞𝐬𝐢𝐝𝐞𝐧𝐭 𝐨𝐟 𝐆𝐞𝐧𝐞𝐫𝐢𝐜𝐬 𝐛𝐮𝐬𝐢𝐧𝐞𝐬𝐬 Hikma…
Liked by Mike Awada, PhD, MBA
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Excited to announce that I’ll be joining the Northrop Grumman team as a principal chemist. Looking forward to the opportunity to contribute to their…
Excited to announce that I’ll be joining the Northrop Grumman team as a principal chemist. Looking forward to the opportunity to contribute to their…
Liked by Mike Awada, PhD, MBA
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