Publications by authors named "Melissa A Pinard"

19 Publications

  • Page 1 of 1

Crystal Structure of Cleaved Serp-1, a Myxomavirus-Derived Immune Modulating Serpin: Structural Design of Serpin Reactive Center Loop Peptides with Improved Therapeutic Function.

Biochemistry 2018 02 6;57(7):1096-1107. Epub 2018 Feb 6.

Department of Medicine, Divisions of Cardiovascular Medicine and Rheumatology, University of Florida , Gainesville, Florida 32610-0277, United States.

The Myxomavirus-derived protein Serp-1 has potent anti-inflammatory activity in models of vasculitis, lupus, viral sepsis, and transplant. Serp-1 has also been tested successfully in a Phase IIa clinical trial in unstable angina, representing a "first-in-class" therapeutic. Recently, peptides derived from the reactive center loop (RCL) have been developed as stand-alone therapeutics for reducing vasculitis and improving survival in MHV68-infected mice. However, both Serp-1 and the RCL peptides lose activity in MHV68-infected mice after antibiotic suppression of intestinal microbiota. Here, we utilize a structure-guided approach to design and test a series of next-generation RCL peptides with improved therapeutic potential that is not reduced when the peptides are combined with antibiotic treatments. The crystal structure of cleaved Serp-1 was determined to 2.5 Å resolution and reveals a classical serpin structure with potential for serpin-derived RCL peptides to bind and inhibit mammalian serpins, plasminogen activator inhibitor 1 (PAI-1), anti-thrombin III (ATIII), and α-1 antitrypsin (A1AT), and target proteases. Using in silico modeling of the Serp-1 RCL peptide, S-7, we designed several modified RCL peptides that were predicted to have stronger interactions with human serpins because of the larger number of stabilizing hydrogen bonds. Two of these peptides (MPS7-8 and -9) displayed extended activity, improving survival where activity was previously lost in antibiotic-treated MHV68-infected mice (P < 0.0001). Mass spectrometry and kinetic assays suggest interaction of the peptides with ATIII, A1AT, and target proteases in mouse and human plasma. In summary, we present the next step toward the development of a promising new class of anti-inflammatory serpin-based therapeutics.
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http://dx.doi.org/10.1021/acs.biochem.7b01171DOI Listing
February 2018

Cryoannealing-induced space-group transition of crystals of the carbonic anhydrase psCA3.

Acta Crystallogr F Struct Biol Commun 2016 07 28;72(Pt 7):573-7. Epub 2016 Jun 28.

Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, 1200 Newell Drive, PO Box 100245, Gainesville, FL 32610, USA.

Cryoannealing has been demonstrated to improve the diffraction quality and resolution of crystals of the β-carbonic anhydrase psCA3 concomitant with a change in space group. After initial flash-cooling in a liquid-nitrogen cryostream an X-ray diffraction data set from a psCA3 crystal was indexed in space group P21212 and was scaled to 2.6 Å resolution, but subsequent cryoannealing studies revealed induced protein rearrangements in the crystal contacts, which transformed the space group to I222, with a corresponding improvement of 0.7 Å in resolution. Although the change in diffraction resolution was significant, only minor changes in the psCA3 structure, which retained its catalytic `open' conformation, were observed. These findings demonstrate that cryoannealing can be successfully utilized to induce higher diffraction-quality crystals while maintaining enzymatically relevant conformations and may be useful as an experimental tool for structural studies of other enzymes where the initial diffraction quality is poor.
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http://dx.doi.org/10.1107/S2053230X16009286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933009PMC
July 2016

Carbon Dioxide "Trapped" in a β-Carbonic Anhydrase.

Biochemistry 2015 Nov 16;54(43):6631-8. Epub 2015 Oct 16.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida , Gainesville, Florida 32610, United States.

Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/dehydration of CO2/HCO3(-) with rates approaching diffusion-controlled limits (kcat/KM ∼ 10(8) M(-1) s(-1)). This family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a β-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryo-cooled crystallography. The structure has been refined to 1.6 Å resolution with R(cryst) and R(free) values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2 captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2 binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2 binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dimers or higher-order oligomeric states, and the CO2 bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.
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http://dx.doi.org/10.1021/acs.biochem.5b00987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745652PMC
November 2015

A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX.

Acta Crystallogr F Struct Biol Commun 2015 Oct 23;71(Pt 10):1352-8. Epub 2015 Sep 23.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.

Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.
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http://dx.doi.org/10.1107/S2053230X1501239XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601603PMC
October 2015

Observed surface lysine acetylation of human carbonic anhydrase II expressed in Escherichia coli.

Protein Sci 2015 Nov 15;24(11):1800-7. Epub 2015 Sep 15.

Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida, 32610.

Acetylation of surface lysine residues of proteins has been observed in Escherichia coli (E. coli), an organism that has been extensively utilized for recombinant protein expression. This post-translational modification is shown to be important in various processes such as metabolism, stress-response, transcription, and translation. As such, utilization of E. coli expression systems for protein production may yield non-native acetylation events of surface lysine residues. Here we present the crystal structures of wild-type and a variant of human carbonic anhydrase II (hCA II) that have been expressed in E. coli and exhibit surface lysine acetylation and we speculate on the effect this has on the conformational stability of each enzyme. Both structures were determined to 1.6 Å resolution and show clear electron density for lysine acetylation. The lysine acetylation does not distort the structure and the surface lysine acetylation events most likely do not interfere with the biological interpretation. However, there is a reduction in conformational stability in the hCA II variant compared to wild type (∼ 4°C decrease). This may be due to other lysine acetylation events that have occurred but are not visible in the crystal structure due to intrinsic disorder. Therefore, surface lysine acetylation events may affect overall protein stability and crystallization, and should be considered when using E. coli expression systems.
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http://dx.doi.org/10.1002/pro.2771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4622213PMC
November 2015

Structural and biophysical characterization of the α-carbonic anhydrase from the gammaproteobacterium Thiomicrospira crunogena XCL-2: insights into engineering thermostable enzymes for CO2 sequestration.

Acta Crystallogr D Biol Crystallogr 2015 Aug 31;71(Pt 8):1745-56. Epub 2015 Jul 31.

Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610, USA.

Biocatalytic CO2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2 sequestration.
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http://dx.doi.org/10.1107/S1399004715012183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528804PMC
August 2015

Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure-Activity Relationships of Glucosyl-Based Sulfamates.

J Med Chem 2015 Aug 13;58(16):6630-8. Epub 2015 Aug 13.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida , 1600 SW Archer Road, PO Box 100245, Gainesville, Florida 32610, United States.

Inhibition of human carbonic anhydrase IX (hCA IX) has shown to be therapeutically advantageous for treating many types of highly aggressive cancers. However, designing selective inhibitors for hCA IX has been difficult due to its high structural homology and sequence similarity with off-target hCAs. Recently, the use of glucosyl sulfamate inhibitors has shown promise as selective inhibitors for hCA IX. In this study, we present five X-ray crystal structures, determined to a resolution of 1.7 Å or better, of both hCA II (a ubiquitous CA) and an engineered hCA IX-mimic in complex with selected glucosyl sulfamates and structurally rationalize mechanisms for hCA IX selectivity. Results from this study have allowed us, for the first time, to empirically "map" key interactions of the hCA IX active site in order to establish parameters needed to design novel hCA IX selective inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00845DOI Listing
August 2015

Structure and inhibition studies of a type II beta-carbonic anhydrase psCA3 from Pseudomonas aeruginosa.

Bioorg Med Chem 2015 Aug 8;23(15):4831-4838. Epub 2015 Jun 8.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA. Electronic address:

Carbonic anhydrases (CAs) are metallo-enzymes that catalyze the reversible hydration of carbon dioxide into bicarbonate and a proton. The β-class CAs (β-CAs) are expressed in prokaryotes, fungi, plants, and more recently have been isolated in some animals. The β-CA class is divided into two subclasses, termed type I and II, defined by pH catalytic activity profile and active site structural configuration. Type I β-CAs display catalytic activity over a broad pH range (6.5-9.0) with the active site zinc tetrahedrally coordinated by three amino acids and a hydroxide/water. In contrast, type II β-CAs are catalytically active only at a pH 8 and higher where they adopt a functional active site configuration like that of type I. However, below pH 8 they are conformationally self-inactivated by the addition of a fourth amino acid coordinating the zinc and thereby displacing the zinc bound solvent. We have determined the structure of psCA3, a type II β-CA, isolated from Pseudomonas aeruginosa (P. aeruginosa) PAO1 at pH 8.3, in its open active state to a resolution of 1.9 Å. The active site zinc is coordinated by Cys42, His98, Cys101 and a water/hydroxide molecule. P. aeruginosa is a multi-drug resistant bacterium and displays intrinsic resistance to most of the currently used antibiotics; therefore, there is a need for new antibacterial targets. Kinetic data confirm that psCA3 belongs to the type II subclass and that sulfamide, sulfamic acid, phenylboronic acid and phenylarsonic acid are micromolar inhibitors. In vivo studies identified that among six tested inhibitors representing sulfonamides, inorganic anions, and small molecules, acetazolamide has the most significant dose-dependent inhibitory effect on P. aeruginosa growth.
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http://dx.doi.org/10.1016/j.bmc.2015.05.029DOI Listing
August 2015

Activity and anion inhibition studies of the α-carbonic anhydrase from Thiomicrospira crunogena XCL-2 Gammaproteobacterium.

Bioorg Med Chem Lett 2015 Nov 6;25(21):4937-4940. Epub 2015 May 6.

Department of Biochemistry and Molecular Biology, University of Florida, College of Medicine, 100 Newell Dr LG-171, Gainesville, FL 32610, United States. Electronic address:

Thiomicrospira crunogena XCL-2 expresses an α-carbonic anhydrase (TcruCA). Sequence alignments reveal that TcruCA displays a high sequence identity (>30%) relative to other α-CAs. This includes three conserved histidines that coordinate the active site zinc, a histidine proton shuttling residue, and opposing hydrophilic and hydrophobic sides that line the active site. The catalytic efficiency of TcruCA is considered moderate relative to other α-CAs (k(cat)/K(M)=1.1×10(7) M(-1) s(-1)), being a factor of ten less efficient than the most active α-CAs. TcruCA is also inhibited by anions with Cl(-), Br(-), and I(-), all showing Ki values in the millimolar range (53-361 mM). Hydrogen sulfide (HS(-)) revealed the highest affinity for TcruCA with a Ki of 1.1 μM. It is predicted that inhibition of TcruCA by HS(-) (an anion commonly found in the environment where Thiomicrospira crunogena is located) is a way for Thiomicrospira crunogena to regulate its carbon-concentrating mechanism (CCM) and thus the organism's metabolic functions. Results from this study provide preliminary insights into the role of TcruCA in the general metabolism of Thiomicrospira crunogena.
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http://dx.doi.org/10.1016/j.bmcl.2015.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358508PMC
November 2015

Probing the surface of human carbonic anhydrase for clues towards the design of isoform specific inhibitors.

Biomed Res Int 2015 24;2015:453543. Epub 2015 Feb 24.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, P.O. Box 100245, Gainesville, FL 32610, USA.

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3(-). Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of "selective drug targetability" and how these observations can be utilized to develop isoform selective CA inhibitors.
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http://dx.doi.org/10.1155/2015/453543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355338PMC
December 2015

Targeting carbonic anhydrase IX activity and expression.

Molecules 2015 Jan 30;20(2):2323-48. Epub 2015 Jan 30.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611, USA.

Metastatic tumors are often hypoxic exhibiting a decrease in extracellular pH (~6.5) due to a metabolic transition described by the Warburg Effect. This shift in tumor cell metabolism alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments; hence hindering treatment of aggressive cancers. As a result, tumors exhibiting this phenotype are directly associated with poor prognosis and decreased survival rates in cancer patients. A key component to this tumor microenvironment is carbonic anhydrase IX (CA IX). Knockdown of CA IX expression or inhibition of its activity has been shown to reduce primary tumor growth, tumor proliferation, and also decrease tumor resistance to conventional anti-cancer therapies. As such several approaches have been taken to target CA IX in tumors via small-molecule, anti-body, and RNAi delivery systems. Here we will review recent developments that have exploited these approaches and provide our thoughts for future directions of CA IX targeting for the treatment of cancer.
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http://dx.doi.org/10.3390/molecules20022323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272707PMC
January 2015

Human carbonic anhydrase II-cyanate inhibitor complex: putting the debate to rest.

Acta Crystallogr F Struct Biol Commun 2014 Oct 25;70(Pt 10):1324-7. Epub 2014 Sep 25.

Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA.

The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using (18)O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.
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http://dx.doi.org/10.1107/S2053230X14018135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188073PMC
October 2014

Structural study of interaction between brinzolamide and dorzolamide inhibition of human carbonic anhydrases.

Bioorg Med Chem 2013 Nov 28;21(22):7210-5. Epub 2013 Aug 28.

Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Box 100245, Gainesville, FL 32610, USA.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and bicarbonate. Their pivotal role in metabolism, ubiquitous nature, and multiple isoforms (CA I-XIV) has made CAs an attractive drug target in clinical applications. The usefulness of CA inhibitors (CAIs) in the treatment of glaucoma and epilepsy are well documented. In addition several isoforms of CAs (namely, CA IX) also serve as biological markers for certain tumors, and therefore they have the potential for useful applications in the treatment of cancer. This is a structural study on the binding interactions of the widely used CA inhibitory drugs brinzolamide (marketed as Azopt®) and dorzolamide (marketed as Trusopt®) with CA II and a CA IX-mimic, which was created via site-directed mutagenesis of CA II cDNA such that the active site resembles that of CA IX. Also the inhibition of CA II and CA IX and molecular docking reveal brinzolamide to be a more potent inhibitor among the other catalytically active CA isoforms compared to dorzolamide. The structures show that the tail end of the sulfonamide inhibitor is critical in forming stabilizing interactions that influence tight binding; therefore, for future drug design it is the tail moiety that will ultimately determine isoform specificity.
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http://dx.doi.org/10.1016/j.bmc.2013.08.033DOI Listing
November 2013

The malignant brain tumor (MBT) domain protein SFMBT1 is an integral histone reader subunit of the LSD1 demethylase complex for chromatin association and epithelial-to-mesenchymal transition.

J Biol Chem 2013 Sep 8;288(38):27680-27691. Epub 2013 Aug 8.

Department of Biochemistry and Molecular Biology; Genetics and Genomics Graduate Program, Genetics Institute, University of Florida, Gainesville, Florida 32610. Electronic address:

Chromatin readers decipher the functional readouts of histone modifications by recruiting specific effector complexes for subsequent epigenetic reprogramming. The LSD1 (also known as KDM1A) histone demethylase complex modifies chromatin and represses transcription in part by catalyzing demethylation of dimethylated histone H3 lysine 4 (H3K4me2), a mark for active transcription. However, none of its currently known subunits recognizes methylated histones. The Snai1 family transcription factors are central drivers of epithelial-to-mesenchymal transition (EMT) by which epithelial cells acquire enhanced invasiveness. Snai1-mediated transcriptional repression of epithelial genes depends on its recruitment of the LSD1 complex and ensuing demethylation of H3K4me2 at its target genes. Through biochemical purification, we identified the MBT domain-containing protein SFMBT1 as a novel component of the LSD1 complex associated with Snai1. Unlike other mammalian MBT domain proteins characterized to date that selectively recognize mono- and dimethylated lysines, SFMBT1 binds di- and trimethyl H3K4, both of which are enriched at active promoters. We show that SFMBT1 is essential for Snai1-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGFβ. Carcinogenic metal nickel is a widespread environmental and occupational pollutant. Nickel alters gene expression and induces EMT. We demonstrate the nickel-initiated effects are dependent on LSD1-SFMBT1-mediated chromatin modification. Furthermore, in human cancer, expression of SFMBT1 is associated with mesenchymal markers and unfavorable prognosis. These results highlight a critical role of SFMBT1 in epigenetic regulation, EMT, and cancer.
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http://dx.doi.org/10.1074/jbc.M113.482349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779763PMC
September 2013

Rapid crystallization of glycine using metal-assisted and microwave-accelerated evaporative crystallization: the effect of engineered surfaces and sample volume.

Nano Biomed Eng 2012 ;4(3):125-131

Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane, Baltimore, MD 21251, USA.

Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC), is a new approach to crystallization of drug compounds, amino acids, DNA and proteins. In this work, we report our additional findings on the effect of engineered surfaces and sample volume on the rapid crystallization of glycine. With the use of hydrophilic functionalized surfaces and the MA-MAEC technique, glycine crystals ~1 mm in size were grown in 35 seconds with 100% selectivity for the α-form.The use of moderately hydrophobic surfaces resulted in the growth of glycine crystals only at room temperature. An increase in volume of initial glycine solution (5-100 μL) resulted in an increase in crystal size without a significant increase in total crystallization time. Raman spectroscopy and powder X-ray diffraction results demonstrated that the glycine crystals grown on engineered surfaces were structurally identical to those grown using conventional evaporative crystallization.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548239PMC
http://dx.doi.org/10.5101/nbe.v4i3.p125-131DOI Listing
January 2012

Rapid crystallization of L-arginine acetate on engineered surfaces using metal-assisted and microwave-accelerated evaporative crystallization().

CrystEngComm 2012 Jan 1;14(14):4557-4561. Epub 2012 May 1.

University of Florida, Department of Biochemistry and Molecular Biology, 1600 SW Archer Road, Gainesville, FL 32610, USA.

We report the application of our newly described crystallization technique, which employs silver island films (SIFs) and microwave heating, to rapid crystallization of L-arginine acetate (LAA). Using our technique, LAA crystals (~ 1.2 mm in length) were grown from a 20 μl solution in 1 min on surface functionalized SIFs. In control experiments (glass slides and at room temperature) the growth of LAA crystals (0.1-0.3 mm) took ~ 55 min.
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http://dx.doi.org/10.1039/C2CE25380ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3413293PMC
January 2012

Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

Cryst Growth Des 2010 Nov;10(11):4706-4709

Morgan State University, Department of Chemistry, Baltimore, Maryland 21251, United States.

We describe a platform technology, called metal-assisted and microwave-assisted evaporative crystallization (MA-MAEC), based on the combined use of silver nanoparticles and microwave heating for selective and rapid crystallization of small molecules. In this regard, the crystallization of a model small molecule (glycine) was achieved in several seconds. Glycine crystals grown on silver nanostructures with and without microwave heating were found to be larger than those grown on blank glass slides. The MA-MAEC technique has the potential to selectively grow the desired polymorphs of small molecules "on-demand" in a fraction of the time as compared to the conventional evaporative crystallization.
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http://dx.doi.org/10.1021/cg101059cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994586PMC
November 2010

Rapid and Sensitive Colorimetric ELISA using Silver Nanoparticles, Microwaves and Split Ring Resonator Structures.

Nano Biomed Eng 2010 Jan;2(3):155-164

Morgan State University, Department of Chemistry, Baltimore, MD, 21251, USA.

We report a new approach to colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) that reduces the total assay time to < 2 min and the lower-detection-limit by 100-fold based on absorbance readout. The new approach combines the use of silver nanoparticles, microwaves and split ring resonators (SRR). The SRR structure is comprised of a square frame of copper thin film (30 µm thick, 1 mm wide, overall length of ~9.4 mm on each side) with a single split on one side, which was deposited onto a circuit board (2×2 cm(2)). A single micro-cuvette (10 µl volume capacity) was placed in the split of the SRR structures. Theoretical simulations predict that electric fields are focused in and above the micro-cuvette without the accumulation of electrical charge that breaks down the copper film. Subsequently, the walls and the bottom of the micro-cuvette were coated with silver nanoparticles using a modified Tollen's reaction scheme. The silver nanoparticles served as a mediator for the creation of thermal gradient between the bioassay medium and the silver surface, where the bioassay is constructed. Upon exposure to low power microwave heating, the bioassay medium in the micro-cuvette was rapidly and uniformly heated by the focused electric fields. In addition, the creation of thermal gradient resulted in the rapid assembly of the proteins on the surface of silver nanoparticles without denaturing the proteins. The proof-of-principle of the new approach to ELISA was demonstrated for the detection of a model protein (biotinylated-bovine serum albumin, b-BSA). In this regard, the detection of b-BSA with bulk concentrations (1 µM to 1 pM) was carried out on commercially available 96-well high throughput screening (HTS) plates and silver nanoparticle-deposited SRR structures at room temperature and with microwave heating, respectively. While the room temperature bioassay (without microwave heating) took 70 min to complete, the identical bioassay took < 2 min to complete using the SRR structures (with microwave heating). A lower detection limit of 0.01 nM for b-BSA (100-fold lower than room temperature ELISA) was observed using the SRR structures.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2953801PMC
http://dx.doi.org/10.5101/nbe.v2i3.p155-164DOI Listing
January 2010

Ultrafast and sensitive bioassay using split ring resonator structures and microwave heating.

Appl Phys Lett 2010 Aug;97(9)

In this paper, we have reported that split ring resonators (SRRs) structures can be used for bioassay applications in order to further improve the assay time and sensitivity. The proof-of-principle demonstration of the ultrafast bioassays was accomplished by using a model biotin-avidin bioassay. While the identical room temperature bioassay (without microwave heating) took 70 min to complete, the identical bioassay took less than 2 min to complete by using SRR structures (with microwave heating). A lower detection limit of 0.01 nM for biotinylated-bovine serum albumin (100-fold lower than the room temperature bioassay) was observed by using SRR structures.
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http://dx.doi.org/10.1063/1.3484958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945734PMC
August 2010