Search for Invisible Axion Dark Matter in the 3.3-4.2 µeV Mass Range.

We report the results from a haloscope search for axion dark matter in the 3.3-4.2 µeV mass range. This search excludes the axion-photon coupling predicted by one of the benchmark models of "invisible" axion dark matter, the Kim-Shifman-Vainshtein-Zakharov model. This sensitivity is achieved using a large-volume cavity, a superconducting magnet, an ultra low noise Josephson parametric amplifier, and sub-Kelvin temperatures. The validity of our detection procedure is ensured by injecting and detecting blind synthetic axion signals.

Extended Search for the Invisible Axion with the Axion Dark Matter Experiment.

This Letter reports on a cavity haloscope search for dark matter axions in the Galactic halo in the mass range 2.81-3.31 µeV. This search utilizes the combination of a low-noise Josephson parametric amplifier and a large-cavity haloscope to achieve unprecedented sensitivity across this mass range. This search excludes the full range of axion-photon coupling values predicted in benchmark models of the invisible axion that solve the strong CP problem of quantum chromodynamics.

Search for Invisible Axion Dark Matter with the Axion Dark Matter Experiment.

This Letter reports the results from a haloscope search for dark matter axions with masses between 2.66 and 2.81 µeV. The search excludes the range of axion-photon couplings predicted by plausible models of the invisible axion. This unprecedented sensitivity is achieved by operating a large-volume haloscope at subkelvin temperatures, thereby reducing thermal noise as well as the excess noise from the ultralow-noise superconducting quantum interference device amplifier used for the signal power readout. Ongoing searches will provide nearly definitive tests of the invisible axion model over a wide range of axion masses.

Piezoelectrically Tuned Multimode Cavity Search for Axion Dark Matter.

The µeV axion is a well-motivated extension to the standard model. The Axion Dark Matter eXperiment (ADMX) collaboration seeks to discover this particle by looking for the resonant conversion of dark-matter axions to microwave photons in a strong magnetic field. In this Letter, we report results from a pathfinder experiment, the ADMX "Sidecar," which is designed to pave the way for future, higher mass, searches. This testbed experiment lives inside of and operates in tandem with the main ADMX experiment. The Sidecar experiment excludes masses in three widely spaced frequency ranges (4202-4249, 5086-5799, and 7173-7203 MHz). In addition, Sidecar demonstrates the successful use of a piezoelectric actuator for cavity tuning. Finally, this publication is the first to report data measured using both the TM_{010} and TM_{020} modes.

Dark matter axion search using a Josephson Traveling wave parametric amplifier.

We describe the first implementation of a Josephson Traveling Wave Parametric Amplifier (JTWPA) in an axion dark matter search. The operation of the JTWPA for a period of about two weeks achieved sensitivity to axion-like particle dark matter with axion-photon couplings above 10-13 Ge V-1 over a narrow range of axion masses centered around 19.84 µeV by tuning the resonant frequency of the cavity over the frequency range of 4796.7-4799.5 MHz. The JTWPA was operated in the insert of the axion dark matter experiment as part of an independent receiver chain that was attached to a 0.56-l cavity. The ability of the JTWPA to deliver high gain over a wide (3 GHz) bandwidth has engendered interest from those aiming to perform broadband axion searches, a longstanding goal in this field.

Axion Dark Matter Experiment: Detailed design and operations.

Axion dark matter experiment ultra-low noise haloscope technology has enabled the successful completion of two science runs (1A and 1B) that looked for dark matter axions in the 2.66-3.1 µeV mass range with Dine-Fischler-Srednicki-Zhitnisky sensitivity [Du et al., Phys. Rev. Lett. 120, 151301 (2018) and Braine et al., Phys. Rev. Lett. 124, 101303 (2020)]. Therefore, it is the most sensitive axion search experiment to date in this mass range. We discuss the technological advances made in the last several years to achieve this sensitivity, which includes the implementation of components, such as the state-of-the-art quantum-noise-limited amplifiers and a dilution refrigerator. Furthermore, we demonstrate the use of a frequency tunable microstrip superconducting quantum interference device amplifier in run 1A, and a Josephson parametric amplifier in run 1B, along with novel analysis tools that characterize the system noise temperature.

A small peptide derived from Flt-1 (VEGFR-1) functions as an angiogenic inhibitor.

Vascular endothelial growth factor (VEGF) is an angiogenic stimulator which functions through two endothelial specific tyrosine kinase receptors, Flt-1 and Flk-1. In this work, we show that an 11-amino acid peptide derived from the second immunoglobulin-like domain of Flt-1 functions as an angiogenic inhibitor in chick chorioallantoic membrane and inhibited VEGF-induced vascular permeability in Miles' assay without binding to VEGF directly. Circular dichroism and nuclear magnetic resonance analyses indicate that this peptide forms a stable extended structure in solution, presumably beta-sheet structure and is most likely existing as a dimer. Our results suggest that this small peptide functions as an angiogenic inhibitor by inhibiting VEGF function through a non-VEGF binding mechanism.

Comparison of the solution conformations of a cell-adhesive peptide LBE and its reverse sequence EBL.

T-cell adhesion is mediated by an ICAM-1/LFA-1 interaction; this interaction plays a crucial role in T-cell activation during immune response. LBE peptide, which is derived from the beta-subunit of LFA-1, has been shown to inhibit ICAM-1/LFA-1-mediated T-cell adhesion. In this work, we studied the solution conformations of LBE peptide and its reverse sequence (EBL) by NMR, CD and molecular dynamics simulations. Reverse peptides have been used as controls in biological studies. The effect of reversing the sequence of LBE to EBL peptides on their respective conformations is important in understanding their biological properties in vitro or in vivo. The NMR studies for these peptides were carried out in water and in TFE/water solvent systems. In 40% TFE/water, both peptides exhibited helical conformation. CD studies suggested that the LBE exhibits 30% helical conformation, while the EBL exhibits 20% helical conformation. From the NMR and MD simulation studies, it was evident that the peptides exhibited a stable helical conformation; a stable helical structure was found at Leu6 to Leu15 for LBE and at Gly9 to Leu17 for EBL. The helical conformations of LBE and EBL may be in equilibrium with other possible conformers; the other conformers contain loop and turn structures. Both peptides bind to divalent cations because the LBE is derived from the cation-binding region of the LFA-1. This study shows that reversing the peptide sequence did not alter the secondary structure of the corresponding sequence. Hence, caution must be exercised when using reverse peptides as controls in biological studies. This report will improve our ability to design a better inhibitor of ICAM-1/LFA-1 interaction.

Secondary structure of the HAV peptide which regulates cadherin-cadherin interaction.

Cadherins are calcium-binding proteins which are responsible for cell-cell adhesion in biological systems. Cadherins are involved in embryo compaction, neurite growth, cellular differentiation and formation of biological barriers (i.e., intestinal and blood brain barriers). A short linear peptide, LRAHAVDVNG-NH2 (Peptide 1), which contains His-Ala-Val sequence and is derived from the N-cadherin sequence has been shown to inhibit embryo compaction and neurite growth; this is caused by inhibition of the cadherin-cadherin interactions. Peptide 1 was synthesized and its solution conformation was determined by proton nuclear magnetic resonance, circular dichroism and molecular dynamics simulations. These studies indicated that the peptide has an extended structure from residue Leu1 to Asp7, possibly a beta-sheet structure, followed by a beta-turn from Asp7 to Gly10. The X-ray crystal structure of a sequence similar to that of peptide 1 in hemagglutinin indicated that it also has a beta-sheet structure around the HAV sequence, followed by a beta-turn.

Solution structure of a cyclic RGD peptide that inhibits platelet aggregation.

Peptides containing the Arg-Gly-Asp (RGD) sequence can inhibit platelet aggregation. Incorporation of this sequence into a cyclic peptide results in specific binding to a particular integrin. Studies of cyclic RGD peptides show that residues surrounding the RGD sequence have important effects on the selectivity of the peptide to bind with glycoprotein IIb/IIIa (GPIIb/IIIa). In this paper, we elucidate the conformation of cyclo(2,10)Ac-Gly1-Pen2-Gly3-His4-Arg5-Gly6-Asp7 -Leu8-Arg9-Cys10-Ala11-NH2 (1) by NMR and molecular dynamics simulations. This peptide inhibits platelet aggregation in a manner similar to that reported for cyclo(2,10)Gly1-Pen2-Gly3-His4-Arg5-Gly6-Asp7-Le u8-Arg9-Cys10-Ala11-OH (6) (Cheng, S. et al. J. Med. Chem. 1994, 37, 1-8), which is shown to be selective for the GPIIb/IIIa receptor. The cyclic peptide 1 exhibited a major and a minor conformer in solution. In the major conformer, the His4-Arg5-Gly6-Asp7 segment encompasses a 4-->1 hydrogen bond with a distorted type II beta-turn, and the minor conformer has turn-extended-turn. A comparison between the major conformation of this peptide and those of other cyclic RGD peptides suggests the importance of a hydrophobic residue adjacent to the RGD sequence.

Effect of conformation on the rate of deamidation of vancomycin in aqueous solutions.

The instability of vancomycin, a glycopeptide antibiotic, limits its shelf-life because the deamidation of its asparagine residue results in the formation of a zwitterion with limited aqueous solubility. Analysis of the pH-rate profile for vancomycin indicates that the deamidation reaction is notably sensitive to the ionic state of the molecule. This observation results in a hypothesis in which the ionic state of vancomycin may influence the conformation of the molecule and therefore affect its reactivity. Two-dimensional nuclear magnetic resonance (NMR), homonuclear Hartmann-Hahn (HOHAHA) and rotating frame Overhauser enhancement spectroscopy (ROESY) information combined with molecular dynamic simulations were used to estimate the apparent conformation of vancomycin in aqueous solution at pH 4 and pH 9 where the molecule exists primarily as a monocation and monoanion, respectively. The apparent conformation for vancomycin at pH 4 is compact, and the proximity of the backbone amide nitrogen to the side chain carbonyl carbon of asparagine is favorable for the rapid formation of the cyclic imide intermediate, thus increasing its reactivity. The apparent conformation for vancomycin at pH 9, however, is expanded in comparison with the conformation at pH 4, and the increase in distance between the reacting atoms leads to slower cyclic imide formation and thus decreased intrinsic reactivity. That cyclic imide formation was rate limiting at both pH values was confirmed by cyclic imide isolation and stability estimation. It becomes apparent from the analysis of the pH-rate and conformational profiles of vancomycin that the deamidation rate of vancomycin is largely influenced by the ionization state of the N-methyl leucine nitrogen.

Ultraviolet absorption and vibrational spectra of 2-fluoro-5-bromopyridine.

The ultraviolet absorption spectrum in the range 340-185 nm in the vapour and solution phase has been measured for 2-fluoro-5-bromopyridine. Three fairly intense band systems identified as the pi* <-- pi transitions II, III and IV have been observed. A detailed vibronic analysis of the vapor and solution spectra is presented. The first system of bands is resolved into about sixty-two distinct vibronic bands in the vapour-phase spectrum. The 0,0 band is located at 35944 cm(-1). Two well-developed progressions, in which the excited state frequencies nu'25 (283 cm(-1)) and nu'19 (550 cm(-1)) are excited by several quanta, have been observed. The corresponding excited state vibrational and anharmonicity constants are found to be omega'i = 292 cm(-1), x'ii = 4.5 cm(-1) (i = 25) and omega'i = 563.8 cm(-1), x'ii = 6.9 cm(-1) (i = 19). The other two band systems show no vibronic structure, the band maxima being located at 48346 and 52701 cm(-1), respectively. The oscillator strength of the band systems in different solutions and the excited state dipole moments associated with the first two transitions have been determined by the solvent-shift method. The infrared spectrum in the region 4000-130 cm(-1) and the laser Raman spectrum of the molecule in the liquid state have been measured and a complete vibrational assignment of the observed frequencies is given. A correlation of the ground and excited state fundamental frequencies observed in the UV absorption spectrum with the Raman or infrared frequencies is presented.

Effect of conformation on the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH to its cyclic imide degradation product.

The objective of this study was to explain the increased propensity for the conversion of cyclo-(1,7)-Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly-OH (1), a vitronectin-selective inhibitor, to its cyclic imide counterpart cyclo-(1,7)-Gly-Arg-Gly-Asu-Ser-Pro-Asp-Gly-OH (2). Therefore, we present the conformational analysis of peptides 1 and 2 by NMR and molecular dynamic simulations (MD). Several different NMR experiments, including COSY, COSY-Relay, HOHAHA, NOESY, ROESY, DQF-COSY and HMQC, were used to: (a) identify each proton in the peptides; (b) determine the sequential assignments; (c) determine the cis-trans isomerization of X-Pro peptide bond; and (d) measure the NH-HCalpha coupling constants. NOE- or ROE-constraints were used in the MD simulations and energy minimizations to determine the preferred conformations of cyclic peptides 1 and 2. Both cyclic peptides 1 and 2 have a stable solution conformation; MD simulations suggest that cyclic peptide 1 has a distorted type I beta-turn at Arg2-Gly3-Asp4-Ser5 and cyclic peptide 2 has a pseudo-type I beta-turn at Ser5-Pro6-Asp7-Gly1. A shift in position of the type I beta-turn at Arg2-Gly3-Asp4-Ser5 in peptide 1 to Ser5-Pro6-Asp7-Gly1 in peptide 2 occurs upon formation of the cyclic imide at the Asp4 residue. Although the secondary structure of cyclic peptide 1 is not conducive to succinimide formation, the reaction proceeds via neighbouring group catalysis by the Ser5 side chain. This mechanism is also supported by the intramolecular hydrogen bond network between the hydroxyl side chain and the backbone nitrogen of Ser5. Based on these results, the stability of Asp-containing peptides cannot be predicted by conformational analysis alone; the influence of anchimeric assistance by surrounding residues must also be considered.

The effect of conformation on membrane permeability of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide.

PURPOSE: To determine the different conformations of the acyloxyalkoxy-linked cyclic prodrug 1 of the model hexapeptide 2 in solution and to investigate the relationship between these solution conformations and the cellular permeability characteristics of this prodrug. METHODS: Two-dimensional Homonuclear Hartmann-Hahn spectroscopy, Rotating-Frame Overhouser effect spectroscopy, circular dichroism and molecular dynamics simulations were used to find the solution conformers of cyclic prodrug 1. RESULTS: Our spectroscopic findings suggest that cyclic prodrug 1 exhibits a major and a minor conformer in solution. The major conformer appears to have a well-defined secondary structure, which involves a beta-turn and 4-->1 intramolecular hydrogen bond, creating a compact structure with a reduced average hydrodynamic radius compared to the model hexapeptide 2. CONCLUSIONS: The increased ability of cyclic prodrug 1 to permeate membranes compared to the model hexapeptide 2 could be due to reduction in the average hydrodynamic radius of the molecule facilitating paracellular flux and/or the reduction in the hydrogen bonding potential facilitating transcellular flux.

The effect of conformation on the membrane permeation of coumarinic acid- and phenylpropionic acid-based cyclic prodrugs of opioid peptides.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu5]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3-6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.

The effect of conformation of the acyloxyalkoxy-based cyclic prodrugs of opioid peptides on their membrane permeability.

In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the acyloxyalkoxy-based cyclic prodrugs 3 and 4 of the opioid peptides [Leu5]-enkephalin(1, H-Tyr-GLY-Gly-Phe-Leu-OH) and DADLE(2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, were substrates for apically polarized efflux systems and therefore less able to permeate the cell monolayers than were the opioid peptides themselves. In an attempt to explain how structure may influence the recognition of these cyclic prodrugs as substrates by the apically polarized efflux systems, we have determined the possible solution conformations of 3 and 4 using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as computational studies indicate that cyclic prodrug 4 exhibits a major and a minor conformer in a ratio of 3:2 where both conformers exhibit gamma and beta-turn structures. Spectroscopic, as well as molecular dynamics, studies indicate that the difference between the two conformers involves a cis/trans inversion occurring at the amide bond between the promoiety and Tyr1. The major conformer has a trans amide bond between the promoiety and Tyr1, whereas the minor conformer has a cis amide bond. The spectroscopic data indicate that cyclic prodrug 3 has a structure similar to that of the major conformer in cyclic prodrug 4. It has recently been reported that a particular arrangement of polar groups and spatial separation distances is required for substrate recognition by P-glycoprotein. When the conformation of the acyloxyalkoxy linker was investigated in the major and minor conformers of cyclic prodrug 4, with respect to distances between the polar functional groups, this ideal fixed spatial orientation was observed. Interestingly this same spatial orientation of polar functional groups was not observed for other cyclic prodrugs prepared by our laboratory using different chemical linkers (coumarinic acid and phenylpropionic acid) but the same opioid peptides that had previously been shown not to be substrates for the apically polarized efflux systems. Therefore, we hypothesize that the structure and/or the flexibility of the acyloxyalkoxy linker itself allows cyclic prodrugs 3 and 4 to adopt conformations that permit ideal arrangement of polar groups in the linker and their fixed spatial orientation. This possibly induces the substrate activity of cyclic prodrugs 3 and 4 for the apically polarized efflux systems.

A Ca2+ binding cyclic peptide derived from the alpha-subunit of LFA-1: inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion.

The objective of this work is to study the conformation of cyclic peptide (1), cyclo (1, 12) Pen1-Gly2-Val3-Asp4-Val5-Asp6-Gln7-+ ++Asp8-Gly9-Glu10-Thr11-Cys12, in the presence and absence of calcium. Cyclic peptide 1 is derived from the divalent cation binding sequence of the alpha-subunit of LFA-1. This peptide has been shown to inhibit ICAM-1-LFA-1 mediated T-cell adhesion. In order to understand the structural requirements for this biologically active peptide, its solution structure was studied by nuclear magnetic resonance (NMR), circular dichroism (CD) and molecular dynamics simulations. This cyclic peptide exhibits two types of possible conformations in solution. Structure I is a loop-turn-loop type of structure, which is suitable to bind cations such as EF hand proteins. Structure II is a more extended structure with beta-hairpin bend at Asp4-Val5-Asp6-Gln7. There is evidence that alterations in the conformation of LFA-1 upon binding to divalent cations cause LFA-1 to bind to ICAM-1. To understand this mechanism, the cation-binding properties of the peptide were studied by CD and NMR. CD studies indicated that the peptide binds to calcium and forms a 1 : 1 (peptide: calcium) complex at low calcium concentrations and multiple types of complexes at higher cation concentrations. NMR studies indicated that the conformation of the peptide is not significantly altered upon binding to calcium. The peptide can inhibit T-cell adhesion by directly binding to ICAM-1 or by disrupting the interaction of the alpha and beta-subunits of LFA-1 protein. This study will help us to understand the mechanism(s) of action of this peptide and will improve our ability to design a better inhibitor of T-cell adhesion.