Directing growth cones of optic axons growing with laser scissors and laser tweezers

We have combined a laser scissors and a laser tweezers to study, (1) the response of nerve fiber growth cones to laserinduced damage on single axons, and (2) localized microfluidic flow generated by laser-driven spinning birefringent particles. In the laser scissors study, sub-axotomy damage elicits a growth cone response whether damage is on the same or an adjacent axon. In laser tweezers study, the axon growth cones turn in response to the optically driven microfluidic flow. In summary, both the laser scissors and the laser tweezers studies elicit growth cone turning responses

Synthesis and in vitro biological evaluation of new P2X7R radioligands [11C]halo-GSK1482160 analogs

The reference standards halo-GSK1482160 (F-, Br-, and I-) and their corresponding precursors desmethyl-halo-GSK1482160 (F-, Br-, and I-) were synthesized from (S)-1-methyl-5-oxopyrrolidine-2-carboxylic acid or (S)-5-oxopyrrolidine-2-carboxylic acid and 2-halo-3-(trifluoromethyl)benzylamine (F-, Br-, and I-) in one step with 45–93% yields. The target tracers [11C]halo-GSK1482160 (F-, Br-, and I-) were prepared from desmethyl-halo-GSK1482160 (F-, Br-, and I-) with [11C]CH3OTf under basic conditions (NaOH-Na2CO3, solid, w/w 1:2) through N-[11C]methylation and isolated by HPLC combined with SPE in 40–50% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity (AM) at end of bombardment (EOB) was 370–740 GBq/μmol. The potency of halo-GSK1482160 (F-, Br-, and I-) in comparison with GSK1482160 (Cl-) was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity Ki values for halo-GSK1482160 (F-, Br-, and I-) and GSK1482160 (Cl-) are 54.2, 2.5, 1.9 and 3.1 nM, respectively

Synthesis and preliminary biological evaluation of radiolabeled 5-BDBD analogs as new candidate PET radioligands for P2X4 receptor

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer’s disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD analog, [11C]9) and 5-(3-Bromophenyl)-1-[11C]methyl-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one (N-[11C]Me-5-BDBD, [11C]8c) were prepared from their corresponding desmethylated precursors with [11C]CH3OTf through N-[11C]methylation and isolated by HPLC combined with SPE in 30–50% decay corrected radiochemical yields with 370–1110 GBq/µmol specific activity at EOB. 5-(3-[18F]Fluorophenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]F-5-BDBD, [18F]5a) and 5-(3-(2-[18F]fluoroethoxy)phenyl)-1,3-dihydro-2H-benzofuro[3,2-e][1,4]diazepin-2-one ([18F]FE-5-BDBD, [18F]11) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[18F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5–25% decay corrected radiochemical yields with 111–740 GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD

Synthesis and preliminary biological evaluation of [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate for the fractalkine receptor (CX3CR1)

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate (5) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucine (6) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [11C]methyl (2-amino-5-(benzylthio)thiazolo[4,5-d]pyrimidin-7-yl)-d-leucinate ([11C]5) was prepared from the acid precursor with [11C]CH3OTf through O-[11C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [11C]CO2 and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ∼40-min from EOB. The radioligand depletion experiment of [11C]5 did not display specific binding to CX3CR1, and the competitive binding assay of ligand 5 found much lower CX3CR1 binding affinity

Characterization of 11C-GSK1482160 for Targeting the P2X7 Receptor as a Biomarker for Neuroinflammation

The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood–brain barrier penetration, and the ability to be radiolabeled with 11C. We report the initial physical and biologic characterization of this novel ligand. Methods: 11C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association–disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity 11C-GSK1482160, receptor density and Kd were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min−1⋅nM−1, 0.2547 ± 0.0155 min−1, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. Conclusion: The current study provides compelling data that support the suitability of 11C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation

Subseafloor microbial communities in hydrogen-rich vent fluids from hydrothermal systems along the Mid-Cayman Rise

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Environmental Microbiology 18 (2016): 1970–1987, doi:10.1111/1462-2920.13173.Warm fluids emanating from hydrothermal vents can be used as windows into the rocky subseafloor habitat and its resident microbial community. Two new vent systems on the Mid-Cayman Rise each exhibits novel geologic settings and distinctively hydrogen-rich vent fluid compositions. We have determined and compared the chemistry, potential energy yielding reactions, abundance, community composition, diversity, and function of microbes in venting fluids from both sites: Piccard, the world's deepest vent site, hosted in mafic rocks; and Von Damm, an adjacent, ultramafic-influenced system. Von Damm hosted a wider diversity of lineages and metabolisms in comparison to Piccard, consistent with thermodynamic models that predict more numerous energy sources at ultramafic systems. There was little overlap in the phylotypes found at each site, although similar and dominant hydrogen-utilizing genera were present at both. Despite the differences in community structure, depth, geology, and fluid chemistry, energetic modelling and metagenomic analysis indicate near functional equivalence between Von Damm and Piccard, likely driven by the high hydrogen concentrations and elevated temperatures at both sites. Results are compared with hydrothermal sites worldwide to provide a global perspective on the distinctiveness of these newly discovered sites and the interplay among rocks, fluid composition and life in the subseafloor.National Aeronautics and Space Administration Grant Number: NNX09AB756; Alfred P. Sloan Foundation; NSF Grant Number: OCE10618

Synthesis and preliminary biological evaluation of a novel P2X7R radioligand [18F]IUR-1601

The reference standard IUR-1601 ((S)-N-(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from tert-butyl (S)-5-oxopyrrolidine-2-carboxylate, fluoroethylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 12% in three steps. The target tracer [18F]IUR-1601 ((S)-N-(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-[18F]fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from desmethyl-GSK1482160 with 2-[18F]fluoroethyl tosylate, prepared from 1,2-ethylene glycol-bis-tosylate and K[18F]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 1–3% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370 GBq/μmol. The potency of IUR-1601 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity Ki values for IUR-1601 and GSK1482160 are 4.31 and 5.14 nM, respectively

Synthesis and initial in vitro characterization of a new P2X7R radioligand [18F]IUR-1602

The overexpression of P2X7R is associated with neuroinflammation and plays an important role in various neurodegenerative diseases. The [18F]fluoropropyl derivative of GSK1482160, [18F]IUR-1602, has been first prepared and examined as a new potential P2X7R radioligand. The reference standard IUR-1602 was synthesized from tert-butyl (S)-5-oxopyrrolidine-2-carboxylate, fluoropropylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 13% in three steps. The target tracer [18F]IUR-1602 was synthesized from desmethyl-GSK1482160 with 3-[18F]fluoropropyl tosylate, prepared from propane-1,3-diyl bis(4-methylbenzenesulfonate) and K[18F]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 2–7% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370 GBq/μmol. The potency of IUR-1602 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity Ki values for IUR-1602 and GSK1482160 are 23.6 and 3.07 nM, respectively. The initial in vitro evaluation results, 8-fold less potency of [18F]IUR-1602 compared to [11C]GSK1482160, prevent further in vivo evaluation of [18F]IUR-1602 in animals and human

Molecular Hydrogen in the Damped Ly alpha Absorber of Q1331+170

We used HST/STIS to obtain the spectrum of molecular hydrogen associated with the damped Ly$\alpha$ system at $z_{\rm abs}=1.7765$ toward the quasar Q1331+170 at $z_{\rm em}=2.084$. Strong ${\rm H}_2$ absorption was detected, with a total ${\rm H}_2$ column density of $N({\rm H}_2)=(4.45\pm 0.36)\times 10^{19} {\rm cm^{-2}}$.The molecular hydrogen fraction is $f_{{\rm H}_2}=\frac{2N_{\rm H_2}}{N_{\rm HI}+2N_{\rm H_2}}=(5.6\pm 0.7)$, which is the greatest value reported so far in any redshifted damped Ly$\alpha$ system. This results from the combined effect of a relatively high dust-to-gas ratio, a low gas temperature, and an extremely low ambient UV radiation field. Based on the observed population of $J$ states, we estimate the photo-absorption rate to be $R_{\rm abs}=(7.6\pm 2.4)\times 10^{-13} {\rm s^{-1}}$, corresponding to a local UV radiation field of $J(1000{\rm \AA})\approx 2.1\times 10^{-3} J_{1000{\rm \AA},\odot}$, where $J_{1000{\rm \AA},\odot}$ is the UV intensity at $1000 \AA$ in the solar neighborhood. This is comparable with the metagalactic UV background intensity at this redshift, and implies an extremely low star formation rate in the absorber's environment. The observed CO-to-H$_2$ column density ratio is $\frac{N_{\rm CO}}{N_{\rm H_2}}<2.5\times 10^{-7}$, which is similar to the value measured for diffuse molecular clouds in the Galactic ISM. Finally, applying the inferred physical conditions to the observed C I fine structure excitation (Songaila {\it et al.} 1994), we estimate the cosmic microwave background temperature to be $T_{\rm CMB}=(7.2\pm 0.8) {\rm K}$ at $z=1.77654$, consistent with the predicted value of $7.566 {\rm K}$ from the standard cosmology.Comment: Accepted for publication, Astrophysical Journal. Abstract abbreviate