Identification of ER-000444793, a Cyclophilin D-independent inhibitor of mitochondrial permeability transition, using a high-throughput screen in cryopreserved mitochondria

Growing evidence suggests persistent mitochondrial permeability transition pore (mPTP) opening is a key pathophysiological event in cell death underlying a variety of diseases. While it has long been clear the mPTP is a druggable target, current agents are limited by off-target effects and low therapeutic efficacy. Therefore identification and development of novel inhibitors is necessary. To rapidly screen large compound libraries for novel mPTP modulators, a method was exploited to cryopreserve large batches of functionally active mitochondria from cells and tissues. The cryopreserved mitochondria maintained respiratory coupling and ATP synthesis, Ca(2+) uptake and transmembrane potential. A high-throughput screen (HTS), using an assay of Ca(2+)-induced mitochondrial swelling in the cryopreserved mitochondria identified ER-000444793, a potent inhibitor of mPTP opening. Further evaluation using assays of Ca(2+)-induced membrane depolarisation and Ca(2+) retention capacity also indicated that ER-000444793 acted as an inhibitor of the mPTP. ER-000444793 neither affected cyclophilin D (CypD) enzymatic activity, nor displaced of CsA from CypD protein, suggesting a mechanism independent of CypD inhibition. Here we identified a novel, CypD-independent inhibitor of the mPTP. The screening approach and compound described provides a workflow and additional tool to aid the search for novel mPTP modulators and to help understand its molecular nature

Report on 240Am(n,x) surrogate cross section test measurement

The main goal of the test measurement was to determine the feasibility of the {sup 243}Am(p,t) reaction as a surrogate for {sup 240}Am(n,f). No data cross section data exists for neutron induced reactions on {sup 240}Am; the half-life of this isotope is only 2.1 days making direct measurements difficult, if not impossible. The 48-hour experiment was conducted using the STARS/LIBERACE experimental facility located at the 88 Inch Cyclotron at Lawrence Berkeley National Laboratory in August 2011. A description of the experiment and results is given. The beam energy was initially chosen to be 39 MeV in order to measure an equivalent neutron energy range from 0 to 20 MeV. However, the proton beam was not stopped in the farady cup and the beam was deposited in the surrounding shielding material. The shielding material was not conductive, and a beam current, needed for proper tuning of the beam as well as experimental monitoring, could not be read. If the {sup 240}Am(n,f) surrogate experiment is to be run at LBNL, simple modifications to the beam collection site will need to be made. The beam energy was reduced to 29 MeV, which was within an energy regime of prior experiments and tuning conditions at STARS/LIBERACE. At this energy, the beam current was successfully tuned and measured. At 29 MeV, data was collected with both the {sup 243}Am and {sup 238}U targets. An example particle identification plot is shown in Fig. 1. The triton-fission coincidence rate for the {sup 243}Am target and {sup 238}U target were measured. Coincidence rates of 0.0233(1) cps and 0.150(6) cps were observed for the {sup 243}Am and {sup 238}U targets, respectively. The difference in count rate is largely attributed to the available target material - the {sup 238}U target contains approximately 7 times more atoms than the {sup 243}Am. A proton beam current of {approx}0.7 nA was used for measurements on both targets. Assuming a full experimental run under similar conditions, an estimate for the run time needed was made. Figure 2 shows the number of days needed as a function of acceptable uncertainty for a measurement of 1-20 MeV equivalent neutron energy, binned into 200 keV increments. A 5% measurement will take 3 days for U, but 20 days for Am. It may be difficult to be the sole user of the LBNL cyclotron, or another facility, for such an extended period. However, a 10% measurement will take 19 hours for U, and 5 days for Am. Such a run period is more reasonable and will allow for the first ever measurement of the {sup 240}Am(n,f) cross section. We also anticipate 40% more beam time being available at Texas A&M Cyclotron Institute compared to LBNL in FY2012. The increased amount of beam time will allow us to accumulate better statistics then what would have been available at LBNL

Strongly aligned gas-phase molecules at Free-Electron Lasers

We demonstrate a novel experimental implementation to strongly align molecules at full repetition rates of free-electron lasers. We utilized the available in-house laser system at the coherent x-ray imaging beamline at the Linac Coherent Light Source. Chirped laser pulses, i. e., the direct output from the regenerative amplifier of the Ti:Sa chirped pulse amplification laser system, were used to strongly align 2,5-diiodothiophene molecules in a molecular beam. The alignment laser pulses had pulse energies of a few mJ and a pulse duration of 94 ps. A degree of alignment of \left = 0.85 was measured, limited by the intrinsic temperature of the molecular beam rather than by the available laser system. With the general availability of synchronized chirped-pulse-amplified near-infrared laser systems at short-wavelength laser facilities, our approach allows for the universal preparation of molecules tightly fixed in space for experiments with x-ray pulses.Comment: 10 pages, 5 figure

Accelerator Testing of the General Antiparticle Spectrometer, a Novel Approach to Indirect Dark Matter Detection

We report on recent accelerator testing of a prototype general antiparticle spectrometer (GAPS). GAPS is a novel approach for indirect dark matter searches that exploits the antideuterons produced in neutralino-neutralino annihilations. GAPS captures these antideuterons into a target with the subsequent formation of exotic atoms. These exotic atoms decay with the emission of X-rays of precisely defined energy and a correlated pion signature from nuclear annihilation. This signature uniquely characterizes the antideuterons. Preliminary analysis of data from a prototype GAPS in an antiproton beam at the KEK accelerator in Japan has confirmed the multi-X-ray/pion star topology and indicated X-ray yields consistent with prior expectations. Moreover our success in utilizing solid rather than gas targets represents a significant simplification over our original approach and offers potential gains in sensitivity through reduced dead mass in the target area.Comment: 18 pages, 9 figures, submitted to JCA

Photoreversible interconversion of a phytochrome photosensory module in the crystalline state.

A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in Thermosynechococcus elongatus assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on Fobs - Fobs difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion

Spatial competition in a global disturbance minimum; the seabed under an Antarctic ice shelf

The marine habitat beneath Antarctica's ice shelves spans ∼1.6 million km2, and life in this vast and extreme environment is among Earth's least accessible, least disturbed and least known, yet likely to be impacted by climate-forced warming and environmental change. Although competition among biota is a fundamental structuring force of ecological communities, hence ecosystem functions and services, nothing was known of competition for resources under ice shelves, until this study. Boreholes drilled through a ∼ 200 m thick ice shelf enabled collections of novel sub-ice-shelf seabed sediment which contained fragments of biogenic substrata rich in encrusting (lithophilic) macrobenthos, principally bryozoans – a globally-ubiquitous phylum sensitive to environmental change. Analysis of sub-glacial biogenic substrata, by stereo microscopy, provided first evidence of spatial contest competition, enabling generation of a new range of competition measures for the sub-ice-shelf benthic space. Measures were compared with those of global open-water datasets traversing polar, temperate and tropical latitudes (and encompassing both hemispheres). Spatial competition in sub-ice-shelf samples was found to be higher in intensity and severity than all other global means. The likelihood of sub-ice-shelf competition being intraspecific was three times lower than for open-sea polar continental shelf areas, and competition complexity, in terms of the number of different types of competitor pairings, was two-fold higher. As posited for an enduring disturbance minimum, a specific bryozoan clade was especially competitively dominant in sub-ice-shelf samples compared with both contemporary and fossil assemblage records. Overall, spatial competition under an Antarctic ice shelf, as characterised by bryozoan interactions, was strikingly different from that of open-sea polar continental shelf sites, and more closely resembled tropical and temperate latitudes. This study represents the first analysis of sub-ice-shelf macrobenthic spatial competition and provides a new ecological baseline for exploring, monitoring and comparing ecosystem response to environmental change in a warming world