Successful Flash-Cooling of Xenon Derivatized Myoglobin Crystals

This paper demonstrates for the first time a method for preparing cryocooled xenon-derivatized protein crystals. The method is based upon the hypothesis and subsequent observation that the diffusion of a xenon atom from a tight binding site following depressurization occurs on a timescale of minutes. We have observed significant changes in diffraction intensities from myoglobin crystals for up to 5 min following depressurization from 1 MPa of xenon. In accordance with this observation, a xenon-derivatized myoglobin crystal was cryocooled at ~95 K within 20 s of complete depressurization. A crystallographic data set was then collected to 2.0 Å resolution and isomorphous and anomalous difference Patterson maps revealed the presence of a well ordered xenon site with an occupancy of approximately 0.5. Phasing statistics for this site were of good quality and demonstrate the practicality of this method. The ability to cryocool xenon-derivatized crystals will make this heavy-atom substitution method even more useful for single-isomorphous-replacement and multiple-isomorphous-replacement phasing of macromolecules

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Electron paramagnetic resonance (EPR) studies of succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans have been undertaken in the purified and membrane-bound states. Spectroscopic “signatures” accounting for the three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S), cytochromeb, flavin, and protein-bound ubisemiquinone radicals have been obtained in air-oxidized, succinate-reduced, and dithionite-reduced preparations at 4–10 K. Spectra obtained at 170 K in the presence of excess succinate showed a signal typical of that of a flavin radical, but superimposed with another signal. The superimposed signal originated from two bound ubisemiquinones, as shown by spectral simulations. Power saturation measurements performed on the air-oxidized enzyme provided evidence for a weak magnetic dipolar interaction operating between the oxidized 3Fe-4S cluster and the oxidized cytochrome b. Power saturation experiments performed on the succinate- and dithionite-reduced forms of the enzyme demonstrated that the 4Fe-4S cluster is coupled weakly to both the 2Fe-2S and the 3Fe-4S clusters. Quantitative interpretation of these power saturation experiments has been achieved through redox calculations. They revealed that a spin-spin interaction between the reduced 3Fe-4S cluster and the cytochrome b (oxidized) may also exist. These findings form the first direct EPR evidence for a close proximity (≤2 nm) of the high potential 3Fe-4S cluster, situated in the succinate dehydrogenase part of the enzyme, and the low potential, low spin b-heme in the membrane anchor of the enzyme

A simple device for studying macromolecular crystals under moderate gas pressures (0.1-10 MPa)

A simple device for studying crystalline samples under moderate gas pressure (0.1-10 MPa) has been developed. The device employs a modified Cajon ultra-torr fitting to ensure a gas-tight seal around an X-ray capillary. The cell accommodates standard X-ray capillaries that require no modification. The device is straightforward to utilize and samples can be mounted with routine techniques and pressurized in a matter of seconds. In a subsequent development, a complete purging and pressurization system has been designed and constructed for use on beamline 7-1 at the Stanford Synchrotron Radiation Laboratory. This paper describes the construction of both the pressure cell and the delivery system and presents results of the use of this cell for the preparation of xenon derivatives to be used in phase determination by the multiple isomorphous replacement method

Universal WBC reduction

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75583/1/j.1537-2995.2000.40060751.x.pd

Coordinating the impact of structural genomics on the human α-helical transmembrane proteome

Given the recent successes in determining membrane-protein structures, we explore the tractability of determining representatives for the entire human membrane proteome. This proteome contains 2,925 unique integral α-helical transmembrane-domain sequences that cluster into 1,201 families sharing more than 25% sequence identity. Structures of 100 optimally selected targets would increase the fraction of modelable human α-helical transmembrane domains from 26% to 58%, providing structure and function information not otherwise available

Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards Nematotoxic Fungal Galectin CGL2

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galβ1,4Fucα1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galβ1,4Fucα1,6GlcNAc trisaccharide at 1.5 Å resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms

Plasticity of the β-Trefoil Protein Fold in the Recognition and Control of Invertebrate Predators and Parasites by a Fungal Defence System

Discrimination between self and non-self is a prerequisite for any defence mechanism; in innate defence, this discrimination is often mediated by lectins recognizing non-self carbohydrate structures and so relies on an arsenal of host lectins with different specificities towards target organism carbohydrate structures. Recently, cytoplasmic lectins isolated from fungal fruiting bodies have been shown to play a role in the defence of multicellular fungi against predators and parasites. Here, we present a novel fruiting body lectin, CCL2, from the ink cap mushroom Coprinopsis cinerea. We demonstrate the toxicity of the lectin towards Caenorhabditis elegans and Drosophila melanogaster and present its NMR solution structure in complex with the trisaccharide, GlcNAcβ1,4[Fucα1,3]GlcNAc, to which it binds with high specificity and affinity in vitro. The structure reveals that the monomeric CCL2 adopts a β-trefoil fold and recognizes the trisaccharide by a single, topologically novel carbohydrate-binding site. Site-directed mutagenesis of CCL2 and identification of C. elegans mutants resistant to this lectin show that its nematotoxicity is mediated by binding to α1,3-fucosylated N-glycan core structures of nematode glycoproteins; feeding with fluorescently labeled CCL2 demonstrates that these target glycoproteins localize to the C. elegans intestine. Since the identified glycoepitope is characteristic for invertebrates but absent from fungi, our data show that the defence function of fruiting body lectins is based on the specific recognition of non-self carbohydrate structures. The trisaccharide specifically recognized by CCL2 is a key carbohydrate determinant of pollen and insect venom allergens implying this particular glycoepitope is targeted by both fungal defence and mammalian immune systems. In summary, our results demonstrate how the plasticity of a common protein fold can contribute to the recognition and control of antagonists by an innate defence mechanism, whereby the monovalency of the lectin for its ligand implies a novel mechanism of lectin-mediated toxicity

A Selective HDAC 1/2 Inhibitor Modulates Chromatin and Gene Expression in Brain and Alters Mouse Behavior in Two Mood-Related Tests

Psychiatric diseases, including schizophrenia, bipolar disorder and major depression, are projected to lead global disease burden within the next decade. Pharmacotherapy, the primary – albeit often ineffective – treatment method, has remained largely unchanged over the past 50 years, highlighting the need for novel target discovery and improved mechanism-based treatments. Here, we examined in wild type mice the impact of chronic, systemic treatment with Compound 60 (Cpd-60), a slow-binding, benzamide-based inhibitor of the class I histone deacetylase (HDAC) family members, HDAC1 and HDAC2, in mood-related behavioral assays responsive to clinically effective drugs. Cpd-60 treatment for one week was associated with attenuated locomotor activity following acute amphetamine challenge. Further, treated mice demonstrated decreased immobility in the forced swim test. These changes are consistent with established effects of clinical mood stabilizers and antidepressants, respectively. Whole-genome expression profiling of specific brain regions (prefrontal cortex, nucleus accumbens, hippocampus) from mice treated with Cpd-60 identified gene expression changes, including a small subset of transcripts that significantly overlapped those previously reported in lithium-treated mice. HDAC inhibition in brain was confirmed by increased histone acetylation both globally and, using chromatin immunoprecipitation, at the promoter regions of upregulated transcripts, a finding consistent with in vivo engagement of HDAC targets. In contrast, treatment with suberoylanilide hydroxamic acid (SAHA), a non-selective fast-binding, hydroxamic acid HDAC 1/2/3/6 inhibitor, was sufficient to increase histone acetylation in brain, but did not alter mood-related behaviors and had dissimilar transcriptional regulatory effects compared to Cpd-60. These results provide evidence that selective inhibition of HDAC1 and HDAC2 in brain may provide an epigenetic-based target for developing improved treatments for mood disorders and other brain disorders with altered chromatin-mediated neuroplasticity.Stanley Medical Research InstituteNational Institutes of Health (U.S.) (R01DA028301)National Institutes of Health (U.S.) (R01DA030321

Uganda's experience in Ebola virus disease outbreak preparedness, 2018-2019.

BACKGROUND: Since the declaration of the 10th Ebola Virus Disease (EVD) outbreak in DRC on 1st Aug 2018, several neighboring countries have been developing and implementing preparedness efforts to prevent EVD cross-border transmission to enable timely detection, investigation, and response in the event of a confirmed EVD outbreak in the country. We describe Uganda's experience in EVD preparedness. RESULTS: On 4 August 2018, the Uganda Ministry of Health (MoH) activated the Public Health Emergency Operations Centre (PHEOC) and the National Task Force (NTF) for public health emergencies to plan, guide, and coordinate EVD preparedness in the country. The NTF selected an Incident Management Team (IMT), constituting a National Rapid Response Team (NRRT) that supported activation of the District Task Forces (DTFs) and District Rapid Response Teams (DRRTs) that jointly assessed levels of preparedness in 30 designated high-risk districts representing category 1 (20 districts) and category 2 (10 districts). The MoH, with technical guidance from the World Health Organisation (WHO), led EVD preparedness activities and worked together with other ministries and partner organisations to enhance community-based surveillance systems, develop and disseminate risk communication messages, engage communities, reinforce EVD screening and infection prevention measures at Points of Entry (PoEs) and in high-risk health facilities, construct and equip EVD isolation and treatment units, and establish coordination and procurement mechanisms. CONCLUSION: As of 31 May 2019, there was no confirmed case of EVD as Uganda has continued to make significant and verifiable progress in EVD preparedness. There is a need to sustain these efforts, not only in EVD preparedness but also across the entire spectrum of a multi-hazard framework. These efforts strengthen country capacity and compel the country to avail resources for preparedness and management of incidents at the source while effectively cutting costs of using a "fire-fighting" approach during public health emergencies