Omega-3 fatty acids (n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive ...reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in Humans. However, the n-3 PUFAs deficiency-mediated mechanisms affecting the development of the central nervous system are poorly understood. Active microglial engulfment of synapses regulates brain development. Impaired synaptic pruning is associated with several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development in mice. Our results show that maternal dietary n-3 PUFA deficiency increases microglia-mediated phagocytosis of synaptic elements in the rodent developing hippocampus, partly through the activation of 12/15-lipoxygenase (LOX)/12-HETE signaling, altering neuronal morphology and affecting cognitive performance of the offspring. These findings provide a mechanistic insight into neurodevelopmental defects caused by maternal n-3 PUFAs dietary deficiency.
Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using ...time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a₃ iron atom is in a ferryl (Fe4+ = O2−) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.
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The dominant feature of large-scale mass transfer in the modern ocean is the Atlantic meridional overturning circulation (AMOC). The geometry and vigour of this circulation influences global climate ...on various timescales. Palaeoceanographic evidence suggests that during glacial periods of the past 1.5 million years the AMOC had markedly different features from today
; in the Atlantic basin, deep waters of Southern Ocean origin increased in volume while above them the core of the North Atlantic Deep Water (NADW) shoaled
. An absence of evidence on the origin of this phenomenon means that the sequence of events leading to global glacial conditions remains unclear. Here we present multi-proxy evidence showing that northward shifts in Antarctic iceberg melt in the Indian-Atlantic Southern Ocean (0-50° E) systematically preceded deep-water mass reorganizations by one to two thousand years during Pleistocene-era glaciations. With the aid of iceberg-trajectory model experiments, we demonstrate that such a shift in iceberg trajectories during glacial periods can result in a considerable redistribution of freshwater in the Southern Ocean. We suggest that this, in concert with increased sea-ice cover, enabled positive buoyancy anomalies to 'escape' into the upper limb of the AMOC, providing a teleconnection between surface Southern Ocean conditions and the formation of NADW. The magnitude and pacing of this mechanism evolved substantially across the mid-Pleistocene transition, and the coeval increase in magnitude of the 'southern escape' and deep circulation perturbations implicate this mechanism as a key feedback in the transition to the '100-kyr world', in which glacial-interglacial cycles occur at roughly 100,000-year periods.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
NendoU from SARS-CoV-2 is responsible for the virus’s ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature ...structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.
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•The first room-temperature structure of NendoU from SARS-CoV-2 is solved at an XFEL•Designed RNA substrates are cleaved within microcrystals of NendoU•High B factors indicate varying flexibility between trimers of the NendoU hexamer•Alternating trimer flexibility suggests a binding-change mechanism
NendoU from SARS-CoV-2 is a protein responsible for the virus’s ability to evade the immune system. Jernigan et al. report the room-temperature structure of NendoU, finding indications that it functions by an alternate-switch mechanism. This sets a benchmark for studies targeting this enzyme in treating coronavirus infections.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction ...of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe–C–O angle of ∼142°. In contrast, in the synchrotron structure, the Fe–CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a₃ iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a₃ iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a₃, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.
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Abstract
The Simons Observatory is a ground-based cosmic microwave background experiment that consists of three 0.4 m small-aperture telescopes and one 6 m Large Aperture Telescope, located at an ...elevation of 5300 m on Cerro Toco in Chile. The Simons Observatory Large Aperture Telescope Receiver (LATR) is the cryogenic camera that will be coupled to the Large Aperture Telescope. The resulting instrument will produce arcminute-resolution millimeter-wave maps of half the sky with unprecedented precision. The LATR is the largest cryogenic millimeter-wave camera built to date, with a diameter of 2.4 m and a length of 2.6 m. The coldest stage of the camera is cooled to 100 mK, the operating temperature of the bolometric detectors with bands centered around 27, 39, 93, 145, 225, and 280 GHz. Ultimately, the LATR will accommodate 13 40 cm diameter optics tubes, each with three detector wafers and a total of 62,000 detectors. The LATR design must simultaneously maintain the optical alignment of the system, control stray light, provide cryogenic isolation, limit thermal gradients, and minimize the time to cool the system from room temperature to 100 mK. The interplay between these competing factors poses unique challenges. We discuss the trade studies involved with the design, the final optimization, the construction, and ultimate performance of the system.
Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active ...sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a 3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a 3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO–CO complex, where CO is dissociated from the heme a 3 iron and moved to a temporary binding site midway between the CuB and the heme a 3 iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a 3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.
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Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with ...continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 , leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.
A 3D-printed modular droplet injector successfully delivered microcrystals of human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin with electrical stimulation in a serial crystallography experiment at 120 Hz repetition rate.
The Behavior Rating Inventory of Executive Function–Adult Version (BRIEF-A) is a standardized rating scale of subjective executive functioning. We provide univariate and multivariate base rates (BRs) ...for scale/index scores in the clinical range (T scores ≥65), reliable change, and inter-rater information not included in the Professional Manual. Participants were adults (ages = 18–90 years) from the BRIEF-A self-report (N = 1,050) and informant report (N = 1,200) standardization samples, as well as test–retest (n = 50 for self, n = 44 for informant) and inter-rater (n = 180) samples. Univariate BRs of elevated T scores were low (self-report = 3.3%–15.4%, informant report = 4.5%–16.3%). Multivariate BRs revealed the common occurrence of obtaining at least one elevated T-score across scales (self-report = 26.5%–37.3%, informant report = 22.7%–30.3%), whereas virtually none had elevated scores on all scales. Test–retest scores were highly correlated (self = .82–.94; informant = .91–.96). Inter-rater correlations ranged from .44 to .68. Significant (p < .05) test–retest T-score differences ranged from 7 to 12 for self-report, from 6 to 8 for informant report, and from 16 to 21 points for inter-rater T-score differences. Applications of these findings are discussed.
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