Structure and absolute configuration of natural fungal product beauveriolide I, isolated from Cordyceps javanica, determined by 3D electron diffraction.

Transcription Factor cFOS Mediated Foam Cell Formation in Diabetes Associated Atherosclerosis

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme-like phenotype, characterized by upregulation of heme oxygenase-1 (HO-1) and the ATP-binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF-α under inflammatory stimulation. The upregulation of HO-1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low-density lipoproteins (oxLDL)-treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high-fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis.

As little as 1 microliter of serum-free supernatant from Mo(t), an established lymphocyte line, when added to a 500-microliters incubation of macrophages derived from human monocytes, significantly decreased the receptor-mediated uptake and degradation of three cholesterol-rich molecules: low density lipoprotein (LDL); LDL complexed to dextran sulfate; and LDL modified by malondialdehyde (MDA-LDL). In contrast, the receptor-mediated uptake and degradation of mannosyl bovine serum albumin was increased three-fold. The Mo(t) supernatant did not contain competitive inhibitors of the cholesterol-rich ligands, and it did not alter macrophage receptor-independent endocytosis, protein synthesis, or phagocytosis of heat-killed yeast. The effect of the Mo(t) supernatant was specific for macrophages and was abolished by preincubation of the supernatant with trypsin, which indicates that the active substances are protein in nature. The decrease in the uptake and degradation of MDA-LDL induced by preincubating the macrophages with Mo(t) supernatant appeared to result from a decrease in the number of receptors for this ligand at the cell surface. The isolation of these lymphokines should offer new insights into macrophage receptor-mediated endocytosis, and may yield substances useful in preventing foam cell formation in atherosclerosis.

Beauverolides are hydrophobic cyclodepsipeptides that inhibit sterol O-acyltransferases and calmodulin, thereby reducing senile plaques in Alzheimer’s disease and preventing foam cell formation in atherosclerosis. Other sterol O-acyltransferase inhibitors suffer from low bioavailability, and evidence of the distribution of beauverolides in bodily fluids is lacking. We aimed to determine the optimal formulation of beauverolides for administration to experimental animals and to determine if the orally administered beauverolides could cross the gastrointestinal barrier and be secreted in urine. We found that beauverolides formed gels in aqueous solutions and we developed a formula for their peroral administration. Administration of the beauverolide gel pellets to experimental mice revealed that beauverolides cross the gastrointestinal barrier, circulate untouched in the blood, and are excreted in the urine. Using liquid chromatography-mass spectrometry analyses, we show that the administered beauverolides circulate in mouse blood and are excreted from the body 24 h after their administration.

The absolute structure of the 3D MOF anhydrous zinc (II) tartrate with space group I222 has been determined for both [Zn(L-TAR)] and [Zn(D-TAR)] by electron diffraction using crystals of sub-micron dimensions. Dynamical refinement gives a strong difference in R factors for the correct and inverted structures. These anhydrous MOFs may be prepared phase pure from mild hydrothermal conditions. Powder X-ray diffraction indicates that isostructural or pseudo-isostructural phases can be similarly prepared for several other M2+ = Mg, Mn, Co, Ni and Cu. I222 is a relatively uncommon space group since it involves intersecting two-fold axes that place constraints on molecular crystals. However, in the case of MOFs the packing is dominated by satisfying the octahedral coordination centers. These MOFs are dense 3D networks with chiral octahedral metal centers that may be classed as Δ (for L-TAR) or Λ (for D-TAR).

Structure analysis using 3D electron diffraction (3D ED, aka ADT, cRED, MicroED) data has become increasingly popular among chemists and materials scientists for its ability to solve crystal structures from single nanocrystals. Despite substantial progress in this method, it is still generally considered to provide relatively low-accuracy structure models, unsuitable for all but basic crystallographic analysis. Recent advances in data acquisition, data processing in PETS2 [1], including determination of the exact experimental geometry, and dynamical refinement in Jana2020/Dyngo [2] with modeling of the apparent crystal thickness distribution, have enabled 3D ED to answer questions about the finest details of structure, including partially occupied hydrogen positions or charge density analysis. Figure 1 shows methyl disorder in acetaminophen and charge density analysis in L-tyrosine obtained from 3D ED data. It is also possible to determine the absolute structure [3]. For this purpose, the so-called z-score has been introduced for enantiopure materials [4]. However, if certain conditions are fulfilled, the classical Flack parameter determination can be applied in electron crystallography. These advances bring electron crystallography to the same level of accuracy as X-ray diffraction, with the ability to see light atoms alongside heavy ones due to a smaller increase in scattering potential with atomic number and greater sensitivity to the absolute structure of structures consisting of only light atoms.

3D electron diffraction (3D ED) has undergone impressive development in the last decade. However, its accuracy and reproducibility have never been tested, up to now, in different laboratories on the same batch of samples. This paper reports a round robin on three test structures, two inorganic and one organic, solved and refined with 3D ED in seven different laboratories employing different transmission electron microscopes, with different acceleration voltages, different methodologies and different detectors. The results of the round robin show a remarkable accuracy of the technique that, in the case of kinematical refinement, is around 0.05 Å error on atomic positions for the inorganic samples and 0.15 Å for the beam-sensitive organic crystal. Dynamical refinement further improves the accuracy. The analysis of diverse samples and numerous data sets again confirms that dynamical refinement is a well established procedure, significantly reducing the refinement R factors, improving the accuracy of the structure models in most cases, and providing fine structural details, such as hydrogen-atom positions and the absolute structure, for both inorganic and organic samples.

Low-density lipoprotein (LDL) and cholesterol homeostasis in the peripheral blood is maintained by specialized cells, such as macrophages. Macrophages express a variety of scavenger receptors (SR) that interact with lipoproteins, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1). These cells also have several cholesterol transporters, including ATP-binding cassette transporter ABCA1, ABCG1, and SR-BI, that are involved in reverse cholesterol transport. Lipids internalized by phagocytosis are transported to late endosomes/lysosomes, where lysosomal acid lipase (LAL) digests cholesteryl esters releasing free cholesterol. Free cholesterol in turn is processed by acetyl-CoA acetyltransferase (ACAT1), an enzyme that transforms cholesterol to cholesteryl esters. The endoplasmic reticulum serves as a depot for maintaining newly synthesized cholesteryl esters that can be processed by neutral cholesterol ester hydrolase (NCEH), which generates free cholesterol that can exit via cholesterol transporters. In atherosclerosis, pro-inflammatory stimuli upregulate expression of scavenger receptors, especially LOX-1, and downregulate expression of cholesterol transporters. ACAT1 is also increased, while NCEH expression is reduced. This results in deposition of free and esterified cholesterol in macrophages and generation of foam cells. Moreover, other cell types, such as endothelial (ECs) and vascular smooth muscle cells (VSMCs), can also become foam cells. In this review, we discuss known pathways of foam cell formation in atherosclerosis.

Monocyte recruitment and foam cell formation in atherosclerosis

Dynamical refinement is a well established method for refining crystal structures against 3D electron diffraction (ED) data and its benefits have been discussed in the literature [Palatinus, Petříček & Corrêa, (2015). Acta Cryst. A71, 235-244; Palatinus, Corrêa et al. (2015). Acta Cryst. B71, 740-751]. However, until now, dynamical refinements have only been conducted using the independent atom model (IAM). Recent research has shown that a more accurate description can be achieved by applying the transferable aspherical atom model (TAAM), but this has been limited only to kinematical refinements [Gruza et al. (2020). Acta Cryst. A76, 92-109; Jha et al. (2021). J. Appl. Cryst. 54, 1234-1243]. In this study, we combine dynamical refinement with TAAM for the crystal structure of 1-methyluracil, using data from precession ED. Our results show that this approach improves the residual Fourier electrostatic potential and refinement figures of merit. Furthermore, it leads to systematic changes in the atomic displacement parameters of all atoms and the positions of hydrogen atoms. We found that the refinement results are sensitive to the parameters used in the TAAM modelling process. Though our results show that TAAM offers superior performance compared with IAM in all cases, they also show that TAAM parameters obtained by periodic DFT calculations on the refined structure are superior to the TAAM parameters from the UBDB/MATTS database. It appears that multipolar parameters transferred from the database may not be sufficiently accurate to provide a satisfactory description of all details of the electrostatic potential probed by the 3D ED experiment.

3D electron diffraction (3DED) has established itself as a powerful technique to elucidate atomic structures of nano-sized crystals 1 .One of the most commonly encountered issues in the 3DED field is beam damage to crystals under electron exposure, which causes diffraction intensity loss and a decay in data resolution from sensitive crystals 2 .Here we implement the technique of dose-symmetric tomography (DST) 3 employed in the field of cryo-electron tomography (cryoET) into low-dose electron diffraction tomography (LD-EDT) 4 to further improve the signal-to-noise ratio in 3DED.Starting the acquisition in the low-tilt region, which often provides high-resolution data due to lower apparent thickness, assures that these data are recorded while the crystal is not beam yet damaged (Figure 1).The high-tilt frames of the damaged crystal are used for unit cell determination only.Damage-free low-tilt data from multiple particles is then merged for structure determination (Figure 2).We present results obtained on two test samples Sr 5 CuGe 9 O 24 and Mn-formiate.Results on Sr 5 CuGe 9 O 24 , containing 9 independent cation and 13 independent oxygen positions, show that it is possible to get an accurate structure by solely using frames in the +/-10 range from 3 particles.Model accuracy often improves with data completeness by merging more particles, but this is not always the case.Particles that yield only very weak diffraction intensities generate difficulties in the rescaling process and tend to worsen the data quality.The same is true for thick crystals subjected to higher dynamical scattering effects.For Mn-formiate the high tilt diffraction frames clearly showed beam damage effects and it was possible to reduce the range to +/-8 for the structure solution in SIR2014.All non-hydrogen atom positions were directly obtained with a high accuracy (average distance to the DRX refined positions of 0.1 ).Dynamical refinement is possible on dose symmetric electron diffraction tomography (DS-EDT) data but requires a certain amount of data completeness.Instead of a tilt range of around 100 in standard 3D ED, DS-EDT only needs a tilt range of 20 or less on an individual crystal to obtain exploitable data.At the same signal-to-noise ratio, the necessary dose can therefore be reduced by an order of magnitude.

Zeolites with large cavities that are accessible via wide pore windows are desirable but very rare. They have been dominantly used as catalysts in industry. Here we report a novel porous germanosilicate SCM-25, the zeolite structure containing ordered meso-cavities (29.9 × 7.6 × 6.0 Å3) interconnected by 10- and 12-ring channels. SCM-25 was synthesized as nanosized crystals by using a simple organic structure-directing agent (OSDA). Three-dimensional (3D) electron diffraction shows that SCM-25 crystallizes in the orthorhombic space group Cmmm with a = 14.62 Å, b = 51.82 Å, c = 13.11 Å, which is one of the zeolites with the largest unit cell dimensions. We demonstrate that 3D electron diffraction is a powerful technique for determining the complex structure of SCM-25, including the disorders and distributions of framework atoms silicon and germanium. SCM-25 has a high surface area (510 m2/g) and high thermal stability (700 °C). Furthermore, we propose a potential postsynthetic strategy for the preparation of zeolites with ordered meso-cavities by applying the ADOR (assembly–disassembly–organization–reassembly) approach.

All crystalline materials in nature, whether inorganic, organic, or biological, macroscopic or microscopic, have their own chemical and physical properties, which strongly depend on their atomic structures. Therefore, structure determination is extremely important in chemistry, physics, materials science, etc. In the past centuries, many techniques have been developed for structure determination. The most widely used one is X-ray crystallography (single-crystal X-ray diffraction (SCXRD) and powder X-ray diffraction (PXRD)), and it remains the most important technique for structure determination of crystalline materials. Although SCXRD and PXRD are successful in many cases, a number of reasons limit their applications, such as SCXRD for nanosized crystals, intergrowth, and defects and PXRD for complex structures, multiphasic samples, impurities, peak overlaps, etc. Another most valuable technique for structure determination is electron crystallography (EC). With the electron as a probe, EC alone can also be used for structure determination, especially for crystals that are too small to be studied by SCXRD or too complex for PXRD. As electrons interact much more strongly with matter than X-rays do, both electron diffraction (ED) patterns and high-resolution transmission electron microscopy (HRTEM) images can be obtained from nanosized crystals. However, collecting a complete set of ED patterns or recording a good HRTEM image requires considerable expertise on the operation of electron microscopes and crystallography. The strong interactions between electrons and materials can also lead to dynamical effects and beam damage. These difficulties make structure determination from ED patterns and HRTEM images not straightforward. Recently, two three-dimensional (3D) electron diffraction techniques, automated electron diffraction tomography (ADT) and rotation electron diffraction (RED), have been developed, which perform the data collection in an automated manner. Although the dynamical effects in the newly developed 3D electron diffraction techniques (ADT, RED) are reduced significantly, for some structures there are still problems with obtaining an initial model because of beam damage. The X-ray diffraction and EC methods discussed above are both powerful techniques but have their own limitations. In many complicated cases, one technique alone is not enough to solve the crystal structure, and different techniques that supply complementary structural information have to support each other for the complete structure determination. In this Account, we provide a summary of the advantages and disadvantages of X-ray diffraction (PXRD and SCXRD) and EC (HRTEM and ED) for structure determination and include a review of applications of X-ray diffraction and EC for solving complex structure problems such as peak overlap, impurities, pseudosymmetry and twinning, disordered frameworks, locating guests, aperiodic structures, etc. Some of the latest advances in structure determination are also presented briefly, namely, revealing hydrogen positions by ED, protein crystal structure solution by 3D electron diffraction, and structure determination using an X-ray free electron laser (XFEL).

Atherosclerosis is the product of excessive lipid accumulation and inflammation in the artery wall. The lipid that tops every list of suspects is cholesterol, which is the primary lipid in low-density lipoproteins (LDL). Cholesterol exists either as a simple molecule or as cholesterol esters, in which the hydroxyl group is linked to a fatty acyl moiety. In cells, cholesterol esters are synthesized in an intracellular reaction catalyzed by acyl coenzyme A (CoA):cholesterol acyltransferase (ACAT) enzymes.1,2 Owing to the discoveries of cholesterol esters in arterial lesions in 19103 and of ACAT activity in the mid 1900s,4 inhibiting ACAT has been considered as a strategy for preventing or treating atherosclerosis. Over the past 25 years, interest in ACAT inhibitors has waxed and waned as new studies advance knowledge in the field. Prominently reported and disappointing results from a recent human trial of an ACAT inhibitor5 dampened enthusiasm for this potential therapy. However, a study by Bell et al in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology 6 revives the idea of targeting ACAT enzymes and highlights a key unanswered question: Can ACAT2-specific inhibitors lower plasma cholesterol and treat or prevent atherosclerosis? See page 1814 After the discovery of the ACAT reaction, two rationales for inhibiting ACAT emerged. One, in retrospect, was perhaps overly simplistic: blocking cholesterol esterification in macrophages would diminish macrophage “foam cell” formation and thereby decrease atherosclerotic lesion development. The other was to decrease hepatic and intestinal cholesterol ester formation, resulting in decreases in plasma levels …

The effect of the acceleration voltage on the quality of structure determination by 3D-electron diffraction