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The Multiple-DataSet (MDS)1 data collection strategy was examined to determine if the method could be used to produce SAD data sets capable of phasing the structure with a lower X-ray dose. The tests were carried out using data collected on zinc-free Insulin crystals at SER-CAT 22ID beamline. In each experiment, a standard data set was collected followed by the collection of N low-exposure data sets collected using 1/Nth exposures to keep the total radiation dose the same in all cases.The anomalous signal was analyzed and a comparison was made between the standard data set and the merged low-exposure data set by MDS. The study showed that the anomalous signal (as defined by the Ras index2 and peak heights of sulfurs from Bijvoet difference Fourier maps) for the merged low-exposure (MDS) data set was significantly higher compared to the signal observed for the standard data set. The analysis was then repeated merging fewer low-exposure data sets (e.g. N-1, N-2) and the anomalous signal in these cases was found to be comparable or higher than that observed for the standard set. This implies that, by using the MDS strategy, a crystal can be exposed to a lower radiation dose and can still provide sufficient anomalous signal for phasing. Thus, the MDS strategy may offer a better data collection option for radiation sensitive crystals and crystals with a lower symmetry, since it may provide enough data to solve the structure before significant radiation decay occurs. This may be more significant considering the highly intense beams currently available at synchrotron sources such as APS (Advanced Photon Source) after the recent upgrade.

The high levels of flux at a fourth-generation synchrotron are shown to have significant beam heating effects with increasing risk of radiation damage X-ray crystallography technique is widely used to determine the three-dimensional structures of macromolecules and it is very important to know and how it might affect an X- ray diffraction experiments and the resulting structures. There are two types of radiation damages (global and specific).The radiation damage process is dose dependent and there is no technique available to prevent. X- ray crystallography can be used to monitor the damages by collecting consecutive data sets in the same protein crystal to track the progression of damages. Damage incurred during data collection in macromolecular crystallography (MX) limits the information that can be obtained from a single crystal, and it may also prevent getting the solution of structure. Each year, hundreds of scientists are using SER-CAT to conduct X-ray diffraction experiments many of which are directly related to human diseases. Their efficiency and quality of the data collected for various high impact scientific projects will be depending upon the how precisely we study this radiation damage and provide a general data collection strategy especially for the increased intensity (after APS-U) to SER-CAT user community. To study the consequences and progression of radiation damages, 10 consecutive datasets were collected using single trypsin crystal and analyzed. The results for both global and specific damage effects will be presented.

To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.

During the APS Dark period, SER-CAT like other CATs used this time as an opportunity to update the 20-year-old Resource. This included upgrading components of the optic chain for 22ID-D (adding new functionality), decommissioning 22BM (only the hutch remains) and building a second fixed wavelength undulator beamline 22ID-E. These activities were more than moving old components out and new components in since over the past 20 years there has been substantial increases in computing processor and memory speed, data transfer speed, data storage capacity and motor speed and accuracy. In addition, vastly improved detectors coupled with automation have had a great impact on the speed of data collection and structure factor accuracy (Sulfur SAD and better maps). To keep pace with the increased speed of data collection SER-CAT has developed a high-performance 368 processor cloud with load leveling consisting of both cluster and PC based processors. Remembering back to 2002 when it took ∼9 minutes on 22ID to hand mount the crystal, align it, and evacuate the hutch. Since then, much has changed. Today using robotics, automation, high precision goniometers and photon counting detectors we can produce 2 - 3 data sets in the same time it took to mount/align your crystal back in 2002. The presentation will cover the Dark Period upgrades to the SER-CAT Resource and the new opportunities these upgrades represent.

The question of when two skew Young diagrams produce the same skew Schur function has been well-studied. We investigate the same question in the case of stable Grothendieck polynomials, which are the K-theoretic analogues of the Schur functions. We prove a necessary condition for two skew shapes to give rise to the same dual stable Grothendieck polynomial. We also provide a necessary and sufficient condition in the case where the two skew shapes are ribbons.

Cytosine methylation of genomic DNA controls gene expression and maintains genome stability. How a specific DNA sequence is targeted for methylation by a methyitransferase is largely unknown. Here, we show that histone H3 tails lacking iysine 4 （K4） methylation function as an allosteric activator for methyltransferase Dnmt3a by binding to its plant homeodomain （PHD）. In vitro, histone H3 peptides stimulated the methylation activity of Dnmt3a up to 8-fold, in a manner reversely correlated with the level of K4 methylation. The biological significance of allosteric regulation was manifested by molecular modeling and identification of key residues in both the PHD and the catalytic domain of Dnmt3a whose mutations impaired the stimulation of methylation activity by H3 peptides but not the binding of H3 peptides. Significantly, these mutant Dnmt3a proteins were almost inactive in DNA methylation when expressed in mouse embryonic stem cells while their recruitment to genomic targets was unaltered. We therefore propose a two-step mechanism for de novo DNA methylation - first recruitment of the methyltransferase probably assisted by a chromatin- or DNA-binding factor, and then allosteric activation depending on the interaction between Dnmt3a and the histone tails - the latter might serve as a checkpoint for the methylation activity.

Current-induced spin-orbit torques (SOTs) are of interest for fast and energy-efficient manipulation of magnetic order in spintronic devices. To be deterministic, however, switching of perpendicularly magnetized materials by SOT requires a mechanism for in-plane symmetry breaking. Existing methods to do so involve the application of an in-plane bias magnetic field, or incorporation of in-plane structural asymmetry in the device, both of which can be difficult to implement in practical applications. Here, we report bias-field-free SOT switching in a single perpendicular CoTb layer with an engineered vertical composition gradient. The vertical structural inversion asymmetry induces strong intrinsic SOTs and a gradient-driven Dzyaloshinskii–Moriya interaction (g-DMI), which breaks the in-plane symmetry during the switching process. Micromagnetic simulations are in agreement with experimental results, and elucidate the role of g-DMI in the deterministic switching processes. This bias-field-free switching scheme for perpendicular ferrimagnets with g-DMI provides a strategy for efficient and compact SOT device design. Switching of ferrimagnets by current-induced spin-orbit torque is promising for spintronics, due to their high-speed dynamics and small macroscopic magnetization. Switching of perpendicularly magnetized materials, however, requires a bias field for symmetry breaking. Here, Zheng et al demonstrate field-free current-induced switching of perpendicular ferrimagnets, using a compositional gradient-driven Dzyaloshinskii–Moriya interaction.

Cellular proteins continuously undergo non-enzymatic covalent modifications (NECMs) that accumulate under normal physiological conditions and are stimulated by changes in the cellular microenvironment. Glycation, the hallmark of diabetes, is a prevalent NECM associated with an array of pathologies. Histone proteins are particularly susceptible to NECMs due to their long half-lives and nucleophilic disordered tails that undergo extensive regulatory modifications; however, histone NECMs remain poorly understood. Here we perform a detailed analysis of histone glycation in vitro and in vivo and find it has global ramifications on histone enzymatic PTMs, the assembly and stability of nucleosomes, and chromatin architecture. Importantly, we identify a physiologic regulation mechanism, the enzyme DJ-1, which functions as a potent histone deglycase. Finally, we detect intense histone glycation and DJ-1 overexpression in breast cancer tumors. Collectively, our results suggest an additional mechanism for cellular metabolic damage through epigenetic perturbation, with implications in pathogenesis. Proteins continuously undergo non-enzymatic modifications such as glycation, which accumulate under physiological conditions but can be enhanced in disease. Here the authors characterise histone glycation, provide evidence that it affects chromatin, particularly in breast cancer, and identify DJ-1 as a deglycase.

Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood–brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.