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We demonstrate ultrafast-electron diffraction using an electro-optic comb-generator, emitting a 3 GHz train of ps pulses at 257 nm. This new operation mode improves electron coherence and reduces space-charge effects.

Raw meat pet foods can pose health risks to pets and humans. High-pressure processing (HPP) was used in a previous study to demonstrate its effectiveness in achieving a 5-log reduction of Salmonella, E. coli STEC, and L. monocytogenes in commercially available raw pet foods and maintaining the 5-log reduction throughout shelf-life with frozen storage being more effective than refrigerated. L. monocytogenes, being more HPP resistant, could potentially regrow when stored at refrigeration temperatures and required further optimization. Chicken-based raw diet pet food was inoculated with 7–8 log CFU/g cocktails of Salmonella spp., E. coli STEC, or L. monocytogenes and stored at 4 °C for 24 h before the addition of either 0.7% or 1.0% w/v lactic acid fermentate (LAF) and HPP treated at 586 MPa for 2, 3, and 4 min after 24 or 72 h storage at 4 °C. HPP-treated products were stored frozen (−10 to −16 °C) up to 21 days with microbiological analyses on days 1, 3, 5, 7, 14, and 21. All HPP-and LAF-treated samples demonstrated a 5-log reduction of Salmonella spp., E. coli STEC, and L. monocytogenes. Samples without LAF and HPP treated after 24 h storage at 4 °C resulted in an average 4.02 log cfu/g reduction of L. monocytogenes with 2 min HPP hold time while longer HPP hold times at 4 min improved L. monocytogenes reduction by 0.35 log cfu/g. E. coli was found to be more HPP resistant in this study than L. monocytogenes and the addition of LAF had a significant impact on the overall pathogen survival during post-HPP storage. Based on qualitative enrichment data for each pathogen, the use of LAF resulted in more complete inactivation compared to samples without LAF. The use of 1% LAF in combination with 586 MPa for 4 min was found to be most effective for the inactivation of Salmonella spp., E. coli STEC, and L. monocytogenes. The findings are significant as it provides both formulation and processing controls to ensure the safety of raw diet pet foods.

Cs2H12MgO14Se2, monoclinic, P121/a1 (No. 14), a = 9.513(1) Å, b = 13.037(1) Å, c = 6.4730(8) Å, β = 106.244(8)°, V = 770.7 Å3, Z = 2, Rgt(F) = 0.020, wRref(F2) = 0.036, T = 293 K.

Cs2H12MgO14S2, monoclinic, P121/a1 (No. 14), a = 9.338(2) Å, b = 12.849(4) Å, c = 6.361(2) Å, β = 107.07(2)°, V = 729.6 Å3, Z = 2, Rgt(F) = 0.017, wRref(F2) = 0.041, T = 293 K.

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.

Abstract Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene. The neuropathology of HD is characterized by the decline of a specific neuronal population within the brain, the striatal medium spiny neurons (MSNs). The origins of this extreme vulnerability remain unknown. Human induced pluripotent stem cell (hiPS cell)-derived MSNs represent a powerful tool to study this genetic disease. However, the differentiation protocols published so far show a high heterogeneity of neuronal populations in vitro. Here, we compared two previously published protocols to obtain hiPS cell-derived striatal neurons from both healthy donors and HD patients. Patch-clamp experiments, immunostaining and RT-qPCR were performed to characterize the neurons in culture. While the neurons were mature enough to fire action potentials, a majority failed to express markers typical for MSNs. Voltage-clamp experiments on voltage-gated sodium (Nav) channels revealed a large variability between the two differentiation protocols. Action potential analysis did not reveal changes induced by the HD mutation. This study attempts to demonstrate the current challenges in reproducing data of previously published differentiation protocols and in generating hiPS cell-derived striatal MSNs to model a genetic neurodegenerative disorder in vitro.

For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.

Reliable sample delivery and efficient use of limited beam time have remained bottlenecks for serial crystallography (SX). Using a high-intensity polychromatic X-ray beam in combination with a newly developed charge-integrating JUNGFRAU detector, we have applied the method of fixed-target SX to collect data at a rate of 1 kHz at a synchrotron-radiation facility. According to our data analysis for the given experimental conditions, only about 3 000 diffraction patterns are required for a high-quality diffraction dataset. With indexing rates of up to 25%, recording of such a dataset takes less than 30 s.

Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, `naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.

Apart from the most prominent symptoms in Autism spectrum disorder (ASD), namely deficits in social interaction, communication and repetitive behavior, patients often show abnormal sensory reactivity to environmental stimuli. Especially potentially painful stimuli are reported to be experienced in a different way compared to healthy persons. In our present study, we identified an ASD patient carrying compound heterozygous mutations in the voltage-gated sodium channel (VGSC) Nav1.8, which is preferentially expressed in sensory neurons. We expressed both mutations, p.I1511M and p.R512∗, in a heterologous expression system and investigated their biophysical properties using patch-clamp recordings. The results of these experiments reveal that the p.R512∗ mutation renders the channel non-functional, while the p.I1511M mutation showed only minor effects on the channel’s function. Behavioral experiments in a Nav1.8 loss-of-function mouse model additionally revealed that Nav1.8 may play a role in autism-like symptomatology. Our results present Nav1.8 as a protein potentially involved in ASD pathophysiology and may therefore offer new insights into the genetic basis of this disease.