Crystals in the community and the classroom.

The growing pressure on school curricula has meant crystals and the science of crystallography have been cut from or made optional for many educational programs. This omission is a serious disservice to the history and understanding of modern sciences, given that crystallography underpins many of the greatest advancements in science over the past century, is a critical component of many modern research papers and patents, and has 29 Nobel Prizes awarded in the field. This contribution describes a simple activity to target classroom and public engagement with crystallography, using marshmallows or equivalent sweets/candy to represent atoms and cocktail sticks to represent bonds, together with examples of how crystals are studied and how they are useful. Though it has a simple basis, this activity can be extended in numerous ways to reflect the aims of the demonstrator, and a few of these are described.

A similar injury profile observed in franchise men's and women's cricket in England and Wales: injury surveillance analysis from the first three 'The Hundred' competitions.

Objectives: To describe the injury profile of a novel format cricket competition ('The Hundred') and compare injury incidence and prevalence between the men's and women's competitions. Methods: Medical staff prospectively collected injury data from the eight men's and women's teams during the 2021-2023 competitions. Injury definitions and incidence calculations followed the international consensus statement. Results: In the men's competition, 164 injuries were recorded, compared with 127 in the women's competition. Tournament injury incidence was 36.6 (95% CI 31.4 to 42.7) and 32.5 (95% CI 27.3 to 38.7)/100 players/tournament in the men's and women's competition, respectively. Non-time-loss incidence (men's 26.6 (95% CI 22.2 to 31.8), women's 24.6 (95% CI 20.1 to 30.0)/100 players/tournament) was higher than time-loss incidence (men's 10.0 (95% CI 7.5 to 13.5), women's 7.9 (95% CI 5.6 to 11.3)/100 players/tournament). Injury prevalence was 2.9% and 3.6% in the men's and women's competitions, respectively. Match fielding was the most common activity at injury in both competitions. The thigh and hand were the most common body location time-loss injury in the men's and women's competitions, respectively. Conclusion: A similar injury profile was observed between the men's and women's competition. Preventative strategies targeting thigh injuries in the men's competition and hand injuries in the women's competition would be beneficial. Compared with published injury rates, 'The Hundred' men's presents a greater risk of injury than Twenty20 (T20), but similar to one-day cricket, with 'The Hundred' women's presenting a similar injury risk to T20 and one-day cricket. Additional years of data are required to confirm these findings.

Atomic structure of a nudivirus occlusion body protein determined from a 70-year-old crystal sample.

Infectious protein crystals are an essential part of the viral lifecycle for double-stranded DNA Baculoviridae and double-stranded RNA cypoviruses. These viral protein crystals, termed occlusion bodies or polyhedra, are dense protein assemblies that form a crystalline array, encasing newly formed virions. Here, using X-ray crystallography we determine the structure of a polyhedrin from Nudiviridae. This double-stranded DNA virus family is a sister-group to the baculoviruses, whose members were thought to lack occlusion bodies. The 70-year-old sample contains a well-ordered lattice formed by a predominantly α-helical building block that assembles into a dense, highly interconnected protein crystal. The lattice is maintained by extensive hydrophobic and electrostatic interactions, disulfide bonds, and domain switching. The resulting lattice is resistant to most environmental stresses. Comparison of this structure to baculovirus or cypovirus polyhedra shows a distinct protein structure, crystal space group, and unit cell dimensions, however, all polyhedra utilise common principles of occlusion body assembly.

Alkyne Derivatives of SARS-CoV-2 Main Protease Inhibitors Including Nirmatrelvir Inhibit by Reacting Covalently with the Nucleophilic Cysteine.

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic properties.

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

Bone health and asymmetry in elite female cricketers.

The aim of this study is to determine if bone mineral density (BMD) and bone asymmetry differs between female cricket fast bowlers, spin bowlers and batters. BMD was determined at the total body, lumbar spine, and proximal femurs in 12 fast bowlers, 13 batters and 11 spin bowlers from pre-season DXA scans. High Z-scores at the total body, lumbar spine, and proximal femur were observed in all cricketers (mean Z-scores: +1.4 to +3.3) compared with a general age matched reference population. Fast bowlers had significantly greater BMD on the contralateral side of the lumbar spine compared with the ipsilateral side (p = 0.001, 5.9-12.1%). No asymmetry was found between hips in all groups. All cricket positions demonstrated high BMD at all measured sites. The lumbar spine of fast bowlers is asymmetric, with significantly greater BMD on the contralateral side of the spine, particularly at L4, possibly in response to the asymmetric lumbar loading patterns observed in bowling.HighlightsElite female cricketers demonstrate high BMD at total body, lumbar spine and proximal femur sites, regardless of playing position compared with a general age and ethnic group matched reference population.Fast bowlers have greater BMD on the contralateral (opposite bowling arm) side of the lumbar spine compared with the ipsilateral side, while a symmetrical pattern was observed in spin bowlers and batters.No asymmetry in BMD or section modulus between hips was observed at any proximal femur site for any cricket position.

Childhood gender-typed behaviour, sexual orientation, childhood abuse and post-traumatic stress disorder: a prospective birth-cohort study.

ALSPAC birth-cohort data were analysed to assess prospective associations between childhood gender nonconformity (CGN), childhood/adolescent abuse, and adulthood PTSD symptoms. Structural equation models assessed whether abuse mediated the relationship between CGN and PTSD. Sex and sexual orientation differences were investigated. For females, higher parent-rated CGN at 30, 42 and 57-months was associated with mother-reported abuse, self-reported physical/psychological abuse, and/or self-reported sexual abuse. Higher CGN at 30-months was associated with more PTSD symptoms at 23 years. Self-rated CGN in males and females, and parent-rated CGN in males, were not associated with abuse or PTSD. Sexual minority identification was associated with higher CGN and abuse and for females, PTSD symptoms. In females, the relationship between greater CGN at 30-months and PTSD symptoms was separately mediated by each abuse variable. Self-reported sexual abuse was no longer a significant mediator after sexual orientation adjustment. Self-reported physical/psychological abuse significantly mediated the association alone when it was entered together with mother-reported abuse, even after sexual orientation adjustment. In conclusion, childhood gender nonconformity in females may increase the risk for adult PTSD symptoms, possibly mediated by childhood abuse. In females, mediation of the relationship between CGN and PTSD by sexual abuse may be particularly relevant for sexual minority individuals.

Detecting Injury Risk Factors with Algorithmic Models in Elite Women's Pathway Cricket.

This exploratory retrospective cohort analysis aimed to explore how algorithmic models may be able to identify important risk factors that may otherwise not have been apparent. Their association with injury was then assessed with more conventional data models. Participants were players registered on the England and Wales Cricket Board women's international development pathway (n=17) from April 2018 to August 2019 aged between 14-23 years (mean 18.2±1.9) at the start of the study period. Two supervised learning techniques (a decision tree and random forest with traditional and conditional algorithms) and generalised linear mixed effect models explored associations between risk factors and injury. The supervised learning models did not predict injury (decision tree and random forest area under the curve [AUC] of 0.66 and 0.72 for conditional algorithms) but did identify important risk factors. The best-fitting generalised linear mixed effect model for predicting injury (Akaike Information Criteria [AIC]=843.94, conditional r-squared=0.58) contained smoothed differential 7-day load (P<0.001), average broad jump scores (P<0.001) and 20 m speed (P<0.001). Algorithmic models identified novel injury risk factors in this population, which can guide practice and future confirmatory studies can now investigate.

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline.

The mounting of microcrystals (<10 µm) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise. Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

Redox Metabolism Measurement in Mammalian Cells and Tissues by LC-MS.

Cellular redox state is highly dynamic and delicately balanced between constant production of reactive oxygen species (ROS), and neutralization by endogenous antioxidants, such as glutathione. Physiologic ROS levels can function as signal transduction messengers, while high levels of ROS can react with and damage various molecules eliciting cellular toxicity. The redox state is reflective of the cell's metabolic status and can inform on regulated cell-state transitions or various pathologies including aging and cancer. Therefore, methods that enable reliable, quantitative readout of the cellular redox state are imperative for scientists from multiple fields. Liquid-chromatography mass-spectrometry (LC-MS) based methods to detect small molecules that reflect the redox balance in the cell such as glutathione, NADH, and NADPH, have been developed and applied successfully, but because redox metabolites are very labile, these methods are not easily standardized or consolidated. Here, we report a robust LC-MS method for the simultaneous detection of several redox-reactive metabolites that is compatible with parallel global metabolic profiling in mammalian cells. We performed a comprehensive comparison between three commercial hydrophilic interaction chromatography (HILIC) columns, and we describe the application of our method in mammalian cells and tissues. The presented method is easily applicable and will enable the study of ROS function and oxidative stress in mammalian cells by researchers from various fields.

Injury and Player Availability in Women's International Pathway Cricket from 2015 to 2019.

This prospective cohort study aimed to describe injury and illness epidemiology within women's international pathway cricket, understanding what influences player availability in this unique context where players are contracted part-time. Approximately 8.4% of players were impacted by injury or illness during the year, with an average 2.3% of players completely unavailable on any given day. Most medical complaints occurred during training (111.2 injuries/100 players per year). Of all complaints, medical illness had the highest overall incidence (45.0 complaints/100 players), followed by hand injuries (24.7 injuries/100 players). Gradual onset injuries were most common. Overall average match time-loss complaint prevalence rate was 4.1% and average match time-loss injury incidence rate was 7.0 injuries/1000 days of play. Fielding (56.4 injuries/100 players per year) was the activity resulting in the highest average overall and time-loss injury incidence rates, though 'other' activities (e. g. those occurring outside of cricket participation) collectively accounted for 78.3 injuries/100 players per year. The high incidence of medical illness relative to other complaints may be a distinct feature of the women's cricket international pathway compared to other cricket samples. The high occurrence of injuries arising from 'other' activities, likely due to part-time participation, presents an opportunity for targeted injury prevention strategies.• The first study on an international women's cricket pathway, contributes to the empirical base for specific injury risks associated with the women's cricket game, which is an emerging research area for a developing sport.• Some of the findings may be a distinct feature of the women's cricket international pathway, highlighting potential opportunities for targeted prevention strategies.• With the upcoming development of an elite domestic structure these preliminary findings will provide a good starting point for physiotherapy and medical staff working in these contexts.

Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography.

Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.

Adherence to treatment for polycystic ovarian syndrome: A systematic review.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is one of the most prevalent endocrine disorders of women of reproductive age. Treatment plans for this chronic condition frequently include long-term use of a combination of medication and lifestyle interventions. However, treatment outcomes are dependent on adherence to treatment regimens. This study aimed to systematically review the literature for reported adherence to treatments for PCOS. METHODS: A systematic search of Embase, Cochrane, PubMed, CINAHL, PsychINFO, SCOPUS, and International Pharmaceutical Abstracts from inception until January 2019 utilizing the terms PCOS, adherence, and patient compliance was conducted. A total of 179 possible articles were identified. RESULTS: Fourteen articles reporting adherence data were included in the review. Self-report was the most commonly reported method of measuring adherence. Adherence to lifestyle interventions, such as prescribed diets and physical activity, was reported in ten studies and adherence to medications was reported in seven studies, with some reporting both. CONCLUSIONS: Minimal data are available regarding factors associated with adherence in patients with PCOS. Diverse methods of adherence assessment are utilized. Future studies of PCOS treatments should effectively assess and report adherence data as it is essential to evaluating the effectiveness of PCOS treatments and is critically needed to guide clinician efforts to facilitate optimal outcomes for patients.

Direct measurement of X-ray-induced heating of microcrystals.

Temperature control is a key aspect of macromolecular crystallography, with the technique of cryocooling routinely being used to mitigate X-ray-induced damage. Beam-induced heating could cause the temperature of crystals to rise above the glass transition temperature, greatly increasing the rate of damage. X-ray-induced heating of ruby crystals of 20-40 µm in size has been quantified non-invasively by monitoring the emission wavelengths of X-ray-induced fluorescence during exposure to the X-ray beam. For the beam sizes and dose rates typically used in macromolecular crystallography, the temperature rises are of the order of 20 K. The temperature changes observed are compared with models in the literature and can be used as a validation tool for future models.

Childhood gender-typed behavior and emotional or peer problems: a prospective birth-cohort study.

BACKGROUND: Retrospective and cross-sectional studies often report associations between childhood gender nonconformity and greater emotional and peer difficulties. This study used the ALSPAC birth cohort to investigate relationships between childhood gender-typed behavior and peer and emotional problems throughout childhood and adolescence. METHODS: A total of 11,192 participants had at least one measure of parent-rated gender-typed behavior in infancy; 7,049 participants had a measure of child-rated gender-typed behavior at 8.5 years. Separate linear mixed regression models were fitted to assess whether parent-rated and child-rated gender-typed behaviors were associated with emotional and peer problems across childhood and adolescence (6-16 years old). The effect of adding covariates (self-esteem, abuse, bullying, feeling accepted by peers, and feeling different) on these relationships was assessed. RESULTS: For boys, more gender-nonconforming behavior was associated with greater subsequent emotional and peer problems in childhood and adolescence. Adjusting for self-esteem, relational bullying victimization, feeling different, or feeling accepted by peers reduced some of these associations. In contrast, for girls, more gender-nonconforming behavior was associated with fewer emotional problems in childhood and adolescence. In girls, self-reported gender-nonconforming behavior was also associated with fewer parent-rated peer problems but parent-rated gender-nonconforming behavior was associated with more parent-rated peer problems; this latter association was partly explained by self-esteem, bullying, and abuse victimization. These associations were statistically significant but small. CONCLUSIONS: Overall, more female-typical behaviors were generally associated with greater subsequent emotional and peer problems, for both boys and girls. Future studies should investigate factors that reduced these associations, as well as potential negative effects of female-typical behaviors or advantages of male-typical ones. As this was a 14-year longitudinal study, the relationships between gender-role behaviors and emotional/peer problems warrant further research despite the small association sizes.

Exploiting Microbeams for Membrane Protein Structure Determination.

A reproducible, and sample independent means of predictably obtaining large, well-ordered crystals has proven elusive in macromolecular crystallography. In the structure determination pipeline, crystallisation often proves to be a rate-limiting step, and the process of obtaining even small or badly ordered crystals can prove time-consuming and laborious. This is particularly true in the field of membrane protein crystallography and this is reflected in the limited number of unique membrane protein structures deposited in the protein data bank (less than 650 by June 2016 - http://blanco.biomol.uci.edu/mpstruc ). Over recent years the requirement for, and time and cost associated with obtaining, large crystals has been partially alleviated through the development of beamline instrumentation allowing data collection, and structure solution, from ever-smaller crystals. Advances in several areas have led to a step change in what might be considered achievable during a synchrotron trip over the last decade. This chapter will briefly review the current status of the field, the tools available to ease data collection and processing, and give some examples of exploitation of these for membrane protein microfocus macromolecular crystallography.

In vacuo X-ray data collection from graphene-wrapped protein crystals.

The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.

Application of in situ diffraction in high-throughput structure determination platforms.

Macromolecular crystallography (MX) is the most powerful technique available to structural biologists to visualize in atomic detail the macromolecular machinery of the cell. Since the emergence of structural genomics initiatives, significant advances have been made in all key steps of the structure determination process. In particular, third-generation synchrotron sources and the application of highly automated approaches to data acquisition and analysis at these facilities have been the major factors in the rate of increase of macromolecular structures determined annually. A plethora of tools are now available to users of synchrotron beamlines to enable rapid and efficient evaluation of samples, collection of the best data, and in favorable cases structure solution in near real time. Here, we provide a short overview of the emerging use of collecting X-ray diffraction data directly from the crystallization experiment. These in situ experiments are now routinely available to users at a number of synchrotron MX beamlines. A practical guide to the use of the method on the MX suite of beamlines at Diamond Light Source is given.

Solid-state interconversions: unique 100 % reversible transformations between the ground and metastable states in single-crystals of a series of nickel(II) nitro complexes.

The solid-state, low-temperature linkage isomerism in a series of five square planar group 10 phosphino nitro complexes have been investigated by a combination of photocrystallographic experiments, Raman spectroscopy and computer modelling. The factors influencing the reversible solid-state interconversion between the nitro and nitrito structural isomers have also been investigated, providing insight into the dynamics of this process. The cis-[Ni(dcpe)(NO2)2] (1) and cis-[Ni(dppe)(NO2)2] (2) complexes show reversible 100 % interconversion between the η(1)-NO2 nitro isomer and the η(1)-ONO nitrito form when single-crystals are irradiated with 400 nm light at 100 K. Variable temperature photocrystallographic studies for these complexes established that the metastable nitrito isomer reverted to the ground-state nitro isomer at temperatures above 180 K. By comparison, the related trans complex [Ni(PCy3)2(NO2)2] (3) showed 82 % conversion under the same experimental conditions at 100 K. The level of conversion to the metastable nitrito isomers is further reduced when the nickel centre is replaced by palladium or platinum. Prolonged irradiation of the trans-[Pd(PCy3)2(NO2)2] (4) and trans-[Pt(PCy3)2(NO2)2] (5) with 400 nm light gives reversible conversions of 44 and 27 %, respectively, consistent with the slower kinetics associated with the heavier members of group 10. The mechanism of the interconversion has been investigated by theoretical calculations based on the model complex [Ni(dmpe)Cl(NO2)].

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.