Density Matching for Microencapsulation of Field Responsive Suspensions of Non-Brownian Microparticles.

When forming composite microcapsules through the emulsification of a dispersed phase laden with microparticles, one will find that the microparticles become irreversibly embedded in the resulting microcapsule membrane. This phenomenon, known as Pickering stabilization, is detrimental when the end function of the microcapsules relies on the mobility of encapsulated microparticles within the capsule core. In this work, a robust microencapsulation route using density matching of non-Brownian microparticles in a binary solvent is shown to easily and effectively encapsulate particles, with >90% of particles retaining mobility within the microcapsules, without the necessity for prior chemical/physical modifications to the microparticles. This is proposed as a generalized method to be used for all manner of particle chemistries, shapes, and sizes.

Characterization of the genetic determinants of context-specific DNA methylation in primary monocytes.

To better understand inter-individual variation in sensitivity of DNA methylation (DNAm) to immune activity, we characterized effects of inflammatory stimuli on primary monocyte DNAm (n = 190). We find that monocyte DNAm is site-dependently sensitive to lipopolysaccharide (LPS), with LPS-induced demethylation occurring following hydroxymethylation. We identify 7,359 high-confidence immune-modulated CpGs (imCpGs) that differ in genomic localization and transcription factor usage according to whether they represent a gain or loss in DNAm. Demethylated imCpGs are profoundly enriched for enhancers and colocalize to genes enriched for disease associations, especially cancer. DNAm is age associated, and we find that 24-h LPS exposure triggers approximately 6 months of gain in epigenetic age, directly linking epigenetic aging with innate immune activity. By integrating LPS-induced changes in DNAm with genetic variation, we identify 234 imCpGs under local genetic control. Exploring shared causal loci between LPS-induced DNAm responses and human disease traits highlights examples of disease-associated loci that modulate imCpG formation.

A common NFKB1 variant detected through antibody analysis in UK Biobank predicts risk of infection and allergy.

Infectious agents contribute significantly to the global burden of diseases through both acute infection and their chronic sequelae. We leveraged the UK Biobank to identify genetic loci that influence humoral immune response to multiple infections. From 45 genome-wide association studies in 9,611 participants from UK Biobank, we identified NFKB1 as a locus associated with quantitative antibody responses to multiple pathogens, including those from the herpes, retro-, and polyoma-virus families. An insertion-deletion variant thought to affect NFKB1 expression (rs28362491), was mapped as the likely causal variant and could play a key role in regulation of the immune response. Using 121 infection- and inflammation-related traits in 487,297 UK Biobank participants, we show that the deletion allele was associated with an increased risk of infection from diverse pathogens but had a protective effect against allergic disease. We propose that altered expression of NFKB1, as a result of the deletion, modulates hematopoietic pathways and likely impacts cell survival, antibody production, and inflammation. Taken together, we show that disruptions to the tightly regulated immune processes may tip the balance between exacerbated immune responses and allergy, or increased risk of infection and impaired resolution of inflammation.

Multi-ancestry meta-analysis of host genetic susceptibility to tuberculosis identifies shared genetic architecture.

The heritability of susceptibility to tuberculosis (TB) disease has been well recognized. Over 100 genes have been studied as candidates for TB susceptibility, and several variants were identified by genome-wide association studies (GWAS), but few replicate. We established the International Tuberculosis Host Genetics Consortium to perform a multi-ancestry meta-analysis of GWAS, including 14,153 cases and 19,536 controls of African, Asian, and European ancestry. Our analyses demonstrate a substantial degree of heritability (pooled polygenic h2 = 26.3%, 95% CI 23.7-29.0%) for susceptibility to TB that is shared across ancestries, highlighting an important host genetic influence on disease. We identified one global host genetic correlate for TB at genome-wide significance (p<5 × 10-8) in the human leukocyte antigen (HLA)-II region (rs28383206, p-value=5.2 × 10-9) but failed to replicate variants previously associated with TB susceptibility. These data demonstrate the complex shared genetic architecture of susceptibility to TB and the importance of large-scale GWAS analysis across multiple ancestries experiencing different levels of infection pressure.

Yield stress-enabled microencapsulation of field responsive microparticle suspensions.

Try and encapsulate microparticles inside the cores of microcapsules and you will often find that particles adhere to the liquid-liquid interface in a phenomenon known as Pickering stabilization. Particles will remain irreversibly trapped and embedded within the subsequently formed microcapsule membrane. In cases where the encapsulant particles must remain suspended inside the microcapsule core to retain their desired properties or behaviours, Pickering stabilization is detrimental. Here we demonstrate a general procedure using yield stress materials as the core material, where the yield stress of the gel is strong enough to suspend particles against sedimentation, but weak enough to allow spatial manipulation of encapsulant particles using an external field. This external field imparts enough force on particles to disrupt the supporting network and allow particle mobility after encapsulation.

Microrollers flow uphill as granular media.

Pour sand into a container and only the grains near the top surface move. The collective motion associated with the translational and rotational energy of the grains in a thin flowing layer is quickly dissipated as friction through multibody interactions. Alternatively, consider what will happen to a bed of particles if one applies a torque to each individual particle. In this paper, we demonstrate an experimental system where torque is applied at the constituent level through a rotating magnetic field in a dense bed of microrollers. The net result is the grains roll uphill, forming a heap with a negative angle of repose. Two different regimes have been identified related to the degree of mobility or fluidisation of the particles in the bulk. Velocimetry of the near surface flowing layer reveals the collective motion of these responsive particles scales in a similar way to flowing bulk granular flows. A simple granular model that includes cohesion accurately predicts the apparent negative coefficient of friction. In contrast to the response of active or responsive particles that mimic thermodynamic principles, this system results in macroscopic collective behavior that has the kinematics of a purely dissipative granular system.

Intracranial Empyema in Children: A Single-center Retrospective Case Series.

We conducted a retrospective, observational study of 42 children with intracranial empyema admitted to a pediatric neurosurgical center over a 9-year period. Intracranial empyema is rare, but causes significant morbidity and mortality. Twenty-eight cases had neurosurgical source control, more commonly for subdural collections. Streptococcus anginosus group bacteria are important pathogens in subdural empyema, whose isolation predicts more complicated postoperative courses.

Pulmonary Cystic Echinococcosis in a Child Presenting in the United Kingdom with Fever and Chest Pain: A Brief Report and Discussion on Management.

Cystic echinococcosis is a zoonosis caused by the larvae of Echinococcus granulosus . Pulmonary disease may be asymptomatic until the cyst ruptures or becomes secondarily infected. We report a case of pulmonary cystic echinococcosis presenting in the United Kingdom, with discussion on management: optimum antihelminthic agent, length of treatment and type of operative intervention. Treatment should be individualized to the clinical scenario.

Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography.

Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.

Altered IL-6 signalling and risk of tuberculosis disease: a meta-analysis and Mendelian randomisation study.

IL-6 responses are ubiquitous in Mycobacterium tuberculosis (Mtb) infections, but their role in determining human tuberculosis (TB) disease risk is unknown. We used single nucleotide polymorphisms (SNPs) in and near the IL-6 receptor (IL6R) gene, focusing on the non-synonymous variant, rs2228145, associated with reduced classical IL-6 signalling, to assess the effect of altered IL-6 activity on TB disease risk. We identified 16 genome wide association studies (GWAS) of TB disease collating 17,982 cases of TB disease and 972,389 controls across 4 continents. Meta-analyses and Mendelian randomisation analyses revealed that reduced classical IL-6 signalling was associated with lower odds of TB disease, a finding replicated using multiple, independent SNP instruments and 2 separate exposure variables. Our findings establish a causal relationship between IL-6 signalling and the outcome of Mtb infection, suggesting IL-6 antagonists do not increase the risk of TB disease and should be investigated as adjuncts in treatment.

IL7 genetic variation and toxicity to immune checkpoint blockade in patients with melanoma.

Treatment with immune checkpoint blockade (ICB) frequently triggers immune-related adverse events (irAEs), causing considerable morbidity. In 214 patients receiving ICB for melanoma, we observed increased severe irAE risk in minor allele carriers of rs16906115, intronic to IL7. We found that rs16906115 forms a B cell-specific expression quantitative trait locus (eQTL) to IL7 in patients. Patients carrying the risk allele demonstrate increased pre-treatment B cell IL7 expression, which independently associates with irAE risk, divergent immunoglobulin expression and more B cell receptor mutations. Consistent with the role of IL-7 in T cell development, risk allele carriers have distinct ICB-induced CD8+ T cell subset responses, skewing of T cell clonality and greater proportional repertoire occupancy by large clones. Finally, analysis of TCGA data suggests that risk allele carriers independently have improved melanoma survival. These observations highlight key roles for B cells and IL-7 in both ICB response and toxicity and clinical outcomes in melanoma.

Three-Dimensional Sag Tracking in Falling Liquid Films.

Coating defects often arise during application in the flash stage, which constitutes the ∼10 min interval immediately following film application when the solvent evaporates. Understanding the transient rheology and kinematics of a coating system is necessary to avoid defects such as sag, which results in undesirable appearance. A new technique named variable angle inspection microscopy (VAIM) aimed at measuring these phenomena was developed and is summarized herein. The essence of this new, non-invasive, rheological technique is the measurement of a flow field in response to a known gravitational stress. VAIM was used to measure the flow profile through a volume of a liquid thin film at an arbitrary orientation. Flow kinematics of the falling thin film was inferred from particle tracking measurements. Initial benchmarking measurements in the absence of drying tracked the velocity of silica probe particles in ∼140 µm thick films of known viscosity, much greater than water, at incline angles of 5° and 10°. Probe particles were tracked through the entire thickness of the film and at speeds as high as ∼100 µm/s. The sag flow field was well resolved in ∼10 µm thick cross sections, and in general the VAIM measurements were highly reproducible. Complementary profilometer measurements of film thinning were utilized to predict sag velocities with a known model. The model predictions showed good agreement with measurements, which validated the effectiveness of this new method in relating material properties and flow kinematics.

Genome-wide association study of leprosy in Malawi and Mali.

Leprosy is a chronic infection of the skin and peripheral nerves caused by Mycobacterium leprae. Despite recent improvements in disease control, leprosy remains an important cause of infectious disability globally. Large-scale genetic association studies in Chinese, Vietnamese and Indian populations have identified over 30 susceptibility loci for leprosy. There is a significant burden of leprosy in Africa, however it is uncertain whether the findings of published genetic association studies are generalizable to African populations. To address this, we conducted a genome-wide association study (GWAS) of leprosy in Malawian (327 cases, 436 controls) and Malian (247 cases, 368 controls) individuals. In that analysis, we replicated four risk loci previously reported in China, Vietnam and India; MHC Class I and II, LACC1 and SLC29A3. We further identified a novel leprosy susceptibility locus at 10q24 (rs2015583; combined p = 8.81 × 10-9; OR = 0.51 [95% CI 0.40 - 0.64]). Using publicly-available data we characterise regulatory activity at this locus, identifying ACTR1A as a candidate mediator of leprosy risk. This locus shows evidence of recent positive selection and demonstrates pleiotropy with established risk loci for inflammatory bowel disease and childhood-onset asthma. A shared genetic architecture for leprosy and inflammatory bowel disease has been previously described. We expand on this, strengthening the hypothesis that selection pressure driven by leprosy has shaped the evolution of autoimmune and atopic disease in modern populations. More broadly, our data highlights the importance of defining the genetic architecture of disease across genetically diverse populations, and that disease insights derived from GWAS in one population may not translate to all affected populations.

Natural Killer cells demonstrate distinct eQTL and transcriptome-wide disease associations, highlighting their role in autoimmunity.

Natural Killer cells are innate lymphocytes with central roles in immunosurveillance and are implicated in autoimmune pathogenesis. The degree to which regulatory variants affect Natural Killer cell gene expression is poorly understood. Here we perform expression quantitative trait locus mapping of negatively selected Natural Killer cells from a population of healthy Europeans (n = 245). We find a significant subset of genes demonstrate expression quantitative trait loci specific to Natural Killer cells and these are highly informative of human disease, in particular autoimmunity. A Natural Killer cell transcriptome-wide association study across five common autoimmune diseases identifies further novel associations at 27 genes. In addition to these cis observations, we find novel master-regulatory regions impacting expression of trans gene networks at regions including 19q13.4, the Killer cell Immunoglobulin-like Receptor region, GNLY, MC1R and UVSSA. Our findings provide new insights into the unique biology of Natural Killer cells, demonstrating markedly different expression quantitative trait loci from other immune cells, with implications for disease mechanisms.

BIRC6 modifies risk of invasive bacterial infection in Kenyan children.

Invasive bacterial disease is a major cause of morbidity and mortality in African children. Despite being caused by diverse pathogens, children with sepsis are clinically indistinguishable from one another. In spite of this, most genetic susceptibility loci for invasive infection that have been discovered to date are pathogen specific and are not therefore suggestive of a shared genetic architecture of bacterial sepsis. Here, we utilise probabilistic diagnostic models to identify children with a high probability of invasive bacterial disease among critically unwell Kenyan children with Plasmodium falciparum parasitaemia. We construct a joint dataset including 1445 bacteraemia cases and 1143 severe malaria cases, and population controls, among critically unwell Kenyan children that have previously been genotyped for human genetic variation. Using these data, we perform a cross-trait genome-wide association study of invasive bacterial infection, weighting cases according to their probability of bacterial disease. In doing so, we identify and validate a novel risk locus for invasive infection secondary to multiple bacterial pathogens, that has no apparent effect on malaria risk. The locus identified modifies splicing of BIRC6 in stimulated monocytes, implicating regulation of apoptosis and autophagy in the pathogenesis of sepsis in Kenyan children.

Bacterial infections are a major cause of severe illness and death in African children. Understanding which children are at risk of life-threatening infection and why, is key to designing new tools to help protect them. Some risk is likely inherited, but scientists do not know which genes are responsible. Genome-wide association studies (GWAS) may be one way to identify bacterial infection risk genes. GWAS look for genetic differences associated with a particular disease. But previous GWAS studies have failed to find genes linked with bacterial infections in African children because they were too small. Malaria is another frequent cause of life-threatening illness in African children. It can be hard for clinicians to determine if a child's illness is caused by malaria, a bacterial infection, or both. Many children in Africa have malaria parasites in their blood, but they do not always cause disease. Most children with suspected severe malaria are treated with antibiotics in case of bacterial infection. Clinicians may then conduct further testing to determine the illness's actual cause. Scientists may be able to use this data on children with suspected malaria to study bacterial infections. Gilchrist et al. show that children with an unusual alteration in the BIRC6 gene are at increased risk of bacterial infections. In the experiments, Gilchrist et al. used computer modeling to identify a subset of children with likely bacterial infections among 2,200 children admitted to a hospital in Kenya with a high fever and malaria parasites. By combining information on this subset of children with data on children with confirmed bacterial infections and healthy children, Gilchrist created a sample of 5,400 children for a GWAS. The analyses found that children with a variation in the BIRC6 gene on chromosome 2 had a higher risk of bacterial infections. This genetic change is linked with the production of a modified form of BIRC6 in infection-fighting immune cells called monocytes. More studies will help scientists understand how this change might contribute to severe bacterial infections. Learning more may help scientists develop new treatment strategies and identify children most at risk.

Precision, reliability and application of the Wilkins Rate of Reading Test.

BACKGROUND: The Wilkins Rate of Reading Test (WRRT) enables rapid measurement of reading speed using text passages that have no semantic content and demand minimal word recognition skills. It is suited to applications where the primary interest is in the influence of visual and ocular motor factors on reading rate. METHODS: We obtained estimates of precision and reliability of WRRT from four data samples (A-D) collected independently by the authors: (A) n = 118 adults; (B) n = 90 adults; (C) n = 787 children; (D) n = 134 children. Each participant was asked to read aloud as quickly and accurately as possible, for 1 min, and results were recorded as number of words read correctly per minute (wcpm). RESULTS: Estimates of precision are given by the within-subjects standard deviation sw , and reliability by the intraclass correlation coefficient for single measurements r1 . For each sample these estimates were (A) sw  = 11.5 wcpm, r1  = 0.85; (B) sw  = 3.8 wcpm, r1  = 0.98; (C) sw  = 6.7 wcpm, r1  = 0.93; (D) sw  = 6.2 wcpm, r1  = 0.94. CONCLUSION: The reliability of WRRT reflects large variation in reading rate between individuals compared to within-individual variability, indicating that it is a good test for discriminating differences in reading speed between individuals. The precision of the test varies from 3.8 wcpm to 11.5 wcpm among samples, and the pooled value of 7.2 wcpm, provides a basis for setting a population-wide criterion for minimum detectable change of reading rate in individuals over time. Nevertheless, a preferable way of monitoring change in an individual would be to use a criterion determined from estimates of that individual's baseline variation in WRRT scores.

Aged layered double hydroxide nanosheet-polyvinyl alcohol dispersions for enhanced gas barrier coating performance.

Whilst applying a coating layer to a polymer film is a routine approach to enhance the gas barrier properties of the film, it is counter-intuitive to consider that the gas barrier performance of the film would improve by ageing the coating dispersion for weeks before application. Herein, we report that the oxygen barrier performance of a 12 µm PET film coated with a dispersion of inorganic nanosheets in polyvinyl alcohol can be significantly enhanced by ageing this coating dispersion for up to 8 weeks before application. We found up to a 37-fold decrease in the oxygen transmission rate (OTR) of the PET coated film using aged dispersions of [Mg0.66Al0.33(OH)2](NO3)0.33 layered double hydroxide nanosheets (Mg2Al-LDH NS) in polyvinyl alcohol (PVA) compared to the film coated with an equivalent freshly prepared LDH/PVA dispersion. A limiting OTR value of 0.31 cc m-2 day-1 was achieved using the PET film coated with a 3 week aged LDH NS/PVA dispersion. X-ray diffraction experiments show that the degree of in plane alignment of LDH NS on the PET film surface increased significantly from 70.6 ± 0.6 to 86.7 ± 0.6 (%) (100% represents complete alignment of LDH NS platelets on the film surface) for the 4 week aged dispersion compared to the freshly prepared layer. We postulate that when the Mg2Al-LDH NS are aged in PVA the coiled PVA aggregates start to unwrap and attach onto the Mg2Al-LDH NS through hydrogen bonding and eventually form a hydrogen bonded ordered network that facilitates the alignment of nanosheet dispersions during the coating process. Our results suggest that the ageing of inorganic nanosheet dispersions in PVA or other potential hydrogen bonding adhesive systems could be a general approach to improve the alignment of the nanosheets on the polymer film surface once applied and thus improve their performance characteristics for barrier coating applications.

Preparing Lamellae from Vitreous Biological Samples using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography.

Presented here is a protocol for preparing cryo-lamellae from plunge-frozen grids of Plasmodium falciparum-infected human erythrocytes, which could easily be adapted for other biological samples. The basic principles for preparing samples, milling, and viewing lamellae are common to all instruments and the protocol can be followed as a general guide to on-grid cryo-lamella preparation for cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET). Electron microscopy grids supporting the cells are plunge-frozen into liquid nitrogen-cooled liquid ethane using a manual or automated plunge freezer, then screened on a light microscope equipped with a cryo-stage. Frozen grids are transferred into a cryo-scanning electron microscope equipped with a focused ion beam (cryoFIB-SEM). Grids are routinely sputter coated prior to milling, which aids dispersal of charge build-up during milling. Alternatively, an e-beam rotary coater can be used to apply a layer of carbon-platinum to the grids, the exact thickness of which can be more precisely controlled. Once inside the cryoFIB-SEM an additional coating of an organoplatinum compound is applied to the surface of the grid via a gas injection system (GIS). This layer protects the front edge of the lamella as it is milled, the integrity of which is critical for achieving uniformly thin lamellae. Regions of interest are identified via SEM and milling is carried out in a step-wise fashion, reducing the current of the ion beam as the lamella reaches electron transparency, in order to avoid excessive heat generation. A grid with multiple lamellae is then transferred to a transmission electron microscope (TEM) under cryogenic conditions for tilt-series acquisition. A robust and contamination-free workflow for lamella preparation is an essential step for downstream techniques, including cellular cryoEM, cryoET, and sub-tomogram averaging. Development of these techniques, especially for lift-out and milling of high-pressure frozen samples, is of high-priority in the field.

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events - e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Correlative Multi-scale Cryo-imaging Unveils SARS-CoV-2 Assembly and Egress.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events â€" e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.