Limb development genes underlie variation in human fingerprint patterns.

Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized "pattern-block" correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning.

Accurate structure prediction of biomolecular interactions with AlphaFold 3.

The introduction of AlphaFold 21 has spurred a revolution in modelling the structure of proteins and their interactions, enabling a huge range of applications in protein modelling and design2-6. Here we describe our AlphaFold 3 model with a substantially updated diffusion-based architecture that is capable of predicting the joint structure of complexes including proteins, nucleic acids, small molecules, ions and modified residues. The new AlphaFold model demonstrates substantially improved accuracy over many previous specialized tools: far greater accuracy for protein-ligand interactions compared with state-of-the-art docking tools, much higher accuracy for protein-nucleic acid interactions compared with nucleic-acid-specific predictors and substantially higher antibody-antigen prediction accuracy compared with AlphaFold-Multimer v.2.37,8. Together, these results show that high-accuracy modelling across biomolecular space is possible within a single unified deep-learning framework.

Vaccine-Induced Protection from Homologous Tier 2 SHIV Challenge in Nonhuman Primates Depends on Serum-Neutralizing Antibody Titers.

Passive administration of HIV neutralizing antibodies (nAbs) can protect macaques from hard-to-neutralize (tier 2) chimeric simian-human immunodeficiency virus (SHIV) challenge. However, conditions for nAb-mediated protection after vaccination have not been established. Here, we selected groups of 6 rhesus macaques with either high or low serum nAb titers from a total of 78 animals immunized with recombinant native-like (SOSIP) Env trimers. Repeat intrarectal challenge with homologous tier 2 SHIVBG505 led to rapid infection in unimmunized and low-titer animals. High-titer animals, however, demonstrated protection that was gradually lost as nAb titers waned over time. An autologous serum ID50 nAb titer of ∼1:500 afforded more than 90% protection from medium-dose SHIV infection. In contrast, antibody-dependent cellular cytotoxicity and T cell activity did not correlate with protection. Therefore, Env protein-based vaccination strategies can protect against hard-to-neutralize SHIV challenge in rhesus macaques by inducing tier 2 nAbs, provided appropriate neutralizing titers can be reached and maintained.

Estimating disease heritability from complex pedigrees allowing for ascertainment and covariates.

We propose TetraHer, a method for estimating the liability heritability of binary phenotypes. TetraHer has five key features. First, it can be applied to data from complex pedigrees that contain multiple types of relationships. Second, it can correct for ascertainment of cases or controls. Third, it can accommodate covariates. Fourth, it can model the contribution of common environment. Fifth, it produces a likelihood that can be used for significance testing. We first demonstrate the validity of TetraHer on simulated data. We then use TetraHer to estimate liability heritability for 229 codes from the tenth International Classification of Diseases (ICD-10). We identify 107 codes with significant heritability (p < 0.05/229), which can be used in future analyses for investigating the genetic architecture of human diseases.

The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis.

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.

Factors associated with COVID-19-related death using OpenSAFELY.

Coronavirus disease 2019 (COVID-19) has rapidly affected mortality worldwide1. There is unprecedented urgency to understand who is most at risk of severe outcomes, and this requires new approaches for the timely analysis of large datasets. Working on behalf of NHS England, we created OpenSAFELY-a secure health analytics platform that covers 40% of all patients in England and holds patient data within the existing data centre of a major vendor of primary care electronic health records. Here we used OpenSAFELY to examine factors associated with COVID-19-related death. Primary care records of 17,278,392 adults were pseudonymously linked to 10,926 COVID-19-related deaths. COVID-19-related death was associated with: being male (hazard ratio (HR) 1.59 (95% confidence interval 1.53-1.65)); greater age and deprivation (both with a strong gradient); diabetes; severe asthma; and various other medical conditions. Compared with people of white ethnicity, Black and South Asian people were at higher risk, even after adjustment for other factors (HR 1.48 (1.29-1.69) and 1.45 (1.32-1.58), respectively). We have quantified a range of clinical factors associated with COVID-19-related death in one of the largest cohort studies on this topic so far. More patient records are rapidly being added to OpenSAFELY, we will update and extend our results regularly.

Antibody-dependent cellular cytotoxicity, infected cell binding and neutralization by antibodies to the SIV envelope glycoprotein.

Antibodies specific for diverse epitopes of the simian immunodeficiency virus envelope glycoprotein (SIV Env) have been isolated from rhesus macaques to provide physiologically relevant reagents for investigating antibody-mediated protection in this species as a nonhuman primate model for HIV/AIDS. With increasing interest in the contribution of Fc-mediated effector functions to protective immunity, we selected thirty antibodies representing different classes of SIV Env epitopes for a comparison of antibody-dependent cellular cytotoxicity (ADCC), binding to Env on the surface of infected cells and neutralization of viral infectivity. These activities were measured against cells infected with neutralization-sensitive (SIVmac316 and SIVsmE660-FL14) and neutralization-resistant (SIVmac239 and SIVsmE543-3) viruses representing genetically distinct isolates. Antibodies to the CD4-binding site and CD4-inducible epitopes were identified with especially potent ADCC against all four viruses. ADCC correlated well with antibody binding to virus-infected cells. ADCC also correlated with neutralization. However, several instances of ADCC without detectable neutralization or neutralization without detectable ADCC were observed. The incomplete correspondence between ADCC and neutralization shows that some antibody-Env interactions can uncouple these antiviral activities. Nevertheless, the overall correlation between neutralization and ADCC implies that most antibodies that are capable of binding to Env on the surface of virions to block infectivity are also capable of binding to Env on the surface of virus-infected cells to direct their elimination by ADCC.

MHC Class I Ligands of Rhesus Macaque Killer Cell Ig-like Receptors.

Definition of MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as an animal model for infectious diseases, reproductive biology, and transplantation. To provide a more complete foundation for studying NK cell responses, rhesus macaque KIRs representing common allotypes of lineage II KIR genes were tested for interactions with MHC class I molecules representing diverse Macaca mulatta (Mamu)-A, -B, -E, -F, -I, and -AG alleles. KIR-MHC class I interactions were identified by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. Ligands for 12 KIRs of previously unknown specificity were identified that fell into three general categories: interactions with multiple Mamu-Bw4 molecules, interactions with Mamu-A-related molecules, including allotypes of Mamu-AG and the hybrid Mamu-B*045:03 molecule, or interactions with Mamu-A1*012:01. Whereas most KIRs found to interact with Mamu-Bw4 are inhibitory, most of the KIRs that interact with Mamu-AG are activating. The KIRs that recognize Mamu-A1*012:01 belong to a phylogenetically distinct group of macaque KIRs with a 3-aa deletion in the D0 domain that is also present in human KIR3DL1/S1 and KIR3DL2. This study more than doubles the number of rhesus macaque KIRs with defined MHC class I ligands and identifies interactions with Mamu-AG, -B*045, and -A1*012. These findings support overlapping, but nonredundant, patterns of ligand recognition that reflect extensive functional diversification of these receptors.

Evaluating indirect genetic effects of siblings using singletons.

Estimating effects of parental and sibling genotypes (indirect genetic effects) can provide insight into how the family environment influences phenotypic variation. There is growing molecular genetic evidence for effects of parental phenotypes on their offspring (e.g. parental educational attainment), but the extent to which siblings affect each other is currently unclear. Here we used data from samples of unrelated individuals, without (singletons) and with biological full-siblings (non-singletons), to investigate and estimate sibling effects. Indirect genetic effects of siblings increase (or decrease) the covariance between genetic variation and a phenotype. It follows that differences in genetic association estimates between singletons and non-singletons could indicate indirect genetic effects of siblings if there is no heterogeneity in other sources of genetic association between singletons and non-singletons. We used UK Biobank data to estimate polygenic score (PGS) associations for height, BMI and educational attainment in self-reported singletons (N = 50,143) and non-singletons (N = 328,549). The educational attainment PGS association estimate was 12% larger (95% C.I. 3%, 21%) in the non-singleton sample than in the singleton sample, but the height and BMI PGS associations were consistent. Birth order data suggested that the difference in educational attainment PGS associations was driven by individuals with older siblings rather than firstborns. The relationship between number of siblings and educational attainment PGS associations was non-linear; PGS associations were 24% smaller in individuals with 6 or more siblings compared to the rest of the sample (95% C.I. 11%, 38%). We estimate that a 1 SD increase in sibling educational attainment PGS corresponds to a 0.025 year increase in the index individual's years in schooling (95% C.I. 0.013, 0.036). Our results suggest that older siblings may influence the educational attainment of younger siblings, adding to the growing evidence that effects of the environment on phenotypic variation partially reflect social effects of germline genetic variation in relatives.

Fetal alleles predisposing to metabolically favorable adiposity are associated with higher birth weight.

BACKGROUND: Higher birthweight is associated with higher adult body mass index (BMI). Alleles that predispose to greater adult adiposity might act in fetal life to increase fetal growth and birthweight. Whether there are fetal effects of recently identified adult metabolically favorable adiposity alleles on birthweight is unknown. AIM: We aimed to test the effect on birthweight of fetal genetic predisposition to higher metabolically favorable adult adiposity and compare that with the effect of fetal genetic predisposition to higher adult BMI. METHODS: We used published genome wide association study data (n = upto 406 063) to estimate fetal effects on birthweight (adjusting for maternal genotype) of alleles known to raise metabolically favorable adult adiposity or BMI. We combined summary data across single nucleotide polymorphisms (SNPs) with random effects meta-analyses. We performed weighted linear regression of SNP-birthweight effects against SNP-adult adiposity effects to test for a dose-dependent association. RESULTS: Fetal genetic predisposition to higher metabolically favorable adult adiposity and higher adult BMI were both associated with higher birthweight (3 g per effect allele (95% CI: 1-5) averaged over 14 SNPs; P = 0.002; 0.5 g per effect allele (95% CI: 0-1) averaged over 76 SNPs; P = 0.042, respectively). SNPs with greater effects on metabolically favorable adiposity tended to have greater effects on birthweight (R2 = 0.2912, P = 0.027). There was no dose-dependent association for BMI (R2 = -0.0019, P = 0.602). CONCLUSIONS: Fetal genetic predisposition to both higher adult metabolically favorable adiposity and BMI is associated with birthweight. Fetal effects of metabolically favorable adiposity-raising alleles on birthweight are modestly proportional to their effects on future adiposity, but those of BMI-raising alleles are not.

Deep learning -- promises for 3D nuclear imaging: a guide for biologists.

For the past century, the nucleus has been the focus of extensive investigations in cell biology. However, many questions remain about how its shape and size are regulated during development, in different tissues, or during disease and aging. To track these changes, microscopy has long been the tool of choice. Image analysis has revolutionized this field of research by providing computational tools that can be used to translate qualitative images into quantitative parameters. Many tools have been designed to delimit objects in 2D and, eventually, in 3D in order to define their shapes, their number or their position in nuclear space. Today, the field is driven by deep-learning methods, most of which take advantage of convolutional neural networks. These techniques are remarkably adapted to biomedical images when trained using large datasets and powerful computer graphics cards. To promote these innovative and promising methods to cell biologists, this Review summarizes the main concepts and terminologies of deep learning. Special emphasis is placed on the availability of these methods. We highlight why the quality and characteristics of training image datasets are important and where to find them, as well as how to create, store and share image datasets. Finally, we describe deep-learning methods well-suited for 3D analysis of nuclei and classify them according to their level of usability for biologists. Out of more than 150 published methods, we identify fewer than 12 that biologists can use, and we explain why this is the case. Based on this experience, we propose best practices to share deep-learning methods with biologists.

Consistency, completeness and external validity of ethnicity recording in NHS primary care records: a cohort study in 25 million patients' records at source using OpenSAFELY.

BACKGROUND: Ethnicity is known to be an important correlate of health outcomes, particularly during the COVID-19 pandemic, where some ethnic groups were shown to be at higher risk of infection and adverse outcomes. The recording of patients' ethnic groups in primary care can support research and efforts to achieve equity in service provision and outcomes; however, the coding of ethnicity is known to present complex challenges. We therefore set out to describe ethnicity coding in detail with a view to supporting the use of this data in a wide range of settings, as part of wider efforts to robustly describe and define methods of using administrative data. METHODS: We describe the completeness and consistency of primary care ethnicity recording in the OpenSAFELY-TPP database, containing linked primary care and hospital records in > 25 million patients in England. We also compared the ethnic breakdown in OpenSAFELY-TPP with that of the 2021 UK census. RESULTS: 78.2% of patients registered in OpenSAFELY-TPP on 1 January 2022 had their ethnicity recorded in primary care records, rising to 92.5% when supplemented with hospital data. The completeness of ethnicity recording was higher for women than for men. The rate of primary care ethnicity recording ranged from 77% in the South East of England to 82.2% in the West Midlands. Ethnicity recording rates were higher in patients with chronic or other serious health conditions. For each of the five broad ethnicity groups, primary care recorded ethnicity was within 2.9 percentage points of the population rate as recorded in the 2021 Census for England as a whole. For patients with multiple ethnicity records, 98.7% of the latest recorded ethnicities matched the most frequently coded ethnicity. Patients whose latest recorded ethnicity was categorised as Other were most likely to have a discordant ethnicity recording (32.2%). CONCLUSIONS: Primary care ethnicity data in OpenSAFELY is present for over three quarters of all patients, and combined with data from other sources can achieve a high level of completeness. The overall distribution of ethnicities across all English OpenSAFELY-TPP practices was similar to the 2021 Census, with some regional variation. This report identifies the best available codelist for use in OpenSAFELY and similar electronic health record data.

Effect of Passive Administration of Monoclonal Antibodies Recognizing Simian Immunodeficiency Virus (SIV) V2 in CH59-Like Coil/Helical or ß-Sheet Conformations on Time of SIVmac251 Acquisition.

The monoclonal antibodies (MAbs) NCI05 and NCI09, isolated from a vaccinated macaque that was protected from multiple simian immunodeficiency virus (SIV) challenges, both target an overlapping, conformationally dynamic epitope in SIV envelope variable region 2 (V2). Here, we show that NCI05 recognizes a CH59-like coil/helical epitope, whereas NCI09 recognizes a ß-hairpin linear epitope. In vitro, NCI05 and, to a lesser extent, NCI09 mediate the killing of SIV-infected cells in a CD4-dependent manner. Compared to NCI05, NCI09 mediates higher titers of antibody-dependent cellular cytotoxicity (ADCC) to gp120-coated cells, as well as higher levels of trogocytosis, a monocyte function that contributes to immune evasion. We also found that passive administration of NCI05 or NCI09 to macaques did not affect the risk of SIVmac251 acquisition compared to controls, demonstrating that these anti-V2 antibodies alone are not protective. However, NCI05 but not NCI09 mucosal levels strongly correlated with delayed SIVmac251 acquisition, and functional and structural data suggest that NCI05 targets a transient state of the viral spike apex that is partially opened, compared to its prefusion-closed conformation. IMPORTANCE Studies suggest that the protection against SIV/simian-human immunodeficiency virus (SHIV) acquisition afforded by the SIV/HIV V1 deletion-containing envelope immunogens, delivered by the DNA/ALVAC vaccine platform, requires multiple innate and adaptive host responses. Anti-inflammatory macrophages and tolerogenic dendritic cells (DC-10), together with CD14+ efferocytes, are consistently found to correlate with a vaccine-induced decrease in the risk of SIV/SHIV acquisition. Similarly, V2-specific antibody responses mediating ADCC, Th1 and Th2 cells expressing no or low levels of CCR5, and envelope-specific NKp44+ cells producing interleukin 17 (IL-17) also are reproducible correlates of decreased risk of virus acquisition. We focused on the function and the antiviral potential of two monoclonal antibodies (NCI05 and NCI09) isolated from vaccinated animals that differ in antiviral function in vitro and recognize V2 in a linear (NCI09) or coil/helical (NCI05) conformation. We demonstrate that NCI05, but not NCI09, delays SIVmac251 acquisition, highlighting the complexity of antibody responses to V2.

Exotoxin S secreted by internalized Pseudomonas aeruginosa delays lytic host cell death.

The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.

The mismatched nucleotides encoded in vaccinia virus flip-and-flop hairpin telomeres serve an essential role in virion maturation.

Poxvirus genomes consist of a linear duplex DNA that ends in short inverted and complementary hairpin structures. These elements also encode loops and mismatches that likely serve a role in genome packaging and perhaps replication. We constructed mutant vaccinia viruses (VACV) where the native hairpins were replaced by altered forms and tested effects on replication, assembly, and virulence. Our studies showed that structure, not sequence, likely determines function as one can replace an Orthopoxvirus (VACV) hairpin with one copied from a Leporipoxvirus with no effect on growth. Some loops can be deleted from VACV hairpins with little effect, but VACV bearing too few mismatches grew poorly and we couldn't recover viruses lacking all mismatches. Further studies were conducted using a mutant bearing only one of six mismatches found in wild-type hairpins (SΔ1Δ3-6). This virus grew to ~20-fold lower titers, but neither DNA synthesis nor telomere resolution was affected. However, the mutant exhibited a particle-to-PFU ratio 10-20-fold higher than wild-type viruses and p4b/4b core protein processing was compromised, indicating an assembly defect. Electron microscopy showed that SΔ1Δ3-6 mutant development was blocked at the immature virus (IV) stage, which phenocopies known effects of I1L mutants. Competitive DNA binding assays showed that recombinant I1 protein had less affinity for the SΔ1Δ3-6 hairpin than the wild-type hairpin. The SΔ1Δ3-6 mutant was also attenuated when administered to SCID-NCR mice by tail scarification. Mice inoculated with viruses bearing wild-type hairpins exhibited a median survival of 30-37 days, while mice infected with SΔ1Δ3-6 virus survived >70 days. Persistent infections favor genetic reversion and genome sequencing detected one example where a small duplication near the hairpin tip likely created a new loop. These observations show that mismatches serve a critical role in genome packaging and provide new insights into how VACV "flip and flop" telomeres are arranged.

A Note on Modelling Bidirectional Feedback Loops in Mendelian Randomization Studies.

Structural equation models (SEMs) involving feedback loops may offer advantages over standard instrumental variables estimators in terms of modelling causal effects in the presence of bidirectional relationships. In the following note, we show that in the case of a single "exposure" and "outcome" variable, modelling relationships using a SEM with a simple bidirectional linear feedback loop offers no advantage over traditional instrumental variables estimators in terms of consistency (i.e. both approaches yield consistent estimates of the causal effect, provided that causal estimates are obtained in both directions). In the case of finite samples, traditional IV estimators and SEM exhibited similar power across many of the conditions we examined, although which method performed best depended on the residual correlation between variables and the strength of the instruments. In particular, the power of SEM was insensitive to the residual correlation between variables, whereas the power of the Wald estimator/2SLS improved (deteriorated) relative to SEM as the magnitude of the residual correlation increased (decreased) assuming a positive causal effect of the exposure on the outcome. The power of SEM improved relative to the Wald estimator/2SLS as the instruments explained more residual variance in the "outcome" variable.

Prostagram magnetic resonance imaging in a screening population: Prostate Imaging-Reporting and Data System or Likert?

OBJECTIVE: To compare biopsy recommendation rates and accuracy of the Prostate Imaging-Reporting and Data System, version 2 (PI-RADSv2) with the Likert scale for detection of clinically significant and insignificant prostate cancer in men screened within the Imperial Prostate 1 Prostate Cancer Screening Trial Using Imaging (IP1-PROSTAGRAM). PATIENTS AND METHODS: Men aged 50-69 years were screened with Prostagram MRI. Scans were prospectively reported using both PI-RADSv2 (excluding dynamic contrast-enhanced sequence score) and 5-point Likert scores by expert uro-radiologists. Systematic and targeted transperineal biopsy was recommended if the scan was scored ≥ 3, based on either reporting system. The proportion of patients recommended for biopsy and detection rates for Grade Groups (GGs) 1 and ≥ 2 were compared. Receiver operating characteristic (ROC) analysis was performed to compare performance. RESULTS: A total of 406 men underwent Prostagram MRI. The median (interquartile range) age and prostate-specific antigen level were 57 (53-61) years and 0.91 (0.56-1.74) ng/mL, respectively. At MRI score ≥ 3, more patients were recommended for biopsy based on Likert criteria (94/406; 23%, 95% confidence interval [CI] 19.2%-27.6%) compared to PI-RADSv2 (72/406; 18%, 95% CI 14.2%-21.9%; P = 0.03). For MRI scores ≥ 4, PI-RADSv2 and Likert scales led to 43/406 (11%, 95% CI 7.9%-14.1%) and 35/406 (9%, 95% CI 6.2%-11.9%) men recommended for biopsy (P = 0.40). For GG ≥ 2 detection, PIRADSv2 and Likert detected 22% (95% CI 11.4%-30.8%, 14/72) and 16% (95% CI 9.5%-25.3%, 15/94), respectively (P = 0.56). For GG1 cancers detection these were 11% (95% CI 4.3%-19.6%, seven of 72) vs 11% (95% CI 4.7%-17.8%, nine of 94; P = 1.00). The accuracy of PI-RADSv2 and Likert scale was similar (area under the ROC curve 0.64 vs 0.65, P = 0.95). CONCLUSIONS: In reporting non-contrast-enhanced Prostagram MRI in a screening population, the PI-RADSv2 and Likert scoring systems were equally accurate; however, Likert scale use led to more men undergoing biopsy without a subsequent increase in significant cancer detection rates. To improve reporting of Prostagram MRI, either the PI-RADSv2 or a modified Likert scale or a standalone scoring system should be developed.