The SHDRA syndrome-associated gene TMEM260 encodes a protein-specific O-mannosyltransferase.

Mutations in the TMEM260 gene cause structural heart defects and renal anomalies syndrome, but the function of the encoded protein remains unknown. We previously reported wide occurrence of O-mannose glycans on extracellular immunoglobulin, plexin, transcription factor (IPT) domains found in the hepatocyte growth factor receptor (cMET), macrophage-stimulating protein receptor (RON), and plexin receptors, and further demonstrated that two known protein O-mannosylation systems orchestrated by the POMT1/2 and transmembrane and tetratricopeptide repeat-containing proteins 1-4 gene families were not required for glycosylation of these IPT domains. Here, we report that the TMEM260 gene encodes an ER-located protein O-mannosyltransferase that selectively glycosylates IPT domains. We demonstrate that disease-causing TMEM260 mutations impair O-mannosylation of IPT domains and that TMEM260 knockout in cells results in receptor maturation defects and abnormal growth of 3D cell models. Thus, our study identifies the third protein-specific O-mannosylation pathway in mammals and demonstrates that O-mannosylation of IPT domains serves critical functions during epithelial morphogenesis. Our findings add a new glycosylation pathway and gene to a growing group of congenital disorders of glycosylation.

Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans.

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

Racgap1 knockdown results in cells with multiple cilia due to cytokinesis failure.

Most mammalian cells have a single primary cilium that acts as a signalling hub in mediating cellular functions. However, little is known about the mechanisms that result in aberrant supernumerary primary cilia per cell. In this study, we re-analysed a previously published whole-genome siRNA-based reverse genetic screen for genes mediating ciliogenesis to identify knockdowns that permit multi-ciliation. We identified siRNA knockdowns that caused significant formation of supernumerary cilia, validated candidate hits in different cell-lines and confirmed that RACGAP1, a component of the centralspindlin complex, was the strongest candidate hit at the whole-genome level. Following loss of RACGAP1, mother centrioles were specified correctly prior to ciliogenesis and the cilia appeared normal. Live cell imaging revealed that increased cilia incidence was caused by cytokinesis failure which led to the formation of multinucleate cells with supernumerary cilia. This suggests that the signalling mechanisms for ciliogenesis are unable to identify supernumerary centrosomes and therefore allow ciliation of duplicated centrosomes as if they were in a new diploid daughter cell. These results, demonstrating that aberrant ciliogenesis is de-coupled from cell cycle regulation, have functional implications in diseases marked by centrosomal amplification.

Cosmetic and functional impact of fascia lata harvest for use in surgery for stress urinary incontinence.

INTRODUCTION: There has been increased interest in using autologous tissues since the Food and Drug Administration banned transvaginal mesh for pelvic organ prolapse in 2019. Our study aims to assess patients' perspective of functional and cosmetic impact on the fascia lata harvest site in patients undergoing fascia lata harvest for the treatment of stress urinary incontinence (SUI). METHODS: This is a prospective survey study of a retrospective cohort of patients who underwent a fascia lata pubovaginal sling between 2017 and 2022. Participants completed a survey regarding the functional and cosmetic outcomes of the harvest site. RESULTS: Seventy-two patients met the inclusion criteria. Twenty-nine patients completed the survey for a completion rate of 40.3%. For functional symptoms, 24.1% (7/29) of patients reported leg discomfort, 10.3% (3/29) reported leg weakness, 10.3% (3/29) reported a bulge, 17.2% (5/29) reported scar pain, 14.8% (4/27) reported scar numbness, and 17.2% (5/29) reported paresthesia at the scar. For cosmetic outcomes, 72.4% (21/29) reported an excellent or good scar appearance. On the PGI-I, 75.9% (22/29) reported their condition as very much better (48.3%, 14/29) or much better (27.6%, 8/29). CONCLUSIONS: The majority of patients reported being satisfied with the functional and cosmetic outcomes of their harvest site as well as satisfied with the improvement in their SUI. Less than 25% of patients report harvest site symptoms, including leg weakness, scar bulging, scar pain, scar numbness, or paresthesia in the scar. This is important in the context of appropriate preoperative discussion and counseling regarding fascia lata harvest.

Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.

Decades ago it was proposed that exocytosis involves invagination of the target membrane, resulting in a highly localized site of contact between the bilayers destined to fuse. The vesicle protein synaptotagmin-I (syt) bends membranes in response to Ca(2+), but whether this drives localized invagination of the target membrane to accelerate fusion has not been determined. Previous studies relied on reconstituted vesicles that were already highly curved and used mutations in syt that were not selective for membrane-bending activity. Here, we directly address this question by utilizing vesicles with different degrees of curvature. A tubulation-defective syt mutant was able to promote fusion between highly curved SNARE-bearing liposomes but exhibited a marked loss of activity when the membranes were relatively flat. Moreover, bending of flat membranes by adding an N-BAR domain rescued the function of the tubulation-deficient syt mutant. Hence, syt-mediated membrane bending is a critical step in membrane fusion.

Dispatch Categories as Indicators of Out-of-Hospital Time Critical Interventions and Associated Emergency Department Outcomes.

OBJECTIVES: Emergency medical services (EMS) systems increasingly grapple with rising call volumes and workforce shortages, forcing systems to decide which responses may be delayed. Limited research has linked dispatch codes, on-scene findings, and emergency department (ED) outcomes. This study evaluated the association between dispatch categorizations and time-critical EMS responses defined by prehospital interventions and ED outcomes. Secondarily, we proposed a framework for identifying dispatch categorizations that are safe or unsafe to hold in queue. METHODS: This retrospective, multi-center analysis encompassed all 9-1-1 responses from 8 accredited EMS systems between 1/1/2021 and 06/30/2023, utilizing the Medical Priority Dispatch System (MPDS). Independent variables included MPDS Protocol numbers and Determinant levels. EMS treatments and ED diagnoses/dispositions were categorized as time-critical using a multi-round consensus survey. The primary outcome was the proportion of EMS responses categorized as time-critical. A non-parametric test for trend was used to assess the proportion of time-critical responses Determinant levels. Based on group consensus, Protocol/Determinant level combinations with at least 120 responses (∼1 per week) were further categorized as safe to hold in queue (<1% time-critical intervention by EMS and <5% time-critical ED outcome) or unsafe to hold in queue (>10% time-critical intervention by EMS or >10% time-critical ED outcome). RESULTS: Of 1,715,612 EMS incidents, 6% (109,250) involved a time-critical EMS intervention. Among EMS transports with linked outcome data (543,883), 12% had time-critical ED outcomes. The proportion of time-critical EMS interventions increased with Determinant level (OMEGA: 1%, ECHO: 38%, p-trend < 0.01) as did time-critical ED outcomes (OMEGA: 3%, ECHO: 31%, p-trend < 0.01). Of 162 unique Protocols/Determinants with at least 120 uses, 30 met criteria for safe to hold in queue, accounting for 8% (142,067) of incidents. Meanwhile, 72 Protocols/Determinants met criteria for unsafe to hold, accounting for 52% (883,683) of incidents. Seven of 32 ALPHA level Protocols and 3/17 OMEGA level Protocols met the proposed criteria for unsafe to hold in queue. CONCLUSIONS: In general, Determinant levels aligned with time-critical responses; however, a notable minority of lower acuity Determinant level Protocols met criteria for unsafe to hold. This suggests a more nuanced approach to dispatch prioritization, considering both Protocol and Determinant level factors.

A patient's perspective on the side effects of tyrosine kinase inhibitors in the treatment of advanced and metastatic gastrointestinal stromal tumors.

Aim: To provide the gastrointestinal stromal tumor patient's perspective on side effects of tyrosine kinase inhibitors and compare this with that of healthcare professionals. Materials & methods: Semi-structured interviews were conducted with 19 patients with an advanced or metastatic gastrointestinal stromal tumor, as well as six healthcare professionals, and five patients participated in a focus group. Thematic analysis was used to interpret the data. Results: Most participants (n = 29) reported gastrointestinal symptoms followed by tiredness (n = 25), edema (n = 22), muscle cramps (n = 21), skin problems (n = 21), eye problems (n = 11) and trouble sleeping (n = 10). Patients, but not healthcare professionals, reported cognitive problems or symptoms of depression. Conclusion: These results underline the importance of including the patient's perspective, as there is a gap in symptom reporting between patients and healthcare professionals.

In this study, the authors report on the side effects of targeted therapies used in the treatment of gastrointestinal stromal tumors from the patient's perspective and draw comparisons with reports from healthcare professionals. The authors conducted interviews with both patients and healthcare professionals. Most participants reported gastrointestinal symptoms followed by tiredness, fluid retention, muscle cramps, skin problems, eye problems and trouble sleeping. Gastrointestinal stromal tumor patients reported cognitive problems and symptoms of depression, which were not reported by healthcare professionals. In conclusion, the authors' results highlight the importance of including the patient's perspective, as there is a gap in symptom reporting between patients and healthcare professionals.

Erratum: Strong constraints on aerosol-cloud interactions from volcanic eruptions.

This corrects the article DOI: 10.1038/nature22974.

Strong constraints on aerosol-cloud interactions from volcanic eruptions.

Aerosols have a potentially large effect on climate, particularly through their interactions with clouds, but the magnitude of this effect is highly uncertain. Large volcanic eruptions produce sulfur dioxide, which in turn produces aerosols; these eruptions thus represent a natural experiment through which to quantify aerosol-cloud interactions. Here we show that the massive 2014-2015 fissure eruption in Holuhraun, Iceland, reduced the size of liquid cloud droplets-consistent with expectations-but had no discernible effect on other cloud properties. The reduction in droplet size led to cloud brightening and global-mean radiative forcing of around -0.2 watts per square metre for September to October 2014. Changes in cloud amount or cloud liquid water path, however, were undetectable, indicating that these indirect effects, and cloud systems in general, are well buffered against aerosol changes. This result will reduce uncertainties in future climate projections, because we are now able to reject results from climate models with an excessive liquid-water-path response.

Uncovering the burden of hidden ciliopathies in the 100 000 Genomes Project: a reverse phenotyping approach.

BACKGROUND: The 100 000 Genomes Project (100K) recruited National Health Service patients with eligible rare diseases and cancer between 2016 and 2018. PanelApp virtual gene panels were applied to whole genome sequencing data according to Human Phenotyping Ontology (HPO) terms entered by recruiting clinicians to guide focused analysis. METHODS: We developed a reverse phenotyping strategy to identify 100K participants with pathogenic variants in nine prioritised disease genes (BBS1, BBS10, ALMS1, OFD1, DYNC2H1, WDR34, NPHP1, TMEM67, CEP290), representative of the full phenotypic spectrum of multisystemic primary ciliopathies. We mapped genotype data 'backwards' onto available clinical data to assess potential matches against phenotypes. Participants with novel molecular diagnoses and key clinical features compatible with the identified disease gene were reported to recruiting clinicians. RESULTS: We identified 62 reportable molecular diagnoses with variants in these nine ciliopathy genes. Forty-four have been reported by 100K, 5 were previously unreported and 13 are new diagnoses. We identified 11 participants with unreportable, novel molecular diagnoses, who lacked key clinical features to justify reporting to recruiting clinicians. Two participants had likely pathogenic structural variants and one a deep intronic predicted splice variant. These variants would not be prioritised for review by standard 100K diagnostic pipelines. CONCLUSION: Reverse phenotyping improves the rate of successful molecular diagnosis for unsolved 100K participants with primary ciliopathies. Previous analyses likely missed these diagnoses because incomplete HPO term entry led to incorrect gene panel choice, meaning that pathogenic variants were not prioritised. Better phenotyping data are therefore essential for accurate variant interpretation and improved patient benefit.

Molecular diagnoses in the congenital malformations caused by ciliopathies cohort of the 100,000 Genomes Project.

BACKGROUND: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians. METHODS: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants. RESULTS: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene. CONCLUSION: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data.

Decision regret among couples experiencing infertility: a mixed methods longitudinal cohort study.

BACKGROUND: Decisions for how to resolve infertility are complex and may lead to regret. We examined whether couples and individuals who sought a consultation from a reproductive specialist for infertility later expressed decisional regret about their family-building choices and whether regret was associated with parental role, family-building paths, or outcomes. METHODS: This longitudinal mixed methods study included women and their partners who completed a questionnaire prior to their initial consultation with a reproductive specialist and 6 years later. The six-year questionnaire included the Ottawa Decision Regret Scale referencing "the decisions you made about how to add a child to your family." A score of 25+ indicates moderate-to-severe regret. Additional items invited reflections on family-building decisions, treatments, and costs. A systematic content analysis assessed qualitative themes. RESULTS: Forty-five couples and 34 individuals participated in the six-year questionnaire (76% retention rate), Half (n = 61) of participants expressed no regret, which was similar by role (median 0 for women and supporting partners, F = .08; p = .77). One in 5 women and 1 in 7 partners expressed moderate-to-severe regret. Women who did not pursue any treatment had significantly higher regret (median 15; F = 5.6, p < 0.01) compared to those who pursued IVF (median 0) or other treatments (median 0). Women who did not add a child to their family had significantly higher regret (median 35; F = 10.1, p < 0.001) than those who added a child through treatment (median 0), through fostering/adoption (median 0), or naturally (median 5). Among partners, regret scores were not associated with family-building paths or outcomes. More than one-quarter of participants wished they had spent less money trying to add a child to their family. Qualitative themes included gratitude for parenthood despite the burdensome process of family-building as well as dissatisfaction or regret about the process. Results should be confirmed in other settings to increase generalizability. CONCLUSION: This longitudinal study provides new insight into the burden of infertility. For women seeking parenthood, any of the multiple paths to parenthood may prevent future decision regret. Greater psychosocial, financial, and decision support is needed to help patients and their partners navigate family-building with minimal regret.

When people experience infertility, there are many decisions that can be challenging, such as whether to seek fertility treatments, to pursue fostering/adoption, and how to manage costs. With each decision, there is an opportunity for regret. The goal of this study was to look at whether people who were experiencing infertility and made an appointment with a doctor who specializes in infertility felt any regret about their decisions 6 years later. We also looked at whether different roles (that is, women seeking pregnancy or their supporting partners), different family-building paths (that is, medical treatments or not), or different outcomes (that is, adding a child to their family or not) were associated with different levels of regret. Results showed that half of the 120 people in the study did not have any regret 6 years after meeting with a specialty doctor. However, some patients did have regret, including 20% of women and 14% of partners who expressed moderate-to-severe regret. Women who did not add a child to their family in the six years during the study reported higher regret compared to women who did add a child to their family. There were no such differences among partners. About 25% of participants wished they had tried more, fewer, or different treatments. More than 25% wished they spent less money to try to add a child to their family. For people who want to add a child to their family, there are multiple ways to become a parent, any of which may be linked to lower decision regret. Decision regret is experienced differently between women seeking to add a child to their family and their partners. Would-be parents need more emotional, financial, and decision making support to help them navigate family-building with minimal regret.

WHEN REPRODUCTION IS NO LONGER AUTONOMOUS: FEELING RESPECTED BY MATERNITY CARE PROVIDERS MODERATES THE ASSOCIATION BETWEEN AUTONOMY IN DECISION MAKING AND BIRTH-RELATED PTSD SYMPTOMS IN A COMMUNITY SAMPLE OF POSTPARTUM BLACK WOMEN.

Black individuals are at particularly high risk for birth-related posttraumatic stress disorder (PTSD) symptoms, in part due to a lack of opportunity to lead maternity care decisions. Maternal care providers need evidence-based ways to reduce pregnant persons' risk for birth-related PTSD symptoms despite reduced autonomy in decision making resulting from heightened restrictions on reproductive rights. We investigated whether a potential relation between autonomy in decision making and birth-related PTSD symptoms would be moderated by being mistreated or feeling respected by maternity care providers in a community sample of Black women (N = 52; Mage = 28.2 years, SDage = 5.7 years) seeking maternity care at a public hospital in the southeastern United States. At six weeks postpartum, participants completed measures assessing autonomy in decision making, current birth-related PTSD symptoms, number of mistreatment events, and feelings of respect from providers during pregnancy, childbirth, and the postpartum period. Autonomy in decision making was negatively correlated with birth-related PTSD symptoms, r=-.43, p < .01. An interaction between autonomy in decision making and mistreatment by providers was trending toward significance, B=-.23, SE=.14, p = .10. Autonomy in decision making and feeling respected by maternity care provider interacted to predict birth-related PTSD symptoms, B = .05, SE=.01, p < .01. Feeling respected by providers may buffer against the negative effects of lack of autonomy in decision making on birth-related PTSD symptoms, highlighting the importance of providers' ability to convey respect to pregnant patients when they cannot lead care decisions.

Novel SIX6 mutations cause recessively inherited congenital cataract, microcornea, and corneal opacification with or without coloboma and microphthalmia.

Purpose: To investigate the molecular basis of recessively inherited congenital cataract, microcornea, and corneal opacification with or without coloboma and microphthalmia in two consanguineous families. Methods: Conventional autozygosity mapping was performed using single nucleotide polymorphism (SNP) microarrays. Whole-exome sequencing was completed on genomic DNA from one affected member of each family. Exome sequence data were also used for homozygosity mapping and copy number variation analysis. PCR and Sanger sequencing were used to confirm the identification of mutations and to screen further patients. Evolutionary conservation of protein sequences was assessed using CLUSTALW, and protein structures were modeled using PyMol. Results: In family MEP68, a novel homozygous nucleotide substitution in SIX6 was found, c.547G>C, that converts the evolutionarily conserved aspartic acid residue at the 183rd amino acid in the protein to a histidine, p.(Asp183His). This residue mapped to the third helix of the DNA-binding homeobox domain in SIX6, which interacts with the major groove of double-stranded DNA. This interaction is likely to be disrupted by the mutation. In family F1332, a novel homozygous 1034 bp deletion that encompasses the first exon of SIX6 was identified, chr14:g.60975890_60976923del. Both mutations segregated with the disease phenotype as expected for a recessive condition and were absent from publicly available variant databases. Conclusions: Our findings expand the mutation spectrum in this form of inherited eye disease and confirm that homozygous human SIX6 mutations cause a developmental spectrum of ocular phenotypes that includes not only the previously described features of microphthalmia, coloboma, and congenital cataract but also corneal abnormalities.

Health-related quality of life scores of metastatic pancreatic cancer patients responsive to first line chemotherapy compared to newly derived EORTC QLQ-C30 reference values.

BACKGROUND: Metastatic pancreatic cancer (mPC) and its treatments significantly impact health-related quality of life (HRQoL). POLO, a randomized, double-blind, placebo-controlled phase 3 trial evaluated the efficacy of olaparib as maintenance therapy in germline BRCA mutated mPC patients who had not progressed during ≥16 weeks of first-line platinum-based chemotherapy. HRQoL was assessed using the EORTC QLQ-C30. To enhance score interpretation, we derived reference values for treatment-naïve mPC patients from the literature. METHODS: A targeted literature review identified EORTC QLQ-C30 baseline values in treatment-naïve mPC patients. Reference values were calculated by deriving means from studies meeting inclusion criteria, with scores from 0 to 100 (higher scores indicate better QoL/functioning but worse symptoms). For POLO patients, means were calculated using pooled baseline data across study arms. RESULTS: Four studies met inclusion criteria. Depending on the specific scale, sample sizes ranged from n = 466 to n = 639. Compared to newly derived reference values, POLO patients reported markedly better HRQoL scores at baseline across most scales, with eight scales showing differences of ≥10 points. POLO patients' HRQoL scores were often close to or better than general population norm data. CONCLUSIONS: This is the first study to systematically derive EORTC QLQ-C30 reference values for mPC. POLO patients had better HRQoL scores than those in the literature and similar to general population data. Comparatively high HRQoL of POLO patients are likely due to effects of prior first-line treatment and resolution of chemotherapy-related symptoms, response shift, or a combination. Newly derived reference values can enhance interpretation of mPC patients' HRQoL. TRIAL REGISTRATION: The POLO trial was registered on 9 July 2014 with ClinicalTrials.gov as NCT02184195.

Novel loss-of-function mutation in HERC2 is associated with severe developmental delay and paediatric lethality.

BACKGROUND: The HERC2 gene encodes a 527 kDa E3 ubiquitin protein ligase that has key roles in cell cycle regulation, spindle formation during mitosis, mitochondrial functions and DNA damage responses. It has essential roles during embryonic development, particularly for neuronal and muscular functions. To date, missense mutations in HERC2 have been associated with an autosomal recessive neurodevelopmental disorder with some phenotypical similarities to Angelman syndrome, and a homozygous deletion spanning HERC2 and OCA2 causing a more severe neurodevelopmental phenotype. METHODS AND RESULTS: We ascertained a consanguineous family with a presumed autosomal recessive severe neurodevelopmental disorder that leads to paediatric lethality. In affected individuals, we identified a homozygous HERC2 frameshift variant that results in a premature stop codon and complete loss of HERC2 protein. Functional characterisation of this variant in fibroblasts, from one living affected individual, revealed impaired mitochondrial network and function as well as disrupted levels of known interacting proteins such as XPA. CONCLUSION: This study extends the genotype-phenotype correlation for HERC2 variants to include a distinct lethal neurodevelopmental disorder, highlighting the importance of further characterisation for HERC2-related disorders.

Protective immunity in recurrent Staphylococcus aureus infection reflects localized immune signatures and macrophage-conferred memory.

Staphylococcus aureus is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Our prior studies discovered a role for protective innate memory against recurrent methicillin-resistant S. aureus (MRSA) SSSI. In the present study, the dynamics and mechanisms of this response were explored in recurrent SSSI in WT mice. Priming by prior infection reduced skin lesion severity and MRSA burden, and protected against dissemination at day 7 but not day 2. Cytokine and cellular signatures in SSSI differed at day 2 versus 7, and were distinct in skin versus blood or spleen. Cytokines associated with protection in skin included increased IL-17, IL-6, monokine inducible by IFN-γ (MIG), and RANTES, while increased IP-10 correlated with protection from dissemination. Cellular signatures of protection included increased Th17, M1 macrophage, and dendritic cell populations in abscesses, and total macrophages in lymph nodes. Priming potentiated S. aureus-specific phagocytic killing by bone marrow-derived macrophages in vitro, and their adoptive transfer into naïve skin afforded protective efficacy in vivo. Present findings indicate that protective immunity in recurrent S. aureus infection is locally targeted, and involves specific memory conferred by macrophages. These insights provide targets for vaccine and immunotherapeutic development against MRSA.

Psychometric properties of the updated EORTC module for assessing quality of life in patients with lung cancer (QLQ-LC29): an international, observational field study.

BACKGROUND: The European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-Lung Cancer 13 (QLQ-LC13) assesses quality of life (QOL) in patients with lung cancer and was the first EORTC module developed for use in international clinical trials. Since its publication in 1994, major treatment advances with possible effects on QOL have occurred. These changes called for an update of the module and its international psychometric validation. We aimed to investigate the scale structure and psychometric properties of the updated lung cancer module, QLQ-LC29, in patients with lung cancer. METHODS: This international, observational field study was done in 19 hospitals across 12 countries. Patients aged older than 18 years with a confirmed diagnosis of lung cancer and no other previous primary tumour, and who were mentally fit with sufficient language skills to understand and complete the questionnaire were included. Patients were asked during a hospital visit to fill in the paper versions of the core questionnaire EORTC QLQ-C30 plus QLQ-LC29, and investigators selected half of these patients to complete the questionnaire again 2-4 weeks later. Our primary aim was to assess the scale structure and psychometric properties of EORTC QLQ-LC29. We analysed scale structure using confirmatory factor analysis; reliability using Cronbach's α value (internal consistency) and intra-class coefficient (test-retest reliability); sensitivity using independent t tests stratified by Karnofsky performance status; and responsiveness to change over time by ANOVA. This study is registered with ClinicalTrials.gov, NCT02745691. FINDINGS: Between April 12, 2016, and Sept 26, 2018, 523 patients with a confirmed diagnosis of either non-small-cell lung cancer (n=442) or small-cell lung cancer (n=81) were recruited. Confirmatory factor analysis provided a solution composed of five multi-item scales (coughing, shortness of breath, fear of progression, hair problems, and surgery-related symptoms) plus 15 single symptom or side-effect items: χ2=370·233, root mean square error of approximation=0·075, and comparative-fit index=0·901. Cronbach's α for internal consistencies of all multi-item scales were above the threshold of 0·70. Intra-class coefficients for test-retest reliabilities ranged between 0·82 and 0·97. Three (shortness of breath, fear of progression, and hair problems) of the five multi-item scales showed responsiveness to change over time (p values <0·05), as did nine of 15 single symptom items. Four (coughing, shortness of breath, fear of progression, and surgery-related symptoms) of the five multi-item scales and ten of the 15 single symptom items were sensitive to known group differences (ie, lower vs higher Karnofsky performance status). INTERPRETATION: Results determined the psychometric properties of the updated lung cancer module, which is ready for use in international clinical studies. FUNDING: EORTC Quality of Life Group.

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.

Mutations in GPAA1, Encoding a GPI Transamidase Complex Protein, Cause Developmental Delay, Epilepsy, Cerebellar Atrophy, and Osteopenia.

Approximately one in every 200 mammalian proteins is anchored to the cell membrane through a glycosylphosphatidylinositol (GPI) anchor. These proteins play important roles notably in neurological development and function. To date, more than 20 genes have been implicated in the biogenesis of GPI-anchored proteins. GPAA1 (glycosylphosphatidylinositol anchor attachment 1) is an essential component of the transamidase complex along with PIGK, PIGS, PIGT, and PIGU (phosphatidylinositol-glycan biosynthesis classes K, S, T, and U, respectively). This complex orchestrates the attachment of the GPI anchor to the C terminus of precursor proteins in the endoplasmic reticulum. Here, we report bi-allelic mutations in GPAA1 in ten individuals from five families. Using whole-exome sequencing, we identified two frameshift mutations (c.981_993del [p.Gln327Hisfs∗102] and c.920delG [p.Gly307Alafs∗11]), one intronic splicing mutation (c.1164+5C>T), and six missense mutations (c.152C>T [p.Ser51Leu], c.160_161delinsAA [p.Ala54Asn], c.527G>C [p.Trp176Ser], c.869T>C [p.Leu290Pro], c.872T>C [p.Leu291Pro], and c.1165G>C [p.Ala389Pro]). Most individuals presented with global developmental delay, hypotonia, early-onset seizures, cerebellar atrophy, and osteopenia. The splicing mutation was found to decrease GPAA1 mRNA. Moreover, flow-cytometry analysis of five available individual samples showed that several GPI-anchored proteins had decreased cell-surface abundance in leukocytes (FLAER, CD16, and CD59) or fibroblasts (CD73 and CD109). Transduction of fibroblasts with a lentivirus encoding the wild-type protein partially rescued the deficiency of GPI-anchored proteins. These findings highlight the role of the transamidase complex in the development and function of the cerebellum and the skeletal system.