Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport.

Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.

Roles for CEP170 in cilia function and dynein-2 assembly.

Primary cilia are essential eukaryotic organelles required for signalling and secretion. Dynein-2 is a microtubule-motor protein complex and is required for ciliogenesis via its role in facilitating retrograde intraflagellar transport (IFT) from the cilia tip to the cell body. Dynein-2 must be assembled and loaded onto IFT trains for entry into cilia for this process to occur, but how dynein-2 is assembled and how it is recycled back into a cilium remain poorly understood. Here, we identify centrosomal protein of 170 kDa (CEP170) as a dynein-2-interacting protein in mammalian cells. We show that loss of CEP170 perturbs intraflagellar transport and hedgehog signalling, and alters the stability of dynein-2 holoenzyme complex. Together, our data indicate a role for CEP170 in supporting cilia function and dynein-2 assembly.

Molecular signatures of antibody responses derived from a systems biology study of five human vaccines.

Many vaccines induce protective immunity via antibodies. Systems biology approaches have been used to determine signatures that can be used to predict vaccine-induced immunity in humans, but whether there is a 'universal signature' that can be used to predict antibody responses to any vaccine is unknown. Here we did systems analyses of immune responses to the polysaccharide and conjugate vaccines against meningococcus in healthy adults, in the broader context of published studies of vaccines against yellow fever virus and influenza virus. To achieve this, we did a large-scale network integration of publicly available human blood transcriptomes and systems-scale databases in specific biological contexts and deduced a set of transcription modules in blood. Those modules revealed distinct transcriptional signatures of antibody responses to different classes of vaccines, which provided key insights into primary viral, protein recall and anti-polysaccharide responses. Our results elucidate the early transcriptional programs that orchestrate vaccine immunity in humans and demonstrate the power of integrative network modeling.

Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2.

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. The motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport, respectively, in the cilium. WDR34 (also known as DYNC2I2), a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome and asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases, making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34-knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity and Hedgehog signalling were also affected.

Multiple interactions of the dynein-2 complex with the IFT-B complex are required for effective intraflagellar transport.

The dynein-2 complex must be transported anterogradely within cilia to then drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Here, we screened for potential interactions between the dynein-2 and IFT-B complexes and found multiple interactions among the dynein-2 and IFT-B subunits. In particular, WDR60 (also known as DYNC2I1) and the DYNC2H1-DYNC2LI1 dimer from dynein-2, and IFT54 (also known as TRAF3IP1) and IFT57 from IFT-B contribute to the dynein-2-IFT-B interactions. WDR60 interacts with IFT54 via a conserved region N-terminal to its light chain-binding regions. Expression of the WDR60 constructs in WDR60-knockout (KO) cells revealed that N-terminal truncation mutants lacking the IFT54-binding site fail to rescue abnormal phenotypes of WDR60-KO cells, such as aberrant accumulation of the IFT machinery around the ciliary tip and on the distal side of the transition zone. However, a WDR60 construct specifically lacking just the IFT54-binding site substantially restored the ciliary defects. In line with the current docking model of dynein-2 with the anterograde IFT trains, these results indicate that extensive interactions involving multiple subunits from the dynein-2 and IFT-B complexes participate in their connection.

Homologous and Heterologous Covid-19 Booster Vaccinations.

BACKGROUND: Although the three vaccines against coronavirus disease 2019 (Covid-19) that have received emergency use authorization in the United States are highly effective, breakthrough infections are occurring. Data are needed on the serial use of homologous boosters (same as the primary vaccine) and heterologous boosters (different from the primary vaccine) in fully vaccinated recipients. METHODS: In this phase 1-2, open-label clinical trial conducted at 10 sites in the United States, adults who had completed a Covid-19 vaccine regimen at least 12 weeks earlier and had no reported history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection received a booster injection with one of three vaccines: mRNA-1273 (Moderna) at a dose of 100 µg, Ad26.COV2.S (Johnson & Johnson-Janssen) at a dose of 5×1010 virus particles, or BNT162b2 (Pfizer-BioNTech) at a dose of 30 µg. The primary end points were safety, reactogenicity, and humoral immunogenicity on trial days 15 and 29. RESULTS: Of the 458 participants who were enrolled in the trial, 154 received mRNA-1273, 150 received Ad26.COV2.S, and 153 received BNT162b2 as booster vaccines; 1 participant did not receive the assigned vaccine. Reactogenicity was similar to that reported for the primary series. More than half the recipients reported having injection-site pain, malaise, headache, or myalgia. For all combinations, antibody neutralizing titers against a SARS-CoV-2 D614G pseudovirus increased by a factor of 4 to 73, and binding titers increased by a factor of 5 to 55. Homologous boosters increased neutralizing antibody titers by a factor of 4 to 20, whereas heterologous boosters increased titers by a factor of 6 to 73. Spike-specific T-cell responses increased in all but the homologous Ad26.COV2.S-boosted subgroup. CD8+ T-cell levels were more durable in the Ad26.COV2.S-primed recipients, and heterologous boosting with the Ad26.COV2.S vaccine substantially increased spike-specific CD8+ T cells in the mRNA vaccine recipients. CONCLUSIONS: Homologous and heterologous booster vaccines had an acceptable safety profile and were immunogenic in adults who had completed a primary Covid-19 vaccine regimen at least 12 weeks earlier. (Funded by the National Institute of Allergy and Infectious Diseases; DMID 21-0012 ClinicalTrials.gov number, NCT04889209.).

Supply chain logistics - the role of the Golgi complex in extracellular matrix production and maintenance.

The biomechanical and biochemical properties of connective tissues are determined by the composition and quality of their extracellular matrix. This, in turn, is highly dependent on the function and organisation of the secretory pathway. The Golgi complex plays a vital role in directing matrix output by co-ordinating the post-translational modification and proteolytic processing of matrix components prior to their secretion. These modifications have broad impacts on the secretion and subsequent assembly of matrix components, as well as their function in the extracellular environment. In this Review, we highlight the role of the Golgi in the formation of an adaptable, healthy matrix, with a focus on proteoglycan and procollagen secretion as example cargoes. We then discuss the impact of Golgi dysfunction on connective tissue in the context of human disease and ageing.

Semiparametric Bayesian inference for optimal dynamic treatment regimes via dynamic marginal structural models.

Considerable statistical work done on dynamic treatment regimes (DTRs) is in the frequentist paradigm, but Bayesian methods may have much to offer in this setting as they allow for the appropriate representation and propagation of uncertainty, including at the individual level. In this work, we extend the use of recently developed Bayesian methods for Marginal Structural Models to arrive at inference of DTRs. We do this (i) by linking the observational world with a world in which all patients are randomized to a DTR, thereby allowing for causal inference and then (ii) by maximizing a posterior predictive utility, where the posterior distribution has been obtained from nonparametric prior assumptions on the observational world data-generating process. Our approach relies on Bayesian semiparametric inference, where inference about a finite-dimensional parameter is made all while working within an infinite-dimensional space of distributions. We further study Bayesian inference of DTRs in the double robust setting by using posterior predictive inference and the nonparametric Bayesian bootstrap. The proposed methods allow for uncertainty quantification at the individual level, thereby enabling personalized decision-making. We examine the performance of these methods via simulation and demonstrate their utility by exploring whether to adapt HIV therapy to a measure of patients' liver health, in order to minimize liver scarring.

A Bayesian non-stationary heteroskedastic time series model for multivariate critical care data.

We propose a multivariate GARCH model for non-stationary health time series by modifying the observation-level variance of the standard state space model. The proposed model provides an intuitive and novel way of dealing with heteroskedastic data using the conditional nature of state-space models. We follow the Bayesian paradigm to perform the inference procedure. In particular, we use Markov chain Monte Carlo methods to obtain samples from the resultant posterior distribution. We use the forward filtering backward sampling algorithm to efficiently obtain samples from the posterior distribution of the latent state. The proposed model also handles missing data in a fully Bayesian fashion. We validate our model on synthetic data and analyze a data set obtained from an intensive care unit in a Montreal hospital and the MIMIC dataset. We further show that our proposed models offer better performance, in terms of WAIC than standard state space models. The proposed model provides a new way to model multivariate heteroskedastic non-stationary time series data. Model comparison can then be easily performed using the WAIC.

Acquisition of Gonococcal AniA-NorB Pathway by the Neisseria meningitidis Urethritis Clade Confers Denitrifying and Microaerobic Respiration Advantages for Urogenital Adaptation.

Neisseria meningitidis historically has been an infrequent and sporadic cause of urethritis and other urogenital infections. However, a nonencapsulated meningococcal clade belonging to the hyperinvasive clonal complex 11.2 lineage has recently emerged and caused clusters of urethritis cases in the United States and other countries. One of the genetic signatures of the emerging N. meningitidis urethritis clade (NmUC) is a chromosomal gene conversion event resulting in the acquisition of the Neisseria gonorrhoeae denitrification apparatus-the N. gonorrhoeae alleles encoding the nitrite reductase AniA, the nitric oxide (NO) reductase NorB, and the intergenic promoter region. The biological importance of the N. gonorrhoeae AniA-NorB for adaptation of the NmUC to a new environmental niche is investigated herein. We found that oxygen consumption, nitrite utilization, and NO production were significantly altered by the conversion event, resulting in different denitrifying aerobic and microaerobic growth of the clade. Further, transcription of aniA and norB in NmUC isolates differed from canonical N. meningitidis, and important polymorphisms within the intergenic region, which influenced aniA promoter activity of the NmUC, were identified. The contributions of three known meningococcal regulators (NsrR, FNR, and NarQP) in controlling the denitrification pathway and endogenous NO metabolism were distinct. Overall, transcription of aniA was dampened relative to canonical N. meningitidis, and this correlated with the lower NO accumulation in the clade. Denitrification and microaerobic respiration were bolstered, and protection against host-derived NO was likely enhanced. The acquisition of the N. gonorrhoeae denitrification pathway by the NmUC supports the clade's adaptation and survival in a microaerobic urogenital environment.

Inducible Mega-Mediated Macrolide Resistance Confers Heteroresistance in Streptococcus pneumoniae.

In Streptococcus pneumoniae (Spn), the 5.4 to 5.5 kb Macrolide Genetic Assembly (Mega) encodes an efflux pump (Mef[E]) and a ribosomal protection protein (Mel) conferring antibiotic resistance to commonly used macrolides in clinical isolates. We found the macrolide-inducible Mega operon provides heteroresistance (more than 8-fold range in MICs) to 14- and 15-membered ring macrolides. Heteroresistance is commonly missed during traditional clinical resistance screens but is highly concerning as resistant subpopulations can persist despite treatment. Spn strains containing the Mega element were screened via Etesting and population analysis profiling (PAP). All Mega-containing Spn strains screened displayed heteroresistance by PAP. The heteroresistance phenotype was linked to the mRNA expression of the mef(E)/mel operon of the Mega element. Macrolide induction uniformly increased Mega operon mRNA expression across the population, and heteroresistance was eliminated. A deletion of the 5' regulatory region of the Mega operon results in a mutant deficient in induction as well as in heteroresistance. The mef(E)L leader peptide sequence of the 5' regulatory region was required for induction and heteroresistance. Treatment with a noninducing 16-membered ring macrolide antibiotic did not induce the mef(E)/mel operon or eliminate the heteroresistance phenotype. Thus, inducibility of the Mega element by 14- and 15-membered macrolides and heteroresistance are linked in Spn. The stochastic variation in mef(E)/mel expression in a Spn population containing Mega provides the basis for heteroresistance.

A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function.

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER-Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.

Bayesian sample size calculations for comparing two strategies in SMART studies.

In the management of most chronic conditions characterized by the lack of universally effective treatments, adaptive treatment strategies (ATSs) have grown in popularity as they offer a more individualized approach. As a result, sequential multiple assignment randomized trials (SMARTs) have gained attention as the most suitable clinical trial design to formalize the study of these strategies. While the number of SMARTs has increased in recent years, sample size and design considerations have generally been carried out in frequentist settings. However, standard frequentist formulae require assumptions on interim response rates and variance components. Misspecifying these can lead to incorrect sample size calculations and correspondingly inadequate levels of power. The Bayesian framework offers a straightforward path to alleviate some of these concerns. In this paper, we provide calculations in a Bayesian setting to allow more realistic and robust estimates that account for uncertainty in inputs through the 'two priors' approach. Additionally, compared to the standard frequentist formulae, this methodology allows us to rely on fewer assumptions, integrate pre-trial knowledge, and switch the focus from the standardized effect size to the MDD. The proposed methodology is evaluated in a thorough simulation study and is implemented to estimate the sample size for a full-scale SMART of an internet-based adaptive stress management intervention on cardiovascular disease patients using data from its pilot study conducted in two Canadian provinces.

A time-dependent Poisson-Gamma model for recruitment forecasting in multicenter studies.

Forecasting recruitments is a key component of the monitoring phase of multicenter studies. One of the most popular techniques in this field is the Poisson-Gamma recruitment model, a Bayesian technique built on a doubly stochastic Poisson process. This approach is based on the modeling of enrollments as a Poisson process where the recruitment rates are assumed to be constant over time and to follow a common Gamma prior distribution. However, the constant-rate assumption is a restrictive limitation that is rarely appropriate for applications in real studies. In this paper, we illustrate a flexible generalization of this methodology which allows the enrollment rates to vary over time by modeling them through B-splines. We show the suitability of this approach for a wide range of recruitment behaviors in a simulation study and by estimating the recruitment progression of the Canadian Co-infection Cohort.

The evolution of SARS-CoV-2 seroprevalence in Canada: a time-series study, 2020-2023.

BACKGROUND: During the first year of the COVID-19 pandemic, the proportion of reported cases of COVID-19 among Canadians was under 6%. Although high vaccine coverage was achieved in Canada by fall 2021, the Omicron variant caused unprecedented numbers of infections, overwhelming testing capacity and making it difficult to quantify the trajectory of population immunity. METHODS: Using a time-series approach and data from more than 900 000 samples collected by 7 research studies collaborating with the COVID-19 Immunity Task Force (CITF), we estimated trends in SARS-CoV-2 seroprevalence owing to infection and vaccination for the Canadian population over 3 intervals: prevaccination (March to November 2020), vaccine roll-out (December 2020 to November 2021), and the arrival of the Omicron variant (December 2021 to March 2023). We also estimated seroprevalence by geographical region and age. RESULTS: By November 2021, 9.0% (95% credible interval [CrI] 7.3%-11%) of people in Canada had humoral immunity to SARS-CoV-2 from an infection. Seroprevalence increased rapidly after the arrival of the Omicron variant - by Mar. 15, 2023, 76% (95% CrI 74%-79%) of the population had detectable antibodies from infections. The rapid rise in infection-induced antibodies occurred across Canada and was most pronounced in younger age groups and in the Western provinces: Manitoba, Saskatchewan, Alberta and British Columbia. INTERPRETATION: Data up to March 2023 indicate that most people in Canada had acquired antibodies against SARS-CoV-2 through natural infection and vaccination. However, given variations in population seropositivity by age and geography, the potential for waning antibody levels, and new variants that may escape immunity, public health policy and clinical decisions should be tailored to local patterns of population immunity.

Survival Modelling For Data From Combined Cohorts: Opening the Door to Meta Survival Analyses and Survival Analysis using Electronic Health Records.

Non-parametric estimation of the survival function using observed failure time data depends on the underlying data generating mechanism, including the ways in which the data may be censored and/or truncated. For data arising from a single source or collected from a single cohort, a wide range of estimators have been proposed and compared in the literature. Often, however, it may be possible, and indeed advantageous, to combine and then analyze survival data that have been collected under different study designs. We review non-parametric survival analysis for data obtained by combining the most common types of cohort. We have two main goals: (i) To clarify the differences in the model assumptions, and (ii) to provide a single lens through which some of the proposed estimators may be viewed. Our discussion is relevant to the meta analysis of survival data obtained from different types of study, and to the modern era of electronic health records.

Catalytic Ammonia Oxidation to Dinitrogen by a Nickel Complex.

We report a nickel complex for catalytic oxidation of ammonia to dinitrogen under ambient conditions. Using the aryloxyl radical 2,4,6-tri-tert-butylphenoxyl (t Bu3 ArO⋅) as a H atom acceptor to cleave the N-H bond of a coordinated NH3 ligand up to 56 equiv of N2 per Ni center can be generated. Employing the N-oxyl radical 2,2,6,6-(tetramethylpiperidin-1-yl)oxyl (TEMPO⋅) as the H-atom acceptor, up to 15 equiv of N2 per Ni center are formed. A bridging Ni-hydrazine product identified by isotopic nitrogen (15 N) studies and supported by computational models indicates the N-N bond forming step occurs by bimetallic homocoupling of two paramagnetic [Ni]-NH2 fragments. Ni-mediated hydrazine disproportionation to N2 and NH3 completes the catalytic cycle.

Infection With the US Neisseria meningitidis Urethritis Clade Does Not Lower Future Risk of Urethral Gonorrhea.

BACKGROUND: Cross-protective immunity between Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng) may inform gonococcal vaccine development. Meningococcal serogroup B (MenB) outer membrane vesicle (OMV) vaccines confer modest protection against gonorrhea. However, whether urethral Nm infection protects against gonorrhea is unknown. We examined gonorrhea risk among men with US Nm urethritis clade (US_NmUC) infections. METHODS: We conducted a retrospective cohort study of men with urethral US_NmUC (n = 128) between January 2015 and April 2018. Using diagnosis date as the baseline visit, we examined Ng status at return visits to compute urethral Ng risk. We compared these data to 3 referent populations: men with urethral Ng (n = 253), urethral chlamydia (Ct) (n = 251), and no urethral Ng or Ct (n = 255). We conducted sensitivity analyses to assess varied approaches to censoring, missing data, and anatomical site of infection. We also compared sequences of protein antigens in the OMV-based MenB-4C vaccine, US_NmUC, and Ng. RESULTS: Participants were primarily Black (65%) and heterosexual (82%). Over follow-up, 91 men acquired urethral Ng. Men with urethral US_NmUC had similar Ng risk to men with prior urethral Ng (adjusted hazard ratio [aHR]: 1.27; 95% CI: .65-2.48). Men with urethral US_NmUC had nonsignificantly increased Ng risk compared with men with urethral Ct (aHR: 1.51; 95% CI: .79-2.88), and significantly increased Ng risk compared with men without urethral Ng or Ct (aHR: 3.55; 95% CI: 1.27-9.91). Most of the protein antigens analyzed shared high sequence similarity. CONCLUSIONS: Urethral US_NmUC infection did not protect against gonorrhea despite substantial sequence similarities in shared protein antigens.

Cytoplasmic dynein-2 at a glance.

Cytoplasmic dynein-2 is a motor protein complex that drives the movement of cargoes along microtubules within cilia, facilitating the assembly of these organelles on the surface of nearly all mammalian cells. Dynein-2 is crucial for ciliary function, as evidenced by deleterious mutations in patients with skeletal abnormalities. Long-standing questions include how the dynein-2 complex is assembled, regulated, and switched between active and inactive states. A combination of model organisms, in vitro cell biology, live-cell imaging, structural biology and biochemistry has advanced our understanding of the dynein-2 motor. In this Cell Science at a Glance article and the accompanying poster, we discuss the current understanding of dynein-2 and its roles in ciliary assembly and function.

Impact of antibiotic allergy labels on patient outcomes in a tertiary paediatric hospital.

AIMS: Antibiotic allergies are reported in 5-15% of children. This study aimed to evaluate the impact of common ß-lactam antibiotic allergy labels (AALs) on hospital treatment, focusing on length of stay and appropriateness of antibiotic prescribing. METHODS: This was a retrospective cohort study over 21 months at the Royal Children's Hospital Melbourne, Australia. A subset of children with the most common ß-lactam allergies, and who required admission for intravenous antibiotics over a 12-month period, was analysed for appropriateness of prescribing. Non-allergic patients were matched to evaluate associations between AALs and hospital treatment. RESULTS: There were 98 912 children admitted over the study period, of whom 938 (1%) had at least one AAL on first admission. Of all encounters, 5145 (2.5%) were for children with AALs. The most common AALs were to amoxicillin and amoxicillin-clavulanic acid combinations (40.8%), cefalexin (14.4%) and trimethoprim-sulfamethoxazole (9.7%). For the subset, there were 66 admissions for children who required intravenous antibiotics. Documentation was adequate for 27% of AALs. Inappropriate prescribing occurred in almost half (47%). Hospital stay was longer for children with AALs (median 4.7 days; IQR 2.3-9.2) compared to non-allergic controls (median 3.9 days; IQR 1.9-6.8; P = .02). Children with AALs were more likely to receive restricted antibiotics (aOR 3.03; 95% CI, 1.45-6.30; P = .003). CONCLUSION: This is the first study to demonstrate high rates of inappropriate prescribing in children with AALs. Children with AALs were significantly more likely to receive restricted antibiotics and had a longer length of stay compared with non-allergic controls.