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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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

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., (© 2024. The Author(s).)

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., Competing Interests: Competing interests D.J.S. is an Editor for Journal of Cell Science and played no role in the editorial handling of this paper. The authors declare no other competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interests D.J.S. is an Editor for Journal of Cell Science and played no role in the editorial handling of this paper. The authors declare no other competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Purpose: American Indians/Alaska Native (AI/AN) persons are disproportionately affected by hepatitis C virus (HCV). The Northwest Portland Area Indian Health Board Indian Country Extension for Community Healthcare Outcomes (ECHO) telehealth clinic supports primary care providers (PCPs) in treating HCV. We evaluated the extent to which Indian Country ECHO increases access to HCV treatment and holistically serves AI/AN patients., Methods: We conducted a retrospective descriptive analysis of Indian Country ECHO treatment recommendations from 2017 to 2021. Recommendations were classified into the following categories: HCV treatment with direct-acting antiviral medication, prevention, substance use disorder treatment, lab or imaging orders, pharmacological considerations, behavior changes, other, and referral. Subanalysis of treatment recommendations was completed for patients with cirrhosis., Findings: Of the 776 patients from 77 Indian Health System facilities who presented at Indian Country ECHO, 718 (93%) received treatment recommendations. Most patients (93%) received recommendations for HCV treatment by their PCP; only 3% received a recommendation for referral to a hepatologist or liver transplant center for additional care. Most patients received at least 1 recommendation beyond the scope of HCV treatment provision. Cirrhosis criteria were met by 8% of patients, of which 80% received recommendations for HCV treatment by their PCP and 25% received recommendations for referral to a specialist for additional care., Conclusions: Most patients presented at the Indian Country ECHO received recommendations for HCV treatment by their PCP, along with recommendations beyond the scope of HCV. Indian Country ECHO telehealth clinic provides comprehensive recommendations to effectively integrate evidence-based HCV treatment with holistic care at the primary care level., (© 2022 National Rural Health Association.)

Left ventricular assist devices (LVADs) have revolutionized the management of patients with advanced heart failure refractory to medical therapy. Current indications of LVADs include Bridge to Transplantation (BTT), Destination Therapy (DT) for long-term use, Bridge to the Decision (BTD) used as a temporary measure, and lastly Bridge to Recovery (BTR). Here, we briefly review the clinical evidence and the molecular mechanisms behind myocardial recovery following LVAD placement. We also share institutional protocols used at 2 major medical centers in the USA., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Introduction: We used interrupted time series (ITS) analysis to determine whether e-scooter shares' introduction in September 2017 increased serious scooter-related injury across the United States., Methods: Using the National Electronic Injury Surveillance System, we queried emergency department visits involving motorized scooter-related injuries from January 2010-December 2019. Cases originating where e-scooter shares launched between September 1, 2017-December 1, 2019 (intervention period) were considered exposed. The first month of launch (September 2017) was chosen as the time point for pre- and post-intervention analysis. The primary outcome was change in hospitalizations following scooter injury in association with the month/year launch., Results: This analysis includes 2754 unweighted encounters, representing 102614 estimated injuries involving motorized scooters nationwide. Hospitals within 20 miles of e-scooter shares also experienced a significant monthly increase of 0.24 scooter-related injury hospitalizations/1000 product-related injury hospitalizations ([0.17,0.31]) compared to a non-significant change in hospitalizations of 0.02 [-0.05,0.09] for control hospitals., Conclusion: An increase in serious motorized scooter injuries coincides with e-scooter shares' introduction in the US. Future works should explore effective polices to improve public safety., (Copyright © 2021. Published by Elsevier Inc.)

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., Competing Interests: Competing interests D.J.S. is an Editor for Journal of Cell Science; all other authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing., (© 2021 Stevenson et al.)

In a warned reaction time task, the warning stimulus (S1) initiates a process of temporal preparation, which promotes a speeded response to the impending target stimulus (S2). According to the multiple trace theory of temporal preparation (MTP), participants learn the timing of S2 by storing a memory trace on each trial, which contains a temporal profile of the events on that trial. On each new trial, S1 serves as a retrieval cue that implicitly and associatively activates memory traces created on earlier trials, which jointly drive temporal preparation for S2. The idea that S1 assumes this role as a retrieval cue was tested across eight experiments, in which two different S1s were associated with two different distributions of S1-S2 intervals: one with predominantly short and one with predominantly long intervals. Experiments differed regarding the S1 features that made up a pair, ranging from highly distinct (e.g., tone and flash) to more similar (e.g., red and green flash) and verbal (i.e., "short" vs "long"). Exclusively for pairs of highly distinct S1s, the results showed that the S1 cue modified temporal preparation, even in participants who showed no awareness of the contingency. This cueing effect persisted in a subsequent transfer phase, in which the contingency between S1 and the timing of S2 was broken - a fact participants were informed of in advance. Together, these findings support the role of S1 as an implicit retrieval cue, consistent with MTP., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aim: To evaluate the frequency and predictors of poor outcome in febrile children presenting to the Emergency Department., Methods: Retrospective observational study from the Emergency Department of The Royal Children's Hospital, Melbourne, Australia. All children with presenting complaint of fever or triage temperature >38°C over a 6-month period were included. Poor outcome was defined as: new organ dysfunction or the requirement for organ support therapy (inotrope infusion, mechanical ventilation, renal replacement therapy and extra-corporeal life support). Predictors evaluated were as follows: initial vital signs, blood tests and clinical scores. Odds ratio, sensitivity, specificity and area under the receiver-operating characteristics curve were calculated for each predictor variable., Results: Between Jan-June 2019, 6217 children met inclusion criteria. Twenty-seven (0.4%) developed new organ dysfunction, 10 (0.2%) required organ support therapy (inotrope infusion in 5, mechanical ventilation in 6, renal replacement therapy in 1, extra-corporeal life support in 1). Odds of new organ dysfunction, requirement for inotropic support and mechanical ventilation were higher with abnormal initial vital signs, blood tests and clinical scores, though overall test characteristics were poor due to infrequency., Conclusion: Poor outcomes were uncommon among febrile children presenting to the Emergency Department. Vital signs, blood tests and clinical scores were poor predictors., (©2020 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Objective: Fluid bolus therapy (FBT) is recommended as the initial form of acute circulatory support for many disease processes. The epidemiology of its use in the ED is unknown. The aim of this study was to assess indications, use patterns and short-term effects of FBT in a paediatric ED., Methods: Retrospective observational study in the ED of the Royal Children's Hospital, Melbourne, Australia (annual census >90 000) using hospital electronic medical record data for all patients who received an i.v. fluid bolus in the ED over the calendar year 2018 were included in the study. We extracted demographics, indications, volume, content of FBT as well as subsequent vital sign and biochemical changes., Results: One thousand five hundred and thirty-nine fluid boluses were administered to 1343/90 000 children (1.5%), 1185 received 1, 123 received 2, 32 received 3, and 3 received 4 boluses. Fluid bolus volume of 10 mL/kg was used in 45.3%, 20 mL/kg in 35.7%, 500 mL in 6.4% and 1000 mL in 7.1%. The fluid content was 0.9% saline in 99.9% of cases. The most common indications for FBT were: vomiting/diarrhoea (23%), acute febrile illness (11%) and pneumonia or sepsis (10% each). FBT was associated with a reduction in median heart rate by 6 beats per minute (P < 0.01), reduction in mean blood pressure by 3 mmHg (P < 0.01), and reduction in venous lactate by 0.2 mmol/L (P < 0.01)., Conclusions: Fluid bolus therapy is a commonly used intervention in the paediatric ED, most often for dehydration. Variability in indications, dose and effects of FBT warrant further exploration., (© 2019 Australasian College for Emergency Medicine.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: With increased access to genetic testing, variants of uncertain significance (VUS) where pathogenicity is uncertain are being increasingly identified. More than 85% Osteogenesis Imperfecta (OI) patients have pathogenic variants in COL1A1/A2. However, when a VUS is identified, there are no pathways in place for determining significance., Objective: Define a diagnostic pathway to confirm pathogenicity, providing patients with definitive genetic diagnosis, accurate recurrence risks, and prenatal testing options., Methods: Functional studies on collagen secretion from cultured patient fibroblasts combined with detailed phenotyping and segregation family studies., Results: We demonstrate data from a family with a VUS identified in type I collagen. FAMILY-1: Six-year-old boy with failure-to-gain weight, talipes, fractures, on and off treatment with Pamidronate as diagnosis of OI uncertain. Transiliac bone biopsy at 2 years of age demonstrated active new bone formation within periosteum; bone cortices were normal thickness but increased porosity. Trabecular bone showed features of advanced osteoporosis. Genetic testing identified a de novo COL1A1 c.206&#95;208delTGT, p.Leu69del variant. Sibling with similar phenotype but no fractures as yet, tested positive for variant raising concerns regarding her diagnosis, and management. Results from three independent experiments (cell immunofluorescence, collagen secretion assay by Western Blot, and unbiased proteomics) from cultured patient fibroblasts demonstrate COL1A1 c.206&#95;208delTGT, p.Leu69del variant causing a substantial defect to collagen extracellular matrix assembly confirming variant pathogenicity., Conclusion: Access to genetic testing in OI is increasing as advances in genetic technologies decreases cost; a clinical diagnostic pathway needs to be implemented for managing variants identified by such testing., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Metazoans require efficient and ordered secretion of extracellular matrix (ECM) to coordinate cell and tissue function. Many ECM proteins are atypically large and their demand during key stages of development presents a major challenge to the canonical secretion machinery. While many of the molecular players in this pathway are known, little is understood about how they are integrated in time and space. Recent advances in gene engineering and super-resolution microscopy have underscored the spatiotemporal organisation of the endoplasmic reticulum (ER)-Golgi interface. These findings are challenging long-held models of vesicular transport of large matrix proteins, such as procollagen, and are implicating less well-defined carriers and direct interconnections between organelles. Here, we discuss current models describing the dynamics and mechanisms of ER-Golgi transport., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A subset of Rab GTPases have been implicated in cilium formation in cultured mammalian cells [1-6]. Rab11 and Rab8, together with their GDP-GTP exchange factors (GEFs), TRAPP-II and Rabin8, promote recruitment of the ciliary vesicle to the mother centriole and its subsequent maturation, docking, and fusion with the cell surface [2-5]. Rab23 has been linked to cilium formation and membrane trafficking at mature cilia [1, 7, 8]; however, the identity of the GEF pathway activating Rab23, a member of the Rab7 subfamily of Rabs, remains unclear. Longin-domain-containing complexes have been shown to act as GEFs for Rab7 subfamily GTPases [9-12]. Here, we show that Inturned and Fuzzy, proteins previously implicated as planar cell polarity (PCP) effectors and in developmentally regulated cilium formation [13, 14], contain multiple longin domains characteristic of the Mon1-Ccz1 family of Rab7 GEFs and form a specific Rab23 GEF complex. In flies, loss of Rab23 function gave rise to defects in planar-polarized trichome formation consistent with this biochemical relationship. In cultured human and mouse cells, Inturned and Fuzzy localized to the basal body and proximal region of cilia, and cilium formation was compromised by depletion of either Inturned or Fuzzy. Cilium formation arrested after docking of the ciliary vesicle to the mother centriole but prior to axoneme elongation and fusion of the ciliary vesicle and plasma membrane. These findings extend the family of longin domain GEFs and define a molecular activity linking Rab23-regulated membrane traffic to cilia and planar cell polarity., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Secretion and assembly of collagen are fundamental to the function of the extracellular matrix. Defects in the assembly of a collagen matrix lead to pathologies including fibrosis and osteogenesis imperfecta. Owing to the size of fibril-forming procollagen molecules it is assumed that they are transported from the endoplasmic reticulum to the Golgi in specialized large COPII-dependent carriers. Here, analyzing endogenous procollagen and a new engineered GFP-tagged form, we show that transport to the Golgi occurs in the absence of large (>350 nm) carriers. Large GFP-positive structures were observed occasionally, but these were nondynamic, are not COPII positive, and are labeled with markers of the ER. We propose a short-loop model of COPII-dependent ER-to-Golgi traffic that, while consistent with models of ERGIC-dependent expansion of COPII carriers, does not invoke long-range trafficking of large vesicular structures. Our findings provide an important insight into the process of procollagen trafficking and reveal a short-loop pathway from the ER to the Golgi, without the use of large carriers., (© 2019 McCaughey et al.)

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport., Competing Interests: LV, NS, KH, DS No competing interests declared, (© 2018, Vuolo et al.)

The export of newly synthesized proteins from the endoplasmic reticulum is fundamental to the ongoing maintenance of cell and tissue structure and function. After co-translational translocation into the ER, proteins destined for downstream intracellular compartments or secretion from the cell are sorted and packaged into transport vesicles by the COPII coat protein complex. The fundamental discovery and characterization of the pathway has now been augmented by a greater understanding of the role of COPII in diverse aspects of cell function. We now have a deep understanding of how COPII contributes to the trafficking of diverse cargoes including extracellular matrix molecules, developmental signalling proteins, and key metabolic factors such as lipoproteins. Structural and functional studies have shown that the COPII coat is both highly flexible and subject to multiple modes of regulation. This has led to new discoveries defining roles of COPII in development, autophagy, and tissue organization. Many of these newly emerging features of the canonical COPII pathway are placed in a context of procollagen secretion because of the fundamental interest in how a coat complex that typically generates 80-nm transport vesicles can package a cargo reported to be over 300 nm. Here we review the current understanding of COPII and assess the current consensus on its role in packaging diverse cargo proteins.

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies, which are characterised by debilitating developmental defects that affect many tissues. Here, we report a new role for regulator of calcineurin 2 (RCAN2) in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia after serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in the control of cilia length that are seen upon RNAi of giantin itself. Together, these data define RCAN2 as a regulator of cilia function that can compensate for the loss of giantin function., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Background: Cole-Carpenter syndrome (CCS) is commonly classified as a rare Osteogenesis Imperfecta (OI) disorder. This was following the description of two unrelated patients with very similar phenotypes who were subsequently shown to have a heterozygous missense mutation in P4HB ., Objectives: Here, we report a 3-year old female patient with severe OI who on exome sequencing was found to carry the same missense mutation in P4HB as reported in the original cohort. We discuss the genetic heterogeneity of CCS and underlying mechanism of P4HB in collagen production., Methods: We undertook detailed clinical, radiological and molecular phenotyping in addition, to analysis of collagen in cultured fibroblasts and electron microscopic examination in the patient reported here., Results: The clinical phenotype appears consistent in patients reported so far but interestingly, there also appears to be a definitive phenotypic clue (crumpling metadiaphyseal fractures of the long tubular bones with metaphyseal sclerosis which are findings that are uncommon in OI) to the underlying genotype ( P4HB variant)., Discussion: P4HB (Prolyl 4-hydroxylase, betasubunit) encodes for PDI (Protein Disulfide isomerase) and in cells, in its tetrameric form, catalyses formation of 4-hydroxyproline in collagen. The recurrent variant in P4HB , c.1178A>G, p.Tyr393Cys, sits in the C-terminal reactive centre and is said to interfere with disulphide isomerase function of the C-terminal reactive centre. P4HB catalyses the hydroxylation of proline residues within the X-Pro-Gly repeats in the procollagen helical domain. Given the inter-dependence of extracellular matrix (ECM) components in assembly of a functional matrix, our data suggest that it is the organisation and assembly of the functional ECM that is perturbed rather than the secretion of collagen type I per se., Conclusions: We provide additional evidence of P4HB as a cause of a specific form of OI-CCS and expand on response to treatment with bisphosphonates in this rare disorder., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The Golgi is the cellular hub for complex glycosylation, controlling accurate processing of complex proteoglycans, receptors, ligands and glycolipids. Its structure and organisation are dependent on golgins, which tether cisternal membranes and incoming transport vesicles. Here, we show that knockout of the largest golgin, giantin, leads to substantial changes in gene expression but only limited effects on Golgi structure. Notably, 22 Golgi-resident glycosyltransferases, but not glycan-processing enzymes or the ER glycosylation machinery, are differentially expressed following giantin ablation. This includes near-complete loss of function of GALNT3 in both mammalian cell and zebrafish models. Giantin-knockout zebrafish exhibit hyperostosis and ectopic calcium deposits, recapitulating phenotypes of hyperphosphatemic familial tumoral calcinosis, a disease caused by mutations in GALNT3. These data reveal a new feature of Golgi homeostasis: the ability to regulate glycosyltransferase expression to generate a functional proteoglycome., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The Golgi is essential for glycosylation of newly synthesised proteins including almost all cell-surface and extracellular matrix proteoglycans. Giantin, encoded by the golgb1 gene, is a member of the golgin family of proteins that reside within the Golgi stack, but its function remains elusive. Loss of function of giantin in rats causes osteochondrodysplasia; knockout mice show milder defects, notably a cleft palate. In vitro , giantin has been implicated in Golgi organisation, biosynthetic trafficking, and ciliogenesis. Here we show that loss of function of giantin in zebrafish, using either morpholino or knockout techniques, causes defects in cilia function. Giantin morphants have fewer cilia in the neural tube and those remaining are longer. Mutants have the same number of cilia in the neural tube but these cilia are also elongated. Scanning electron microscopy shows that loss of giantin results in an accumulation of material at the ciliary tip, consistent with a loss of function of retrograde intraflagellar transport. Mutants show milder defects than morphants consistent with adaptation to loss of giantin., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Ionotropic glutamate receptor (iGluR) trafficking and function underpin excitatory synaptic transmission and plasticity and shape neuronal networks. It is well established that the transcription, translation, and endocytosis/recycling of iGluRs are all regulated by neuronal activity, but much less is known about the activity dependence of iGluR transport through the secretory pathway. Here, we use the kainate receptor subunit GluK2 as a model iGluR cargo to show that the assembly, early secretory pathway trafficking, and surface delivery of iGluRs are all controlled by neuronal activity. We show that the delivery of de novo kainate receptors is differentially regulated by modulation of GluK2 Q/R editing, PKC phosphorylation, and PDZ ligand interactions. These findings reveal that, in addition to short-term regulation of iGluRs by recycling/endocytosis and long-term modulation by altered transcription/translation, the trafficking of iGluRs through the secretory pathway is under tight activity-dependent control to determine the numbers and properties of surface-expressed iGluRs., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Uveal melanoma is a rare melanoma variant with no effective therapies once metastases develop. Although durable cancer regression can be achieved in metastatic cutaneous melanoma with immunotherapies that augment naturally existing antitumor T-cell responses, the role of these treatments for metastatic uveal melanoma remains unclear. We sought to define the relative immunogenicity of these two melanoma variants and determine whether endogenous antitumor immune responses exist against uveal melanoma., Experimental Design: We surgically procured liver metastases from uveal melanoma (n = 16) and cutaneous melanoma (n = 35) patients and compared the attributes of their respective tumor cell populations and their infiltrating T cells (TIL) using clinical radiology, histopathology, immune assays, and whole-exomic sequencing., Results: Despite having common melanocytic lineage, uveal melanoma and cutaneous melanoma metastases differed in their melanin content, tumor differentiation antigen expression, and somatic mutational profile. Immunologic analysis of TIL cultures expanded from these divergent forms of melanoma revealed cutaneous melanoma TIL were predominantly composed of CD8(+) T cells, whereas uveal melanoma TIL were CD4(+) dominant. Reactivity against autologous tumor was significantly greater in cutaneous melanoma TIL compared with uveal melanoma TIL. However, we identified TIL from a subset of uveal melanoma patients which had robust antitumor reactivity comparable in magnitude with cutaneous melanoma TIL. Interestingly, the absence of melanin pigmentation in the parental tumor strongly correlated with the generation of highly reactive uveal melanoma TIL., Conclusions: The discovery of this immunogenic group of uveal melanoma metastases should prompt clinical efforts to determine whether patients who harbor these unique tumors can benefit from immunotherapies that exploit endogenous antitumor T-cell populations. Clin Cancer Res; 22(9); 2237-49. ©2015 AACR., (©2015 American Association for Cancer Research.)

Rip11 is a Rab11 effector protein that has been shown to be important in controlling the trafficking of several intracellular cargoes, including the fatty acid transporter FAT/CD36, V-ATPase and the glucose transporter GLUT4. We have previously demonstrated that Rip11 translocates to the plasma membrane in response to insulin and here we examine the basis of this regulated phenomenon in more detail. We show that Rip11 rapidly recycles between the cell interior and surface, and that the ability of insulin to increase the appearance of Rip11 at the cell surface involves an inhibition of Rip11 internalisation from the plasma membrane. By contrast the hormone has no effect on the rate of Rip11 translocation towards the plasma membrane. The ability of insulin to inhibit Rip11 internalisation requires dynamin and class I PI3-kinases, but is independent of the activation of the protein kinase Akt; characteristics which are very similar to the mechanism by which insulin inhibits GLUT4 endocytosis., (Copyright © 2015. Published by Elsevier Inc.)

The optimal T-cell attributes for adoptive cancer immunotherapy are unclear. Recent clinical trials of ex vivo-expanded tumor-infiltrating lymphocytes indicated that differentiated T effector cells can elicit durable antitumor responses in some patients with cancer, with their antitumor activity tightly correlated with their persistence in the host. Thus, there is great interest in the definition of intrinsic biomarkers that can predict the conversion of short-lived tumor antigen-specific T effector cells into long-lived T memory cells. Long-term persistence of ex vivo-expanded tumor-specific CD8+ T effector clones has been reported in refractory metastatic melanoma patients after adoptive T-cell transfer. By using highly homogeneous clone populations from these preparations, we performed a comparative transcriptional profiling to define preinfusion molecular attributes that can be ascribed to an effector-to-memory transition. Through this route, we discovered that preinfusion T-cell clones that expressed the IL7 receptor (IL7R) and c-myc were more likely to persist longer after adoptive transfer to patients. The predictive value of these two biomarkers was strengthened by using IL7R protein, IL7-induced pSTAT5, and c-myc mRNA expression to prospectively identify human tumor-specific T effector clones capable of engraftment into immunodeficient mice. Overall, our findings reveal IL7R and c-myc expression as intrinsic biomarkers that can predict the fate of CD8+ T effector cells after adoptive transfer., (©2015 American Association for Cancer Research.)

Purpose: Adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) can mediate durable cancer regression in selected patients with metastatic melanoma. However, the tumor antigens associated with these favorable responses remain unclear. We hypothesized that a clinical strategy involving the iterative adoptive transfer of selected autologous antigen-specific T-cell clones could help systematically define immunologic targets associated with successful cancer therapy, without the interpretative ambiguity of transferring polyclonal populations. Here, we evaluated the clinical efficacy of CD8(+) T-cell clones specific for the melanocyte differentiation antigens (MDA), gp100 and MART-1, respectively., Experimental Design: We conducted two consecutive phase II clinical trials involving the adoptive transfer of highly selected autologous antigen-specific CD8(+) T-cell clones against gp100 and MART-1, respectively. Fifteen patients with HLA-A2(+) treatment-refractory metastatic melanoma received highly avid MDA-specific CD8(+) T-cell clones specific for either gp100 (n = 10) or MART-1 (n = 5) with or without intravenous interleukin-2 (IL2) after a lymphodepleting myeloablative preparative regimen., Results: Of the 15 treated patients, we observed immune-mediated targeting of skin melanocytes in 11 patients (73%) and clonal engraftment in eight patients (53%) after cell transfer. There were only transient minor tumor regressions observed, but no objective tumor responses based on Response Evaluation Criteria in Solid Tumor (RECIST) criteria., Conclusions: Despite successful clonal repopulation and evidence of in vivo antigen targeting, the poor therapeutic efficacy after the adoptive transfer of autologous MDA-specific T cells raises significant concerns regarding future immunotherapy efforts targeting this class of tumor antigens., (©2014 American Association for Cancer Research.)

Cytoplasmic dynein-2 is the motor for retrograde intraflagellar transport (IFT), and mutations in dynein-2 are known to cause skeletal ciliopathies. Here, we define for the first time the composition of the human cytoplasmic dynein-2 complex. We show that the proteins encoded by the ciliopathy genes WDR34 and WDR60 are bona fide dynein-2 intermediate chains and are both required for dynein-2 function. In addition, we identify TCTEX1D2 as a unique dynein-2 light chain that is itself required for cilia function. We define several subunits common to both dynein-1 and dynein-2, including TCTEX-1 (also known as DYNLT1) and TCTEX-3 (also known as DYNLT3), roadblock-1 (also known as DYNLRB1) and roadblock-2 (also known as DYNLRB2), and LC8-1 and LC8-2 light chains (DYNLL1 and DYNLL2, respectively). We also find that NudCD3 associates with dynein-2 as it does with dynein-1. By contrast, the common dynein-1 regulators dynactin, LIS1 (also known as PAFAH1B1) and BICD2 are not found in association with dynein-2. These data explain why mutations in either WDR34 or WDR60 cause disease, as well as identifying TCTEX1D2 as a candidate ciliopathy gene., (© 2014. Published by The Company of Biologists Ltd.)

Background Information: The centrosome is the primary microtubule-organising centre of animal cells and it has crucial roles in several fundamental cellular functions, including cell division, cell polarity, and intracellular transport. The mechanisms responsible for this are not completely understood., Results: The poorly characterised protein CEP126 localises to the centrosome, pericentriolar satellites and the base of the primary cilium. Suppression of CEP126 expression results in dispersion of the pericentriolar satellites and disruption of the radial organisation of the microtubules, and induces disorganisation of the mitotic spindle. Moreover, CEP126 depletion or the transfection of a CEP126 truncation mutant in hTERT-RPE-1 and IMCD3 cells impairs the formation of the primary cilium., Conclusions: We propose that CEP126 is a regulator of microtubule organisation at the centrosome that acts through modulation of the transport of pericentriolar satellites, and consequently, of the organisation of cell structure., (© 2014 The Authors. Biology of the Cell published by Wiley-VCH Verlag GmbH & Co. KGaA on behalf of Société de Biologie Cellulaire Francaise.)

Purpose: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies., Experimental Design: From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4(+) and CD8(+) T-cell infiltrate., Results: Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8(+) and CD4(+) T-cell infiltrates., Conclusion: Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies., (©2014 American Association for Cancer Research.)

We recently demonstrated that dynein and kinesin motors drive multiple aspects of endosomal function in mammalian cells. These functions include driving motility, maintaining morphology (notably through providing longitudinal tension to support vesicle fission), and driving cargo sorting. Microtubule motors drive bidirectional motility during traffic between the endoplasmic reticulum (ER) and Golgi. Here, we have examined the role of microtubule motors in transport carrier motility, morphology, and domain organization during ER-to-Golgi transport. We show that, consistent with our findings for endosomal dynamics, microtubule motor function during ER-to-Golgi transport of secretory cargo is required for motility, morphology, and cargo sorting within vesicular tubular carriers en route to the Golgi. Our data are consistent with previous findings that defined roles for dynein-1, kinesin-1 (KIF5B) and kinesin-2 in this trafficking step. Our high resolution tracking data identify some intriguing aspects. Depletion of kinesin-1 reduces the number of motile structures seen, which is in line with other findings relating to the role of kinesin-1 in ER export. However, those transport carriers that were produced had a much greater run length suggesting that this motor can act as a brake on anterograde motility. Kinesin-2 depletion did not significantly reduce the number of motile transport carriers but did cause a similar increase in run length. These data suggest that kinesins act as negative regulators of ER-to-Golgi transport. Depletion of dynein not only reduced the number of motile carriers formed but also caused tubulation of carriers similar to that seen for sorting nexin-coated early endosomes. Our data indicated that the previously observed anterograde-retrograde polarity of transport carriers in transit to the Golgi from the ER is maintained by microtubule motor function., (© 2014. Published by The Company of Biologists Ltd.)

Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

Picornaviruses replicate their genomes in association with cellular membranes. While enteroviruses are believed to utilize membranes of the early secretory pathway, the origin of the membranes used by foot-and-mouth disease virus (FMDV) for replication are unknown. Secretory-vesicle traffic through the early secretory pathway is mediated by the sequential acquisition of two distinct membrane coat complexes, COPII and COPI, and requires the coordinated actions of Sar1, Arf1 and Rab proteins. Sar1 is essential for generating COPII vesicles at endoplasmic reticulum (ER) exit sites (ERESs), while Arf1 and Rab1 are required for subsequent vesicle transport by COPI vesicles. In the present study, we have provided evidence that FMDV requires pre-Golgi membranes of the early secretory pathway for infection. Small interfering RNA depletion of Sar1 or expression of a dominant-negative (DN) mutant of Sar1a inhibited FMDV infection. In contrast, a dominant-active mutant of Sar1a, which allowed COPII vesicle formation but inhibited the secretory pathway by stabilizing COPII coats, caused major disruption to the ER-Golgi intermediate compartment (ERGIC) but did not inhibit infection. Treatment of cells with brefeldin A, or expression of DN mutants of Arf1 and Rab1a, disrupted the Golgi and enhanced FMDV infection. These results show that reagents that block the early secretory pathway at ERESs have an inhibitory effect on FMDV infection, while reagents that block the early secretory pathway immediately after ER exit but before the ERGIC and Golgi make infection more favourable. Together, these observations argue for a role for Sar1 in FMDV infection and that initial virus replication takes place on membranes that are formed at ERESs.

The correct formation of primary cilia is central to the development and function of nearly all cells and tissues. Cilia grow from the mother centriole by extension of a microtubule core, the axoneme, which is then surrounded with a specialized ciliary membrane that is continuous with the plasma membrane. Intraflagellar transport moves particles along the length of the axoneme to direct assembly of the cilium and is also required for proper cilia function. The microtubule motor, cytoplasmic dynein-2 mediates retrograde transport along the axoneme from the tip to the base; dynein-2 is also required for some aspects of cilia formation. In most cells, the Golgi lies adjacent to the centrioles and key components of the cilia machinery localize to this organelle. Golgi-localized proteins have also been implicated in ciliogenesis and in intraflagellar transport. Here, we show that the transmembrane Golgi matrix protein giantin (GOLGB1) is required for ciliogenesis. We show that giantin is not required for the Rab11-Rabin8-Rab8 pathway that has been implicated in the early stages of ciliary membrane formation. Instead we find that suppression of giantin results in mis-localization of WDR34, the intermediate chain of dynein-2. Highly effective depletion of giantin or WDR34 leads to an inability of cells to form primary cilia. Partial depletion of giantin or of WDR34 leads to an increase in cilia length consistent with the concept that giantin acts through dynein-2. Our data implicate giantin in ciliogenesis through control of dynein-2 localization.

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse.

Many microtubule motors have been shown to couple to endosomal membranes. These motors include dynein in addition to many different kinesin family members. Sorting nexins (SNXs) are central to the organization and function of endosomes. These proteins can actively shape endosomal membranes and couple directly or indirectly to the minus-end microtubule motor dynein. Motor proteins acting on endosomes drive their motility, dictate their morphology and affect cargo segregation. We have used well-characterized members of the SNX family to elucidate motor coupling using high-resolution light microscopy coupled with depletion of specific microtubule motors. Endosomal domains labelled with SNX1, SNX4 and SNX8 couple to discrete combinations of dynein and kinesin motors. These specific combinations govern the structure and motility of each SNX-coated membrane in addition to the segregation of distinct functional endosomal subdomains. Taken together, our data show that these key features of endosome dynamics are governed by the same set of opposing microtubule motors. Thus, microtubule motors help to define the mosaic layout of endosomes that underpins cargo sorting.

ER-to-Golgi transport of proteins destined for the extracellular space or intracellular compartments depends on the COPII vesicle coat and is constitutive in all translationally active cells. Nevertheless, there is emerging evidence that this process is regulated on a cell- and tissue-specific basis, which means that components of the COPII coat will be of differential importance to certain cell types. The COPII coat consists of an inner layer, Sec23/24 and an outer shell, Sec13/31. We have shown previously that knock-down of Sec13 results in concomitant loss of Sec31. In zebrafish and cultured human cells this leads to impaired trafficking of large cargo, namely procollagens, and is causative for defects in craniofacial and gut development. It is now widely accepted that the outer COPII coat is key to the architecture and stability of ER export vesicles containing large, unusual cargo proteins. Here, we investigate zebrafish eye development following Sec13 depletion. We find that photoreceptors degenerate or fail to develop from the onset. Impaired collagen trafficking from the retinal pigment epithelium and defects in overall retinal lamination also seen in Sec13-depleted zebrafish might have been caused by increased apoptosis and reduced topical proliferation in the retina. Our data show that the outer layer of the COPII coat is also necessary for the transport of large amounts of cargo proteins, in this case rhodopsin, rather than just large cargo as previously thought.

The ability to deplete specific proteins from cells has transformed cell biology. Targeting of gene transcripts using RNA interference has allowed for a highly refined approach to the analysis of gene function that has been applied to all aspects of cell biology. Developments of the technology have reached a point where it is now a relatively trivial task to assess the role of an individual protein in a particular cell function. RNAi also allows for genome-wide screening as a discovery step toward the identification of new components of cellular pathways and machines. The technique has been applied extensively to the analysis of Golgi complex function, leading to significant insight into the biology of this complex organelle. Here, we describe the commonly used options for targeting individual genes for both transient and stable knockdown. We consider the alternative methods for introducing these reagents into cells and outline methods that we and others have used widely for validation of specificity and efficacy of gene targeting., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: The incidence of Clostridium difficile-associated disease (CDAD) in the adult population doubled in the past decade, with increasing morbidity and mortality; however, little research has been performed in the pediatric population. We characterized C difficile infection in the pediatric population, with emphasis on the surgical population., Methods: At a university-based children's hospital, we reviewed 231 patient (birth to 18 years of age) records containing a diagnosis of CDAD between January 1, 2002, and December 31, 2008., Results: Clostridium difficile-associated disease incidence increased from 250 per 100,000 hospitalizations in 2002 to 580 per 100,000 hospitalizations in 2008. No fatalities or surgical interventions were attributable to CDAD. Eighty-seven percent of patients received antibiotics within 2 months of diagnosis. Fifty-two percent of patients underwent operative intervention within 2 months of diagnosis; of these, 89% percent received previous antibiotic therapy and 57% were immunosuppressed. The most common antecedent procedures were bone marrow biopsy and line placement for myelodysplastic diseases (40%), followed by renal transplant (11%)., Conclusions: Pediatric CDAD incidence doubled during the study period but was not associated with death or operative intervention. A substantial number of CDAD cases were associated with previous operative procedures, particularly in immunosuppressed patients and those who received prior antibiotics., (Copyright © 2012 Elsevier Inc. All rights reserved.)