Evolution of Pulmonary Contusions in Patients With Severe Rib Fractures: Cause for Concern?

INTRODUCTION: The progression of pulmonary contusions remains poorly understood. This study aimed to measure the radiographic change in pulmonary contusions over time and evaluate the association of the radiographic change with clinical outcomes and surgical stabilization of rib fractures (SSRF). METHODS: This retrospective cohort study included adults admitted with three or more displaced rib fractures or flail segment on trauma CT and when a chest CT was repeated within one week after trauma. Radiographic severity of pulmonary contusions was assessed using the Blunt Pulmonary Contusion Score (BPC18). Logistic regression was performed to evaluate the relation between SSRF and worsening contusions on repeat CT, adjusted for potential confounders. RESULTS: Of 231 patients, 56 (24%) had a repeat CT scan. Of these, 55 (98%) had pulmonary contusion on the first CT scan with a median BPC18 score of 5 (P25-P75 3-7). Repeat CTs showed an overall decrease of the median BPC18 score to 4 (P25-P75 2-6, P = .02), but demonstrated a worsening of the pulmonary contusion in 16 patients (29%). All repeat CTs conducted within 12 hours post-injury demonstrated increasing BPC18. Radiographic worsening of pulmonary contusions was not associated with SSRF, nor with worse respiratory outcomes or intensive care length of stay, compared to patients with radiographically stable or improving contusions. DISCUSSION: In patients with severe rib fracture patterns who undergo repeat imaging, pulmonary contusions are prevalent and become radiographically worse within at least the first 12 hours after injury. No association between radiographic worsening and clinical outcomes was found.

Preterm birth and exercise capacity: what do we currently know?

Objectives: The long-term cardiopulmonary outcomes following preterm birth during the surfactant era remain unclear. Respiratory symptoms, particularly exertional symptoms, are common in preterm children. Therefore, cardiopulmonary exercise testing may provide insights into the pathophysiology driving exertional respiratory symptoms in those born preterm. This review aims to outline the current knowledge of cardiopulmonary exercise testing in the assessment of children born preterm in the surfactant era. Design: This study is a narrative literature review. Methods: Published manuscripts concerning the assessment of pulmonary outcomes using cardiopulmonary exercise testing in preterm children (aged <18 years) were reviewed. Search terms related to preterm birth, bronchopulmonary dysplasia, and exercise were entered into electronic databases, including Medline, PubMed, and Google Scholar. Reference lists from included studies were scanned for additional manuscripts. Results: Preterm children have disrupted lung development with significant structural and functional lung disease and increased respiratory symptoms. The association between these (resting) assessments of respiratory health and exercise capacity is unclear; however, expiratory flow limitation and an altered ventilatory response (rapid, shallow breathing) are seen during exercise. Due to the heterogeneity of participants, treatments, and exercise protocols, the effect of the aforementioned limitations on exercise capacity in children born preterm is conflicting and poorly understood. Conclusion: Risk factors for reduced exercise capacity in those born preterm remain poorly understood; however, utilizing cardiopulmonary exercise testing to its full potential, the pathophysiology of exercise limitation in survivors of preterm birth will enhance our understanding of the role exercise may play. The role of exercise interventions in mitigating the risk of chronic disease and premature death following preterm birth has yet to be fully realized and should be a focus of future robust randomized controlled trials.

Oscillometry and spirometry are not interchangeable when assessing the bronchodilator response in children and young adults born preterm.

INTRODUCTION: The European Respiratory Society Oscillometry Taskforce identified that clinical correlates of bronchodilator responses are needed to advance oscillometry in clinical practice. The understanding of bronchodilator-induced oscillometry changes in preterm lung disease is poor. Here we describe a comparison of bronchodilator assessments performed using oscillometry and spirometry in a population born very preterm and explore the relationship between bronchodilator-induced changes in respiratory function and clinical outcomes. METHODS: Participants aged 6-23 born ≤32 (N = 288; 132 with bronchopulmonary dysplasia) and ≥37 weeks' gestation (N = 76, term-born controls) performed spirometry and oscillometry. A significant bronchodilator response (BDR) to 400 µg salbutamol was classified according to published criteria. RESULTS: A BDR was identified in 30.9% (n = 85) of preterm-born individuals via spirometry and/or oscillometry, with poor agreement between spirometry and oscillometry definitions (k = 0.26; 95% confidence interval [CI] 0.18-0.40, p < .001). Those born preterm with a BDR by oscillometry but not spirometry had increased wheeze (33% vs. 11%, p = .010) and baseline resistance (Rrs5 z-score mean difference (MD) = 0.86, 95% CI 0.07-1.65, p = .025), but similar baseline spirometry to the group without a BDR (forced expiratory volume in 1 s [FEV1 ] z-score MD = -0.01, 95% CI -0.66 to 0.68, p > .999). Oscillometry was more feasible than spirometry (95% success rate vs. 85% (FEV1 ), 69% (forced vital capacity) success rate, p < .001), however being born preterm did not affect test feasibility. CONCLUSION: In the preterm population, oscillometry is a feasible and clinically useful supportive test to assess the airway response to inhaled salbutamol. Changes measured by oscillometry reflect related but distinct physiological changes to those measured by spirometry, and thus these tests should not be used interchangeably.

Efficacy of algaecides for the proactive treatment of overwintering cyanobacteria.

Once established within a water resource, harmful algal blooms (HABs) can occur seasonally with an intense and rapid onset, giving water resource managers limited time to respond to lessen risks. An attractive strategy to decrease human, ecological, and economic risks from HABs is to implement proactive algaecide treatments applied to overwintering cyanobacteria (i.e., akinetes and quiescent vegetative cells) in sediments prior to the formation of a HAB; however, this approach is novel and very limited efficacy data exist. Therefore, the specific objectives of this research were to 1) evaluate copper- and peroxide-based algaecides, applied as single and repeat treatments at the bench scale, to identify effective proactive treatments, and 2) compare correlations between cell density and other response measurements (i.e., in vivo chlorophyll a and phycocyanin concentrations and percent benthic coverage), to identify informative metrics to assess overwintering cyanobacteria responses. Twelve treatment scenarios using copper- and peroxide-based algaecides were applied to sediments containing overwintering cyanobacteria prior to a 14 d incubation under favorable growth conditions. Responses of cyanobacteria in the planktonic (i.e., cell density, in vivo chlorophyll a and phycocyanin concentrations) and benthic (percent coverage) phases after a 14 d incubation were evaluated in treatments and controls. The HAB-forming cyanobacteria present after a 14 d incubation were: Aphanizomenon, Dolichospermum, Microcystis, Nostoc, and Planktonthrix. Successive treatments of copper sulfate (CuSulfate) followed by sodium carbonate peroxyhydrate (PeroxiSolid) (second algaecide applied after 24 h) as well as repeat applications of a single algaecide, PeroxiSolid (second treatment applied after 24 h) resulted in statistically significant (p ≤ 0.05; α = 0.05) declines in cell density relative to untreated controls. Planktonic cyanobacteria responses measured in terms of phycocyanin concentrations were strongly correlated with cyanobacteria density measurements (Pearson's correlation coefficient (r) = 0.89). Chlorophyll a concentrations and percent benthic coverage did not correlate with planktonic cyanobacteria density measurements (r = 0.37 and -0.49, respectively) and therefore, were unreliable metrics for cyanobacterial responses in this study. These data provide initial evidence of the efficacy of algaecides for treating overwintering cells in sediments and contribute to our overarching hypothesis that proactive treatments may delay the onset and intensity of HABs in impacted waterbodies.

Inhaled corticosteroids to improve lung function in children (aged 6-12 years) who were born very preterm (PICSI): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Despite the substantial burden of lung disease throughout childhood in children who were born very preterm, there are no evidence-based interventions to improve lung health beyond the neonatal period. We tested the hypothesis that inhaled corticosteroid improves lung function in this population. METHODS: PICSI was a randomised, double-blind, placebo-controlled trial at Perth Children's Hospital (Perth, WA, Australia) to assess whether fluticasone propionate, an inhaled corticosteroid, improves lung function in children who had been born very preterm (<32 weeks of gestation). Eligible children were aged 6-12 years and did not have severe congenital abnormalities, cardiopulmonary defects, neurodevelopmental impairment, diabetes, or any glucocorticoid use within the preceding 3 months. Participants were randomly assigned (1:1) to receive 125 µg fluticasone propionate or placebo twice daily for 12 weeks. Participants were stratified for sex, age, bronchopulmonary dysplasia diagnosis, and recent respiratory symptoms using the biased-coin minimisation technique. The primary outcome was change in pre-bronchodilator forced expiratory volume in 1 s (FEV1) after 12 weeks of treatment. Data were analysed by intention-to-treat (ie, all participants who were randomly assigned and took at least the tolerance dose of the drug). All participants were included in the safety analyses. This trial is registered at the Australian and New Zealand Clinical Trials Registry, number 12618000781246. FINDINGS: Between Oct 23, 2018, and Feb 4, 2022, 170 participants were randomly assigned and received at least the tolerance dose (83 received placebo and 87 received inhaled corticosteroid). 92 (54%) participants were male and 78 (46%) were female. 31 participants discontinued treatment before 12 weeks (14 in the placebo group and 17 in the inhaled corticosteroid group), mostly due to the impact of the COVID-19 pandemic. When analysed by intention-to-treat, the change in pre-bronchodilator FEV1 Z score over 12 weeks was -0·11 (95% CI -0·21 to 0·00) in the placebo group and 0·20 (0·11 to 0·30) in the inhaled corticosteroid group (imputed mean difference 0·30, 0·15-0·45). Three of 83 participants in the inhaled corticosteroid group had adverse events requiring treatment discontinuation (exacerbation of asthma-like symptoms). One of 87 participants in the placebo group had an adverse event requiring treatment discontinuation (inability to tolerate the treatment with dizziness, headaches, stomach pains, and worsening of a skin condition). INTERPRETATION: As a group, children born very preterm have only modestly improved lung function when treated with inhaled corticosteroid for 12 weeks. Future studies should consider individual phenotypes of lung disease after preterm birth and other agents to improve management of prematurity-associated lung disease. FUNDING: Australian National Health and Medical Research Council, Telethon Kids Institute, and Curtin University.

Early Surgical Stabilization of Rib Fractures is Feasible in Patients With Non-Urgent Operative Pelvic Injuries.

INTRODUCTION: The feasibility of prioritizing surgical stabilization of rib fractures (SSRF) in patients with other injuries is unknown. The purpose of this study was to evaluate the timing and outcomes of SSRF between patients with and without non-urgent operative pelvic injuries. PATIENTS AND METHODS: In this retrospective observational study, all patients between 2010 and 2020 who underwent SSRF (SSRF group) and those who underwent SSRF and non-urgent operative management of pelvic fractures (SSRF + P group) were included. Demographics, injury characteristics, operative details, and outcomes were compared between the 2 groups. RESULTS: Over 11 years, 154 SSRF patients were identified, with 143 patients in the SSRF group (93%) and 11 patients in the SSRF + P group (7%). Median number of rib fractures (7 vs 9, P = .04), total number of fractures (11 vs 15, P < .01), and flail segment (54% vs 91%, P = .02) were higher in SSRF + P group. Median time to SSRF was similar (0 vs 1 day, P = .20) between the 2 groups. Median time to pelvic fixation was 3 days in SSRF + P group and 8 out of 11 patients (73%) underwent SSRF prior to pelvic fixation. Median operative time (137 vs 178 mins, P = .14) and median number of ribs plated (4 vs 5, P = .05) were higher in SSRF + P group. There was no difference in SSRF-related complications, pelvic fracture-related complications from operative positioning, rates of pneumonia, or mortality between the 2 groups. CONCLUSIONS: SSRF can be performed early in patients with non-urgent operative pelvic injuries without a difference in pelvic fracture-related complications, SSRF-related complications, pneumonia, or mortality.

Risk factors for poorer respiratory outcomes in adolescents and young adults born preterm.

RATIONALE: The respiratory outcomes for adult survivors of preterm birth in the postsurfactant era are wide-ranging with prognostic factors, especially those encountered after the neonatal period, poorly understood. OBJECTIVES: To obtain comprehensive 'peak' lung health data from survivors of very preterm birth and identify neonatal and life-course risk factors for poorer respiratory outcomes in adulthood. METHODS: 127 participants born ≤32 weeks gestation (64%, n=81 with bronchopulmonary dysplasia (BPD), initially recruited according to a 2 with-BPD:1 without-BPD strategy), and 41 term-born controls completed a lung health assessment at 16-23 years, including lung function, imaging and symptom review. Risk factors assessed against poor lung health included neonatal treatments, respiratory hospitalisation in childhood, atopy and tobacco smoke exposure. MEASUREMENTS AND MAIN RESULTS: Young adults born prematurely had greater airflow obstruction, gas trapping and ventilation inhomogeneity, in addition to abnormalities in gas transfer and respiratory mechanics, compared with term. Beyond lung function, we observed greater structural abnormalities, respiratory symptoms and inhaled medication use. A previous respiratory admission was associated with airway obstruction; mean forced expiratory volume in 1 s/forced vital capacity z-score was -0.561 lower after neonatal confounders were accounted for (95% CI -0.998 to -0.125; p=0.012). Similarly, respiratory symptom burden was increased in the preterm group with a respiratory admission, as was peribronchial thickening (6% vs 23%, p=0.010) and bronchodilator responsiveness (17% vs 35%, p=0.025). Atopy, maternal asthma and tobacco smoke exposure did not influence lung function or structure at 16-23 years in our preterm cohort. CONCLUSIONS: Even after accounting for the neonatal course, a respiratory admission during childhood remained significantly associated with reduced peak lung function in the preterm-born cohort, with the largest difference seen in those with BPD. A respiratory admission during childhood should, therefore, be considered a risk factor for long-term respiratory morbidity in those born preterm, especially for individuals with BPD.

Prevalence of chronic respiratory diseases in Aboriginal children: A whole population study.

BACKGROUND: The burden of bronchiectasis is disproportionately high in Aboriginal adults, with early mortality. Bronchiectasis precursors, that is, protracted bacterial bronchitis (PBB) and chronic suppurative lung disease (CSLD), often commence in early childhood. We previously reported a 10% prevalence of PBB in Aboriginal children aged 0 to 7 years, however there are no data on prevalence of chronic lung diseases in older children. Our study aimed to determine the prevalence of PBB, CSLD, bronchiectasis, and asthma in Aboriginal children living in four communities. METHODS: A whole-population cross-sectional community co-designed study of Aboriginal children aged <18-years in four remote communities in Western Australia across two-time points, a month apart. Children were assessed by pediatric respiratory clinicians with spirometry undertaken (when possible) between March-September 2021. Children with respiratory symptoms were followed up via medical record audit from either the local medical clinic or via a respiratory specialist clinic through to March 2022 to establish a final diagnosis. FINDINGS: We recruited 392 (91.6%) of those in the selected communities; median age = 8.4 years (interquartile range [IQR] 5.1-11.5). Seventy children (17.9%) had a chronic respiratory pathology or abnormal spirometry results. PBB was confirmed in 30 (7.7%), CSLD = 13 (3.3%), bronchiectasis = 5 (1.3%) and asthma = 17 (4.3%). The prevalence of chronic wet cough significantly increased with increasing age. INTERPRETATION: The prevalence of PBB, CSLD and bronchiectasis is high in Aboriginal children and chronic wet cough increases with age. This study highlights the high disease burden in Aboriginal children and the urgent need for strategies to address these conditions.

Rib fixation in patients with severe rib fractures and pulmonary contusions: Is it safe?

BACKGROUND: Pulmonary contusion has been considered a contraindication to surgical stabilization of rib fractures (SSRFs). This study aimed to evaluate the association between pulmonary contusion severity and outcomes after SSRF. We hypothesized that outcomes would be worse in patients who undergo SSRF compared with nonoperative management, in presence of varying severity of pulmonary contusions. METHODS: This retrospective cohort study included adults with three or more displaced rib fractures or flail segment. Patients were divided into those who underwent SSRF versus those managed nonoperatively. Severity of pulmonary contusions was assessed using the Blunt Pulmonary Contusion 18 (BPC18) score. Outcomes (pneumonia, tracheostomy, mechanical ventilation days, intensive care unit (ICU) length of stay, hospital length of stay, mortality) were retrieved from patients' medical records. Comparisons were made using Fisher's exact and Kruskal-Wallis tests, and correction for potential confounding was done with regression analyses. RESULTS: A total of 221 patients were included; SSRF was performed in 148 (67%). Demographics and chest injury patterns were similar in SSRF and nonoperatively managed patients. Surgical stabilization of rib fracture patients had less frequent head and abdominal/pelvic injuries ( p = 0.017 and p = 0.003). Higher BPC18 score was associated with worse outcomes in both groups. When adjusted for ISS, the ICU stay was shorter (adjusted ß , -2.511 [95% confidence interval, -4.87 to -0.16]) in patients with mild contusions who underwent SSRF versus nonoperative patients. In patients with moderate contusions, those who underwent SSRF had fewer ventilator days (adjusted ß , -5.19 [95% confidence interval, -10.2 to -0.17]). For severe pulmonary contusions, outcomes did not differ between SSRF and nonoperative management. CONCLUSION: In patients with severe rib fracture patterns, higher BPC18 score is associated with worse respiratory outcomes and longer ICU and hospital admission duration. The presence of pulmonary contusions is not associated with worse SSRF outcomes, and SSRF is associated with better outcomes for patients with mild to moderate pulmonary contusions. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level IV.

Methods Used to Evaluate the Immediate Effects of Airway Clearance Techniques in Adults with Cystic Fibrosis: A Systematic Review and Meta-Analysis.

This review reports on methods used to evaluate airway clearance techniques (ACT) in adults with CF and examined data for evidence of any effect. Sixty-eight studies described ACT in adequate detail and were included in this review. Frequently reported outcomes were sputum expectoration (72%) and spirometric lung function (60%). Compared with cough alone, following any ACT, there was a trend for greater sputum wet weight, however FEV1 was not different. The mean (95% CI) within-group effect for sputum wet weight following any ACT was 12.43 g (9.28 to 15.58) (n = 30 studies) and for FEV1 was 0.03 L (-0.17 to 0.24) (n = 14 studies). Meta-regression demonstrated that, when compared with cough alone, greater sputum wet weight was reported in groups that received additional ACT by between 2.45 and 3.94 g (F3,66 = 2.97, p = 0.04). These data suggest the addition of ACT to cough alone may optimise sputum clearance; however, FEV1 lacked sensitivity to detect this change. Importantly, this review highlights the lack of appropriate measures to assess ACT efficacy.

Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.

Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type Chlamydomonas with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating.

PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility.

We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified Chlamydomonas pacrg mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA b and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating.

The Central Apparatus of Cilia and Eukaryotic Flagella.

The motile cilium is a complex organelle that is typically comprised of a 9+2 microtubule skeleton; nine doublet microtubules surrounding a pair of central singlet microtubules. Like the doublet microtubules, the central microtubules form a scaffold for the assembly of protein complexes forming an intricate network of interconnected projections. The central microtubules and associated structures are collectively referred to as the central apparatus (CA). Studies using a variety of experimental approaches and model organisms have led to the discovery of a number of highly conserved protein complexes, unprecedented high-resolution views of projection structure, and new insights into regulation of dynein-driven microtubule sliding. Here, we review recent progress in defining mechanisms for the assembly and function of the CA and include possible implications for the importance of the CA in human health.

Microtubule binding protein PACRG plays a role in regulating specific ciliary dyneins during microtubule sliding.

The complex waveforms characteristic of motile eukaryotic cilia and flagella are produced by the temporally and spatially regulated action of multiple dynein subforms generating sliding between subsets of axonemal microtubules. Multiple protein complexes have been identified that are associated with the doublet microtubules and that mediate regulatory signals between key axonemal structures, such as the radial spokes and central apparatus, and the dynein arm motors; these complexes include the N-DRC, MIA, and CSC complexes. Previous studies have shown that PACRG (parkin co-regulated gene) forms a complex that is anchored to the axonemal doublet microtubules. Loss of PACRG causes defects in ciliary motility and cilia related diseases. Here, we use an in vitro microtubule sliding assay to demonstrate that PACRG and its interactors are part of a signaling pathway that includes the central apparatus, radial spokes and specific inner dynein arm subforms to control dynein-driven microtubule sliding. Using a biochemical approach, our studies also indicate that PACRG interacts with the radial spokes. © 2016 Wiley Periodicals, Inc.

FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.

Aqueous cationic, anionic and non-ionic multi-walled carbon nanotubes, functionalised with minimal framework damage, for biomedical application.

The use of a thermochemical grafting approach provides a versatile means to functionalise as-synthesised, bulk multi-walled carbon nanotubes (MWNTs) without altering their inherent structure. The associated retention of properties is desirable for a wide range of commercial applications, including for drug delivery and medical purposes; it is also pertinent to studies of intrinsic toxicology. A systematic series of water-compatible MWNTs, with diameter around 12 nm have been prepared, to provide structurally-equivalent samples predominantly stabilised by anionic, cationic, or non-ionic groups. The surface charge of MWNTs was controlled by varying the grafting reagents and subsequent post-functionalisation modifications. The degree of grafting was established by thermal analysis (TGA). High resolution transmission electron microscope (HRTEM) and Raman measurements confirmed that the structural framework of the MWNTs was unaffected by the thermochemical treatment, in contrast to a conventional acid-oxidised control which was severely damaged. The effectiveness of the surface modification was demonstrated by significantly improved solubility and stability in both water and cell culture medium, and further quantified by zeta-potential analysis. The grafted MWNTs exhibited relatively low bioreactivity on transformed human alveolar epithelial type 1-like cells (TT1) following 24 h exposure as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase release (LDH) assays. The exposure of TT1 cells to MWNTs suppressed the release of the inflammatory mediators, interleukin 6 (IL-6) and interleukin 8 (IL-8). TEM cell uptake studies indicated efficient cellular entry of MWNTs into TT1 cells, via a range of mechanisms. Cationic MWNTs showed a more substantial interaction with TT1 cell membranes than anionic MWNTs, demonstrating a surface charge effect on cell uptake.

A differential cargo-loading model of ciliary length regulation by IFT.

BACKGROUND: During the assembly and maintenance of cilia, precursor proteins need to be transported from the cell body into the organelle. Intraflagellar transport (IFT) is assumed to be the predominant protein transport pathway in cilia, but it remains largely unknown how ciliary proteins use IFT to reach their destination sites in the cilium and whether the amount of cargo transported by IFT is regulated. RESULTS: Single-particle imaging showed that DRC4, a structural protein of the axoneme, moves in association with IFT particles inside Chlamydomonas reinhardtii cilia. IFT is required for DRC4 transport both into and within the cilium. DRC4 cargoes dissociate from IFT trains at the tip as well as at various sites along the length of the cilium. Unloaded DRC4 diffuses before docking at its axonemal assembly site. In growing cilia, DRC4 transport by IFT was strongly increased over the steady-state level, and the frequency decreased linearly with the increasing ciliary length. The frequency of DRC4 transport was similarly elevated in short growth-arrested cilia and remained high even when the amount of DRC4 available in the cell body was reduced. CONCLUSIONS: DRC4 is a bona fide cargo of IFT. Incompletely assembled cilia trigger an increase in the amount of DRC4 cargo transported by IFT particles, and DRC4 transport is downregulated as cilia approach their steady-state length. We propose a model in which ciliary length is controlled by regulating the amount of cargo transported by IFT particles.

Methods for analysis of calcium/calmodulin signaling in cilia and flagella.

The axonemal microtubules of cilia/flagella act as a scaffold for assembly of the protein complexes that ultimately regulate dynein activity to control the size and shape of ciliary bends. Despite our general understanding of the contribution of microtubule sliding to ciliary and flagellar motility, many questions regarding the regulation of dynein remain unanswered. For example, we know that the second messenger calcium plays an important role in modulating dynein activity in response to extracellular cues, but it remains unclear how calcium-binding proteins anchored to the axoneme contribute to this regulation. Recent work has focused on determining the identity and specific functions of these axonemal calcium-binding proteins. Here, we review our current knowledge of calcium-mediated motility and highlight key experiments that have substantially aided our understanding of calcium signaling within the axoneme.

The CSC connects three major axonemal complexes involved in dynein regulation.

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.

A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus.

Virtually all motile eukaryotic cilia and flagella have a '9+2' axoneme in which nine doublet microtubules surround two singlet microtubules. Associated with the central pair of microtubules are protein complexes that form at least seven biochemically and structurally distinct central pair projections. Analysis of mutants lacking specific projections has indicated that each may play a unique role in the control of flagellar motility. One of these is the C1d projection previously shown to contain the proteins FAP54, FAP46, FAP74 and FAP221/Pcdp1, which exhibits Ca(2+)-sensitive calmodulin binding. Here we report the isolation and characterization of a Chlamydomonas reinhardtii null mutant for FAP46. This mutant, fap46-1, lacks the C1d projection and has impaired motility, confirming the importance of this projection for normal flagellar movement. Those cells that are motile have severe defects in phototaxis and the photoshock response, underscoring a role for the C1d projection in Ca(2+)-mediated flagellar behavior. The data also reveal for the first time that the C1d projection is involved in the control of interdoublet sliding velocity. Our studies further identify a novel C1d subunit that we term C1d-87, give new insight into relationships between the C1d subunits, and provide evidence for multiple sites of calmodulin interaction within the C1d projection. These results represent significant advances in our understanding of an important but little studied axonemal structure.