CDK11(p58) is required for centriole duplication and Plk4 recruitment to mitotic centrosomes

Background

CDK11^p58 is a mitotic protein kinase, which has been shown to be required for different mitotic events such as centrosome maturation, chromatid cohesion and cytokinesis.

Methodology/Principal Findings

In addition to these previously described roles, our study shows that CDK11^p58 inhibition induces a failure in the centriole duplication process in different human cell lines. We propose that this effect is mediated by the defective centrosomal recruitment of proteins at the onset of mitosis. Indeed, Plk4 protein kinase and the centrosomal protein Cep192, which are key components of the centriole duplication machinery, showed reduced levels at centrosomes of mitotic CDK11-depleted cells. CDK11^p58, which accumulates only in the vicinity of mitotic centrosomes, directly interacts with the centriole-associated protein kinase Plk4 that regulates centriole number in cells. In addition, we show that centriole from CDK11 defective cells are not able to be over duplicated following Plk4 overexpression.

Conclusion/Significance

We thus propose that CDK11 is required for centriole duplication by two non-mutually-exclusive mechanisms. On one hand, the observed duplication defect could be caused indirectly by a failure of the centrosome to fully maturate during mitosis. On the other hand, CDK11^p58 could also directly regulate key centriole components such as Plk4 during mitosis to trigger essential mitotic centriole modifications, required for centriole duplication during subsequent interphase.     

Nuclei of Non-Muscle Cells Bind Centrosome Proteins upon Fusion with Differentiating Myoblasts

Background

In differentiating myoblasts, the microtubule network is reorganized from a centrosome-bound, radial array into parallel fibres, aligned along the long axis of the cell. Concomitantly, proteins of the centrosome relocalize from the pericentriolar material to the outer surface of the nucleus. The mechanisms that govern this relocalization are largely unknown.

Methodology

In this study, we perform experiments in vitro and in cell culture indicating that microtubule nucleation at the centrosome is reduced during myoblast differentiation, while nucleation at the nuclear surface increases. We show in heterologous cell fusion experiments, between cultures of differentiating mouse myoblasts and human cells of non-muscular origin, that nuclei from non-muscle cells recruit centrosome proteins once fused with the differentiating myoblasts. This recruitment still occurs in the presence of cycloheximide and thus appears to be independent of new protein biosynthesis.

Conclusions

Altogether, our data suggest that nuclei of undifferentiated cells have the dormant potential to bind centrosome proteins, and that this potential becomes activated during myoblast differentiation.     

Centrosome abnormalities during a Chlamydia trachomatis infection are caused by dysregulation of the normal duplication pathway

The presence of supernumerary centrosomes in cells infected with Chlamydia trachomatis may provide a mechanism to explain the association of C. trachomatis genital infection with cervical cancer. We show that the amplified centrosomal foci induced during a chlamydial infection contain both centriolar and pericentriolar matrix markers, demonstrating that they are bona fide centrosomes. As there were multiple immature centrioles but approximately one mature centriole per cell, aborted cytokinesis alone cannot account for centrosome amplification during a chlamydial infection. Production of supernumerary centrosomes required the kinase activities of Cdk2 and Plk4, which are known regulators of centrosome duplication, and progression through S-phase, which is the stage in the cell cycle when duplication of the centrosome occurs. These requirements indicate that centrosome amplification during a chlamydial infection depends on the host centrosome duplication pathway, which normally produces a single procentriole from each template centriole. However, C. trachomatis induces a loss of numerical control so that multiple procentrioles are formed per template.     

The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.     

Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.     

An intact centrosome is required for the maintenance of polarization during directional cell migration

Background

Establishing and maintaining polarization is critical during cell migration. It is known that the centrosome contains numerous proteins whose roles of organizing the microtubule network range include nucleation, stabilization and severing. It is not known whether the centrosome is necessary to maintain polarization. Due to its role as the microtubule organizing center, we hypothesize that the centrosome is necessary to maintain polarization in a migrating cell. Although there have been implications of its role in cell migration, there is no direct study of the centrosome''s role in maintaining polarization. In this study we ablate the centrosome by intracellular laser irradiation to understand the role of the centrosome in two vastly different cell types, human osteosarcoma (U2OS) and rat kangaroo kidney epithelial cells (PtK). The PtK cell line has been extensively used as a model for cytoskeletal dynamics during cell migration. The U2OS cell line serves as a model for a complex, single migrating cell.

Methodology/Principal Findings

In this study we use femtosecond near-infrared laser irradiation to remove the centrosome in migrating U2OS and PtK2 cells. Immunofluorescence staining for centrosomal markers verified successful irradiation with 94% success. A loss of cell polarization is observed between 30 and 90 minutes following removal of the centrosome. Changes in cell shape are correlated with modifications in microtubule and actin organization. Changes in cell morphology and microtubule organization were quantified revealing significant depolarization resulting from centrosome irradiation.

Conclusions/Significance

This study demonstrates that the centrosome is necessary for the maintenance of polarization during directed cell migration in two widely different cell types. Removal of the centrosome from a polarized cell results in the reorganization of the microtubule network into a symmetric non-polarized phenotype. These results demonstrate that the centrosome plays a critical role in the maintenance of cytoskeletal asymmetry during cell migration.     

The Role of Statins in Prevention and Treatment of Community Acquired Pneumonia: A Systematic Review and Meta-Analysis

Background

Emerging epidemiological evidence suggests that statins may reduce the risk of community-acquired pneumonia (CAP) and its complications.

Purpose

Performed a systematic review to address the role of statins in the prevention or treatment of CAP.

Data Source

Ovid MEDLINE, Cochrane, EMBASE, ISI Web of Science, and Scopus from inception through December 2011 were searched for randomized clinical trials, cohort and case-control studies.

Study Selection

Two authors independently reviewed studies that examined the role of statins in CAP.

Data Extraction

Data about study characteristics, adjusted effect-estimates and quality characteristics was extracted.

Data Synthesis

Eighteen studies corresponding to 21 effect-estimates (eight and 13 of which addressed the preventive and therapeutic roles of statins, respectively) were included. All studies were of good methodological quality. Random-effects meta-analyses of adjusted effect-estimates were used. Statins were associated with a lower risk of CAP, 0.84 (95% CI, 0.74–0.95), I² = 90.5% and a lower short-term mortality in patients with CAP, 0.68 (95% CI, 0.59–0.78), I² = 75.7%. Meta-regression did not identify sources of heterogeneity. A funnel plot suggested publication bias in the treatment group, which was adjusted by a novel regression method with a resultant effect-estimate of 0.85 (95% CI, 0.77–0.93). Sensitivity analyses using the rule-out approach showed that it is unlikely that the results were due to an unmeasured confounder.

Conclusions

Our meta-analysis reveals a beneficial role of statins for the risk of development and mortality associated with CAP. However, the results constitute very low quality evidence as per the GRADE framework due to observational study design, heterogeneity and publication bias.     

Centriolar satellite– and hMsd1/SSX2IP-dependent microtubule anchoring is critical for centriole assembly

Centriolar satellites are numerous electron-dense granules dispersed around the centrosome. Mutations in their components are linked to various human diseases, but their molecular roles remain elusive. In particular, the significance of spatial communication between centriolar satellites and the centrosome is unknown. hMsd1/SSX2IP localizes to both the centrosome and centriolar satellites and is required for tethering microtubules to the centrosome. Here we show that hMsd1/SSX2IP-mediated microtubule anchoring is essential for proper centriole assembly and duplication. On hMsd1/SSX2IP knockdown, the centriolar satellites become stuck at the microtubule minus end near the centrosome. Intriguingly, these satellites contain many proteins that normally localize to the centrosome. Of importance, microtubule structures, albeit not being anchored properly, are still required for the emergence of abnormal satellites, as complete microtubule depolymerization results in the disappearance of these aggregates from the vicinity of the centrosome. We highlighted, using superresolution and electron microscopy, that under these conditions, centriole structures are faulty. Remarkably, these cells are insensitive to Plk4 overproduction–induced ectopic centriole formation, yet they accelerate centrosome reduplication upon hydroxyurea arrest. Finally, the appearance of satellite aggregates is cancer cell specific. Together our findings provide novel insights into the mechanism of centriole assembly and microtubule anchoring.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

The Evolutionary History of Protein Domains Viewed by Species Phylogeny

Background

Protein structural domains are evolutionary units whose relationships can be detected over long evolutionary distances. The evolutionary history of protein domains, including the origin of protein domains, the identification of domain loss, transfer, duplication and combination with other domains to form new proteins, and the formation of the entire protein domain repertoire, are of great interest.

Methodology/Principal Findings

A methodology is presented for providing a parsimonious domain history based on gain, loss, vertical and horizontal transfer derived from the complete genomic domain assignments of 1015 organisms across the tree of life. When mapped to species trees the evolutionary history of domains and domain combinations is revealed, and the general evolutionary trend of domain and combination is analyzed.

Conclusions/Significance

We show that this approach provides a powerful tool to study how new proteins and functions emerged and to study such processes as horizontal gene transfer among more distant species.     

Clinical Features,Etiology and Outcomes of Community-Acquired Pneumonia in Patients with Chronic Obstructive Pulmonary Disease

Background

Community-acquired pneumonia (CAP) is a frequent complication of chronic obstructive pulmonary disease (COPD), but previous studies are often contradictory.

Objectives

We aimed to ascertain the characteristics and outcomes of CAP in patients with COPD as well as to determine the risk factors for mortality and Pseudomonas aeruginosa pneumonia in COPD patients with CAP. We also describe the etiology and outcomes of CAP in COPD patients receiving chronic oxygen therapy at home and those receiving inhaled steroids.

Methods

An observational analysis of a prospective cohort of hospitalized adults with CAP (1995–2011) was performed.

Results

We documented 4121 CAP episodes, of which 983 (23.9%) occurred in patients with COPD; the median FEV1 value was 50%, and 57.8% were classified as stage III or IV in the GOLD classification. Fifty-eight per cent of patients were receiving inhaled steroids, and 14.6% chronic oxygen therapy at home. Patients with COPD presented specific clinical features. S. pneumoniae was the leading causative organism overall, but P. aeruginosa was more frequent in COPD (3.4 vs. 0.5%; p<0.001). Independent risk factors for case-fatality rate in patients with COPD were multilobar pneumonia, P. aeruginosa pneumonia, and high-risk PSI classes. Prior pneumococcal vaccination was found to be protective. FEV₁ was an independent risk factor for P. aeruginosa pneumonia.

Conclusions

CAP in patients with COPD presents specific characteristics and risk factors for mortality. Prior pneumococcal vaccine has a beneficial effect on outcomes. P. aeruginosa pneumonia is associated with low FEV1 values and poor prognosis.     

A Proximal Centriole-Like Structure Is Present in Drosophila Spermatids and Can Serve as a Model to Study Centriole Duplication

Most animals have two centrioles in spermatids (the distal and proximal centrioles), but insect spermatids seem to contain only one centriole (Fuller 1993), which functionally resembles the distal centriole. Using fluorescent centriolar markers, we identified a structure near the fly distal centriole that is reminiscent of a proximal centriole (i.e., proximal centriole-like, or PCL). We show that the PCL exhibits several features of daughter centrioles. First, a single PCL forms near the proximal segment of the older centriole. Second, the centriolar proteins SAS-6, Ana1, and Bld10p/Cep135 are in the PCL. Third, PCL formation depends on SAK/PLK4 and SAS-6. Using a genetic screen for PCL defect, we identified a mutation in the gene encoding the conserved centriolar protein POC1, which is part of the daughter centriole initiation site (Kilburn et al. 2007) in Tetrahymena. We conclude that the PCL resembles an early intermediate structure of a forming centriole, which may explain why no typical centriolar structure is observed under electron microscopy. We propose that, during the evolution of insects, the proximal centriole was simplified by eliminating the later steps in centriole assembly. The PCL may provide a unique model to study early steps of centriole formation.THE centriole is a cylindrical structure rich in microtubules, which are organized in a ninefold symmetry. As the template of the ciliary axoneme, the centriole transmits its symmetry to the cilium. Dividing cells contain two centrosomes at the cell poles, each containing a pair of centrioles (mother and daughter centrioles) surrounded by a thick layer of pericentriolar material (PCM). Upon differentiation, the mother centriole of each pair becomes a basal body, which acts as a template for the cilium (Azimzadeh and Bornens 2007). The function of the daughter centriole is less clear. For example, in animal spermatids, the mother centriole, known as the distal centriole, becomes a basal body and gives rise to the sperm flagellum (Krioutchkova and Onishchenko 1999; Sathananthan et al. 2001). The daughter centriole in spermatids, known as the proximal centriole, is attached to the nucleus.Unlike other animal groups, multiple ultrastructural studies of insect sperm find only one centriole that has the canonical structure of microtubules organized in a ninefold symmetry (Anderson 1967; Tates 1971; Tokuyasu 1975a,b). This centriole forms the flagellum and is therefore the homolog to the vertebrate distal centriole. During spermatid differentiation (Figure 2A), a structure, called the centriolar adjunct (CA), appears transiently around this centriole (Friedlander and Wahrman 1971; Tates 1971; Tokuyasu 1975a,b; Wilson et al. 1997). The centriolar adjunct is a very dynamic structure, which shrinks during spermatid differentiation to form a collar around the distal centriole and then disappears (Tates 1971; Wilson et al. 1997). Studies using light microscopy found that γ-tubulin localizes and redistributes around the centriole in a way similar to the CA (Wilson et al. 1997), suggesting that it may serve as its marker. Earlier electron microscopy (EM) studies describe the appearance of a centriole inside the centriolar adjunct after it obtains the collar-shape structure (Anderson 1967; Phillips 1970). But this structure is amorphous and does not exhibit the morphological features of a centriole. Interestingly, the earliest intermediate observed during centriole formation is described as an amorphous structure (Anderson 1967; Dippell 1968; Sorokin 1968; Allen 1969).Open in a separate windowFigure 2.—Ana1 labels a novel structure appearing near the mother centriole in spermatids. (A) Diagram depicting the different stages of spermatid development based on the observations of Tates (1971). (M, mitochondria; N, nucleus; Ax, axoneme). The basal body or giant centriole (Cen) is surrounded by the centriolar adjunct (CA) and, near it, we can follow the formation of the PCL. (B) We use phase-contrast pictures (unfixed testis) to determine the spermatid stage. The onion stage (stage S13) is characterized by a round nucleus (N) of the same size as the mitochondrial derivatives (M). The cell body of intermediate spermatids (stages 15 and 16) elongates, forming short protrusions (arrows), but the nucleus remains round. In late spermatid development (stage 17), the nucleus becomes oval. Ana1-GFP labels the giant centriole (Cen), and in intermediate spermatids a bulge forms on one side and becomes individualized as PCL in late spermatid development. (C) Staining with anti-γ-tubulin antibody shows that the PCL labeled by Ana1 is an entity different from the γ-tubulin collar that is reminiscent of the centriolar adjunct (CA). (D) Antibody against Ana1 labels the V-shape pair of giant centrioles in primary spermatocytes (left) and the giant centriole and PCL in spermatids (right) in flies expressing Ana1-GFP. (E) In wild-type primary spermatocytes, anti-Ana1 antibody stains the endogenous protein in the giant centrioles and colocalizes with γ-tubulin staining (left). In spermatids, the antibody labels the PCL, demonstrating that its formation is not due to centriolar protein overexpression (right).Centriole duplication provides the cell with a mechanism for tightly controlling the number of centrosomes and cilia. In most cells, the centriole duplicates once per cell cycle and a single new centriole is formed in the vicinity of each mother centriole. The mechanism ensuring that only one daughter centriole forms in the vicinity of the mother centriole is not known (Strnad and Gonczy 2008). Two major limiting factors hinder the investigation of this process: (1) the difficulty of distinguishing between the mother centriole and the forming daughter centriole and (2) the short time that it takes for the process to reach completion, which in turn hinders the identification of intermediates. Few model systems are currently available for studying this process (Pelletier et al. 2006; Kleylein-Sohn et al. 2007).Here, we demonstrate that fly spermatids contain a novel structure that is labeled by centriolar proteins and that forms in the vicinity of the proximal end of the mother centriole. Because it is reminiscent of the vertebrate proximal centriole but no morphological signatures of a centriole have been observed, we propose to call it proximal centriole-like (PCL). While studying the pan-centriolar protein Ana1, we found that it labeled the PCL. The PCL forms before γ-tubulin is redistributed as a collar, showing that it is a distinct entity. We then found that the formation of the PCL depends on the proteins SAK/PLK4 and SAS-6, which are essential early in daughter centriole formation, but not on SAS-4, which in worms is required later in the process. These observations indicate that the PCL represents an early intermediate structure in centriole formation. We also tested the involvement of the centriolar protein Bld10p/Cep135, which was found in Chlamydomonas and humans to be a component of the centriole cartwheel and wall (Hiraki et al. 2007; Kleylein-Sohn et al. 2007). We found that Bld10p is recruited to the PCL only later in the process and is not required for PCL formation. We performed a genetic screen finding that the Drosophila ortholog of POC1 is essential for the formation of normal PCL. POC1 was identified previously in a proteomic screen as a centriolar protein and is localized to the early intermediate structure in centriole/basal body formation (Keller et al. 2005, 2008; Kilburn et al. 2007). We propose to use PCL formation as a model to study the molecular pathway for centriole initiation. Our results suggest that POC1, like PLK4 and SAS-6, plays an important role early in centriole formation whereas Bld10p function is required later as SAS-4 is.     

Current management of patients hospitalized with community-acquired pneumonia across Europe: outcomes from REACH

Background

Data describing real-life management and treatment of community-acquired pneumonia (CAP) in Europe are limited. REACH (http://{"type":"clinical-trial","attrs":{"text":"NCT01293435","term_id":"NCT01293435"}}NCT01293435) was a retrospective, observational study collecting data on the management of EU patients hospitalized with CAP.The purpose of this study was to understand patient and disease characteristics in patients hospitalized with CAP and to review current clinical practices and outcomes.

Methods

Patients were aged ≥18 years, hospitalized with CAP between March 2010 and February 2011, and requiring in-hospital treatment with intravenous antibiotics. An electronic Case Report Form was used to collect patient, disease and treatment variables, including type of CAP, medical history, treatment setting, antibiotics administered and clinical outcomes.

Results

Patients (N = 2,039) were recruited from 128 centres in ten EU countries (Belgium, France, Germany, Greece, Italy, the Netherlands, Portugal, Spain, Turkey, UK). The majority of patients were aged ≥65 years (56.4%) and had CAP only (78.8%). Initial antibiotic treatment modification occurred in 28.9% of patients and was more likely in certain groups (patients with comorbidities; more severely ill patients; patients with healthcare-associated pneumonia, immunosuppression or recurrent episodes of CAP). Streamlining (de-escalation) of therapy occurred in 5.1% of patients. Mean length of hospital stay was 12.6 days and overall mortality was 7.2%.

Conclusion

These data provide a current overview of clinical practice in patients with CAP in EU hospitals, revealing high rates of initial antibiotic treatment modification. The findings may precipitate reassessment of optimal management regimens for hospitalized CAP patients.     

PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified c entrosomal P 4.1‐a ssociated p rotein (CPAP), a human homologue of SAS‐4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage‐specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho‐resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle.     

Inflammatory parameters predict etiologic patterns but do not allow for individual prediction of etiology in patients with CAP – Results from the German competence network CAPNETZ

Background

Aim of this study was to evaluate the correlation of inflammatory markers procalcitonin (PCT), C-reactive protein (CRP) and leukocyte count (WBC) with microbiological etiology of CAP.

Methods

We enrolled 1337 patients (62 ± 18 y, 45% f) with proven CAP. Extensive microbiological workup was performed. In all patients PCT, CRP, WBC and CRB-65 score were determined. Patients were classified according to microbial diagnosis and CRB-65 score.

Results

In patients with typical bacterial CAP, levels of PCT, CRP and WBC were significantly higher compared to CAP of atypical or viral etiology. There were no significant differences in PCT, CRP and WBC in patients with atypical or viral etiology of CAP. In contrast to CRP and WBC, PCT markedly increased with severity of CAP as measured by CRB-65 score (p < 0.0001). In ROC analysis for discrimination of patients with CRB-65 scores > 1, AUC for PCT was 0.69 (95% CI 0.66 to 0.71), which was higher compared to CRP and WBC (p < 0.0001). CRB-65, PCT, CRP and WBC were higher (p < 0.0001) in hospitalised patients in comparison to outpatients.

Conclusion

PCT, CRP and WBC are highest in typical bacterial etiology in CAP but do not allow individual prediction of etiology. In contrast to CRP and WBC, PCT is useful in severity assessment of CAP.     

Pandemic Influenza (H1N1) 2009 Pneumonia: CURB-65 Score for Predicting Severity and Nasopharyngeal Sampling for Diagnosis Are Unreliable

Background

From the first case reports of pandemic influenza (H1N1) 2009 it was clear that a significant proportion of infected individuals suffered a primary viral pneumonia. The objective of this study was twofold; to assess the utility of the CURB-65 community acquired pneumonia (CAP) severity index in predicting pneumonia severity and ICU admission, and to assess the relative sensitivity of nasopharyngeal versus lower respiratory tract sampling for the detection of pandemic influenza (H1N1) CAP.

Methods

A retrospective cohort study of 70 patients hospitalised for pandemic influenza (H1N1) 2009 in an adult tertiary referral hospital. Characteristics evaluated included age, pregnancy status, sex, respiratory signs and symptoms, smoking and alcohol history, CURB-65 score, co-morbidities, disabling sequelae, length of stay and in-hospital mortality outcomes. Laboratory features evaluated included lymphocyte count, C-reactive protein (CRP), nasopharyngeal and lower respiratory tract pandemic influenza (H1N1) 2009 PCR results.

Results

Patients with pandemic (H1N1) 2009 influenza CAP differed significantly from those without pneumonia regarding length of stay, need for ICU admission, CRP and the likelihood of disabling sequelae. The CURB-65 score did not predict CAP severity or the need for ICU admission (only 2/11 patients admitted to ICU had CURB-65 scores of 2 or 3). Nasopharyngeal specimens for PCR were only 62.9% sensitive in CAP patients compared to 97.8% sensitivity for lower respiratory tract specimens.

Conclusions

The CURB-65 score does not predict severe pandemic influenza (H1N1) 2009 CAP or need for ICU admission. Lower respiratory tract specimens should be collected when pandemic (H1N1) 2009 influenza CAP is suspected.     

Regulated HsSAS-6 levels ensure formation of a single procentriole per centriole during the centrosome duplication cycle

Centrosome duplication involves the formation of a single procentriole next to each centriole, once per cell cycle. The mechanisms governing procentriole formation and those restricting its occurrence to one event per centriole are poorly understood. Here, we show that HsSAS-6 is necessary for procentriole formation and that it localizes asymmetrically next to the centriole at the onset of procentriole formation. HsSAS-6 levels oscillate during the cell cycle, with the protein being degraded in mitosis and starting to accumulate again at the end of the following G1. Our findings indicate that APC(Cdh1) targets HsSAS-6 for degradation by the 26S proteasome. Importantly, we demonstrate that increased HsSAS-6 levels promote formation of more than one procentriole per centriole. Therefore, regulated HsSAS-6 levels normally ensure that each centriole seeds the formation of a single procentriole per cell cycle, thus playing a fundamental role in driving the centrosome duplication cycle and ensuring genome integrity.     

Targeting of β-Arrestin2 to the Centrosome and Primary Cilium: Role in Cell Proliferation Control

Background

The primary cilium is a sensory organelle generated from the centrosome in quiescent cells and found at the surface of most cell types, from where it controls important physiological processes. Specific sets of membrane proteins involved in sensing the extracellular milieu are concentrated within cilia, including G protein coupled receptors (GPCRs). Most GPCRs are regulated by β-arrestins, βarr1 and βarr2, which control both their signalling and endocytosis, suggesting that βarrs may also function at primary cilium.

Methodology/Principal Findings

In cycling cells, βarr2 was observed at the centrosome, at the proximal region of the centrioles, in a microtubule independent manner. However, βarr2 did not appear to be involved in classical centrosome-associated functions. In quiescent cells, both in vitro and in vivo, βarr2 was found at the basal body and axoneme of primary cilia. Interestingly, βarr2 was found to interact and colocalize with 14-3-3 proteins and Kif3A, two proteins known to be involved in ciliogenesis and intraciliary transport. In addition, as suggested for other centrosome or cilia-associated proteins, βarrs appear to control cell cycle progression. Indeed, cells lacking βarr2 were unable to properly respond to serum starvation and formed less primary cilia in these conditions.

Conclusions/Significance

Our results show that βarr2 is localized to the centrosome in cycling cells and to the primary cilium in quiescent cells, a feature shared with other proteins known to be involved in ciliogenesis or primary cilium function. Within cilia, βarr2 may participate in the signaling of cilia-associated GPCRs and, therefore, in the sensory functions of this cell “antenna”.     

Variability in pediatric infectious disease consultants' recommendations for management of community-acquired pneumonia

Background

Community-acquired pneumonia (CAP) is a common childhood infection. CAP complications, such as parapneumonic empyema (PPE), are increasing and are frequently caused by antibiotic-resistant organisms. No clinical guidelines currently exist for management of pediatric CAP and no published data exist about variations in antibiotic prescribing patterns. Our objectives were to describe variation in CAP clinical management for hospitalized children by pediatric infectious disease consultants and to examine associations between recommended antibiotic regimens and local antibiotic resistance levels.

Methods

We surveyed pediatric members of the Emerging Infections Network, which consists of 259 pediatric infectious disease physicians. Participants responded regarding their recommended empiric antibiotic regimens for hospitalized children with CAP with and without PPE and their recommendations for duration of therapy. Participants also provided information about the prevalence of penicillin non-susceptible S. pneumoniae and methicillin-resistant S. aureus (MRSA) in their community.

Results

We received 148 responses (57%). For uncomplicated CAP, respondents were divided between recommending beta-lactams alone (55%) versus beta-lactams in combination with another class (40%). For PPE, most recommended a combination of a beta-lactam plus an anti-MRSA agent, however, they were divided between clindamycin (44%) and vancomycin (57%). The relationship between reported antibiotic resistance and empiric regimen was mixed. We found no relationship between aminopenicillin use and prevalence of penicillin non-suscepetible S. pneumoniae or clindamycin use and clindamycin resistance, however, respondents were more likely to recommend an anti-MRSA agent when MRSA prevalence increased.

Conclusions

Substantial variability exists in recommendations for CAP management. Development of clinical guidelines via antimicrobial stewardship programs and dissemination of data about local antibiotic resistance patterns represent opportunities to improve care.