The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.     

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1)

Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.     

Histamine H(1) receptor signaling regulates effector T cell responses and susceptibility to coxsackievirus B3-induced myocarditis

Susceptibility to autoimmune myocarditis has been associated with histamine release by mast cells during the innate immune response to coxsackievirus B3 (CVB3) infection. To investigate the contribution of histamine H(1) receptor (H(1)R) signaling to CVB3-induced myocarditis, we assessed susceptibility to the disease in C57BL/6J (B6) H(1)R(-/-) mice. No difference was observed in mortality between CVB3-infected B6 and H(1)R(-/-) mice. However, analysis of their hearts revealed a significant increase in myocarditis in H(1)R(-/-) mice that is not attributed to increased virus replication. Enhanced myocarditis susceptibility correlated with a significant expansion in pathogenic Th1 and Vγ4(+) γδ T cells in the periphery of these animals. Furthermore, an increase in regulatory T cells was observed, yet these cells were incapable of controlling myocarditis in H(1)R(-/-) mice. These data establish a critical role for histamine and H(1)R signaling in regulating T cell responses and susceptibility to CVB3-induced myocarditis in B6 mice.     

Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles

Objective

To explore the capacity of human CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes to phagocyte iron-oxide nanoparticles in vitro.

Methods

Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.

Results

Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14⁺⁺CD16^- monocytes displayed a significantly higher uptake compared to CD14⁺CD16⁺⁺ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14⁺CD16⁺⁺ and CD14⁺⁺CD16^- monocytes differently.

Conclusion

Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.     

Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.     

Drought Effects in Climate Change Manipulation Experiments: Quantifying the Influence of Ambient Weather Conditions and Rain-out Shelter Artifacts

Extreme drought events challenge ecosystem functioning. Ecological response to drought is studied worldwide in a growing number of field experiments by rain-out shelters. Yet, few meta-analyses face severe challenges in the comparability of studies. This is partly because build-up of drought stress in rain-out shelters is modified by ambient weather conditions. Rain-out shelters can further create confounding effects (radiation, temperature), which may influence plant responses. Yet, a quantification of ecophysiological effects within rain-out shelters under opposing ambient weather conditions and of microclimatological artifacts is missing. Here, we examined phytometers—standardized potted individuals of Plantago lanceolata—under rain-out shelter, rain-out shelter artifact control, and ambient control during opposing outside microclimatological conditions. Furthermore, we tested for artifacts of rain-out shelters on plant responses in a long-term semi-natural grassland experiment. Phytometer plants below the rain-out shelters showed lower stomatal conductance, maximum quantum efficiency, and leaf water potential during warm ambient conditions with high evaporative demand than during cold conditions with low evaporative demand. Plant performance was highly correlated with ambient temperature and vapor pressure deficit (VPD). Rain-out shelter artifacts on plant responses were nonsignificant. Rain-out shelters remain a viable tool for studying ecosystem responses to drought. However, drought manipulations using rain-out shelters are strongly modified by ambient weather conditions. Attributing the results from rain-out shelter studies to drought effects and comparability among studies and study years therefore requires the quantification of the realized drought stress, for example, by relating ecosystem responses to measured microclimatological parameters such as air temperature and VPD.     

Usefulness of 1,3 Beta-<Emphasis Type="SmallCaps">d</Emphasis>-Glucan Detection in non-HIV Immunocompromised Mechanical Ventilated Critically Ill Patients with ARDS and Suspected <Emphasis Type="Italic">Pneumocystis jirovecii</Emphasis> Pneumonia

Introduction

Pneumocystis jirovecii pneumonia (PCP) is a major cause of disease in immunocompromised individuals. Diagnosis is typically obtained by microscopy and/or PCR. For ambiguous PCR results, we evaluated the new biomarker 1,3-Beta-d-Glucan (BDG).

Methods

BDG serum levels were assessed and correlated to PCR results in immunosuppressed patients with ARDS.

Results

11 (22%) out of 50 patients had suspected PCP. APACHE II (26 vs. 24; p < 0.002), SOFA score (16 vs. 14; p < 0.010) and mortality rate (34 vs. 69% p < 0.004; 34 vs. 80% p < 0.003) were significantly altered in patients with positive (pPCR) and slightly positive (spPCR) PCJ PCR as compared to patients with no-PCP (nPCP). BDG levels were significantly lower in patients with nPCP (86; 30–315 pg/ml) than in patients with pPCR (589; 356–1000 pg/ml; p < 0.001) and spPCP (398; 297–516 pg/ml; p < 0.004) referring to the cutoff in this study for PCP of 275 pg/ml. An overall sensitivity (S) of 92% (95% CI 86–96%) and specificity (SP) of 84% (95% CI 79–85%) for PCP were found for the BDG Fungitell assay. In detail, S of 98% (95% CI 94–100%) and SP of 86% (95% CI 82–92%) for pPCP and S of 98% (95% CI 96–100%) and SP of 88% (95% CI 86–96%) for spPCO were found.

Conclusion

Serum BDG levels were strongly elevated in PCP, and the negative predictive value is high. BDG could be used as a preliminary test for patients with suspected PCP, especially in patients with slightly positive PCR results.

     

Some observations of a correlation between the symmetry of large heavy-atom complexes and their binding sites on proteins

Two large, electron dense heavy-metal complexes were found to bind to crystals of heavy riboflavin synthase. The crystallographic analysis by difference Fourier methods shows that the tungsten cluster compound [(W3O2(O2CCH3)6]2+, which has trigonal symmetry, binds to a site on the 3-fold axis of the protein. The heteropolytungstate compound [NaP5W30O110]14-, which has pentagonal symmetry, binds to a site on the 5-fold axis of the protein.     

Central projections of fibers in the auditory and tensor nerves of cicadas (Homoptera: Cicadidae)

Summary The auditory and tensor nerves of cicadas are mixed nerves containing both afferent and efferent elements. In 17-year cicadas, and in Okanagana rimosa, the auditory nerve contains afferents from body hairs, from the detensor tympani-chordotonal organ, and some 1300–1500 afferents from the hearing organ. Within the fused metathoracic-abdominal ganglionic complex the receptors from both the auditory and tensor nerves form a neuropilar structure that reveals the metameric organization of this complex. A few fibers run anteriorly, projecting into the meso and prothoracic ganglia. Within the ganglionic complex a division of auditory nerve afferents into a dense intermediate and a more diffuse ventral neuropile is observed. In addition, a dorsal motor neuropile is outlined by arborizations of the timbal motor neuron. This neuron is one of several efferent cell types associated with the auditory nerve, and there is an indication that several efferent fibers innervate the timbal muscle. There is anatomical evidence for a possible neuronal coupling between the bilaterally symmetrical large timbal motor neurons. In general, central projections from the auditory and tensor nerves support evidence of a structural layering within the CNS of insects.