Cellular Sites Research Articles

Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome.

The adenomatous polyposis coli (APC) tumor suppressor is a multifunctional regulator of Wnt signaling and acts as a mobile scaffold at different cellular sites. APC was recently found to stimulate microtubule (MT) growth at the interphase centrosome; however, little is known about its dynamics and localization at this site. To address this, we analysed APC dynamics in fixed and live cells by fluorescence microscopy. In detergent-extracted cells, we discovered that APC was only weakly retained at the centrosome during interphase suggesting a rapid rate of exchange. This was confirmed in living cells by fluorescence recovery after photobleaching (FRAP), which identified two pools of green fluorescent protein (GFP)-APC: a major rapidly exchanging pool (~86%) and minor retained pool (~14%). The dynamic exchange rate of APC was unaffected by C-terminal truncations implicating a targeting role for the N-terminus. Indeed, we mapped centrosome localization to N-terminal armadillo repeat (ARM) domain amino acids 334–625. Interestingly, the rate of APC movement to the centrosome was stimulated by intact MTs, and APC dynamics slowed when MTs were disrupted by nocodazole treatment or knockdown of γ-tubulin. Thus, the rate of APC recycling at the centrosome is enhanced by MT growth, suggesting a positive feedback to stimulate its role in MT growth.

New aspects of p66Shc in ischaemia reperfusion injury and other cardiovascular diseases.

This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Tissue‐Specific Seasonal Changes in Mitochondrial Membrane Composition and Respiratory Function in the Golden‐Mantled Ground Squirrel

Mammals that hibernate, such as the golden‐mantled ground squirrel (Callospermophilus lateralis; GMGS), cease to feed, reduce metabolic rate, and lower body temperature during the winter months, surviving almost exclusively on the oxidation of lipids from endogenous fat stores. Whether mitochondria, the cellular sites of oxidative metabolism, undergo adaptive changes in response to hypometabolism and decreasing ambient temperature to facilitate this remarkable phenotype is unclear. It has been postulated that changes in mitochondrial membrane composition reported in response to cold exposure in some species may facilitate maintenance of respiratory enzyme function with decreasing temperature. However, no studies to date have investigated the function and membrane fatty acid composition of mitochondria from different tissues from the warm summer months, to fall and winter in a hibernator. We hypothesize that tissue‐specific differences in mitochondrial respiration occur across seasons in the GMGS that may parallel distinct changes in mitochondrial membrane composition. We compared the respiratory capacity, oxidative enzyme activity, and membrane composition of GMGS mitochondria isolated from liver, heart, skeletal muscle, and brown adipose tissue (BAT) from summer, fall, and winter (torpid) seasons. Maximal mitochondrial oxidative phosphorylation (OXPHOS)‐supported respiration was determined by high‐resolution respirometry at 37°C, 25°C, and 15°C in the presence of saturating concentrations of ADP and respiratory substrates. Mitochondrial membrane fatty acid composition was determined by gas chromatography on phospholipid fractions obtained from isolated mitochondria. Maximal OXPHOS assayed at 37°C tended to increase from summer to winter in liver, heart and BAT, and decrease in skeletal muscle, with tissue‐ and season‐specific changes in pyruvate versus fatty acid (palmitoylcarnitine) oxidation capacity. OXPHOS capacity was uniformly suppressed by decreasing assay temperature in all tissue mitochondria from torpid GMGS in the winter, despite widely variable changes in mitochondrial membrane composition across tissues and seasons. Interestingly, a consistent relative decrease in mitochondrial membrane docosahexaenoic acid (DHA; 22:6n3) was seen from summer to winter in all tissues, the biological relevance of which merits further investigation. Taken together, these findings argue against a consistent relationship between changes in mitochondrial membrane composition and respiratory function across seasons in GMGS, but highlight distinct tissue‐ and season‐specific differences that may have important biological effects that remain to be elucidated.

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK’s (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.

Amyloid precursor-like protein 1 (APLP1) exhibits stronger zinc-dependent neuronal adhesion than amyloid precursor protein and APLP2.

The amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein 1 (APLP1) and APLP2, are metalloproteins with a putative role both in synaptogenesis and in maintaining synapse structure. Here, we studied the effect of zinc on membrane localization, adhesion, and secretase cleavage of APP, APLP1, and APLP2 in cell culture and rat neurons. For this, we employed live-cell microscopy techniques, a microcontact printing adhesion assay and ELISA for protein detection in cell culture supernatants. We report that zinc induces the multimerization of proteins of the amyloid precursor protein family and enriches them at cellular adhesion sites. Thus, zinc facilitates the formation of de novo APP and APLP1 containing adhesion complexes, whereas it does not have such influence on APLP2. Furthermore, zinc-binding prevented cleavage of APP and APLPs by extracellular secretases. In conclusion, the complexation of zinc modulates neuronal functions of APP and APLPs by (i) regulating formation of adhesion complexes, most prominently for APLP1, and (ii) by reducing the concentrations of neurotrophic soluble APP/APLP ectodomains. Earlier studies suggest a function of the amyloid precursor protein (APP) family proteins in neuronal adhesion. We report here that adhesive function of these proteins is tightly regulated by zinc, most prominently for amyloid precursor-like protein 1 (APLP1). Zinc-mediated APLP1 multimerization, which induced formation of new neuronal contacts and decreased APLP1 shedding. This suggests that APLP1 could function as a zinc receptor processing zinc signals to stabilized or new neuronal contacts.

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied the UL116 gene product, a putative open reading frame identified by in silico analysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of the UL116 gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex. HCMV infection can cause severe disease in immunocompromised adults and infants infected in utero The dissection of the HCMV entry machinery is important to understand the mechanism of viral infection and to identify new vaccine antigens. The gH/gL/gO and gH/gL/UL128/UL130/UL131 (Pentamer) complexes play a key role in HCMV cell entry and tropism. Both complexes are formed by an invariant gH/gL scaffold on which the other subunits assemble. Here, we show that the UL116 gene product is expressed in infected cells and forms a heterodimer with gH. The gH/UL116 complex is carried on the infectious virions, although in smaller amounts than gH/gL complexes. No gH/UL116/gL ternary complex formed in transfected cells, suggesting that the gH/UL116 complex is independent from gL. This new gH-based gL-free complex represents a potential target for a protective HCMV vaccine and opens new perspectives on the comprehension of the HCMV cell entry mechanism and tropism.

HIV-1 cellular and tissue replication patterns in infected humanized mice.

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human.

Renal epithelium regulates erythropoiesis via HIF-dependent suppression of erythropoietin.

The adult kidney plays a central role in erythropoiesis and is the main source of erythropoietin (EPO), an oxygen-sensitive glycoprotein that is essential for red blood cell production. Decreases of renal pO2 promote hypoxia-inducible factor 2-mediated (HIF-2-mediated) induction of EPO in peritubular interstitial fibroblast-like cells, which serve as the cellular site of EPO synthesis in the kidney. It is not clear whether HIF signaling in other renal cell types also contributes to the regulation of EPO production. Here, we used a genetic approach in mice to investigate the role of renal epithelial HIF in erythropoiesis. Specifically, we found that HIF activation in the proximal nephron via induced inactivation of the von Hippel-Lindau tumor suppressor, which targets the HIF-α subunit for proteasomal degradation, led to rapid development of hypoproliferative anemia that was associated with a reduction in the number of EPO-producing renal interstitial cells. Moreover, suppression of renal EPO production was associated with increased glucose uptake, enhanced glycolysis, reduced mitochondrial mass, diminished O2 consumption, and elevated renal tissue pO2. Our genetic analysis suggests that tubulointerstitial cellular crosstalk modulates renal EPO production under conditions of epithelial HIF activation in the kidney.

FTIR Biospectroscopy Investigation on Cisplatin Cytotoxicity in Three Pairs of Sensitive and Resistant Cell Line.

Fourier Transformed Infrared Spectroscopy (FTIR) has extensively been used for biological applications. Cisplatin is one the most useful antineoplastic chemotherapy drugs for a variety of different human cancers. One of the clinical problems in its application, which would consequently affect the therapeutic outcome of its application, is the occurrence of resistance to this agent. In this project three different pairs of sensitive and resistant cell lines of human ovarian A2780 and its resistant pair of A2780-CP, human ovarian OV2008 and its resistant pair of C13, and finally human lung carcinoma of HTB56 and its resistant pair of HTB56-CP were grown in the laboratory under the standard procedure. Saline was exposed to control cells, whereas 1, 5 and 10 µg/mL of cisplatin was exposed to experimental cells, for 1 h. Cells were then collected and lyophilized from which spectra were taken. According to our results, we could not trigger a well-recognized cells biomolecular band at 1015 cm(-1), being modified after exposure to cisplatin in all cell lines. On the other hand, there was a clear dose-dependent increase in protein β-sheet structure related peaks shift in resistant cell lines after exposure to cisplatin. This would probably indicate an easier protein interaction site for cisplatin in the resistant cell lines, which would probably inhibit cisplatin from binding to DNA, as the cytotoxic target. As a conclusion, FTIR biospectroscopy has proven its potency to identify the interactions, as well as the false engagement cellular sites for cisplatin in sensitive and resistant cell lines.

Rab32 and Rab38 genes in chordate pigmentation: an evolutionary perspective.

BackgroundThe regulation of cellular membrane trafficking in all eukaryotes is a very complex mechanism, mostly regulated by the Rab family proteins. Among all membrane-enclosed organelles, melanosomes are the cellular site for synthesis, storage and transport of melanin granules, making them an excellent model for studies on organelle biogenesis and motility. Specific Rab proteins, as Rab32 and Rab38, have been shown to play a key role in melanosome biogenesis. We analysed the Rab32 and Rab38 genes in the teleost zebrafish and in the cephalochordate amphioxus, gaining insight on their evolutionary history following gene and genome duplications.ResultsWe studied the molecular evolution of Rab supergroup III in deuterostomes by phylogenetic reconstruction, intron and synteny conservation. We discovered a novel amino acid stretch, named FALK, shared by three related classes belonging to Rab supergroup III: Rab7L1, Rab32LO and Rab32/Rab38. Among these, we demonstrated that the Rab32LO class, already present in the last common eukaryotic ancestor, was lost in urochordates and vertebrates. Synteny shows that one zebrafish gene, Rab38a, which is expressed in pigmented cells, retained the linkage with tyrosinase, a protein essential for pigmentation. Moreover, the chromosomal linkage of Rab32 or Rab38 with a member of the glutamate receptor metabotropic (Grm) family has been retained in all analysed gnathostomes, suggesting a conserved microsynteny in the vertebrate ancestor. Expression patterns of Rab32 and Rab38 genes in zebrafish, and Rab32/38 in amphioxus, indicate their involvement in development of pigmented cells and notochord.ConclusionsPhylogenetic, intron conservation and synteny analyses point towards an evolutionary scenario based on a duplication of a single invertebrate Rab32/38 gene giving rise to vertebrate Rab32 and Rab38. The expression patterns of Rab38 paralogues highlight sub-functionalization event. Finally, the discovery of a chromosomal linkage between the Rab32 or Rab38 gene with a Grm opens new perspectives on possible conserved bystander gene regulation across the vertebrate evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0596-1) contains supplementary material, which is available to authorized users.

Metformin is not just an antihyperglycaemic drug but also has protective effects on the vascular endothelium.

Metformin, a synthetic dimethyl biguanide, has been in clinical use for over 55years, and today is considered the first-choice drug for the treatment of type 2 diabetes used by an estimated 125million people worldwide. Metformin is orally effective, not metabolized, excreted unchanged by the kidney, relatively free of side effects and well tolerated by the majority of patients. Of importance is that the United Kingdom Prospective Diabetes Study 20-year study of type 2 diabetics, completed in 1998, compared patients treated with insulin, sulfonylureas and metformin and concluded that metformin provided vascular protective actions. Cardiovascular disease is the primary basis for the high morbidity and mortality that is associated with diabetes and that metformin proved to be protective resulted in a dramatic increase in its use. The vascular protective actions of metformin are thought to be secondary to the antihyperglycaemic effects of metformin that are mediated via activation of AMP kinase and subsequent inhibition of hepatic gluconeogenesis, fatty acid oxidation as well as an insulin sensitizing action in striated muscle and adipose tissue. As reflected by a number of clinical studies, patients treated with metformin also have improvement in endothelial function as measured by the use of plethysmography and measurement of flow-mediated vasodilatation. These data as well as data from animal studies are supportive that metformin has a direct protective action on the vascular endothelium. In this review article, we discuss the pharmacology of metformin and critique the literature as to its cellular sites and mechanism(s) of action.

Nanocarriers Based Anticancer Drugs: Current Scenario and Future Perceptions.

Anticancer therapies mostly depend on the ability of the bioactives to reach their designated cellular and subcellular target sites, while minimizing accumulation and side effects at non specific sites. The development of nanotechnology based drug delivery systems that are able to modify the biodistribution, tissue uptake and pharmacokinetics of therapeutic agents is considered of great importance in biomedical research and treatment therapy. Controlled releases from nanocarriers can significantly enhance the therapeutic effect of a drug. Nanotechnology has the potential to revolutionize in cancer diagnosis and therapy. Targeted nano medicines either marketed or under development, are designed for the treatment of various types of cancer. Nanocarriers are able to reduce cytotoxic effect of the active anticancer drugs by increasing cancer cell targeting in comparison to conventional formulations. The newly developed nano devices such as quantum dots, liposomes, nanotubes, nanoparticles, micelles, gold nanoparticles, carbon nanotubes and solid lipid nanoparticles are the most promising applications for various cancer treatments. This review is focused on currently available information regarding pharmaceutical nanocarriers for cancer therapy and imaging.

The Heritage of 30 Years of Mobile Communications in the UK

The first mobile phone networks were launched in the UK in 1985. In the 30 years that have elapsed since then, the mobile phone has evolved from an expensive device owned by the few to a mass-market smartphone that is now regarded as an essential part of modern-day life. Behind the scenes, the mobile networks have undergone four major technology evolutions that have transformed their functionality from providing a basic telephone service to offering very high-speed connectivity for data applications. Yet, much of the heritage of this relatively young but transformational mobile technology is poorly documented and in great danger of being completely lost. Each new generation of mobile network replaces and ultimately erases the evidence of its predecessor. Equally, manufacturers are so focused on the release of their next handset that this leaves little scope or motivation for preserving and recording older models. The perception remains that the mobile phone is a new and young technology which is still considered by many to be a recent phenomenon. This results in a very casual attitude to the preservation of cellular mobile communications heritage, hence raising the significant risk that no suitable historical records will be available for future generations. This paper therefore begins to address this deficit by examining, through a UK perspective, two key but highly visible parts of the mobile phone story, namely, the cellular radio site installations that adorn roadsides, rooftops and hillsides and the mobile phones themselves as important historical artefacts.

Expression mediated by three partial sequences of the human tyrosine hydroxylase promoter in vivo

The use of viral vectors to transfect postmitotic neurons has provided an important research tool, and it offers promise for treatment of neurologic disease. The utility of vectors is enhanced by the use of selective promoters that permit control of the cellular site of expression. One potential clinical application is in the neurorestorative treatment of Parkinson’s disease by the induction of new axon growth. However, many of the genes with an ability to restore axons have oncogenic potential. Therefore, clinical safety would be enhanced by restriction of expression to neurons affected by the disease, particularly dopamine neurons. To achieve this goal we have evaluated in vivo three partial sequences of the promoter for human tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis. All sequences induced expression in dopamine neurons. None of them induced expression in glia or in nondopaminergic neurons in striatum or cortex. We conclude that these sequences have potential use for targeting dopamine neurons in research and clinical applications.

Local expression of SOD1G93A mutant protein triggers neuromuscular junction dismantlement

The alteration of Reactive Oxygen Species (ROS) homeostasis plays a causal role in several chronic pathology such as aging and neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). Although it is recognized that axon and synapses are first cellular sites of degeneration in ALS disease, controversy exists on whether pathological events initially begin at the NMJs and then, in a dying back phenomena, contribute to motor neuron degeneration. Moreover, the precise molecular mechanisms of pathology-associated deterioration in neuromuscular system have remained elusive (1). Here we provide evidences that muscle specific accumulation of SOD1G93A in the transgenic mice model MLC/SOD1G93A (2) induces mitochondria dysfunction and triggers NMJ dismantlement. Further, we demonstrate that treatment of MLC/SOD1G93A mice with Trolox, a potent antioxidant, is sufficient to rescue mitochondria and NMJ defects in the MLC/SOD1G93A mice, stabilizing muscle-nerve connection. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 revealed the activation of specific Protein Kinase as a downstream player of NMJ dismantlement. Overall our data demonstrate that muscle specific expression of SOD1G93A mutation causes mitochondrial impairment and NMJ dismantlement, suggesting that muscle defects and NMJs alteration precede motor neuron degeneration rather than resulting from it.

The CDKN2A/p16(INK) (4a) 5'UTR sequence and translational regulation: impact of novel variants predisposing to melanoma.

Many variants of uncertain functional significance in cancer susceptibility genes lie in regulatory regions, and clarifying their association with disease risk poses significant challenges. We studied 17 germline variants (nine of which were novel) in the CDKN2A 5'UTR with independent approaches, which included mono and bicistronic reporter assays, Western blot of endogenous protein, and allelic representation after polysomal profiling to investigate their impact on CDKN2A mRNA translation regulation. Two of the novel variants (c.-27del23, c.-93-91delAGG) were classified as causal mutations (score ≥3), along with the c.-21C>T, c.-34G>T, and c.-56G>T, which had already been studied by a subset of assays. The novel c.-42T>A as well as the previously described c.-67G>C were classified as potential mutations (score 1 or 2). The remaining variants (c.-14C>T, c.-20A>G, c.-25C>T+c.-180G>A, c.-30G>A, c.-40C>T, c.-45G>A, c.-59C>G, c.-87T>A, c.-252A>T) were classified as neutral (score 0). In conclusion, we found evidence that nearly half of the variants found in this region had a negative impact on CDKN2A mRNA translation, supporting the hypothesis that 5'UTR can act as a cellular Internal Ribosome Entry Site (IRES) to modulate p16(INK) (4a) translation.

Ultrasound-stimulated microbubble enhancement of radiation treatments: endothelial cell function and mechanism.

Endothelial cell death caused by novel microbubble-enhanced ultrasound cancer therapy leads to secondary tumour cell death. In order to characterize and optimize these treatments, the molecular mechanisms resulting from the interaction with endothelial cells were investigated here. Endothelial cells (HUVEC) were treated with ultrasound-stimulated microbubbles (US/MB), radiation (XRT), or a combination of US/MB+XRT. Effects on cells were evaluated at 0, 3, 6, and 24 hours after treatment. Experiments took place in the presence of modulators of sphingolipid-based signalling including ceramide, fumonisin B1, monensin, and sphingosine-1-phosphate. Experimental outcomes were evaluated using histology, TUNEL, clonogenic survival methods, immuno-fluorescence, electron microscopy, and endothelial cell blood-vessel-like tube forming assays. Fewer cells survived after treatment using US/MB+XRT compared to either the control or XRT. The functional ability to form tubes was only reduced in the US/ MB+XRT condition in the control, the ceramide, and the sphingosine-1-phosphate treated groups. The combined treatment had no effect on tube forming ability in either the fumonisin B1 or in the monensin exposed groups, since both interfere with ceramide production at different cellular sites. In summary, experimental results supported the role of ceramide signalling as a key element in cell death initiation with treatments using US/MB+XRT to target endothelial cells.

Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes.

Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.

Memory Formation Shaped by Astroglia.

Astrocytes, the most heterogeneous glial cells in the central nervous system (CNS), execute a multitude of homeostatic functions and contribute to memory formation. Consolidation of synaptic and systemic memory is a prolonged process and hours are required to form long-term memory. In the past, neurons or their parts have been considered to be the exclusive cellular sites of these processes, however, it has now become evident that astrocytes provide an important and essential contribution to memory formation. Astrocytes participate in the morphological remodeling associated with synaptic plasticity, an energy-demanding process that requires mobilization of glycogen, which, in the CNS, is almost exclusively stored in astrocytes. Synaptic remodeling also involves bidirectional astroglial-neuronal communication supported by astroglial receptors and release of gliosignaling molecules. Astroglia exhibit cytoplasmic excitability that engages second messengers, such as Ca2+, for phasic, and cyclic adenosine monophosphate (cAMP), for tonic signal coordination with neuronal processes. The detection of signals by astrocytes and the release of gliosignaling molecules, in particular by vesicle-based mechanisms, occurs with a significant delay after stimulation, orders of magnitude longer than that present in stimulus–secretion coupling in neurons. These particular arrangements position astrocytes as integrators ideally tuned to support time-dependent memory formation.

P70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung.

The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-β-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung.