The activation of microglia and the infiltration of macrophages are hallmarks of neuroinflammation after acute brain injuries, including traumatic brain injury (TBI). The two myeloid populations ...share many features in the post-injury inflammatory response, thus, being antigenically indistinguishable. Recently Tmem119, a type I transmembrane protein specifically expressed by microglia under physiological conditions, was proposed as a tool to differentiate resident microglia from blood-borne macrophages, not expressing it. However, the validity of Tmem119 as a specific marker of resident microglia in the context of acute brain injury, where microglia are activated and macrophages are recruited, needs validation. Our purpose was to investigate Tmem119 expression and distribution in relation to the morphology of brain myeloid cells present in the injured area after TBI. Mice underwent sham surgery or TBI by controlled cortical impact (CCI). Brains from sham-operated, or TBI mice, were analyzed by
hybridization to identify the cells expressing
, and by Western blot and quantitative immunofluorescence to measure Tmem119 protein levels in the entire brain regions and single cells. The morphology of Iba1+ myeloid cells was analyzed at different times (4 and 7 days after TBI) and several distances from the contused edge in order to associate Tmem119 expression with morphological evolution of active microglia.
hybridization indicated an increased
RNA along with increased microglial complement C1q activation in the contused area and surrounding regions. On the contrary, the biochemical evaluation showed a drop in Tmem119 protein levels in the same areas. The Tmem119 immunoreactivity decreased in Iba1+ myeloid cells found in the contused cortex at both time points, with the cells showing the hypertrophic ameboid morphology having no Tmem119 expression. The Tmem119 was present on ramifications of resident microglia and its presence was decreased as a consequence of microglial activation in cortical areas close to contusion. Based on the data, we conclude that the decrease of Tmem119 in reactive microglia may depend on the process of microglial activation, which involves the retracting of their branchings to acquire an ameboid shape. The Tmem119 immunoreactivity decreases in reactive microglia to similar levels than the blood-borne macrophages, thus, failing to discriminate the two myeloid populations after TBI.
Lower respiratory tract infections are among the most common causes of death worldwide. Main pathogens leading to these severe infections are viruses and gram-positive bacteria that activate ...toll-like receptor (TLR)-mediated immune responses via pathogen-associated molecular patterns. One protective factor induced during infection is Chitinase-3-like 1 (CHI3L1), which exerts various functions, e.g. in host cell proliferation and bacterial counteraction, and has been proposed as a biomarker in several acute and chronic inflammatory conditions. MicroRNAs (miR) have become important regulators of inflammation and infection and are considered therapeutic targets in recent years. However, it is not known whether microRNAs play a role in the regulation of CHI3L1 expression in TLR-mediated respiratory epithelial cell inflammation. In this study, we analysed the pre- and post-transcriptional regulation of CHI3L1 by TLRs in bronchial epithelial cells. Therefore, we stimulated BEAS-2B cells with the bacterial TLR2-ligand lipoteichoic acid or the viral dsRNA analogue poly(I:C). We observed an increase in the expression of CHI3L1, which was dependent on TNF-α-mediated NF-κB activation in TLR2- and TLR3-activated cells. Moreover, TLR2 and − 3 stimulation caused downregulation of the microRNA miR-149-5p, an effect that could be suppressed by inhibiting NF-κB translocation into the nucleus. Luciferase reporter assays identified a direct interaction of miR-149-5p with the CHI3L1 3´untranslated region. This interaction was confirmed by inhibition and overexpression of miR-149-5p in BEAS-2B cells, which altered the expression levels of CHI3L1 mRNA. In summary, miR-149-5p directly regulates CHI3L1 in context of TLR-mediated airway epithelial cell inflammation and may be a potential therapeutic target in inflammation and other diseases.
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•Activation of TLR2 and TLR3 increases CHI3L1 and down-regulates miR-149-5p in bronchial epithelial cells.•Both changes are dependent on TNF-α-mediated NF-κB translocation to the nucleus.•miR-149-5p directly targets CHI3L1 3‵untranslated region.•Absence or presence of miR-149-5p function alters expression levels of CHI3L1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Tuft cells are a rare population of chemosensory cells at the mucosal surface epithelia of hollow organs. Their name-giving morphological feature is an apical tuft of stiff microvilli. Accordingly, ...the actin-binding protein, villin, was identified as one of the first tuft cell markers in immunohistochemical analysis. Unfortunately, villin expression is not restricted to tuft cells, but is also prominent e.g. in enterocytes, which limits the use of this gene as a marker and as an experimental tool to genetically target tuft cells. Here, we report that the villin-related protein, advillin, is a specific tuft cell marker in the gastro-intestinal and biliary tract epithelia. In situ hybridization and immunohistochemistry revealed that advillin expression, unlike villin, was restricted to solitary cholinergic tuft cells in the mucosal linings of the small and large intestine, and in the gall bladder. In the glandular stomach, villin and advillin mRNA were present in all epithelial cells, while detectable protein levels were confined to solitary tuft cells. Advillin expression was no longer detectable in the mucosa of the intestinal and biliary tract from
Pou2f3
deficient mice that lack tuft cells. Finally, crossing
Avil-Cre
transgenic mice with a double-fluorescent reporter mouse line resulted in specific targeting of gastro-intestinal and biliary tuft cells. Our analysis introduces advillin as a selective marker and tool in histological and functional analysis of the alimentary tract tuft cell system.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The gallbladder stores bile between meals and empties into the duodenum upon demand and is thereby exposed to the intestinal microbiome. This exposure raises the need for antimicrobial factors, among ...them, mucins produced by cholangiocytes, the dominant epithelial cell type in the gallbladder. The role of the much less frequent biliary tuft cells is still unknown. We here show that propionate, a major metabolite of intestinal bacteria, activates tuft cells via the short-chain free fatty acid receptor 2 and downstream signaling involving the cation channel transient receptor potential cation channel subfamily M member 5. This results in corelease of acetylcholine and cysteinyl leukotrienes from tuft cells and evokes synergistic paracrine effects upon the epithelium and the gallbladder smooth muscle, respectively. Acetylcholine triggers mucin release from cholangiocytes, an epithelial defense mechanism, through the muscarinic acetylcholine receptor M3. Cysteinyl leukotrienes cause gallbladder contraction through their cognate receptor CysLTR1, prompting emptying and closing. Our results establish gallbladder tuft cells as sensors of the microbial metabolite propionate, initiating dichotomous innate defense mechanisms through simultaneous release of acetylcholine and cysteinyl leukotrienes.
The fungus Ustilago maydis is a pathogen that establishes a biotrophic interaction with Zea mays. The interaction with the plant host is largely governed by more than 300 novel, secreted protein ...effectors, of which only four have been functionally characterized. Prerequisite to examine effector function is to know where effectors reside after secretion. Effectors can remain in the extracellular space, i.e. the plant apoplast (apoplastic effectors), or can cross the plant plasma membrane and exert their function inside the host cell (cytoplasmic effectors). The U. maydis effectors lack conserved motifs in their primary sequences that could allow a classification of the effectome into apoplastic/cytoplasmic effectors. This represents a significant obstacle in functional effector characterization. Here we describe our attempts to establish a system for effector classification into apoplastic and cytoplasmic members, using U. maydis for effector delivery.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
This study offered insight on the intermingling of discourse communities in the learning environment by examining experiences of composition instructors from a regional Southern university and their ...reflections of teaching students who struggled to navigate from home discourse to academic discourse. Merriam's basic qualitative research design served as the methodological design. Although interview was the primary method of collecting data on instructors' experiences, three types of data were collected to form a holistic view of the phenomenon: semi-structured in-depth interviews, researcher's field notes, and member checks of the transcriptions. The population consisted of ten, full-time faculty members who have taught composition courses each semester for the past five years. The sample population was employed at a regional Southern university. The data analysis consisted of coding for patterns and themes. The analysis offered findings that demonstrated various discourse communities existed in the learning environment. Underrepresented student populations were present in the classroom with nontraditional students as the most mentioned. Essay errors were corrected by instructor feedback with grammatical with mechanical errors were reported to be the most common error on composition essays. The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.
The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external ...medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.
Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there ...have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.
Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there ...have to date been few metabolomic studies of this parasite. Using super(1)H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.
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