TLR2 controls random motility, while TLR7 regulates chemotaxis of microglial cells via distinct pathways

Microglial cells are the pathologic sensor of the brain, and any pathologic event triggers microglial activation, which involves migration of these cells to a lesion site. Employing different migration assays, we show that ligands for toll-like receptor (TLR) 2 stimulate random motility, while TLR7 ligands are chemoattractants. The subtype specificity of the TLR ligands was verified by using different TLR-deficient (TLRKO) mouse lines. PI3K and Rac inhibition impairs both TLR2- and TLR7-stimulated microglial migration. In contrast, Akt phosphorylation is only required for the TLR2-, but not for the TLR7-stimulated pathway. Interestingly, P2Y12 receptor signaling is involved in the TLR2 activation-induced microglial migration but not TLR7. Furthermore, TLR7 mRNA expression is down-regulated by TLR2 and TLR7 activation. We conclude that TLRs control the migratory behavior of microglia in a distinct manner

Fabrication of Chitin Nanofiber-Reinforced PLA Nanocomposites by an Environmentally Friendly Process

Polylactic acid (PLA) reinforced with chitin nanofibers was produced from a mixture of a colloidal suspension of PLA particles with chitin nanofiber suspension. The dispersion medium was solely water, which was removed by filtration and drying. Nanocomposites were obtained by compression molding of the filtrates. Static tensile test and dynamic mechanical analysis were performed to evaluate the reinforcement as a function of nanofiber content. Chitin nanofibers delivered reinforcement similar to cellulose nanofibers, being especially effective at up to 70 wt% fiber load. The ultimate tensile modulus and strength reached 7.7 GPa and 110 MPa, respectively, at a nanofiber content of 70 wt%

Bradykinin-induced microglial migration mediated by B1-bradykinin receptors depends on Ca2+ influx via reverse-mode activity of the Na+/Ca2+ exchanger

Bradykinin (BK) is produced and acts at the site of injury and inflammation. In the CNS, migration of microglia toward the lesion site plays an important role pathologically. In the present study, we investigated the effect of BK on microglial migration. Increased motility of cultured microglia was mimicked by B1 receptor agonists and markedly inhibited by a B1 antagonist but not by a B2 receptor antagonist. BK induced chemotaxis in microglia isolated from wild-type and B2-knock-out mice but not from B1-knock-out mice. BK-induced motility was not blocked by pertussis toxin but was blocked by chelating intracellular Ca2+ or by low extracellular Ca2+, implying that Ca2+ influx is prerequisite. Blocking the reverse mode of Na+/Ca2+ exchanger (NCX) completely inhibited BK-induced migration. The involvement of NCX was further confirmed by using NCX+/- mice; B1-agonist-induced motility and chemotaxis was decreased compared with that in NCX+/+ mice. Activation of NCX seemed to be dependent on protein kinase C and phosphoinositide 3-kinase, and resultant activation of intermediate-conductance (IK-type) Ca2+-dependent K+ currents (I(K(Ca))) was activated. Despite these effects, BK did not activate microglia, as judged from OX6 staining. Using in vivo lesion models and pharmacological injection to the brain, it was shown that microglial accumulation around the lesion was also dependent on B1 receptors and I(K(Ca)). These observations support the view that BK functions as a chemoattractant by using the distinct signal pathways in the brain and, thus, attracts microglia to the lesion site in vivo

Stimulation of Na⁺/H⁺ Exchanger Isoform 1 Promotes Microglial Migration

Regulation of microglial migration is not well understood. In this study, we proposed that Na+/H+ exchanger isoform 1 (NHE-1) is important in microglial migration. NHE-1 protein was co-localized with cytoskeletal protein ezrin in lamellipodia of microglia and maintained its more alkaline intracellular pH (pHi). Chemoattractant bradykinin (BK) stimulated microglial migration by increasing lamellipodial area and protrusion rate, but reducing lamellipodial persistence time. Interestingly, blocking NHE-1 activity with its potent inhibitor HOE 642 not only acidified microglia, abolished the BK-triggered dynamic changes of lamellipodia, but also reduced microglial motility and microchemotaxis in response to BK. In addition, NHE-1 activation resulted in intracellular Na+ loading as well as intracellular Ca2+ elevation mediated by stimulating reverse mode operation of Na+/Ca2+ exchange (NCXrev). Taken together, our study shows that NHE-1 protein is abundantly expressed in microglial lamellipodia and maintains alkaline pHi in response to BK stimulation. In addition, NHE-1 and NCXrev play a concerted role in BK-induced microglial migration via Na+ and Ca2+ signaling. © 2013 Shi et al

One-step isolation and biochemical characterization of a highlyactive plant PSII monomeric core

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009)

Activation of Toll-like receptor 5 in microglia modulates their function and triggers neuronal injury

Microglia are the primary immune-competent cells of the central nervous system (CNS) and sense both pathogen- and host-derived factors through several receptor systems including the Toll-like receptor (TLR) family. Although TLR5 has previously been implicated in different CNS disorders including neurodegenerative diseases, its mode of action in the brain remained largely unexplored. We sought to determine the expression and functional consequences of TLR5 activation in the CNS. Quantitative real-time PCR and immunocytochemical analysis revealed that microglia is the major CNS cell type that constitutively expresses TLR5. Using Tlr5(-/-) mice and inhibitory TLR5 antibody we found that activation of TLR5 in microglial cells by its agonist flagellin, a principal protein component of bacterial flagella, triggers their release of distinct inflammatory molecules, regulates chemotaxis, and increases their phagocytic activity. Furthermore, while TLR5 activation does not affect tumor growth in an ex vivo GL261 glioma mouse model, it triggers microglial accumulation and neuronal apoptosis in the cerebral cortex in vivo. TLR5-mediated microglial function involves the PI3K/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, as specific inhibitors of this signaling pathway abolish microglial activation. Taken together, our findings establish TLR5 as a modulator of microglial function and indicate its contribution to inflammatory and injurious processes in the CNS

Novel transparent nanocomposite films based on chitosan and bacterial cellulose

New nanocomposite films based on different chitosan matrices (two chitosans with different DPs and one water soluble derivative) and bacterial cellulose were prepared by a fully green procedure by casting a water based suspension of chitosan and bacterial cellulose nanofibrils. The films were characterized by several techniques, namely SEM, AFM, X-ray diffraction, TGA, tensile assays and visible spectroscopy. They were highly transparent, flexible and displayed better mechanical properties than the corresponding unfilled chitosan films. These new renewable nanocomposite materials also presented reasonable thermal stability and low O(2) permeability.FCT - SFRH/BD/41388/ 2007FCT - SFRH/BPD/38515/200

Addressing the unmet clinical need for low-volume assays in early diagnosis of pancreatic cancer

The incidental detection of pancreatic cysts, an opportunity for the early detection of pancreatic cancer, is increasing, owing to an aging population and improvements in imaging technology. The classification of pancreatic cystic precursors currently relies on imaging and cyst fluid evaluations, including cytology and protein and genomic analyses. However, there are persistent limitations that obstruct the accuracy and quality of information for clinicians, including the limited volume of the complex, often acellular, and proteinaceous milieu that comprises pancreatic cyst fluid. The constraints of currently available clinical assays lead clinicians to the subjective and inconsistent application of diagnostic tools, which can contribute to unnecessary surgery and missed pancreatic cancers. Herein, we describe the pathway toward pancreatic cyst classification and diagnosis, the volume requirements for several clinically available diagnostic tools, and some analytical and diagnostic limitations for each assay. We then discuss current and future work on novel markers and methods, and how to expand the utility of clinical pancreatic cyst fluid samples. Results of ongoing studies applying SERS as a detection mode suggest that 50 µL of pancreatic cyst fluid is more than sufficient to accurately rule out non-mucinous pancreatic cysts with no malignant potential from further evaluation. This process is expected to leave sufficient fluid to analyze a follow-up, rule-in panel of markers currently in development that can stratify grades of dysplasia in mucinous pancreatic cysts and improve clinical decision-making