RhoA GTPase Activation by TLR2 and TLR3 Ligands: Connecting via Src to NF- B

Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as NF-κB, C/EBP and serum response factor. The multifaceted host cell activation triggered by Toll-like receptors (TLRs) in response to soluble and particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered recruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-κB activation, while RhoA was dispensable for type I interferon generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and -independent TLR signaling and acts as a molecular switch downstream of TLR-Src initiated pathways

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5–240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro

Circadian dynamics in measures of cortical excitation and inhibition balance

Several neuropsychiatric and neurological disorders have recently been characterized as dysfunctions arising from a ‘final common pathway’ of imbalanced excitation to inhibition within cortical networks. How the regulation of a cortical E/I ratio is affected by sleep and the circadian rhythm however, remains to be established. Here we addressed this issue through the analyses of TMS-evoked responses recorded over a 29h sleep deprivation protocol conducted in young and healthy volunteers. Spectral analyses of TMS-evoked responses in frontal cortex revealed non-linear changes in gamma band evoked oscillations, compatible with an influence of circadian timing on inhibitory interneuron activity. In silico inferences of cell-to-cell excitatory and inhibitory connectivity and GABA/Glutamate receptor time constant based on neural mass modeling within the Dynamic causal modeling framework, further suggested excitation/inhibition balance was under a strong circadian influence. These results indicate that circadian changes in EEG spectral properties, in measure of excitatory/inhibitory connectivity and in GABA/glutamate receptor function could support the maintenance of cognitive performance during a normal waking day, but also during overnight wakefulness. More generally, these findings demonstrate a slow daily regulation of cortical excitation/inhibition balance, which depends on circadian-timing and prior sleep-wake history

Early brainstem [18F]THK5351 uptake is linked to cortical hyper-excitability in healthy aging

BACKGROUND: Neuronal hyper-excitability characterizes the early stages of Alzheimer's disease (AD). In animals, early misfolded tau and amyloid-beta (Aβ) protein accumulation, both central to AD neuropathology, promote cortical excitability and neuronal network dysfunction. In healthy humans, misfolded tau and Aβ aggregates are first detected, respectively, in the brainstem and frontomedial and temporobasal cortices, decades prior to the onset of AD cognitive symptoms. Whether cortical excitability is related to early brainstem tau, and its associated neuroinflammation, and cortical Aβ aggregations remains unknown. METHODS: We probed frontal cortex excitability, using transcranial magnetic stimulation combined with electroencephalography, in a sample of 64 healthy late middle-aged individuals (50-69 y; 45 women). We assessed whole-brain [18F]THK5351 positron emission tomography (PET) uptake as a proxy measure of tau/neuroinflammation, and whole-brain Aβ burden with [18F]Flutemetamol or [18F]Florbetapir radiotracers. RESULTS: We find that higher [18F]THK5351 uptake in a brainstem monoaminergic compartment is associated with increased cortical excitability (r = .29, p = .02). By contrast, [18F]THK5351 PET signal in the hippocampal formation, although strongly correlated with brainstem signal in whole-brain voxel-based quantification analyses (pFWE-corrected < .001), was not significantly associated with cortical excitability (r = .14, p = .25). Importantly, no significant association was found between early Aβ cortical deposits and cortical excitability (r = -.20, p = .11). CONCLUSION: These findings reveal potential brain substrates for increased cortical excitability in preclinical AD and may constitute functional in vivo correlates of early brainstem tau accumulation and neuroinflammation in humans. TRIAL REGISTRATION: EudraCT 2016-001436-35. FUNDING: F.R.S.-FNRS Belgium, Wallonie-Bruxelles International, ULiège, Fondation Simone et Pierre Clerdent, European Regional Development Fund

Recent Advances in Graph Partitioning

We survey recent trends in practical algorithms for balanced graph partitioning together with applications and future research directions

New Noncovalent Inhibitors of Penicillin-Binding Proteins from Penicillin-Resistant Bacteria

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.Eur-Intafa

Lung Epithelial Injury by B. Anthracis Lethal Toxin Is Caused by MKK-Dependent Loss of Cytoskeletal Integrity

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn2+-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax

Functional neuroimaging (fMRI, PET and MEG): what do we measure?

peer reviewedFunctional cerebral imaging techniques allow the in vivo study of human cognitive and sensorimotor functions in physiological or pathological conditions. In this paper, we review the advantages and limitations of functional magnetic resonance imaging (fMRI), positron emission tomography (PET) and magnetoencephalography (MEG). fMRI and PET measure haemodynamic changes induced by regional changes in neuronal activity. These techniques have a high spatial resolution (a few millimeters), but a poor temporal resolution (a few seconds to several minutes). Electroencephalogram (EEG) and MEG measure the neuronal electrical or magnetic activity with a high temporal resolution (i.e., milliseconds) albeit with a poorer spatial resolution (i.e., a few millimeters to one centimeter). The combination of these different neuroimaging techniques allows studying different components of the brain's activity (e.g., neurovascular coupling, electromagnetic activity) with both a high temporal and spatial resolution