C5a impairs phagosomal maturation in the neutrophil through phosphoproteomic remodeling.

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signaling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signaling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. An assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification, which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the PI3K VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signaling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and mechanisms of immune failure in critical illness.AJTW was a Gates Cambridge Scholar supported by the Gates Cambridge Trust from 2015-2018. ACM is supported by a Clinical Research Career Development Fellowship from the Wellcome Trust (WT 2055214/Z/16/Z). Grants to ACM from the Academy of Medical Sciences and European Society for Intensive Care Medicine supported this work. The study was also supported by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre (IS-BRC-1215-20001) and the Medical Research Council SHIELD antimicrobial resistance consortium (MR/ N02995X/1

Reconstructing the three-dimensional GABAergic microcircuit of the striatum

A system's wiring constrains its dynamics, yet modelling of neural structures often overlooks the specific networks formed by their neurons. We developed an approach for constructing anatomically realistic networks and reconstructed the GABAergic microcircuit formed by the medium spiny neurons (MSNs) and fast-spiking interneurons (FSIs) of the adult rat striatum. We grew dendrite and axon models for these neurons and extracted probabilities for the presence of these neurites as a function of distance from the soma. From these, we found the probabilities of intersection between the neurites of two neurons given their inter-somatic distance, and used these to construct three-dimensional striatal networks. The MSN dendrite models predicted that half of all dendritic spines are within 100 mu m of the soma. The constructed networks predict distributions of gap junctions between FSI dendrites, synaptic contacts between MSNs, and synaptic inputs from FSIs to MSNs that are consistent with current estimates. The models predict that to achieve this, FSIs should be at most 1% of the striatal population. They also show that the striatum is sparsely connected: FSI-MSN and MSN-MSN contacts respectively form 7% and 1.7% of all possible connections. The models predict two striking network properties: the dominant GABAergic input to a MSN arises from neurons with somas at the edge of its dendritic field; and FSIs are interconnected on two different spatial scales: locally by gap junctions and distally by synapses. We show that both properties influence striatal dynamics: the most potent inhibition of a MSN arises from a region of striatum at the edge of its dendritic field; and the combination of local gap junction and distal synaptic networks between FSIs sets a robust input-output regime for the MSN population. Our models thus intimately link striatal micro-anatomy to its dynamics, providing a biologically grounded platform for further study

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Funder: NHLI FoundationFunder: NIHR Imperial Biomedical Research Centre; FundRef: http://dx.doi.org/10.13039/501100013342Funder: National Heart Lung and Blood InstituteFunder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: National Institute of Biomedical Imaging and Bioengineering; FundRef: http://dx.doi.org/10.13039/100000070Funder: Gates Cambridge ScholarshipFunder: NIH/OXCAM FellowshipObjectives: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. Methods: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. Results: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. Conclusions: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy

Condensin I Recruitment to Base Damage-Enriched DNA Lesions Is Modulated by PARP1

Condensin I is important for chromosome organization and segregation in mitosis. We previously showed that condensin I also interacts with PARP1 in response to DNA damage and plays a role in single-strand break repair. However, whether condensin I physically associates with DNA damage sites and how PARP1 may contribute to this process were unclear. We found that condensin I is preferentially recruited to DNA damage sites enriched for base damage. This process is dictated by PARP1 through its interaction with the chromosome-targeting domain of the hCAP-D2 subunit of condensin I

Aerosolized Delivery of Antifungal Agents

Pulmonary infections caused by Aspergillus species are associated with significant morbidity and mortality in immunocompromised patients. Although the treatment of pulmonary fungal infections requires the use of systemic agents, aerosolized delivery is an attractive option in prevention because the drug can concentrate locally at the site of infection with minimal systemic exposure. Current clinical evidence for the use of aerosolized delivery in preventing fungal infections is limited to amphotericin B products, although itraconazole, voriconazole, and caspofungin are under investigation. Based on conflicting results from clinical trials that evaluated various amphotericin B formulations, the routine use of aerosolized delivery cannot be recommended. Further research with well-designed clinical trials is necessary to elucidate the therapeutic role and risks associated with aerosolized delivery of antifungal agents. This article provides an overview of aerosolized delivery systems, the intrapulmonary pharmacokinetic properties of aerosolized antifungal agents, and key findings from clinical studies

Identifying Drug Effects via Pathway Alterations using an Integer Linear Programming Optimization Formulation on Phosphoproteomic Data

Understanding the mechanisms of cell function and drug action is a major endeavor in the pharmaceutical industry. Drug effects are governed by the intrinsic properties of the drug (i.e., selectivity and potency) and the specific signaling transduction network of the host (i.e., normal vs. diseased cells). Here, we describe an unbiased, phosphoproteomicbased approach to identify drug effects by monitoring drug-induced topology alterations. With the proposed method, drug effects are investigated under several conditions on a cell-type specific signaling network. First, starting with a generic pathway made of logical gates, we build a cell-type specific map by constraining it to fit 13 key phopshoprotein signals under 55 experimental cases. Fitting is performed via a formulation as an Integer Linear Program (ILP) and solution by standard ILP solvers; a procedure that drastically outperforms previous fitting schemes. Then, knowing the cell topology, we monitor the same key phopshoprotein signals under the presence of drug and cytokines and we re-optimize the specific map to reveal the drug-induced topology alterations. To prove our case, we make a pathway map for the hepatocytic cell line HepG2 and we evaluate the effects of 4 drugs: 3 selective inhibitors for the Epidermal Growth Factor Receptor (EGFR) and a non selective drug. We confirm effects easily predictable from the drugs’ main target (i.e. EGFR inhibitors blocks the EGFR pathway) but we also uncover unanticipated effects due to either drug promiscuity or the cell’s specific topology. An interesting finding is that the selective EGFR inhibitor Gefitinib is able to inhibit signaling downstream the Interleukin-1alpha (IL-1α) pathway; an effect that cannot be extracted from binding affinity based approaches. Our method represents an unbiased approach to identify drug effects on a small to medium size pathways and is scalable to larger topologies with any type of signaling perturbations (small molecules, 3 RNAi etc). The method is a step towards a better picture of drug effects in pathways, the cornerstone in identifying the mechanisms of drug efficacy and toxicity

H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast

<p>Abstract</p> <p>Background</p> <p>The packaging of DNA into chromatin regulates transcription from initiation through 3' end processing. One aspect of transcription in which chromatin plays a poorly understood role is the co-transcriptional splicing of pre-mRNA.</p> <p>Results</p> <p>Here we provide evidence that H2B monoubiquitylation (H2BK123ub1) marks introns in <it>Saccharomyces cerevisiae</it>. A genome-wide map of H2BK123ub1 in this organism reveals that this modification is enriched in coding regions and that its levels peak at the transcribed regions of two characteristic subgroups of genes. First, long genes are more likely to have higher levels of H2BK123ub1, correlating with the postulated role of this modification in preventing cryptic transcription initiation in ORFs. Second, genes that are highly transcribed also have high levels of H2BK123ub1, including the ribosomal protein genes, which comprise the majority of intron-containing genes in yeast. H2BK123ub1 is also a feature of introns in the yeast genome, and the disruption of this modification alters the intragenic distribution of H3 trimethylation on lysine 36 (H3K36me3), which functionally correlates with alternative RNA splicing in humans. In addition, the deletion of genes encoding the U2 snRNP subunits, Lea1 or Msl1, in combination with an <it>htb-K123R </it>mutation, leads to synthetic lethality.</p> <p>Conclusion</p> <p>These data suggest that H2BK123ub1 facilitates cross talk between chromatin and pre-mRNA splicing by modulating the distribution of intronic and exonic histone modifications.</p

New technologies for examining neuronal ensembles in drug addiction and fear

Correlational data suggest that learned associations are encoded within neuronal ensembles. However, it has been difficult to prove that neuronal ensembles mediate learned behaviours because traditional pharmacological and lesion methods, and even newer cell type-specific methods, affect both activated and non-activated neurons. Additionally, previous studies on synaptic and molecular alterations induced by learning did not distinguish between behaviourally activated and non-activated neurons. Here, we describe three new approaches—Daun02 inactivation, FACS sorting of activated neurons and c-fos-GFP transgenic rats — that have been used to selectively target and study activated neuronal ensembles in models of conditioned drug effects and relapse. We also describe two new tools — c-fos-tTA mice and inactivation of CREB-overexpressing neurons — that have been used to study the role of neuronal ensembles in conditioned fear

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

ICAR: endoscopic skull‐base surgery

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