A phosphatidylinositol phosphate kinase inhibits Ras activation and regulates chemorepulsion in Dictyostelium discoideum.

During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.

Improved Resting-State Functional MRI Using Multi-Echo Echo-Planar Imaging on a Compact 3T MRI Scanner with High-Performance Gradients.

In blood-oxygen-level-dependent (BOLD)-based resting-state functional (RS-fMRI) studies, usage of multi-echo echo-planar-imaging (ME-EPI) is limited due to unacceptable late echo times when high spatial resolution is used. Equipped with high-performance gradients, the compact 3T MRI system (C3T) enables a three-echo whole-brain ME-EPI protocol with smaller than 2.5 mm isotropic voxel and shorter than 1 s repetition time, as required in landmark fMRI studies. The performance of the ME-EPI was comprehensively evaluated with signal variance reduction and region-of-interest-, seed- and independent-component-analysis-based functional connectivity analyses and compared with a counterpart of single-echo EPI with the shortest TR possible. Through the multi-echo combination, the thermal noise level is reduced. Functional connectivity, as well as signal intensity, are recovered in the medial orbital sulcus and anterior transverse collateral sulcus in ME-EPI. It is demonstrated that ME-EPI provides superior sensitivity and accuracy for detecting functional connectivity and/or brain networks in comparison with single-echo EPI. In conclusion, the high-performance gradient enabled high-spatial-temporal resolution ME-EPI would be the method of choice for RS-fMRI study on the C3T.

Aerosols, airflow, and airspace contamination during laparoscopy.

Laparoscopic surgery has been undermined throughout the COVID-19 pandemic by concerns that it may generate an infectious risk to the operating team through aerosolization of peritoneal particles. There is anyway a need for increased awareness and understanding of the occupational hazard for surgical teams regarding unfiltered escape of pollutants generated by surgical smoke and other microbials. Here, the aerosol-generating nature of this access modality was confirmed through repeatable real-time methodology both qualitatively and quantitively to inform best practice and additional engineering solutions to optimize the operating room environment.

Laparoscopic surgery has been undermined throughout the COVID-19 pandemic by concerns that it may generate an infectious risk to the operating team through aerosolization of peritoneal particles. There is anyway a need for increased awareness and understanding of the occupational hazard for surgical teams regarding unfiltered escape of pollutants generated by surgical smoke and other microbials. Here, the aerosol-generating nature of this access modality was confirmed through repeatable real-time methodology both qualitatively and quantitively to inform best practice and additional engineering solutions to optimize the operating room environment.

Solving the problems of gas leakage at laparoscopy.

Gas leakage during minimally invasive surgery is an aerosolization hazard. Sensitive optical and thermographic imaging can demonstrate and differentiate between mechanistic categories, enabling engineering solutions to fortify surgical care against pollutants and pathogens affecting operating room teams. Areas for improvement.

Induction of calcitonin gene-related peptide expression in rats by cortical spreading depression.

OBJECTIVE: The neuropeptide calcitonin gene-related peptide (CGRP) has now been established as a key player in migraine. However, the mechanisms underlying the reported elevation of CGRP in the serum and cerebrospinal fluid of some migraineurs are not known. A candidate mechanism is cortical spreading depression (CSD), which is associated with migraine with aura and traumatic brain injury. The aim of this study was to investigate whether CGRP gene expression may be induced by experimental CSD in the rat cerebral cortex. METHODS: CSD was induced by topical application of KCl and monitored using electrophysiological methods. Quantitative PCR and ELISA were used to measure CGRP mRNA and peptide levels in discrete ipsilateral and contralateral cortical regions of the rat brain 24 hours following CSD events and compared with sham treatments. RESULTS: The data show that multiple, but not single, CSD events significantly increase CGRP mRNA levels at 24 hours post-CSD in the ipsilateral rat cerebral cortex. Increased CGRP was observed in the ipsilateral frontal, motor, somatosensory, and visual cortices, but not the cingulate cortex, or contralateral cortices. CSD also induced CGRP peptide expression in the ipsilateral, but not contralateral, cortex. CONCLUSIONS: Repeated CSD provides a mechanism for prolonged elevation of CGRP in the cerebral cortex, which may contribute to migraine and post-traumatic headache.

The cyclopentenone prostaglandin 15d-PGJ2 inhibits the NLRP1 and NLRP3 inflammasomes.

Inflammasomes are cytosolic protein complexes that respond to diverse danger signals by activating caspase-1. The sensor components of the inflammasome, often proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family, detect stress, danger stimuli, and pathogen-associated molecular patterns. We report that the eicosanoid 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2) and related cyclopentenone PGs inhibit caspase-1 activation by the NLR family leucine-rich repeat protein (NLRP)1 and NLRP3 inflammasomes. This inhibition was independent of the well-characterized role of 15d-PGJ2 as a peroxisome proliferator receptor-γ agonist, its activation of NF erythroid 2-related factor 2, or its anti-inflammatory function as an inhibitor of NF-κB. Instead, 15d-PGJ2 prevents the autoproteolytic activation of caspase-1 and the maturation of IL-1ß through induction of a cellular state inhibitory to caspase-1 proteolytic function. The eicosanoid does not directly modify or inactivate the caspase-1 enzyme. Rather, inhibition is dependent on de novo protein synthesis. In a mouse peritonitis model of gout, using monosodium urate crystals to activate NLRP3, 15d-PGJ2 caused a significant inhibition of cell recruitment and associated IL-1ß release. Furthermore, in a murine anthrax infection model, 15d-PGJ2 reversed anthrax lethal toxin-mediated NLRP1-dependent resistance. The findings reported in this study suggest a novel mechanism for the anti-inflammatory properties of the cyclopentenone PGs through inhibition of caspase-1 and the inflammasome.

Arsenic trioxide and other arsenical compounds inhibit the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes.

Inflammasomes are large cytoplasmic multiprotein complexes that activate caspase-1 in response to diverse intracellular danger signals. Inflammasome components termed nucleotide-binding oligomerization domain-like receptor (NLR) proteins act as sensors for pathogen-associated molecular patterns, stress, or danger stimuli. We discovered that arsenicals, including arsenic trioxide and sodium arsenite, inhibited activation of the NLRP1, NLRP3, and NAIP5/NLRC4 inflammasomes by their respective activating signals, anthrax lethal toxin, nigericin, and flagellin. These compounds prevented the autoproteolytic activation of caspase-1 and the processing and secretion of IL-1ß from macrophages. Inhibition was independent of protein synthesis induction, proteasome-mediated protein breakdown, or kinase signaling pathways. Arsenic trioxide and sodium arsenite did not directly modify or inhibit the activity of preactivated recombinant caspase-1. Rather, they induced a cellular state inhibitory to both the autoproteolytic and substrate cleavage activities of caspase-1, which was reversed by the reactive oxygen species scavenger N-acetylcysteine but not by reducing agents or NO pathway inhibitors. Arsenicals provided protection against NLRP1-dependent anthrax lethal toxin-mediated cell death and prevented NLRP3-dependent neutrophil recruitment in a monosodium urate crystal inflammatory murine peritonitis model. These findings suggest a novel role in inhibition of the innate immune response for arsenical compounds that have been used as therapeutics for a few hundred years.

Extracellular Hsp90 mediates an NF-κB dependent inflammatory stromal program: implications for the prostate tumor microenvironment.

BACKGROUND: The tumor microenvironment (TME) plays an essential role in supporting and promoting tumor growth and progression. An inflammatory stroma is a widespread hallmark of the prostate TME, and prostate tumors are known to co-evolve with their reactive stroma. Cancer-associated fibroblasts (CAFs) within the reactive stroma play a salient role in secreting cytokines that contribute to this inflammatory TME. Although a number of inflammatory mediators have been identified, a clear understanding of key factors initiating the formation of reactive stroma is lacking. METHODS: We explored whether tumor secreted extracellular Hsp90 alpha (eHsp90α) may initiate a reactive stroma. Prostate stromal fibroblasts (PrSFs) were exposed to exogenous Hsp90α protein, or to conditioned medium (CM) from eHsp90α-expressing prostate cancer cells, and evaluated for signaling, motility, and expression of prototypic reactive markers. In tandem, ELISA assays were utilized to characterize Hsp90α-mediated secreted factors. RESULTS: We report that exposure of PrSFs to eHsp90 upregulates the transcription and protein secretion of IL-6 and IL-8, key inflammatory cytokines known to play a causative role in prostate cancer progression. Cytokine secretion was regulated in part via a MEK/ERK and NF-κB dependent pathway. Secreted eHsp90α also promoted the rapid and durable activation of the oncogenic inflammatory mediator signal transducer and activator of transcription (STAT3). Finally, eHsp90 induced the expression of MMP-3, a well-known mediator of fibrosis and the myofibroblast phenotype. CONCLUSIONS: Our results provide compelling support for eHsp90α as a transducer of signaling events culminating in an inflammatory and reactive stroma, thereby conferring properties associated with prostate cancer progression.

Dissecting host-microbe interactions with modern functional genomics.

Interrogation of host-microbe interactions has long been a source of both basic discoveries and benefits to human health. Here, we review the role that functional genomics approaches have played in such efforts, with an emphasis on recent examples that have harnessed technological advances to provide mechanistic insight at increased scale and resolution. Finally, we discuss how concurrent innovations in model systems and genetic tools have afforded opportunities to interrogate additional types of host-microbe relationships, such as those in the mammalian gut. Bringing these innovations together promises many exciting discoveries ahead.

Functional genetics reveals modulators of anti-microtubule drug sensitivity.

Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for ~1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.

Transkingdom Network Analysis (TkNA): a systems framework for inferring causal factors underlying host-microbiota and other multi-omic interactions.

We present Transkingdom Network Analysis (TkNA), a unique causal-inference analytical framework that offers a holistic view of biological systems by integrating data from multiple cohorts and diverse omics types. TkNA helps to decipher key players and mechanisms governing host-microbiota (or any multi-omic data) interactions in specific conditions or diseases. TkNA reconstructs a network that represents a statistical model capturing the complex relationships between different omics in the biological system. It identifies robust and reproducible patterns of fold change direction and correlation sign across several cohorts to select differential features and their per-group correlations. The framework then uses causality-sensitive metrics, statistical thresholds and topological criteria to determine the final edges forming the transkingdom network. With the subsequent network's topological features, TkNA identifies nodes controlling a given subnetwork or governing communication between kingdoms and/or subnetworks. The computational time for the millions of correlations necessary for network reconstruction in TkNA typically takes only a few minutes, varying with the study design. Unlike most other multi-omics approaches that find only associations, TkNA focuses on establishing causality while accounting for the complex structure of multi-omic data. It achieves this without requiring huge sample sizes. Moreover, the TkNA protocol is user friendly, requiring minimal installation and basic familiarity with Unix. Researchers can access the TkNA software at https://github.com/CAnBioNet/TkNA/ .

Model-based iterative reconstruction for direct imaging with point spread function encoded echo planar MRI.

BACKGROUND: Echo planar imaging (EPI) is a fast measurement technique commonly used in magnetic resonance imaging (MRI), but is highly sensitive to measurement non-idealities in reconstruction. Point spread function (PSF)-encoded EPI is a multi-shot strategy which alleviates distortion, but acquisition of encodings suitable for direct distortion-free imaging prolongs scan time. In this work, a model-based iterative reconstruction (MBIR) framework is introduced for direct imaging with PSF-EPI to improve image quality and acceleration potential. METHODS: An MBIR platform was developed for accelerated PSF-EPI. The reconstruction utilizes a subspace representation, is regularized to promote local low-rankedness (LLR), and uses variable splitting for efficient iteration. Comparisons were made against standard reconstructions from prospectively accelerated PSF-EPI data and with retrospective subsampling. Exploring aggressive partial Fourier acceleration of the PSF-encoding dimension, additional comparisons were made against an extension of Homodyne to direct PSF-EPI in numerical experiments. A neuroradiologists' assessment was completed comparing images reconstructed with MBIR from retrospectively truncated data directly against images obtained with standard reconstructions from non-truncated datasets. RESULTS: Image quality results were consistently superior for MBIR relative to standard and Homodyne reconstructions. As the MBIR signal model and reconstruction allow for arbitrary sampling of the PSF space, random sampling of the PSF-encoding dimension was also demonstrated, with quantitative assessments indicating best performance achieved through nonuniform PSF sampling combined with partial Fourier. With retrospective subsampling, MBIR reconstructs high-quality images from sub-minute scan datasets. MBIR was shown to be superior in a neuroradiologists' assessment with respect to three of five performance criteria, with equivalence for the remaining two. CONCLUSIONS: A novel image reconstruction framework is introduced for direct imaging with PSF-EPI, enabling arbitrary PSF space sampling and reconstruction of diagnostic-quality images from highly accelerated PSF-encoded EPI data.

Neuropathologic changes associated with stereoelectroencephalography depth electrode placement.

BACKGROUND: The aim of this study was to detail the neuropathologic changes resulting from the surgical placement of stereoelectroencephalography (SEEG) leads in an initial small group of epilepsy cases and to raise awareness of this iatrogenic pathology, especially to those medical providers who specialize in the care of epilepsy patients. METHODS: Five consecutive patients who underwent epilepsy resection surgery following SEEG monitoring at OSF Saint Francis Medical Center were included in our report. Resection specimens were examined grossly and entirely submitted for microscopic evaluation by a neuropathologist. Seizure-related pathologies, as well as histologic changes related to SEEG electrode placement, were documented. RESULTS: The patient cohort included two females and three males, with an age range of 9 to 47 years. Neuropathologic examination revealed one or more seizure-related pathologies in each patient's resection specimen. In addition, all brain resection specimens showed multiple microinfarcts, which appeared to correlate with the placement and size of SEEG electrodes. Patchy leptomeningeal chronic inflammation was also seen in most cases. CONCLUSIONS: SEEG electrode placement is an effective procedure for determining epileptogenic regions and guiding subsequent resection surgeries in medically refractory epilepsy. Multiple microinfarcts and chronic inflammation are commonly seen in brain resection specimens following SEEG electrode insertion, but studies detailing these iatrogenic histopathologic changes are lacking. The clinical significance and long-term implications of multiple small foci of electrode-induced injury that remain in the patient's brain after resection of the epileptogenic focus are unknown and may provide a welcome area for future study.

Locally Low-Rank Denoising of Multi-Echo Functional MRI Data With Application in Resting-State Analysis.

OBJECTIVES: Locally low-rank (LLR) denoising of functional magnetic resonance imaging (fMRI) time series image data is extended to multi-echo (ME) data. The proposed method extends the capabilities of non-physiologic noise suppression beyond single-echo applications with a dedicated ME algorithm. MATERIALS AND METHODS: Following an institutional review board (IRB) approved protocol, resting-state fMRI data were acquired in 7 healthy subjects. A compact 3T scanner enabled whole-brain acquisition of multiband ME fMRI data at high spatial resolution (1.4 × 1.4 × 2.8 mm 3 ) with a 1810 ms repetition time (TR). Image data were denoised with ME-LLR preceding functional processing. The results of connectivity maps generated from denoised data were compared with maps generated with equivalent processing of non-denoised images. To assess ME-LLR as a method to reduce scan time, comparisons were made between maps computed from image data with full and retrospectively truncated durations. Assessments were completed with seed-based connectivity analyses using echo-combined image data. In a feasibility assessment, nondenoised and denoised full-duration echo-combined data were equivalently processed with independent component analysis (ICA) and compared. RESULTS: ME-LLR denoising yielded strengthened resting-state network connectivity maps after nuisance regression and seed-based connectivity analysis. In assessing ME-LLR as a scan reduction mechanism, maps generated from denoised data at half scan time showed comparable quality with maps generated from full-duration, non-denoised data, at both single subject and group levels. ME-LLR substantially increased temporal signal-to-noise ratio (tSNR) for image data respective to each individual echo and for image data after nuisance regression. Among echo-specific image volumes, increases in tSNR yielded by ME-LLR were most pronounced for image data with the longest echo time and thereby lowest SNR. ICA showed resting-state networks consistently identified between non-denoised and denoised data, with clearer demarcation of networks for ME-LLR. CONCLUSIONS: ME-LLR is demonstrated to suppress non-physiologic noise, enhance functional connectivity map quality, and could potentially facilitate scan time reduction in ME-fMRI.