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Anti-CRISPR AcrIE2 binds the type I-E CRISPR-Cas complex but does not block DNA binding.

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Anti-CRISPR AcrIE2 binds the type I-E CRISPR-Cas complex but does not block DNA binding.

J Mol Biol. 2020 Dec 15;:166759

Authors: Mejdani M, Pawluk A, Maxwell KL, Davidson AR

Abstract
Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage.

PMID: 33338493 [PubMed - as supplied by publisher]



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Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.

Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.

Nano Lett. 2020 Dec 10;:

Authors: Overchuk M, Harmatys KM, Sindhwani S, Rajora MA, Koebel A, Charron DM, Syed AM, Chen J, Pomper MG, Wilson BC, Chan WCW, Zheng G

Abstract
Limited tumor nanoparticle accumulation remains one of the main challenges in cancer nanomedicine. Here, we demonstrate that subtherapeutic photodynamic priming (PDP) enhances the accumulation of nanoparticles in subcutaneous murine prostate tumors ∼3-5-times without inducing cell death, vascular destruction, or tumor growth delay. We also found that PDP resulted in an ∼2-times decrease in tumor collagen content as well as a significant reduction of extracellular matrix density in the subendothelial zone. Enhanced nanoparticle accumulation combined with the reduced extravascular barriers improved therapeutic efficacy in the absence of off-target toxicity, wherein 5 mg/kg of Doxil with PDP was equally effective in delaying tumor growth as 15 mg/kg of Doxil. Overall, this study demonstrates the potential of PDP to enhance tumor nanomedicine accumulation and alleviate tumor desmoplasia without causing cell death or vascular destruction, highlighting the utility of PDP as a minimally invasive priming strategy that can improve therapeutic outcomes in desmoplastic tumors.

PMID: 33301689 [PubMed - as supplied by publisher]



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Prioritizing genes for systematic variant effect mapping.

Prioritizing genes for systematic variant effect mapping.

Bioinformatics. 2020 Dec 10;:

Authors: Kuang D, Truty R, Weile J, Johnson B, Nykamp K, Araya C, Nussbaum RL, Roth FP

Abstract
MOTIVATION: When rare missense variants are clinically interpreted as to their pathogenicity, most are classified as variant(s) of uncertain significance (VUS). Although functional assays can provide strong evidence for variant classification, such results are generally unavailable. Multiplexed assays of variant effect can generate experimental 'variant effect maps' that score nearly all possible missense variants in selected protein targets for their impact on protein function. However, these efforts have not always prioritized proteins for which variant effect maps would have the greatest impact on clinical variant interpretation.
RESULTS: Here we mined databases of clinically interpreted variants and applied three strategies, each building on the previous, to prioritize genes for systematic functional testing of missense variation. The strategies ranked genes 1) by the number of unique missense VUS that had been reported to ClinVar; 2) by movability- and reappearance-weighted impact scores, to give extra weight to reappearing, movable VUS; and 3) by difficulty-adjusted impact scores, to account for the more resource-intensive nature of generating variant effect maps for longer genes. Our results could be used to guide systematic functional testing of missense variation towards greater impact on clinical variant interpretation.
AVAILABILITY: Source code available at: https://github.com/rothlab/mave-gene-prioritization.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PMID: 33300982 [PubMed - as supplied by publisher]



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Quantifying the influence of mutation detection on tumour subclonal reconstruction.

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Quantifying the influence of mutation detection on tumour subclonal reconstruction.

Nat Commun. 2020 12 07;11(1):6247

Authors: Liu LY, Bhandari V, Salcedo A, Espiritu SMG, Morris QD, Kislinger T, Boutros PC

Abstract
Whole-genome sequencing can be used to estimate subclonal populations in tumours and this intra-tumoural heterogeneity is linked to clinical outcomes. Many algorithms have been developed for subclonal reconstruction, but their variabilities and consistencies are largely unknown. We evaluate sixteen pipelines for reconstructing the evolutionary histories of 293 localized prostate cancers from single samples, and eighteen pipelines for the reconstruction of 10 tumours with multi-region sampling. We show that predictions of subclonal architecture and timing of somatic mutations vary extensively across pipelines. Pipelines show consistent types of biases, with those incorporating SomaticSniper and Battenberg preferentially predicting homogenous cancer cell populations and those using MuTect tending to predict multiple populations of cancer cells. Subclonal reconstructions using multi-region sampling confirm that single-sample reconstructions systematically underestimate intra-tumoural heterogeneity, predicting on average fewer than half of the cancer cell populations identified by multi-region sequencing. Overall, these biases suggest caution in interpreting specific architectures and subclonal variants.

PMID: 33288765 [PubMed - indexed for MEDLINE]



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Bead-based multiplex detection of dengue biomarkers in a portable imaging device.

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Bead-based multiplex detection of dengue biomarkers in a portable imaging device.

Biomed Opt Express. 2020 Nov 01;11(11):6154-6167

Authors: Yuan X, Garg S, Haan K, Fellouse FA, Gopalsamy A, Tykvart J, Sidhu SS, Varma MM, Pal P, Hillan EM, Dou JJ, Aitchison JS

Abstract
Dengue is one of the most rapidly spreading mosquito-borne viral diseases in the world. Differential diagnosis is a crucial step for the management of the disease and its epidemiology. Point-of-care testing of blood-borne dengue biomarkers provides an advantageous approach in many health care settings, and the ability to follow more than one biomarker at once could significantly improve the management of the disease. Bead-based multiplex technologies (suspension array) can measure multiple biomarker targets simultaneously by using recognition molecules immobilized on microsphere beads. The overarching objective of our work is to develop a portable detection device for the simultaneous measurement of multiple biomarkers important in dengue diagnosis, monitoring and treatment. Here, we present a bead-based assay for the detection of one of the four serotypes of dengue virus non-structural protein (DENV-NS1) as well as its cognate human IgG. In this system, the fluorescent microspheres containing the classification fluorophore and detection fluorophore are imaged through a microfluidic chip using an infinity-corrected microscope system. Calibration curves were plotted for median fluorescence intensity against known concentrations of DENV-NS1 protein and anti-NS1 human IgG. The limit of quantitation was 7.8 ng/mL and 15.6 ng/mL, respectively. The results of this study demonstrate the feasibility of the multiplex detection of dengue biomarkers and present its analytical performance parameters. The proposed imaging device holds potential for point-of-care testing of biomarkers on a highly portable system, and it may facilitate the diagnosis and prevention of dengue as well as other infectious diseases.

PMID: 33282481 [PubMed]



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Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

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Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Mol Cell. 2020 12 17;80(6):1104-1122.e9

Authors: Hekman RM, Hume AJ, Goel RK, Abo KM, Huang J, Blum BC, Werder RB, Suder EL, Paul I, Phanse S, Youssef A, Alysandratos KD, Padhorny D, Ojha S, Mora-Martin A, Kretov D, Ash PEA, Verma M, Zhao J, Patten JJ, Villacorta-Martin C, Bolzan D, Perea-Resa C, Bullitt E, Hinds A, Tilston-Lunel A, Varelas X, Farhangmehr S, Braunschweig U, Kwan JH, McComb M, Basu A, Saeed M, Perissi V, Burks EJ, Layne MD, Connor JH, Davey R, Cheng JX, Wolozin BL, Blencowe BJ, Wuchty S, Lyons SM, Kozakov D, Cifuentes D, Blower M, Kotton DN, Wilson AA, Mühlberger E, Emili A

Abstract
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

PMID: 33259812 [PubMed - indexed for MEDLINE]



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diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition.

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diaPASEF: parallel accumulation-serial fragmentation combined with data-independent acquisition.

Nat Methods. 2020 Dec;17(12):1229-1236

Authors: Meier F, Brunner AD, Frank M, Ha A, Bludau I, Voytik E, Kaspar-Schoenefeld S, Lubeck M, Raether O, Bache N, Aebersold R, Collins BC, Röst HL, Mann M

Abstract
Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

PMID: 33257825 [PubMed - in process]



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Canadian recommendations for training and performance in basic perioperative point-of-care ultrasound: recommendations from a consensus of Canadian anesthesiology academic centres.

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Canadian recommendations for training and performance in basic perioperative point-of-care ultrasound: recommendations from a consensus of Canadian anesthesiology academic centres.

Can J Anaesth. 2020 Nov 24;:

Authors: Meineri M, Arellano R, Bryson G, Arzola C, Chen R, Collins P, Denault A, Desjardins G, Fayad A, Funk D, Hegazy AF, Kim H, Kruger M, Kruisselbrink R, Perlas A, Prabhakar C, Syed S, Sidhu S, Tanzola R, Van Rensburg A, Talab H, Vegas A, Bainbridge D

Abstract
Point-of-care ultrasound (POCUS) uses ultrasound at the bedside to aid decision-making in acute clinical scenarios. The increased use of ultrasound for regional anesthesia and vascular cannulation, together with more anesthesiologists trained in transesophageal echocardiography have contributed to the widespread use of POCUS in perioperative care. Despite the support of international experts, the practice of POCUS in perioperative care is variable as Canadian guidelines for anesthesiologists do not currently exist. Using a Delphi process of online surveys and a face-to-face national Canadian meeting, we developed a consensus statement for basic POCUS (bPOCUS) performance and training with a group of national experts from all Canadian universities. The group of experts consisted of 55 anesthesiologists from 12 Canadian universities considered local leaders in the field. An initial exploratory online survey of 47 statements was conducted. These statements were derived from previous generic guidelines or consensus conferences, or were based on current literature. Fourteen statements reached full consensus, 19 had 90-100% agreement, and 14 had less than 90% agreement. Eight new statements were proposed during the national meeting, and all statements without full agreement were discussed. A second online survey included 42 modified or new statements. From this second survey, 16 statements obtained full consensus, 39 had very good agreement, and one had good agreement. The final document includes 56 statements that define the scope of practice and necessary training for perioperative bPOCUS. The statements include five bPOCUS domains: cardiac, lung, airway, gastric, and abdomen. The use of bPOCUS is evolving and will play a significant role in perioperative medicine. This consensus statement aims to define a Canadian national standard on which curricula may be based. It also provides a framework to allow further development of bPOCUS in the perioperative setting.

PMID: 33236278 [PubMed - as supplied by publisher]



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Digital microfluidic isolation of single cells for -Omics.

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Digital microfluidic isolation of single cells for -Omics.

Nat Commun. 2020 11 11;11(1):5632

Authors: Lamanna J, Scott EY, Edwards HS, Chamberlain MD, Dryden MDM, Peng J, Mair B, Lee A, Chan C, Sklavounos AA, Heffernan A, Abbas F, Lam C, Olson ME, Moffat J, Wheeler AR

Abstract
We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.

PMID: 33177493 [PubMed - indexed for MEDLINE]



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A scalable device-less biomaterial approach for subcutaneous islet transplantation.

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A scalable device-less biomaterial approach for subcutaneous islet transplantation.

Biomaterials. 2020 Nov 02;:120499

Authors: Vlahos AE, Talior-Volodarsky I, Kinney SM, Sefton MV

Abstract
The subcutaneous space has been shown to be a suitable site for islet transplantation, however an abundance of islets is required to achieve normoglycemia, often requiring multiple donors. The loss of islets is due to the hypoxic conditions islets experience during revascularization, resulting in apoptosis. Therefore, to reduce the therapeutic dosage required to achieve normoglycemia, pre-vascularization of the subcutaneous space has been pursued. In this study, we highlight a biomaterial-based approach using a methacrylic acid copolymer coating to generate a robust pre-vascularized subcutaneous cavity for islet transplantation. We also devised a simple, but not-trivial, procedure for filling the cavity with an islet suspension in collagen. We show that the pre-vascularized site can support a marginal mass of islets to rapidly return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. Furthermore, immunocompetent Sprague Daley rats remained normoglycemia for up to 70 days until they experienced graft destabilization as they outgrew their implants. This work highlights methacrylic acid-based biomaterials as a suitable pre-vascularization strategy for the subcutaneous space that is scalable and doesn't require exogenous cells or growth factors.

PMID: 33168223 [PubMed - as supplied by publisher]



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