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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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CD200 expression marks leukemia stem cells in human AML.

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CD200 expression marks leukemia stem cells in human AML.

Blood Adv. 2020 Nov 10;4(21):5402-5413

Authors: Ho JM, Dobson SM, Voisin V, McLeod J, Kennedy JA, Mitchell A, Jin L, Eppert K, Bader G, Minden MD, Dick JE, Wang JCY

Abstract
The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34- and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34-, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.

PMID: 33147339 [PubMed - as supplied by publisher]



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Genetic profiling of protein burden and nuclear export overload.

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Genetic profiling of protein burden and nuclear export overload.

Elife. 2020 Nov 04;9:

Authors: Kintaka R, Makanae K, Namba S, Kato H, Kito K, Ohnuki S, Ohya Y, Andrews BJ, Boone C, Moriya H

Abstract
Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload.

PMID: 33146608 [PubMed - as supplied by publisher]



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Intensive Care Unit-Acquired Weakness: Not just Another Muscle Atrophying Condition.

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Intensive Care Unit-Acquired Weakness: Not just Another Muscle Atrophying Condition.

Int J Mol Sci. 2020 Oct 22;21(21):

Authors: Lad H, Saumur TM, Herridge MS, Dos Santos CC, Mathur S, Batt J, Gilbert PM

Abstract
Intensive care unit-acquired weakness (ICUAW) occurs in critically ill patients stemming from the critical illness itself, and results in sustained disability long after the ICU stay. Weakness can be attributed to muscle wasting, impaired contractility, neuropathy, and major pathways associated with muscle protein degradation such as the ubiquitin proteasome system and dysregulated autophagy. Furthermore, it is characterized by the preferential loss of myosin, a distinct feature of the condition. While many risk factors for ICUAW have been identified, effective interventions to offset these changes remain elusive. In addition, our understanding of the mechanisms underlying the long-term, sustained weakness observed in a subset of patients after discharge is minimal. Herein, we discuss the various proposed pathways involved in the pathophysiology of ICUAW, with a focus on the mechanisms underpinning skeletal muscle wasting and impaired contractility, and the animal models used to study them. Furthermore, we will explore the contributions of inflammation, steroid use, and paralysis to the development of ICUAW and how it pertains to those with the corona virus disease of 2019 (COVID-19). We then elaborate on interventions tested as a means to offset these decrements in muscle function that occur as a result of critical illness, and we propose new strategies to explore the molecular mechanisms of ICUAW, including serum-related biomarkers and 3D human skeletal muscle culture models.

PMID: 33105809 [PubMed - indexed for MEDLINE]



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TPR is required for the efficient nuclear export of mRNAs and lncRNAs from short and intron-poor genes.

TPR is required for the efficient nuclear export of mRNAs and lncRNAs from short and intron-poor genes.

Nucleic Acids Res. 2020 Oct 22;:

Authors: Lee ES, Wolf EJ, Ihn SSJ, Smith HW, Emili A, Palazzo AF

Abstract
While splicing has been shown to enhance nuclear export, it has remained unclear whether mRNAs generated from intronless genes use specific machinery to promote their export. Here, we investigate the role of the major nuclear pore basket protein, TPR, in regulating mRNA and lncRNA nuclear export in human cells. By sequencing mRNA from the nucleus and cytosol of control and TPR-depleted cells, we provide evidence that TPR is required for the efficient nuclear export of mRNAs and lncRNAs that are generated from short transcripts that tend to have few introns, and we validate this with reporter constructs. Moreover, in TPR-depleted cells reporter mRNAs generated from short transcripts accumulate in nuclear speckles and are bound to Nxf1. These observations suggest that TPR acts downstream of Nxf1 recruitment and may allow mRNAs to leave nuclear speckles and properly dock with the nuclear pore. In summary, our study provides one of the first examples of a factor that is specifically required for the nuclear export of intronless and intron-poor mRNAs and lncRNAs.

PMID: 33091126 [PubMed - as supplied by publisher]



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Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

Chemical-Genetic Interactions with the Proline Analog L-Azetidine-2-Carboxylic Acid in Saccharomyces cerevisiae.

G3 (Bethesda). 2020 Oct 20;:

Authors: Berg MD, Zhu Y, Isaacson J, Genereaux J, Loll-Krippleber R, Brown GW, Brandl CJ

Abstract
Non-proteinogenic amino acids, such as the proline analog L-azetidine-2-carboxylic acid (AZC), are detrimental to cells because they are mis-incorporated into proteins and lead to proteotoxic stress. Our goal was to identify genes that show chemical-genetic interactions with AZC in Saccharomyces cerevisiae and thus also potentially define the pathways cells use to cope with amino acid mis-incorporation. Screening the yeast deletion and temperature sensitive collections, we found 72 alleles with negative chemical-genetic interactions with AZC treatment and 12 alleles that suppress AZC toxicity. Many of the genes with negative chemical-genetic interactions are involved in protein quality control pathways through the proteasome. Genes involved in actin cytoskeleton organization and endocytosis also had negative chemical-genetic interactions with AZC. Related to this, the number of actin patches per cell increases upon AZC treatment. Many of the same cellular processes were identified to have interactions with proteotoxic stress caused by two other amino acid analogs, canavanine and thialysine, or a mistranslating tRNA variant that mis-incorporates serine at proline codons. Alleles that suppressed AZC-induced toxicity functioned through the amino acid sensing TOR pathway or controlled amino acid permeases required for AZC uptake. Further suggesting the potential of genetic changes to influence the cellular response to proteotoxic stress, overexpressing many of the genes that had a negative chemical-genetic interaction with AZC suppressed AZC toxicity.

PMID: 33082270 [PubMed - as supplied by publisher]



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