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Recent Publications

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

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 - in process]



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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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The structure and function of deubiquitinases: lessons from budding yeast.

The structure and function of deubiquitinases: lessons from budding yeast.

Open Biol. 2020 Oct;10(10):200279

Authors: Suresh HG, Pascoe N, Andrews B

Abstract
Protein ubiquitination is a key post-translational modification that regulates diverse cellular processes in eukaryotic cells. The specificity of ubiquitin (Ub) signalling for different bioprocesses and pathways is dictated by the large variety of mono-ubiquitination and polyubiquitination events, including many possible chain architectures. Deubiquitinases (DUBs) reverse or edit Ub signals with high sophistication and specificity, forming an integral arm of the Ub signalling machinery, thus impinging on fundamental cellular processes including DNA damage repair, gene expression, protein quality control and organellar integrity. In this review, we discuss the many layers of DUB function and regulation, with a focus on insights gained from budding yeast. Our review provides a framework to understand key aspects of DUB biology.

PMID: 33081638 [PubMed - in process]



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The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

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The GATOR-Rag GTPase pathway inhibits mTORC1 activation by lysosome-derived amino acids.

Science. 2020 10 16;370(6514):351-356

Authors: Hesketh GG, Papazotos F, Pawling J, Rajendran D, Knight JDR, Martinez S, Taipale M, Schramek D, Dennis JW, Gingras AC

Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.

PMID: 33060361 [PubMed - indexed for MEDLINE]



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Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways.

Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways.

J Biol Chem. 2020 Oct 15;:

Authors: Kim JH, Seo Y, Jo M, Jeon H, Kim YS, Kim EJ, Seo D, Lee WH, Kim SR, Yachie N, Zhong Q, Vidal M, Roth FP, Suk K

Abstract
Kinases are critical components of intracellular signaling pathways and have been extensively investigated in regards to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid S. cerevisiae yeast deletion mutants identified approximately 400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration, and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

PMID: 33060198 [PubMed - as supplied by publisher]



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An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Int J Mol Sci. 2020 Oct 07;21(19):

Authors: Choi UY, Joshi HP, Payne S, Kim KT, Kyung JW, Choi H, Cooke MJ, Kwon SY, Roh EJ, Sohn S, Shoichet MS, Han I

Abstract
Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

PMID: 33036383 [PubMed - as supplied by publisher]



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DNA-Controlled Encapsulation of Small Molecules in Protein Nanoparticles.

DNA-Controlled Encapsulation of Small Molecules in Protein Nanoparticles.

J Am Chem Soc. 2020 Oct 06;:

Authors: Ngo W, Stordy B, Lazarovits J, Raja EK, Etienne CL, Chan WCW

Abstract
A nanoparticle can hold multiple types of therapeutic and imaging agents for disease treatment and diagnosis. However, controlling the storage of molecules in nanoparticles is challenging, because nonspecific intermolecular interactions are used for encapsulation. Here, we used specific DNA interactions to store molecules in nanoparticles. We made nanoparticles containing DNA anchors to capture DNA-conjugated small molecules. By changing the sequences and stoichiometry of DNA anchors, we can control the amount and ratio of molecules with different chemical properties in the nanoparticles. We modified the cytotoxicity of our nanoparticles to cancer cells by changing the ratio of encapsulated drugs (mertansine and doxorubicin). Specifically controlling the storage of multiple types of molecules allows us to optimize the properties of combination drug and imaging nanoparticles.

PMID: 33022172 [PubMed - as supplied by publisher]



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An efficient KRAB domain for CRISPRi applications in human cells.

An efficient KRAB domain for CRISPRi applications in human cells.

Nat Methods. 2020 Oct 05;:

Authors: Alerasool N, Segal D, Lee H, Taipale M

Abstract
Clustered regularly interspaced short palindromic repeat interference (CRISPRi), based on the fusion of inactive Cas9 (dCas9) to the Krüppel-associated box (KRAB) repressor, is a powerful platform for silencing gene expression. However, it suffers from incomplete silencing of target genes. We assayed 57 KRAB domains for their repressive potency and identified the ZIM3 KRAB domain as an exceptionally potent repressor. We establish that ZIM3 KRAB-dCas9 fusion silences gene expression more efficiently than existing platforms.

PMID: 33020655 [PubMed - as supplied by publisher]



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Constraint-induced movement therapy promotes motor recovery after neonatal stroke in the absence of neural precursor activation.

Constraint-induced movement therapy promotes motor recovery after neonatal stroke in the absence of neural precursor activation.

Eur J Neurosci. 2020 Oct 03;:

Authors: Adams KV, Mahmud N, Green-Holland M, Vonderwalde I, Umebayashi D, Sachewsky N, Coles BL, van der Kooy D, Morshead CM

Abstract
Neonatal stroke is a leading cause of long-term disability and currently available rehabilitation treatments are insufficient to promote recovery. Activating neural precursor cells (NPCs) in adult rodents, in combination with rehabilitation, can accelerate functional recovery following stroke. Here, we describe a novel method of constraint-induced movement therapy (CIMT) in a rodent model of neonatal stroke that leads to improved functional outcomes, and asked whether the recovery was correlated with expansion of NPCs. A hypoxia/ischemia (H/I) injury was induced on postnatal day 8 (PND8) via unilateral carotid artery ligation followed by systemic hypoxia. One week and two weeks post-H/I, CIMT was administered in the form of 3 botulinum toxin (Botox) injections, which induced temporary paralysis in the unaffected limb. Functional recovery was assessed using the foot fault task. NPC proliferation was assessed using the neurosphere assay and EdU immunohistochemistry. We found that neonatal H/I injury alone expands the NPC pool by >2.5 fold relative to controls. We determined that Botox injections as a means to provide CIMT results in significant functional motor recovery following H/I. However, CIMT does not lead to enhanced NPC activation or migration into the injured parenchyma in vivo. At the time of functional recovery increased numbers of proliferating inflammatory cells were found within the injured motor cortex. Together, these findings suggest that NPC activation following CIMT does not account for the observed functional improvement and suggests that CIMT-mediated modification of the CNS inflammatory response may play a role in the motor recovery.

PMID: 33010080 [PubMed - as supplied by publisher]



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