Development of a Novel HEK293 Cell Model Lacking to Study the Pharmacology of Endogenous -Encoded Equilibrative Nucleoside Transporter Subtype 2.

Equilibrative nucleoside transporters (ENTs) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogs used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of coexpressed ENT1.

Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.

Genome editing technologies based on DNA-dependent polymerases (DDPs) could offer several benefits compared with other types of editors to install diverse edits. Here, we develop click editing, a genome writing platform that couples the advantageous properties of DDPs with RNA-programmable nickases to permit the installation of a range of edits, including substitutions, insertions and deletions. Click editors (CEs) leverage the 'click'-like bioconjugation ability of HUH endonucleases with single-stranded DNA substrates to covalently tether 'click DNA' (clkDNA) templates encoding user-specifiable edits at targeted genomic loci.

Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

Background: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs.

Multidisciplinary management of a pregnancy complicated by Glanzmann thrombasthenia: A case report.

Background: Glanzmann thrombasthenia (GT) is a rare, autosomal recessive disorder of platelet glycoprotein IIb-IIIa receptors. Pregnant patients with GT are at increased risk of maternal and fetal bleeding. There is a paucity of literature on the peripartum management of patients.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer.

Lipid phosphate phosphatase-2 promotes tumor growth through increased c-Myc expression.

LPP2 is one of three enzymes in the lipid phosphate phosphatase family (LPP1-3) that dephosphorylate extracellular and intracellular bioactive lipid phosphates and pyrophosphates. LPP2 increases cell growth and LPP2 expression is elevated in a variety of malignancies, implying that LPP2 is a pro-tumorigenic factor. LPP2 expression in human breast tumors and normal breast tissue was measured by qPCR.

CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing.

Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences.

CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems.

Identification of Drug Resistance Genes Using a Pooled Lentiviral CRISPR/Cas9 Screening Approach.

In addition to advancing the development of gene-editing therapeutics, CRISPR/Cas9 is transforming how functional genetic studies are carried out in the lab. By increasing the ease with which genetic information can be inserted, deleted, or edited in cell and organism models, it facilitates genotype-phenotype analysis. Moreover, CRISPR/Cas9 has revolutionized the speed at which new genes underlying a particular phenotype can be identified through its application in genomic screens.

Matrix metalloproteinase-2 mediates ribosomal RNA transcription by cleaving nucleolar histones.

Cell proliferation and survival require continuous ribosome biogenesis and protein synthesis. Genes encoding ribosomal RNA are physically located in a specialized substructure within the nucleus known as the nucleolus, which has a central role in the biogenesis of ribosomes. Matrix metalloproteinase-2 was previously detected in the nucleus, however, its role there is elusive.

Tripeptide IRW Upregulates NAMPT Protein Levels in Cells and Obese C57BL/6J Mice.

Nicotinamide adenine dinucleotide (NAD) plays a vital role in cellular processes that govern human health and disease. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in NAD biosynthesis. Thus, boosting NAD level via an increase in NAMPT levels is an attractive approach for countering the effects of aging and metabolic disease.

Improving Access to Palliative Care at a VHA Hospital.

Objective: Explore veteran-specific factors impacting the acceptance of palliative care services at a Veterans Health Administration hospital.

Methods: Prospective, focused one-on-one interviews were conducted with 18 inpatient veterans with an initial consult to receive palliative care services. Domains impacting reception of outpatient palliative care management were evaluated including knowledge deficit, emotional barriers, physical barriers, psychosocial barriers, and physical support.

In Vitro Assays for Comparing the Specificity of First- and Next-Generation CRISPR/Cas9 Systems.

CRISPR/Cas9 has revolutionized the ability to edit cellular DNA and is poised to transform the treatment of genetic diseases. One of the major concerns regarding its therapeutic use is the potential for off-target DNA cleavage, which could have detrimental consequences in vivo. To circumvent this, a number of strategies have been employed to develop next-generation CRISPR/Cas9 systems with improved specificity.

Methods for Measuring CRISPR/Cas9 DNA Cleavage in Cells.

The CRISPR/Cas9 system has transformed how gene knockout and knock-in studies are performed in the lab, and it is poised to revolutionize medicine. However, one of the present limitations of this technology is its imperfect specificity. While CRISPR/Cas9 can be programmed to cut a specific DNA target sequence with relative precision, off-target sequence cleavage can occur in large genomes.

CRISPR Lights up In Situ Protein Evolution.

In this issue of Cell Chemical Biology, Erdogan et al. (2020) describe a new CRISPR/Cas9-based strategy for performing directed evolution of mammalian proteins in situ. Using this technique to select functional mRuby3 variants within lysosomes, they identify mCRISPRed, a fluorescent protein that displays robust stability and activity at low pH.

The utility of thrombophilia testing in patients with newly diagnosed portal vein thrombosis.

: Thrombophilia testing is frequently performed in both seemingly provoked and unprovoked portal vein thrombosis (PVT), yet the clinical implications of these expensive laboratory tests are unknown. We investigated the frequency of clinical management changes in patients with newly diagnosed PVT. This is a retrospective analysis of adult patients with a newly diagnosed PVT at a single institution.

Hop Testing Lacks Strong Association With Key Outcome Variables After Primary Anterior Cruciate Ligament Reconstruction: A Systematic Review.

Background: Single-legged hop tests are commonly used assessments in return to sport (RTS) testing after anterior cruciate ligament reconstruction (ACLR). Although these tests are commonly used, their predictive validity has not yet been established.

Purpose: To determine the strength of association between hop testing and RTS, knee reinjury, subjective report of knee function, and posttraumatic knee osteoarthritis (PTOA) after primary ACLR.

The Necessity for Post-Maneuver Restrictions in the Treatment of Benign Paroxysmal Positional Vertigo: An Updated Meta-Analysis of the Literature.

Objective: Many studies have published conflicting results regarding the necessity of post-maneuver postural restrictions following treatment of benign paroxysmal positional vertigo (BPPV). The purpose of this meta-analysis is to complete an updated, comprehensive review to determine best practice following a repositioning maneuver (RM).

Data Sources: PubMed, CINAHL, and Embase were searched through July 2016.

Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.

Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2',4'-BNA[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNA incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA-DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, "zipped" conformation.

Whole-exome sequencing in evaluation of patients with venous thromboembolism.

Genetics play a significant role in venous thromboembolism (VTE), yet current clinical laboratory-based testing identifies a known heritable thrombophilia (factor V Leiden, prothrombin gene mutation G20210A, or a deficiency of protein C, protein S, or antithrombin) in only a minority of VTE patients. We hypothesized that a substantial number of VTE patients could have lesser-known thrombophilia mutations. To test this hypothesis, we performed whole-exome sequencing (WES) in 64 patients with VTE, focusing our analysis on a novel 55-gene extended thrombophilia panel that we compiled.

Application of phospho-CyTOF to characterize immune activation in patients with sickle cell disease in an ex vivo model of thrombosis.

Sickle cell disease (SCD) is a genetic disease caused by mutations in the beta globin gene, and inflammation plays a key role in driving many aspects of disease pathology. Early immune activation is believed to be associated with hemodynamic stresses and thrombus formation as cells traffic through blood vessels. We applied an extracorporeal perfusion system to model these effects ex vivo, and combined this with a phospho-CyTOF workflow to comprehensively evaluate single-cell signatures of early activation across all major circulating immune subsets.