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Mashup Score: 2A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells - 3 hour(s) ago
The transformative potential of gene editing technologies hinges on the development of safe and effective delivery methods. In this study, we developed a temperature-sensitive and interferon-silent Sendai virus (ts SeV) as a novel delivery vector for CRISPR-Cas9 and for efficient gene editing in sensitive human cell types without inducing IFN responses. ts SeV demonstrates unprecedented transduction efficiency in human CD34+ hematopoietic stem and progenitor cells (HSPCs) including transduction of the CD34+/CD38-/CD45RA-/CD90+(Thy1+)/CD49fhigh stem cell enriched subpopulation. The frequency of CCR5 editing exceeded 90% and bi-allelic CCR5 editing exceeded 70% resulting in significant inhibition of HIV-1 infection in primary human CD14+ monocytes. These results demonstrate the potential of the ts SeV platform as a safe, efficient, and flexible addition to the current gene-editing tool delivery methods, which may help to further expand the possibilities in personalized medicine and the t
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Mashup Score: 1
ABSTRACT Sepsis and chronic infections with Pseudomonas aeruginosa, a leading ESKAPE bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum sensing (QS) transcription factor MvfR by interrogating five days post-infection its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs ATP generation, oxidative phosphorylation (OXPHOS), and antioxidant response, elevates the production of reactive oxygen species, and promotes
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Mashup Score: 0Genome-guided, field-deployable loop-mediated isothermal amplification (LAMP) assay for specific detection of Dickeya dadantii - 3 hour(s) ago
Potatoes, among the most economically significant crops worldwide, are susceptible to various plant pathogens that significantly impact their propagation, production, storage, and distribution. Soft rot disease, caused primarily by Dickeya and Pectobacterium, results in substantial economic losses to the agricultural industry annually. In this study, we developed a rapid, reliable, and field-deployable loop-mediated isothermal amplification (LAMP) assay for detecting D. dadantii, a common soft rot causing bacteria. The D. dadantii-specific LAMP primers were designed targeting a highly conserved genomic region within D. dadantii, the TetR/AcrR family transcriptional regulator CDS and its flanking sequences. This assay was thoroughly validated with the members of inclusivity (nine strains of D. dadantii) and exclusivity panels (85 strains, including all Dickeya species, related taxa, and host DNA), detecting no false positives or negatives. The limit of detection (LOD) was established by
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Mashup Score: 1Observing one-divalent-metal-ion dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo - 4 hour(s) ago
Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The superfamily of His-Me nucleases is renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how this single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for
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Mashup Score: 2Loss of Intracellular Fibroblast Growth Factor 14 (iFGF14) Increases the Excitability of Mature Hippocampal and Cortical Pyramidal Neurons - 4 hour(s) ago
Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia type 27 (SCA27), a multisystem disorder associated with progressive deficits in motor coordination and cognitive function. Mice (Fgf14-/-) lacking iFGF14 display similar phenotypes, and we have previously shown that the deficits in motor coordination reflect reduced excitability of cerebellar Purkinje neurons, owing to the loss of iFGF14-mediated regulation of the voltage-dependence of inactivation of the fast transient component of the voltage-gated Na+ (Nav) current, INaT. Here, we present the results of experiments designed to test the hypothesis that loss of iFGF14 also attenuates the intrinsic excitability of mature hippocampal and cortical pyramidal neurons. Current-clamp recordings from adult mouse hippocampal CA1 pyramidal neurons in acute in vitro slices, however, revealed that repetitive firing rates were higher in Fgf14-/-, than in wild type (WT),
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Mashup Score: 0Exploring sex-specific neuroendocrine influences on the sensorimotor-association axis in single individuals - 5 hour(s) ago
Human neuroimaging studies consistently show multimodal patterns of variability along a key principle of macroscale cortical organization – the sensorimotor-association (S-A) axis. However, little is known about day-to-day fluctuations in functional activity along this axis within an individual, including sex-specific neuroendocrine factors contributing to such transient changes. We leveraged data from two densely sampled healthy young adults, one female and one male, to investigate intra-individual daily variability along the S-A axis, which we computed as our measure of functional cortical organization by reducing the dimensionality of functional connectivity matrices. Daily variability was greatest in temporal limbic and ventral prefrontal regions in both participants, and was more strongly pronounced in the male subject. Next, we probed local- and system-level effects of steroid hormones and self-reported perceived stress on functional organization. Our findings revealed modest eff
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Mashup Score: 3A gentle palette of plasma membrane dyes - 15 hour(s) ago
Plasma membrane stains are one of the most important organelle markers for unambiguous assignments of individual cells and monitoring membrane morphology and dynamics. The state-of-the-art PM stains are bright, specific, fluorogenic, and compatible with super-resolution imaging. However, when recording membrane dynamics, particularly under light-intensive microscopes, PM is prone to photodynamic damages due to its phospholipid bilayer nature. Here we developed PK Mem dyes tailored for time-lapse fluorescence imaging. By integrating triplet-state quenchers into the MemBright dyes featuring cyanine chromophores and amphiphilic zwitterion anchors, PK Mem dyes exhibited a three-fold reduction in phototoxicity and a more than four-fold improvement in photostability in imaging experiments. These dyes enable 2D and 3D imaging of live or fixed cancer cell lines and a wide range of primary cells, at the same time pair well with various fluorescent markers. PK Mem dyes can be applied to neuronal
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Mashup Score: 1
Liquid-like protein condensates perform diverse physiological functions from the catalysis of biochemical reactions to the assembly of functional biomolecular complexes. Our previous work showed that VASP, a processive actin polymerase, forms condensates that polymerize and bundle actin. Semi-rigid actin filaments polymerized inside condensates, where they partitioned to the inner condensate surface to minimize their curvature. As filaments accumulated at this surface, their collective rigidity overcame the surface tension of the condensate, deforming it into a rod-like shape, filled with a bundle of parallel filaments. These findings suggest a potentially important role for protein condensates in the growth and organization of actin networks. Here we show that this behavior is inherent to all actin-binding condensates and does not require proteins with specific polymerase activity. In particular, we found that condensates composed of Lamellipodin, a protein that binds actin but is not
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Mashup Score: 1Phosphatidylinositol (4,5)-bisphosphate drives the formation of EGFR and EphA2 complexes - 16 hour(s) ago
Receptor tyrosine kinases (RTKs) regulate many cellular functions and are important targets in pharmaceutical development, particularly in cancer treatment. EGFR and EphA2 are two key RTKs that are associated with oncogenic phenotypes. Several studies have reported functional interplay between these receptors, but the mechanism of interaction is still unresolved. Here we utilize a time-resolved fluorescence spectroscopy called PIE-FCCS to resolve EGFR and EphA2 interactions in live cells. We tested the role of ligands and found that EGF, but not ephrin A1 (EA1), stimulated hetero-multimerization between the receptors. To determine the effect of anionic lipids, we targeted phospholipase C (PLC) activity to alter the abundance of phosphatidylinositol (4,5)-bisphosphate (PIP2). We found that higher PIP2 levels increased homo-multimerization of both EGFR and EphA2, as well as hetero-multimerization. This study provides a direct characterization of EGFR and EphA2 interactions in live cells
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Mashup Score: 9Reconstruction of artificial nuclei with nuclear import activity in living mouse oocytes - 16 hour(s) ago
The cell nucleus is a dynamic structure repeating disassembly and reformation during mitosis. Reformation of the nucleus is essential for cell proliferation, and therefore, the factors required for nuclear reformation are fundamental for eukaryotes. Although various factors have been identified in in vitro systems using frog egg extracts and in vivo imaging of somatic cells, little is known about the factors required for the formation of functional nuclear structures in living mouse eggs. To identify such factors, we used a reconstruction approach to construct an artificial nucleus around DNA in mouse eggs. T4 phage DNA (166 kbp) was microinjected into living mouse oocytes. Amounts of DNA injected and injection timing were examined to determine the conditions appropriate for the formation of functional nuclei. Microinjection of 100-500 ng/ul DNA during metaphase through telophase of the second meiosis, but not the subsequent interphase, was important for the formation of artificial nuc
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A temperature-sensitive and interferon-silent Sendai virus vector for CRISPR-Cas9 delivery and gene editing in primary human cells https://t.co/p0t4lPvy09 #bioRxiv