We narrowed to 997 results for: lars

... efficiency. Detecting certain cellular changes with an intermolecular probe is often either impractical..., detect cellular processes and signaling cascades, quantify mechanical tension (a molecular “spring”)...the possibilities can seem overwhelming given the large assortment of fluorescent proteins available. Fortunately...plasmids will be most useful for constructing intermolecular FRET probes, where the donor and acceptor fluorophores...fused to two separate proteins. (In contrast, intramolecular FRET probes contain the donor and acceptor ...these biosensors later on). Experiments using intermolecular FRET probes are typically used for studying...proximity of the two proteins can be inferred. Intermolecular FRET can be experimentally difficult to achieve...

...studying other systems in which there is large scale cellular movement throughout an organism. Studying...Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during...body. In contrast, these dynamic processes are largely absent in adult organisms, where tissues structures...fluorescent proteins can and have been used to monitor cellular movements in the immune system. The techniques...microscopy from PLoS Pathogens - Kamenyeva et al, 2015. Cellular trafficking studies enabled by fluorescent proteins...organs and visualize how cells interact after a particular stimulus. Using an epifluorescence microscope...

... (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal (IT), and ...to transduce the same cell, can accommodate larger, modular genetic payloads. Since most systemic vectors...S., Siverts, L. A., Larsen, R., Gore, B. B., Szelenyi, E. R., Trader, C., Balaram, P., … Tasic, B. (2021...distributing widely throughout the organism to target large or diffuse biological structures. Of primary interest...brain along with high off-target expression, particularly in the liver, when administered intravenously...Hinderer et al, 2018). The optimal dose for each particular experiment is determined by the capsid and promoter... capsids cross the BBB via interactions with a similar protein called LY6C1. This protein is expressed...

...two appear on an agarose gel at notably higher molecular weights than expected, given the size of the plasmid...observed to occur more frequently in plasmids with large inserts (>7 kb)(Berg et al., 1989), direct repeats...just as well as monomers. However, due to their larger size, multimers may exhibit reduced transformation... & Sherratt, 1984; Summers et al., 1993). These larger plasmids are also maintained at lower copy numbers...monomeric version. However, undigested multimers are larger than monomers and will therefore run higher on ...reads (<300 bp) which cannot distinguish between large repeat sequences within a plasmid, it cannot be ...sequencing. These longer reads are able to detect large repeat sequences within a plasmid and, therefore...

...space. Zebrafish have become a popular model organism because their larval stage lends itself well to studies...to be hyperpolarized. The chloride pump NpHR brings in negative chloride ions which hyperpolarize the cell...studies of neuroscience. The larvae of zebrafish are translucent and allow for noninvasive live imaging... Furthermore, during the first 2 weeks of life, larvae already exhibit distinct behaviors such as spontaneous...positive or more negative.  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive...positive over a certain threshold, it becomes depolarized and will fire a signal. The non-specific cation...both cause ion movement that results in cell depolarization. They therefore function as a neuron “on switch...

...flow”) has emerged as a particularly useful tool for those wishing to run larger panels: five laser spectral...This tool is of particular value when designing a panel.     Figure 3: Similarity index across ...and a low similarity index. In figure 3, we can see that PerCP and PE/Cy5 have a similarity index of 0.87...molecules are added during that particular batch. This can present particular difficulty in unmixing if your...detector to detect one fluorophore and therefore one cellular marker. However, emission spectra of certain fluorophores...fluorophores. This allows for the creation of much larger panels, as fluorophores that cannot be distinguished...you want to use in a given experiment. For very large panels (upwards of 30-40 colors), spectral flow ...

...into the genome, and its large DNA packaging capacity is well suited for larger transgenes. Unfortunately...ongoing. Moving forward with ocular gene therapy The pipeline of AAV-based ocular gene therapies is impressive...genetic diseases such as hemophilia, muscular dystrophy, spinal muscular atrophy, neurodegenerative diseases...life-threatening (cystic fibrosis, hemophilia, muscular dystrophy)? AAV gene therapy started in the mid... in the form of Luxturna™. A better vector for ocular gene therapy: Adeno-Associated Virus (AAV) While...cells, AAV quickly became the vector of choice for ocular gene therapy. AdV and LV have been used successfully...successfully in preclinical and clinical settings for ocular conditions. However, AdV was abandoned early on...

...mdx Mice With Large Truncated Dystrophins Generated by Recombination Using rAAV6. Molecular Therapy 19:36...Reassembly of fragmented genomes is the key to fitting large genes into AAV First, some history. In 2008, the...reproducing these results and determined that even when larger vector genomes are packaged, the physical size ... the genomes is still ~4.7kb. But despite this, larger functional transgene products were produced following...in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom ...deliver the mini-dystrophin gene to a mouse model of muscular dystrophy. While mini-dystrophin expression levels... 6 kb mini-dystrophin gene to a mouse model of muscular dystrophy. Three different splice site donor and...

...classification system relies on a combination of sequence similarity, phylogenetic analysis, neighborhood analysis... the same class and type don’t share sequence similarity but are grouped due to mechanisms of action and...completely different! They are merely linked by a similar function. After analysis, CRISPR classifications...nearly 100% in archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas... ensure every unit of the complex expresses at similar levels. It was only recently that researchers began... type of CRISPR overall. Type I systems degrade large sections of DNA thanks to the dual action power ...

...diffusing in the extracellular space; they also have low immunogenicity and cellular toxicity. AAV vectors...brain’s function is knowing its architecture, in particular the connections between different brain regions...brain tissue is injected with compounds that intracellularly spread away from the site of application and...Schmued and Fallon, 1986) are used. To facilitate cellular uptake of these dyes in vivo, microinjection, ...but these methods can have a negative impact on cellular health. Another limitation of traditional tracing...using these viruses to mark neuronal connectivity a large majority of neuronal cells are labeled overtime ...also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011), adenovirus...

...viability. Unlike intracellular labeling, dye staining can be done on live cells. For cellular proliferation... epitopes of interest that are found in the extracellular space. The targeted epitopes can be motifs within... glycosylation patterns. Surface labeling is particularly useful for classifying and sorting cells by ...technique that combines a marker with a signal. For clarity, Addgene uses 'labeling' to refer to antibodies...commonly used in western blotting or ELISAs.  A popular method for indirect labeling employs a primary ...further (non-lineage) markers alongside, e.g., for cellular activation. In addition, FACS achieves greater... T cell. Created using biorender.com.   Intracellular labeling Oftentimes, a marker of interest is...

...many postdocs this will lead to a raise in salary, particularly given that this has precipitated increases...institutional responses to the FLSA ruling. Similarly, from purely a salary perspective, while this is a move in...Many thanks to our guest blogger, David T. Riglar. David T Riglar is a postdoc at Harvard Medical School....post was contributed by guest blogger, David T. Riglar Advocating for Science Symposium and Workshop 2016...a joint American Society for Biochemistry and Molecular Biology/ FoR Hack Day during which several projects...continue their careers in science-related fields. In particular, two recent events have the potential to influence.... The topic of unionization continues to be a polarizing one. High-profile universities remain outspoken...

... the cellular level, it cannot be used for subcellular analysis such as morphology or subcellular localization...antibodies. It’s also particularly useful for intracellular staining where binding of large immune complexes...sources that allow users to assess a cell’s size, granularity, and target protein composition. It is used for...sheath fluid and transported through tubes or capillaries to a laser. As the cells move through the fluidics...Figure 1).   Figure 1: Cells expressing a particular surface receptor are stained with a fluorescent...information about the relative size of the cell; larger cells produce more FSC. Light that scatters sideways... different colors. This lends flow cytometry particularly well to complex experiments looking at a variety...

... as proxies for a cellular process or directly measure a cellular process. Cellular phenomena such as ...cell!)—ATP. There are biosensors for intracellular, extracellular, and even the ADP:ATP ratio. Most of... measure real-time cellular reactions to stimuli and as well as changes in cellular dynamics during so-called...biosensor is the thing that you care about within your cellular or animal system. This ‘thing’ could be a neurotransmitter... these ions, calcium is by far one of the most popular, with dozens of sensors developed for it. Calcium...These ion sensors can be targeted to different cellular locations or tissues depending on the biology ...of different factors, in different or the same cellular location, all at once! Small molecules What type...

...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...luminescence. Intracellular Calcium Sensor Yang, J. et al. (7) wished to trigger intracellular Ca++ release...photoactivatable, optogenetic accuators to depolarize or hyperpolarize membranes. Traditional FRET reporters... sensor, membrane depolarization increases the BRET signal whereas hyperpolarization decreases the signal...orange fluorescent protein, CyOFP1. CyOFP1 was molecularly evolved from mNeptune2 through 33 mutations and...brightness and improved maturation time compared to similar orange fluorescent proteins. In their work, the...

... less selective if used in intracellular labeling because the intracellular compartment may contain cross-reacting...for the same clone. This can be particularly useful when designing larger multicolour panels, where different...approaches available broadly map onto the five pillars of antibody validation (see: Uhlen et al., 2016...for other sample types and for non-surface (intracellular) antigens. Unfortunately, antibodies frequently...protocol. This requires use of one of the five pillars of antibody validation, and it is recommended to...likely to express the target at a robust level, similar to that in samples of interest. If you are working...This can involve cell surface labeling and/or intracellular labeling (or both). It is important to test ...

...challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017) and ...be used to simultaneously target cellular RNA to specific intracellular locations using Cas9 and track ... satisfying ingredients reagents permeate the molecular biologist’s kitchen lab - and scientists using...molecule. Fluorescent RNA aptamers are used to track cellular processes by transforming cells with plasmids ...binding affinity to a specific target, and the molecular processes that make aptamer biology a powerful...a miniaturized version of Spinach that retains similar levels of fluorescence when bound to DFHBI despite...

...more efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified fairly...unmodified plasmid into a larger band (not shown). As a footnote; a similar approach can be used to identify... plasmid (or other). This is particularly relevant in cases where a large plasmid with multiple functional... to introduce a point mutation in a large construct. Particularly, if a pair of unique restriction site...the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data (see Xia...homologous ends recombine in vivo to give the final circular plasmid product pictured here. B) Screening of...portion of the plasmid containing the mutation). Similar results can be obtained with PCR screening using...

...combining them with a molecular species with high affinity for an endogenous molecular target, meaning the.... You may find yourself defaulting to the most popular or familiar options without even realizing it. ...attached to the target through binding of the molecular species. Administration of these dyes results ...fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent... dyes/ligands Effects on protein function and cellular components: It's important to assess how dye-conjugation...affects protein function and its propensity to bind cellular components. Some dyes target specific organelles.... Other dyes may prove challenging to stain intracellular compartments in in vivo model systems, so extra...

...for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter...for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional...Primer on the Molecular Expressions Website Other major microscopy manufacturers have similar information...color contrast, to an image and thereby provide molecular or structural specificity to the viewer. Finally...thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished by...entire axial volume of a sample. Therefore, when a large number of optical slices are acquired all fluorophores...thin sheet of excitation light that propagates perpendicular to an imaging objective that collects fluorescence...