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  1. Marker Gene Monthly Newsletter March 2020

    Marker Gene acquired by Abcam plc to extend assay and labelling capabilities

    OSCC Cells On March 4, Marker Gene was acquired by Abcam plc, a global life sciences company that identifies, develops, and distributes high-quality biological reagents and tools crucial to research, drug discovery and diagnostics. Marker Gene brings its deep expertise in the areas of biology, organic synthesis and fluorescence chemistry to enhance Abcam’s current in-house capabilities. Our experience in the creation of detection tools, including proprietary assay development technologies and labeling capabilities, are a compelling addition to the Abcam portfolio. It’s an exciting time for our team at Marker Gene to be joining Abcam as it expands its antibody-conjugation strategy. We are looking forward to leveraging our skills and experience to help drive the creation of a broad range of new products.Our products and services are still available to order from our website and via our other sales channels.

    Marker Gene Technologies, Inc and COVID-19

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  2. Marker Gene Monthly Newsletter February 2020 - Volume 20, Number 2

    Multiple Drug Resistance in Oral Squamous Cell Carcinoma

    OSCC CellsOral squamous cell carcinoma (OSCC), the sixth most common malignancy, is an aggressive type of cancer that affects the head and neck areas. The major risk factors for OSCC are tobacco smoking and excessive alcohol consumption. It is often difficult to treat, continuing to have a 5-year survival rate of about 50% for the past 30 years. Surgical resection and chemotherapies are the most common methods of treatment, although multiple drug resistance often reduces the effect of antineoplastic drugs.

    Recent work from the laboratories of Drs. Hiroyuki Shimomura and Tomonori Sasahir of Nara Medical University (Japan) have implicated the overexpression of the condensin complex subunit 2, also known as NCAPH, as a recurrent factor in OSCC. NCAPH is a large protein complex involved in chromosome condensation during cell division. It plays a pivotal role in chromosome-wide gene regulation by controlling chromosome assembly and separation in the mitotic and meiotic cell cycles of proliferative cells. This overexpression resulted in resistance to cisplatin, carboplatin, and nedaplatin in OSCC cells, which was measured using the MarkerGene™ Multiple Drug Resistance Microtiterplate Assay Kit (M1580). This assay measures the efflux of a fluorescent dye that binds to cell surface ATP-binding cassette (ABC) transporters in a similar manner to many chemotherapeutic drugs.

    In addition, platinum anticancer drug therapy combined with siRNA silencing of NCAPH decreased MDR. Interestingly they found no association between NCAPH levels and resistance to paclitaxel, docetaxel or 5-fluorouracil. Recent research has suggested that extracellular vesicles such as exosomes may be involved in the drug resistance of OSCC cells. NCAPH may therefore induce MDR in oral cancer cells by modulating exosome formation. The implications of monitoring NCAPH as a potential diagnostic or even for use in potential therapeutic discovery applications to combat MDR are significant.

    Also in this issue:

    Monitoring Drug Treatment in Osteoarthritis

    Novel Mutations Identified in Gaucher Disease

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  3. Marker Gene Monthly Newsletter January 2020 - Volume 20, Number 1

    Membrane Tension Orchestrates Cell Migration

    membraneCell movement is critical in development, wound healing, immune response and in metastasis of cancer cells. While much is known about the creation of adhesive sites and associated motor proteins in attachment and growth of the leading edge of cells, less is known about how those anchored portions and trailing membranes are decoupled and absorbed to allow net progress forward in an oriented manner. As shown by the Caswell lab at the University of Manchester and co-researchers the retraction of the rear membrane occurs in a positive feedback response or mechano-sensing to the lowered membrane tension brought about after the protrusion of the leading-edge via integrin-based attachment and subsequent F-actin based extension. In response to the lowered membrane tension, caveolae begin to accumulate in the trailing portion of the cell which in turn triggers RhoA guanidine nucleotide exchange factor Ect2 leading to F-actin based contraction of the trailing portion of the cells. All this was demonstrated in a series of elegant live cell experiments, including the use of a fluorescent strain gauge (Flipper-TR™) in the plasma membrane that exhibits fluorescent lifetime changes in strained membranes. With Flipper-TR™ they were able to document a differential in the front-rear membrane tension in motile cells. In response to lowered membrane tension, the rear membrane was gathered in or endocytosed via plasma membrane invaginations controlled by caveolin to form dozens of plasma membrane blebs, then vesicles, called caveolae. These caveolae recycle the plasma membrane components to the leading edge. By altering the osmolarity of the media they were able to de-couple the polarity in tension and the net movement of the cells and associated protein responses. Most of the light microscopy fluorescent studies were done on live 2-D and 3-D cultures in the presence of Marker Gene’s Opti-Klear™ Live Cell Imaging Buffer (M1898) , which provides: low background fluorescence, fluorescent reagent protection, an energy source, stable pH, ideal osmolarity for hours long imaging sessions in the absence of CO2. 

    Also in this issue:

    FAM222A Gene Linked to Alzheimer's Disease Brain Atrophy

    Autophagy Staining Methods

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  4. Marker Gene Monthly Newsletter December 2019 - Volume 19, Number 12

    Dysfunctional Mitochondria-Lysosome Contact Sites Linked to GBA mutation in Parkinson's Disease

    Mito-Lyso contactMitochondria and lysosomes perform the critical functions of producing energy and breaking down excess or worn-out cell components. These organelles are essential for maintaining cellular homeostasis and regulate their respective functions via mitochondria-lysosome contact sites. Contact sites between the two organelles form and release through the tethering/untethering function of the protein RAB7, a small GTP binding protein. Dysfunction in both organelles and their contact sites has been observed in various human diseases, such as Parkinson’s Disease (PD) and several lysosomal storage diseases.

    Dimitri Krainc and co-workers at Northwestern’s Feinberg School of Medicine presented their work in which they demonstrated direct physical mitochondria-lysosome contact sites. These contact sites form in the cell body, axon, and dendrites of human iPSC-derived dopaminergic neurons. Furthermore, heterozygous glucocerebrosidase (GBA) gene mutations in the neurons of PD patients result in dysfunctional mitochondria-lysosome contact sites, thus affecting mitochondrial dynamics in the axon and cell body. Their work provides important insight into the relationship between mitochondria and lysosomes, as well as Parkinson’s disease pathogenesis. Marker Gene provides a number of kits for determining lysosomal metabolic health and mitochondrial health that may prove useful for these studies. 

     

    Also In this Issue:

    Genome-Wide Screens Tie Lysosomal Dysfunction To Alzheimer's Disease

    Intracellular Organelle Targeted Substrates

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  5. Marker Gene Monthly Newsletter November 2019 - Volume 19, Number 11

    Hydrophobic Interactions in DNA Hybridization

    DNA-ProteinThe consensus theory for DNA base-pair binding is that it is controlled by hydrogen bonding. But recent work from the laboratories of Bengt Norden and co-workers at the Department of Chemistry and Chemical Engineering, Chalmers University-Sweden as well as collaborators at UC-Berkeley, Univ. of Minnesota and Tokyo Institute of Technology (Japan) have put forward a new theory of DNA binding and interactions with proteins. Their work describes the effect of hydrophobic interactions in base stacking and protein catalysis. They've found that the DNA nitrogen bases, which are hydrophobic in nature, try to group together to minimize their exposure to the aqueous environment. This makes the DNA interior "dry" so that hydrogen bonds can exert full recognition power. In addition, DNA modifying proteins contain hydrophobic sequences that faciliate opening of the double helix for repair or for reading and manipulating the genome.

    They used an optical tweezers method of monitoring the softening of base-pairing when DNA was treated with different percentages of hydrophobic, water-miscible ethers, such as PEG-400 and diglyme. The changes were monitored using force spectrocopy data with a bulky ruthenium-based fluorescent dye that intercalates the DNA (μ-bidppz(phen)4Ru2]4+; a semi-rigid binuclear derivative of the “light switch ” monomer [Ru(phen)2dppz]2+) and reports back on strand distortion holes in the DNA helix. The implications for this new theory of hydrophobic catalysis by DNA modifying enzymes has the potential to influence the development of new or improved DNA-modifying proteins and enzymes. Marker Gene provides a number of ruthenium dyes that may be useful for such studies. 

    Also in this issue:

    Protein Gels Turn 50!

    Neural Stem Cell Differentiation

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  6. Marker Gene Monthly Newsletter October 2019 - Volume 19, Number 10

    CRISPR Gets an Upgrade

    Prime EditingCRISPR-Cas9 has revolutionized the field of molecular biology in the past few years, providing the ability to efficiently target and alter gene expression in living organisms. However, some issues still remain; the double stranded breaks produced with CRISPR-Cas9 gene editing can lead to multiple products, translocations, and activation of p53. It also lacks the ability to precisely edit genes at the level of base pairs, which is needed to alter the vast majority of pathogenic alleles.

    Prime editing, a new gene editing technique developed by David Liu’s Lab at the Broad Institute, improves upon CRISPR by utilizing a modified Cas9 enzyme fused to reverse transcriptase, that when linked to a guide RNA can precisely target any gene for editing, without double strand breaks. Earlier versions of this technique known as “base editing” could only change C to G, G to C, A to G or G to A, however prime editing offers the ability to perform all twelve possible base conversions. With its ability to also correct insertions and deletions, prime editing has the capacity to correct up to almost 90% of the 75,122 known pathogenic genetic variants. While further studies are needed to bring this powerful tool to the clinic, the “search-and-replace” capability of prime editing represents a huge step forward in precision genetic manipulation. 

    Also in this issue:

    Fluorescent Molecular Imaging Turns 20

    Mathematical Model for Quantification of Isothermic Amplification

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  7. Marker Gene Newsletter Volume 19(9): September, 2019

    RNA Hitchhikes on Lysosomes for Trafficking

    lysosomesSome cells, such as neurons, can be quite long; with axons more than a meter in length. And the ability to synthesize proteins at distant sites requires RNA to be transported throughout the cell. This transport is controlled by RNA-binding proteins (RBPs), which self-organize into structures called RNA granules. Mutations in these RBPs have been implicated in a number of neurological diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).

    Recent work from the laboratories of Jennifer Lippincott-Schwartz at the Howard Hughes Medical Institute's Janelia Reseach Campus and co-workers at the University of Cambridge and the University of Toronto have discovered that the transport of the RNA granules involves their binding to lysosomes for long-range transport. These lysosomes are in turn coupled to motor proteins and the microtubule network in the cell for long range trafficking. As they move around the cell, the RNA piggybacks on the lysosomes for transport. One RBP in particular, Annexin A11 (ANXA11), when mutated in ALS, disrupts docking between the RNA granules and lysosomes and consequently impedes RNA granule transport in neurons. The authors used a number of antibody techniques to pinpoint the RNA-lysosome transport in WT and disease states including LAMP1 staining for lysosomes, ANAX11 probes of RNA granule-lysosome binding, Sec61 for the ER, TOMM20 for mitochondria, SiT for Golgi, SKL for peroxisome, Rab5 for early endosome, Rab7 for late endosome, Rab11a for recycling endosome and Ensconsin for microtubule assemblies. The implications of this work in discovering a new mechanistic function of lysosomes may provide a new lead for the discovery of ALS-associated therapeutics and for other related diseases. Marker Gene provides a number of lysosome functional stains for monitoring lysosomal activity including the LysoLive™ kits and probes that may be useful in these quests. 

    Also in this issue:

    CMOS Sensors Turn 25

    First Responders to Researchers in Crisis

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  8. Marker Gene Monthly Newsletter August 2019 - Volume 19, Number 8

    NIR Luciferin Analogs for in vivo Imaging

    NIR luciferinFirefly luciferase is a well known marker gene used for ultrasensitive detection of ATP in cell viability assays when employing the natural substrate D-Luciferin (M0237). It has also been used in conjunction with other substrate analogs like D-Luciferin-6-O-β-D-galactopyranoside (M1087) for detection of additional enzymes such as cloned lacZ β-Galactosidase activity. These enzymatic visible light emission processes (EM: 482 nm) do not require external light input and therefore have an extremely high signal-to-noise ratio.

    But whole animal imaging via visible light is particularly challenging when compared to imaging in cell culture or tissue sections. Not only is the biology substantially more complex in vivo, but the tissue depth precludes the use of many probes due to light scattering and the absorption of light by endogenous proteins and molecules at shorter wavelengths. To ameliorate the latter challenge, D-luciferin analogs have been developed that emit in the far-red and near infrared region of the spectrum, where tissue absorption is less. These include the 7-allyl-D-luciferin analog (EM: 605 nm), amino-naphthyl naphtho[2,1]thiazole luciferin (NH2-NpLH2) (EM: 730 nm), the extended conjugation AkaLumine® (EM: 675 nm) and the infra-luciferin iLH2 (EM: 706 nm).

    Marker Gene has also developed a mutant luciferase gene that when expressed and reacted with the common substrate D-luciferin (M0237), emits a long wavelength light with an emission peak at 619 nm. It is possible that with the use of these new luciferin analogs, the emission wavelengths can maybe be extended even further into the IR and improve whole body imaging studies.

    Also in this issue:

    GFP Turns 25!

    Bacterial Growth in Lysosomes

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  9. Marker Gene Monthly Newsletter July 2019 - Volume 19, Number 7

    Mitochondrial Heterogeneity Analysis

    mitochondriaMitochondria are well known for their bioenergetic function in cells, but they are also involved in many other cell processes such as hormone synthesis, ion-flux and apoptosis which have been implicated in aging, cancer and cell death. Recent studies have pointed to the fact that mitochondria are not all the same, even in a single cell, but have differences in size, function and activity.

    To investigate this heterogeneity, recent work from the laboratories of Dori Woods and co-workers at the Northeastern University have developed an ingenious method of isolating individual mitochondria using a FACS technique they named FAMS for fluorescence-activated mitochondrial sorting and then characterizing them on a single organelle basis. This method utilizes several stains and methods to quantitate mitochondrial size, membrane potential, redox activity, and ATP levels as well as the presence of mDNA and resulting proteomic analysis. Their methods were validated by measuring proteomic profiles for the isolated mitochondria, electron microscopic analysis as well as knock-down of redox behavior with FCCP and antibody staining. Among the stains and methods employed were MitoTracker™ Green (MTG, see M1151), JC-1 (M1842), TMRM (see M1690) a luciferase based ATP assay (see M0626), MitoTracker™ Red CMXRos (MTR) as well as several specific labeled antibodies. The combination of these methods made it possible to characterize the complex heterogeneity of mitochondria isolated from various tissues and disease states. The use of FAMS to help characterize mitochondrial subpopulations and how they may contribute to disease, cell aging and cell fate are significant. 

    Also in this issue:

    The Father of Fluorescence, Sir George G. Stokes

    Small Molecule Promotes Lysosomal Transport

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  10. Marker Gene Monthly Newsletter June 2019 - Volume 19, Number 6

    Cre Recombinase in Plants

    Recombinant PlantsGenome engineering in plants has been limited by the ability to deliver biomaterials such as DNA, RNA or proteins across the plant cell wall which is designed to protect the plant from exogenous substances or infection. One way around this limitation is the use of plant protoplasts, where the plant cell wall is degraded by enzymes, in order to facilitate transfections. But the ability to regenerate whole plants from these recombinant protoplasts is often inefficient or impossible. Agrobacterium mediated transfections are also widely used but have been less successful when utilizing Cre recombinase as a genome engineering technique, due to variable expression or unexpected incorporations into the genome.

    Recent work from the laboratories of Yoshio Kato at the National Institute of Industrial Science and Technology and co-workers at Kobe and Tokyo Universities has demonstrated the first efficient electroporation-mediated protein delivery of Cre recombinase to achieve nucleic acid-free genome engineering of Arabidopsis thaliana T87 plant cells with an intact cell wall. As part of the process they utilized our pCAMBIA vectors (pCAMBIA 1305.2) for plasmid construction of a reporter cell line to quantify delivery efficiency by monitoring b-glucuronidase (GUS) expression. Marker Gene provides a variety of pCAMBIA vectors that can be utilized for these techniques. Since Cre recombinase can induce site-specific recombinations, this technology holds the promise for conditional knock out of lethal genes or removal of selection markers from genetically engineered plants. 

    Also in this issue:

    GCase Expression in Sunflowers

    GUS Assay for Recombinant Plant Analysis

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