scholarly journals Trehalose amide glycolipids as Mincle ligands: Towards new vaccine adjuvants

2021 ◽  
Author(s):  
◽  
Amy Lynch
Keyword(s):  
Fluorescent Protein ◽  
Vaccine Adjuvants ◽  
Protecting Group ◽  
Reporter Cell ◽  
Straight Chain ◽  
Adjuvant Activity ◽  
Amide Bonds ◽  
Gfp Reporter ◽  
Comparable Activity ◽  
Green Fluorescent

<p>The development of new vaccines to respond to infectious diseases requires new vaccine adjuvants, which improve vaccine efficacy and shape the immune response. Trehalose glycolipids, consisting of α,α'-trehalose esterified at the 6- and 6'- positions with lipids, exhibit adjuvant activity by binding and activating Macrophage inducible C-type lectin (Mincle). However, the adjuvant activity of trehalose glycolipids could potentially be improved by substituting the ester linkages for more physiologically stable amide bonds. This thesis presents a short protecting group free route to trehalose amide glycolipids, thus allowing for the synthesis of the straight chain glycolipid amides 1a-e in four steps and in excellent (53-61%) overall yields (Figure 1). Amide glycolipids 1a-e were demonstrated to be Mincle agonists with comparable activity to their ester counterparts, as determined using a green fluorescent protein (GFP) reporter cell line assay. A second generation of trehalose amide glycolipids, the lipidated brartemicin amide analogues 2a-c, were subsequently synthesised (Figure 1). This report is the first example of trehalose amide glycolipids acting as Mincle agonists, and further studies into the potential of the amides as vaccine adjuvants will be undertaken in due course.</p>

scholarly journals Trehalose amide glycolipids as Mincle ligands: Towards new vaccine adjuvants

2021 ◽  
Author(s):  
◽  
Amy Lynch
Keyword(s):  
Fluorescent Protein ◽  
Vaccine Adjuvants ◽  
Protecting Group ◽  
Reporter Cell ◽  
Straight Chain ◽  
Adjuvant Activity ◽  
Amide Bonds ◽  
Gfp Reporter ◽  
Comparable Activity ◽  
Green Fluorescent

<p>The development of new vaccines to respond to infectious diseases requires new vaccine adjuvants, which improve vaccine efficacy and shape the immune response. Trehalose glycolipids, consisting of α,α'-trehalose esterified at the 6- and 6'- positions with lipids, exhibit adjuvant activity by binding and activating Macrophage inducible C-type lectin (Mincle). However, the adjuvant activity of trehalose glycolipids could potentially be improved by substituting the ester linkages for more physiologically stable amide bonds. This thesis presents a short protecting group free route to trehalose amide glycolipids, thus allowing for the synthesis of the straight chain glycolipid amides 1a-e in four steps and in excellent (53-61%) overall yields (Figure 1). Amide glycolipids 1a-e were demonstrated to be Mincle agonists with comparable activity to their ester counterparts, as determined using a green fluorescent protein (GFP) reporter cell line assay. A second generation of trehalose amide glycolipids, the lipidated brartemicin amide analogues 2a-c, were subsequently synthesised (Figure 1). This report is the first example of trehalose amide glycolipids acting as Mincle agonists, and further studies into the potential of the amides as vaccine adjuvants will be undertaken in due course.</p>

scholarly journals Regulation of RamA by RamR in Salmonella enterica Serovar Typhimurium: Isolation of a RamR Superrepressor

2012 ◽  
Vol 56 (11) ◽  
pp. 6037-6040 ◽  
Author(s):  
Vito Ricci ◽  
Stephen J. W. Busby ◽  
Laura J. V. Piddock
Keyword(s):  
Transcription Factor ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Promoter Sequence ◽  
Palindromic Sequence ◽  
Content Type ◽  
Gfp Reporter ◽  
Serovar Typhimurium ◽  
Green Fluorescent

ABSTRACTRamA is a transcription factor involved in regulating multidrug resistance inSalmonella entericaserovar Typhimurium SL1344. Green fluorescent protein (GFP) reporter fusions were exploited to investigate the regulation of RamA expression by RamR. We show that RamR represses theramApromoter by binding to a palindromic sequence and describe a superrepressor RamR mutant that binds to theramApromoter sequence more efficiently, thus exhibiting aramAinactivated phenotype.

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1819-1828 ◽  
Author(s):  
James A. Carroll ◽  
Philip E. Stewart ◽  
Patricia Rosa ◽  
Abdallah F. Elias ◽  
Claude F. Garon
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Epifluorescence Microscopy ◽  
Reporter System ◽  
Environmental Signals ◽  
Gfp Reporter ◽  
Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

Evaluation of Disinfection Efficacy by a Green Fluorescent Protein (GFP) Reporter System

2011 ◽  
Vol 2011 (3) ◽  
pp. 243-248
Author(s):  
Ting Lu ◽  
Yanping Chen ◽  
Jorge W. Santo Domingo ◽  
Daniel B. Oerther
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter System ◽  
Gfp Reporter ◽  
Green Fluorescent

scholarly journals Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820 ◽  
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cns Infection ◽  
Gfp Expression ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
Differential Gene ◽  
Green Fluorescent

ABSTRACT Synthetic green fluorescent protein (GFP) was used as a reporter to detect differential gene expression in the pathogenic fungusCryptococcus neoformans. Promoters from the C. neoformans actin, GAL7, or mating-type alpha pheromone (MFα1) genes were fused to GFP, and the resulting reporter genes were used to assess gene expression in serotype A C. neoformans. Yeast cells containing an integrated pACT::GFP construct demonstrated that the actin promoter was expressed during vegetative growth on yeast extract-peptone-dextrose medium. In contrast, yeast cells containing the inducible GAL7::GFP or MFα1::GFP reporter genes expressed significant GFP activity only during growth on galactose medium or V-8 agar, respectively. These findings demonstrated that the GAL7 and MFα1 promoters from a serotype D C. neoformans strain function when introduced into a serotype A strain. Because the MFα1 promoter is induced by nutrient deprivation and the MATα locus containing the MFα1 gene has been linked with virulence, yeast cells containing the pMFα1::GFP reporter gene were analyzed for GFP expression in the central nervous system (CNS) of immunosuppressed rabbits. In fact, significant GFP expression from the MFα1::GFP reporter gene was detected after the first week of a CNS infection. These findings suggest that there are temporal, host-specific cues that regulate gene expression during infection and that the MFα1 gene is induced during the proliferative stage of a CNS infection. In conclusion, GFP can be used as an effective and sensitive reporter to monitor specific C. neoformans gene expression in vitro, and GFP reporter constructs can be used as an approach to identify a novel gene(s) or to characterize known genes whose expression is regulated during infection.

scholarly journals Genetic identity of thermosensory relay neurons in the lateral parabrachial nucleus

2016 ◽  
Vol 310 (1) ◽  
pp. R41-R54 ◽  
Author(s):  
Joel C. Geerling ◽  
Minjee Kim ◽  
Carrie E. Mahoney ◽  
Stephen B. G. Abbott ◽  
Lindsay J. Agostinelli ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Preoptic Area ◽  
Fluorescent Protein ◽  
Parabrachial Nucleus ◽  
Glutamatergic Neurons ◽  
Lateral Parabrachial Nucleus ◽  
Reporter Mice ◽  
Gfp Reporter ◽  
Green Fluorescent ◽  
Relay Neurons

The parabrachial nucleus is important for thermoregulation because it relays skin temperature information from the spinal cord to the hypothalamus. Prior work in rats localized thermosensory relay neurons to its lateral subdivision (LPB), but the genetic and neurochemical identity of these neurons remains unknown. To determine the identity of LPB thermosensory neurons, we exposed mice to a warm (36°C) or cool (4°C) ambient temperature. Each condition activated neurons in distinct LPB subregions that receive input from the spinal cord. Most c-Fos+ neurons in these LPB subregions expressed the transcription factor marker FoxP2. Consistent with prior evidence that LPB thermosensory relay neurons are glutamatergic, all FoxP2+ neurons in these subregions colocalized with green fluorescent protein (GFP) in reporter mice for Vglut2, but not for Vgat. Prodynorphin ( Pdyn)-expressing neurons were identified using a GFP reporter mouse and formed a caudal subset of LPB FoxP2+ neurons, primarily in the dorsal lateral subnucleus (PBdL). Warm exposure activated many FoxP2+ neurons within PBdL. Half of the c-Fos+ neurons in PBdL were Pdyn+, and most of these project into the preoptic area. Cool exposure activated a separate FoxP2+ cluster of neurons in the far-rostral LPB, which we named the rostral-to-external lateral subnucleus (PBreL). These findings improve our understanding of LPB organization and reveal that Pdyn- IRES- Cre mice provide genetic access to warm-activated, FoxP2+ glutamatergic neurons in PBdL, many of which project to the hypothalamus.

scholarly journals GATA Motifs Regulate Early Hematopoietic Lineage-Specific Expression of the Gata2 Gene

2005 ◽  
Vol 25 (16) ◽  
pp. 7005-7020 ◽  
Author(s):  
Maki Kobayashi-Osaki ◽  
Osamu Ohneda ◽  
Norio Suzuki ◽  
Naoko Minegishi ◽  
Tomomasa Yokomizo ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Regulatory Domain ◽  
Gata Factor ◽  
Specific Expression ◽  
Developmental Potential ◽  
Gfp Reporter ◽  
Transgenic Embryos ◽  
Green Fluorescent ◽  
Definitive Hematopoiesis

ABSTRACT Transcription factor GATA-2 is essential for definitive hematopoiesis, which developmentally emerges from the para-aortic splanchnopleura (P-Sp). The expression of a green fluorescent protein (GFP) reporter placed under the control of a 3.1-kbp Gata2 gene regulatory domain 5′ to the distal first exon (IS) mirrored that of the endogenous Gata2 gene within the P-Sp and yolk sac (YS) blood islands of embryonic day (E) 9.5 murine embryos. The P-Sp- and YS-derived GFP+ fraction of flow-sorted cells dissociated from E9.5 transgenic embryos contained far more CD34+/c-Kit+ cells than the GFP− fraction did. When cultured in vitro, the P-Sp GFP+ cells generated both immature hematopoietic and endothelial cell clusters. Detailed transgenic mouse reporter expression analyses demonstrate that five GATA motifs within the 3.1-kbp Gata2 early hematopoietic regulatory domain (G2-EHRD) were essential for GFP expression within the dorsal aortic wall, where hemangioblasts, the earliest precursors possessing both hematopoietic and vascular developmental potential, are thought to reside. These results thus show that the Gata2 gene IS promoter is regulated by a GATA factor(s) and selectively marks putative hematopoietic/endothelial precursor cells within the P-Sp.

scholarly journals Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

Reproduction ◽  
2013 ◽  
Vol 145 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Shahin Eghbalsaied ◽  
Kamran Ghaedi ◽  
Götz Laible ◽  
Sayed Morteza Hosseini ◽  
Mohsen Forouzanfar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Gfp Expression ◽  
Exogenous Dna ◽  
Bovine Sperm ◽  
Gfp Fluorescence ◽  
Gfp Reporter ◽  
Green Fluorescent

Transgenic mammals have been produced using sperm as vectors for exogenous DNA (sperm-mediated gene transfer (SMGT)) in combination with artificial insemination. Our study evaluated whether SMGT could also be achieved in combination with IVF to efficiently produce transgenic bovine embryos. We assessed binding and uptake of fluorescently labelled plasmids into sperm in the presence of different concentrations of dimethyl sulphoxide or lipofectamine. Live motile sperm displayed a characteristic punctuate fluorescence pattern across their entire surface, while uniform postacrosomal fluorescence was only apparent in dead sperm. Association with sperm or lipofection reagent protected exogenous DNA from DNase I digestion. Following IVF, presence and expression of episomal and non-episomal green fluorescent protein (GFP)-reporter plasmids was monitored in oocytes and embryos. We found no evidence of intracellular plasmid uptake and none of the resulting zygotes (n=96) and blastocysts were GFP positive by fluorescence microscopy or genomic PCR (n=751). When individual zona-free oocytes were matured, fertilised and continuously cultured in the presence of episomal reporter plasmids until the blastocyst stage, most embryos (38/68=56%) were associated with the exogenous DNA. Using anti-GFP immunocytochemistry (n=48) or GFP fluorescence (n=94), no GFP expression was detected in blastocysts. By contrast, ICSI resulted in 18% of embryos expressing the GFP reporter. In summary, exposure to DNA was an inefficient technique to produce transgenic bovine sperm or blastocysts in vitro.

scholarly journals Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability

2000 ◽  
Vol 149 (5) ◽  
pp. 1027-1038 ◽  
Author(s):  
Nataliya Shulga ◽  
Nima Mosammaparast ◽  
Richard Wozniak ◽  
David S. Goldfarb
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Transport Channel ◽  
Diffusion Channel ◽  
Gfp Reporter ◽  
Green Fluorescent ◽  
Gfp Reporters

The vertebrate nuclear pore complex (NPC) harbors an ∼10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Δ and nup170-Δ cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36–126 kD and were found to be greater than wild-type in nup188-Δ and nup170-Δ cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.

scholarly journals Gene editing in bacteria via SpCas9/sgRNA ribonucleoprotein complexes

2021 ◽  
Author(s):  
Ruben Dario Arroyo-Olarte ◽  
Ricardo Neftali Bravo-Rodriguez ◽  
Edgar Morales-Rios
Keyword(s):  
Green Fluorescent Protein ◽  
Homologous Recombination ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
High Toxicity ◽  
Guide Rna ◽  
Ribonucleoprotein Complexes ◽  
Gfp Reporter ◽  
Blue Fluorescent Protein ◽  
Green Fluorescent

Gene editing has been revolutionized by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas technology in a variety of organisms. In bacteria, however, CRISPR-Cas still holds many caveats such as high toxicity of Cas9 and off-target editing effects. In this work we develop a system for the incorporation of Cas9/single guide RNA ribonucleoprotein complexes in bacteria and their successful application for gene editing via homologous recombination. Targeting of a green fluorescent protein (GFP) reporter allows for easy verification of gene-edition via conversion to blue-fluorescent protein (BFP), mediated by the well characterized 196T > C (Tyr66His) mutation.

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