In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichia coli expressing infrared fluorescent protein in mice

Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.

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Fluorescent proteins for in vivo imaging, where's the biliverdin?

Biochemical Society Transactions ◽

10.1042/bst20200444 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2657-2667

Author(s):

Felipe Montecinos-Franjola ◽

John Y. Lin ◽

Erik A. Rodriguez

Keyword(s):

Hydrogen Peroxide ◽

In Vivo Imaging ◽

Near Infrared ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Imaging ◽

Infrared Light ◽

Red Fluorescent Protein ◽

Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

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CAPACIDAD PROBIÓTICA DE BACTERIAS LÁCTICAS AISLADAS DE CHICHA DE MOLLE

Revista de la Sociedad Química del Perú ◽

10.37761/rsqp.v83i4.212 ◽

2017 ◽

Vol 83 (4) ◽

pp. 391-402

Author(s):

Jhanina Rodríguez Carrasco ◽

Paula García-Godos Alcázar

Keyword(s):

Escherichia Coli ◽

Candida Albicans ◽

Salmonella Typhimurium ◽

Lactobacillus Plantarum ◽

Leuconostoc Mesenteroides

La investigación tuvo como objetivos aislar e identificar bacterias lácticas (BAL), evaluar la capacidad probiótica in vitro e in vivo de bacterias lácticas aisladas de chicha de molle, para ello se muestreó chichas de molle elaboradas artesanalmente de las provincias de Huanta y Huamanga, aislando 55 cepas BAL e identificando a Lactobacillus plantarum, Lactobacillus maltaromicus y Leuconostoc mesenteroides en base a la coloración Gram, producción de gas, gluconato y fermentación de azúcares. Para evaluar la capacidad probiótica in vitro se realizaron pruebas de antagonismo entre BAL con cuatro microorganismos patógenos (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Staphylococus aureus ATCC 25923 y Candida albicans ATCC 90028), mostrándose que 14 de las 55 cepas BAL producen sustancias inhibitorias de amplio espectro; asimismo, se evaluó la capacidad de tolerancia a condiciones gastrointestinales de cepas BAL, realizando ensayos a diferentes pHs , diferentes concentraciones de sales biliares y extracto gástrico artificial, resultando 25 cepas BAL con capacidad de tolerancia gastrointestinal y se seleccionaron cuatro cepas con mayor diámetro de halos de inhibición y cepas tolerantes a condiciones gastrointestinales siendo las cepas: BL-1 (Lactobacillus plantarum), BL-26 (Lactobacillus maltaromicus), BL-27 (Lactobacillus plantarum) y BL-53 (Lactobacillus maltaromicus), a las cuales se evaluaron la capacidad probiótica in vivo en 20 ratas para luego realizar recuento de BAL en el intestino a los 21 días, encontrándose en el grupo de estudio con BAL a 60x1019 UFC/ mL, mientras en el tratamiento con BAL más yacón a 25x1024 UFC/mL y los tratamientos de yacón y control a 50x1014 UFC/mL de BAL obteniéndose una de ganancia de peso en ratas en el grupo de estudio de BAL más yacón de 46 g, mientras con bacterias lácticas se tuvo 24 g y 16 g en el grupo control y extracto de yacón. En consecuencia esta investigación demuestra que la toma diaria de bebidas fermentadas tradicionales favorece el incremento de Lactobacillus en la microbiota intestinal.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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