Synthetic lipid-containing scaffolds enhance production by co-localizing enzymes

AbstractSubcellular organization is critical for isolating, concentrating, and protecting biological activities. Natural subcellular organization is often achieved using co-localization of proteins on scaffold molecules, thereby enhancing metabolic fluxes and enabling co-regulation. Synthetic scaffolds extend these benefits to new biological processes, and are typically constructed from proteins or nucleic acids. To expand the range of available building materials, we use a minimal set of components from the lipid-encapsulated bacteriophage Φ6 to form synthetic lipid-containing scaffolds (SLSs) inE. coli. Analysis of diffusive behavior by tracking particles in live cells indicates that SLSs are >20 nm in diameter; furthermore, density measurements demonstrate that SLSs contain a mixture of lipids and proteins. The fluorescent proteins mCitrine and mCerulean can be co-localized to SLSs. To test for effects on enzymatic production, we localized two enzymes involved in indigo biosynthesis to SLSs. We observed a scaffold-dependent increase in indigo production, showing that SLSs can enhance metabolic reactions.

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Genetically-encoded fluorescent biosensor for rapid detection of protein expression

10.1101/2020.07.30.229633 ◽

2020 ◽

Author(s):

Matthew G Eason ◽

Antonia T Pandelieva ◽

Marc M Mayer ◽

Safwat T Khan ◽

Hernan G Garcia ◽

...

Keyword(s):

Protein Expression ◽

Real Time ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Live Cells ◽

Spatiotemporal Resolution ◽

E Coli ◽

Fluorescent Biosensor ◽

Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

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Chemical Composition and Antibacterial and Antioxidant Activity of a Citrus Essential Oil and Its Fractions

Molecules ◽

10.3390/molecules26102888 ◽

2021 ◽

Vol 26 (10) ◽

pp. 2888

Author(s):

Carmen M. S. Ambrosio ◽

Gloria L. Diaz-Arenas ◽

Leidy P. A. Agudelo ◽

Elena Stashenko ◽

Carmen J. Contreras-Castillo ◽

...

Keyword(s):

Antioxidant Activity ◽

Antibacterial Activity ◽

Chemical Composition ◽

Essential Oil ◽

Biological Activities ◽

E Coli ◽

Beneficial Bacterium ◽

Minor Compounds ◽

Antibacterial And Antioxidant Activity ◽

High Relative Amount

Essential oils (EOs) from Citrus are the main by-product of Citrus-processing industries. In addition to food/beverage and cosmetic applications, citrus EOs could also potentially be used as an alternative to antibiotics in food-producing animals. A commercial citrus EO—Brazilian Orange Terpenes (BOT)—was fractionated by vacuum fractional distillation to separate BOT into various fractions: F1, F2, F3, and F4. Next, the chemical composition and biological activities of BOT and its fractions were characterized. Results showed the three first fractions had a high relative amount of limonene (≥10.86), even higher than the whole BOT. Conversely, F4 presented a larger relative amount of BOT’s minor compounds (carvone, cis-carveol, trans-carveol, cis-p-Mentha-2,8-dien-1-ol, and trans-p-Mentha-2,8-dien-1-ol) and a very low relative amount of limonene (0.08–0.13). Antibacterial activity results showed F4 was the only fraction exhibiting this activity, which was selective and higher activity on a pathogenic bacterium (E. coli) than on a beneficial bacterium (Lactobacillus sp.). However, F4 activity was lower than BOT. Similarly, F4 displayed the highest antioxidant activity among fractions (equivalent to BOT). These results indicated that probably those minor compounds that detected in F4 would be more involved in conferring the biological activities for this fraction and consequently for the whole BOT, instead of the major compound, limonene, playing this role exclusively.

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Biosynthesis of resveratrol derivatives and evaluation of their anti-inflammatory activity

Applied Biological Chemistry ◽

10.1186/s13765-021-00607-4 ◽

2021 ◽

Vol 64 (1) ◽

Author(s):

Yoojin Chong ◽

Hye Lim Lee ◽

Jihyeon Song ◽

Youngshim Lee ◽

Bong-Gyu Kim ◽

...

Keyword(s):

Biological Activities ◽

Stilbene Synthase ◽

Inflammatory Activity ◽

E Coli ◽

Prephenate Dehydrogenase ◽

Coa Ligase ◽

Final Yield ◽

Resveratrol Derivatives ◽

Anti Inflammatory ◽

Tyrosine Biosynthesis

AbstractResveratrol is a typical plant phenolic compound whose derivatives are synthesized through hydroxylation, O-methylation, prenylation, and oligomerization. Resveratrol and its derivatives exhibit anti-neurodegenerative, anti-rheumatoid, and anti-inflammatory effects. Owing to the diverse biological activities of these compounds and their importance in human health, this study attempted to synthesize five resveratrol derivatives (isorhapontigenin, pterostilbene, 4-methoxyresveratrol, piceatannol, and rhapontigenin) using Escherichia coli. Two-culture system was used to improve the final yield of resveratrol derivatives. Resveratrol was synthesized in the first E. coli cell that harbored genes for resveratrol biosynthesis including TAL (tyrosine ammonia lyase), 4CL (4-coumaroyl CoA ligase), STS (stilbene synthase) and genes for tyrosine biosynthesis such as aroG (deoxyphosphoheptonate aldolase) and tyrA (prephenate dehydrogenase). Thereafter, culture filtrate from the first cell was used for the modification reaction carried out using the second E. coli harboring hydroxylase and/or O-methyltransferase. Approximately, 89.8 mg/L of resveratrol was synthesized and using the same, five derivatives were prepared with a conversion rate of 88.2% to 22.9%. Using these synthesized resveratrol derivatives, we evaluated their anti-inflammatory activity. 4-Methoxyresveratrol, pterostilbene and isorhapontigenin showed the anti-inflammatory effects without any toxicity. In addition, pterostilbene exhibited the enhanced anti-inflammatory effects for macrophages compared to resveratrol.

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Silver Nanoparticles from Oregano Leaves’ Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses

International Journal of Molecular Sciences ◽

10.3390/ijms22073539 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3539

Author(s):

Anastasia Meretoudi ◽

Christina N. Banti ◽

Panagiotis K. Raptis ◽

Christina Papachristodoulou ◽

Nikolaos Kourkoumelis ◽

...

Keyword(s):

Silver Nanoparticles ◽

Contact Lenses ◽

Attenuated Total Reflection ◽

Bacterial Strains ◽

Scanning Calorimetry ◽

X Ray ◽

E Coli ◽

Polymer Hydrogels ◽

20 Nm ◽

And Staphylococcus Aureus

The oregano leaves’ extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl–methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV–Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.

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GABA transporter function, oligomerization state, and anchoring: correlates with subcellularly resolved FRET

The Journal of General Physiology ◽

10.1085/jgp.200910314 ◽

2009 ◽

Vol 134 (6) ◽

pp. 489-521 ◽

Cited By ~ 28

Author(s):

Fraser J. Moss ◽

P.I. Imoukhuede ◽

Kimberly Scott ◽

Jia Hu ◽

Joanna L. Jankowsky ◽

...

Keyword(s):

Actin Cytoskeleton ◽

Fluorescent Proteins ◽

Resonance Energy ◽

High Order ◽

Live Cells ◽

Gaba Uptake ◽

Cell Periphery ◽

Wild Type ◽

Gaba Transporter ◽

Amplitude Component

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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Betalains in Edible Fruits of Three Cactaceae Taxa—Epiphyllum, Hylocereus, and Opuntia—Their LC-MS/MS and FTIR Identification and Biological Activities Evaluation

Plants ◽

10.3390/plants10122669 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2669

Author(s):

Michaela Barkociová ◽

Jaroslav Tóth ◽

Katarzyna Sutor ◽

Natalia Drobnicka ◽

Slawomir Wybraniec ◽

...

Keyword(s):

Antimicrobial Activity ◽

Antioxidant Activity ◽

Food Industry ◽

Biological Activities ◽

Ornamental Plants ◽

Reducing Power ◽

Activity Assay ◽

Natural Pigment ◽

E Coli ◽

Ft Ir

Epiphyllum, Hylocereus, and Opuntia plants belong to the Cactaceae family. They are mostly known as ornamental plants but also for their edible fruits, which can potentially be sources of betalains, such as betanin, a natural pigment used in the food industry, e.g., under the European label code E 162. The aim of this work was the identification of betalains (using LC-MS/MS), evaluation of total betalain content (spectrophotometrically), analysis of functional groups (using FT-IR), evaluation of antioxidant activity (using DPPH, ABTS, FRAP, DCFH-DA, and reducing power methods) and evaluation of antimicrobial activity (S. aureus, E. coli, and C. albicans) in fruits of Epiphyllum, Hylocereus, and Opuntia taxa. A total of 20 betalains were identified in the studied Cactaceae fruits. The Epiphyllum pink hybrid had the highest values of total betalains amongst all samples. The highest antioxidant activity was observed in the Epiphyllum pink hybrid, in Opuntia zacuapanensis and O. humifusa fruits. The antimicrobial activity assay showed that cacti fruits were not able to effectively inhibit the growth of E. coli, S. aureus, or C. albicans. Our results prove that these fruits are good sources of natural pigments—betalains. They do not contain toxic compounds in significant amounts and they exhibit antioxidant activity.

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Synthesis, characterisation and antibacterial evaluation of some novel 1-phenyl-1h-tetrazole-5-thiol derivatives

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v10i2.394 ◽

2019 ◽

Vol 10 (2) ◽

pp. 1136-1142

Author(s):

Athraa H Mekky ◽

Sajida M Thamir

Keyword(s):

Staphylococcus Aureus ◽

Biological Activities ◽

Aromatic Aldehydes ◽

Maximum Activity ◽

Phenacyl Bromide ◽

Alkylation Reaction ◽

E Coli ◽

Percentage Yield ◽

And Staphylococcus Aureus ◽

Antibacterial Evaluation

The aim of the study is to synthesise and. Characterize — some novel, "tetrazole., -5-thiol” derivatives. Firstly, the “1-phenyl-1H7-tetrazole-5-thiol” (A1) has been., synthesised by the reaction of phenylisothiocyanate with NaN3 in water as a solvent. Secondly, the tetrazole-5-thiol derivatives (A2-A4) were synthesised by the alkylation reaction of the compound (A1) with chloroacetone, phenacyl bromide and chloromethyl acetate respectively. The resulted percentage yield was relatively high (92%, %95, %90 respectively). Compound (A5) was synthesised by the reaction of ethyl acetate tetrazole derivative (A4) with hydrazine. Moreover, the derivatives (A6-A11) were synthesised by the reaction of the (A5) with various substituted aromatic aldehydes. Moreover, compounds (A12-A13) have been synthesised by the cyclization reaction of compound A5 with acetylacetone and phenyl acetylacetone respectively. The produced compounds have been identified by IR, 13C-NMR and 1H-NMR Spectroscopy, and the quantities of various of the physical data (melting point, the shape of crystal and color). Finally, the compounds were examined for their biological activities alongside two kinds of bacteria (E. Coli and Staphylococcus aureus). Compounds A2, A4, A8, A11 and A13 showed the highest inhibition activity against E. Coli. Compounds A2, A9, A10and A13 showed the maximum activity against Staphylococcus aureus.

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Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

10.1101/500298 ◽

2018 ◽

Cited By ~ 1

Author(s):

Shama Sograte-Idrissi ◽

Nazar Oleksiievets ◽

Sebastian Isbaner ◽

Mariana Eggert-Martinez ◽

Jörg Enderlein ◽

...

Keyword(s):

Fluorescent Proteins ◽

Super Resolution ◽

Resolving Power ◽

Acquisition Time ◽

Efficient Manner ◽

Target Dna ◽

Multiplexed Imaging ◽

Dna Strands ◽

Linkage Error ◽

20 Nm

AbstractDNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time.

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