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PLoS Biology Apr 2020The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to...
The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.
Topics: Animals; CRISPR-Cas Systems; Cells, Cultured; Dependovirus; Epitopes; Female; Gene Editing; Gene Knock-In Techniques; Genes, Reporter; Genetic Vectors; Genome; Green Fluorescent Proteins; Male; Mice, Transgenic; Microscopy, Fluorescence; Molecular Imaging; Neurons; Organ Culture Techniques; Proteins; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spatio-Temporal Analysis
PubMed: 32275651
DOI: 10.1371/journal.pbio.3000665 -
Molecules (Basel, Switzerland) Apr 2021Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP) to phosphate and, in this way, controls numerous biosynthetic reactions that... (Review)
Review
Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP hydrolysis, (b) continuous and fixed-time measurements of PP synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.
Topics: Diphosphates; Enzyme Assays; Humans; Hydrolysis; Inorganic Pyrophosphatase; Luciferases; Phosphates
PubMed: 33919593
DOI: 10.3390/molecules26082356 -
Pharmacology 2015Aberrant epigenetic alterations in the genome such as DNA methylation play a significant role in cancer development. Green tea catechins have been reported to modulate... (Review)
Review
Aberrant epigenetic alterations in the genome such as DNA methylation play a significant role in cancer development. Green tea catechins have been reported to modulate epigenetic processes. This review aims to synthesize evidence on the modulation of DNA methylation by green tea catechins. Green tea catechins have been reported to reverse DNA methylation of tumor suppressor genes and increase transcription of these genes. Green tea catechins and especially epigallocatechin gallate modulate DNA methylation by attenuating the effect of DNA methyltransferase 1 (DNMT1). However, the exact mechanism of DNMT1 inhibition is not delineated. Suggested mechanisms include direct enzymatic inhibition, indirect enzymatic inhibition, reduced DNMT1 expression and translation. The possible effect of green tea catechins on other pathways of DNA methylation, i.e. methyl-CpG binding domain proteins, has not been investigated. Furthermore, the link between redox properties and epigenetic modulation by green tea catechins has not been defined either. Since green tea catechins are natural compounds with a rather acceptable safety profile, further research on their action as inhibitors of DNA methylation seems worthwhile.
Topics: Catechin; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Humans; Tea
PubMed: 25792496
DOI: 10.1159/000375503 -
Frontiers in Nutrition 2022The green plum is a native fruit of Australia that grows on the tree . This study aimed to confirm the high level of folate in green plums by analyzing a large number of...
The green plum is a native fruit of Australia that grows on the tree . This study aimed to confirm the high level of folate in green plums by analyzing a large number of ripe samples from multiple locations and to understand how folate vitamers change as the fruit grows through maturity stages. This study analyzed green plums for five vitamers of folate, Hfolate, 5-CH-Hfolate, 5-CHO-Hfolate, 10-CHO-PteGlu, and PteGlu (folic acid) using a stable isotope dilution assay on a liquid chromatograph mass spectrometer (LC-MS). Green plums were tested from four locations, two harvests and five maturity stages. Another 11 ripe samples, each from different tree clumps from one location, were also tested as were ripe red-colored green plums. The results show the 5-CH-Hfolate in green plum increases and accumulates in the fruit through development, ripening and senescence. The ripe green plums contain between 82.4 ± 5.5 and 149.4 ± 10.7 μg/100 g Fresh Weight (FW). The red-colored green plums are even higher in folate, with total folate measured as 192.5 ± 7.0 and 293.7 ± 27.4 μg/100 g FW, and further analysis of them is suggested. There is some variation in amounts of folate between fruit from different locations and sets of trees, but all ripe green plums tested are considered good dietary sources of folate.
PubMed: 36313068
DOI: 10.3389/fnut.2022.1006393 -
Plants (Basel, Switzerland) May 2022Kale sprouts contain health-promoting compounds that could be increased by applying plant nutrients or exogenous phytohormones during pre-harvest. The effects of...
Kale sprouts contain health-promoting compounds that could be increased by applying plant nutrients or exogenous phytohormones during pre-harvest. The effects of selenium (Se), sulfur (S), and methyl jasmonate (MeJA) on lutein, glucosinolate, and phenolic accumulation were assessed in kale sprouts. Red Russian and Dwarf Green kale were chamber-grown using different treatment concentrations of Se (10, 20, 40 mg/L), S (30, 60, 120 mg/L), and MeJA (25, 50, 100 µM). Sprouts were harvested every 24 h for 7 days to identify and quantify phytochemicals. The highest lutein accumulation occurred 7 days after S 120 mg/L (178%) and Se 40 mg/L (199%) treatments in Red Russian and Dwarf Green kale sprouts, respectively. MeJA treatment decreased the level of most phenolic levels, except for kaempferol and quercetin, where increases were higher than 70% for both varieties when treated with MeJA 25 µM. The most effective treatment for glucosinolate accumulation was S 120 mg/L in the Red Russian kale variety at 7 days of germination, increasing glucoraphanin (262.4%), glucoerucin (510.8%), 4-methoxy-glucobrassicin (430.7%), and glucoiberin (1150%). Results show that kales treated with Se, S, and MeJA could be used as a functional food for fresh consumption or as raw materials for different industrial applications.
PubMed: 35567272
DOI: 10.3390/plants11091271 -
Journal of Animal Physiology and Animal... May 2022Numerous natural and synthetic substances have effects on reproduction through several mechanisms. This review aims to summarize the impact of green tea (GT), yucca... (Review)
Review
Numerous natural and synthetic substances have effects on reproduction through several mechanisms. This review aims to summarize the impact of green tea (GT), yucca schidigera (YS) extract, curcuma longa (CL), adenosine 3',5'-cyclic monophosphate (cAMP) and isobutyl-1-methyl-xanthine (IBMX) stimulators on rabbit reproduction performance. To obtain a comprehensive overview of this topic, the keywords "reproduction," "substances," "spermatogenesis," "embryogenesis,"hormonal profil", "green tea", "yucca schidigera" were searched in such databases as WOS and PubMed to obtain relevant information. Spermatozoa profile was positively effected by the GT and YS, however, cAMP inhibitors stimulated spermatozoa motility resulted in positive or negative effects depending on the doses. Similarly, embryogenesis and hormonal profile were positively influenced by the GT, YS, cAMP and IBMX in a proper administration dose. Further research is needed to improve current knowledge about these substances to identify potential effects on the other reproduction parameters. Furthermore, future studies should combine GT, YS and CL with different plant extracts to determine their effects on spermatozoa status, embryogenesis as well as hormonal profile as key outcomes. This review summarizes current knowledge about effect of natural and synthetic substances on rabbit reproduction.
Topics: 1-Methyl-3-isobutylxanthine; Animals; Male; Rabbits; Reproduction; Yucca
PubMed: 34542913
DOI: 10.1111/jpn.13641 -
Microbial Cell Factories Jan 2022Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully...
BACKGROUND
Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803).
RESULTS
To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L and 75 mg L, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers.
CONCLUSION
A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.
Topics: Bacterial Proteins; Butanols; Light; Metabolic Engineering; Pentanols; Photoreceptors, Microbial; Photosynthesis; Promoter Regions, Genetic; Synechocystis
PubMed: 34991586
DOI: 10.1186/s12934-021-01732-x -
Chemosphere Oct 2020The herbicides diuron, hexazinone, and sulfometuron-methyl present a potential risk of environmental contamination and are widely used for weed control in sugarcane...
The herbicides diuron, hexazinone, and sulfometuron-methyl present a potential risk of environmental contamination and are widely used for weed control in sugarcane cultivation. Our objectives were to measure the tolerance of Canavalia ensiformes (L.) DC., Stilizobium aterrimum L., Raphanus sativus L., Crotalaria spectabilis Röth, Lupinus albus L., and Pennisetum glaucum (L.) R. Br. To the herbicides diuron, hexazinone, and sulfometuron-methyl to assess the capacity of these species to extract and accumulate the herbicides in their tissues. Before sowing the green manure species, the soils were individually contaminated with the three C-radiolabeled herbicides. C-diuron and C-sulfometuron-methyl showed higher values remaining in the soil (>90%) for all species of green manure compared to hexazinone (<80%). The green manure species analyzed showed greater potential to remedy soils contaminated with hexazinone than the other herbicides. C. ensiformes showed high phytoextraction of hexazinone when compared to the other species, removing 11.2% of the pollutant from the soil, followed by L. albus (8.6%), S. aterrimum (7.3%), R. sativus (4.8%), C. spectabilis (2.5%), and P. glaucum (1.1%). The results indicate that the phytoextraction of diuron, hexazinone and sulfometuron-methyl is dependent on the species of green manure and can be an important tool for the decontamination of areas polluted by these herbicides.
Topics: Biodegradation, Environmental; Diuron; Herbicides; Manure; Saccharum; Soil; Soil Pollutants; Sulfonylurea Compounds; Triazines
PubMed: 32447109
DOI: 10.1016/j.chemosphere.2020.127059 -
Food Research International (Ottawa,... Sep 2022This study aimed to evaluate the volatile compounds of chocolates made of Brazilian cocoas and statistically track them according to the products' sensorial profile in...
This study aimed to evaluate the volatile compounds of chocolates made of Brazilian cocoas and statistically track them according to the products' sensorial profile in order to relate them to consumers' acceptance by preference map methodology. The intensity of the chocolate, acidity, woody, smoked, green, floral, burned, musty, and cocoa notes from chocolates produced with cocoa from different Brazilian states were analyzed by a trained panel and by 128 consumers. Samples from Côte d'Ivoire, which is known for its high-quality chocolate, were evaluated for comparison. Solid-phase microextraction headspace sampling/gas chromatography-mass spectrometry was employed to evaluate the samples' volatile compounds. One hundred volatile compounds were identified within the samples. The results from the preference maps showed that the maximum preference was found for chocolate made of cocoa from Rondônia, Bahia, and Espírito Santo and Côte d'Ivoire and organic samples from Pará. The ideal sample point was characterized by intense chocolate, floral, and woody notes and mild green and burned notes. The presence of furfural, 3-methyl butanal, phenethyl acetate, 2-phenyl-5-methyl-2-hexenal, methyl pyrazine, phenethyl acetate, 2-phenyl-5-methyl-2-hexenal, and tetramethyl pyrazine were shown to be important for consumer acceptance in the ideal product, whereas the presence of (Z)-2-heptenal and 2-pentyl furan may increase consumer rejection. 2,3-Methyl pyrazine, methyl pyrazine, and 2,3-butanediol, which are important volatile compounds previously reported in the literature, were statistically tracked to both positive and negative sample attributes and must be better explored concerning consumers' acceptance of chocolates.
Topics: Brazil; Cacao; Chocolate; Pyrazines; Taste; Volatile Organic Compounds
PubMed: 35940809
DOI: 10.1016/j.foodres.2022.111618