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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 -
FEMS Microbiology Reviews Mar 2021The production of bulk chemicals mostly depends on exhausting petroleum sources and leads to emission of greenhouse gases. Within the last decades the urgent need for... (Review)
Review
The production of bulk chemicals mostly depends on exhausting petroleum sources and leads to emission of greenhouse gases. Within the last decades the urgent need for alternative sources has increased and the development of bio-based processes received new attention. To avoid the competition between the use of sugars as food or fuel, other feedstocks with high availability and low cost are needed, which brought acetogenic bacteria into focus. This group of anaerobic organisms uses mixtures of CO2, CO and H2 for the production of mostly acetate and ethanol. Also methanol, a cheap and abundant bulk chemical produced from methane, is a suitable substrate for acetogenic bacteria. In methylotrophic acetogens the methyl group is transferred to the Wood-Ljungdahl pathway, a pathway to reduce CO2 to acetate via a series of C1-intermediates bound to tetrahydrofolic acid. Here we describe the biochemistry and bioenergetics of methanol conversion in the biotechnologically interesting group of anaerobic, acetogenic bacteria. Further, the bioenergetics of biochemical production from methanol is discussed.
Topics: Acetates; Bacteria; Energy Metabolism; Ethanol; Green Chemistry Technology; Methanol
PubMed: 32901799
DOI: 10.1093/femsre/fuaa040 -
Medicine and Science in Sports and... Nov 2019Aerobic training and green tea extract can be used to reduce the risk of prostate cancer. The goal of this study was to evaluate the effects of 8-wk aerobic exercise...
INTRODUCTION
Aerobic training and green tea extract can be used to reduce the risk of prostate cancer. The goal of this study was to evaluate the effects of 8-wk aerobic exercise training and administration of green tea extract on the level of nuclear factor kappa B (NF-kB), cyclooxygenase-2 (COX-2) and p53 tumor suppressor protein (p53) in prostate of rats which were stimulated by N-methyl-N-nitrosourea to induce the prostate cancer.
METHODS
Sixty adult male Wistar rats were assigned into six groups including healthy control, cancer control (CCt), cancer training (CTr: 45 min·d at low to moderate intensity, five times per week, 8 wk), cancer extract (CEx: 1.34 mL of green tea extract, three times per week, 8 wk), cancer training+ cancer extract (CTr + CEx) and sham groups. Rats were sacrificed 48 h after the last intervention session, and the prostate tissue was isolated to measure the levels of NF-kB, COX-2, and p53.
RESULTS
The NF-kB level in CCt group was increased significantly compared to the healthy control (P = 0.02). In the CTr group, NF-kB level was decreased significantly compared to the CCt and CEx groups (P = 0.001 and 0.05, respectively). In addition, the levels of P53 protein were reduced in CTr, CEx, and CTr + CEx groups compared to CCt group (P = 0.001, 0.02 and 0.004, respectively). No significant changes were found in the level of COX-2 between groups.
CONCLUSIONS
These results suggest that a long-term exercise training combined with the intake of green tea extract may reduce levels of NF-kB and p53 in rats with prostate cancer. Given the importance of recognizing complementary therapies in this regard, future studies are warranted.
Topics: Animals; Anticarcinogenic Agents; Body Weight; Cyclooxygenase 2; Male; Methylnitrosourea; NF-kappa B; Organ Size; Physical Conditioning, Animal; Plant Extracts; Prostate; Prostatic Neoplasms; Rats, Wistar; Tea; Tumor Suppressor Protein p53
PubMed: 31626054
DOI: 10.1249/MSS.0000000000002054 -
Food Chemistry Mar 2022Matcha tea contains only the softer parts of the tea leaves and is finely ground. Therefore, extraction of the flavanols for analysis by HPLC is possible by a simpler...
Matcha tea contains only the softer parts of the tea leaves and is finely ground. Therefore, extraction of the flavanols for analysis by HPLC is possible by a simpler protocol compared to the ISO 14502-2 method. 21 different simplified extraction methods were screened and five of them gave equal results as the ISO 14502-2 method. The simplest and fastest method consists of extraction by ethanol + water (7 + 3, v + v) at room temperature with ultrasonication. This method was validated by determining accuracy, intraday and interday repeatability. The simplified method was successfully applied to four traditional matcha teas and two powdered green teas from Japan. This method paves the way for time-saving, energy-saving and accurate analyses of flavanols in matcha tea.
Topics: Antioxidants; Camellia sinensis; Catechin; Chromatography, High Pressure Liquid; Polyphenols; Tea
PubMed: 34863606
DOI: 10.1016/j.foodchem.2021.131628 -
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 -
Angewandte Chemie (International Ed. in... Apr 2022D-Luciferin (D-LH ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method...
D-Luciferin (D-LH ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH analogues with ≈51 % yield. The method can synthesize the novel 5'-methyl-D-LH and 4',5'-dimethyl-D-LH , which have never been synthesized or found in nature. 5'-Methyl-D-LH emits brighter and longer wavelength light than the D-LH . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.
Topics: Firefly Luciferin; Luciferases, Firefly; Luciferins; Luminescence; Luminescent Measurements; Pesticides
PubMed: 35138676
DOI: 10.1002/anie.202116908