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Plastic pollution is a global problem affecting the environment and, consequently, people’s well-being. Careful and timely end-of-life plastic recycling is certainly a way, albeit a partial one, to remedy the problem. The immediate identification and selection of the different types of plastic materials in the recycling process certainly facilitate its recovery and reuse, allowing the damage caused by plastic emission into the environment to be limited. Recently, new technologies for automatic sorting of plastics based upon fluorescent tagging have been considered. This article reports the synthesis and characterization of fluorescent copolymers of poly(methyl methacrylate) (PMMA) that could be potentially used as fluorescent markers of commercial PMMA. Poly(methylmetacrylate-co-2-(9-carbazolyl)ethyl methacrylate) (P(MMA-co-CEMA)) and poly(methylmetacrylate-co-7-methacryloyloxycoumarin) (P(MMA-co-MAOC)) samples containing a small number of fluorescent units (<4%) were synthesized by free-radical polymerization. All copolymer samples show chemico-physical properties like those of pure PMMA and produce fluorescence emission under 290 nm wavelength excitation. P(MMA-co-CEMA)s and P(MMA-co-MAOC)s were also tested as fluorescent dyes for PMMA identification. The experimental results demonstrate that PMMA/P(MMA-co-CEMA) and PMMA/P(MMA-co-MAOC) blends prepared using 1% by weight of fluorescent copolymer show a homogeneous morphology completely similar to pure PMMA and are still optically active.

Summary Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein–protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co‐transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all‐in‐one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single‐tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high‐throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS‐guided GA‐mediated AioFFP vector toolkit is cost‐effective, easy to use and will promote functional genomics research in plants.

The agriculture sector is the building block of an economy with more than 60% of the world population depending on it for livelihood. Among the many crops, rice is the most important income source. It is the staple food for more than half of the world population. In spite of its huge demand, rice production has been dwindling due to various constraints. Chitosan nanoparticles (ChNP) are an excellent choice for agricultural applications owing to its non-toxic, biodegradable nature. Chitosan is an interesting polymer and is then partially or fully deacetylated chitin. In the present study, the effectiveness of ChNP as a growth promoter in improving the yield and biological activity of rice has been analyzed. 1 mg/ml of ChNP was applied as a seed, soil, foliar and combination treatments and the growth and yield parameters were measured to understand the best mode of application. The combination treatment of seed, soil and the foliar application was found to be most efficient. The cellular uptake of ChNP was also studied to deduce the mechanism of action. The soil toxicity of ChNP was studied prior to application and was found to be non-toxic.

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana , rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

Mitophagy, a selective clearance of damaged or superfluous mitochondria via autophagy machinery and lysosomal degradation, is an evolutionarily conserved process essential for various physiological functions, including cellular differentiation and immune responses. Defects in mitophagy are implicated in numerous human diseases, such as neurodegenerative disorders, cancer, and metabolic conditions. Despite significant advancements in mitophagy research over recent decades, novel and robust methodologies are necessary to elucidate its molecular mechanisms comprehensively. In this study, we present a detailed protocol for quantitatively assessing mitophagy through flow cytometry using a mitochondria‐targeted fluorescent mitophagy receptor, GFP‐BNIP3L/NIX. This method offers a rapid alternative to conventional microscopy or immunoblotting techniques for analyzing mitophagy activity. Additionally, this approach can theoretically be adapted to utilize any fluorescent‐tagged selective autophagy receptor, enabling the direct and rapid analysis of various types of receptor‐mediated selective autophagy. Mitophagy, the selective degradation of damaged mitochondria, is crucial for cellular health and function. This study introduces a rapid and efficient flow cytometry method using GFP‐BNIP3L/NIX to accurately quantify mitophagy. Our approach can be adapted to use any fluorescent‐tagged selective autophagy receptor, enabling direct and rapid analysis of various types of receptor‐mediated selective autophagy in diverse biological conditions.

Cellulose nanofibers (CNFs) have great potential to be a layer in packaging materials because of their good barrier properties. When paper is coated with CNFs, they are difficult to distinguish from the base sheet. This issue creates challenges when trying to determine where CNFs migrate relative to the paper fibers during coating and drying. A three-dimensional analysis is possible by using confocal laser scanning microscopy (CLSM) if CNFs can be tagged with fluorescently active groups. In this study, CNFs were fluorescently tagged through adsorption of fluorescent dyes such as fluorescein isothiocyanate (FITC) and thioflavin by mixing with CNFs in their native suspension followed by purification. The adsorbed dye remained attached during typical coating procedures, low pH values, and high ionic strengths, but not for high pH and in contact with acetone. CNFs were also covalently tagged with FITC following methods reported in the literature as a comparison to already established methods for tagging cellulose nanocrystals. Images of never dried samples indicated that covalently tagging CNFs altered the state of the fines dispersion, while dye adsorption did not. Coatings of the adsorbed dye tagged CNFs on paper were successfully imaged by CLSM since the concentration of dye in the water phase was low enough to provide a good contrast between regions of CNFs and paper. With this method, the location and potential migration of CNFs coated on paper were successfully determined for the first time to the best of our knowledge. CNF based coatings with solids larger than 2.8% were found to have a distinct layer of CNFs at the paper surface with little CNFs penetrating into the paper structure, but lower solids result in significant penetration into the paper.

The increasingly intense consumption of plastics and, above all, their improper disposal in the environment are causing serious environmental concerns. Great efforts have been made for the development of new methods aimed at facilitating and speeding up the identification and sorting of different materials in the plastic recycling process. In this field, new strategies based on fluorescent tagging have been developed. This work concerns the synthesis and characterization of new fluorescent copolymers of polyethylene (PE) and polystyrene (PS), which are among the most produced and consumed plastic materials. The synthesized copolymers are potentially suitable for use as fluorescent markers of PE and PS. Ethylene-co-N-pentenyl carbazole (P(E-co-PK)) and styrene-co-4-(N-carbazolyl)methyl styrene (P(S-co-SK)) copolymers were prepared by Ziegler–Natta and free radical polymerization, respectively. If excited at 300 nm, both P(E-co-PK)s and P(S-co-SK)s give fluorescence emissions resulting in them being optically active. Moreover, due to the low amount of fluorescent units, they show chemico-physical properties such as those of their corresponding homopolymers (PE and PS). P(E-co-PK)s and P(S-co-SK)s have been also tested as fluorescent markers of PE and PS. The experimental results demonstrate that from PE/P(E-co-PK) and PS/P(S-co-SK) blends prepared using only 1% by weight of fluorescent copolymer, distinguishable fluorescent emissions can be still detected.

A set of tools for the genetic manipulation of the osmotolerant yeast Zygosaccharomyces rouxii was developed. Auxotrophic mutants (ura3 leu2, ura3 ade2, ura3 leu2 ade2) derived from the CBS 732 type strain were prepared. Centromeric and episomal Z. rouxii/Escherichia coli shuttle plasmids with different marker genes (ScURA3, ZrLEU2, ZrADE2) and with multiple cloning sites were constructed, together with a plasmid enabling green fluorescent protein-tagging. A system for repeatable targeted gene deletion in Z. rouxii was established, involving first the integration of a PCR-generated loxP-kanMX-loxP cassette and second the removal of kanMX from the genome using a Z. rouxii plasmid harbouring cre recombinase.

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

Here, we constructed stable, chromosomal, constitutively expressed, green and red fluorescent protein (GFP and RFP) as reporters in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei. Using bioinformatic approaches and other experimental analyses, we identified P0253 and P1 as potent promoters that drive the optimal expression of fluorescent reporters in single copy in B. anthracis and Burkholderia spp. as well as their surrogate strains, respectively. In comparison, Y. pestis and its surrogate strain need two chromosomal copies of cysZK promoter (P2cysZK) for optimal fluorescence. The P0253‐, P2cysZK‐, and P1‐driven GFP and RFP fusions were first cloned into the vectors pRP1028, pUC18R6KT‐mini‐Tn7T‐Km, pmini‐Tn7‐gat, or their derivatives. The resultant constructs were delivered into the respective surrogates and subsequently into the select agent strains. The chromosomal GFP‐ and RFP‐tagged strains exhibited bright fluorescence at an exposure time of less than 200 msec and displayed the same virulence traits as their wild‐type parental strains. The utility of the tagged strains was proven by the macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high‐throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages. The utility of chromosomally integrated and constitutively expressed green and red fluorescent protein tagged Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei strains was proven by macrophage infection assays and lactate dehydrogenase release analysis. Such strains will be extremely useful in high‐throughput screens for novel compounds that could either kill these organisms, or interfere with critical virulence processes in these important bioweapon agents and during infection of alveolar macrophages.