A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

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.

Chitosan nanoparticles as a rice growth promoter: evaluation of biological activity.

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.

Selective visual staining of polyurethane microplastics by novel colorimetric and near-infrared (NIR) fluorescent dye: Application to environmental water and natural soil samples.

Microplastics can cause environmental pollution and ecosystem destruction as well as human health problems. Among the types of microplastics, polyurethane (PU) is particularly resistant to heat and difficult to decompose, causing disposal problems, and is evaluated as one of the most hazardous polymers. We present a novel colorimetric and near-infrared (NIR) fluorescence dye, (E)-N-(2-((4-(diphenylamino)benzylidene)amino)phenyl)- 7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (DPNA), designed for selective visual PU microplastic staining. The intramolecular charge transfer (ICT) properties of DPNA are demonstrated through density functional theory (DFT) calculations along with solvatochromic shift. DPNA exhibits red color and red fluorescence emission, showing promising potential as a staining dye. To achieve selective PU microplastic staining, we establish an optimized experimental procedure with the staining dye DPNA by evaluating the staining efficiency under different staining solvent compositions and staining times. DPNA can distinguish PU by both red fluorescence signal and red coloration among different types of microplastics. In addition, DPNA well stain fresh PUs with diverse sizes and at various pH range of 5-9, and the aged PUs can also be dyed as effectively as the fresh PU. Most importantly, DPNA selectively stains PU among 11 types of microplastics and 5 types of natural particles in environmental water and soil with and without any pre-treatments. The adsorption mechanism of DPNA on PU microplastic is demonstrated through field emission scanning electron microscopes (FE-SEM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), and non-covalent interaction (NCI)-reduced density gradient (RDG) analyses, and proposed that intermolecular hydrogen bonding has a significant effect.

Biochemical Characterization of Recombinant Enterococcus faecalis EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against Candida albicans.

Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV88-His6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV88-His6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV88-His6, we revealed that EntV88-His6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV88-His6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro. The combination of EntV88-His6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants.

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.

A Dictyostelium discoideum mitochondrial fluorescent tagging vector that does not affect respiratory function.

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function.

Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.

We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.

Fluorescent Labeling of Proteins.

Many single-molecule experimental techniques exploit fluorescence as a tool to investigate conformational dynamics, molecular interactions, or track the movement of proteins in order to gain insight into their biological functions. A prerequisite to these experimental approaches is to graft one or more fluorophores on the protein of interest with the desired photophysical properties. Here, we describe procedures for efficient methods used to covalently attach fluorophores to proteins. Alternative direct and indirect labeling strategies are also described.

Identification of microplastics using Raman spectroscopy: Latest developments and future prospects.

Widespread microplastic pollution is raising growing concerns as to its detrimental effects upon living organisms. A realistic risk assessment must stand on representative data on the abundance, size distribution and chemical composition of microplastics. Raman microscopy is an indispensable tool for the analysis of very small microplastics (<20 µm). Still, its use is far from widespread, in part due to drawbacks such as long measurement time and proneness to spectral distortion induced by fluorescence. This review discusses each drawback followed by a showcase of interesting and easily available solutions that contribute to faster and better identification of microplastics using Raman spectroscopy. Among discussed topics are: enhanced signal quality with better detectors and spectrum processing; automated particle selection for faster Raman mapping; comprehensive reference libraries for successful spectral matching. A last section introduces non-conventional Raman techniques (non-linear Raman, hyperspectral imaging, standoff Raman) which permit more advanced applications such as real-time Raman detection and imaging of microplastics.

Novel tetraphenylethylene diol amphiphile with aggregation-induced emission: self-assembly, cell imaging and tagging property.

In this study, we reported a novel tetraphenylethylene diol amphiphile (2) with aggregation-induced emission property which self-assembled to form unique 3D cubic superstructure. This TPE amphiphile with strong bluish green emission exhibited high biocompatibility and improved cellular uptake. Moreover, this highly fluorescent molecule was conjugated with gold nanoparticles and used as a fluorescent tagging agent. Altogether, results supported the cell-imaging and fluorescent tagging properties of tetraphenylethylene diol amphiphile.

Construction and characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agents, Bacillus anthracis, Yersinia pestis, Burkholderia mallei, and Burkholderia pseudomallei.

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.

Invasion of rhizobial infection thread by non-rhizobia for colonization of Vigna radiata root nodules.

Legumes develop symbiotic relationships with Rhizobium by a complex exchange of signals. Despite the high specificity between symbiotic partners, the presence of non-rhizobial bacteria in root nodules has been reported. To investigate how these rhizobacteria enter root nodules, fluorescently tagged Pseudomonas fluorescens and Klebsiella pneumoniae were co-inoculated with host-nodulating Ensifer adhaerens to Vigna radiata seedlings and root hair infection was monitored using confocal microscopy at 5 days post inoculation. Pseudomonas fluorescens and K. pneumoniae invaded the root hair only when co-inoculated with E. adhaerens. Recovery of inoculated tagged strains and confirmation through CLSM and 16S rRNA gene sequencing confirmed that the test rhizobacteria occupied nodules. We hereby report with the help of confocal microscopy that rhizobacteria migrate along the length of host-nodulating rhizobial strain and become localized in root nodules. We further report isolation of eight non-rhizobial bacterial genera, predominantly Bacillus spp. and Paenibacillus spp., from nodules of field-grown V. radiata.