Zhao Y et al. (2011) Science "An expanded palette of genetically encoded Ca indicators."

Nagai T, Teramoto T, Araki S, Zhao Y, Chang YF, Nakano M, Campbell RE, Ishihara T, Abdelfattah AS, Fujiwara M, Wu J

[Science, 2011]

Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.

Daehnel K et al. (2007) Parasite Immunol "Filaria/Wolbachia activation of dendritic cells and development of Th1-associated responses is dependent on Toll-like receptor 2 in a mouse model of ocular onchocerciasis (river blindness)."

Pearlman E, Gillette-Ferguson I, Heinzel FP, Diaconu E, Hise AG, Harling MJ, Daehnel K

[Parasite Immunol, 2007]

Toll-like receptors (TLRs) regulate dendritic cell function and activate signals that mediate the nature of the adaptive immune response. The current study examined the role of TLRs in dendritic cell activation and in regulating T cell and antibody responses to antigens from the filarial parasites Onchocerca volvulus and Brugia malayi, which cause river blindness and lymphatic filariasis, respectively. Bone-marrow-derived CD11c(+) cells from C57BL/6 and TLR4(-/-) mice produced high levels of IL-6 and RANTES, and showed elevated surface CD40 expression, whereas CD11c(+) cells from myeloid differentiation factor 88(-/-) (MyD88(-/-)), TLR2(-/-) and TLR2/4(-/-) mice were not activated. Similarly, IFN-gamma production by splenocytes from immunized TLR2(-/-) mice was significantly impaired compared with splenocytes from C57BL/6 and TLR4(-/-) mice. In contrast, there was no difference among these strains in Th2-associated responses including IL-5 production by splenocytes from immunized animals, serum IgE and IgG(1), or eosinophil infiltration into the corneal stroma. Neutrophil recruitment to the cornea and CXC chemokine production was inhibited in immunized TLR2(-/-) mice compared with C57BL/6 and TLR4(-/-) mice. Taken together, these findings demonstrate an essential role for TLR2 in filaria-induced dendritic cell activation, IFN-gamma production and neutrophil migration to the cornea, but does not affect filaria-induced Th2-associated responses.

Vicencio J et al. (2019) Genetics "Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans."

Ceron J, Serrat X, Martinez-Fernandez C, Vicencio J

[Genetics, 2019]

CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry, or wrmScarlet in <i>Caenorhabditis elegans</i> This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, ssDNA donors (200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a dsDNA donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal crRNAs and PCR products. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic sgRNAs, but did not observe a significant increase in efficiency. To streamline the approach, we combined all Step 1 and Step 2 reagents in a single injection and were successful in 3 of 5 loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.

Kuramochi T et al. (1994) Mol Biochem Parasitol "Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans."

Kuramochi T, Hirawake H, Furushima R, Komuniecki R, Kojima S, Kita K, Aoki T, Takamiya S

[Mol Biochem Parasitol, 1994]

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly(A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.

Dickinson DJ et al. (2015) Genetics "Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette."

Heppert JK, Goldstein B, Dickinson DJ, Pani AM, Higgins CD

[Genetics, 2015]

A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.

Nonet ML (2023) Genetics "Rapid generation of C. elegans single-copy transgenes combining RMCE and drug selection."

Nonet ML

[Genetics, 2023]

I outline a streamlined method to insert large, single-copy transgenes into the C. elegans genome using Recombination-Mediated Cassette Exchange (RMCE) that relies solely on drug selection yielding a homozygous fluorescent protein (FP) marked transgene in 3 generations (8 days) at high efficiency (&gt;1 insertion per 2 injected P0 animals). Landing sites for this approach are available on four chromosomes in several configurations which yield lines marked in distinct cell types. An array of vectors permit creating transgenes using a variety of selection methods (HygR, NeoR, PuroR, and unc-119) that yield lines expressing different colored FP tagged lines (BFP, GFP, mNG, and Scarlet). Although these transgenes retain a plasmid backbone and a selection marker, the inclusion of these sequences typically does not alter the expression of several cell specific promoters tested. However, in certain orientations promoters exhibits crosstalk with adjacent transcription units. In cases where crosstalk is problematic, the loxP-flanked fluorescent marker, plasmid backbone and hygR gene can be excised by crossing through germline Cre expressing lines also created using this technique. Finally, genetic and molecular reagents designed to facilitate customization of both targeting vectors and landing sites are also described. Together, the rRMCE toolbox provides a platform for developing further innovative uses of RMCE to create complex genetically engineered tools.

Salas AL et al. (2016) Phytomedicine "Biological activities of polyphenols-enriched propolis from Argentina arid regions."

Salas AL, Isla MI, Alberto MR, Cuello AS, Zampini IC, Schmeda-Hirschmann G, Rios JL, Maldonado L

[Phytomedicine, 2016]

BACKGROUND: Propolis is a bioactive natural product collected by honeybees (Apis mellifera) from plant sources. PURPOSE: This study was undertaken to determine the effect of propolis extracts from arid region of Argentina, on the activity/expression of pro-inflammatory enzymes, and as potential free radical scavenger, antifungal and anthelmintic agent as well as to get a first insight into the polyphenolic profile of the active fractions. STUDY DESIGN/METHODS: Two propolis samples were collected in different time from hives located in Tucuman, Argentina. They are representative of the collection time of the raw material for phytotherapeutical purposes. Ethanolic extracts from both propolis were obtained. The PEEs were analyzed for total polyphenol (TP), non-flavonoid phenols (NFP) and flavonoid (FP) content followed by HPLC-DAD analysis and identification of components by HPLC-MS/MS(n). The potentiality as anti-inflammatory (LOX, COX, iNOS enzymes), antioxidant, antifungal and nematicidal was determined. RESULTS: PEEs contain high levels of TP, NFP and FP, including cinnamic acid, caffeic acid prenyl ester, caffeoyl dihydrocaffeate and caffeic acid 3,4-dihydroxyphenethyl ester, liquiritigenin, 2',4'-dihydroxychalcone and 2',4'-dihydroxy-3'-methoxychalcone. The PEEs in vitro reduced the activity of LOX and COX-2. Pretreatment of RAW 264.7 cells with PEEs before the induction of inflammatory state, inhibited NO overproduction and the iNOS protein expression was significantly decreased. The PEEs exhibited antioxidant, antifungal (Candida sp.) and nematicidal effect (C. elegans). CONCLUSION: These findings show the potential use of characterized PEEs from arid regions of Argentina as phytomedicine.

Lee LW et al. (2010) Gene "Vectors for co-expression of two genes in Caenorhabditis elegans."

Lee LW, Lo SJ, Lo HW

[Gene, 2010]

To meet the increasing need of simultaneously co-expressing two different genes in the same cell of transgenic Caenorhabditis elegans, here, we report the establishment of dicistronic vectors that contain an intercistronic region (ICR) of the C. elegans operon, CEOP5428. In these vectors, a green fluorescence protein (GFP) and a red FP (RFP) genes were placed in the first and second cistrons, respectively, which were separated by the ICR. Driven by the fibrillarin (fib-1) or myo-2 promoter, the GFP- and RFP-fusion proteins were consistently co-expressed in the entire worm cells or in the pharynx muscle cells of the transgenic worms, respectively. Our work demonstrates that ICR-containing dicistronic vectors could be developed into versatile co-expression systems in C. elegans for functional analysis of genes of interest.

Mita M et al. (2019) Anal Chem "Green fluorescent protein-based glucose indicators report glucose dynamics in living cells."

Harada K, Kitaguchi T, Sugawara I, Ito M, Mita M, Tsuboi T, Ueda H

[Anal Chem, 2019]

Glucose is the most important energy source for living animals. Here, we developed a series of single fluorescent protein (FP)-based glucose indicators, named as &quot;Green Glifons&quot;, to understand the hierarchal and mutual relationships between molecules involved in energy metabolism. Three indicators showed a different EC50 for glucose (50 M, 600 M and 4,000 M), producing a ~7-fold change in fluorescence intensity in response to glucose. The indicators could visualize glucose dynamics in the cytoplasm, plasma membrane, nucleus and mitochondria of living HeLa cells and in vivo, in the pharyngeal muscle of C. elegans and could measure murine blood glucose levels. Finally, the indicators were applicable to dual-color imaging, revealing the dynamic interplay between glucose and Ca2+ in mouse pancreatic MIN6 m9 cells. We propose that these indicators will facilitate and contribute to in vivo and multi-color imaging of energy metabolism.

Feng S et al. (2019) Commun Biol "Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses."

Muller JD, Kohler J, Coto Villa D, Varshney A, Feng S, Huang B, Modavi C, Zhou S, Van Hoven MK, Farah F, Ali N

[Commun Biol, 2019]

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP_1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.