Fluorescent Protein mCherry Research Articles

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

Highly stable Saccharomyces cerevisiae L-BC capsids with versatile packing potential.

Virus-like particles (VLPs) are promising nanoscaffolds in development of vaccines and nanodelivery systems. Along with efficient production in various expression systems, they also offer extensive functionalization options. Nevertheless, the ultimate integrity of VLPs is an important burden for the applicability in nanobiotechnology. In this study, we characterize the Saccharomyces cerevisiae L-BC VLPs synthesized and purified from Escherichia coli and Saccharomyces cerevisiae cells. The particles exhibited prominent size stability in buffers within a range of ionic strength conditions, pH environment and presence of magnesium ions during the long-term storage at temperatures up to 37°C. Bacteria-derived particles exhibited alleviated stability in acidic pH values, higher ionic strength and temperature compared to yeast-derived particles. Taking advantage of gene engineering, 120 copies of red fluorescent protein mCherry were successfully encapsulated into both preparations of L-BC VLPs, while passive diffusion enabled encapsulation of antimicrobial peptide nisin into the yeast-derived unmodified VLPs. Our findings indicate that L-BC VLPs generally exhibit high long-term stability under various conditions, while yeast-derived L-BC VLPs are more stable under the elevated temperatures than bacteria-derived particles. Stability studies and encapsulation of particles by different molecules involving alternative strategies delineate the L-BC VLP potential to be developed into versatile nanodelivery system.

Multiple Posterior Insula Projections to the Brainstem Descending Pain Modulatory System.

The insular cortex is an important hub for sensory and emotional integration. It is one of the areas consistently found activated during pain. While the insular's connections to the limbic system might play a role in the aversive and emotional component of pain, its connections to the descending pain system might be involved in pain intensity coding. Here, we used anterograde tracing with viral expression of mCherry fluorescent protein, to examine the connectivity of insular axons to different brainstem nuclei involved in the descending modulation of pain in detail. We found extensive connections to the main areas of descending pain control, namely, the periaqueductal gray (PAG) and the raphe magnus (RMg). In addition, we also identified an extensive insular connection to the parabrachial nucleus (PBN). Although not as extensive, we found a consistent axonal input from the insula to different noradrenergic nuclei, the locus coeruleus (LC), the subcoereuleus (SubCD) and the A5 nucleus. These connections emphasize a prominent relation of the insula with the descending pain modulatory system, which reveals an important role of the insula in pain processing through descending pathways.

Reverse genetic approaches allowing the characterization of the rabies virus street strain belonging to the SEA4 subclade

Rabies virus (RABV) is the causative agent of rabies, a lethal neurological disease in mammals. RABV strains can be classified into fixed strains (laboratory strains) and street strains (field/clinical strains), which have different properties including cell tropism and neuroinvasiveness. RABV Toyohashi strain is a street strain isolated in Japan from an imported case which had been bitten by rabid dog in the Philippines. In order to facilitate molecular studies of RABV, we established a reverse genetics (RG) system for the study of the Toyohashi strain. The recombinant virus was obtained from a cDNA clone of Toyohashi strain and exhibited similar growth efficiency as the original virus in cultured cell lines. Both the original and recombinant strains showed similar pathogenicity with high neuroinvasiveness in mice, and the infected mice developed a long and inconsistent incubation period, which is characteristic of street strains. We also generated a recombinant Toyohashi strain expressing viral phosphoprotein (P protein) fused with the fluorescent protein mCherry, and tracked the intracellular dynamics of the viral P protein using live-cell imaging. The presented reverse genetics system for Toyohashi strain will be a useful tool to explore the fundamental molecular mechanisms of the replication of RABV street strains.

Bright New Resources for Syphilis Research: Genetically Encoded Fluorescent Tags for Treponema pallidum and Sf1Ep Cells.

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the invitro evaluation of antibiotic efficacy, performance of controlled studies to assess differential treponemal gene expression, and generation of loss-of-function mutants to evaluate the contribution of specific genetic loci to T. pallidum virulence. Building on this progress, we engineered the T. pallidum SS14 strain to express a red-shifted green fluorescent protein (GFP) and Sf1Ep cells to express mCherry and blue fluorescent protein (BFP) for enhanced visualization. These new resources improve microscopy- and cell sorting-based applications for T. pallidum, better capturing the physical interaction between the host and pathogen, among other possibilities. Continued efforts to develop and share new tools and resources are required to help our overall knowledge of T. pallidum biology and syphilis pathogenesis reach that of other bacterial pathogens, including spirochetes.

Trajectories of Solitons Movement in the Potential Field of pPF1 Plasmid with Non-Zero Initial Velocity

Nonlinear conformational distortions, such as, for example, locally unwound regions of the DNA double helix, named open states, are considered in this work as solitons moving in the potential field of this molecule. It is believed that open states play an important role in the processes of transcription, replication, denaturation, as well as in the transmission of structural changes and information along the DNA molecule. This work examines the problem of the movement of kink-like solitons (kinks) in the potential field of the pPF1 plasmid, the sequence of which includes the genes of the fluorescent proteins Egfp and mCherry, separated by a small intermediate region. The results of calculations of energy profiles of the main and complementary sequences of the plasmid, as well as 2D and 3D trajectories of kinks with a non-zero initial velocity are presented. It has been shown that two types of kinks can be activated in the pPF1 plasmid, which can be considered as two types of quasiparticles that have their own energy, mass, velocity and move in the potential field of the plasmid. It was found that the lowest energy required to form these kinks is observed in the intermediate region located between the fluorescent protein genes. It was shown that the nature of the motion of kinks does not depend on the value of their initial velocity. It was shown that there are threshold values of the torsion field, upon reaching which the behavior of kinks changes sharply: from damped oscillations within the intermediate region to forward motion and penetration into neighboring regions. These values have been calculated. It turned out that for the first kink moving in the main sequence potential field, the threshold value is 4.95×10–22 J, and for the second kink – 4.20 × 10–22 J.

The Stop Codon after the nsp3 Gene of Ross River Virus (RRV) Is Not Essential for Virus Replication in Three Cell Lines Tested, but RRV Replication Is Attenuated in HEK 293T Cells.

A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.

Long-range gene editing of LMNA

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Hartstichting (Dutch Heart Foundation) Background Mutations in the LMNA gene commonly affect the cardiac muscle tissue, which leads to the development of a severe dilated cardiomyopathy with life-threatening arrhythmias. The lack of response to conventional heart failure therapy in these patients indicates the urgent need for new therapies. Gene editing has the potential to become a curative treatment for these patients by correcting the genetic cause of the disease. Prime editing is a promising new gene editing technology, which uses a prime editing guide RNA (pegRNA) to target a specific genetic locus and creates edits using a reverse-transcriptase enzyme linked to a Cas9-nickase. Purpose Conventional prime-editing technology targets one mutation per pegRNA. However, with more than 600 disease-causing mutations described in LMNA, the feasibility of applying this technology to each mutation is limited. Therefore, we aim to target not a single, but a set of mutations with one single pegRNA. Methods The pegRNAs are optimised by selecting the optimal target regions, improving their stability and 3D structure. The in vitro editing efficiency is assessed using the fluoPEER reporter plasmid, transfected into HEK293T cells. Using flow cytometry, expression of mCherry fluorescent protein is detected following successful editing of the target mutation. Results Flow-cytometry analysis of treated cells revealed an editing efficiency of 12% with a long-range pegRNA targeting a full exon, compared to 15% with a conventional short pegRNA targeting the same mutation <10bp downstream of the Cas9 nick site. This editing efficiency using the same long-range pegRNA was retained at 9% when editing at a distant position >100bp downstream of the nick site. To increase the editing efficiency, we improved the stability of the pegRNAs by the addition of different 3’ structural motifs. This resulted in an increased editing efficiencies of 15% and 17% for nearby and distant mutations, respectively. Conclusion We show that it is possible to edit different mutations within a range of up to 120 bp with a single pegRNA. By optimisations of the pegRNA 3D structure and optimal selection of target locations we aim to further improve the editing efficiency of this system. This work could eventually provide a prime editing therapy able to cure disease in a subset of patients carrying different mutations in the same genetic region. For LMNA-associated diseases this would substantially reduce the number of pegRNAs that have to be developed to cover all mutations, greatly improving the feasibility of prime editing therapy.

Inhibition of somatostatinergic interneurons of the lateral parafacial region reduces the ventilatory response to hypercapnia of mice

The lateral parafacial region (pFL), which contains excitatory and inhibitory neurons and interneurons, generates active expiration in response to hypercapnia. Inhibition of inhibitory somatostatinergic (Sst)-positive neurons in parafacial region increases pulmonary ventilation, while hypercapnia inhibits some of them intrinsically. However, the role of pFL Sst-positive interneurons (with local projections) in regulating pulmonary ventilation during hypercapnia is unclear. We hypothesized that the pFL Sst-positive interneurons control the pulmonary ventilation of mice under normocapnia and hypercapnia, as part of a local neural network. Herein, we evaluated the impact of pFL Sst-positive interneurons inhibition on pulmonary ventilation of non-anesthetized mice during normocapnia and hypercapnia. We used genetically modified mice, expressing the cre recombinase in Sst-positive neurons, and injected unilaterally a cre-dependent adeno-associated (AAV) retrograde virus in the pFL region to express the FLP recombinase, followed by a cre- and FLP-dependent AAV to express the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (hM4D-Gi) in the pFL Sst-positive interneurons. We used whole-body plethysmography to measure the respiratory frequency (fR), tidal volume (VT), pulmonary ventilation (VE), as well as the duration of the respiratory cycle, inspiration (DI) and expiration (DE) at normocapnia and hypercapnia (7% CO2) after activation of hM4D-Gi in the pFL Sst-positive interneurons using JHU 37160 (JHU; 0.1 mg/kg; i.p.). Data are expressed as mean ± standard deviation. By analyzing the expression of mCherry fluorescent protein, we found a significant number of Sst-positive interneurons (73.43 ± 10.52) in the pFL region (n=14). JHU administration did not affect the ventilatory parameters of Sst(cre/cre) (n= 9) and Sst(cre/cre + hM4D-Gi) (n= 14) groups at normocapnia [(Δ fR: 6.5 ± 33.56 vs. -14.86 ± 49.03 cpm; p= 0.42) (Δ VT: 0.87 ± 1.15 vs. -0.97 ± 2.58 μL·g−1; p= 0.19) (Δ VE: 235.8 ± 388.8 vs. -315.9 ± 725.1 μL·g−1·min−1; p= 0.16) (Δ duration of the respiratory cycle: -13 ± 45.11 vs. 12.34 ± 53.07 ms; p= 0.39) (Δ DI: 10.36 ± 11.23 vs. 14.7 ± 16.38 ms; p= 0.62) (Δ DE: -23.36 ± 42.42 vs. -2.41± 53.58 ms; p= 0.48)]. On the other hand, JHU administration attenuated the hypercapnia-induced responses of all ventilatory parameters, such as fR (Δ: 44.57 ± 56.66 vs. 126± 21.28 cpm; p= 0.0002), VT (Δ: 1.32 ± 3.35 vs. 4.16 ± 2.56 μL·g−1; p= 0.04), VE (Δ: 948.7 ± 1407.1 vs. 2183 ± 909.4 μL·g−1·min−1; p= 0.03), duration of the respiratory cycle (Δ: -44.57 ± 46.31 vs. -122.1 ± 30.7 ms; p= 0.0002), DI (Δ: 6.79 ± 16.36 vs. -13.03 ± 10.19 ms; p= 0.003) and DE (Δ: -51.37 ± 37.50 vs. -109.1 ± 32.78 ms; p= 0.001) in the Sst(cre/cre + hM4D-Gi) compared with the Sst(cre/cre) group. Thus, we conclude that the pFL Sst-positive interneurons control the ventilatory parameters of mice during hypercapnia, but not during normocapnia. FAPESP, CNPq and CAPES. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inhibition of glutamatergic interneurons of the lateral parafacial region of mice attenuates the ventilatory response to hypercapnia

The lateral parafacial region (pFL) generates the expiratory motor activity during hypercapnia. It was demonstrated that this region contains excitatory neurons and interneurons (with local projections), with a population of glutamatergic neurons that are silent under resting conditions, but become active at the end of expiration, simultaneously with active expiration, in response to hypercapnia/acidosis. However, the role of pFL glutamatergic interneurons in regulating pulmonary ventilation during hypercapnia is unclear. Herein, we hypothesized that the inhibition of pFL glutamatergic interneurons attenuates the ventilatory responses to hypercapnia. Therefore, we evaluated the effects of the chemogenetic inhibition of pFL glutamatergic interneurons of non-anesthetized mice on ventilatory parameters during resting and hypercapnia. We used genetically modified mice expressing the enzyme cre recombinase in glutamatergic VGLUT2-positive neurons (CEUA 1143/2022), and injected unilaterally in the pFL region a cre-dependent adeno-associated retrograde virus to express the FLP recombinase, followed by a cre- and FLP-dependent virus to express the inhibitory Designer Receptors Exclusively Activated by Designer Drugs (hM4D-Gi) only in pFL glutamatergic interneurons. Using whole-body plethysmography, we measured respiratory frequency (fR), tidal volume (VT), pulmonary ventilation (VE), duration of respiratory cycle, inspiration (DI) and expiration (DE) at normocapnia and hypercapnia (7% CO2) after activating the hM4D-Gi in the pFL glutamatergic interneurons with JHU 37160 (0.1 mg/kg; i.p.). Data are expressed as mean ± standard deviation. By analyzing the expression of mCherry fluorescent protein, we found a significant number of glutamatergic interneurons in the pFL region (68.22 ± 11.7; n=18). At normocapnia, JHU injection did not change the ventilatory parameters of VGLUT2(cre/cre + hM4D-Gi) (n=18) and VGLUT2(cre/cre) (n=12) mice [(ΔfR: -2.4±51 vs 0.2±48 cpm; p=0.88) (ΔVT: -1.3±4 vs -0.2±1 μL·g−1; p=0.39) (ΔVE: -39 ±1198 vs -71±607 μL·g−1·min−1; p=0.72) (ΔCycle duration: 5.3±41 vs -1.9±45 ms; p=0.65) (ΔDI: 6.6±14 vs 6±20 ms; p=0.92) (ΔDE: -1.3±35 vs -1.6±31 ms; p=0.97)]. On the other hand, JHU injection attenuated the hypercapnia-induced ventilatory responses of VGLUT2(cre/cre + hM4D-Gi) (n=18) mice when compared to VGLUT2(cre/cre) (n=9) [(ΔfR: 67±69 vs 157±41 cpm; p=0.0003) (ΔVT: 3.8±5 vs 11.9±4 μL·g−1; p<0.0001) (ΔVE: 2052±2197 vs 5374±1773 μL·g−1·min−1; p<0.0001) (Δcycle duration: -46±50 vs -107±49 ms; p=0.002) (ΔDI: -3.7±16 vs -21±19 ms; p=0.011) (ΔDE: -42±41 vs -86±36 ms; p=0.01)]. In conclusion, our data demonstrate that the pFL glutamatergic interneurons are important for the ventilatory response of mice to hypercapnia. FAPESP, CNPq and CAPES. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

Dirac Cones and Room Temperature Polariton Lasing Evidenced in an Organic Honeycomb Lattice.

Artificial 1D and 2D lattices have emerged as a powerful platform for the emulation of lattice Hamiltonians, the fundamental study of collective many-body effects, and phenomena arising from non-trivial topology. Exciton-polaritons, bosonic part-light and part-matter quasiparticles, combine pronounced nonlinearities with the possibility of on-chip implementation. In this context, organic semiconductors embedded in microcavities have proven to be versatile candidates to study nonlinear many-body physics and bosonic condensation, and in contrast to most inorganic systems, they allow the use at ambient conditions since they host ultra-stable Frenkel excitons. A well-controlled, high-quality optical lattice is implemented that accommodates light-matter quasiparticles. The realized polariton graphene presents with excellent cavity quality factors, showing distinct signatures of Dirac cone and flatband dispersions as well as polariton lasing at room temperature. This is realized by filling coupled dielectric microcavities with the fluorescent protein mCherry. The emergence of a coherent polariton condensate at ambient conditions are demonstrated, taking advantage of coupling conditions as precise and controllable as in state-of-the-art inorganic semiconductor-based systems, without the limitations of e.g. lattice matching in epitaxial growth. This progress allows straightforward extension to more complex systems, such as the study of topological phenomena in 2D lattices including topological lasers and non-Hermitian optics.

Gentisic acid prevents colorectal cancer metastasis via blocking GPR81-mediated DEPDC5 degradation

BackgroundMetastasis driven by epithelial-mesenchymal transition (EMT) remains a significant contributor to the poor prognosis of colorectal cancer (CRC), and requires more effective interventions. GPR81 signaling has been linked to tumor metastasis, while lacks an efficient specific inhibitor. PurposeOur study aimed to investigate the effect and mechanism of Gentisic acid on colorectal cancer (CRC) metastasis. Study designA lung metastasis mouse model induced by tail vein injection and a subcutaneous graft tumor model were used. Gentisic acid (GA) was administered by an intraperitoneal injection. HCT116 was treated with lactate to establish an in vitro model. MethodsMC38 cells with mCherry fluorescent protein were injected into tail vein to investigate lung metastasis ability in vivo. GA was administered by intraperitoneal injection for 3 weeks. The therapeutic effect was evaluated by survival rates, histochemical analysis, RT-qPCR and live imaging. The mechanism was explored using small interfering RNA (siRNA), Western blotting, RT-qPCR and immunofluorescence. ResultsGA had a therapeutic effect on CRC metastasis and improved survival rates and pathological changes in dose-dependent manner. GA emerged as an GPR81 inhibitor, effectively suppressed EMT and mTOR signaling in CRC induced by lactate both in vivo and in vitro. Mechanistically, GA halted lactate-induce degradation of DEPDC5 through impeding the activation of Chaperone-mediated autophagy (CMA). ConclusionCMA-mediated DEPDC5 degradation is crucial for lactate/GPR81-induced CRC metastasis, and GA may be a promising candidate for metastasis by inhibiting GPR81 signaling.

Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Creation of Knock-In Alleles of Insulin Receptor Tagged by Fluorescent Proteins mCherry or EYFP in Fruit Fly Drosophila melanogaster.

The insulin/insulin-like growth factor-like signaling (IIS) pathway is highly conserved across metazoans and regulates numerous physiological functions, including development, metabolism, fecundity, and lifespan. The insulin receptor (InR), a crucial membrane receptor in the IIS pathway, is known to be ubiquitously expressed in various tissues, albeit at generally low levels, and its subcellular localization remains incompletely characterized. In this study, we employed CRISPR-mediated mutagenesis in the fruit fly Drosophila to create knock-in alleles of InR tagged with fluorescent proteins (InR::mCherry or InR::EYFP). By inserting the coding sequence of the fluorescent proteins mCherry or EYFP near the end of the coding sequence of the endogenous InR gene, we could trace the natural InR protein through their fluorescence. As an example, we investigated epithelial cells of the male accessory gland (AG), an internal reproductive organ, and identified two distinct patterns of InR::mCherry localization. In young AG, InR::mCherry accumulated on the basal plasma membrane between cells, whereas in mature AG, it exhibited intracellular localization as multiple puncta, indicating endocytic recycling of InR during cell growth. In the AG senescence accelerated by the mutation of Diuretic hormone 31 (Dh31), the presence of InR::mCherry puncta was more pronounced compared to the wild type. These findings raise expectations for the utility of the newly created InR::mCherry/EYFP alleles for studying the precise expression levels and subcellular localization of InR. Furthermore, this fluorescently tagged allele approach can be extended to investigate other membrane receptors with low abundance, facilitating the direct examination of their true expression and localization.

Single- and two-photon-induced Förster resonance energy transfer in InP-mCherry bioconjugates.

Indium phosphide (InP) quantum dots (QDs) have recently garnered considerable interest in the design of bioprobes due to their non-toxic nature and excellent optical properties. Several attempts for the conjunction of InP QDs with various entities such as organic dyes and dye-labeled proteins have been reported, while that with fluorescent proteins remains largely uncharted. This study reports the development of a Förster resonance energy transfer pair comprising glutathione-capped InP/GaP/ZnS QDs [InP(G)] and the fluorescent protein mCherry. Glutathione on InP(G) undergoes effective bioconjugation with mCherry consisting of a hexahistidine tag, and the nonradiative energy transfer is investigated using steady-state and time-resolved measurements. Selective one-photon excitation of InP(G) in the presence of mCherry shows a decay of the emission of the QDs and a concomitant growth of acceptor emission. Time-resolved investigations prove the nonradiative transfer of energy between InP(G) and mCherry. Furthermore, the scope of two-photon-induced energy transfer between InP(G) and mCherry is investigated by exciting the donor in the optical transparency range. The two-photon absorption is confirmed by the quadratic relationship between the emission intensity and the excitation power. In general, near-infrared excitation provides a path for effective light penetration into the tissues and reduces the photodamage of the sample. The two-photon-induced energy transfer in such assemblies could set the stage for a wide range of biological and optoelectronic applications in the foreseeable future.

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting.

Oncogenic fusion genes are attractive therapeutic targets because of their tumor-specific expression and central "driver" roles in various human cancers. However, oncogenic fusions involving transcription factors such as PAX3-FOXO1 in alveolar fusion gene-positive rhabdomyosarcoma (FP-RMS) have been difficult to inhibit due to the apparent lack of tractable drug-like binding sites comparable to that recognized by Gleevec (imatinib mesylate) on the BCR-ABL1 tyrosine kinase fusion protein. Toward the identification of novel small molecules that selectively target PAX3-FOXO1, we used CRISPR-Cas9-mediated knock-in to append the pro-luminescent HiBiT tag onto the carboxy terminus of the endogenous PAX3-FOXO1 fusion protein in two human FP-RMS cell lines (RH4 and SCMC). HiBiT is an 11-amino acid peptide derived from the NanoLuc luciferase that produces a luminescence signal which is ~100-fold brighter than firefly or Renilla luciferases through high-affinity binding to a complementary NanoLuc peptide fragment called LgBiT. To facilitate single-cell clonal isolation of knock-ins, the homology-directed repair template encoding HiBiT was followed by a P2A self-cleaving peptide for coexpression of an mCherry fluorescent protein as a fluorescence-activated cell sorter (FACS)-selectable marker. HiBiT tagging thus allows highly sensitive luminescence detection of endogenous PAX3-FOXO1 levels permitting quantitative high-throughput screening of large compound libraries for the discovery of PAX3-FOXO1 inhibitors and degraders.

Developing a trans-multisynaptic tracer to map the neural circuit of recovered sciatic nerve after treatment with nerve growth factor

Nerve growth factor (NGF) has been shown to support the survival and differentiation of neurons. In this study, we first developed a retrograde trans-multisynaptic tracer PRV580 expressing the mCherry fluorescent protein based on pseudorabies virus Bartha strain to map the neural circuit of sciatic nerve. Secondly, the newly developed PRV580 was used to map the neural circuit of the recovering sciatic nerve upon treatment with NGF. Our results showed that red signals from PRV580 were observed in various brain regions. Among these regions, many areas of the pyramidal system and the extra-pyramidal system had been mapped, accounting for as much as 56.8 % of the total inputs. Furthermore, we found that NGF could significantly increase the ratio of total input (29.05 %) compared to PBS (3.65 %), indicating that NGF indeed can aid in the repair of injured sciatic nerve. These findings indicated that NGF has therapeutic ability for the treatment of peripheral nerve injuries and virus-based tracers can be used to monitor the recovery.