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In the framework of simplified quantum chemistry methods, we introduce the eXact integral simplified time-dependent density functional theory (XsTD-DFT). This method is based on the simplified time-dependent density functional theory (sTD-DFT), where all semi-empirical two-electron integrals are replaced by exact one- and two-center two-electron integrals, while other approximations from sTD-DFT are kept. The performance of this new parameter-free XsTD-DFT method was benchmarked on excited state and (non)linear response properties, including ultra-violet/visible absorption, first hyperpolarizability, and two-photon absorption (2PA). For a set of 77 molecules, the results from the XsTDA approach were compared to the TDA data. XsTDA/B3LYP excitation energies only deviate on average by 0.14 eV from TDA while drastically cutting computational costs by a factor of 20 or more depending on the energy threshold chosen. The absolute deviations of excitation energies with respect to the full scheme are decreasing with increasing system size, showing the suitability of XsTDA/XsTD-DFT to treat large systems. Comparing XsTDA and its predecessor sTDA, the new scheme generally improves excitation energies and oscillator strengths, in particular, for charge transfer states. TD-DFT first hyperpolarizability frequency dispersions for a set of push-pull π-conjugated molecules are faithfully reproduced by XsTD-DFT, while the previous sTD-DFT method provides redshifted resonance energy positions. Excellent performance with respect to the experiment is observed for the 2PA spectrum of the enhanced green fluorescent protein. The obtained robust accuracy similar to TD-DFT at a fraction of the computational cost opens the way for a plethora of applications for large systems and in high throughput screening studies. [ABSTRACT FROM AUTHOR]

Chemical phenomena involving near-degenerate electronic states, such as conical intersections or avoided crossing, can be properly described using quasi-degenerate perturbation theory. This study proposed a highly scalable quasi-degenerate second-order N-electron valence state perturbation theory (QD-NEVPT2) using the local pair-natural orbital (PNO) method. Our recent study showed an efficient implementation of the PNO-based state-specific NEVPT2 method using orthonormal localized virtual molecular orbitals (LVMOs) as an intermediate local basis. This study derived the state-coupling (or off-diagonal) terms to implement QD-NEVPT2 in an alternative manner to enhance efficiency based on the internally contracted basis and PNO overlap matrices between different references. To facilitate further acceleration, a local resolution-of-the-identity (RI) three-index integral generation algorithm was developed using LMOs and LVMOs. Although the NEVPT2 theory is considered to be less susceptible to the intruder-state problem (ISP), this study revealed that it can easily suffer from ISP when calculating high-lying excited states. We ameliorated this instability using the imaginary level shift technique. The PNO-QD-NEVPT2 calculations were performed on small organic molecules for the 30 lowest-lying states, as well as photoisomerization involving the conical intersection of 1,1-dimethyldibenzo[b,f] silepin with a cis-stilbene skeleton. These calculations revealed that the PNO-QD-NEVPT2 method yielded negligible errors compared to the canonical QD-NEVPT2 results. Furthermore, we tested its applicability to a large photoisomerization system using the green fluorescent protein model and the ten-state calculation of the large transition metal complex, showcasing that off-diagonal elements can be evaluated at a relatively low cost. [ABSTRACT FROM AUTHOR]

Photoexcitation of green fluorescent protein (GFP) triggers long-range proton transfer along a "wire" of neighboring protein residues, which, in turn, activates its characteristic green fluorescence. The GFP proton wire is one of the simplest, most well-characterized models of biological proton transfer but remains challenging to simulate due to the sensitivity of its energetics to the surrounding protein conformation and the possibility of non-classical behavior associated with the movement of lightweight protons. Using a direct dynamics variational multiconfigurational Gaussian wavepacket method to provide a fully quantum description of both electrons and nuclei, we explore the mechanism of excited state proton transfer in a high-dimensional model of the GFP chromophore cluster over the first two picoseconds following excitation. During our simulation, we observe the sequential starts of two of the three proton transfers along the wire, confirming the predictions of previous studies that the overall process starts from the end of the wire furthest from the fluorescent chromophore and proceeds in a concerted but asynchronous manner. Furthermore, by comparing the full quantum dynamics to a set of classical trajectories, we provide unambiguous evidence that tunneling plays a critical role in facilitating the leading proton transfer. [ABSTRACT FROM AUTHOR]

The methylotrophic yeast Komagataella phaffii is a popular host system for the pharmaceutical and biotechnological production of recombinant proteins. CRISPR-Cas9 and its derivative CRISPR interference (CRISPRi) offer a promising avenue to further enhance and exploit the full capabilities of this host. MAD7 and its catalytically inactive variant "dead" MAD7 (dMAD7) represent an interesting alternative to established CRISPR-Cas9 systems and are free to use for industrial and academic research. CRISPRi utilizing dMAD7 does not introduce double-strand breaks but only binds to the DNA to regulate gene expression. Here, we report the first use of dMAD7 in K. phaffii to regulate the expression of the enhanced green fluorescent protein (eGFP). A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. Integration loci/events of investigated strains were assessed through whole genome sequencing. Additionally, RNA-sequencing experiments corroborated the whole genome sequencing results and showed a significantly reduced expression of eGFP in strains containing a dMAD7 plasmid, among others. Our findings conclusively demonstrate the utility of dMAD7 in K. phaffii through successfully regulating eGFP expression. • We report the first use of dMAD7 in K. phaffii to regulate the expression of enhanced green fluorescent protein (eGFP). • A reduction of eGFP fluorescence level (up to 88 %) was achieved in random integration experiments using dMAD7 plasmids. • Whole genome sequencing and RNA-sequencing were used to assess the integration events and to characterize the strains. • Our findings conclusively demonstrate the utility of the dMAD7 system in K. phaffii. [ABSTRACT FROM AUTHOR]

Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron emitters [99mTc]TcO 4 − and [123I]I− and β−-particle emitter [188Re]ReO 4 −. Studies were carried out using genetically modified cells that permitted direct radionuclide comparisons. Triple-negative MDA-MB-231 breast cancer cells expressing the human sodium iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO 4 −, [123I]I−, and [188Re]ReO 4 − was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear absorbed doses using the Medical Internal Radiation Dose (MIRD) formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared with X-ray–treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and sodium iodide symporter–expressing organs was extrapolated to reference human adult models using OLINDA/EXM. [99mTc]TcO 4 − and [123I]I− reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO 4 − reduced survival fraction in hNIS-expressing and parental cells. [123I]I− required 2.4- and 1.5-fold lower decays/cell to achieve 37% survival compared with [99mTc]TcO 4 − and [188Re]ReO 4 −, respectively, after 72 hours of incubation. Additionally, [99mTc]TcO 4 −, [123I]I−, and [188Re]ReO 4 − had superior cell killing effectiveness in vitro compared with X-rays. In vivo, X-ray led to a greater median survival compared with [188Re]ReO 4 − and [123I]I− (54 days vs 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO 4 − to a 1 g tumor were 13.8- and 11.2-fold greater than for [123I]I− in female and male models, respectively. This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO 4 −, [123I]I−, and [188Re]ReO 4 − for the first time. We further demonstrate the tumor-controlling effects of [123I]I− and [188Re]ReO 4 − in comparison with external beam radiation therapy. [ABSTRACT FROM AUTHOR]

Self-labeling protein (SLP) tags, such as HaloTag, have gained considerable interest as advanced tools for live cell labeling. However, the chloroalkane-based substrates that can be directly used for protein labeling are limited. Here, we report two bioorthogonal small molecule linkers, chloroalkane-tetrazine (CA-Tz) and chloroalkane-azide (CA-N3), which can penetrate cell membranes and facilitate click chemistry-based labeling in live cells. We compare their labeling capability using two clickable silicon rhodamine dyes (SiR-PEG3-TCO and SiR-PEG4-DBCO). Confocal imaging results demonstrate that using CA-Tz and SiR-PEG3-TCO dye exhibits superior intracellular labeling with low nonspecific signals. We subsequently compared the photostability of SiR dyes with that of green fluorescent proteins (mEmerald). Total internal reflection fluorescence (TIRF) imaging indicates that SiR dyes exhibit superior photostability under identical excitation conditions, making them suitable for long-term cell imaging. Furthermore, SiR dyes labeling also shows high structure retention for the fourth-order super-resolution optical fluctuation imaging (SOFI) compared to fluorescent proteins. This study presents clickable HaloTag linkers as effective tools for live cell labeling and imaging, highlighting the high-quality labeling of chloroalkane linkers and clickable dyes for live cell imaging. [ABSTRACT FROM AUTHOR]

Disruption of inhibitory interneurons is common in the epileptic brain and is hypothesized to play a pivotal role in epileptogenesis. Abrupt disruption and loss of interneurons is well-characterized in status epilepticus models of epilepsy, however, status epilepticus is a relatively rare cause of epilepsy in humans. How interneuron disruption evolves in other forms of epilepsy is less clear. Here, we explored how somatostatin (SST) interneuron disruption evolves in quadruple transgenic Gli1-CreERT2, Ptenfl/fl, SST-FlpO, and frt-eGFP mice. In these animals, epilepsy develops following deletion of the mammalian target of rapamycin (mTOR) negative regulator phosphatase and tensin homolog (Pten) from a subset of dentate granule cells, while downstream Pten-expressing SST neurons are fate-mapped with green fluorescent protein (GFP). The model captures the genetic complexity of human mTORopathies, in which mutations can be restricted to excitatory neuron lineages, implying that interneuron involvement is later developing and secondary. In dentate granule cell (DGC)-Pten knockouts (KOs), the density of fate-mapped SST neurons was reduced in the hippocampus, but their molecular phenotype was unchanged, with similar percentages of GFP+ cells immunoreactive for SST and parvalbumin (PV). Surviving SST neurons in the dentate gyrus had larger somas, and the density of GFP+ processes in the dentate molecular layer was unchanged despite SST cell loss and expansion of the molecular layer, implying compensatory sprouting of surviving cells. The density of Znt3-immunolabeled puncta, a marker of granule cell presynaptic terminals, apposed to GFP+ processes in the hilus was increased, suggesting enhanced granule cell input to SST neurons. Finally, the percentage of GFP+ cells that were FosB positive was significantly increased, implying that surviving SST neurons are more active. Together, findings suggest that somatostatin-expressing interneurons exhibit a combination of pathological (cell loss) and adaptive (growth) responses to hyperexcitability and seizures driven by upstream Pten KO excitatory granule cells. Illustration and summary of key study findings. The left portion of the illustration depicts DGC and SST interneuron interaction in normal conditions. The right portion depicts changes in the setting of DGC-Pten KO which are supported by the following findings in this study: (1) eGFP-SST cell density was reduced in multiple hippocampal regions. Remaining hilar eGFP-SST cells exhibited larger soma sizes. (2) Preserved eGFP-SST processes in the hilus and molecular layer, coupled with cell loss and an enlarged molecular layer, suggests sprouting. (3) Increased apposition of Znt-3 puncta to eGFP-SST processes suggests increased DGC innervation of SST interneurons. Created in BioRender.com. BioRender.com/h33m167. [ABSTRACT FROM AUTHOR]

To detect cellular activities deep within the body using magnetic resonance platforms, magnetosomes are the ideal model of genetically-encoded nanoparticles. These membrane-bound iron biominerals produced by magnetotactic bacteria are highly regulated by approximately 30 genes; however, the number of magnetosome genes that are essential and/or constitute the root structure upon which biominerals form is largely undefined. To examine the possibility that key magnetosome genes may interact in a foreign environment, we expressed mamI and mamL as fluorescent fusion proteins in mammalian cells. Localization and potential protein-protein interaction(s) were investigated using confocal microscopy and fluorescence correlation spectroscopy (FCS). Enhanced green fluorescent protein (EGFP)-MamI and the red fluorescent Tomato-MamL displayed distinct intracellular localization, with net-like and punctate fluorescence, respectively. Remarkably, co-expression revealed co-localization of both fluorescent fusion proteins in the same punctate pattern. An interaction between MamI and MamL was confirmed by co-immunoprecipitation. In addition, changes in EGFP-MamI distribution were accompanied by acquisition of intracellular mobility which all Tomato-MamL structures displayed. Analysis of extracts from these cells by FCS was consistent with an interaction between fluorescent fusion proteins, including an increase in particle radius. Co-localization and interaction of MamI and MamL demonstrate that select magnetosome proteins may associate in mammalian cells. [ABSTRACT FROM AUTHOR]

The article discusses molecular biological studies on the interactions between bromoviruses and plants, focusing on factors influencing virus replication, movement, infection, and host defense responses. It highlights the establishment of gene manipulation systems for bromoviruses and the analysis of viral and host factors required for infection in plants. The research also delves into the role of specific plant genes in regulating viral infection and explores the potential for developing new disease control technologies. The study emphasizes the importance of understanding virus-plant interactions to combat viral diseases effectively. [Extracted from the article]

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research. [Display omitted] • LNPs an efficient in vitro transfection tool of CRISPR-Cas9 RNAs in GSCs, an alterantive to commercial transfecion reagents. • The CRISPR-Cas9 LNP platform enables efficient in vivo gene editing of a difficult to treat model of mesenchymal GSCs. • Proposing CRISPR-Cas9 LNPs as an in vivo screening tool for therapeutic target identification in GSCs. [ABSTRACT FROM AUTHOR]

This study aims to develop an experimental treatment model effective against oxidative stress in the acute period of severe burns and to analyze the mechanisms of healing large wound defects. Five rats, including 2 females and 3 males, were used as donors to obtain adipose-derived stem cells (ADSC) from the inguinal fat pad. The stem cells were labeled with green fluorescent protein. The study included four groups of 17 rats, each with grade 3 scalding burns on 30 % of their body surface, and a control group of 10 rats with an equal number of males and females. After early excision, 106 ADSC-derived stem cells were administered subdermally to the burned wound and autografted to the stem cell group (n = 17). The early excision group (n = 17) received early excision and autograft, with 2 ml of normal saline injected subdermally into the burn wound edge. The PLM group (n = 17) was treated with a polylactic membrane (PLM) dressing after the burn. No treatment was given to the burn group (n = 17). Ten rats from all groups were sacrificed on the 4th day post-burn for oxidative stress evaluation. The control group (n = 10) was sacrificed on day 4. Blood and tissue samples were collected post-sacrifice. Oxidative stress and inflammation in the blood, as well as cell damage in the skin, liver, kidneys, and lungs, were investigated histopathologically and biochemically on the 4th day post-burn. On the 70th day after burn, wound healing was examined macroscopically and histopathologically. On the 4th day, oxidative stress results showed that the levels of Total Oxidative Capacity (TOC) in the blood were lowest in the stem cell (7.4 [6–8.8]), control (6.7 [5.9–7.6]), and early excision (7.5 [6.6–8.5]) groups, with no significant difference between them. The burn group (14.7 [12.5–16.9]) had the highest TOC levels. The PLM group (9.7 [8.6–10.7]) had lower TOC levels than the burn group but higher levels than the other groups. Histopathological examination on the 4th day revealed low liver caspase-3 immunoreactivity in the stem cell and early excision groups among the burn groups. Caspase-3 immunoreactivity levels were as follows: stem cell group (20 [10–30]), early excision group (25 [15–50]), PLM group (70 [50–100]), control group (0), and burn group (80 [60–120]). Other oxidative stress and end-organ damage outcomes were consistent with these results. All rats in the stem cell group had burn wounds that healed completely by the 70th day. Examination of the skin and its appendages from the stem cell group with an immunofluorescence microscope demonstrated green coloration, indicating incorporation of stem cells. Stem cells may have the potential to form new skin and its appendages, providing better healing for large skin defects. Early excision treatment, by removing local necrotic tissues after extensive and deep burns, can prevent end-organ damage due to systemic oxidative stress and inflammation. We also believe that when these two treatments are used together, they can achieve the best results. • Systemic inflammation and oxidative stress occur during the acute phase of severe burns, causing damage to end organs. • Wound healing is an important problem in patients with severe burns if there is not enough space to harvest the skin autograft. • Early excision treatment, which involves clearing local necrotic tissue after extensive and deep severe burns, can prevent end-organ damage. • The stem cells may have the potential to form new skin and its appendages providing better healing for large skin defects. • Combining these two treatments may yield the best results. [ABSTRACT FROM AUTHOR]

Introduction: Senecavirus A (SVA), belonging to the genus Senecavirus in the family Picornaviridae, is an emerging pathogen causing vesicular disease in pigs. The main clinical manifestations of SVA infection include high mortality in neonatal piglets, skin ulceration, and vesicular lesions. So far, there is no commercially available vaccines or drugs against SVA. Construction of SVA infectious clones carrying reporter genes will help understand the characteristics of SVA and promote vaccine development. Methods: In this study, we established a reverse genetics system for a local SVA isolate and used it to rescue a recombinant SVA, rSVA-eGFP, expressing the enhanced green fluorescent protein (eGFP) by inserting eGFP, GSG linker and the P2A sequence between 2A and 2B genes. Results: We found that rSVA-eGFP exhibited a high replication efficiency comparable to the parental virus, was able to express the eGFP reporter efficiently and stable in maintaining the reporter gene up to six rounds of serial passages in BHK-21 cells. In mice, rSVA-eGFP also showed similar replication kinetics and pathogenicity to the parental virus, both causing mild lung lesions. In addition, a high-throughput viral neutralization assay was developed using eGFP as a surrogate readout in a fluorescence-based direct titration (FBT) assay based on rSVA-eGFP, facilitating rapid and accurate determination of the neutralizing antibody (nAb) titers. Discussion: The successful establishment of an SVA reverse genetics system and the rescue of rSVA-eGFP would create a powerful tool for future studies of SVA replication mechanisms and pathogenicity as well as for antiviral development. [ABSTRACT FROM AUTHOR]

Background: Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions. Methods: The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA). Results: In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation. Conclusions: This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system. [ABSTRACT FROM AUTHOR]

The article focuses on the promising potential of RNA editing as a therapeutic approach for genetic disorders, highlighted through the story of a mother advocating for treatment options for her daughter with Rett syndrome. Topics include the historical context of Rett syndrome and its genetic basis, the current advancements and research funding in RNA editing therapies, and the advantages of RNA editing over traditional DNA-based therapies, including safety and temporary effects.

SUMMARY Boron (B) is an important limiting factor for plant growth and yield in saline soils, but the underlying molecular mechanisms remain poorly understood. In this study, we found that appropriate B supply obviously complemented rapeseed (Brassica napus L.) growth under salinity accompanied by higher biomass production and less reactive oxygen species accumulation. Determination of Na+ content in shoots and roots indicated that B significantly repressed root‐to‐shoot Na+ translocation, and non‐invasive micro‐tests of root xylem sap demonstrated that B increased xylem Na+ unloading in the roots of rapeseed plants under salinity. Comparative transcriptomic profiling revealed that B strongly upregulated BnaHKT1s expression, especially BnaA2.HKT1, in rapeseed roots exposed to salinity. In situ hybridizations analysis showed that BnaA2.HKT1 was significantly induced in root stelar tissues by high B (HB) under salinity. Green fluorescent protein and yeast heterologous expression showed that BnaA2.HKT1 functioned as a plasma membrane‐localized Na+ transporter. Knockout of BnaA2.HKT1 by CRISPR/Cas9 resulted in hypersensitive of rapeseed plants to salinity even under HB condition, with higher shoot Na+ accumulation and lower biomass production. By contrast, overexpression of BnaA2.HKT1 ameliorated salinity‐induced growth inhibition under B deficiency and salinity. Overall, our results proposed that B functioned as a positive regulator for the rapeseed growth and seed production under salt stress through facilitating BnaA2.HKT1‐mediated root xylem Na+ unloading. This study may also provide an alternative strategy for the improvement of crop growth and development in saline soils. [ABSTRACT FROM AUTHOR]

Spreading depolarization (SD) causes a massive neuronal/glial depolarization, disturbs ionic homeostasis and deranges neuronal network function. The metabolic burden imposed by SD may also generate marked amounts of reactive oxygen species (ROS). Yet, proper optical tools are required to study this aspect with spatiotemporal detail. Therefore, we earlier generated transgenic redox indicator mice. They express in excitatory projection neurons the cytosolic redox-sensor roGFP, a reduction/oxidation sensitive green fluorescent protein which is ratiometric by excitation and responds reversibly to redox alterations. Using adult male roGFPc mice, we analyzed SD-related ROS production in CA1 stratum pyramidale of submerged slices. SD was induced by K+ microinjection, O2 withdrawal or mitochondrial uncoupling (FCCP). The extracellular DC potential deflection was accompanied by a spreading wavefront of roGFP oxidation, confirming marked neuronal ROS generation. Hypoxia-induced SD was preceded by a moderate oxidation, which became intensified as the DC potential deflection occurred. Upon K+-induced SD, roGFP oxidation slowly recovered within 10-15 min in some slices. Upon FCCPor hypoxia-induced SD, recovery was limited. Withdrawing extracellular Ca2+ markedly dampened the SD-related roGFP oxidation and improved its reversibility, confirming a key-role of neuronal Ca2+ load in SD-related ROS generation. Neither mitochondrial uncoupling, nor inhibition of NADPH oxidase or xanthine oxidase abolished the SD-related roGFP oxidation. Therefore, ROS generation during SD involves mitochondria as well as non-mitochondrial sources. This first-time analysis of SD-related ROS dynamics became possible based on quantitative redox imaging in roGFP mice, an advanced approach, which will contribute to further decipher the molecular understanding of SD in brain pathophysiology. [ABSTRACT FROM AUTHOR]

Introduction: Tobacco vein mottling virus (TVMV) is a member of the family Potyviridae. The 3' untranslated region (3'UTR) of viral genomic RNA has been reported to significantly impact viral infection. Nevertheless, the role of the TVMV 3'UTR during viral infection remains unknown. Methods: Here, a 3'UTR-GFP expression vector was transiently expressed in Nicotiana benthamiana, in which the 3'UTR of TVMV was introduced upstream of the green fluorescent protein (GFP) gene. Transcriptome sequencing was performed to analyze the genes associated with plant resistance. The effect of the TVMV 3'UTR on GFP expression was studied using an Agrobacteriummediated transient expression assay, revealing that the TVMV 3'UTR significantly inhibited GFP expression. Transcriptome analysis of differentially expressed genes in 3'UTR-GFP in N. benthamiana was performed to elucidate the why the TVMV 3'UTR inhibited GFP expression. Results: Eighty genes related to plant disease resistance were differentially expressed, including 29 upregulated and 51 downregulated genes. Significantly upregulated genes included those encoding the calcium-binding protein CML24, leucine-rich repeat receptor-like tyrosine-protein kinase, and respiratory burst oxidase homolog protein E. The significantly downregulated genes included calcium-binding protein 7, ethylene-responsive transcription factor 10, endoglucanase 5, and receptor-like protein kinase. Discussion: These findings indicate that the 3'UTR of TVMV may inhibit the expression of GFP gene by inducing the expression of plant resistance genes. This study provides a theoretical basis for further research on the function and mechanism of the TVMV 3'UTR. [ABSTRACT FROM AUTHOR]

Background: Kidney fibrosis is a hallmark of chronic kidney disease (CKD) and compromises the viability of transplanted human bone marrow-derived mesenchymal stromal cells (BM-MSCs). Hence, BM-MSCs were genetically-engineered to express the anti-fibrotic and renoprotective hormone, human relaxin-2 (RLX) and green fluorescent protein (BM-MSCs-eRLX + GFP), which enabled BM-MSCs-eRLX + GFP delivery via a single intravenous injection. Methods: BM-MSCs were lentiviral-transduced with human relaxin-2 cDNA and GFP, under a eukaryotic translation elongation factor-1α promoter (BM-MSCs-eRLX + GFP) or GFP alone (BM-MSCs-eGFP). The ability of BM-MSCs-eRLX + GFP to differentiate, proliferate, migrate, produce RLX and cytokines was evaluated in vitro, whilst BM-MSC-eRLX + GFP vs BM-MSCs-eGFP homing to the injured kidney and renoprotective effects were evaluated in preclinical models of ischemia reperfusion injury (IRI) and high salt (HS)-induced hypertensive CKD in vivo. The long-term safety of BM-MSCs-RLX + GFP was also determined 9-months after treatment cessation in vivo. Results: When cultured for 3- or 7-days in vitro, 1 × 106 BM-MSCs-eRLX + GFP produced therapeutic RLX levels, and secreted an enhanced but finely-tuned cytokine profile without compromising their proliferation or differentiation capacity compared to naïve BM-MSCs. BM-MSCs-eRLX + GFP were identified in the kidney 2-weeks post-administration and retained the therapeutic effects of RLX in vivo. 1–2 × 106 BM-MSCs-eRLX + GFP attenuated the IRI- or therapeutically abrogated the HS-induced tubular epithelial damage and interstitial fibrosis, and significantly reduced the HS-induced hypertension, glomerulosclerosis and proteinuria. This was to an equivalent extent as RLX and BM-MSCs administered separately but to a broader extent than BM-MSCs-eGFP or the angiotensin-converting enzyme inhibitor, perindopril. Additionally, these renoprotective effects of BM-MSCs-eRLX + GFP were maintained in the presence of perindopril co-treatment, highlighting their suitability as adjunct therapies to ACE inhibition. Importantly, no major long-term adverse effects of BM-MSCs-eRLX + GFP were observed. Conclusions: BM-MSCs-eRLX + GFP produced greater renoprotective and therapeutic efficacy over that of BM-MSCs-eGFP or ACE inhibition, and may represent a novel and safe treatment option for acute kidney injury and hypertensive CKD. [ABSTRACT FROM AUTHOR]

Genetically encoded fluorescent biosensors became indispensable tools for biological research, enabling real-time observation of physiological processes in live cells. Recent protein engineering efforts have resulted in the generation of a large variety of fluorescent biosensors for a wide range of biologically relevant processes, from small ions to enzymatic activity and signaling pathways. However, biosensors for imaging sulfate ions, the fourth most abundant physiological anion, in mammalian cells are still lacking. Here, we report the development and characterization of a green fluorescent biosensor for sulfate named Thyone. Thyone, derived through structure-guided design from bright green fluorescent protein mNeonGreen, exhibited a large negative fluorescence response upon subsecond association with sulfate anion with an affinity of 11 mM in mammalian cells. By integrating mutagenesis analyses with molecular dynamics simulations, we elucidated the molecular mechanism of sulfate binding and revealed key amino acid residues responsible for sulfate sensitivity. High anion selectivity and sensitivity of Thyone allowed for imaging of sulfate anion transients mediated by sulfate transporter heterologously expressed in cultured mammalian cells. We believe that Thyone will find a broad application for assaying the sulfate transport in mammalian cells via anion transporters and exchangers. A genetically encoded green fluorescent sensor, Thyone, enables real-time imaging of sulfate anion dynamics in mammalian cells to probe the sulfate transport activity of the SLC26A2 protein. [ABSTRACT FROM AUTHOR]

Background: Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom. Results: Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system. Conclusions: Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications. [ABSTRACT FROM AUTHOR]

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial–temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function. [ABSTRACT FROM AUTHOR]

Polysaccharide‐based hydrogels are promising for biomedical applications such as drug delivery owing to their biocompatibility, biodegradability, and bioactivities. In particular, there is a need for hydrogels with injectability for minimally invasive therapies and controlled sustained release for sustained drug effect. Hydrogels made from carboxymethyl chitosan (CMC) and tannic acid (TA) (CMC‐TA) contain dynamic covalent bonds owing to autoxidation between CMC and TA, and interact with proteins via TA, elucidation of the detailed mechanism of dynamic covalent bonding in CMC‐TA hydrogels will facilitate stable loading and zero‐order release of proteins. Herein, the physical properties of oxidized CMC‐TA (Oxi‐CMC‐TA) prepared for the encapsulation and sustained release of proteins are explored. Following incubation at 37 °C for 24 h, CMC‐TA undergoes secondary crosslinking by autoxidation to produce Oxi‐CMC‐TA. Notably, CMC‐TA is viscoelastic and shear‐thinning, allowing for injection and 3D bioprinting. Indeed, CMC‐TA can be 3D‐printed with green fluorescent protein (GFP) encapsulated in its matrix. Oxi‐CMC‐TA also exhibits an affinity for protein owing to its gallol groups, enabling Oxi‐CMC‐TA to collect GFP in aqueous solutions against a concentration gradient. Moreover, Oxi‐CMC‐TA releases GFP over 15 days. Injectable, 3D‐printable, protein‐collecting, and zero‐order sustained‐releasing Oxi‐CMC‐TA has the potential to make a significant contribution to drug delivery. [ABSTRACT FROM AUTHOR]

Host‐specific pathogens have long been suggested to act as a major driver of species diversity in tropical forests. However, determining the degree of host specificity of potential pathogens coupled to key cellular characteristics of infection is difficult and time‐consuming. These challenges have delayed progress in relating the functional specificity of pathogenic fungi to ecological consequences. We tested the pathogenicity of 27 (of 215) fungi that were isolated from surface sterilized roots of seedlings from four common tree species in a diverse subtropical forest. Inoculation experiments showed that six fungi exhibited strong pathogenicity on the host seedlings. Five of these only infected their specific hosts (i.e. host‐specific pathogen species). Green fluorescent protein labelling revealed that in three host‐specific pathogens fungal hyphae were able to grow into the vascular tissues only in their specific host plant, in contrast to other fungal‐host combinations that exhibit non‐pathogenic interactions. This fluorescent labelling technique allows direct tracking of the progress of fungal infection in different host tissues. Synthesis. By coupling the green fluorescent protein technique with standard host inoculation experiments, we determined the developmental differences between infections by pathogenic and non‐pathogenic fungi in a relatively simple and straightforward way. Our work provides a useful tool for rapidly screening and categorizing host–fungal interactions, reflecting the functional basis of host specificity of pathogenic fungi. More broadly, this technique can contribute to understanding the roles of pathogens in species coexistence and biodiversity maintenance in forest communities. [ABSTRACT FROM AUTHOR]

Background: Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications. Methods: In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors. Results: Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies. Conclusions: This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles. [ABSTRACT FROM AUTHOR]

Low Earth Orbit (LEO) has emerged as a unique environment for evaluating altered stem cell properties in microgravity. LEO has become increasingly accessible for research and development due to progress in private spaceflight. Axiom Mission 2 (Ax-2) was launched as the second all-private astronaut mission to the International Space Station (ISS). Frozen human induced pluripotent stem cells (hiPSCs) expressing green fluorescent protein (GFP) under the SOX2 promoter, as well as fibroblasts differentiated from SOX2-GFP hiPSCs, were sent to the ISS. Astronauts then thawed and seeded both cell types into commercially available 96-well plates, which provided surface tension that reduced fluid movement out of individual wells and showed that hiPSCs or hiPSC-derived fibroblasts could survive either in suspension or attached to a Matrigel substrate. Furthermore, both cell types could be transfected with red fluorescent protein (RFP)-expressing plasmid. We demonstrate that hiPSCs and hiPSC-fibroblasts can be thawed in microgravity in off-the-shelf, commercially-available cell culture hardware, can associate into 3D spheroids or grow adherently in Matrigel, and can be transfected with DNA. This lays the groundwork for future biomanufacturing experiments in space. [ABSTRACT FROM AUTHOR]

Backgound Information Results Conclusion and Significance Toxoplasma gondii has a relict plastid, the apicoplast, to which nuclear‐encoded proteins are targeted after synthesis in the cytosol. Proteins exclusively found in the apicoplast use a Golgi‐independent route for trafficking, while dually targeted proteins found in both the apicoplast and the mitochondrion use a Golgi‐dependent route. For apicoplast targeting, N‐terminal signal sequences have been shown to direct the localization of different reporters. In this study, we use chimeric proteins to dissect out the roles of N‐terminal sequences and coding sequences in apicoplast localization and the choice of the trafficking route.We show that when the N‐termini of a dually targeted protein, TgTPx1/2, or of an apicoplast protein, TgACP, are fused with the reporter protein, enhanced green fluorescent protein (eGFP) or endogenous proteins, TgSOD2, TgSOD3, TgACP, or TgTPx1/2, the chimeric proteins exhibit flexibility in apicoplast targeting depending on the coding sequences. Further, the chimeras that are localized to the apicoplast use different trafficking pathways depending on the combination of the N‐terminal signals and the coding sequences.This report shows, for the first time, that in addition to the N‐terminal signal sequences, targeting and trafficking signals also reside within the coding sequences of apicoplast proteins. [ABSTRACT FROM AUTHOR]

The evaluation of the Z/E photoisomerization efficiency is an essential task to design photoactive molecular devices. Nevertheless, this photoreactivity can be correctly described only by applying extensive and expensive computational methods. In this study, a predictive tool to screen the photoinduced Z/E isomerization efficiency of molecular switches is presented, based on three key properties: the structure of the ground state minimum, the nature of the electronic transition populating the optically bright state, and the presence of crossings between the optically bright state and the one lower in energy. Our methodology allows evaluating these properties by few and affordable calculations, potentially enabling the screening of large sets of photoswitches. After presenting the formal aspects, the tool is applied to model systems of paradigmatic classes of photoswitches (retinal, green fluorescent protein, hemithioindigo, chiroptical, and stilbene compounds) including derivatives. A comparison with the available experimental data is performed to validate our approach. Cis-trans photoisomerization is a key process for many processes in biology and materials science, but only careful and time-consuming quantum chemistry methods can describe such reaction in detail. Here, a predictive tool is presented requiring few and affordable calculations, evaluating the efficiency of paradigmatic and modified photoswitches. [ABSTRACT FROM AUTHOR]

Genetic modification in vivo could provide direct functions of genes that could potentially contribute to diverse areas of research including genetics, developmental biology, and physiology. It has been reported that genes of interest could be introduced via recombinant adenovirus type 5 (Ad5) in poultry. Successful gene delivery to mammal fetuses in utero promises substantial progress in clinical and developmental biology, but it is limited because of difficulties in injecting specific sites and invasiveness. On the other hand, developing avian embryos are easily accessible by making a window on the eggshell. Therefore, the objective of this study is to determine permissive embryonic stages for gene transfer into specific avian tissue/organs by injection of Ad5 containing the green fluorescent protein (GFP) gene into blood vessels. At 2 d of post-injection, a strong GFP signal was predominantly identified in the heart of chicken embryos injected at Hamilton-Hamburger (HH) 14, 15, 16 and17 stages with the percentages (44%, 53%, 25%, and 14%, respectively) of GFP positive embryos. In quail embryos, the injection at the HH 15 resulted in heart-specific expression of GFP. Western blot analysis revealed that GFP was exclusively expressed in the avian hearts. These results suggest that the GFP gene is specifically delivered to the avian embryonic hearts when Ad5 is injected through the blood vessel at HH 14-17. This adenoviral transduction of genes of interest in avian embryonic hearts can provide new models for understanding functions of genetic factors on embryonic heart development and unravel genetic etiology of congenital heart diseases. [ABSTRACT FROM AUTHOR]

Aureobasidium pullulans is a ubiquitous polymorphic black yeast with industrial and agricultural applications. It has recently gained attention amongst cell biologists for its unconventional mode of proliferation in which multinucleate yeast cells make multiple buds within a single cell cycle. Here, we combine a chemical transformation method with genome-targeted homologous recombination to yield ∼60 transformants/μg of DNA in just 3 days. This protocol is simple, inexpensive, and requires no specialized equipment. We also describe vectors with codon-optimized green and red fluorescent proteins for A. pullulans and use these tools to explore novel cell biology. Quantitative imaging of a strain expressing cytosolic and nuclear markers showed that although the nuclear number varies considerably among cells of similar volume, total nuclear volume scales with cell volume over an impressive 70-fold size range. The protocols and tools described here expand the toolkit for A. pullulans biologists and will help researchers address the many other puzzles posed by this polyextremotolerant and morphologically plastic organism. [ABSTRACT FROM AUTHOR]

Pear chlorotic leaf spot‐associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single‐stranded, negative‐sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens‐mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self‐interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell‐to‐cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N‐terminal 1–89 amino acids of the P5 were responsible for the self‐interaction ability and RSS activity, for which the signal peptide at positions 1–19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement. [ABSTRACT FROM AUTHOR]

Background/Objectives: The development of polymer–lipid hybrid nanoparticles (PLNs) is a promising area of research, as it can help increase the stability of cationic lipid carriers. Hybrid PLNs are core–shell nanoparticle structures that combine the advantages of both polymer nanoparticles and liposomes, especially in terms of their physical stability and biocompatibility. Natural polymers such as polyhydroxyalkanoate (PHA) can be used as a matrix for the PLNs' preparation. Methods: In this study, we first obtained stable cationic hybrid PLNs using a cationic liposome (CL) composed of a polycationic lipid 2X3 (1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and the hydrophobic polymer mcl-PHA, which was produced by the soil bacterium Pseudomonas helmantisensis P1. Results: The new polymer-lipid carriers effectively encapsulated and delivered model mRNA-eGFP (enhanced green fluorescent protein mRNA) to BHK-21 cells. We then evaluated the role of mcl-PHA in increasing the stability of cationic PLNs in ionic solutions using dynamic light scattering data, electrophoretic mobility, and transmission electron microscopy techniques. Conclusions: The results showed that increasing the concentration of PBS (phosphate buffered saline) led to a decrease in the stability of the CLs. At high concentrations of PBS, the CLs aggregate. In contrast, the presence of isotonic PBS did not result in the aggregation of PLNs, and the particles remained stable for 120 h when stored at +4 °C. The obtained results show that PLNs hold promise for further in vivo studies on nucleic acid delivery. [ABSTRACT FROM AUTHOR]

Complementation remains a foundation for demonstrating molecular Koch's postulates. While this is frequently achieved using plasmids, limitations such as increased gene copy number and the need for antibiotic supplementation to avoid plasmid loss can restrict their use. Chromosomal integration systems using the Tn7 transposon provide an alternative to plasmids for complementation and facilitate the stable insertion of genes at the chromosomal attTn7 site without the need for selection pressure. Here, we enhanced the utility of mini-Tn7 insertion vectors by the addition of inducible (Pcym) and constitutive (PcL and PrpsM) promoters, allowing different ial transcriptional control of genes integrated into the chromosome. We validated the utility of these promoters by cloning the gfp gene, encoding green fluorescent protein, downstream of each promoter and integrating a mini-Tn7 construct harboring these elements into the attTn7 site on the chromosome of the Escherichia coli K-12 strain MG1655. The PcL and PrpsM promoters provided equivalent levels of GFP expression and offered flexibility based on the target host strain. Activation of the tightly regulated Pcym promoter with its inducer cumate resulted in tunable expression of GFP in a dose-dependent manner. We further demonstrated the tight control of the Pcym promoter using the toxic impCAB genes, and the expression of which is detrimental to E. coli viability. Together, these modified mini-Tn7 vectors allowing differential control of genes integrated into the chromosome at a conserved site offer an efficient system for complementation where plasmid use is restricted. IMPORTANCE Chromosomal integration using mini-Tn7 vectors provides an efficient means to insert genes into the chromosome of many gram-negative bacteria. Insertion occurs at a conserved site and allows for the stable integration of genes in single copy. While this system has multiple benefits for enabling complementation, a cornerstone for fulfilling molecular Koch's postulates, greater flexibility for controlled gene expression would enhance its utility. Here, we have added to the function of mini-Tn7 vectors by the addition of inducible and constitutive promoters and demonstrated their capacity to drive the controlled expression of target genes integrated into the chromosome. In addition to complementation, these modified vectors offer broad application for other approaches including chromosomal tagging, in vivo expression, metabolic engineering, and synthetic biology. [ABSTRACT FROM AUTHOR]

Alignment of picornavirus proteinase/polymerase sequences reveals this family evolved into five 'supergroups'. Interestingly, the nature of the 2A region of the picornavirus polyprotein is highly correlated with this phylogeny. Viruses within supergroup 4, the Paavivirinae, have complex 2A regions with many viruses encoding multiple 2ANPGP sequences. In vitro transcription/translation analyses of a synthetic polyprotein comprising green fluorescent protein (GFP) linked to β-glucuronidase (GUS) via individual 2ANPGPs showed two main phenotypes: highly active 2ANPGP sequences—similar to foot-and-mouth disease virus 2ANPGP—and, surprisingly, a novel phenotype of some 2ANPGP sequences which apparently terminate translation at the C-terminus of 2ANPGP without detectable re-initiation of downstream sequences (GUS). Probing databases with the short sequences between 2ANPGPs did not reveal any potential 'accessory' functions. The novel, highly active, 2A-like sequences we identified substantially expand the toolbox for biomedical/biotechnological co-expression applications. [ABSTRACT FROM AUTHOR]

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescenttagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release. [ABSTRACT FROM AUTHOR]

Substitution of lost neurons by neurotransplantation would be a possible management of advanced degenerative cerebellar ataxias in which insufficient cerebellar reserve remains. In this study, we examined the volume and structure of solid embryonic cerebellar grafts in adult Lurcher mice, a model of olivocerebellar degeneration, and their healthy littermates. Grafts taken from enhanced green fluorescent protein (EGFP)-positive embryos were injected into the cerebellum of host mice. Two or six months later, the brains were examined histologically. The grafts were identified according to the EGFP fluorescence in frozen sections and their volumes were estimated using the Cavalieri principle. For gross histological evaluation, graft-containing slices were processed using Nissl and hematoxylin–eosin staining. Adjustment of the volume estimation approach suggested that it is reasonable to use all sections without sampling, but that calculation of values for up to 20% of lost section using linear interpolation does not constitute substantial error. Mean graft volume was smaller in Lurchers than in healthy mice when examined 6 months after the transplantation. We observed almost no signs of graft destruction. In some cases, compact grafts disorganized the structure of the host's cerebellar cortex. In Lurchers, the grafts had a limited contact with the host's cerebellum. Also, graft size was of greater variability in Lurchers than in healthy mice. The results are in compliance with our previous findings that Lurcher phenotype-associated factors have a negative effect on graft development. These factors can hypothetically include cerebellar morphology, local tissue milieu, or systemic factors such as immune system abnormalities. [ABSTRACT FROM AUTHOR]

The recently discovered methodologies to cultivate and genetically manipulate Treponema pallidum subsp. pallidum (T. pallidum) have significantly helped syphilis research, allowing the in vitro 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. [ABSTRACT FROM AUTHOR]

The leave-one-out (LOO) green fluorescent protein (GFP) approach to biosensor design combines computational protein design with split protein reconstitution. LOO-GFPs reversibly fold and gain fluorescence upon encountering the target peptide, which can be redefined by computational design of the LOO site. Such an approach can be used to create reusable biosensors for the early detection of emerging biological threats. Enlightening biophysical inferences for nine LOO-GFP biosensor libraries are presented, with target sequences from dengue, influenza, or HIV, replacing beta strands 7, 8, or 11. An initially low hit rate was traced to components of the energy function, manifesting in the over-rewarding of over-tight side chain packing. Also, screening by colony picking required a low library complexity, but designing a biosensor against a peptide of at least 12 residues requires a high-complexity library. This double-bind was solved using a "piecemeal" iterative design strategy. Also, designed LOO-GFPs fluoresced in the unbound state due to unwanted dimerization, but this was solved by fusing a fully functional prototype LOO-GFP to a fiber-forming protein, Drosophila ultrabithorax, creating a biosensor fiber. One influenza hemagglutinin biosensor is characterized here in detail, showing a shifted excitation/emission spectrum, a micromolar affinity for the target peptide, and an unexpected photo-switching ability. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a long-term autoimmune condition marked by persistent inflammation of the joints and various systemic complications, including endothelial dysfunction, atherosclerosis, and pulmonary fibrosis. Oxidative stress is a key contributor to the pathogenesis of RA, potentially exacerbating vascular damage and promoting pro-angiogenic and profibrotic processes. Objective: This study aims to investigate the effects of sera from RA patients on human umbilical vein endothelial cells (HUVECs), focusing on the induction of oxidative stress, endothelial cell proliferation, migration, and collagen type I synthesis. Methods: Twenty-eight serum samples were collected from RA patients and healthy donors (HDs). HUVECs were exposed to these sera, and intracellular reactive oxygen species (ROS) levels were fluorescently detected using H2DCF-DA. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was evaluated through a scratch wound assay, and collagen type I synthesis was measured using a lentiviral vector expressing the green fluorescent protein (GFP) under the control of the human COL1A1 gene promoter. Results: Exposure to RA sera resulted in a significant increase in intracellular ROS levels in HUVECs compared to HD sera, indicating an elevated state of oxidative stress. RA sera also promoted endothelial cell proliferation and migration, suggesting a pro-angiogenic stimulus. Additionally, RA sera significantly increased collagen type I synthesis in HUVECs, implicating a potential role in profibrotic processes associated with RA. Conclusion: The results of this study emphasize the importance of circulating factors in RA sera in promoting oxidative stress, endothelial dysfunction, and pro-angiogenic and profibrotic phenotypes in endothelial cells. These processes may contribute to the vascular and fibrotic complications observed in RA, highlighting the necessity for additional research into focused therapeutic approaches to alleviate these effects. [ABSTRACT FROM AUTHOR]

In the present study, we demonstrated that the introduction of a 1,4-diethyl-1,2,3,4-tetrahydroquinoxalin moiety into the arylidene part of GFP chromophore-derived compounds results in the formation of environment-sensitive fluorogens. The rationally designed and synthesized compounds exhibit remarkable solvent- and pH-dependence in fluorescence intensity. The solvent-dependent variation in fluorescence quantum yield makes it possible to use some of the proposed compounds as polarity sensors suitable for selective endoplasmic reticulum fluorescent labeling in living cells. Moreover, the pH-dependent emission intensity variation of other fluorogens makes them selective fluorescent labels for the lysosomes in living cells. [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenases (ALDHs) constitute a diverse superfamily of NAD(P)+-dependent enzymes pivotal in oxidizing endogenous and exogenous aldehydes to carboxylic acids. Beyond metabolic roles, ALDHs participate in essential biological processes, including differentiation, embryogenesis and the DNA damage response, while also serving as markers for cancer stem cells (CSCs). Aldehyde dehydrogenase 1B1 (ALDH1B1) is a mitochondrial enzyme involved in the detoxification of lipid peroxidation by-products and metabolism of various aldehyde substrates. This study examines the potential role of ALDH1B1 in human lung adenocarcinoma and its association with the CSC phenotype. To this end, we utilized the lung adenocarcinoma cell line A549, engineered to stably express the human ALDH1B1 protein tagged with green fluorescent protein (GFP). Overexpression of ALDH1B1 led to notable changes in cell morphology, proliferation rate and clonogenic efficiency. Furthermore, ALDH1B1-overexpressing A549 cells exhibited enhanced resistance to the chemotherapeutic agents etoposide and cisplatin. Additionally, ALDH1B1 overexpression correlated with increased migratory potential and epithelial–mesenchymal transition (EMT), mediated by the upregulation of transcription factors such as SNAI2, ZEB2 and TWIST1, alongside the downregulation of E-cadherin. Moreover, Spearman's rank correlation coefficient analysis using data from 507 publicly available lung adenocarcinoma clinical samples revealed a significant correlation between ALDH1B1 and various molecules implicated in CSC-related signaling pathways, including Wnt, Notch, hypoxia, Hedgehog, retinoic acid, Hippo, NF-κΒ, TGF-β, PI3K/PTEN-AKT and glycolysis/gluconeogenesis. These findings provide insights into the role of ALDH1B1 in lung tumor progression and its relation to the lung CSC phenotype, thereby offering potential therapeutic targets in the clinical management of lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Purpose: Hair cell damage is a common side effect caused by the anticancer drug cisplatin (CDDP), which reduces patient quality of life. One CDDP resistance mechanism that occurs in recurrent cancers is heavy metal detoxification by metallothionein-2(mt2). Here, we show that in zebrafish larvae, dexamethasone (DEX) reduces CDDP-induced hair cell damage by enhancing mt2 expression. Methods: Transgenic zebrafish (cldn: gfp; atoh1: rfp) that express green and red fluorescent proteins in neuromasts and hair cells, respectively, were used. The zebrafish were pretreated with DEX at 52 h post-fertilization (hpf) for 8 h, followed by CDDP treatment for 12 h. The lateral line hair cells of CDDP-treated zebrafish at 72 hpf were observed by fluorescence microscopy. Results: Reporting odds ratio (ROR) analysis using an adverse event database indicated an association between a decrease in CDDP-induced ototoxicity and DEX as an antiemetic treatment for cancer chemotherapy. Pretreatment with DEX protected 72 hpf zebrafish hair cells from CDDP-induced damage. The expression of mt2 mRNA was significantly increased by the combination of 10 µM DEX with CDDP. Gene editing of mt2 reversed the protective effect of DEX against CDDP-induced damage in hair cells. Conclusion: DEX protects hair cells from CDDP-induced damage through increased mt2 expression, which is a resistance mechanism for platinum-based anticancer drugs. [ABSTRACT FROM AUTHOR]

Over the past decades, the heat-inducible (HI) promoter, derived from cI857 phage system has been widely utilized. However, the developments of alternative temperature-controlled promoters are limited. In this study, we explored a novel HI promoter through PCR amplification using the degenerate primer design. BLAST analysis identified this sequence as a partial pls B on Escherichia coli chromosome. Antisense RNAs targeting distinct regions of HI promoter were applied to examine the mechanism. The fluorescence of super-folder green fluorescent protein (sfGFP) driven by the HI promoter showed 5.5-fold increase from 30 to 42 °C, whereas the truncated HI yielded a trace amount of sfGFP. Among various E. coli strains, BL21 exhibited the highest fluorescence, reaching 3976 a.u. at 42 °C. Subsequently, the novel HI promoter was employed to drive T7RNA polymerase within a plasmid-driven T7 (PDT7) plasmid, serving its capability to express sfGFP and carbonic anhydrases (CA), respectively. The maximum intensity of sfGFP reached 38,702 a.u., and CA activity surged to 14286 WAU/mL in W3110 among other strains. Finally, the highest CA activity was 27,521 WAU/mL at pH 9. The promising results from the novel heat-inducible promoter-driven T7RNAP, incorporating the T7 terminator as HI-PDT7-TT, demonstrate potential for expressing more heterogeneous proteins across various chassis in the future. [Display omitted] • A new heat inducible (HI) promoter is explored for enhancing protein production. • Using HI plasmid-driven T7 system (HI-PDT7) improves 5-fold sfGFP in E. coli. • Terminator in HI-PDT7 is essential for plasmid stability and productivity. • HI-PDT7 expressing carbonic anhydrase (CA) has evaluated in different strains. • W3110 exhibits the highest CA at thermo- and alkaline conditions among all strains. [ABSTRACT FROM AUTHOR]

The cuticle covering the outer surface of an eggshell functions as both a physical and chemical barrier against invading microorganisms. Contamination of eggs by microbial pathogens progresses in four stages: bacterial attachment to the egg surface, penetration through the cuticle and eggshell, multiplication within the underlying membranes, and the final stage of contaminating the egg contents. Therefore, it is important to study bacterial count at the first point of contact, i.e., on the surface of the eggs. In this study, we have evaluated the impact of differences in cuticle quality (due to egg washing and hen age) on bacterial load. We compared bacterial adherence on the eggshell surface of white eggs which were either washed (graded) or unwashed (ungraded), collected from Lohmann laying hens of different ages: early (24–28 weeks), mid-lay (44–48 weeks), and late (66–70 weeks). We aimed to determine the impact of hen age and egg washing on differences in cuticle quality and bacterial adherence. Our results indicate that hen age (up to 70 weeks) and commercial egg washing do not significantly impact bacterial adherence on eggshell surfaces. We have developed a novel method using green fluorescent protein (GFP)-expressing Salmonella typhimurium to estimate adherence of bacteria to the eggshell surface, with independent measurement of autofluorescence to quantitate cuticle deposition. S. typhimurium were localized, adhering to cracks visible on the outer cuticle in ungraded eggs, indicating that egg-associated pathogens usually enter the egg interior either through respiratory pores in eggshells or through shell micro-cracks. The results of this study can be utilized to optimize innovative methods for predictive microbiology in order to achieve egg safety. [ABSTRACT FROM AUTHOR]

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species. [ABSTRACT FROM AUTHOR]

The article explores the impact of N1‐methylpseudouridine (m1Ψ) modification levels on mRNA protein expression, immunogenicity, and stability. The study conducted in vitro experiments with different m1Ψ modification ratios in mRNA encoding enhanced green fluorescent protein (EGFP) and found that low m1Ψ ratios increased protein expression, while high ratios decreased it. Additionally, the research suggests that m1Ψ modification reduces mRNA immunogenicity and enhances stability, with a positive correlation between modification ratio and stability. The study highlights the importance of optimizing mRNA modification strategies for various therapeutic applications. [Extracted from the article]

The common clownfish, Amphiprion ocellaris, is an iconic coral reef fish, ubiquitous in the marine aquarium hobby and useful for studying a variety of biological processes (e.g., mutual symbiosis, ultraviolet vision, and protandrous sex change). Recently, CRISPR/Cas9 methods were developed for knocking out specific genes for mechanistic studies. Here, we expand the genetic toolkit for A. ocellaris by creating the first transgenic line using the Tol2 transposon system. Fertilized eggs were co-injected with Tol2 transposase mRNA and a plasmid encoding an elongation factor-1α (Ef1α): green fluorescent protein (GFP) cassette at various concentrations, needle tip dimensions, and timepoints post-fertilization. We compared various injection parameters and sterilization methods to maximize the survival of injected eggs. F0s (n = 10) that were genotyped GFP + were then raised to 6 months of age and crossed with wild-type (WT) females to confirm germline transmission. F1 offspring were also raised and crossed in the same manner. The highly efficient Tol2 transposon system resulted in a 37% rate of transgenesis for surviving eggs amounting to a 2.7% yield of all injected eggs surviving and being GFP + (n = 160). Of these, 10 were raised to adulthood, 8 spawned, and 5/8 (62.5%) produced GFP + offspring. Further, two F1s crossed with WT females produced 54.2% and 44.6% GFP + offspring respectively, confirming the creation of a stable line. This is, to our knowledge, the first generation of a transgenic line in any coral reef fish. The ability to express transgenes of interest in the iconic anemonefish opens the door to a new era of exploration into their fascinating biology. [ABSTRACT FROM AUTHOR]

This study aimed to obtain a lentinan-specific fluorescent probe for the in situ visualization of lentinan. A carbohydrate-binding module with potential lentinan-binding capacity was discovered in a β-1,3-D-glucanase by using the bioinformatics techniques, and further recombinantly expressed. The binding specificity of the expressed protein, which was named as LBM was determined by the microtiter plate assays. The results indicated that LBM displayed a desired specificity for lentinan. The protein exhibited positive binding signals to lentinan, while could not bind to several examined polysaccharides including barley β-glucan, curdlan, or konjac glucomannan. Furthermore, the first lentinan-specific fluorescent probe was successfully constructed by fusing LBM with a green fluorescent protein EmGFP, based on which the in situ visualization of lentinan in Lentinula edodes was realized. It was shown that lentinan was not only presented in the mycelial cell wall, but also amorphously distributed in the interstitial space and inside the cell. The construction of the EmGFP-LBM provided a promising tool for the in situ visualization of lentinan. [ABSTRACT FROM AUTHOR]

Mitochondria undergo fission and fusion, and their coordinated balance is crucial for maintaining mitochondrial homeostasis. In yeast, the dynamin-related protein Dnm1 is a mitochondrial fission factor acting from outside the mitochondria. We recently reported the mitochondrial intermembrane space protein Atg44/mitofissin/Mdi1/Mco8 as a novel fission factor, but the relationship between Atg44 and Dnm1 remains elusive. Here, we show that Atg44 is required to complete Dnm1-mediated mitochondrial fission under homeostatic conditions. Atg44-deficient cells often exhibit enlarged mitochondria with accumulated Dnm1 and rosary-like mitochondria with Dnm1 foci at constriction sites. These mitochondrial constriction sites retain the continuity of both the outer and inner membranes within an extremely confined space, indicating that Dnm1 is unable to complete mitochondrial fission without Atg44. Moreover, accumulated Atg44 proteins are observed at mitochondrial constriction sites. These findings suggest that Atg44 and Dnm1 cooperatively execute mitochondrial fission from inside and outside the mitochondria, respectively. Abbreviation: ATG: autophagy related; CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; ERMES: endoplasmic reticulum-mitochondria encounter structure; GA: glutaraldehyde; GFP: green fluorescent protein; GTP: guanosine triphosphate: IMM: inner mitochondrial membrane; IMS: intermembrane space; OMM: outer mitochondrial membrane; PB: phosphate buffer; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RFP: red fluorescent protein; WT: wild type. [ABSTRACT FROM AUTHOR]

Domesticated strawberry is susceptible to sudden frost episodes, limiting the productivity of this cash crop in regions where they are grown during early spring. In contrast, the ancestral woodland strawberry (Fragaria vesca) has successfully colonized many habitats of the Northern Hemisphere. Thus, this species seems to harbour genetic factors promoting cold tolerance. Screening a germplasm established in the frame of the German Gene Bank for Crop Wild Relatives, we identified, among 70 wild accessions, a pair with contrasting cold tolerance. By following the physiological, biochemical, molecular, and metabolic responses of this contrasting pair, we identified the transcription factor Cold Box Factor 4 and the dehydrin Xero2 as molecular markers associated with superior tolerance to cold stress. Overexpression of green fluorescent protein fusions with Xero2 in tobacco BY-2 cells conferred cold tolerance to these recipient cells. A detailed analysis of the metabolome for the two contrasting genotypes allows the definition of metabolic signatures correlated with cold tolerance versus cold stress. This work provides a proof-of-concept for the value of crop wild relatives as genetic resources to identify genetic factors suitable to increase the stress resilience of crop plants. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults with the lowest survival rates five years post-diagnosis. Oncolytic viruses (OVs) selectively target and damage cancer cells, and for this reason they are being investigated as new therapeutic tools also against GBM. Methods: An oncolytic herpes simplex virus type 1 (oHSV-1) with deletions in the γ34.5 neurovirulence gene and the US12 gene, expressing enhanced green fluorescent protein (EGFP-oHSV-1) as reporter gene was generated and tested for its capacity to infect and kill the murine GL261 glioblastoma (GBM) cell line. Syngeneic mice were orthotopically injected with GL261cells. Seven days post-implantation, EGFP-oHSV-1 was administered intratumorally. Twenty-one days after parental tumor challenge in the opposite brain hemisphere, mice were sacrified and their brains were analysed by immunohistochemistry to assess tumor presence and cell infiltrate. Results: oHSV-1 replicates and induces cell death of GL261 cells in vitro. A single intracranial injection of EGFP-oHSV-1 in established GL261 tumors significantly prolongs survival in all treated mice compared to placebo treatment. Notably, 45% of treated mice became long-term survivors, and rejected GL261 cells upon rechallenge in the contralateral brain hemisphere, indicating an anamnestic antitumoral immune response. Post-mortem analysis revealed a profound modification of the tumor microenvironment with increased infiltration of CD4 + and CD8 + T lymphocytes, intertumoral vascular collapse and activation and redistribution of macrophage, microglia, and astroglia in the tumor area, with the formation of intense fibrotic tissue suggestive of complete rejection in long-term survivor mice. Conclusions: EGFP-oHSV1 demonstrates potent antitumoral activity in an immunocompetent GBM model as a monotherapy, resulting from direct cell killing combined with the stimulation of a protective adaptive immune response. These results open the way to possible application of our strategy in clinical setting. [ABSTRACT FROM AUTHOR]