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Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome. [ABSTRACT FROM AUTHOR]

The fission yeast Schizosaccharomyces pombe produces the hydroxamatetype siderophore ferrichrome (Fc). The biosynthesis of Fc requires the Fc synthase Sib1, the ornithine-N5-oxygenase Sib2, and the N5-hydroxyornithine- N5-transacetylase Sib3. In this study, we demonstrate the critical importance of the His248 residue of Sib3 in Fc production. Cells expressing a sib3H248A mutant allele fail to grow in iron-poor media without Fc supplementation. These sib3H248A mutant cells are consistently unable to promote Fc-dependent growth of Saccharomyces cerevisiae cells in cross-feeding experiments. Green fluorescent protein (GFP)-tagged wild-type Sib3 and mutant Sib3H248A exhibit a pancellular distribution. Coimmunoprecipitation assays revealed that both wild-type and Sib3H248A physically interact with Sib2. Further analysis identified a minimal C-terminal region from amino acids 290--334 of Sib3 that is required for interaction with Sib2. Deletion mapping analysis identified two regions of Sib2 as being required for its association with Sib3. The first region encompasses amino acids 1--135, and the second region corresponds to amino acids 281--358 of Sib2. Taken together, these results describe the first example of a physical interaction between an ornithine-N5-oxygenase and an N5-hydroxyornithine-N5-transacetylase controlling the biosynthesis of a hydroxamate-type siderophore. [ABSTRACT FROM AUTHOR]

Multispecies microbial communities drive most ecosystems on Earth. Chemical and biological interactions within these communities can affect the survival of individual members and the entire community. However, the prohibitively high number of possible interactions within a microbial community has made the characterization of factors that influence community development challenging. Here, we report a Microbial Community Interaction (μCI) device to advance the systematic study of chemical and biological interactions within a microbial community. The μCI creates a combinatorial landscape made up of an array of triangular wells interconnected with circular wells, which each contains either a different chemical or microbial strain, generating chemical gradients and revealing biological interactions. Bacillus cereus UW85 containing green fluorescent protein provided the "target" readout in the triangular wells, and antibiotics or microorganisms in adjacent circular wells are designated the "variables." The μCI device revealed that gentamicin and vancomycin are antagonistic to each other in inhibiting the target B. cereus UW85, displaying weaker inhibitory activity when used in combination than alone. We identified three-member communities constructed with isolates from the plant rhizosphere that increased or decreased the growth of B. cereus. The μCI device enables both strain-level and community-level insight. The scalable geometric design of the μCI device enables experiments with high combinatorial efficiency, thereby providing a simple, scalable platform for systematic interrogation of three-factor interactions that influence microorganisms in solitary or community life. [ABSTRACT FROM AUTHOR]

The RNA‐guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system represents the third family of CRISPR‐Cas systems that are harnessed for genome editing. However, only a few nucleases have demonstrated activity in human cells, and their in vivo therapeutic potential remains uncertain. In this study, a green fluorescent protein (GFP)‐activation assay is conducted to screen a panel of 15 Cas12b orthologs, and four of them exhibited editing activity in mammalian cells. Particularly noteworthy is the NiCas12b derived from Nitrospira sp., which recognizes a "TTN" protospacer adjacent motif (PAM) and facilitates efficient genome editing in various cell lines. Importantly, NiCas12b also exhibits a high degree of specificity, rendering it suitable for therapeutic applications. As proof of concept, the adeno‐associated virus (AAV) is employed to introduce NiCas12b to target the cholesterol regulatory gene proprotein convertase subtilisin/ kexin type 9 (Pcsk9) in the mouse liver. After 4 weeks of injections, an impressive is observed over 16.0% insertion/deletion (indel) efficiency, resulting in a significant reduction in serum cholesterol levels. NiCas12b provides a novel option for both basic research and clinical applications. [ABSTRACT FROM AUTHOR]

Plastids are pivotal target organelles for comprehensively enhancing photosynthetic and metabolic traits in plants via plastid engineering. Plastidial proteins predominantly originate in the nucleus and must traverse membrane-bound multiprotein translocons to access these organelles. This import process is meticulously regulated by chloroplast-targeting peptides (cTPs). Whereas many cTPs have been employed to guide recombinantly expressed functional proteins to chloroplasts, there is a critical need for more efficient cTPs. Here, we performed a comprehensive exploration and comparative assessment of an advanced suite of cTPs exhibiting superior targeting capabilities. We employed a multifaceted approach encompassing computational prediction, in planta expression, fluorescence tracking, and in vitro chloroplast import studies to identify and analyze 88 cTPs associated with Arabidopsis thaliana mutants with phenotypes linked to chloroplast function. These polypeptides exhibited distinct abilities to transport green fluorescent protein (GFP) to various compartments within leaf cells, particularly chloroplasts. A highly efficient cTP derived from Arabidopsis plastid ribosomal protein L35 (At2g24090) displayed remarkable effectiveness in chloroplast localization. This cTP facilitated the activities of chloroplast-targeted RNA-processing proteins and metabolic enzymes within plastids. This cTP could serve as an ideal transit peptide for precisely targeting biomolecules to plastids, leading to advancements in plastid engineering. Plastids are important organelles that can be engineered to enhance photosynthetic and metabolic traits in plants. This study identifies a chloroplast-targeting peptide that is highly efficient in delivering biologically functional proteins to plastids in plants. [ABSTRACT FROM AUTHOR]

Leymus chinensis, a halophytic perennial grass belonging to the Poaceae family, thrives in saline-alkali grasslands and harbors a rich repository of resistance-related genetic resources. This study focused on deciphering the stress-responsive mechanisms of L. chinensis by conducting transcriptomic sequencing under NaHCO3 stress, which resulted in the annotation of a segment corresponding to the 51WRKY gene. The alkali-induced gene LcWRKY40 (QIG37591) was identified by phylogenetic analysis. Real-time quantitative PCR analysis was performed on L. chinensis plants subjected to PEG6000 and alkaline salt (NaHCO3) stress, and the results indicated that the LcWRKY40 gene was upregulated in both the leaves and roots. The localization of the LcWRKY40 protein was confirmed by the use of green fluorescent protein (GFP) fusion technology in transformed rice protoplast cells. The GAL4-driven transformation of the LcWRKY40 gene in INVScI yeast cells, which exhibited enhanced tolerance upon overexpression of the LcWRKY40 gene under mannitol and alkaline salt (NaHCO3) stress conditions. Under drought stress using mannitol, the fresh weight of Nicotiana tabacum overexpressing the LcWRKY40 gene was significantly higher than that of wild-type(WT) tobacco. Through drought and salt alkali stress, we found that overexpressed tobacco at different stages always outperformed the wild type in terms of fresh weight, SOD, MDA, and Fv/Fm. This study provides preliminary insights into the involvement of the LcWRKY40 gene in responding to drought and alkaline salt stresses, highlighting its role in enhancing plant resistance to drought and saline-alkaline conditions. These findings lay the foundation for future molecular breeding strategies aimed at improving grass resistance from different aspects. [ABSTRACT FROM AUTHOR]

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (sRNAs) are pivotal post-transcriptional regulatory factors influencing biological activity. Studies on the rice bacterial blight pathogen Xanthomonas oryzae pathovar oryzae strain PXO99A, previously identified a virulence-associated sRNA, trans3287. A mutant strain lacking this sRNA, named SK01, resulted in markedly diminished virulence towards rice. This study aims to further elucidate the underlying bacterial virulent function of trans3287. The expression of trans3287 was quantified in virulence-inducing and standard nutritional conditions to clarify its production mechanism. The detection of virulence-associated genes revealed that trans3287 regulated the synthesis processes of extracellular polysaccharides, lipopolysaccharides, and the type III secretion system. Moreover, bioinformatics prediction and quantitative PCR indicated a potential direct target of trans3287, PXO_03470, encoding cardiolipin synthase. A dual-plasmid system fusing with GFP tag and protein immunoblotting confirmed that sRNA trans3287 negatively regulated PXO_03470. Bacterial biofilms demonstrated trans3287 regulated the disruption of biofilm integrity through cardiolipin synthase. This study provides preliminary insights into the mechanistic underpinnings of the role of sRNA trans3287 in mediating bacterial virulence through cardiolipin synthase. [ABSTRACT FROM AUTHOR]

Zinc (Zn) shortage is a common micronutrient deficiency affecting plants worldwide, while Zn toxicity may occur when this metal is in excess. Ectomycorrhizal (ECM) fungi are known to be able to modulate the transfer of macro- and microelements, among them Zn, to the plant. However, the underlying mechanisms are not well understood. We identified the HcZnT2 gene from the ECM fungus Hebeloma cylindrosporum, encoding a member of the Cation Diffusion Facilitator (CDF) family including Zn transporters, and analyzed its transcriptional regulation, the transport function by yeast complementation experiments, and its subcellular localization using a GFP fusion protein in yeast. HcZnT2 is highly induced during mycorrhization of Pinus pinaster, and upregulated in presence of the host plant root even without any direct contact. However, HcZnT2 is repressed by Zn excess conditions. By functional expression in yeast, our results strongly support the ability of HcZnT2 to transport Zn and, to a lesser extent, manganese. HcZnT2 localization was associated with the endoplasmic reticulum of yeast. Mycorrhizal gene activation at low external Zn suggests that the Zn transporter HcZnT2 might be important for the early establishment of the ECM symbiosis during Zn deficiency, rather than under Zn excess. HcZnT2 arises as an extremely remarkable candidate playing a key role in Zn homeostasis and regulation in ectomycorrhiza. [ABSTRACT FROM AUTHOR]

Introduction Peripheral nerve injury (PNI) occurs in approximately 3% of all trauma patients and can be challenging to treat, particularly when injury is severe such as with a long-segmental gap. Although peripheral nerves can regenerate after injury, functional recovery is often insufficient, leading to deficits in the quality of life of patients with PNI. Although nerve autografts are the gold standard of care, there are several disadvantages to their use, namely a lack of autologous nerve material for repair. This has led to the pursuit of alternative treatment methods such as axon guidance channels (AGCs). Second-generation AGCs have been shown to be able to deliver growth-enhancing substrates for nerve repair directly to the injury site. Although our laboratory has had success with second-generation AGCs filled with Schwann cells (SCs), SCs have their own set of issues clinically. Because of this, we have begun to utilize SC-derived exosomes as an alternative, as they have the appropriate protein markers, associate to axons in high concentrations, and are able to improve nerve regeneration. However, it is unknown how SC-derived exosomes may react within second-generation AGCs; thus, the aim of the present study was to assess the ability of SC-derived exosomes to be loaded into a second-generation AGC and how they would distribute within it. Materials and Methods A total of 4 dry second-generation AGCs were loaded with SC-derived exosomes that were derived from green fluorescent protein (GFP)-labeled SCs. They were subsequently frozen and sliced before imaging. Results Here, we present findings that SC-derived exosomes can be loaded into second-generation AGCs through our established loading method utilizing negative pressure and are able to survive and equally distribute along the length of the AGC. Conclusions Although only 4 second-generation AGCs were utilized, these findings indicate a potential use for SC-derived exosomes within second-generation AGCs to treat severe PNI. Future research should focus on exploring this in greater detail and in different contexts to assess the ability of SC-derived exosomes to survive at the site of injury and treat PNI. [ABSTRACT FROM AUTHOR]

Significance: Hair-thin multimode optical fiber-based holographic endoscopes have gained considerable interest in modern neuroscience for their ability to achieve cellular and even subcellular resolution during in-vivo deep brain imaging. However, the application of multimode fibers in freely moving animals presents a persistent challenge as it is difficult to maintain optimal imaging performance while the fiber undergoes deformations. Aim: We propose a fiber solution for challenging in-vivo applications with the capability of deep brain high spatial resolution imaging and neuronal activity monitoring in anesthetized as well as awake behaving mice. Approach: We used our previously developed M3CF multimode-multicore fiber to record fluorescently labeled neurons in anesthetized mice. Our M3CF exhibits a cascaded refractive index structure, enabling two distinct regimes of light transport that imitate either a multimode or a multicore fiber. The M3CF has been specifically designed for use in the initial phase of an in-vivo experiment, allowing for the navigation of the endoscope's distal end toward the targeted brain structure. The multicore regime enables the transfer of light to and from each individual neuron within the field of view. For chronic experiments in awake behaving mice, it is crucial to allow for disconnecting the fiber and the animal between experiments. Therefore, we provide here an effective solution and establish a protocol for reconnection of two segments of M3CF with hexagonally arranged corelets. Results: We successfully utilized the M3CF to image neurons in anaesthetized transgenic mice expressing enhanced green fluorescent protein. Additionally, we compared imaging results obtained with the M3CF with larger numerical aperture (NA) fibers in fixed whole-brain tissue. Conclusions: This study focuses on addressing challenges and providing insights into the use of multimode-multicore fibers as imaging solutions for in-vivo applications. We suggest that the upcoming version of the M3CF increases the overall NA between the two cladding layers to allow for access to high resolution spatial imaging. As the NA increases in the multimode regime, the fiber diameter and ring structure must be reduced to minimize the computational burden and invasiveness. [ABSTRACT FROM AUTHOR]

Dendrobium officinale soft rot is a widespread and destructive disease caused by Fusarium oxysporum that can seriously affect yield and quality. To better understand the fungal infection and colonization, we successfully created an F. oxysporum labeled with green fluorescent protein using the Agrobacterium tumefaciens-mediated transformation method. Transformants had varying fluorescence intensities, but their pathogenicity did not differ from that of the wild type. Fluorescence microscopy revealed that F. oxysporum primarily entered the aboveground portion of D. officinale through the leaf margin, stomata, or by direct penetration of the leaf surface. It then colonized the mesophyll and spread along its vascular bundles. D. officinale exhibited typical symptoms of decay and wilting at 14 days postinoculation, accompanied by a pronounced fluorescence signal in the affected area. The initial colonization of F. oxysporum in the subterranean region primarily involved attachment to the root hair and epidermis, which progressed to the medullary vascular bundle. At 14 days postinoculation, the root vascular bundles of D. officinale exhibited significant colonization by F. oxysporum. Macroconidia were also observed in black rot D. officinale tissue. In particular, the entire root was surrounded by a significant number of chlamydospore-producing F. oxysporum mycelia at 28 days postinoculation. This approach allowed for the visualization of the complete infection process of F. oxysporum and provided a theoretical foundation for the development of field control strategies. [ABSTRACT FROM AUTHOR]

Objective: To construct an over-expression lentiviral vector of the dedicator of cytokinesis 4 (DOCK4), and to establish DOCK4 stably over-expressing Neuro-2a cells. Methods: The DOCK4 sequence was searched in the National Center for Biotechnology Information (NCBI) and primers were designed and synthesized; polymerase chain reaction (PCR) method was used to amplify the DOCK4 gene sequences. After digestion with BamHⅠ and AgeⅠ restriction endonucleases, the DOCK4 gene sequences were ligated with the digested lentiviral vector GV492 to construct the GV492-DOCK4 over-expression recombinant plasmid. The positive clones with a similar length to the target gene fragment were screened and identified by PCR method. The GV492-control plasmid and GV492-DOCK4 over-expression recombinant plasmid were transfected into the HEK293T cells, and the lentivirus was collected and titered 48 h after transfection. The Neuro-2a cells were divided into GV492-control group and GV492-DOCK4 group, and the cells were infected with GV492-control lentivirus and GV492-DOCK4 over-expression lentivirus, respectively, and the multiplicity of infection (MOI) was 100. After 72 h of infection, the successfully infected Neuro-2a cells were screened by using puromycin (10 mg·L-1). The growth status of Neuro-2a cells and the expression of green fluorescent protein in various groups were observed under fluorescence microscope. Real-time quantitative PCR (RT-qPCR) and Western blotting methods were used to detect the expression levels of DOCK4 mRNA and DOCK4 protein in the Neuro-2a cells in various groups. Results: The PCR results showed that the gene fragment length of the GV492-DOCK4 over-expression recombinant plasmid was approximately 691 bp. The sequencing results showed that the gene sequence of the GV492-DOCK4 over-expression recombinant plasmid was consistent with the designed over-expression sequence of DOCK4. The titers of the lentiviruses in GV492-control group and GV492-DOCK4 over-expression group were 2. 5×108 TU·mL-1 and 2. 5×108 TU·mL-1, respectively. The fluorescence microscope observation results showed that Neuro-2a cells in various groups grew well and expressed green fluorescent protein. The RT-qPCR results showed that compared with GV492-control group, the expression level of DOCK4 mRNA in the Neuro-2a cells in GV492-DOCK4 group was significantly increased (P<0. 01). The Western blotting results showed the specific bands near the relative molecular mass of 225 000 in various groups. Compared with GV492-control group, the expression level of DOCK4 protein in the Neuro-2a cells in GV492-DOCK4 group was significantly increased (P<0. 01). Conclusion: This study successfully constructs the DOCK4 over-expression lentiviral vector and establishes the Neuro-2a cells stably over-expressing DOCK4. [ABSTRACT FROM AUTHOR]

Background/aim: The application of biocompatible heavy metal-free and cationic Ag2S NIR quantum dots (QDs), which have intense luminosity in the 700-900 nm medical range, as a nonviral gene delivery system paves the way to overcome autofluorescence and easily track the delivery of genes in real time. Materials and methods: The newly developed small and colloidally stable 2-mercaptopropionic acid (MPA)-capped Ag2S aqueous quantum dots electrostatically complexed with linear polyethyleneimine (Ag2S@2MPA/LPEI) were investigated for the first time both as a strong fluorescent probe and as a vector for nonviral gene delivery for the highest tracking of the system and delivery of genes into the nuclei of different cancer cells. The synthesized Ag2S@2MPA/LPEI quantum dots demonstrated strong optical imaging properties and were used to deliver a green fluorescent protein (GFP) plasmid as a standard gene. Results: For Ag2S@2MPA/LPEI-pDNA nanoparticles, an N/P ratio of 20 was the ideal transfection efficiency. Ag2S@2MPA/LPEI was substantially more compatible with HEK 293T cells than the free 25-kDa linear polyethylenimine (LPEI). Next, the transfection efficiency evaluation of pGFP genes with synthesized Ag2S@2MPA/LPEI and LPEI in different cancer cells demonstrated their high potential as a theranostic cancer gene delivery system. Conclusion: This is the first instance of gene transfection and optical imaging carried out in vitro using Ag2S@2MPA/LPEI QDs. Overall, the newly synthesized highly biocompatible and trackable Ag2S@2MPA/LPEI QDs can be an effective and biocompatible theranostic system for cancer gene therapy. [ABSTRACT FROM AUTHOR]

To explore morphological evidence for neuronal circuits between adenosine A2A receptor (A2AR) neurons in the nucleus accumbens (NAc) and orexin neurons. The utilized adeno-associated virus (AAV) encoding humanized Renilla green fluorescent protein (hrGFP) as a tracer to visualize the expression of the A2AR neurons in the NAc and their axon distributions in orexin neurons. The Cre-dependent AAV was injected into the NAc in adenosine A2AR-Cre mice. Immunohistochemistry was then used to visualize hrGFP and their projections in orexin neurons. The data revealed that NAc A2AR neurons, mainly expressed in g-aminobutyric acidergic (GABAergic) projection neurons, projected to the hypothalamus, with apparent contacts of hrGFP containing boutons onto orexin-immunoreactive (IR) soma and dendrites. We also observed strong staining of orexin-IR varicose terminals in the NAc, with apparent contacts of orexin-IR terminals onto A2AR neurons. Furthermore, in the NAc, orexin-IR varicosities contained vesicular glutamate transporter 2 (VGluT2) and synaptophysin (Syp). These data provide morphological evidence for exploring the function between NAc A2AR neurons and orexin neuron in the hypothalamus, particularly in regulating sleep processes. [ABSTRACT FROM AUTHOR]

Significance: Hyperspectral imaging (HSI) of murine tumor models grown in dorsal skinfold window chambers (DSWCs) offers invaluable insight into the tumor microenvironment. However, light loss in a glass coverslip is often overlooked, and particular tissue characteristics are improperly modeled, leading to errors in tissue properties extracted from hyperspectral images. Aim: We highlight the significance of spectral renormalization in HSI of DSWC models and demonstrate the benefit of incorporating enhanced green fluorescent protein (EGFP) excitation and emission in the skin tissue model for tumors expressing genes to produce EGFP. Approach: We employed an HSI system for intravital imaging of mice with 4T1 mammary carcinoma in a DSWC over 14 days. We performed spectral renormalization of hyperspectral images based on the measured reflectance spectra of glass coverslips and utilized an inverse adding-doubling (IAD) algorithm with a two-layer murine skin model, to extract tissue parameters, such as total hemoglobin concentration and tissue oxygenation (StO2). The model was upgraded to consider EGFP fluorescence excitation and emission. Moreover, we conducted additional experiments involving tissue phantoms, human forearm skin imaging, and numerical simulations. Results: Hyperspectral image renormalization and the addition of EGFP fluorescence in the murine skin model reduced the mean absolute percentage errors (MAPEs) of fitted and measured spectra by up to 10% in tissue phantoms, 0.55% to 1.5% in the human forearm experiment and numerical simulations, and up to 0.7% in 4T1 tumors. Similarly, the MAPEs for tissue parameters extracted by IAD were reduced by up to 3% in human forearms and numerical simulations. For some parameters, statistically significant differences (p < 0.05) were observed in 4T1 tumors. Ultimately, we have shown that fluorescence emission could be helpful for 4T1 tumor segmentation. Conclusions: The results contribute to improving intravital monitoring of DWSC models using HSI and pave the way for more accurate and precise quantitative imaging. [ABSTRACT FROM AUTHOR]

Two derivatives of imidazolinones, specifically DMPI {(4Z)-4-(4-(dimethylamino)benzylidene)-2-methyl-1,4-dihydro-5H-imidazolin-5-one} and MMPI {(4Z)-4-(4-methoxybenzylidene)-2-methyl-1-((pyridin-3-yl)methyl)-1,4-dihydro-5H-imidazolin-5-one} are reported which were synthesized under reflux conditions. Both the ligands are characterized with different analytical techniques including NMR spectroscopy, mass spectrometry, IR Spectroscopy. The subsequent elucidation of their solid-state structures was achieved through the single crystal X-ray diffraction method, providing detailed insights into their molecular arrangements. In the crystalline lattice, the asymmetric unit of DMPI comprises a solitary DMPI molecule, whereas MMPI reveals a more intricate arrangement with four molecules within its asymmetric unit. Various types of supramolecular interactions such as C–H⋯O, C–H⋯N, C–H⋯π and π⋯π interactions are observed in the X-ray structures where a trifurcated C–H⋯O bonds are also observed in both the ligands. All these interactions guide the formation of 3D supramolecular architecture in the solid-state of both compounds. Besides these, the 2D fingerprint and Hirshfeld surface analysis computations were served to prove and quantify various supramolecular interactions within the crystal lattice. [ABSTRACT FROM AUTHOR]

Fusarium spp. can cause root rot in alfalfa, leading to the death of the whole plant, which seriously affects the yield and quality of alfalfa. This study used a Fusarium acuminatum strain labeled with green fluorescent protein (GFP) to observe the infection process of F. acuminatum on alfalfa by confocal fluorescence microscopy. The aim of this study was to reveal the infection mechanism of alfalfa Fusarium root rot at the cellular histological level. The results showed that conidia of F. acuminatum attached to the surface of the root and germinated at one day post-inoculation, the mycelium then entered the vascular bundle tissue of the alfalfa root at 5 days post-inoculation, reached the base of the plant stem at 14 days post-inoculation, and colonized the stem of the first and second compound leaf at 28 and 49 days post-inoculation, respectively. Moreover, the experiment, which sprayed a spore suspension, showed that the conidia of F. acuminatum could spread through the air to infect the pericarp and seed coat tissue of the pod. For the first time, we report the infection process of alfalfa Fusarium root rot caused by F. acuminatum and clarify that F. acuminatum can initially infect the root tissue of alfalfa, colonize the bottom stem of the plant through systematic infection, and eventually cause the plant to wilt and die. The results reveal the infection mechanism of F. acuminatum at the cell level via histology and provide theoretical support for the development of control strategies and key control technologies for alfalfa root rot. [ABSTRACT FROM AUTHOR]

Simple Summary: In the medical and pharmaceutical industries, insect cell lines are used to produce useful recombinant proteins, with their production necessitating the genetic engineering of recombinant insect viruses. Recombinant viruses can be used in experiments according to a virus quantification process. However, current virus quantification methods are complicated by their skill and experience requirements and long durations. In this study, we attempted to generate a novel transgenic insect cell line for the prompt and convenient quantification of insect viruses. By introducing a fluorescent gene, Sf9-QE, a transgenic insect cell line that rapidly emits high fluorescence in response to viral infection was generated. Using the Sf9-QE cell line, the quantification completion time was shortened by 4 to 6 days compared to existing insect cell lines, and convenient quantification was also facilitated by fluorescence photometry. The novel transgenic insect cell line generated, Sf9-QE, will be very useful in the production of and research on various medically and pharmaceutically applicable recombinant proteins, greatly contributing to human health and medical care. The following study was conducted to generate a transgenic Sf9 cell line for rapid and easy virus quantification in the baculovirus expression system (BES). The hr3 (homologous region 3) and 39K and p10 promoters were used as the expression structures to induce rapid and intense expression of the enhanced green fluorescent protein gene in cells in response to viral infection. Of 20 transgenic Sf9 cell lines generated using the piggyBac system, the cell line that showed the highest fluorescence expression in the shortest time in response to viral infection was selected and named Sf9-QE. The average diameter of the Sf9-QE cells was around 16 μm, which is 2 μm smaller than the average diameter of Sf9 cells, whereas the rate of cell proliferation was around 1.6 times higher in the Sf9-QE cells. Virus quantification using the Sf9-QE cell line did not produce significantly different results compared to the other cell lines; however, the time required for complete virus quantification was approximately 5.3 to 6.0 days for the Sf9-QE cells, which is around 4 to 6 days shorter than the time required for the other cell lines, enabling convenient and accurate virus quantification via fluorescence photometry within around 6.0 to 6.3 days. The properties of the Sf9-QE cells were stable for up to at least 100 passages. [ABSTRACT FROM AUTHOR]

Objectives: Non-viral mediated plasmid DNA transfection by electroporation (EP) is an established method for gene transfection. In this study, the usefulness of direct EP at an intradermal (i.d.) site (DEP) with implanted electrodes to achieve a high protein expression level was investigated. In addition, DEP application with various intervals with a low application voltage was also evaluated to confirm its effect on protein expression. Methods: Green fluorescent protein (GFP)- and luciferase-encoding DNA were administrated, and GFP and luciferase were evaluated. Results: A higher protein expression level was observed after green fluorescent protein (GFP)- and luciferase-encoding DNA were delivered by i.d. injection followed by DEP application. When luciferase expression was observed with an in vivo imaging system, continuous expression was confirmed over 21 days after i.d. injection followed by DEP at 100 V. This approach provided increased gene expression levels compared with conventional EP methods via the stratum corneum layer. In addition, the effect of application voltage on luciferase expression was investigated; two-time applications (repeated DEP) at 20 V with 5 min intervals showed similar luciferase expression level to single DEP application with 100 V. Histological observations showed the skin became thicker after a single DEP at 100 V, whereas no apparent thickness changes were confirmed after repeated DEP at 20 V with 5 min intervals. Conclusions: This study revealed that direct i.d. voltage application achieved high protein expression levels even at low voltages. Skin is a promising administration site for DNA vaccines, so this approach may be effective for DNA vaccine delivery into skin tissue. [ABSTRACT FROM AUTHOR]

Recombinant AAV (rAAV) vectors are increasingly favored for gene therapy due to their useful features of vectorology, such as transfection of dividing and nondividing cells, the presence of tissue-specific serotypes, and biosafety considerations. This study investigates the impact of commonly used therapeutic drugs—acetaminophen, budesonide, and simvastatin—on rAAV transduction efficiency in HEK-293 cells. Cells were transduced with an AAV mosaic vector under the control of a cytomegalovirus (CMV) promoter encoding green fluorescent protein (GFP). Transduction efficiency was assessed by qPCR and fluorescent microscopy. Analysis of functional interactions between genes potentially involved in rAAV transduction in drug-exposed cells was also performed. This study showed a clear effect of drugs on rAAV transmission. Notably, acetaminophen enhanced transduction efficiency by 9-fold, while budesonide and simvastatin showed 2-fold and 3-fold increases, respectively. The gene analysis illustrates the possible involvement of genes related to cell membranes in the potentiation of rAAV transduction induced by the drugs under investigation. Attention should be paid to S100A8, which is a common drug-modified gene for drugs showing anti-inflammatory effects (budesonide and simvastatin), demonstrating an interaction with the gene encoding the receptor for AAV (HGFR). This study underscores the significance of assessing rAAV pharmacokinetics/pharmacodynamics (PKs/PDs) and drug–gene therapy interactions in optimizing gene therapy protocols. [ABSTRACT FROM AUTHOR]

Leaves play a crucial role as ornamental organs in Spathiphyllum, exhibiting distinct differences across various Spathiphyllum varieties. Leaf development is intricately linked to processes of cell proliferation and expansion, with cell morphology often regulated by plant cell walls, primarily composed of cellulose. Alterations in cellulose content can impact cell morphology, subsequently influencing the overall shape of plant organs. Although cellulases have been shown to affect cellulose levels in plant cells, genetic evidence linking them to the regulation of leaf shape remains limited. This study took the leaves of Spathiphyllum 'Mojo' and its somatic variants as the research objects. We screened four cellulase gene family members from the transcriptome and then measured the leaf cellulose content, cellulase activity, and expression levels of cellulase-related genes. Correlation analysis pinpointed the gene SpGH9A3 as closely associated with leaf shape variations in the mutant. Green fluorescent fusion protein assays revealed that the SpGH9A3 protein was localized to the cell membrane. Notably, the expression of the SpGH9A3 gene in mutant leaves peaked during the early spread stage, resulting in smaller overall leaf size and reduced cellulose content upon overexpression in Arabidopsis. [ABSTRACT FROM AUTHOR]

Fusarium head blight (FHB), a disease inflicted by Fusarium graminearum and F. asiaticum, poses a growing threat to wheat in China, particularly in the face of climate change and evolving agricultural practices. This study unveiled the discovery of the victorivirus FgVV2 from the F. asiaticum strain F16176 and comprehensively characterized the function of the two victoriviruses FaVV1 and FaVV2 in virulence. Through comparative analysis with a virus-free strain, we established that these mycoviruses markedly repress the sexual reproduction and pathogenicity of their fungal hosts. Furthermore, we synthesized the coat protein (CP) genes CP1 from FaVV1 and CP2 from FaVV2, which were fused with the green fluorescent protein (GFP) gene and successfully expressed in Fusarium strains in wild-type isolates of F. asiaticum and F. graminearum. Similar to virus-infected strains, the transformed strains expressing CPs showed a significant decrease in perithecia formation and pathogenicity. Notably, CP2 exhibited a stronger inhibitory effect than CP1, yet the suppression of sexual reproduction in F. graminearum was less pronounced than that in F. asiaticum. Additionally, the pathogenicity of the F. asiaticum and F. graminearum strains expressing CP1 or CP2 was substantially diminished against wheat heads. The GFP-tagged CP1 and CP2 revealed distinct cellular localization patterns, suggesting various mechanisms of interaction with the host. The findings of this study provide a significant research foundation for the study of the interaction mechanisms between FaVV1 and FaVV2 with their hosts, as well as for the exploration and utilization of fungal viral resources. [ABSTRACT FROM AUTHOR]

To present the expression of calsyntenin‐1 (Clstn1) in the brain and investigate the potential mechanism of Clstn1 in lithium–pilocarpine rat seizure models. Thirty‐five male SD adult rats were induced to have seizures by intraperitoneal injection of lithium chloride pilocarpine. Rats exhibiting spontaneous seizures were divided into the epilepsy (EP) group (n = 15), whereas those without seizures were divided into the control group (n = 14). Evaluate the expression of Clstn1 in the temporal lobe of two groups using Western blotting, immunohistochemistry, and immunofluorescence. Additionally, 55 male SD rats were subjected to status epilepticus (SE) using the same induction method. Rats experiencing seizures exceeding Racine's level 4 (n = 48) were randomly divided into three groups: SE, SE + control lentivirus (lentiviral vector expressing green fluorescent protein [LV‐GFP]), and SE + Clstn1‐targeted RNA interference lentivirus (LV‐Clstn1‐RNAi). The LV‐GFP group served as a control for the lentiviral vector, whereas the LV‐Clstn1‐RNAi group received a lentivirus designed to silence Clstn1 expression. These lentiviral treatments were administered via hippocampal stereotactic injection 2 days after SE induction. Seven days after SE, Western blot analysis was performed to evaluate the expression of Clstn1 in the hippocampus and temporal lobe. Meanwhile, we observed the latency of spontaneous seizures and the frequency of spontaneous seizures within 8 weeks among the three groups. The expression of Clstn1 in the cortex and hippocampus of the EP group was significantly increased compared to the control group (p <.05). Immunohistochemistry and immunofluorescence showed that Clstn1 was widely distributed in the cerebral cortex and hippocampus of rats, and colocalization analysis revealed that it was mainly co expressed with neurons in the cytoplasm. Compared with the SE group (11.80 ± 2.17 days) and the SE + GFP group (12.40 ± 1.67 days), there was a statistically significant difference (p <.05) in the latency period of spontaneous seizures (15.14 ± 2.41 days) in the SE + Clstn1 + RNAi group rats. Compared with the SE group (4.60 ± 1.67 times) and the SE + GFP group (4.80 ± 2.05 times), the SE + Clstn1 + RNAi group (2.0 ±.89 times) showed a significant reduction in the frequency of spontaneous seizures within 2 weeks of chronic phase in rats (p <.05). Elevated Clstn1 expression in EP group suggests its role in EP onset. Targeting Clstn1 may be a potential therapeutic approach for EP management. [ABSTRACT FROM AUTHOR]

Strengthening the expression level of integrated genes on the genome is crucial for consistently expressing key enzymes in microbial cell factories for efficient bioproduction in synthetic biology. In comparison to plasmidbased multi-copy expression, the utilization of chromosomal multi-copy genes offers increased stability of expression level, diminishes the metabolic burden on host cells, and enhances overall genetic stability. In this study, we developed the “BacAmp”, a stabilized gene integration expression and copy number amplification system for high-level expression in Bacillus subtilis, which was achieved by employing a combination of repressor and non-natural amino acids (ncAA)-dependent expression system to create a reversible switch to control the key gene recA for homologous recombination. When the reversible switch is turned on, genome editing and gene amplification can be achieved. Subsequently, the reversible switch was turned off therefore stabilizing the gene copy number. The stabilized gene amplification system marked by green fluorescent protein, achieved a 3-fold increase in gene expression by gene amplification and maintained the average gene copy number at 10 after 110 generations. When we implemented the gene amplification system for the regulation of N-acetylneuraminic acid (NeuAc) synthesis, the copy number of the critical gene increased to an average of 7.7, which yielded a 1.3-fold NeuAc titer. Our research provides a new avenue for gene expression in synthetic biology and can be applied in metabolic engineering in B. subtilis. [ABSTRACT FROM AUTHOR]

Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries. Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries. Highthroughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level. In this study, we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin, the inverter part was an orthogonal transcriptional repressor to obtain signal inversion, and the output part was a fluorescent protein to be easily detected. The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo. This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization. An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control. Then, using such a biosensor combined with fluorescence-activated cell sorting, we established a highthroughput screening platform for screening adenosylcobalamin overproducers. This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites. [ABSTRACT FROM AUTHOR]

Objective: To construct a recombinant adenovirus (Ad-DF3-copGFP) containing the DF3/MUC1 promoter transcriptional regulatory sequence and green fluorescent protein (GFP) and to investigate its role in the detection of circulating tumor cells (CTC). Methods: A recombinant adenovirus (Ad - DF3 - copGFP) was prepared and purified. Comparative studies were conducted with a previously constructed and stored recombinant adenovirus (Ad-hTERT-copGFP) in our laboratory. Infection efficiency and non-specific infection rates were evaluated by infecting lung adenocarcinoma A549 and H1299 cells, as well as peripheral blood mononuclear cells (PBMC)from healthy individuals. A549 cells were artificially added to healthy peripheral blood to simulate CTC, and the infection with both recombinant viruses was used to detect simulate CTC for detection rate determination. The Ad-DF3-copGFP and Ad-hTERT- copGFP methods were used to detect CTC in clinical samples from lung cancer patients, and the initial clinical CTC detection performance was evaluated. Results: The recombinant adenovirus (Ad -DF3 - copGFP) was successfully constructed, showing a high infection efficiency for both A549 and H1299 cells. Compared with Ad-hTERT-copGFP, Ad-DF3-copGFP showed a lower non-specific infection rate (P < 0.001). The overall detection rate using the Ad-DF3-copGFP method (77.3%) was higher than that using the AdhTERT-copGFP method (69.6%). In clinical CTC detection, the number of CTC detected by the Ad-DF3-copGFP method [(10.90± 2.42) cells per 4 mL]was significantly higher than that by the Ad-hTERT-copGFP method [(6.20±1.81) cells per 4 mL, P < 0.001]. Conclusion: The recombinant adenovirus (Ad - DF3 - copGFP) is successfully constructed, demonstrating a reliable and efficient detection of CTC, thus providing a new method for CTC detection. [ABSTRACT FROM AUTHOR]

A method is proposed for analysis of the photon-count number distributions (PCD) calculated over the space (pixels) of a stack of fluorescent images obtained in the process of a scanning measurement in fluorescence fluctuation spectroscopy which makes it possible to determine the characteristic brightness and number of molecules of the material being studied. The method is applicable to ergodic systems and is based on a theory of the analysis of Photon Counting Histograms (PCH) developed for single-point measurements. The method is tested on experimentally obtained images of a green fluorescent protein. The obtained results are compared with the results of a single-point experiment and the N&B (Number and Brightness) method, which is most often used for numerical analysis of a stack of fluorescent images obtained in scanning experiments. The estimates of the characteristic brightness and number of molecules of the studied substance are in good agreement with estimates obtained with the aid of the methods of analysis used in this region, which makes it possible to conclude that the theory of the PCH method can be used for analyzing spatial PCDs calculated on the basis of a stack of images. The developed method makes it possible to obtain estimates of the studied parameters based on a selected subregion of a single frame of the image. [ABSTRACT FROM AUTHOR]

Fate mapping and genetic manipulation of renin cells have relied on either noninducible Cre lines that can introduce the developmental effects of gene deletion or bacterial artificial chromosome transgene-based inducible models that may be prone to spurious and/or ectopic gene expression. To circumvent these problems, we generated an inducible mouse model in which CreERT2 is under the control of the endogenous Akr1b7 gene, an independent marker of renin cells that is expressed in a few extrarenal tissues. We confirmed the proper expression of Cre using Akr1b7CreERT2/+;R26RmTmG/+ mice in which Akr1b7+/renin+ cells become green fluorescent protein (GFP)+ upon tamoxifen administration. In embryos and neonates, GFP was found in juxtaglomerular cells, along the arterioles, and in the mesangium, and in adults, GFP was present mainly in juxtaglomerular cells. In mice treated with captopril and a low-salt diet to induce recruitment of renin cells, GFP extended along the afferent arterioles and in the mesangium. We generated Akr1b7CreERT2/+;Ren1cFl/−;R26RmTmG/+ mice to conditionally delete renin in adult mice and found a marked reduction in kidney renin mRNA and protein and mean arterial pressure in mutant animals. When subjected to a homeostatic threat, mutant mice were unable to recruit renin+ cells. Most importantly, these mice developed concentric vascular hypertrophy ruling out potential developmental effects on the vasculature due to the lack of renin. We conclude that Akr1b7CreERT2 mice constitute an excellent model for the fate mapping of renin cells and for the spatial and temporal control of gene expression in renin cells. NEW & NOTEWORTHY: Fate mapping and genetic manipulation are important tools to study the identity of renin cells. Here, we report on a novel Cre mouse model, Akr1b7CreERT2, for the spatial and temporal regulation of gene expression in renin cells. Cre is properly expressed in renin cells during development and in the adult under basal conditions and under physiological stress. Moreover, renin can be efficiently deleted in the adult, leading to the development of concentric vascular hypertrophy. [ABSTRACT FROM AUTHOR]

Agathisflavone is a flavonoid that exhibits anti-inflammatory and anti-oxidative properties. Here, we investigated the neuroprotective effects of agathisflavone on central nervous system (CNS) neurons and glia in the cerebellar slice ex vivo model of neonatal ischemia. Cerebellar slices from neonatal mice, in which glial fibrillary acidic protein (GFAP) and SOX10 drive expression of enhanced green fluorescent protein (EGFP), were used to identify astrocytes and oligodendrocytes, respectively. Agathisflavone (10 μM) was administered preventively for 60 min before inducing ischemia by oxygen and glucose deprivation (OGD) for 60 min and compared to controls maintained in normal oxygen and glucose (OGN). The density of SOX-10+ oligodendrocyte lineage cells and NG2 immunopositive oligodendrocyte progenitor cells (OPCs) were not altered in OGD, but it resulted in significant oligodendroglial cell atrophy marked by the retraction of their processes, and this was prevented by agathisflavone. OGD caused marked axonal demyelination, determined by myelin basic protein (MBP) and neurofilament (NF70) immunofluorescence, and this was blocked by agathisflavone preventative treatment. OGD also resulted in astrocyte reactivity, exhibited by increased GFAP-EGFP fluorescence and decreased expression of glutamate synthetase (GS), and this was prevented by agathisflavone pretreatment. In addition, agathisflavone protected Purkinje neurons from ischemic damage, assessed by calbindin (CB) immunofluorescence. The results demonstrate that agathisflavone protects neuronal and myelin integrity in ischemia, which is associated with the modulation of glial responses in the face of ischemic damage. [ABSTRACT FROM AUTHOR]

Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space group C2) and a newly discovered orthorhombic form (space group P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetric unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the β‐barrel scaffold and a restriction of pH‐dependent shifts of the key residue Lys61, which is crucial for the pH‐dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a bacterial pathogen that colonizes the human stomach, where it can cause a variety of diseases. H. pylori uses a cluster of sheathed flagella for motility, which is required for host colonization in animal models. The flagellar sheath is continuous with the outer membrane and is found in most Helicobacter species identified to date. HP0018 is a predicted lipoprotein of unknown function that is conserved in Helicobacter species that have flagellar sheaths but is absent in Helicobacter species that have sheath-less flagella. Deletion of hp0018 in H. pylori B128 resulted in the formation of long chains of outer membrane vesicles, which were most evident in an aflagellated variant of the Δhp0018 mutant that had a frameshift mutation in fliP. Flagellated cells of the Δhp0018 mutant possessed what appeared to be a normal flagellar sheath, suggesting that HP0018 is not required for sheath formation. Cells of the Δhp0018 mutant were also less helical in shape compared to wild-type cells. A HP0018-superfolder green fluorescent fusion protein expressed in the H. pylori Δhp0018 mutant formed fluorescent foci at the cell poles and lateral sites. Co-immunoprecipitation assays with HP0018 identified two enzymes involved in the modification of the cell wall peptidoglycan, AmiA and MltD, as potential HP0018 interaction partners. HP0018 may modulate the activity of AmiA or MltD, and in the absence of HP0018, the unregulated activity of these enzymes may alter the peptidoglycan layer in a manner that results in an altered cell shape and hypervesiculation. [ABSTRACT FROM AUTHOR]

Background/Aim: Transgenic nude mice expressing green fluorescent protein (GFP), red fluorescent protein (RFP), or cyan fluorescent protein (CFP) were previously developed by our laboratory, AntiCancer Inc. In the present study, we demonstrate imaging of the GFP, RFP, or CFP nude mice with single-nanometer-tuning laser fluorescence excitation with a single instrument. Materials and Methods: Female transgenic C57/B6 nude GFP, RFP, and CFP mice aged six weeks were used. The images were obtained using the UVP Biospectrum Advanced system (Analytik Jena US LLC) with excitation at 480 nm and peak emission at 513 nm for GFP; 520 nm and 605 nm, respectively, for RFP; and 405 nm and 480 nm, respectively, for CFP. Results: For each color transgenic fluorescent mouse, images without background could be obtained individually with the UVP Biospectrum Advanced system. Conclusion: Using a single instrument, brilliant and well-defined images of GFP, RFP, and CFP mice were obtained with single-nanometer-tuning laser fluorescence excitation. This imaging system will be used in future studies to analyze cancer cells in the colored mice that are spectrally distinct in order to determine how stromal cells and cancer interact in the tumor microenvironment. [ABSTRACT FROM AUTHOR]

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research. [ABSTRACT FROM AUTHOR]

We synthesized a "cyborg protein," wherein a synthetic molecule partially substitutes the main peptide chain by linking 2 protein domains with a synthetic oligomer. Green fluorescent protein (GFP) served as the model for constructing the cyborg proteins. We prepared circularly permuted GFP (cpGFP) with new termini between β10 and β11, where the original N- and C-termini were linked by a cleavable peptide loop. The cyborg GFP was constructed from cpGFP by linking the β10 and β11 with oligo(ethylene glycol) (OEG) using maleimide-cysteine couplings, followed by the enzymatic cleavage of the N- and C-termini linking loop by thrombin. With the help of machine learning, we were able to obtain the cpGFP mutants that significantly alter the fluorescence intensity (53% increase) by thrombin treatment, which splits cpGFP into 2 fragments (fragmented GFP), and by heat shock. When the cyborg GFP was constructed using this mutant, the fluorescence intensity increased by 13% after heat treatment, similar to cpGFP (33% increase), and the behavior was significantly different from that of the fragmented GFP. This result suggests the possibility that the OEG chain in the cyborg protein plays a similar role to the peptide in the main chain of the protein. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. [ABSTRACT FROM AUTHOR]

Bacterial adhesion and biofilm maturation is significantly influenced by surface properties, encompassing both bare surfaces and single or multi-layered coatings. Hence, there is an utmost interest in exploring the intricacies of gene regulation in sulfate-reducing bacteria (SRB) on copper and graphene-coated copper surfaces. In this study, Oleidesulfovibrio alaskensis G20 was used as the model SRB to elucidate the pathways that govern pivotal roles during biofilm formation on the graphene layers. Employing a potent reporter green fluorescent protein (GFP) tagged to O. alaskensis G20, the spatial structure of O. alaskensis G20 biofilm on copper foil (CuF), single-layer graphene-coated copper (Cu-GrI), and double-layer graphene-coated copper (Cu-GrII) surfaces was investigated. Biofilm formation on CuF, Cu-GrI, and Cu-GrII surfaces was quantified using CLSM z-stack images within COMSTAT v2 software. The results revealed that CuF, Cu-GrI, and Cu-GrII did not affect the formation of the GFP-tagged O. alaskensis G20 biofilm architecture. qPCR expression showed insignificant fold changes for outer membrane components regulating the quorum-sensing system, and global regulatory proteins between the uncoated and coated surfaces. Notably, a significant expression was observed within the sulfate reduction pathway confined to dissimilatory sulfite reductases on the Cu-GrII surface compared to the CuF and Cu-GrI surfaces. [ABSTRACT FROM AUTHOR]

Background/Aim: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model. Materials and Methods: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6- GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy. Results: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFPstromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow. Conclusion: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression. [ABSTRACT FROM AUTHOR]

β-glucosidase is a key component of cellulase for its function in hydrolyzing cellobiose to glucose in the final step of cellulose degradation. The high-level expression of β-glucosidase is essential for cellulose conversion. Aspergillus niger ATCC 20611 has the potential for efficient protein expression because of its ability to secret enzymes for the industrial production of fructooligosaccharides, but it lacks robust promoters for high-level protein expression. Here, the development of A. niger 20611 as a powerful protein expression system exploited the conserved constitutive promoter Pgpd1 of the glyceraldehyde-3-phosphate dehydrogenase-encoding gene from Trichoerma reesei to drive the expression of the enhanced green fluorescent protein in A. niger ATCC 20611. The mycelium of the transformant AGE9 exhibited intense fluorescence. Then, the promotor Pgpd1 was used to drive the expression of β-glucosidase and the enzyme activity of transformants AGB1 and AGB33 were 1.02 and 0.51 U/mL, respectively. These results demonstrate that the promotor Pgpd1 from T. reesei was applicable for A. niger ATCC 20611. Furthermore, the T. reesei-specific robust promoter Pcdna1 was used to drive the expression of β-glucosidase. The β-glucosidase exhibited a high-level expression with a yield of 15.2 U/mL, which was over 13.9 times higher than that driven by the promoter Pgpd1. The β-glucosidase was thermally stable and accounted for 85% of the total extracellular proteins. Subsequently, the fermentation broth including β-glucosidase was directly added to the cellulase mixture of T. reesei for saccharification of the acid-treated corncob residues and the delignified corncob residues, which increased the saccharification efficiency by 26.21% and 29.51%, respectively. Thus, β-glucosidase exhibited a high level of expression in A. niger ATCC 20611 and enhanced cellulose degradation by addition in vitro. In addition, the robust promoter Pcdna1 of T. reesei could drive the high-level expression of protein in A. niger ATCC 20611. These results demonstrate that the promoters in filamentous fungi could be employed across species in A. niger ATCC 20611 and further facilitated the efficient expression of β-glucosidase to optimize cellulases for efficient cellulose transformation. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved ATG4 cysteine proteases regulate macroautophagy/autophagy through the priming and deconjugation of the Atg8-family proteins. In mammals there are four ATG4 family members (ATG4A, ATG4B, ATG4C, ATG4D) but ATG4D has been relatively understudied. Heightened interest in ATG4D has been stimulated by recent links to human disease. Notably, genetic variations in human ATG4D were implicated in a heritable neurodevelopmental disorder. Genetic analyses in dogs, along with loss-of-function zebrafish and mouse models, further support a neuroprotective role for ATG4D. Here we discuss the evidence connecting ATG4D to neurological diseases and other pathologies and summarize its roles in both autophagy-dependent and autophagy-independent cellular processes. Abbrevation: ATG: autophagy related; BafA1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; BH3: BCL2 homology region 3; CASP3: caspase 3; EV: extracellular vesicle; GABA: gamma aminobutyric acid; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; LIR: LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MYC: MYC proto-oncogene, bHLH transcription factor; PE: phosphatidylethanolamine; PS: phosphatidylserine; QKO: quadruple knockout; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel; SQSTM1: sequestosome 1. [ABSTRACT FROM AUTHOR]

Serological tests are critical tools in the fight against infectious disease. They detect antibodies produced during an adaptive immune response against a pathogen with an immunological reagent, whose antibody binding characteristics define the specificity and sensitivity of the assay. While pathogen proteins have conveniently served as reagents, their performance is limited by the natural grouping of specific and non-specific antibody binding sites, epitopes. An attractive solution is to build synthetic proteins that only contains pathogen-specific epitopes, which could theoretically reach 100% specificity. However, the genesis of de novo proteins remains a challenge. To address the uncertainty of producing a synthetic protein, we have repurposed the beta barrel of fluorescent proteins into a receptacle that can receive several epitope sequences without compromising its ability to be expressed. Here, two versions of a multiepitope protein were built using the receptacle that differ by their grouping of epitopes specific to the parasite Trypanosoma cruzi, the causative agent for Chagas disease. An evaluation of their performance as the capture reagent in ELISAs showed near-complete agreement with recommended diagnostic protocols. The results suggest that a single assay could be developed for the diagnosis of Chagas disease and that this approach could be applied to other diseases. [ABSTRACT FROM AUTHOR]

Transient gene expression is an approach used to study transient genes across various species, with infiltration by Agrobacterium tumefaciens (agroinfiltration) being a commonly used method. Agroinfiltration offers a simple and effective means of delivering transgenes into the plant genome. An alternative method for enhancing the quality and productivity of orchids as ornamental plants is genetic modification through agroinfiltration. Although Agrobacterium‐mediated genetic transformation by immersion has been used on the Phalaenopsis amabilis (L.) Blume species of orchid, transformation efficiency using the immersion technique remains relatively low and the method itself is challenging due to its requirement for aseptic handling. The application of agroinfiltration in P. amabilis has not previously been reported. This study investigates the impact of the injection site, acetosyringone concentration, bacterial density (OD600), and injection volume to determine the optimum conditions for agroinfiltration on P. amabilis. The results demonstrated that injection site had a noticeably distinct impact on transformation effectiveness, with the abaxial position of the leaf being the optimal site for Agrobacterium culture suspension injection. While adjustments in acetosyringone concentration, bacterial density (OD600), and injection volume did not significantly affect transformation efficiency, they did influence the peak time of GFP fluorescence. Acetosyringone at a concentration of 200 µM, an OD600 of 1.0 for Agrobacterium culture, and an injection volume of 500 µL effectively accelerated GFP expression duration.

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48–72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species. [ABSTRACT FROM AUTHOR]

Realistic and modifiable infection models are required to study the pathogenesis of amphibian chytridiomycosis. Understanding the mechanism by which Batrachochytrium dendrobatidis (Bd) can infect and kill diverse amphibians is key to mitigating this pathogen and preventing further loss of biodiversity. In vitro studies of Bd typically rely on a tryptone based growth media, whereas the recent development of a kidney cell-line infection model has provided a more realistic alternative, without the need for live animals. Here we use expression of a fluorescent reporter to enhance the in vitro cell-line based growth assay, and show that transformed Bd cells are able to invade and grow in an amphibian kidney epithelial cell line (A6) as well as in a new system using a lung fibroblast cell line (DWJ). Both Bd and host cells were modified to express reporter fluorescent proteins, enabling immediate and continuous observation of the infection process without the need for destructive sampling for fixation and staining. Plasmid DNA conferring hygromycin resistance and TdTomato (RFP) expression was delivered to Bd zoospores via electroporation, and continuous antibiotic selection after recovery produced stable fluorescent Bd transformants. Host cells (A6 and DWJ) were transfected before each assay using lipofection to deliver plasmid DNA conferring green fluorescent protein (GFP) and containing an empty shRNA expression cassette. Bd RFP expression allowed easy localisation of fungal cells and identification of endobiotic growth was assisted by host GFP expression, by allowing visualization of the space in the host cell occupied by the invading fungal body. In addition to enabling enhanced live imaging, these methods will facilitate future genetic modification and characterisation of specific genes and their effect on Bd virulence. [ABSTRACT FROM AUTHOR]

Hydrogels are useful drug release systems and tissue engineering scaffolds. However, synthetic hydrogels often require harsh gelation conditions and can contain toxic by‐products while naturally derived hydrogels can transmit pathogens and in general have poor mechanical properties. Thus, there is a need for a hydrogel that forms under ambient conditions, is non‐toxic, xeno‐free, and has good mechanical properties. A recombinant spider silk protein‐derived hydrogel that rapidly forms at 37 °C is recently developed. The temperature and gelation times are well‐suited for an injectable in situ polymerising hydrogel, as well as a 3D cell culture scaffold. Here, it is shown that the diffusion rate and the mechanical properties can be tuned by changing the protein concentration and that human fetal mesenchymal stem cells encapsulated in the hydrogels show high survival and viability. Furthermore, mixtures of recombinant spider silk proteins and green fluorescent protein (GFP) form gels from which functional GFP is gradually released, indicating that bioactive molecules are easily included in the gels, maintain activity and can diffuse through the gel. Interestingly, encapsulated ARPE‐19 cells are viable and continuously produce the growth factor progranulin, which is detected in the cell culture medium over the study period of 31 days. [ABSTRACT FROM AUTHOR]

While protein aggregation is a hallmark of many neurodegenerative diseases, acquiring structural information on protein aggregates inside live cells remains challenging. Traditional microscopy does not provide structural information on protein systems. Routinely used fluorescent protein tags, such as Green Fluorescent Protein (GFP), might perturb native structures. Here, we report a counter‐propagating mid‐infrared photothermal imaging approach enabling mapping of secondary structure of protein aggregates in live cells modeling Huntington's disease. By comparing mid‐infrared photothermal spectra of label‐free and GFP‐tagged huntingtin inclusions, we demonstrate that GFP fusions indeed perturb the secondary structure of aggregates. By implementing spectra with small spatial step for dissecting spectral features within sub‐micrometer distances, we reveal that huntingtin inclusions partition into a β‐sheet‐rich core and a ɑ‐helix‐rich shell. We further demonstrate that this structural partition exists only in cells with the [RNQ+] prion state, while [rnq−] cells only carry smaller β‐rich non‐toxic aggregates. Collectively, our methodology has the potential to unveil detailed structural information on protein assemblies in live cells, enabling high‐throughput structural screenings of macromolecular assemblies. [ABSTRACT FROM AUTHOR]

Summary The manipulation of multiple transcription units for simultaneous and coordinated expression is not only key to building complex genetic circuits to accomplish diverse functions in synthetic biology, but is also important in crop breeding for significantly improved productivity and overall performance. However, building constructs with multiple independent transcription units for fine‐tuned and coordinated regulation is complicated and time‐consuming. Here, we introduce the Multiplex Expression Cassette Assembly (MECA) method, which modifies canonical vectors compatible with Golden Gate Assembly, and then uses them to produce multi‐cassette constructs. By embedding the junction syntax in primers that are used to amplify functional elements, MECA is able to make complex constructs using only one intermediate vector and one destination vector via two rounds of one‐pot Golden Gate assembly reactions, without the need for dedicated vectors and a coherent library of standardized modules. As a proof‐of‐concept, we modified eukaryotic and prokaryotic expression vectors to generate constructs for transient expression of green fluorescent protein and β‐glucuronidase in Nicotiana benthamiana, genome editing to block monoterpene metabolism in tomato glandular trichomes, production of betanin in tobacco and synthesis of β‐carotene in Escherichia coli. Additionally, we engineered the stable production of thymol and carvacrol, bioactive compounds from Lamiaceae family plants, in glandular trichomes of tobacco. These results demonstrate that MECA is a flexible, efficient and versatile method for building complex genetic circuits, which will not only play a critical role in plant synthetic biology, but also facilitate improving agronomic traits and pyramiding traits for the development of next‐generation elite crops. [ABSTRACT FROM AUTHOR]

The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon, and a fusion gene expression vector of insulin combined with green fluorescent protein (GFP) was constructed. The optimization of the flax callus culturing was undertaken, and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved. The critical concentration values of hygromycin on the flax hypocotyl development, as well as on its differentiated callus, were explored by the method of antibiotic gradient addition, and the application of antibiotic screening for the verification of positive calluses was assessed. The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed, as confirmed through polymerase chain reaction and Western blotting. In conclusion, we have established a flax callus culture system suitable for insulin expression. By optimizing the conditions of the flax callus induction, transformation, screening, and verification of a transgenic callus, we have provided an effective way to obtain insulin. Moreover, the herein-employed flax callus culture system could provide a feasible, cheap, and environmentally friendly platform for producing bioactive proteins. [ABSTRACT FROM AUTHOR]