Establishment of an Agrobacterium tumefaciens-Mediated Transformation System for Hirsutella sinensis.

Ophiocordyceps sinensis (Berk.) is a complex is formed by Hepialidae larvae and Hirsutella sinensis. Infestation by H. sinensis, interaction with host larvae, and fruiting body development are three crucial processes affecting the formation of O. sinensis. However, research on the molecular mechanism of O. sinensis formation has been hindered by the lack of effective genetic transformation protocols. Therefore, Agrobacterium tumefaciens-mediated transformation (ATMT) was adopted to genetically transform two H. sinensis strains and optimize the transformation conditions. The results revealed that the most suitable Agrobacterium strain for H. sinensis transformation was AGL1, and that the surfactant Triton X-100 could also induce ATMT, although less effectively than acetosyringone (AS). In addition, the endogenous promoters of H. sinensis genes had a stronger ability to drive the expression of the target gene than did the exogenous promoter. The optimal transformation conditions were as follows: AS and hygromycin B concentrations of 100 µM and 50 µg/mL, respectively; A. tumefaciens OD600 of 0.4; cocultivation at 18 °C for 24 h; and H. sinensis used within three passages. The results lay a foundation for the functional study of key regulatory genes involved in the formation of O. sinensis.

First site-specific conjugation method for native goat IgG antibodies via glycan remodeling at the conserved Fc region.

Despite their triumph in treating human diseases, antibody therapies for animals have gained momentum more slowly. However, the first approvals of animal antibodies for osteoarthritic pain in cats and dogs may herald the dawn of a new era. For example, goats are vital to economies around the world for their milk, meat, and hide products. It is therefore imperative to develop therapies to safeguard goats-with antibodies at the forefront. Goat antibodies will be crucial in the development of therapeutic antibodies, for example, as tracers to study antibody distribution in vivo, reagents to develop other therapeutic antibodies, and therapeutic agents themselves (e.g., antibody-drug conjugates). Hamstringing this effort is a still-burgeoning understanding of goat antibodies and their derivatization. Historically, goat antibody conjugates were generated through stochastic chemical modifications, producing numerous attachment sites and modification ratios, thereby deleteriously impacting antigen binding. Site-specific methods exist but often require substantial engineering and have not been demonstrated with goat antibodies. Nevertheless, we present herein a novel method to site-specifically conjugate native goat antibodies: chemo-enzymatic remodeling of the native Fc N-glycan introduces a reactive azide handle, after which click chemistry with strained alkyne partners affords homogeneous conjugates labeled only on the Fc domain. This process is robust, and resulting conjugates retain their antigen binding and specificity. To our knowledge, our report is the first for site-specific conjugation of native goat antibodies. Furthermore, our approach should be applicable to other animal antibodies-even with limited structural information-with similar success.

Parasitism of Hirsutella rhossiliensis on Different Nematodes and Its Endophytism Promoting Plant Growth and Resistance against Root-Knot Nematodes.

The endoparasitic fungus Hirsutella rhossiliensis is an important biocontrol agent of cyst nematodes in nature. To determine the potential parasitism of the fungus on a non-natural host, the pinewood nematode (Bursaphelenchus xylophilus) living in pine trees and the endophytic ability of the fungus on plants, in this paper, we first constructed and utilized a green fluorescent protein (GFP)-tagged H. rhossiliensis HR02 transformant to observe the fungal infection process on B. xylophilus and its colonization on Arabidopsis roots. Then, we compared the fungal parasitism on three species of nematodes with different lifestyles, and we found that the fungal parasitism is correlated with nematode species and stages. The parasitic effect of H. rhossiliensis on adults of B. xylophilus is similar to that on second-stage juveniles (J2) of the root-knot nematode Meloidogyne incognita after 24 h of inoculation, although the virulence of the fungus to second-stage juveniles of M. incognita is stronger than that to those of B. xylophilus and Caenorhabditis elegans. Moreover, the endophytism of H. rhossiliensis was confirmed. By applying an appropriate concentration of H. rhossiliensis conidial suspension (5 × 106 spores/mL) in rhizosphere soil, it was found that the endophytic fungus can promote A. thaliana growth and reproduction, as well as improve host resistance against M. incognita. Our results provide a deeper understanding of the fungus H. rhossiliensis as a promising biocontrol agent against plant-parasitic nematodes.

Tuning Electrostatic Interactions To Control Orientation of GFP Protein Adsorption on Silica Surface.

The adsorption of green fluorescent protein (GFP) on silica surfaces has been the subject of growing interest due to its potential applications in various fields, including biotechnology and biomedicine. In this study, we used all-atom molecular dynamics simulations to investigate the charge-driven adsorption of wild type GFP and its supercharged variants on silica surfaces. The results showed that the positively charged variant of GFP adsorbed on the negatively charged silica surface with minimal loss in its secondary structure. Further studies were conducted to understand the role of surface charge distribution on two other positively charged variants of GFP, and the results showed that the orientation of GFP on silica can be easily tuned by careful mutations of the charged amino acid residues on the GFP. This study provides valuable molecular insights into the role of electrostatic-driven adsorption of GFP and highlights the importance of charge interactions in the adsorption process.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Efficient Agrobacterium-mediated transformation and genome editing of Fagopyrum tataricum.

Fagopyrum tataricum (L.) Gaertn. is an exceptional crop known for its remarkable health benefits, high levels of beneficial polyphenols and gluten-free properties, making it highly sought-after as a functional food. Its self-fertilisation capability and adaptability to challenging environments further contribute to its potential as a sustainable agricultural option. To harness its unique traits, genetic transformation in F. tataricum is crucial. In this study, we optimised the Agrobacterium-mediated transformation protocol for F. tataricum callus, resulting in a transformation rate of regenerated plants of approximately 20%. The protocol's effectiveness was confirmed through successful GUS staining, GFP expression, and the generation of albino plants via FtPDS gene inactivation. These results validate the feasibility of genetic manipulation and highlight the potential for trait enhancement in F. tataricum.

NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms.

(1) Background: Recently, we showed aberrant nuclear/cytoplasmic boundaries/activity-dependent neuroprotective protein (ADNP) distribution in ADNP-mutated cells. This malformation was corrected upon neuronal differentiation by the ADNP-derived fragment drug candidate NAP (davunetide). Here, we investigated the mechanism of NAP nuclear protection. (2) Methods: CRISPR/Cas9 DNA-editing established N1E-115 neuroblastoma cell lines that express two different green fluorescent proteins (GFPs)-labeled mutated ADNP variants (p.Tyr718* and p.Ser403*). Cells were exposed to NAP conjugated to Cy5, followed by live imaging. Cells were further characterized using quantitative morphology/immunocytochemistry/RNA and protein quantifications. (3) Results: NAP rapidly distributed in the cytoplasm and was also seen in the nucleus. Furthermore, reduced microtubule content was observed in the ADNP-mutated cell lines. In parallel, disrupting microtubules by zinc or nocodazole intoxication mimicked ADNP mutation phenotypes and resulted in aberrant nuclear-cytoplasmic boundaries, which were rapidly corrected by NAP treatment. No NAP effects were noted on ADNP levels. Ketamine, used as a control, was ineffective, but both NAP and ketamine exhibited direct interactions with ADNP, as observed via in silico docking. (4) Conclusions: Through a microtubule-linked mechanism, NAP rapidly localized to the cytoplasmic and nuclear compartments, ameliorating mutated ADNP-related deficiencies. These novel findings explain previously published gene expression results and broaden NAP (davunetide) utilization in research and clinical development.

An Undergraduate Laboratory Series Using C. elegans That Prepares Students for Independent Inquiry.

Undergraduate neuroscience laboratories provide valuable opportunities for students to learn about neurobiological systems through active learning. Caenorhabditis elegans (C. elegans) is a valuable model for teaching students how to use a reductionist approach to neuroscientific inquiry. This series of lab modules trains students to utilize foundational laboratory techniques such as worm handling and maintenance, fluorescence imaging, behavioral assays, and Western blot. Upon completing this series of laboratory exercises, students are well prepared to engage in independent research projects using these research techniques. As supported by student survey results, this series of C. elegans laboratory exercises leads to the development of essential research skills, which students may be able to apply to a wide range of future scientific endeavors.

Remyelination and Ageing: Ethical Considerations of Using Surgically Joined Animals in Research.

Remyelination is a key repair process that ensures neurons remain protected following injury. This process is mediated by remyelinating oligodendrocytes in vertebrates, however, similarly to other neurobiological processes, the rate and efficiency of remyelination decreases across age and under pathological conditions. This has largely been attributed to two main contributors: 1) decreased exogenous signals supporting remyelination; and 2) aging of precursor cells that no longer differentiate into remyelinating oligodendrocytes. Here we discuss a key paper by Ruckh et al. (2012) who presented novel evidence that exposure to soluble bloodstream factors of young mice significantly rescues remyelination in old mice following a demyelinating insult. In this paper, a parabiosis approach was used where young and old mice were surgically joined for three weeks before and then left as a pair throughout the experiment. Ruckh and colleagues also offer novel insight into the role played by immune system cells, specifically macrophages, in clearance of myelin debris, a further contributor to remyelination. This paper is a good tool to expose undergraduate neuroscience students to basic molecular processes underlying conduction and transmission, helping them link cellular and network components. It also offers a platform for introducing the practicalities of in vivo research and debating ethical controversies that arise in animal research.

Systemic Colonization of Xanthomonas fragariae Strain YL19 Causing Dry Cavity Rot of Strawberry Crown Tissue in China.

Xanthomonas fragariae usually causes angular leaf spot (ALS) of strawberry, a serious bacterial disease in many strawberry-producing regions worldwide. Recently, a new strain of X. fragariae (YL19) was isolated from strawberry in China and has been shown to cause dry cavity rot in strawberry crown. In this study, we constructed a green fluorescent protein (GFP)-labeled Xf YL19 (YL19-GFP) to visualize the infection process and pathogen colonization in strawberries. Foliar inoculation of YL19-GFP resulted in the pathogen migrating from the leaves to the crown, whereas dip inoculation of wounded crowns or roots resulted in the migration of bacteria from the crowns or roots to the leaves. These two invasion types both resulted in the systematic spread of YL19-GFP, but inoculation of a wounded crown was more harmful to the strawberry plant than foliar inoculation. Results increased our understanding of the systemic invasion of X. fragariae, and the resultant crown cavity caused by Xf YL19.

A Novel Fluorescence-Based Screen of Gene Editing Molecules for Junctional Epidermolysis Bullosa.

Junctional epidermolysis bullosa (JEB) is a severe blistering skin disease caused by mutations in genes encoding structural proteins essential for skin integrity. In this study, we developed a cell line suitable for gene expression studies of the JEB-associated COL17A1 encoding type XVII collagen (C17), a transmembrane protein involved in connecting basal keratinocytes to the underlying dermis of the skin. Using the CRISPR/Cas9 system of Streptococcus pyogenes we fused the coding sequence of GFP to COL17A1 leading to the constitutive expression of GFP-C17 fusion proteins under the control of the endogenous promoter in human wild-type and JEB keratinocytes. We confirmed the accurate full-length expression and localization of GFP-C17 to the plasma membrane via fluorescence microscopy and Western blot analysis. As expected, the expression of GFP-C17mut fusion proteins in JEB keratinocytes generated no specific GFP signal. However, the CRISPR/Cas9-mediated repair of a JEB-associated frameshift mutation in GFP-COL17A1mut-expressing JEB cells led to the restoration of GFP-C17, apparent in the full-length expression of the fusion protein, its accurate localization within the plasma membrane of keratinocyte monolayers as well as within the basement membrane zone of 3D-skin equivalents. Thus, this fluorescence-based JEB cell line provides the potential to serve as a platform to screen for personalized gene editing molecules and applications in vitro and in appropriate animal models in vivo.

Systemic Colonization of Potato Plants by Verticillium dahliae Leads to Infection of Tubers and Sprouting Buds.

A green fluorescent protein (GFP)-tagged isolate of Verticillium dahliae was used to study its colonization in potato plants and tubers. Three-week-old potato plants of the highly susceptible cultivar 'Shepody' were inoculated with a conidial suspension of a GFP-tagged isolate of V. dahliae using a wound inoculation method. Colonization was studied using confocal microscopy combined with tissue sections. Conidia germinated and hyphae grew along the root hairs, elongation zones, and root caps between 24 and 96 h postinoculation (HPI). At 7 days postinoculation (DPI), the pathogen advanced to cortical tissues and grew into the root vascular bundles. At 8 weeks postinoculation (WPI), the stem epidermal cells, cortical tissues, vascular elements, and petioles were fully colonized by the mycelium of V. dahliae. At 11 WPI, the pathogen was detected in the stolon and progeny tubers, as confirmed by both GFP signals in tissues and reisolation of the pathogen on the semiselective NP-10 medium. Progeny potato tubers were harvested from the inoculated potato plants, and the GFP-signal was observed in the epidermal cells and vascular elements of sprouting buds that emerged from the harvested tubers. The infection rate of progeny tubers detected on semiselective NP-10 medium ranged from 34.55 to 55.56%, with an average of 45.31%. In conclusion, we report, for the first time, the entire progression of colonization by V. dahliae in potato plant tissues, progeny tubers, as well as of the sprouting buds that emerged from progeny tubers.

Distinct Impairments Characterizing Different ADNP Mutants Reveal Aberrant Cytoplasmic-Nuclear Crosstalk.

(1) Background: Activity-dependent neuroprotective protein (ADNP) is essential for neuronal structure and function. Multiple de novo pathological mutations in ADNP cause the autistic ADNP syndrome, and they have been further suggested to affect Alzheimer's disease progression in a somatic form. Here, we asked if different ADNP mutations produce specific neuronal-like phenotypes toward better understanding and personalized medicine. (2) Methods: We employed CRISPR/Cas9 genome editing in N1E-115 neuroblastoma cells to form neuron-like cell lines expressing ADNP mutant proteins conjugated to GFP. These new cell lines were characterized by quantitative morphology, immunocytochemistry and live cell imaging. (3) Results: Our novel cell lines, constitutively expressing GFP-ADNP p.Pro403 (p.Ser404* human orthologue) and GFP-ADNP p.Tyr718* (p.Tyr719* human orthologue), revealed new and distinct phenotypes. Increased neurite numbers (day 1, in culture) and increased neurite lengths upon differentiation (day 7, in culture) were linked with p.Pro403*. In contrast, p.Tyr718* decreased cell numbers (day 1). These discrete phenotypes were associated with an increased expression of both mutant proteins in the cytoplasm. Reduced nuclear/cytoplasmic boundaries were observed in the p.Tyr718* ADNP-mutant line, with this malformation being corrected by the ADNP-derived fragment drug candidate NAP. (4) Conclusions: Distinct impairments characterize different ADNP mutants and reveal aberrant cytoplasmic-nuclear crosstalk.

3D bioprinting of in situ vascularized tissue engineered bone for repairing large segmental bone defects.

Large bone defects remain an unsolved clinical challenge because of the lack of effective vascularization in newly formed bone tissue. 3D bioprinting is a fabrication technology with the potential to create vascularized bone grafts with biological activity for repairing bone defects. In this study, vascular endothelial cells laden with thermosensitive bio-ink were bioprinted in situ on the inner surfaces of interconnected tubular channels of bone mesenchymal stem cell-laden 3D-bioprinted scaffolds. Endothelial cells exhibited a more uniform distribution and greater seeding efficiency throughout the channels. In vitro, the in situ bioprinted endothelial cells can form a vascular network through proliferation and migration. The in situ vascularized tissue-engineered bone also resulted in a coupling effect between angiogenesis and osteogenesis. Moreover, RNA sequencing analysis revealed that the expression of genes related to osteogenesis and angiogenesis is upregulated in biological processes. The in vivo 3D-bioprinted in situ vascularized scaffolds exhibited excellent performance in promoting new bone formation in rat calvarial critical-sized defect models. Consequently, in situ vascularized tissue-engineered bones constructed using 3D bioprinting technology have a potential of being used as bone grafts for repairing large bone defects, with a possible clinical application in the future.

Effects of currently used marine antifouling paint biocides on green fluorescent proteins in Anemonia viridis.

Some of the antifouling booster biocides affects the marine ecosystem negatively. The booster biocides that are resistant to degradation are accumulated in the sediment of the oceans. One of the sedentary organisms in the Mediterranean Sea is Anemonia viridis. This study aims at showing the toxicities of common biocides such as irgarol, seanine-211, zinc omadine, and acticide on the fluorescence by GFPs of A. viridis. The decreases in the fluorescence intensities of the GFP were measured within different booster biocide concentrations. The results show that fluorescent intensities of GFP proteins decrease more than 50% when they are exposed to different concentrations of irgarol, zinc omadine, acticide. In conclusion, ecosystem health should be prioritized when new antifouling paint compositions are proposed. From the results, it seems that A. viridis can be considered as a vulnerable organism and it is sensitive to booster biocides within self-polishing antifouling paint formulations.

Revealing heterogeneity in correlation times of EGFP encapsulated in complex coacervate core micelles by analysis of fluorescence anisotropies.

Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores.

Design of a Toolbox of RNA Thermometers.

RNA thermometers are RNA regulatory elements that convert temperature into a functional biological response through a temperature-induced conformational change. These regulatory elements have been investigated in numerous natural contexts and have been designed for synthetic biology as well. A basic challenge has been the design of an RNA thermometer whose final activity in response to temperature matches a prespecified response, in terms of its sensitivity, threshold, and leakiness. This chapter provides a methodology for the design of a toolbox of RNA thermometers. We describe considerations for the conceptual design, a computational assessment, and strategies for experimental synthesis and measurement.

Creation of Markerless Genome Modifications in a Nonmodel Bacterium by Fluorescence-Aided Recombineering.

Metabolic engineering of nonmodel bacteria is often challenging because of the paucity of genetic tools for iterative genome modification necessary to equip bacteria with pathways to produce high-value products. Here, we outline a homologous recombination-based method developed to delete or add genes to the genome of a nonmodel bacterium, Zymomonas mobilis, at the desired locus using a suicide plasmid that contains gfp as a fluorescence marker to track its presence in cells. The suicide plasmid is engineered to contain two 500 bp regions homologous to the DNA sequence immediately flanking the target locus. A single crossover event at one of the two homologous regions facilitates insertion of the plasmid into the genome and subsequent homologous recombination events excise the plasmid from the genome, leaving either the original genotype or the desired modified genotype. A key feature of this plasmid is that Green Fluorescent Protein (GFP) expressed from the suicide plasmid allows easy identification and sorting of cells that have lost the plasmid by use of a fluorescence activated cell sorter. Subsequent PCR amplification of genomic DNA from strains lacking GFP allows rapid identification of the desired genotype, which is confirmed by DNA sequencing. This method provides an efficient and flexible platform for improved genetic engineering of Z. mobilis, which can be easily adapted to other nonmodel bacteria.

Protoplast Isolation, Transfection, and Gene Editing for Soybean (Glycine max ).

Protoplast is a versatile system for conducting cell-based assays, analyzing diverse signaling pathways, studying functions of cellular machineries, and functional genomics screening. Protoplast engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. This system allows the direct delivery of DNA, RNA, or proteins into plant cells and provides a high-throughput system to validate gene-editing reagents. It also facilitates the delivery of homology-directed repair templates (donor molecules) into plant cells, enabling precise DNA edits in the genome. There is a great deal of interest in the plant community to develop these precise edits, as they may expand the potential for developing value-added traits which may be difficult to achieve by other gene-editing applications and/or traditional breeding alone. This chapter provides improved working protocols for isolating and transforming protoplast from immature soybean seeds with 44% of transfection efficiency validated by the green fluorescent protein reporter. We also describe a method for gene editing in soybean protoplasts using single guide RNA molecules.

Assessment of Intracellular GTP Levels Using Genetically Encoded Fluorescent Sensors.

Changes in intracellular GTP levels, even incremental ones, profoundly affect the activity of several GTP-binding proteins ultimately resulting in alteration of several basal cellular phenotypes including cell motility, invasion, and tumorigenesis. However, until recently, no tools were available for GTP quantification in live cells. Therefore, in the current chapter, we describe the methodology for the quantitative assessment of spatiotemporal changes in GTP levels in the cells using genetically encoded fluorescent ratiometric GTP sensors termed GEVALs for GTP evaluators.