RESUMEN

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

Asunto(s)

Recombinasas , Animales , Recombinasas/metabolismo , Recombinasas/genética , Mosaicismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Expresión Génica/genética , Proteína Fluorescente Roja , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos

RESUMEN

Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To address this, a new and efficient method using AAV vectors has been developed to simplify the conversion of systems based on Cre, FLP, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, FLP (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as delivery vectors. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversion, representing a significant improvement in genome engineering techniques.

Asunto(s)

Alelos , Dependovirus , Vectores Genéticos , Animales , Dependovirus/genética , Vectores Genéticos/genética , Ratones , Conversión Génica , Blastocisto/metabolismo , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Recombinación Genética

RESUMEN

Aiming at intramolecular frustrated Lewis pairs (FLPs) based on soft Lewis acidic bismuth centers, a phosphine function was combined with a dichloridobismuthane unit on a phenylene backbone utilizing a scrambling approach. The reaction between two equivalents of BiCl3 and (o-(Ph2P)C6H4)3Bi yielded (o-(Ph2P)C6H4)BiCl2(THF), the structure of which indicated Bi…P interactions and thus a pronounced Lewis acidity at the bismuth center that was confirmed by the Gutmann-Beckett method. However, the system turned out to be insufficient to be utilized for FLP reactivity. Hence, the chloride ligands were exchanged by iodide and C2F5 substituents, respectively. Despite a lower electronegativity the iodide compound exhibits a shorter Bi…P contact, while the C2F5 substituents led to a further decrease of the Lewis acidity, despite their high group electronegativity. DFT calculations rationalized this by a quenching of the Lewis acidity inherent to the σ*(Bi-C) orbital by negative hyperconjugation from occupied p-orbitals at the F atoms. Furthermore, it turned out that the strength of the covalent Bi-X σ-bond is a more important factor than the charge at Bi in determining the energetic accessibility and thus Lewis acidity of the antibonding σ*(Bi-C) orbital.

RESUMEN

CONTEXT: In silico study investigates the activation of sulfur dioxide by newly designed frustrated Lewis pairs, i.e., [P(tBu)3…B(C2NBSHF2)3], where the Lewis acid part is a super Lewis acid. The activation process involves the making of P-S and B-O bonds, leading to the formation of an FLP-SO2 adduct. The calculated results demonstrate that the activation of SO2 by the FLP is almost barrierless and exothermic. Exploration of the impact of the solvent environment on the feasibility and energetics of the reaction has been investigated. The exothermicity is increasing in nonpolar solvents. METHODS: This study focuses on understanding the electronic activity of SO2 activation by FLP with the help of the Minnesota 06 functional, M06-2X (global hybrid functional with 54% HF exchange) along with Pople's basis set, 6-311G (d, p). Principal interacting orbital and extended transition state-natural orbitals for chemical valence studies, giving impactful insight into the favorable orbital interaction and electron transfer in this reaction. Furthermore, useful CDFT descriptors such as reaction force constant and reaction electronic flux profiles along the intrinsic reaction coordinate give insights into the synchronicity and total electronic activity of the reaction.

RESUMEN

KEY MESSAGE: The study demonstrates the successful management of Meloidogyne incognita in eggplant using Mi-flp14 RNA interference, showing reduced nematode penetration and reproduction without off-target effects across multiple generations. Root-knot nematode, Meloidogyne incognita, causes huge yield losses worldwide. Neuromotor function in M. incognita governed by 19 neuropeptides is vital for parasitism and parasite biology. The present study establishes the utility of Mi-flp14 for managing M. incognita in eggplant in continuation of our earlier proof of concept in tobacco (US patent US2015/0361445A1). Mi-flp14 hairpin RNA construct was used for generating 19 independent transgenic eggplant events. PCR and Southern hybridization analysis confirmed transgene integration and its orientation, while RT-qPCR and Northern hybridization established the generation of dsRNA and siRNA of Mi-flp14. In vitro and in vivo bio-efficacy analysis of single-copy events against M. incognita showed reduced nematode penetration and development at various intervals that negatively impacted reproduction. Interestingly, M. incognita preferred wild-type plants over the transgenics even when unbiased equal opportunity was provided for the infection. A significant reduction in disease parameters was observed in transgenic plants viz., galls (40-48%), females (40-50%), egg masses (35-40%), eggs/egg mass (50-55%), and derived multiplication factor (60-65%) compared to wild type. A unique demonstration of perturbed expression of Mi-flp14 in partially penetrated juveniles and female nematodes established successful host-mediated RNAi both at the time of penetration even before the nematodes started withdrawing plant nutrients and later stage, respectively. The absence of off-target effects in transgenic plants was supported by the normal growth phenotype of the plants and T-DNA integration loci. Stability in the bio-efficacy against M. incognita across T1- to T4-generation transgenic plants established the utility of silencing Mi-flp14 for nematode management. This study demonstrates the significance of targeting Mi-flp14 in eggplant for nematode management, particularly to address global agricultural challenges posed by M. incognita.

Asunto(s)

Enfermedades de las Plantas , Plantas Modificadas Genéticamente , Interferencia de ARN , Solanum melongena , Tylenchoidea , Animales , Tylenchoidea/patogenicidad , Tylenchoidea/fisiología , Solanum melongena/genética , Solanum melongena/parasitología , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/prevención & control , Interacciones Huésped-Parásitos/genética

RESUMEN

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or PhiC31 integrase mRNAs along with a compatible donor plasmid into zygotes derived from the seed mice. PITT and its subsequent version, improved PITT (i-PITT), overcome disadvantages of conventional Tg mice such as lack of consistent and reliable expression of the cassettes among different Tg mouse lines, and the PITT approach is superior in terms of cost and labor. One of the limitations of PITT, particularly using Cre-mRNA, is that the approach cannot be used for insertion of conditional expression cassettes using Cre-LoxP site-specific recombination. This is because the LoxP sites in the donor plasmids intended for achieving conditional expression of the transgene will interfere with the PITT recombination reaction with LoxP sites in the landing pad. RESULTS: To enable the i-PITT method to insert a conditional expression cassette, we modified the approach by simultaneously using PhiC31o and FLPo mRNAs. We demonstrate the strategy by creating a model containing a conditional expression cassette at the Rosa26 locus with an efficiency of 13.7%. We also demonstrate that inclusion of FLPo mRNA excludes the insertion of vector backbones in the founder mice. CONCLUSIONS: Simultaneous use of PhiC31 and FLP in i-PITT approach allows insertion of donor plasmids containing Cre-loxP-based conditional expression cassettes.

Asunto(s)

Genoma , Integrasas , Ratones Transgénicos , Animales , Ratones , Integrasas/genética , Integrasas/metabolismo , Transgenes , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , Plásmidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Mutagénesis Insercional

RESUMEN

The strong potential of platinum single atom (PtSA) in gas sensor technology is primarily attributed to its high atomic economy. Nevertheless, it is imperative to conduct further exploration to understand the impact of PtSA on the active sites. In this study, the evolution of PtSA on (100)CeO2 and (111)CeO2 is examined, revealing notable disparities in the position and activity of surface PtSA on different crystal planes. The PtSA in (100)CeO2 surface can enhance the stability of Ce3+ and construct a frustrated Lewis pair (FLP) to form a double active site by combining the steric hindrance effect of oxygen vacancies, which increases the response value from 1.8 to 27 and reduce the response-recovery time from 140-192 s to 25-26 s toward five ppm NO2 at room temperature. Conversely, PtSA tends to bind to terminal oxygen on the surface of (111)CeO2 and become an independent reaction site. The response value of PtSA-(111)CeO2 surface only increased from 1.6 to 3.8. This research underscores the correlation between single atoms and crystal plane effects, laying the groundwork for designing and synthesizing ultra-stable and efficient gas sensors.

RESUMEN

Due to the serious ecological problems caused by the high CO2 content in the atmosphere, reducing atmospheric CO2 has attracted widespread attention from academia and governments. Among the many ways to mitigate CO2 concentration, the capture and comprehensive utilization of CO2 through chemical methods have obvious advantages, whose key is to develop suitable adsorbents and catalysts. Frustrated Lewis pairs (FLPs) are known to bind CO2 through the interaction between unquenched Lewis acid sites/Lewis base sites with the O/C of CO2, simultaneously achieving CO2 capture and activation, which render FLP better potential for CO2 utilization. However, how to construct efficient FLP targeted for CO2 utilization and the mechanism of CO2 activation have not been systematically reported. This review firstly provides a comprehensive summary of the recent advances in the field of CO2 capture, activation, and transformation with the help of FLP, including the construction of homogeneous and heterogeneous FLPs, their interaction with CO2, reaction activity, and mechanism study. We also illustrated the challenges and opportunities faced in this field to shed light on the prospective research.

RESUMEN

Type IVc Pili (T4cP), also known as Tad or Flp pili, are long thin microbial filaments that are made up of small-sized pilins. These appendages serve different functions in bacteria, including attachment, biofilm formation, surface sensing, motility, and host colonization. Despite their relevant role in diverse microbial lifestyles, knowledge about T4cP in bacteria that establish symbiosis with legumes, collectively referred to as rhizobia, is still limited. Sinorhizobium meliloti contains two clusters of T4cP-related genes: flp-1 and flp-2, which are located on the chromosome and the pSymA megaplasmid, respectively. Bundle-forming pili associated with flp-1 are involved in the competitive nodulation of alfalfa plants, but the role of flp-2 remains elusive. In this work, we have performed a comprehensive bioinformatic analysis of T4cP genes in the highly competitive S. meliloti GR4 strain and investigated the role of its flp clusters in pilus biogenesis, motility, and in the interaction with alfalfa. Single and double flp-cluster mutants were constructed on the wild-type genetic background as well as in a flagellaless derivative strain. Our data demonstrate that both chromosomal and pSymA flp clusters are functional in pili biogenesis and contribute to surface translocation and nodule formation efficiency in GR4. In this strain, the presence of flp-1 in the absence of flp-2 reduces the competitiveness for nodule occupation.

RESUMEN

The nematode Caenorhabditis elegans lends itself as an excellent model organism for peptidomics studies. Its ease of cultivation and quick generation time make it suitable for high-throughput studies. The nervous system, with its 302 neurons, is probably the best-known and studied endocrine tissue. Moreover, its neuropeptidergic signaling pathways display numerous similarities with those observed in other metazoans. Here, we describe two label-free approaches for neuropeptidomics in C. elegans: one for discovery purposes, and another for targeted quantification and comparisons of neuropeptide levels between different samples. Starting from a detailed peptide extraction procedure, we here outline the liquid chromatography tandem mass spectrometry (LC-MS/MS) setup and describe subsequent data analysis approaches.

Asunto(s)

Nematodos , Neuropéptidos , Animales , Caenorhabditis elegans/metabolismo , Cromatografía Liquida , Secuencia de Aminoácidos , Espectrometría de Masas en Tándem , Neuropéptidos/metabolismo , Nematodos/metabolismo

RESUMEN

BACKGROUND: The strong connection between gut microbes and human health has been confirmed by an increasing number of studies. Although probiotics have been found to relieve ulcerative colitis, the mechanism varies by the species involved. In this study, the physiological, immune and pathological factors of mice were measured and shotgun metagenomic sequencing was conducted to investigate the potential mechanisms in preventing ulcerative colitis. RESULTS: The results demonstrated that ingestion of Lactobacillus fermentum GLF-217 and Lactobacillus plantarum FLP-215 significantly alleviated ulcerative colitis induced by dextran sulfate sodium (DSS), as evidenced by the increase in body weight, food intake, water intake and colon length as well as the decrease in disease activity index, histopathological score and inflammatory factor. Both strains not only improved intestinal mucosa by increasing mucin-2 and zonula occludens-1, but also improved the immune system response by elevating interleukin-10 levels and decreasing the levels of interleukin-1ß, interleukin-6, tumor necrosis factor-α and interferon-γ. Moreover, L. fermentum GLF-217 and L. plantarum FLP-215 play a role in preventing DSS-induced colitis by regulating the structure of gut microbiota and promoting the formation of short-chain fatty acids. CONCLUSIONS: This study may provide a reference for the prevention strategy of ulcerative colitis. © 2024 Society of Chemical Industry.

Asunto(s)

Colitis Ulcerosa , Microbioma Gastrointestinal , Lactobacillus plantarum , Limosilactobacillus fermentum , Probióticos , Animales , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/prevención & control , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/inmunología , Ratones , Probióticos/administración & dosificación , Probióticos/farmacología , Masculino , Humanos , Sulfato de Dextran/efectos adversos , Colon/microbiología , Colon/inmunología , Colon/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Ratones Endogámicos C57BL , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/inmunología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Mucina 2/metabolismo , Mucina 2/genética , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-6/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Interferón gamma/metabolismo , Interferón gamma/genética , Interferón gamma/inmunología , Proteína de la Zonula Occludens-1/metabolismo , Proteína de la Zonula Occludens-1/genética , Modelos Animales de Enfermedad

RESUMEN

CO2 reduction is appealing for the long-term production of high-value fuels and chemicals. Herein, using density functional theory (DFT) based calculations, we study the CO2 reduction pathway to formic acid using aluminum hydride and phosphine derivatives. Our primary focus is on aluminum hydride derivatives, aimed at improving the efficiency of the CO2 reduction process. Substituents with σ-donating properties at the aluminum center are discovered to lower the activation barriers. We demonstrate how di-tert-butylphosphine oxide (LB-O)/di-tert-butylphosphine sulfide (LB-S)/di-tert-butylphosphanimine (LB-N) work together with aluminum hydride to facilitate CO2 reduction process and generate in-situ frustrated Lewis pairs (FLPs), such as FLP-O, FLP-S, and FLP-N. The activation strain model (ASM) analysis reveals the significance of strain energy in determining activation barriers. EDA-NOCV and PIO analyses elucidate the orbital interactions at the corresponding transition states. Furthermore, the study delves into the activation of various small molecules, such as dihydrogen, acetylene, ethylene, carbon dioxide, nitrous oxide, and acetonitrile, using those in-situ generated FLPs. The study highlights the low activation barriers and emphasizes the potential for small molecule activation in this context.

RESUMEN

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Asunto(s)

Bombyx , Femenino , Animales , Masculino , Bombyx/genética , Proteínas Fluorescentes Verdes/genética , Semen , Animales Modificados Genéticamente , ADN , Células Germinativas

RESUMEN

Recent reports of radical formation within frustrated Lewis pairs (FLPs) suggested that single-electron transfer (SET) could play an important role in their chemistry especially for C-C coupling. In sharp contrast, our extensive dispersion-corrected DFT calculations show that although reactive benzhydryl radical along with phosphine radical cation species can be kinetically generated from bulky phosphines and benzhydryl cation, direct P-C hetero-coupling may lead to bulky phosphonium cation as reactive carbocation transfer reagents to styrene substrates, which is kinetically much more favorable than the recently proposed radical C-C coupling between benzhydryl radical and styrene. Similarly, meta-stable radical cation Mes3 P+ ⋅ salt is also kinetically accessible via SET reactions of Mes3 P and B(C6 F5 )3 with 0.5 equivalent of p-O2 C6 Cl4 .

RESUMEN

There are a number of reporter systems that are useful for gene expression analysis in bacteria. However, at least in Salmonella, a versatile and simple luciferase reporter system that can be integrated precisely behind a promoter or gene of interest on a chromosome is not currently available. The luciferase operon luxCDABE from Photorhabdus luminescens has several advantages, including brightness, wide linear range, absence in most bacteria, stability at high temperature, and no substrate addition required for the assay. Here, a conjugation-mediated site-specific single-copy luciferase fusion system is developed. A reporter plasmid containing the conditional replication origin R6Kgγ, FRT-luxCDABE, and KmR marker was designed to be incorporated into the FRT site behind the promoter or gene of interest on the chromosome in cells expressing FLP. However, when this reporter plasmid was electroporated directly into such a S. enterica strain, no colonies appeared, likely due to the low transformation efficiency of this relatively large plasmid DNA. Meanwhile, the same reporter plasmid was successfully introduced and launched as an insert of an FRT-containing conjugative transfer plasmid from a mating E. coli strain to the same recipient S. enterica strain, as well as Citrobacter koseri. RcsB-dependent inducible luminescence from the constructed wzc-luxCDABE strains was confirmed. This system is feasible for detecting very low levels of transcription, even in Gram-negative bacterial species that are relatively difficult to genetically manipulate.

RESUMEN

Diimino-carbene-supported germylone dimNHCGe does not react with BPh3 and does not activate dihydrogen in the FLP mode in the combination with this borane. However, it reacts with B(C6 F5 )3 to give the zwitterionic borate dimNHCGe-(C6 F4 )BF(C6 F5 )2 . This compound can be converted into the hydroborate dimNHCGe-(C6 F4 )BH(C6 F5 )2 (8) and further into [dimNHCGe-(C6 F4 )B(C6 F5 )2 ]+ (4). Compound 4 is a Ge/B analogue of Stephan's FLP parent P/B compound (C6 H2 Me3 )2 P-C6 F4 -B(C6 F5 )2 but unlike the latter cannot split dihydrogen. Moreover, attempts to prepare a Ge/B analogue of the zwitterion (C6 H2 Me3 )2 HP-C6 F4 -BH(C6 F5 )2 by protonation of borate 8 resulted in immediate elimination of H2.

RESUMEN

Corticotropin-releasing hormone expressing (CRH+) neurons are distributed throughout the brain and play a crucial role in shaping the stress responses. Mouse models expressing site-specific recombinases (SSRs) or reporter genes are important tools providing genetic access to defined cell types and have been widely used to address CRH+ neurons and connected brain circuits. Here, we investigated a recently generated CRH-FlpO driver line expanding the CRH system-related tool box. We directly compared it to a previously established and widely used CRH-Cre line with respect to the FlpO expression pattern and recombination efficiency. In the brain, FlpO mRNA distribution fully recapitulates the expression pattern of endogenous Crh. Combining both Crh locus driven SSRs driver lines with appropriate reporters revealed an overall coherence of respective spatial patterns of reporter gene activation validating CRH-FlpO mice as a valuable tool complementing existing CRH-Cre and reporter lines. However, a substantially lower number of reporter-expressing neurons was discerned in CRH-FlpO mice. Using an additional CRH reporter mouse line (CRH-Venus) and a mouse line allowing for conversion of Cre into FlpO activity (CAG-LSL-FlpO) in combination with intersectional and subtractive mouse genetic approaches, we were able to demonstrate that the reduced number of tdTomato reporter expressing CRH+ neurons can be ascribed to the lower recombination efficiency of FlpO compared to Cre recombinase. This discrepancy particularly manifests under conditions of low CRH expression and can be overcome by utilizing homozygous CRH-FlpO mice. These findings have direct experimental implications which have to be carefully considered when targeting CRH+ neurons using CRH-FlpO mice. However, the lower FlpO-dependent recombination efficiency also entails advantages as it provides a broader dynamic range of expression allowing for the visualization of cells showing stress-induced CRH expression which is not detectable in highly sensitive CRH-Cre mice as Cre-mediated recombination has largely been completed in all cells generally possessing the capacity to express CRH. These findings underscore the importance of a comprehensive evaluation of novel SSR driver lines prior to their application.

RESUMEN

The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C. elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-ß-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.

Asunto(s)

Caenorhabditis elegans , Neuronas , Animales , Músculo Liso , Transducción de Señal , Inmunidad Innata

RESUMEN

Tomato (Solanum lycopersicum) plants are susceptible to infection by root-knot nematodes, which cause severe economic losses. Planting resistant tomato plants can reduce nematode damage; however, the effects of resistant tomato root exudates in suppressing Meloidogyne incognita remain insufficiently understood. Here, we determined that the resistant tomato plant Lycopersicon esculentum cv. Xianke-8 (XK8) alleviates nematode damage by downregulating the expression of the essential parasitic nematode gene Mi-flp-18 to reduce the infection and reproduction of M. incognita. Using gas chromatography-mass spectrometry, we identified vanillin as a unique compound (compared to susceptible tomato cultivars) in XK8 root exudates that acts as a lethal trap and inhibitor of egg hatching. Moreover, the soil application of 0.4-4.0 mmol/kg vanillin significantly reduced galls and egg masses. The parasite gene Mi-flp-18 was downregulated upon treatment with vanillin, both in vitro and in pot experiments. Collectively, our results reveal an effective nematicidal compound that can use in feasible and economical strategies to control RKNs.

Asunto(s)

Solanum lycopersicum , Tylenchoidea , Animales , Exudados de Plantas/farmacología , Exudados de Plantas/química , Solanum lycopersicum/genética , Exudados y Transudados , Raíces de Plantas/genética

RESUMEN

Mammalian display enables the selection of biophysically favorable antibodies from a large IgG antibody library displayed on the plasma membrane of mammalian cells. We constructed and validated a novel mammalian display platform utilizing the commercially available Flp-In CHO cell line as a starting point. We introduced a single copy of a landing pad for Bxb1 integrase-driven recombinase-mediated cassette exchange into the FRT site of the Flp-In CHO line to facilitate the efficient single-copy integration of an antibody display cassette into the genome of the cell line. We then proceeded to demonstrate the ability of our platform to select biophysically favorable antibodies from a library of 1 × 106 displayed antibodies designed to improve the biophysical properties of bococizumab via randomization of problematic hydrophobic surface residues of the antibody. Enrichment of bococizumab variants via fluorescence-activated cell sorting selections was followed by next generation sequencing and thorough characterization of biophysical properties of 10 bococizumab variants that subsequently allowed attribution of the mutations to the biophysical properties of the antibody variants. The mammalian displayed variants exhibited reduced aggregation propensity and polyreactivity, while critically retaining its target binding thereby demonstrating the utility of this valuable tool.