Search Results (129)

Late-spring frost events severely damage low-chill peach blossoms, causing significant yield losses. Although 5-aminolevulinic acid (ALA) enhances cold tolerance through the PpC3H37-PpWRKY18 module, the regulatory mechanism of ALA on PpC3H37 remains to be elucidated. Using yeast one-hybrid screening with the PpC3H37 promoter as bait, we identified PpDof9 as a key interacting transcription factor. A genome-wide analysis revealed 25 PpDof genes in peaches (Prunus persica). These genes exhibited variable physicochemical properties, with most proteins predicted as nuclear-localized. Subcellular localization experiments in tobacco revealed that PpDof9 was localized to the nucleus, consistent with predictions. A synteny analysis indicated nine segmental duplication pairs and tandem duplications on chromosomes 5 and 6, suggesting duplication events drove family expansion. A conserved motif analysis confirmed universal presence of the Dof domain (Motif 1). Promoter cis-element screening identified low-temperature responsive (LTR) elements in 12 PpDofs, including PpDof1, PpDof8, PpDof9, and PpDof25. The quantitative real-time PCR (qRT-PCR) results showed that PpDof1, PpDof8, PpDof9, PpDof15, PpDof16, and PpDof25 were significantly upregulated under low-temperature stress, and this upregulation was further enhanced by ALA pretreatment. Our findings demonstrate ALA-mediated modulation of specific PpDof TFs in cold response and provide candidates (PpDof1, PpDof9, PpDof8, PpDof25) for enhancing floral frost tolerance in peaches. Full article

The innate immune response is an important defense against invading pathogens. Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. However, some viruses have evolved the ability to inhibit the function of STING and evade the host antiviral defenses. Understanding both the mechanism of action and the viruses targets of STING effector is important because of their importance to evade the host antiviral defenses. In this study, the STING (IpSTING) of Ictalurus punctatus was first identified and characterized. Subsequently, the yeast two-hybrid system (Y2HS) was used to screen for proteins from channel catfish virus (CCV, Ictalurid herpesvirus 1) that interact with IpSTING. The ORFs of the CCV were cloned into the pGBKT7 vector and expressed in the AH109 yeast strain. The bait protein expression was validated by autoactivation, and toxicity investigation compared with control (AH109 yeast strain transformed with empty pGBKT7 and pGADT7 vector). Two positive candidate proteins, ORF41 and ORF65, were identified through Y2HS screening as interacting with IpSTING. Their interactions were further validated using co-immunoprecipitation (Co-IP). This represented the first identification of interactions between IpSTING and the CCV proteins ORF41 and ORF65. The data advanced our understanding of the functions of ORF41 and ORF65 and suggested that they might contribute to the evasion of host antiviral defenses. However, the interaction mechanism between IpSTING, and CCV proteins ORF41 and ORF65 still needs to be further explored. Full article

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To address this, we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase, preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation, this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A, the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases. Full article

The control of gene expression by cis-regulatory DNA sequences is a conserved genomic feature. The maize booster1 gene (b1) is a naturally occurring locus that serves as a mechanistic model for the control of gene expression from a distal cis element and a form of allelic interactions called paramutation. Two epi-alleles of b1 produce distinct pigmentation phenotypes correlated with transcriptional enhancement and the silencing of b1. These transcriptional dynamics depend on a hepta-tandem repeat sequence located 100 kb upstream of the b1 locus. In the heterozygous condition, the B′ epi-allele paramutates B-I, heritably converting the B-I epi-allele to the epigenetic state and expression level of B′, producing lightly pigmented plants. To identify b1TR-associated proteins, we used a targeted chromatin immunoprecipitation approach with a stably integrated transgenic b1TR locus. Applying a conservative filtering strategy, we detected several expected factors, including RNA Polymerase II, as well as the novel putative DNA-binding proteins ZAG4 and DDT4. ZAG4 and DDT4 activated GAL expression using b1TR as bait in yeast one-hybrid, supporting their potential interaction with this sequence. The identification of proteins uniquely associated with the UAS::b1TR chromatin provides insight into potential b1 regulatory factors and offers a foundation for future studies to investigate their roles in gene regulation. Full article

Despite broad anti-cancer efficacy as Toll-Like Receptor (TLR) 7/8 agonists, imidazoquinolines remain limited in use via systemic administration or in situ vaccination therapies due to inflammatory toxicity. One approach to address this challenge involves better targeting the action of imidazoquinolines by caging them as glycoconjugate prodrugs. Within cancer cells, imidazoquinoline glycoconjugates are activated by hydrolases prior to efflux by ABC transport proteins, where they then elicit tumoricidal effects from the assistance of bystander immune cells, such as tumor-infiltrating lymphocytes and associated macrophages, in local proximity. While this concept of Bystander-Assisted ImmunoTherapy (BAIT) has been established at a molecular level in vitro, tolerability or efficacy of BAIT has not been reported in vivo. Here, we evaluate the MTD and tumor growth delay efficacy of a lead BAIT prodrug (BAIT628) in a male C57BL/6 mouse TRAMP-C2 prostate cancer model to further establish this methodology. Overall, we find that systemic BAIT628 is well tolerated at over 5-fold the dose-limiting inflammatory toxicity of the parent imidazoquinoline (up to 5 mg/mouse/day I.P. for 10 days). Analyzing serum cytokines reveals that IL-10 production, elicited by the mannoside caging group, likely contributes to the enhanced MTD. Using BAIT628 as an in situ vaccination immunotherapy (seven times over 3 weeks) resulted in significant tumor growth delay and increased survival, both alone and in combination with a murinized α-PD-L1 checkpoint blockade. The tumor histology of tumor-infiltrating immune cell subsets (CD4⁺, CD8⁺, CD11c⁺) reveals significant increases in CD11c⁺ populations, consistent with TLR7/8 agonism. Overall, BAIT628 is well tolerated and exhibits significant efficacy in the TRAMP-C2 model. These results demonstrate how the BAIT approach can optimize imidazoquinolines for in vivo tolerability and subsequent efficacy as cancer immunotherapeutics. Full article

Hypersensitivity reactions are dysregulated and potentially devastating immune responses, characterized by a tendency to become chronic. They target either self-proteins or harmless foreign proteins and are driven by both T and B cells. Although numerous symptomatic treatment options for hypersensitivity reactions have been established over recent decades, only a few antigen-specific, causal approaches capable of specifically targeting the pathogenic autoreactive T and/or B cells have been developed. Among these are cell-based treatment modalities involving chimeric antigen receptor (CAR)- or chimeric autoantibody-receptor (CAAR)-expressing cells. These therapies utilize B- or T-cell antigens, presented as B-cell epitopes or peptide-major histocompatibility complexes (pMHCs) to serve as bait. The latter are coupled to potent activation domains derived from the TCR/CD3 complex itself, such as the zeta or CD3 chains, as well as domains from bona fide co-stimulatory molecules (e.g., CD28, 4-1BB). Recent in vitro and in vivo studies have demonstrated the therapeutic potential of these ATMP-based strategies in eliminating autoreactive lymphocytes and alleviating hypersensitivity reactions. This systematic review provides a comprehensive overview of the current status of antigen-specific CAR and CAAR T-cell therapies, highlighting novel directions as well as the ongoing challenges within this promising research field. Full article

Spotted wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), are invasive vinegar flies of East Asian origin that are an increasingly global threat to the small fruit industry. It is essential that new classes of eco-friendly insecticides and cost-effective strategies for SWD control are developed. Here, we describe the preparation of a strain of RNA interference (RNAi) Saccharomyces cerevisiae expressing shRNA that specifically targets the SWD RNA-binding Fox protein 1 (Rbfox1) gene. The yeast effectively silences the SWD Rbfox1 gene, resulting in significant loss of fly neural activity. Laboratory trials demonstrated that the RNAi yeast can be mixed with soda, which functions as SWD attractive targeted sugar bait (ATSB) that can be delivered in a soda bottle feeder. The ATSB, mixed with yeast that was heat-killed prior to suspension in the ATSB, resulted in 92 ± 1% mortality of SWD flies that consumed it, yet had no impact on non-target dipterans. Rbfox.687 yeast delivered in ATSB feeders may one day be a useful component of integrated SWD control programs. Full article

Previous research has unearthed the integration of the coat protein (CP) gene from alphapartitivirus into plant genomes. Nevertheless, the prevalence of this horizontal gene transfer (HGT) between partitiviruses and cellular organisms remains an enigma. In our investigation, we discovered a novel partitivirus, designated Sclerotinia sclerotiorum alphapartitivirus 1 (SsAPV1), from a hypovirulent strain of Sclerotinia sclerotiorum. Intriguingly, we traced homologs of the SsAPV1 CP to plant genomes, including Helianthus annuus. To delve deeper, we employed the CP and RNA-dependent RNA polymerase (RdRP) sequences of partitiviruses as “bait” to search the NCBI database for similar sequences. Our search unveiled a widespread occurrence of HGT between viruses from all five genera within the family Partitiviridae and other cellular organisms. Notably, numerous CP-like and RdRP-like genes were identified in the genomes of plants, protozoa, animals, fungi, and even, for the first time, in an archaeon. The majority of CP and RdRP genes were integrated into plant and insect genomes, respectively. Furthermore, we detected DNA fragments originating from the SsAPV1 RNA genome in some subcultures of virus-infected strains. It suggested that SsAPV1 RdRP may possesses reverse transcriptase activity, facilitating the integration of viral genes into cellular organism genomes, and this function requires further confirmation. Our study not only offers a hypovirulence-associated partitivirus with implications for fungal disease control but also sheds light on the extensive integration events between partitiviruses and cellular organisms and enhances our comprehension of the origins, evolution, and ecology of partitiviruses, as well as the genome evolution of cellular organisms. Full article

African swine fever virus (ASFV) is known to be very stable within a protein-rich environment and indirect virus transmission can be mediated via oral uptake of different materials. However, experimental studies in pigs have shown that infection by ASFV via the oral route can be difficult to establish. Currently, there is a lack of studies using strict oral inoculations of pigs with different doses of ASFV. Therefore, we aimed to determine the dose of a European genotype II ASFV that is required to establish infection of pigs by the oral route. In this study, 24 pigs were divided into four groups of six. Three of the groups were fed with a low, medium or high dose of the ASFV POL/2015/Podlaskie virus. The pigs in the fourth group served as positive controls and were inoculated intranasally, just once, using the low dose of the virus. All the pigs inoculated intranasally with ASFV succumbed to the infection, while only three of the six pigs that were fed the high dose of the virus became infected. None of the 12 pigs that were fed with either the medium or low dose of the virus became infected, despite receiving up to thirteen doses each. In two of the pigs infected by intranasal inoculation, the presence of a variant form of the ASFV genome was detected. The results obtained in this study underline that ASFV infection is more difficult to establish via the oral route when compared to the intranasal route. The high dose needed in order to establish oral infection could have implications for future strategies using baited vaccines containing infectious live-attenuated ASFV. Full article

Background: Elite-neutralizer-derived HIV-1 envelopes (Envs), which induce broadly neutralizing antibodies (bnAbs), can inform HIV-1 vaccine design by serving as templates for bnAb-eliciting vaccines. Since single Env-based immunizations are insufficient to induce bnAb responses, sequential regimens using multivalent immunogens or Env cocktails hold greater promise. This underscores the need to develop stable Env trimers from diverse HIV-1 strains, particularly clade-C, which accounts for 50% of global infections and over 90% in India and South Africa. While various platforms exist to stabilize soluble Env trimers for use as antigenic baits and vaccines, stabilizing clade C trimers remains challenging. Methods: We stabilized an HIV-1 clade C trimer based on an Env isolated from a pediatric elite neutralizer (AIIMS_329) using multiple platforms, including SOSIP.v8.2, ferritin nanoparticles (NPs) and I53-50 two-component NPs, followed by characterization of their biophysical, antigenic, and immunogenic properties. Results: The stabilized 329 Envs showed binding to multiple HIV-1 bnAbs, with negligible binding to non-neutralizing antibodies. Negative-stain electron microscopy confirmed the native-like conformation of the Envs. Multimerization of 329 SOSIP.v8.2 on ferritin and two-component I53-50 NPs improved the affinity to HIV-1 bnAbs and showed higher immunogenicity in rabbits. Conclusions: The soluble 329 Env protein could serve as an antigenic bait, and multimeric 329 NP Envs are potential vaccine candidates. Full article

Food-baited traps are an important part of early detection programs for invasive tephritid fruit fly species, as they are attractive to both sexes of all targeted species. Torula yeast borax (TYB) mixture is a standard food bait, but its longevity is limited (1–2 weeks). Synthetic food-based lures have been developed, including ammonium acetate, putrescine, and trimethylamine. However, the different formulations of these synthetic lures vary greatly in their attractiveness and longevity. Here, we present the results of field trapping in several Central and South American countries as well as Hawaii that compared captures of Ceratitis capitata, Anastrepha spp., and Bactrocera dorsalis in traps baited with torula yeast borax, which was replaced weekly, versus traps baited with newly developed vial-lures, which contained the same three components noted above and were not replaced over 6–10 weeks of trapping. In all countries, captures of C. capitata in vial-lure-baited traps were equal to or greater than captures in TYB-baited traps. However, the vial-lures attracted fewer B. dorsalis than TYB, and data were inconsistent for Anastrepha spp. The implications of these results for large-scale detection programs are discussed. Full article

The determinate inflorescence trait of Brassica napus L. is associated with various desirable agricultural characteristics. BnTFL1s (BnaA10.TFL1 and BnaC09.TFL1), which encode the transcription factor TERMINAL FLOWER 1 (TFL1), have previously been identified as candidate genes controlling this trait through map-based cloning. However, the mechanism underlying the effects of the BnTFL1 proteins remains unclear. Further, proteins generally interact with each other to fulfill their biological functions. The objective of this study was to construct a cDNA library of the shoot apical meristem (SAM) of B. napus and screen for proteins that interact with BnTFL1s, to better understand its mechanism of action. The recombination efficiency of the yeast two-hybrid (Y2H) library that we constructed was 100%, with insertion fragment lengths ranging from 750 to 2000 bp and a capacity of approximately 1.44 × 10⁷ CFUs (colony-forming units), sufficient for screening protein interactions. Additionally, the bait vector pGBKT7-BnTFL1s was transformed into yeast cells alongside positive and negative controls, demonstrating no toxicity to the yeast cells and no self-activation. This bait was used to screen the SAM cDNA library of B. napus, ultimately identifying two BnTFL1s-interacting proteins: 14-3-3-like protein GF14 omega GRF2. These interactions were verified through one-to-one interaction experiments. This study provides a foundation for further research on the biological functions of the BnTFL1s genes and their regulatory role in inflorescence formation in B. napus, while providing a reference for studying similar mechanisms in other plants. Full article

In the Nicotiana tabacum flower development study, we identified SCI1 (Stigma/style Cell-cycle Inhibitor 1), a regulator of cell proliferation. SCI1 interacts with NtCDKG;2 (N. tabacum Cyclin-Dependent Kinase G;2), a homolog of human CDK11, which is responsible for RanGTP-dependent microtubule stabilization, regulating spindle assembly rate. In a Y2H screening of a cDNA library using NtCDKG;2 as bait, a RanBP1 (Ran-Binding Protein 1) was revealed as its interaction partner. RanBP1 is an essential regulatory protein of the RanGTPase system, contributing to the formation of the Ran gradient, which modulates different important cellular processes. RanBP1 is crucial in the nuclear import/export machinery during interphase and spindle checkpoint formation during cell division. These processes are well studied in animals, but very little is known about them in plants. We confirmed NtCDKG;2 and NtRanBP1 interaction by pairwise Y2H and characterized the localization of both proteins during plant cell division. We demonstrated the presence of NtRanBP1 in the cytoplasm during interphase and its nuclear arrest at mitosis onset. Meanwhile, we showed that NtCDKG;2 is localized in the mitotic spindle during cell division, indicating an analogous function to the human CDK11. We propose that the phosphorylation of the nuclear export signal at RanBP1 by NtCDKG;2 may be responsible for the reported nuclear arrest. Full article

To investigate the dietary effects of chlorogenic acid (CGA) on the growth performance, lipid metabolism, antioxidant activity, and non-specific immunity of Asian swamp eel (Monopterus albus) during the domestication stage, a 28-day feeding experiment was conducted to supplement with CGA at levels of 0 (Cont.), 250 (CGA 0.50%), 500 (CGA 1.00%), and 750 (CGA 1.50%) mg/kg·bw by feeding with yellow mealworm (Tenebrio molitor). Compared with the control group, the addition of 250–750 mg/kg of CGA significantly increased the weight-gain rate (WG) of M. albus, and the CGA 1.0% group displayed the highest value. The content of hemoglobin and high-density lipoprotein in all CGA groups was markedly elevated (p < 0.05), while the triglyceride, glucose, low-density lipoprotein, and glycosylated serum protein levels were lowered (p < 0.05). Among the antioxidant enzymes, the glutathione peroxidase and catalase activity was significantly higher in all experimental groups than that of the control group, whereas the malondialdehyde activity was significantly reduced (p < 0.05). For a non-specific immune enzyme system, the lysozyme and alkaline phosphatase activity in all treatments and the superoxide dismutase and acid phosphatase activity in the CGA 0.5% group was markedly increased (p < 0.05). In conclusion, supplementation with CGA can enhance the growth performance and improve the lipid metabolism, antioxidant capacity, and non-specific immunity of M. albus during the domestication stage, and the optimal CGA supplementation for T. molitor as biocarrier bait is 500 mg/kg, corresponding to 405 mg/kg. Full article

Siniperca species are highly valued freshwater fishes in China. In 2022, the country’s production of farmed Siniperca reached 401 kilotons. With the growing demand for high-quality aquatic products, Siniperca aquaculture offers major economic benefits and promising development prospects. However, the current reliance on live bait in Siniperca farming has resulted in resource wastage, environmental pollution, and disease outbreaks, hindering the healthy and sustainable growth of the industry. This review aims to comprehensively summarize the nutritional requirements of Siniperca in artificial farming, with a focus on proteins, amino acids, lipids, fatty acids, carbohydrates, and micronutrients. We also summarize the progress made in researching alternative protein and lipid sources, feed additives, and the development of artificially formulated feeds to replace live bait. The findings of this review will serve as a reference for further research on the nutritional requirements and development of formulated feeds for Siniperca aquaculture. Full article