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Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.
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Edição de Genes , Inativação Gênica , Oryza , Edição de Genes/métodos , Oryza/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Loci Gênicos , Genoma de Planta , Regiões 5' não Traduzidas/genética , Genes de Plantas , Sequência de Bases , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , FenótipoRESUMO
A newly developed rice guanine base editor (OsGTBE) achieves targeted and efficient G-to-T editing (C-to-A in the opposite strand) in rice. Using OsGTBE to edit endogenous herbicide-resistant loci generated several novel alleles conferring herbicide resistance, highlighting its utility in creating valuable germplasm and enhancing genetic diversity..
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Alelos , Edição de Genes , Resistência a Herbicidas , Oryza , Oryza/genética , Resistência a Herbicidas/genética , Edição de Genes/métodos , Genes de Plantas , Herbicidas/farmacologia , Sequência de BasesRESUMO
With the widespread use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) technologies in plants, large-scale genome editing is increasingly needed. Here, we developed a geminivirus-mediated surrogate system, called Wheat Dwarf Virus-Gate (WDV-surrogate), to facilitate high-throughput genome editing. WDV-Gate has two parts: one is the recipient callus from a transgenic rice line expressing Cas9 and a mutated hygromycin-resistant gene (HygM) for surrogate selection; the other is a WDV-based construct expressing two single guide RNAs (sgRNAs) targeting HygM and a gene of interest, respectively. We evaluated WDV-Gate on six rice loci by producing a total of 874 T0 plants. Compared with the conventional method, the WDV-Gate system, which was characterized by a transient and high level of sgRNA expression, significantly increased editing frequency (66.8% vs. 90.1%), plantlet regeneration efficiency (2.31-fold increase), and numbers of homozygous-edited plants (36.3% vs. 70.7%). Large-scale editing using pooled sgRNAs targeting the SLR1 gene resulted in a high editing frequency of 94.4%, further demonstrating its feasibility. We also tested WDV-Gate on sequence knock-in for protein tagging. By co-delivering a chemically modified donor DNA with the WDV-Gate plasmid, 3xFLAG peptides were successfully fused to three loci with an efficiency of up to 13%. Thus, by combining transiently expressed sgRNAs and a surrogate selection system, WDV-Gate could be useful for high-throughput gene knock-out and sequence knock-in.
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Edição de Genes , Oryza , Edição de Genes/métodos , Sistemas CRISPR-Cas , Oryza/genética , Genoma de Planta , Plantas/genéticaRESUMO
Uveal melanoma (UM), the most common primary malignant tumor of the eye in adults, is difficult-to-treat. UM has a relatively high mortality secondary to distant metastasis and poor overall survival with existing therapies. The oncolytic virus herpes simplex virus type-1 (HSV-1) has been approved for clinical use in melanoma. This double-stranded DNA virus was suspected to directly activate lysis specifically in neoplastic cells. We tested the antitumor efficacy of recombinant oncolytic HSV-1-EGFP (oHSV-EGFP) in UM and characterized the local and systemic antitumor innate immune response in a murine xenograft model. We first determined the efficacy of the oncolytic virus in 92.1, MUM2B and MP41 cell lines. In murine xenograft models, oHSV-EGFP reduced intraocular tumors as well as systemic subcutaneous tumors. A significant change in cytokines was observed in viral infected cells, especially a rise in IFNγ. In vivo analyses showed increased anti-tumorigenic M1 macrophages and decreased pro-tumorigenic M2 macrophages in peripheral blood, and intraocular and distant tumors after intravitreal viral treatment. Increased infiltration of natural killer cells and mature dendritic cells was also detected after viral treatment. In addition, no virus was detected in major organs after the treatment. Our data support that oHSV-EGFP is effective, neoplasm specific, immune active and safe, providing possible clinical translatable options to treat ocular and metastatic UM.
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Modelos Animais de Doenças , Proteínas de Fluorescência Verde/fisiologia , Herpesvirus Humano 1/fisiologia , Ativação de Macrófagos/fisiologia , Melanoma/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Neoplasias Uveais/terapia , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Xenoenxertos , Humanos , Melanoma/metabolismo , Melanoma/patologia , Melanoma/virologia , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Neoplasias Uveais/virologiaRESUMO
We demonstrate the enhancement of femtosecond (fs) laser-induced filaments in air and nitrogen flow fields using a nanosecond (ns) laser. With the ns laser being imposed on the filaments, the length and the emission intensity of the filaments were largely increased. Temporally resolved spectra of the enhanced filaments were obtained. The results show that the ns laser enhanced the short-lifetime fluorescence of nitrogen, which comes from the transition processes of N2 +(B2Σu + - X2Σg +), N2(B3Ðg - A3Σu +) and N2(C3Ðu - B3Ðg). However, it had little effect on the long-lifetime chemiluminescence, which mainly comes from reactions such as N2(A3Σu +) + N2(A3Σu +) â N2(X1Σg +, v = 0) + N2(B3Ðg). A possible explanation of this phenomenon is given, and this phenomenon might have potential applications in instantaneous one-dimensional measurements of various species in gas flow fields.
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As the most common type of tumour in brain, glioma has a high rate of morbidity and mortality and easily penetrates the surrounding normal brain parenchyma. The immunosuppressive microenvironment, which is similar to that in other neoplasms, is believed to participate in the tumorigenesis of glioma. Thus, many experts are seeking to exploit microenvironment as a therapeutic target. In the present study, we focused on microglia polarization to investigate the anti-glioma response of microglia inflammatory activation by Staphylococcus aureus in vitro and in vivo. First, we found that intratumor injection of S. aureus delayed glioma growth in C57/BL6 mice. Then, we showed that inflammatory microglia activated by S. aureus inhibited glioma cell proliferation, migration, and invasion. This inhibition was likely related to the phenotypic switch observed in microglia. In intracranial tumour models, the microglia activated by S. aureus exerted antitumour efficacy and prolonged animal survival. Taken together, our results suggest that microglia activated by S. aureus have antitumour efficacy, which may be a potential therapeutic agent for glioma. SIGNIFICANCE OF THE STUDY: In this study, we mainly demonstrated the antitumour efficacy of microglia after activated by S. aureus. Firstly, we found that intratumor injection of S. aureus inhibited the tumour growth in intracranial orthotopic glioma model. In addition, we found that the microglia around the glioma may exert antitumour efficacy, and its phenotype may be altered by stimulation of S. aureus. Our data manifested that S. aureus did not directly suppress cell proliferative, migration, and invasion capacity, but by activating microglia. And in mice GL261 GBMs, injection of microglia after cocultured with S. aureus inhibited tumour growth and prolonged animal survival.
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Glioblastoma/microbiologia , Glioblastoma/patologia , Inflamação/imunologia , Inflamação/microbiologia , Microglia/imunologia , Staphylococcus aureus/imunologia , Animais , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , FenótipoRESUMO
Ammonia (NH3) is a sustainable fuel with excellent emission characteristics. Hence, it is important to develop diagnostic techniques for NH3 combustion. In this paper, femtosecond laser-induced plasma spectroscopy (FLIPS) was performed in premixed NH3/air flames. The plasma emission spectra induced by the femtosecond laser in the flame and the chemiluminescence spectra of the flame itself were both measured. Through calibration, we found that the spectral intensity ratio of NH (336 nm)/N2 (337 nm) could be used for equivalence ratio measurements in NH3 combustion flow fields. This work is the first attempt at using a femtosecond laser-based technique for NH3 combustion diagnostics.
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We demonstrate a non-contact spatiotemporally resolved comprehensive method for gas flow velocity field measurement: Filamentary Anemometry using femtosecond Laser-extended Electric Discharge (FALED). A faint thin plasma channel was generated in ambient air by focusing an 800-nm laser beam of 45 fs, which was used to ignite a pulsed electric discharge between two electrodes separated over 10 mm. The power supplier provided a maximum voltage up to 5 kV and was operated at a burst mode with a current duration of less than 20 ns and a pulse-to-pulse separation of 40 µs. The laser-guided thin filamentary discharge plasma column was blowing up perpendicularly by an air jet placed beneath in-between the two electrodes. Although the discharge pulse was short, the conductivity of the plasma channel was observed to sustain much longer, so that a sequence of discharge filaments was generated as the plasma channel being blown up by the jet flow. The sequential bright thin discharge filaments can be photographed using a household camera to calculate the flow velocity distribution of the jet flow. For a direct comparison, a flow field measurement using FLEET [Appl. Opt. 50, 5158 (2011)] was also performed. The results indicate that the FALED technique can provide instantaneous nonintrusive flow field velocity measurement with good accuracy.
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Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis. Here, we introduce an "in locus activation" technique in rice that leverages well-characterized STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts. The in locus integration of these STEs into eight rice genes resulted in substantial transcriptional upregulation in the edited plants, with up to 869.1-fold increases in their transcript levels. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes activated simultaneously, significantly enhancing the nicotinamide mononucleotide metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Collectively, our work demonstrates that coupled with STE mining, leveraging genome editing for in locus activation and gene upregulation holds great promise to be widely adopted in fundamental plant research and crop breeding.
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Cytosine and adenosine base editors (CBE and ABE) have been widely used in plants, greatly accelerating gene function research and crop breeding. Current base editors can achieve efficient A-to-G and C-to-T/G/A editing. However, efficient and heritable A-to-Y (A-to-T/C) editing remains to be developed in plants. In this study, a series of A-to-K base editor (AKBE) systems were constructed for monocot and dicot plants. Furthermore, nSpCas9 was replaced with the PAM-less Cas9 variant (nSpRY) to expand the target range of the AKBEs. Analysis of 228 T0 rice plants and 121 T0 tomato plants edited using AKBEs at 18 endogenous loci revealed that, in addition to highly efficient A-to-G substitution (41.0% on average), the plant AKBEs can achieve A-to-T conversion with efficiencies of up to 25.9 and 10.5% in rice and tomato, respectively. Moreover, the rice-optimized AKBE generates A-to-C conversion in rice, with an average efficiency of 1.8%, revealing the significant value of plant-optimized AKBE in creating genetic diversity. Although most of the A-to-T and A-to-C edits were chimeric, desired editing types could be transmitted to the T1 offspring, similar to the edits generated by the traditional ABE8e. Besides, using AKBEs to target tyrosine (Y, TAT) or cysteine (C, TGT) achieved the introduction of an early stop codon (TAG/TAA/TGA) of target genes, demonstrating its potential use in gene disruption.
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Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. While mammalian studies have leveraged in-locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remained unexplored in plants. Here, we introduce the in-locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, which is attributed to the low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence by up to 6.9-fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in-situ visualization using a cross-breeding strategy, and effective immunoprecipitation for protein interaction analysis. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.
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Acid soil syndrome leads to severe yield reductions in various crops worldwide. In addition to low pH and proton stress, this syndrome includes deficiencies of essential salt-based ions, enrichment of toxic metals such as manganese (Mn) and aluminum (Al), and consequent phosphorus (P) fixation. Plants have evolved mechanisms to cope with soil acidity. In particular, STOP1 (Sensitive to proton rhizotoxicity 1) and its homologs are master transcription factors that have been intensively studied in low pH and Al resistance. Recent studies have identified additional functions of STOP1 in coping with other acid soil barriers: STOP1 regulates plant growth under phosphate (Pi) or potassium (K) limitation, promotes nitrate (NO3 -) uptake, confers anoxic tolerance during flooding, and inhibits drought tolerance, suggesting that STOP1 functions as a node for multiple signaling pathways. STOP1 is evolutionarily conserved in a wide range of plant species. This review summarizes the central role of STOP1 and STOP1-like proteins in regulating coexisting stresses in acid soils, outlines the advances in the regulation of STOP1, and highlights the potential of STOP1 and STOP1-like proteins to improve crop production on acid soils.
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Introduction: The biological function and prognosis roles of thymosin ß(TMSB) 10 are still unclear in pan-cancer. Methods: We retrieved The Cancer Genome Atlas and Genotype-tissue expression datasets to obtain the difference of TMSB10 expression between pan-cancer and normal tissues, and analyzed the biological function and prognosis role of TMSB10 in pan-cancer by using cBioPortal Webtool. Results: The expression of TMSB10 in tumor tissues was significantly higher than normal tissues, and showed the potential ability to predict the prognosis of patients in Pan-cancer. It was found that TMSB10 was significantly correlated with tumor microenvironment, immune cell infiltration and immune regulatory factor expression. TMSB10 is involved in the regulation of cellular signal transduction pathways in a variety of tumors, thereby mediating the occurrence of tumor cell invasion and metastasis. Finally, TMSB10 can not only effectively predict the anti-PD-L1 treatment response of cancer patients, but also be used as an important indicator to evaluate the sensitivity of chemotherapy. In vitro, low expression of TMSB10 inhibited clonogenic formation ability, invasion, and migration in glioma cells. Furthermore, TMSB10 may involve glioma immune regulation progression by promoting PD-L1 expression levels via activating STAT3 signaling pathway. Conclusions: Our results show that TMSB10 is abnormally expressed in tumor tissues, which may be related to the infiltration of immune cells in the tumor microenvironment. Clinically, TMSB10 is not only an effective prognostic factor for predicting the clinical treatment outcome of cancer patients, but also a promising biomarker for predicting the effect of tumor immune checkpoint inhibitors (ICIs) and chemotherapy in some cancers.
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Glioma , Timosina , Humanos , Prognóstico , Imunoterapia , Timosina/genética , Inibidores de Checkpoint Imunológico , Microambiente Tumoral/genéticaRESUMO
Glioblastoma (GBM) is a devastating malignant brain tumor. Current therapeutic strategies targeting tumor cells have limited efficacy owing to the immunosuppressive microenvironment. Previous work demonstrated that the targeted Ad5-Ki67/IL-15 could specifically kill tumor cells and decrease the angiogenic capacity in vitro. However, the efficacy of this virus in vivo and its effect on the tumor microenvironment (TME) has not been elucidated. In this study, we found that the Ad5-Ki67/IL-15 treatment down-regulated PD-L1 expression of glioma cells. More importantly, Ad5-Ki67/IL-15 also remodeled the tumor microenvironment via increasing intratumoral T cell infiltration and PD-L1 improvement in a GBM model, as well as the increase of antitumor cytokines, thereby improving the efficacy of GBM treatment. Furthermore, a combination of Ad5-Ki67/IL-15 with PD-L1 blockade significantly inhibits tumor growth in the GBM model. These results provide new insight into the therapeutic effects of targeted oncolytic Ad5-Ki67/IL-15 in patients with GBM, indicating potential clinical applications.
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Background: Inflammation plays a crucial role in the development of diabetic cardiomyopathy (DCM), including inflammation caused by high-glucose and high-lipid (HGHL). Targeting inflammation may provide a useful strategy for preventing and treating DCM. Puerarin has been shown to reduce the inflammation, apoptosis and hypertrophy of cardiomyocytes induced by HGHL, in which this study aims to investigate the underlying mechanisms. Methods: H9c2 cardiomyocytes cultured with HGHL were used to establish a cell model of DCM. Puerarin was then placed to these cells for 24 hours. The effects of HGHL and puerarin on cell viability and apoptosis were investigated by the Cell Proliferation, Toxicity Assay Kit (CCK-8) and flow cytometry. Morphological changes of cardiomyocytes was observed by HE staining. CAV3 proteins in H9c2 cardiomyocytes were altered by transient transfection of CAV3 siRNA. IL-6 was detected by ELISA. The Western blot was performed to determine the CAV3, Bcl-2, Bax, pro-Caspase-3, cleaved-Caspase-3, NF-κB (p65) and p38MAPK proteins. Results: Puerarin treatment reversed the cells viability, hypertrophy in morphology, inflammation (showed by p-p38 and p-p65 and IL-6) and apoptosis-related damage (showed by cleaved-Caspase-3/pro-Caspase-3/Bax, Bcl-2 and flow cytometry) of the H9c2 cardiomyocyte caused by HGHL. Puerarin treatment also restored the decrease of CAV3 proteins of the H9c2 cardiomyocyte caused by HGHL. When silenced the expression of CAV3 proteins with SiRNA, puerarin failed to decreased p-p38 and p-p65 and IL-6, and could not reversed cell viability and morphological damage. In contrast to the simple CAV3 silenced group, the CAV3 silenced with NF-κB pathway or p38MAPK pathway inhibitors, significantly downregulated the p-p38, p-p65 and IL-6. Conclusion: Puerarin upregulated CAV3 protein expression in H9c2 cardiomyocytes and inhibited the NF-κB and p38MAPK pathways, thereby reducing HGHL-induced inflammation and may related to the apoptosis and hypertrophy of cardiomyocytes.
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Gliomas are highly aggressive intracranial tumors that are difficult to resect and have high lethality and recurrence rates. According to WHO grading criteria, glioblastoma with wild-type IDH1 has a poorer prognosis than WHO grade 4 IDH-mutant astrocytomas. To date, no effective therapeutic strategies have been developed to treat glioblastoma. Clinical trials have shown that herpes simplex virus (HSV)-1 is the safest and most efficacious oncolytic virus against glioblastoma, but the molecular antitumor mechanism of action of HSV-1 has not yet been determined. Deletion of the γ34.5 and ICP47 genes from a strain of HSV-1 yielded the oncolytic virus, oHSV-1, which reduced glioma cell viability, migration, and invasive capacity, as well as the growth of microvilli. Infected cell polypeptide 4 (ICP4) expressed by oHSV-1 was found to suppress the expression of the transcription factor Sp1, reducing the expression of host invasion-related genes. In vivo, oHSV-1 showed significant antitumor effects by suppressing the expression of Sp1 and invasion-associated genes, highly expressed in high-grade glioblastoma tissue specimens. These findings indicate that Sp1 may be a molecular marker predicting the antitumor effects of oHSV-1 in the treatment of glioma and that oHSV-1 suppresses host cell invasion through the ICP4-mediated downregulation of Sp1.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Herpesvirus Humano 1 , Terapia Viral Oncolítica , Vírus Oncolíticos , Humanos , Herpesvirus Humano 1/genética , Glioblastoma/metabolismo , Glioma/genética , Vírus Oncolíticos/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Fator de Transcrição Sp1/genéticaRESUMO
In-locus editing of agronomically-important genes to optimize their spatiotemporal expression is becoming an important breeding approach. Compared to intensive studies on mRNA transcription, manipulating protein translation by genome editing has not been well exploited. Here, we found that precise knock-in of a regulating element into the 5'UTR of a target gene could efficiently increase its protein abundance in rice. We firstly screened a translational enhancer (AMVE) from alfalfa mosaic virus using protoplast-based luciferase assays with an 8.5-folds enhancement. Then the chemically modified donor of AMVE was synthesized and targeted inserted into the 5'UTRs of two genes (WRKY71 and SKC1) using CRISPR/Cas9. Following the in-locus AMVE knock-in, we observed up to a 2.8-fold increase in the amount of WRKY71 protein. Notably, editing of SKC1, a sodium transporter, significantly increased salt tolerance in T2 seedlings, indicating the expected regulation of AMVE knock-in. These data demonstrated the feasibility of such in-locus editing to enhance protein expression, providing a new approach to manipulating protein translation for crop breeding.
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Of the more than 370 000 species of higher plants in nature, fewer than 0.1% can be genetically modified due to limitations of the current gene delivery systems. Even for those that can be genetically modified, the modification involves a tedious and costly tissue culture process. Here, we describe an extremely simple cut-dip-budding (CDB) delivery system, which uses Agrobacterium rhizogene to inoculate explants, generating transformed roots that produce transformed buds due to root suckering. We have successfully used CDB to achieve the heritable transformation of plant species in multiple plant families, including two herbaceous plants (Taraxacum kok-saghyz and Coronilla varia), a tuberous root plant (sweet potato), and three woody plant species (Ailanthus altissima, Aralia elata, and Clerodendrum chinense). These plants have previously been difficult or impossible to transform, but the CDB method enabled efficient transformation or gene editing in them using a very simple explant dipping protocol, under non-sterile conditions and without the need for tissue culture. Our work suggests that large numbers of plants could be amenable to genetic modifications using the CDB method.