Rapid and dynamic detection of endogenous proteins through in locus tagging in rice.

Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remains 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, a result attributable to 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 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 analysis of protein interactions. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

Efficient and multiplex gene upregulation in plants through CRISPR-Cas-mediated knockin of enhancers.

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.

In-locus gene silencing in plants using genome editing.

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.

Targeted G-to-T base editing for generation of novel herbicide-resistance gene alleles in rice.

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..

Efficient and heritable A-to-K base editing in rice and tomato.

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.

Ki67-targeted oncolytic adenovirus expressing IL-15 improves intratumoral T cell infiltration and PD-L1 expression in glioblastoma.

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.

STOP1 and STOP1-like proteins, key transcription factors to cope with acid soil syndrome.

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.

Puerarin Attenuates High-Glucose and High-Lipid-Induced Inflammatory Injury in H9c2 Cardiomyocytes via CAV3 Protein Upregulation.

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.

Pan-cancer analysis identifies the correlations of Thymosin Beta 10 with predicting prognosis and immunotherapy response.

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.

Contribution of whole slide imaging-based deep learning in the assessment of intraoperative and postoperative sections in neuropathology.

The pathological diagnosis of intracranial germinoma (IG), oligodendroglioma, and low-grade astrocytoma on intraoperative frozen section (IFS) and hematoxylin-eosin (HE)-staining section directly determines patients' treatment options, but it is a difficult task for pathologists. We aimed to investigate whether whole-slide imaging (WSI)-based deep learning can contribute new precision to the diagnosis of IG, oligodendroglioma, and low-grade astrocytoma. Two types of WSIs (500 IFSs and 832 HE-staining sections) were collected from 379 patients at multiple medical centers. Patients at Center 1 were split into the training, testing, and internal validation sets (3:1:1), while the other centers were the external validation sets. First, we subdivided WSIs into small tiles and selected tissue tiles using a tissue tile selection model. Then a tile-level classification model was established, and the majority voting method was used to determine the final diagnoses. Color jitter was applied to the tiles so that the deep learning (DL) models could adapt to the variations in the staining. Last, we investigated the effectiveness of model assistance. The internal validation accuracies of the IFS and HE models were 93.9% and 95.3%, respectively. The external validation accuracies of the IFS and HE models were 82.0% and 76.9%, respectively. Furthermore, the IFS and HE models can predict Ki-67 positive cell areas with R2 of 0.81 and 0.86, respectively. With model assistance, the IFS and HE diagnosis accuracy of pathologists improved from 54.6%-69.7% and 53.5%-83.7% to 87.9%-93.9% and 86.0%-90.7%, respectively. Both the IFS model and the HE model can differentiate the three tumors, predict the expression of Ki-67, and improve the diagnostic accuracy of pathologists. The use of our model can assist clinicians in providing patients with optimal and timely treatment options.

Targeted insertion of regulatory elements enables translational enhancement in rice.

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.

Oncolytic HSV-1 suppresses cell invasion through downregulating Sp1 in experimental glioblastoma.

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.

High-throughput genome editing in rice with a virus-based surrogate system.

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.

Cut-dip-budding delivery system enables genetic modifications in plants without tissue culture.

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.

[Retracted] MicroRNA­181 serves an oncogenic role in breast cancer via the inhibition of SPRY4.

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the Transwell cell migration assay data shown in Figs. 3B and 9B were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 18: 5603­5613, 2018; DOI: 10.3892/mmr.2018.9572].

The function and mechanism of the JARID2/CCND1 axis in modulating glioma cell growth and sensitivity to temozolomide (TMZ).

A maximal surgical resection followed by radiotherapy and chemotherapy with temozolomide (TMZ) as the representative agent is the standard therapy for gliomas. However, tumor cell resistance to radiotherapy and chemotherapy leads to poor prognosis and high mortality in patients with glioma. In the present study, we demonstrated that JARID2 was downregulated and CCND1 was upregulated within glioma tissues of different grades and glioma cells. In tissue samples, JARID2 was negatively correlated with CCND1. JARID2 overexpression significantly inhibited glioma cell viability, promoted glioma cell apoptosis upon TMZ treatment, and increased p21, cleaved-PARP, and cleaved-caspase3 in TMZ-treated glioma cells. JASPAR tool predicted the possible binding sites between JARID2 and CCND1 promoter regions; through direct binding to CCND1 promoter region, JARID2 negatively regulated CCND1 expression. Under TMZ treatment, JARID2 overexpression inhibited CCND1 expression, promoted glioma cell apoptosis, and increased p21, cleaved-PARP, and cleaved-caspase3 in glioma cells treated with TMZ; meanwhile, CCND1 overexpression exerted opposite effects on glioma cells treated with TMZ and partially reversed the effects of JARID2 overexpression. In conclusion, JARID2 targets and inhibits CCND1. The JARID2/CCND1 axis modulates glioma cell growth and glioma cell sensitivity to TMZ.

Diagnosis and Individualized Treatment of Secondary Central Nervous System Lymphoma: A Case Report.

Non-Hodgkin lymphoma can disseminate to the central nervous system at initiation of treatment for systemic lymphoma or spread during the relapse of systematic lymphoma with CNS involvement, which is defined as secondary central nervous system lymphoma (SCNSL). The incidence of SCNSL depends on the pathological type of lymphoma and is especially high in aggressive lymphoma. SCNSL has a poor prognosis because of the lack of effective treatment regimens. This article presents a rare case of SCNSL; an individualized treatment regimen was designed according to the genetic analyses of the patient tumor and included a Bruton's tyrosine kinase (BTK) inhibitor. After six cycles of treatment and another two cycles of rituximab, most lesions lost their metabolic activity. However, in the final stage of treatment, our patient unfortunately suffered from respiratory failure, which revealed that we should pay attention to Pneumocystis jirovecii pneumonia during ibrutinib treatment.

Long-Term Survival Outcomes and Treatment Experience of 64 Patients With Esthesioneuroblastoma.

Background: Esthesioneuroblastoma (ENB) is a rare sinonasal malignancy, lacking a unified staging system and treatment. Management at a single center was retrospectively evaluated to inform future treatment options and prognostic factors. Methods: Clinical data of 64 consecutive ENB patients, including prognostic factors and treatment methods, were reviewed retrospectively. Data were collected to calculate overall survival (OS) and progression free survival (PFS). Results: The majority of tumors 84.4% were within Kadish C stage, 79.7% were within T3 or T4, and 64.0% were within Hyams grade III or IV. A total of 50 (78.1%) patients received surgery and combined radiotherapy with or without chemotherapy, 10 (15.6%) received surgery with or without chemotherapy alone, and 4 (6.3%) received radiotherapy with or without chemotherapy alone. The majority of patients (79.7%) underwent endoscopic resection (endoscopic and endoscopically assisted). Surgery combined with radiotherapy with or without chemotherapy resulted in significantly better OS (84.4 vs. 50.6%, 84.4 vs. 37.5%) compared to surgery alone and radiotherapy alone (P = 0.0064). Endoscopic surgery group (endoscopic and endoscopically assisted) resulted in significantly better 5-year PFS (61.7 vs. 22.2%) compared to the open surgery group (P < 0.001). Although endoscopic surgery group was not a statistically significant predictor of 5-year OS (P = 0.54), the 5-year OS was 79.3% for the endoscopic surgery group and 76.2% for the open surgery group. A Cox regression analysis identified intracranial extension and surgery combined with radiotherapy as independent factors affecting 5-year OS while cervical lymph node metastasis and Hyams grade IV as independent factors affecting 5-year PFS. Conclusion: Our findings suggest that surgery combined with radiotherapy is the best treatment approach for ENB. For advanced tumors, endoscopic surgery is an effective treatment, and its survival rate is equal to or better than open surgery.