Developing a CRISPR/FrCas9 system for core promoter editing in rice.

Small mutations in the core promoter region of a gene may result in substantial changes in expression strengths. However, targeting TA-rich sequences of core promoters may pose a challenge for Cas9 variants such as SpCas9 and other G-rich PAM-compatible Cas9s. In this study, we engineered a unique FrCas9 system derived from Faecalibaculum rodentium for plant genome editing. Our findings indicate that this system is efficient in rice when the TATA sequence is used as a PAM. In addition, FrCas9 demonstrated activity against all 16 possible NNTA PAMs, achieving an efficiency of up to 35.3% in calli and generating homozygous or biallelic mutations in 31.3% of the T0 transgenic plants. A proof-of-concept experiment to examine editing of the rice WX core promoter confirmed that FrCas9-induced mutations could modify gene expression and amylose content. Multiplex mutations and deletions were produced by bidirectional editing, mediated by FrCas9, using a single palindromic TATA sequence as a PAM. Moreover, we developed FrCas9-derived base editors capable of programmable conversion between A·T and G·C pairs in plants. This study highlights a versatile FrCas9 toolset for plant core promoter editing, offering great potential for the fine-tuning of gene expression and creating of new germplasms. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00157-5.

Conditional knockdown of OsMLH1 to improve plant prime editing systems without disturbing fertility in rice.

BACKGROUND: High-efficiency prime editing (PE) is desirable for precise genome manipulation. The activity of mammalian PE systems can be largely improved by inhibiting DNA mismatch repair by coexpressing a dominant-negative variant of MLH1. However, this strategy has not been widely used for PE optimization in plants, possibly because of its less conspicuous effects and inconsistent performance at different sites. RESULTS: We show that direct RNAi knockdown of OsMLH1 in an ePE5c system increases the efficiency of our most recently updated PE tool by 1.30- to 2.11-fold in stably transformed rice cells, resulting in as many as 85.42% homozygous mutants in the T0 generation. The high specificity of ePE5c is revealed by whole-genome sequencing. To overcome the partial sterility induced by OsMLH1 knockdown of ePE5c, a conditional excision system is introduced to remove the RNAi module by Cre-mediated site-specific recombination. Using a simple approach of enriching excision events, we generate 100% RNAi module-free plants in the T0 generation. The increase in efficiency due to OsMLH1 knockdown is maintained in the excised plants, whose fertility is not impaired. CONCLUSIONS: This study provides a safe and reliable plant PE optimization strategy for improving editing efficiency without disturbing plant development via transient MMR inhibition with an excisable RNAi module of MLH1.

Eliciting Older Cancer Patients' Preferences for Follow-Up Care to Inform a Primary Healthcare Follow-Up Model in China: A Discrete Choice Experiment.

BACKGROUND AND OBJECTIVES: Increasing longevity and advances in treatment have increased the cancer burden in the elderly, resulting in complex follow-up care needs; however, in China, little is known about the follow-up care preferences of these patients. This study quantified older cancer patients' preferences for follow-up care and examined the trade-offs they are willing to make to accept an alternative follow-up model. METHODS: A discrete choice experiment was conducted among inpatients aged over 60 years with breast, prostate, or colorectal cancer, at two large tertiary hospitals in Nantong, China. Preference weights for follow-up care were estimated using mixed logit analysis. Subgroup analysis and latent class analysis were used to explore preference heterogeneity. RESULTS: Complete results were obtained from 422 patients (144 with breast cancer, 133 with prostate cancer, 145 with colorectal cancer), with a mean age of 70.81 years. Older cancer patients stated a preference for follow-up by specialists over primary healthcare (PHC) providers ( ß = -1.18, 95% confidence interval -1.40 to -0.97). The provider of follow-up care services was the most valued attribute among patients with breast cancer (relative importance [RI] 37.17%), while remote contact services were prioritized by patients with prostate (RI 43.50%) and colorectal cancer (RI 33.01%). The uptake rate of an alternative care model integrating PHC increased compared with the baseline setting when patients were provided with preferred services (continuity of care, individualized care plans, and remote contact services). CONCLUSION: To encourage older cancer patients to use PHC-integrated follow-up care, alternative follow-up care models need to be based on patients' preferences before introducing them as a routine option.

Prognostic significance of systemic immune inflammatory index in NSCLC: a meta-analysis.

Aim: The aim of this meta-analysis was to investigate the relationship between the baseline systemic immune inflammatory index (SII) and prognosis in patients with NSCLC. Materials & methods: The relation between pretreatment SII and overall survival, disease-free survival, cancer-specific survival, progression-free survival and recurrence-free survival in NSCLC patients was analyzed combined with hazard ratio and 95% CI. Results: The results showed that high SII was significantly correlated with overall survival and progression-free survival of NSCLC patients, but not with disease-free survival, cancer-specific survival and recurrence-free survival. Conclusion: The study suggests that a higher SII has association with worse prognosis in NSCLC patients. PROSPERO registration number: CRD42022336270.

Application Value of Early Warning Nursing Model with "Evidence-based Concept" as the Core in Peri-anesthesia Period for Gastric Cancer.

Objective: To assess the effectiveness of an evidence-based early warning nursing model in reducing postoperative complications and enhancing care satisfaction for gastric cancer (GC) patients undergoing elective surgery. Methods: A retrospective analysis was performed on patients who received laparoscopic assisted radical gastrectomy in Tumor Hospital Affiliated To Nantong University from January 2020 to January 2022 as the research subjects. According to the anesthesia plan, the patients were divided into a comprehensive (intervention plans that received an early warning care model centered on evidence-based concepts) and a conventional group (routine evidence-based nursing plan). The postoperative anesthesia recovery, complications, pain level at 24 hours after surgery, anxiety at different times after surgery, and nursing satisfaction were compared between the two groups. Results: The postoperative spontaneous breathing time, consciousness recovery time, tracheal removal time, and defecation recovery time in the patients who received the early warning nursing based on the evidence-based concept were lower than those receiving only evidence-based care (P < .05). The total incidence of postoperative complications in the patients who received the early warning nursing based on the evidence-based concept were lower than those receiving only evidence-based care (P < .05). The postoperative pain levels of patients receiving the early warning nursing model were significantly lower than those receiving only evidence-based care (Z = -2.199, P = .028). After the intervention of different peri-anesthesia nursing modes, the anxiety scores of the two groups showed a downward trend with time (Ftime = 8.552, Ptime < .05), and the decrease in the comprehensive were greater than that in the routine group (F groups = 135.100, Ptime < .05), and there were no interaction (Finteraction = 2.424, Pinteraction < .05). Patients in the group that received the comprehensive early warning nursing model had significantly higher satisfaction with peri-anesthesia care compared to those receiving only evidence-based care (Z = -1.965, P < .05). Conclusion: Under the evidence-based early warning nursing model, nurses accurately assess patients' conditions during the peri-anesthesia period, leading to improved care plans that reduce complications and postoperative pain while enhancing patient satisfaction.

Transcriptomics Based Network Analyses and Molecular Docking Highlighted Potentially Therapeutic Biomarkers for Colon Cancer.

In this study, machine learning-based multiple bioinformatics analysis was carried out for the purpose of the deep and efficient mining of high-throughput transcriptomics data from the TCGA database. Compared with normal colon tissue, 2469 genes were significantly differentially expressed in colon cancer tissue. Gene functional annotation and pathway analysis suggested that most DEGs were functionally related to the cell cycle and metabolism. Weighted gene co-expression network analysis revealed a significant module and the enriched genes that were closely related to fatty acid degradation and metabolism. Based on colon cancer progression, the trend analysis highlighted that several gene sets were significantly correlated with disease development. At the same time, the most specific genes were functionally related to cancer cell features such as the high performance of DNA replication and cell division. Moreover, survival analysis and target drug prediction were performed to prioritize reliable biomarkers and potential drugs. In consideration of a combination of different evidence, four genes (ACOX1, CPT2, CDC25C and PKMYT1) were suggested as novel biomarkers in colon cancer. The potential biomarkers and target drugs identified in our study may provide new ideas for colonic-related prevention, diagnosis, and treatment; therefore, our results have high clinical value for colon cancer.

Proteomics-based molecular and functional characteristic profiling of muscle tissue in Triploid crucian carp.

Triploid crucian carp (TCC) is a kind of artificially bred fish with huge economic value to China. It has several excellent characteristics, such as fast growth, strong disease resistance and delicious taste. However, as a regionally specific fish, the underlying molecular mechanisms of these characteristics are largely unknown. In this study, we performed quantitative proteomics on the muscle tissues of TCC and its parents, allotetraploid (♂), red crucian carp (♀) and common carp. Combined with multiple bioinformatic analysis, we found that the taste of TCC can be mainly attributed to umami amino acid-enriched proteins such as PURBA, PVALBI and ATP5F1B, and that its rapid growth can be mainly ascribed to the high expression and regulation of metabolism-related proteins such as NDUFS1, ENO1A and CS. These play significant roles in substrate and energy metabolism, as well as in bias transformation. Subsequently, we identified several proteins, including MDH1AA, GOT1 and DLAT, that may serve as potential regulators of innate immunity by regulating the biosynthesis and transformation of significant antibiotics and antimicrobial peptides. In conclusion, this study can serve as a significant reference for similar investigations and shed light on the molecular and biological functions of individual proteins in TCC muscle tissue.

Development of a highly efficient prime editor 2 system in plants.

Low efficiency has seriously restricted the application of prime editing (PE) systems in plants. In this study, we develop an enhanced plant prime editor 2 system, enpPE2, by stacking various optimization strategies, including updating the PE architecture to PEmax and expressing engineered pegRNA with a structured motif under the control of a composite promoter. In T0 rice plants, enpPE2 exhibits editing frequencies of 64.58% to 77.08%, which are much higher than the frequencies with unmodified pPE2. Our results indicate that the enpPE2 system provides a robust and powerful tool for the precise modification of plant genomes.

Genome editing with type II-C CRISPR-Cas9 systems from Neisseria meningitidis in rice.

Two type II-C Cas9 orthologs (Nm1Cas9 and Nm2Cas9) were recently identified from Neisseria meningitidis and have been extensively used in mammalian cells, but whether these NmCas9 orthologs or other type II-C Cas9 proteins can mediate genome editing in plants remains unclear. In this study, we developed and optimized targeted mutagenesis systems from NmCas9s for plants. Efficient genome editing at the target with N4 GATT and N4 CC protospacer adjacent motifs (PAMs) was achieved with Nm1Cas9 and Nm2Cas9 respectively. These results indicated that a highly active editing system could be developed from type II-C Cas9s with distinct PAM preferences, thus providing a reliable strategy to extend the scope of genome editing in plants. Base editors (BEs) were further developed from the NmCas9s. The editing efficiency of adenine BEs (ABEs) of TadA*-7.10 and cytosine BEs (CBEs) of rat APOBEC1 (rAPO1) or human APOBEC3a (hA3A) were extremely limited, whereas ABEs of TadA-8e and CBEs of Petromyzon marinus cytidine deaminase 1 (PmCDA1) exhibited markedly improved performance on the same targets. In addition, we found that fusion of a single-stranded DNA-binding domain from the human Rad51 protein enhanced the base editing capability of rAPO1-CBEs of NmCas9s. Together, our results suggest that the engineering of NmCas9s or other type II-C Cas9s can provide useful alternatives for crop genome editing.

Phosphoproteomics Identifies Significant Biomarkers Associated with the Proliferation and Metastasis of Prostate Cancer.

The spider peptide toxins HNTX-III and JZTX-I are a specific inhibitor and activator of TTX-S VGSCs, respectively. They play important roles in regulating MAT-LyLu cell metastasis in prostate cancer. In order to identify key biomarkers involved in the regulation of MAT-LyLu cell metastasis, iTRAQ-based quantitative phosphoproteomics analysis was performed on cells treated with HNTX-III, JZTX-I and blank. A total of 554 unique phosphorylated proteins and 1779 distinct phosphorylated proteins were identified, while 55 and 36 phosphorylated proteins were identified as differentially expressed proteins in HNTX-III and JZTX-I treated groups compared with control groups. Multiple bioinformatics analysis based on quantitative phosphoproteomics data suggested that the differentially expressed phosphorylated proteins and peptides were significantly associated with the migration and invasion of prostate tumors. Specifically, the toxins HNTX-III and JZTX-I have opposite effects on tumor formation and metastasis by regulating the expression and phosphorylation level of causal proteins. Herein, we highlighted three key proteins EEF2, U2AF2 and FLNC which were down-regulated in HNTX-III treated cells and up-regulated in JZTX-I treated cells. They played significant roles in cancer related physiological and pathological processes. The differentially expressed phosphorylated proteins identified in this study may serve as potential biomarkers for precision medicine for prostate cancer in the near future.

Origin and Characterization of Extracellular Vesicles Present in the Spider Venom of Ornithoctonus hainana.

Extracellular vesicles (EVs), including exosomes and microvesicles, are membranous vesicles released from nearly all cellular types. They contain various bioactive molecules, and their molecular composition varies depending on their cellular origin. As research into venomous animals has progressed, EVs have been discovered in the venom of snakes and parasitic wasps. Although vesicle secretion in spider venom glands has been observed, these secretory vesicles' origin and biological properties are unknown. In this study, the origin of the EVs from Ornithoctonus hainana venom was observed using transmission electron microscopy (TEM). The Ornithoctonus hainana venom extracellular vesicles (HN-EVs) were isolated and purified by density gradient centrifugation. HN-EVs possess classic membranous vesicles with a size distribution ranging from 50 to 150 nm and express the arthropod EV marker Tsp29Fb. The LC-MS/MS analysis identified a total of 150 proteins, which were divided into three groups according to their potential function: conservative vesicle transport-related proteins, virulence-related proteins, and other proteins of unknown function. Functionally, HN-EVs have hyaluronidase activity and inhibit the proliferation of human umbilical vein endothelial cells (HUVECs) by affecting the cytoskeleton and cell cycle. Overall, this study investigates the biological characteristics of HN-EVs for the first time and sheds new light on the envenomation process of spider venom.

Identification of herbicide resistance OsACC1 mutations via in planta prime-editing-library screening in rice.

Base-editing-library-induced high density nucleotide substitutions have been applied to screen functional mutations in plants. However, due to limitations in the scope and conversion specificity of base editors, many desired mutations at pivotal protein sites may be overlooked. Here, we developed a prime-editing-library-mediated saturation mutagenesis (PLSM) method to substantially increase the diversity of amino acid substitutions at target sites for in planta screening. At six conserved residues of OsACC1, 16 types of herbicide-resistance-endowing mutations were identified. Most of these mutations exhibit reliable tolerance to aryloxyphenoxypropionate herbicides and have not been reported or applied in rice breeding. In addition, the advantage of PLSM was further shown by comparing the base-editing-mediated mutagenesis at the selected targets. The PLSM method established in this study has great potential for the direct evolution of genes related to agronomically important traits for crop improvement.

Development of an efficient plant dual cytosine and adenine editor.

An enhanced CDA-like (eCDAL) was established from Japanese lamprey CDA1-like 4 to achieve a high editing frequency in a broad region as a C-terminal cytosine base editors (CT-CBE). Then, a novel plant dual-base editor version 1(pDuBE1) was developed by integrating TadA-8e into eCDAL. The editing efficiency of pDuBE1 could reach to 87.6%, with frequencies of concurrent A-to-G and C-to-T conversions as high as 49.7% in stably transformed plant cells. Our results showed that pDuBE1 could mediate robust dual editing in plant genome, providing a powerful manipulation tool for precise crop breeding and screening platforms for in planta direct evolution.

Real-world comparison of direct-acting oral anticoagulants and vitamin K antagonists in chronic kidney disease: a systematic review and meta-analysis.

Background: No clear clinical guidelines exist on anticoagulant use for chronic kidney disease (CKD) patients. We compared the efficacy and safety of direct-acting oral anticoagulants (DOACs) vs. vitamin K antagonists (VKA) in patients with CKD by pooling data from real-world observational studies.Research design & methods: This systematic review searched PubMed, Embase, and CENTRAL databases and pooled multivariable-adjusted hazard ratios (HR) of outcomes.Results: Fifteen studies were included. Our results indicated a small but significant reduction in the risk of all-cause mortality (p = 0.01), stroke or systemic embolism (p = 0.03), and major bleeding (p = 0.01) with DOAC as compared to VKA. In subgroup analysis based on the severity of CKD, no difference in the risk of stroke or systemic embolism was noted in any subgroups. The risk of mortality was reduced only in patients with moderate-severe or severe CKD and the risk of major bleeding was reduced only in patients with moderate-severe or moderate CKD.Conclusion: DOACs are associated with only a modest reduction in stroke or systemic embolism, major bleeding, and mortality when compared to VKA in CKD patients. Reduction in mortality and major bleeding with DOAC may only be seen in moderate-to-severe CKD patients.

Preparation of a CaTiO3/Al3+/Pr3+/Sm3+ nanocomposite for enrichment of exosomes in human serum.

Exosomes (30-200 nm) play important roles in intercellular communication. Because their contents differ between healthy individuals and subjects diagnosed with various diseases, exosomes have been regarded as potential sources of biomarkers for clinical diagnosis. However, the accuracy of diagnosis by exosomal biomarkers is highly dependent on the extraction efficiency, yield, and the quality of exosomes. Hence, inexpensive, convenient, and fast exosome separation methods are required. In the present study, the CaTiO3/Al3+/Pr3+/Sm3+ nanocomposite was synthesized and applied in highly selective and efficient separation of exosomes. Notably, the developed material exhibited higher specificity and efficiency than commercially available TiO2. Moreover, CaTiO3/Al3+/Pr3+/Sm3+ could be reused at least three times without any significant decrease in efficiency. The synthesized material was also used for the extraction of exosomes from the serums of patients with Alzheimer's disease (AD) and healthy controls. The exosomes were subjected to two-dimensional gel electrophoresis (2-DE) separation and matrix-assisted laser desorption/ionization-time of flight (MALDI TOF/TOF) mass spectrometry analysis. It was found that five proteins in the exosomes were evidently upregulated, while one protein was downregulated. Among the detected proteins, serum amyloid P-component (SAP) has been reported to be closely related to pathogenesis of AD. The obtained results indicated that the developed method involving separation and analysis of serum exosomes could be used for disease diagnosis or postoperative clinical monitoring.

Effects of graphene oxide on tomato growth in different stages.

The nano-carbon graphene has unique structural and physicochemical properties, which are conducive to various biomedical applications. We assessed the effect of graphene oxide (GO) on tomato plants at the seedling and mature stages in terms of morphological and biochemical indices. GO treatment significantly improved the shoot/stem growth of tomato in a dose-dependent manner by increasing the cortical cells number, cross-sectional area, diameter and vascular-column area. In addition, GO also promoted the morphological development of the root system and increased biomass accumulation. The surface area of root tips and hairs of tomato plants treated with 50 mg/L and 100 mg/L GO were significantly greater compared to the untreated control. At the molecular level, GO induced the expression of root development-related genes (SlExt1 and LeCTR1) and inhibited the auxin-responsive gene (SlIAA3). However, 50 mg/L and 100 mg/L GO significantly increased the root auxin content, which in turn increased the number of fruits and hastened fruit ripening compared to the control plants. Taken together, GO can improve the tomato growth when used at the appropriate concentration, and is a promising nano-carbon material for agricultural use.

Genome editing mediated by SpCas9 variants with broad non-canonical PAM compatibility in plants.

Streptococcus pyogenes Cas9 (SpCas9) is the most widely used genome editing tool in plants. The editing induced by SpCas9 strictly requires a canonical NGG protospacer-adjacent motif (PAM), significantly limiting its scope of application. Recently, five SpCas9 variants, SpCas9-NRRH, SpCas9-NRCH, SpCas9-NRTH, SpG, and SpRY, were developed to recognize non-canonical PAMs in human cells. In this study, these variants were engineered for plant genome editing, and their targeted mutagenesis capabilities were comprehensively examined at various canonical and non-canonical PAM sites in rice (Oryza sativa) by stable transformation. Moreover, both cytosine base editors using a rat APOBEC1 or a human APOBEC3a and adenine base editors using a directly evolved highly compatible TadA∗-8e deaminase were developed from these SpCas9 variants. Our results demonstrated that the developed SpCas9 variants-based base editors readily generated conversions between C∙G and T∙A in the target sites with non-canonical PAMs in transgenic rice lines. Collectively, the toolbox developed in this study substantially expands the scope of SpCas9-mediated genome editing and will greatly facilitate gene disruption and precise editing in plants.

Development of Plant Prime-Editing Systems for Precise Genome Editing.

Prime-editing systems have the capability to perform efficient and precise genome editing in human cells. In this study, we first developed a plant prime editor 2 (pPE2) system and test its activity by generating a targeted mutation on an HPT-ATG reporter in rice. Our results showed that the pPE2 system could induce programmable editing at different genome sites. In transgenic T0 plants, pPE2-generated mutants occurred with 0%-31.3% frequency, suggesting that the efficiency of pPE2 varied greatly at different genomic sites and with prime-editing guide RNAs of diverse structures. To optimize editing efficiency, guide RNAs were introduced into the pPE2 system following the PE3 and PE3b strategy in human cells. However, at the genomic sites tested in this study, pPE3 systems generated only comparable or even lower editing frequencies. Furthemore, we developed a surrogate pPE2 system by incorporating the HPT-ATG reporter to enrich the prime-edited cells. The nucleotide editing was easily detected in the resistant calli transformed with the surrogate pPE2 system, presumably due to the enhanced screening efficiency of edited cells. Taken together, our results indicate that plant prime-editing systems we developed could provide versatile and flexible editing in rice genome.

SpCas9-NG self-targets the sgRNA sequence in plant genome editing.

Streptococcus pyogenes Cas9 (SpCas9)-NG recognizes NGN protospacer adjacent motifs and expands the scope of genome-editing tools. In this study, we found that SpCas9-NG not only targeted the genome but also efficiently self-targeted the single-guide RNA sequence in transfer DNA in transgenic plants, potentially increasing off-target risk by generating new single-guide RNAs. We further showed that the self-target effect of SpCas9-NG could be greatly alleviated by using a modified single-guide RNA scaffold starting with a GCCCC sequence.