The two-step selection model is a widely accepted explanation of the mechanism of root-associated microbial communities. However, few studies have explained it from a time-series succession perspective. We analyzed the time-series succession patterns of bacterial communities in the rhizosphere and endosphere of soybeans using 16S ribosomal RNA (16S rRNA) sequencing. Rhizosphere selection showed temporal variation, with intensity increasing over time and leading to a decrease in the similarity between the rhizosphere and bulk soil communities from 0.42 to 0.17 during the first nine weeks. In contrast, endosphere selection was strong in the early stage but gradually weakening - with similarities between endosphere and bulk soil communities increasing from 0.10 to 0.12, and between endosphere and rhizosphere communities from 0.31 to 0.69. Predicted community functions also showed a gradient shift from bulk soil to rhizosphere and endosphere, further supporting the selection effect. The most representative functional taxa were those exhibiting chemoheterotrophy and aerobic chemoheterotrophy. Operational Taxonomic Units (OTUs) strongly associated with the growth stages, primarily from Proteobacteria, Acidobacteria, and Actinobacteria, showed decreasing abundance in the rhizosphere but increasing abundance in the endosphere. These results indicate that both rhizosphere selection and endosphere selection vary with plant growth. Rhizosphere selection is a gradually strengthening process dominated by inhibition, while endosphere selection is a gradually weakening process dominated by enrichment. The observed differences in temporal succession highlight distinct mechanisms of rhizosphere and endosphere selection, providing important insights into the two-step selection model.

Aliphatic glucosinolates are a large group of plant-specialized metabolites in Brassica vegetables, and some of their degradation products are key nutrients with significant beneficial effects on human health. Increasing the nutritional quality is one of the central research questions and breeding goals for Brassica vegetables. Major progress has been made in understanding transcriptional regulation of aliphatic glucosinolates biosynthesis in the model plant, while little is known about it in Brassica vegetables. In this study, we used cabbage to study the transcriptional regulation of BolBCAT4 genes, the first set of biosynthetic genes in methionine-derived aliphatic glucosinolate metabolism, and identified and functionally validated four upstream positive regulators of BolBCAT4 genes, BolMYB3R, BolbHLH153, BolMED4 and BolERF74, with consistent phenotypic effects of inducing short-chain aliphatic glucosinolates, including glucoraphanin. Our work confirmed the biological functions of BolBCAT4 genes, identified dozens of candidate upstream regulators, and provided valuable regulatory mechanisms and breeding targets for enhancing the nutritional quality of cabbage.

Indolic glucosinolates are a group of plant secondary metabolites found in Brassica vegetables, and their breakdown products could act as important anti-cancer and defense compounds against biotic stresses. Transcriptional regulation plays a key role in modulating the biosynthesis of indolic glucosinolates in the model plant Arabidopsis, but little is known about the transcriptional regulatory landscape of these glucosinolates in Brassica vegetables. In this study, we selected and functionally validated the important biosynthetic gene BolCYP83B1 from the indolic glucosinolate pathway in cabbage. Through a yeast one-hybrid assay, we systemically screened and identified upstream regulators of BolCYP83B1 in cabbage with BolANTs as the top candidates for further functional validation. Two homologs of BolANTs, BolANT1 and BolANT3, were confirmed to bind the promoter of BolCYP83B1 via both a yeast one-hybrid assay and an LUC assay. The overexpression of BolANT3 in cabbage significantly increased the accumulation of indolic glucosinolates, while the virus-induced gene silencing (VIGS) of BolANT3 significantly reduced the accumulation of indolic glucosinolates in cabbage. Our work provides valuable insights into the transcriptional regulatory mechanisms of indolic glucosinolates in Brassica vegetables.

Vertebrate limbs have undergone profound morphological diversification, enabling adaptations to a broad spectrum of ecological niches. In marine mammals, the evolution of highly specialized flipper-like forelimbs represents a profound structural transformation associated with aquatic habitats. This adaptation has been hypothesized to result, in part, from the inhibition of interphalangeal cell apoptosis during limb development, although the underlying genetic mechanism remains poorly understood. This study investigated the evolutionary dynamics and functional consequences of three key bone morphogenetic protein genes, BMP2, BMP4, and BMP7, which regulate apoptosis in interphalangeal mesenchymal stromal cells during embryonic limb development to ensure proper differentiation of interphalangeal tissues. Comparative genomic analysis revealed significantly accelerated evolution for BMP4 and BMP7 in the cetacean ancestral lineage, with two positively selected sites (V79I and H247R) involved in cetacean-specific amino acid substitutions located in the TGF-β propeptide functional domain in BMP4. In vitro assays confirmed that cetacean-specific BMP4 mutations significantly disrupted normal cell apoptosis and proliferation and altered the transcription and protein expression of downstream apoptosis-related factors, including cytochrome c (Cyt c), BCL2 associated X, and B-cell lymphoma 2, within the BMP signaling pathway. The significant influence of BMP4 mutations on apoptotic inhibition highlights a potential role in the development of limb bud mesenchymal tissue and the emergence of the flipper forelimb phenotype in cetaceans.

The use of biocatalysis technology to manufacture rare natural products can solve the contradiction between the market demand for rare natural products in large health industry fields and the protection and sustainable development of wildlife resources. However, the currently available research on fungal endophytes, which are great potential resources for glycoside hydrolase biocatalysts, is still insufficient. In this study, endophytic fungi from Epimedium brevicornum Maxim. were isolated in the Qinling Mountains, identified and tested for their potential to biotransform epimedium extracts into minor epimedium flavonoids. A total of 84 representative morphotype strains were isolated and identified via ITS rDNA sequence analyses and were grouped into 32 taxa. The Shannon‒Wiener index (H’, 3.089) indicated that E. brevicornum Maxim. harboured abundant fungal resources. Ten strains showed strong β-glucosidase activity and exhibited the ability to biotransform major epimedium flavonoids into deglycosylated minor epimedium flavonoids, such as baohuoside I and icaritin, via various glycoside-hydrolysing pathways. Among these strains, strains 8509 and F8889, which were initially characterized as Aspergillus ochraceus and A. protuberus, have the potential for further development in the biotransformation of epimedium extracts into minor epimedium flavonoids because of their excellent biosafety, enzyme activity, and enzymatic characteristics. The enzyme activity of the crude enzyme obtained by freeze-drying from the F8509 fermentation broth supernatant reached 78.24 ± 2.48 U/g. Further research revealed that major glycosylated flavonoids from 100 g/L epimedium extracts were bio-transformed completely into minor deglycosylated flavonoids in 90 min after the addition of 1 g/L crude enzyme. In addition, the liquid phase separation conditions were optimized, and ethyl alcohol and water were ultimately used as the mobile phase for efficient separation of the conversion products at equal flow degrees. This study not only identified a series of candidates for the biotransformation of minor epimedium flavonoids but also provided an efficient purification method. More importantly, this study also demonstrated the important value of endophytes in the biotransformation of rare natural products.

Developing a straightforward and effective strategy to modify antimicrobial peptides (AMPs) is crucial in overcoming the challenges posed by their instability and toxicity. Phosphorylation can reduce toxicity and improve the stability of AMPs. Based on these, we designed a series of peptides and their corresponding phosphorylated forms. The results showed that all phosphorylated peptides displayed reduced toxicity and enhanced stability compared to their unphosphorylated counterparts. Among them, W3BipY8-P stood out as the most promising peptide, exhibiting similar antibacterial activity as its unphosphorylated analog W3BipY8 but with significantly reduced hemolytic activity (19-fold decrease), cytotoxicity (3.3-fold decrease), and an extended serum half-life 6.3 times longer than W3BipY8. W3BipY8-P exerted bactericidal effects by disrupting bacterial membranes. Notably, W3BipY8-P significantly prolonged the survival of bacteria-infected animals while its LD50 was 4.2 times higher than that of W3BipY8. These findings highlight phosphorylation as an effective strategy for improving the antimicrobial properties of AMPs.

Protein tyrosine phosphatase 1B (PTP1B) has been identified as a key drug target for anti-tumor drug development. Oleanolic acid (OA) has been proved to be an inhibitor of PTP1B, but its poor water solubility, low bioavailability and poor activity in vivo limit its clinical efficacy. In this study, a total of 47 new OA derivatives including heteroatom derivatives, ester derivatives, amino substitution derivatives and Schiff base derivatives were designed and synthesized. Among them, OA-Br-1 had stronger inhibition and selectivity on PTP1B than OA, with IC50 value of 7.08 ± 5.05 μM for PTP1B and 222.28 ± 0.11 μM for TCPTP. In addition, OA-Br-1 significantly inhibited the proliferation and induced apoptosis of breast cancer cells, and in vivo nude mice experiments also showed that OA-Br-1 could inhibit the growth of breast tumors. Then network pharmacology was used to predict the targets of OA-Br-1, and the PPI network map between compound - breast cancer - target was constructed. The results showed that the probability value of PTPN1 ranked first among all predicted targets, which was consistent with the results of enzyme activity experiments in vitro. The enrichment results of KEGG pathway and GO functional annotation analysis showed that the effect of OA-Br-1 on breast cancer was significantly correlated with the PI3K/AKT pathway. Subsequent Western Blot results also proved that OA-Br-1 could significantly inhibit the expression of PTP1B, p-PI3K and p-AKT, indicating that OA-Br-1 played an anti-breast cancer role through the PTP1B/PI3K/AKT signaling pathway. Collectively, these findings identify OA-Br-1 as a promising PTP1B inhibitor for breast cancer treatment.

>CRISPR(clustered regularly interspaced short palindromic repeats)-Cas systems, fundamental to prokaryotic adaptive immunity, are divided into two major classes based on their effector protein composition. Class 1 systems, including types Ⅰ, Ⅲ, and Ⅳ, utilize multi-subunit complexes to carry out interference(Wang et al., 2022),

Inflammatory bowel disease remains a high recurrence rate and broad populations all over the world. Liquiritigenin isolated from licorice possess anti-inflammatory and antioxidative activities, suggesting its potential of treating ulcerative colitis (UC). In this study, we explored the effect of oral liquiritigenin on the DSS-induced mice colitis and underlying mechanisms in the aspect of gut microbiome and intestinal barrier dysfunction. The results showed liquiritigenin had protective effects against DSS-induced mice colitis, including attenuating weight loss, disease activity index score elevation, colon length shortening and histological lesions. In addition, the treatment with liquiritigenin significantly reduced the plasma cytokine levels, TNF-α, IL-1β and IL-6 levels as well as the expression of iNOS and COX-2 compared to control group. Liquiritigenin supplementation also led to a restoration of oxidative homeostasis, as indicated by a decrease of myeloperoxidase (MPO) and malondialdehyde (MDA) levels and an increase in reduced glutathione (GSH) and total Superoxide Dismutase (T-SOD) activities in the colitis mice. In regulation of mice gut microbiota, liquiritigenin augmented probiotics abundance (e.g., Akkermansia), decreased harmful bacteria (e.g., Turicibacter), and restored Firmicutes/Bacteroidetes balance. Furthermore, the mRNA levels of tight junction (TJ), including ZO-1, occludin, and claudin-1 were downregulated in colitis mice, whereas these changes were reversed by liquiritigenin. As indicated by TEM images and Endoplasmic reticulum stress (ERs) marker genes expression like GRP78, IRE1α, ATF6 and PERK that mice with colitis treated by oral liquiritigenin trended to attenuate ER stress. Collectively, the ameliorating role of liquiritigenin in gut inflammation can be attributed to intestinal barrier modulation, gut microbiome regulation, and ER stress alleviation. These findings provide a new perspective for developing liquiritigenin as a promising functional compound of food origin for preventing and mitigating UC.

Accurate prediction of drug-target interactions (DTIs) is critical for drug design and optimization in pharmacology. Existing models face challenges such as data sparsity and lack of contextual information, resulting in poor accuracy. Knowledge graphs (KGs) provide a solution by representing relationships in biological data. However, current KG-based DTI methods are limited to static graphs that require time-consuming retraining when knowledge is updated. In this paper, we propose SAGS-DynamicBio, an efficient dynamic embedding model for biological data that integrates semantics and graph structure information. We first generate KGs for the biological knowledge base, representing drugs and targets as entities and interactions as relations. Using KG embedding techniques, we convert each entity and relation into a vector representation. To effectively handle dynamic data, we introduce a semantic perception module based on the attention mechanism, which uses information from neighboring nodes to generate initial representation vectors for new data. Furthermore, we apply graph structure-based representation learning to these initial vectors to satisfy KG’s structural constraints and improve prediction accuracy. To evaluate the effectiveness of our method, we conduct experiments comparing SAGS-DynamicBio with existing KG-based DTI prediction models and generic KGE models. The experimental results show that our method significantly improves the embedding efficiency, reducing the embedding time by 41.5

ABSTRACT Systemic inflammatory response syndrome (SIRS) is a severe inflammatory response that can lead to organ dysfunction and death. Modulating the gut microbiome is a promising therapeutic approach for managing SIRS. This study assesses the therapeutic potential of the Xuanfei Baidu (XFBD) formula in treating SIRS. The results showed that XFBD administration effectively reduced mortality rates and inflammation in SIRS mice. Using 16S rRNA sequencing and fecal microbiota transplantation (FMT), we substantiated that the therapeutic effects of XFBD are partly attributed to gut microbiota modulation. We conducted in vitro experiments to accurately assess the gut microbiome remodeling effects of 51 compounds isolated from XFBD. These compounds exhibited varying abilities to induce a microbial structure that closely resembles that of the healthy control group. By quantifying their impact on microbial structure and clustering their regulatory patterns, we devised multiple gut microbiome remodeling compound (GMRC) cocktails. GMRC cocktail C, comprising aucubin, gentiopicroside, syringic acid, gallic acid, p-hydroxybenzaldehyde, para-hydroxybenzoic acid, and isoimperatorin, demonstrated superior efficacy in treating SIRS compared to a single compound or to other cocktails. Finally, in vitro experiments showcased that GMRC cocktail C effectively rebalanced bacteria composition in SIRS patients. This study underscores XFBD’s therapeutic potential in SIRS and highlights the importance of innovative treatment approaches for this disease by targeting the gut microbiota. IMPORTANCE Developing effective treatment strategies for systemic inflammatory response syndrome (SIRS) is crucial due to its severe and often life-threatening nature. While traditional treatments like dexamethasone have shown efficacy, they also come with significant side effects and limitations. This study makes significant strides by demonstrating that the Xuanfei Baidu (XFBD) formula can substantially reduce mortality rates and inflammation in SIRS mice through effective modulation of the gut microbiota. By quantitatively assessing the impact of 51 compounds derived from XFBD on the gut microbiome, we developed a potent gut microbiome remodeling compound cocktail. This cocktail outperformed individual compounds and other mixtures in efficacy against SIRS. These findings highlight the potential of XFBD as a therapeutic solution for SIRS and underscore the critical role of innovative strategies targeting the gut microbiota in addressing this severe inflammatory condition.

Sheep is an important livestock species raised globally to pro-duce meat,milk,wool,and other by-products.During the Neolithic Revolution,sheep were domesticated in the Fertile Crescent of Southwest Asia around 10,000 years ago(Chessa et al.,2009).Nat-ural and artificial selection has been driving the development of various sheep breeds/ecotypes adapted to different climatic and production conditions.Sheep breeds often differ in coat color,pres-ence or absence of horns,tail shape,etc.Sheep tails can be divided into five main patterns,including fat-long tail,fat-short tail,thin-long tail,thin-short tail,and fat rump(i.e.,'tailless'),which are determined by the length and number of caudal vertebrae as well as the amount of fat deposition.In modern breeding programs,the thin-short tail is considered preferable since other tail patterns have specific issues of concern.For example,fat tails are indicated to be associated with low mating success,animal locomotion,excessive fat deposition,increased production costs,and reduced consumer preference.Similarly,thin long-tailed sheep require tail docking as part of the an-imal management practice,which is a concern for animal welfare.

Prime editors (PEs) are promising genome-editing tools, but effective optimization of prime-editing guide RNA (pegRNA) design remains a challenge owing to the lack of accurate and broadly applicable approaches. Here we develop Optimized Prime Editing Design (OPED), an interpretable nucleotide language model that leverages transfer learning to improve its accuracy and generalizability for the efficiency prediction and design optimization of pegRNAs. Comprehensive validations on various published datasets demonstrate its broad applicability in efficiency prediction across diverse scenarios. Notably, pegRNAs with high OPED scores consistently show significantly increased editing efficiencies. Furthermore, the versatility and efficacy of OPED in design optimization are confirmed by efficiently installing various ClinVar pathogenic variants using optimized pegRNAs in the PE2, PE3/PE3b and ePE editing systems. OPED consistently outperforms existing state-of-the-art approaches. We construct the OPEDVar database of optimized designs from over two billion candidates for all pathogenic variants and provide a user-friendly web application of OPED for any desired edit.

Hair follicle stem cells (HFSCs) are an important basis for hair follicle morphogenesis and hair cycle growth. This cell type also represents an excellent model for studying the gene function and molecular regulation of the hair growth cycle, including proliferation, differentiation, and apoptosis. Basically, the functional investigation of hair growth-regulating genes demands a sufficient amount of HFSCs. However, efficient propagation of HFSCs in goats is a challenging process under the current culture conditions. Here, we investigated the effect of four components, including the Rho-associated protein kinase (ROCK) inhibitor Y-27632, leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and vitamin C, on cell growth and pluripotency in the basal culture medium (DMEM/F12 supplemented with 2% fetal bovine serum). We found that adding Y-27632, LIF, and bFGF independently increased the proliferation and pluripotency of goat HFSCs (gHFSCs), with Y-27632 having the most significant effect (P < 0.001). Fluorescence-activated cell sorting of the cell cycle revealed that Y-27632 promoted gHFSC proliferation by inducing the cell cycle from S to G2/M phase (P < 0.05). We further demonstrated that gHFSCs displayed superior proliferative capacity, clone-forming ability, and differentiation potential in the combined presence of Y-27632 (10 μM) and bFGF (10 ng/mL). We termed this novel culture condition as gHFEM, which stands for goat Hair Follicle Enhanced Medium. Taken together, these results indicate that gHFEM is an optimal condition for in vitro culture of gHFSCs, which will subsequently facilitate the study of HF growth and biology.

Abstract Prime editors (PEs) are promising genome editing tools, but efficiency pre-testing of prime editing guide RNA (pegRNA) design is still laborious and time-consuming due to the lack of accurate and universal approaches. Here, we design a customized attention-based model OPED and train it using transfer learning to improve the accuracy and universality of efficiency prediction and design optimal pegRNAs. We demonstrate its powerful generalization capability across diverse published test datasets. Furthermore, we extend OPED to design optimal pegRNAs and single guide RNAs (sgRNAs) to install various ClinVar human pathogenic variants, and 28 of 30 (93.33%) target sites yield desired variants with few byproducts and practical editing efficiencies of up to 29.30%, 82.84%, and 90.05% for PE2, PE3/PE3b, and ePE systems, respectively. We construct the OPEDVar database of optimal designs from over two billion candidates for all ClinVar variants and provide a user-friendly web application of OPED for any intended edit.

The rapid advances in genome editing technologies have revolutionized the study of gene functions in cell or animal models. The recent generation of double-stranded DNA cleavage-independent base editors has been suitably adapted for interrogation of protein-coding genes on the basis of introducing premature stop codons or disabling the start codons. However, such versions of stop/start codon-oriented genetic tools still present limitations on their versatility, base-level precision, and target specificity. Here, we exploit a newly developed prime editor (PE) that differs from base editors by its adoption of a reverse transcriptase activity, which enables incorporation of various types of precise edits templated by a specialized prime editing guide RNA. Based on such a versatile platform, we established a prime editing-empowered method (PE-STOP) for installation of nonsense substitutions, providing a complementary approach to the present gene-targeting tools. PE-STOP is bioinformatically predicted to feature substantially expanded coverage in the genome space. In practice, PE-STOP introduces stop codons with good efficiencies in human embryonic kidney 293T and N2a cells (with medians of 29% [ten sites] and 25% [four sites] editing efficiencies, respectively), while exhibiting minimal off-target effects and high on-target precision. Furthermore, given the fact that PE installs prime editing guide RNA-templated mutations, we introduce a unique strategy for precise genetic rescue of PE-STOP-dependent nonsense mutation via the same PE platform. Altogether, the present work demonstrates a versatile and specific tool for gene inactivation and for functional interrogation of nonsense mutations.

The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential.

Nitrogen-fixing root nodules are formed by symbiotic association of legume hosts with rhizobia in nitrogen-deprived soils. Successful symbiosis is regulated by signals from both legume hosts and their rhizobial partners. HmuS is a heme degrading factor widely distributed in bacteria, but little is known about the role of rhizobial hmuS in symbiosis with legumes. Here, we found that inactivation of hmuSpSym in the symbiotic plasmid of Mesorhizobium amorphae CCNWGS0123 disrupted rhizobial infection, primordium formation, and nitrogen fixation in symbiosis with Robinia pseudoacacia. Although there was no difference in bacteroids differentiation, infected plant cells were shrunken and bacteroids were disintegrated in nodules of plants infected by the ΔhmuSpSym mutant strain. The balance of defence reaction was also impaired in ΔhmuSpSym strain-infected root nodules. hmuSpSym was strongly expressed in the nitrogen-fixation zone of mature nodules. Furthermore, the HmuSpSym protein could bind to heme but not degrade it. Inactivation of hmuSpSym led to significantly decreased expression levels of oxygen-sensing related genes in nodules. In summary, hmuSpSym of M. amorphae CCNWGS0123 plays an essential role in nodule development and maintenance of bacteroid survival within R. pseudoacacia cells, possibly through heme-binding in symbiosis.

Prime editing is a newly developed CRISPR/Cas system-based genome editing technique. The effector of prime editor (PE) is termed PE2, which is generated by fusing a reverse transcriptase (RT) with a Cas9 H840A nickase. The guide RNA of PE is termed prime editing guide RNA (pegRNA), which consists of a single guide RNA (sgRNA) with a 3' extension containing the RT template (RTT) and primer binding site (PBS). PE can install all 12 types of point mutations, small insertions and deletions and combinations thereof. Since its emergence in 2019, with the high versatility and specificity, PE has been applied to many living organisms, including animals, plants and bacteria. This led to many explorations of PE on gene therapy and genetic improvement in agriculture. In this review, we systematically describe the development, characteristics, optimizations, applications and security of PE. In addition, we discuss the future applications of PE. We expect that this review will help researchers to grasp and better use PE.

The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.