Heat and drought are increasingly concurrent extreme climate events that directly restrict tree growth and development. However, the molecular mechanisms underlying tree responses to combined drought-heat stress remain poorly understood. Here, we demonstrate that combined drought-heat stress causes markedly stronger and more rapid physiological damage in poplar than the individual stress. Under combined stress, the photosynthetic rate declined by 98.2% relative to the control, and leaf chlorosis and tissue damage were evident as early as the second day of treatment. In contrast, photosynthetic rate decreased by approximately 75.19% under heat stress and 61.4% under drought stress. Principal component analysis further indicated that heat dominates physiological variation under combined stress, while drought exacerbates heat-induced injury and accelerates stress symptoms. Time-ordered gene co-expression network (TO-GCN) of combined stresses identified three hierarchical response stages in poplar and revealing that poplar employs specific pathways involved in primary metabolism, hormone signaling, structural remodeling and apoptosis. Integrating TO-GCN and expression pattern analyses, we identified PagMBF1C as a key gene in poplar's response to combined stresses. Functional analyses showed that PagMBF1C-overexpressing lines maintained higher photosynthetic capacity and physiological stability under combined stresses, whereas RNAi-mediated knockdown lines exhibited pronounced sensitivity. Mechanistically, PagMBF1C physically interacts with PagWRKY75 and activates downstream drought and heat-responsive genes of PagPIP1B and PagHsf4. Collectively, this study clarifies the regulatory mechanism of the PagMBF1C-WRKY75 regulatory module in mediating drought and heat tolerance in poplar by reducing stomatal area and the loss of chlorophyll content, providing new strategies for utilizing stress-tolerant germplasm to advance molecular breeding.

AbstractCas12a is an RNA-guided endonuclease that has emerged as a powerful gene-editing tool. We have identified a novel Cas protein, BvCas12a, fromButyricimonas virosawith a 5’-TYTN protospacer adjacent motif (PAM). BvCas12a exhibits double-stranded DNA cleavage activityin vitroand genome editing activity in eukaryotic cells. Though the editing efficiency of BvCas12a is marginally lower than that of AsCas12a, the editing specificity of BvCas12a in eukaryotic cells is comparable or superior to that of AsCas12a. Moreover, BvCas12a exhibits substantial collateral activity and can detect HPV DNA effectively and accurately in conjunction with isothermal amplification, highlighting its potential in nucleic acid diagnostics. Furthermore, we have engineered BvCas12a to create two variants, BvCas12a-R (N549R, T606P) and BvCas12a-RVR (N549R, K555V, C559R, T606P). These variants recognize expanded 5’-YYN and 5’-YN PAMin vitro, respectively. Additionally, they exhibit higher editing activity than wild-type BvCas12a and recognize 5’-YYN PAMin vivoat all sites detected. In conclusion, we have identified a novel BvCas12a protein with high specificity and engineered two variants with broader PAM compatibility and improved genome editing efficiency. These findings offer a potent gene editing tool for application in scientific research, gene therapy, and nucleic acid diagnostics.

Background: CHB patients with NAs still suffer from TCM symptoms which affect the quality of life varying severity. The pathogenesis of CHB in TCM is the accumulation of damp-heat, while there are sub-types of damp heat syndrome. Objective: To evaluate the clinical efficacy and safety of Yinchen Wuling powder combined with NAs on CHB patients of damp-heat and damp-predominant syndrome, while investigate the potential mechanisms and novel applications of classical prescriptions through network pharmacology combined with metabolomics. Design setting participants and intervention: It was a randomized, placebo-controlled clinical study at 3 hospitals in Shanghai. A total of 62 participants diagnosed with damp-heat and damp-predominant syndrome of CHB patients with NAs were randomly allocated to receive either Yinchen Wuling powder or placebo; the therapy consisted of 8.4 g dosage, administered twice a day after meals for a duration of 4 weeks. Blood samples were collected for non-targeted metabolomics research of characteristic metabolites of CHB patients and pathways enriched by YCWLP treatment. The metabolic network and target of YCWLP on CHB patients of damp-heat and damp-predominant syndrome was constructed by the integration of network pharmacology and non-targeted metabolomics.

Drought stress severely limits the growth of perennial trees. Xylem structure is central to water conduction; however, lignin monomer composition driving xylem remodeling for drought adaptation remains enigmatic. By integrating multi-omics analyses, genotype-environment association analysis, and metabolite-based genome-wide association studies, we identified PtoCPK3 as a key gene associated with precipitation-related traits and the aridity index in Populus tomentosa. This variation in association was linked to coniferyl alcohol and ferulic acid, two metabolites related to guaiacyl (G)-lignin monomer biosynthesis. Overexpressing PtoCPK3 enhanced drought tolerance in transgenic poplar by promoting xylem remodeling associated with a decreased lignin S/G ratio. Mechanistically, drought-induced Ca2+ signaling activates PtoCPK3 to phosphorylate PtoERF72 at Ser96, enhancing its activation of PtoWOX13b and direct repression of PtoUGT72AZ2. Synergistic repression of PtoUGT72AZ2 by PtoWOX13b reduces glycosylation of G-monomer precursors, favoring ferulic acid and coniferyl alcohol accumulation. Furthermore, natural variants in PtoCPK3 and PtoERF72 drive geographic divergence, with the PtoCPK3II_PtoERF72CC genotype conferring superior drought resilience via elevated phosphorylation efficiency. This module links drought signaling to xylem remodeling, providing genetic targets for breeding drought-resilient trees.

Importance No oral medication is currently approved for the management of urolithiasis. Guang Jing Qian Cao ( Desmodium styracifolium total flavone capsules; hereinafter, Guang Jing ), a traditional Chinese herbal extract, has shown clinical benefits for urolithiasis, but randomized clinical trials are needed to assess its effectiveness. Objective To evaluate whether Guang Jing improves stone passage rates (SPRs) compared with placebo in adults with urolithiasis. Design, Setting, and Participants This double-blind, placebo-controlled, phase 3 randomized clinical trial was conducted at 34 sites in China from December 2017 to April 2020. Participants included adults (aged 18-70 years) with diagnosed ureteral stones. Data analysis was conducted on November 4, 2020. Intervention Participants were randomized 3:1 to receive oral Guang Jing (0.6 g) or matching placebo 3 times daily for 28 days, in addition to investigator-prescribed background medication. Main Outcomes and Measures The primary outcome was SPR by day 28, confirmed by computed tomography. Secondary outcomes included SPR by day 14, stone migration rate, and stone migration distance. Between-group comparisons were performed using the Cochran-Mantel-Haenszel test for categorical outcomes and t tests for continuous outcomes. Results A total of 606 participants were randomly assigned to receive Guang Jing (n = 458) or placebo (n = 148). Their mean (SD) age was 43.0 (12.0) years, 474 (78.2%) were male, and the mean (SD) stone size was 0.6 (0.1) cm. The SPR by day 28 was significantly higher for the Guang Jing group compared with the placebo group (204 of 457 [44.6%] vs 50 of 148 [33.8%]; relative risk, 1.32 [95% CI, 1.03-1.69]; P = .03), with an absolute risk difference of 10.9 (95% CI, 2.0-19.7) percentage points. No significant between-group differences in SPR by day 14 (Guang Jing vs placebo: 133 [29.1%] vs 33 [22.3%]; P = .14) or stone migration distance (mean [SD], 29.5 [51.8] mm vs 29.7 [43.8] mm; P = .11) were observed. Adverse event rates were similar for the Guang Jing and placebo groups (88 [19.3%] vs 27 [18.2%]). Conclusions and Relevance In this randomized clinical trial, treatment with Guang Jing significantly increased the expulsion of 5- to 10-mm ureteral stones by day 28, with a favorable safety profile. These findings suggest that Guang Jing may be an additional medical expulsive therapy option for appropriately selected patients. Trial Registration Chinese Clinical Trial Registry Identifier: ChiCTR-IIR-17013275

Importance:No oral medication is currently approved for the management of urolithiasis. Guang Jing Qian Cao (Desmodium styracifolium total flavone capsules; hereinafter, Guang Jing), a traditional Chinese herbal extract, has shown clinical benefits for urolithiasis, but randomized clinical trials are needed to assess its effectiveness. Objective:To evaluate whether Guang Jing improves stone passage rates (SPRs) compared with placebo in adults with urolithiasis. Design, Setting, and Participants:This double-blind, placebo-controlled, phase 3 randomized clinical trial was conducted at 34 sites in China from December 2017 to April 2020. Participants included adults (aged 18-70 years) with diagnosed ureteral stones. Data analysis was conducted on November 4, 2020. Intervention:Participants were randomized 3:1 to receive oral Guang Jing (0.6 g) or matching placebo 3 times daily for 28 days, in addition to investigator-prescribed background medication. Main Outcomes and Measures:The primary outcome was SPR by day 28, confirmed by computed tomography. Secondary outcomes included SPR by day 14, stone migration rate, and stone migration distance. Between-group comparisons were performed using the Cochran-Mantel-Haenszel test for categorical outcomes and t tests for continuous outcomes. Results:A total of 606 participants were randomly assigned to receive Guang Jing (n = 458) or placebo (n = 148). Their mean (SD) age was 43.0 (12.0) years, 474 (78.2%) were male, and the mean (SD) stone size was 0.6 (0.1) cm. The SPR by day 28 was significantly higher for the Guang Jing group compared with the placebo group (204 of 457 [44.6%] vs 50 of 148 [33.8%]; relative risk, 1.32 [95% CI, 1.03-1.69]; P = .03), with an absolute risk difference of 10.9 (95% CI, 2.0-19.7) percentage points. No significant between-group differences in SPR by day 14 (Guang Jing vs placebo: 133 [29.1%] vs 33 [22.3%]; P = .14) or stone migration distance (mean [SD], 29.5 [51.8] mm vs 29.7 [43.8] mm; P = .11) were observed. Adverse event rates were similar for the Guang Jing and placebo groups (88 [19.3%] vs 27 [18.2%]). Conclusions and Relevance:In this randomized clinical trial, treatment with Guang Jing significantly increased the expulsion of 5- to 10-mm ureteral stones by day 28, with a favorable safety profile. These findings suggest that Guang Jing may be an additional medical expulsive therapy option for appropriately selected patients. Trial Registration:Chinese Clinical Trial Registry Identifier: ChiCTR-IIR-17013275.

Drought stress limits forest tree growth and adaptation, with xylem vessels critical for hydraulic transport and structural integrity. However, the molecular mechanisms of abscisic acid (ABA)-auxin interaction in regulating vessel morphogenesis under water-deficit conditions remain unclear. Here, we identified PtomiR393a, a drought-responsive microRNA in Populus tomentosa that mediates crosstalk between ABA and auxin signaling pathways under drought stress. Suppressing PtomiR393 enhanced drought tolerance and growth, whereas its overexpression had the opposite effect. Under drought conditions, suppression of PtomiR393 resulted in reduced vessel size (12.18-13.57%) and increased vessel density (27.22-30.14%), while its overexpression exhibited increased vessel size (15.42-16.01%) and reduced vessel density (19.80-20.62%). Functional assays showed that PtomiR393 specifically targets PtoFBL4, an F-box auxin receptor, modulating auxin signaling in response to drought stress. Expression analyses further revealed that PtomiR393 downregulates genes involved in vessel and fiber formation and secondary cell wall biosynthesis by repressing PtoFBL4-mediated auxin signaling. Furthermore, drought-induced ABA signaling activated PtoERF1 expression via PtoAREB13, thereby inhibiting PtomiR393a expression. The study revealed a PtoERF1-PtomiR393a-PtoFBL4 cascade that links ABA-auxin crosstalk and regulates vessel development under drought stress. These findings offer new insights into drought tolerance mechanisms in trees and suggest potential strategies to enhance forest tree resilience to water-deficit conditions.

A comprehensive atlas of genes, cell types, and their spatial distribution across a whole mammalian brain is fundamental for understanding the function of the brain. Here, using single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq techniques, we generated a mouse brain atlas with spatial information for 308 cell clusters at single-cell resolution, involving over 4 million cells, as well as for 29,655 genes. We have identified cell clusters exhibiting preference for cortical subregions and explored their associations with brain-related diseases. Additionally, we pinpointed 155 genes with distinct regional expression patterns within the brainstem and unveiled 513 long non-coding RNAs showing region-enriched expression in the adult brain. Parcellation of brain regions based on spatial transcriptomic information revealed fine structure for several brain areas. Furthermore, we have uncovered 411 transcription factor regulons showing distinct spatiotemporal dynamics during neurodevelopment. Thus, we have constructed a single-cell-resolution spatial transcriptomic atlas of the mouse brain with genome-wide coverage.

Strigolactones (SLs) are a class of carotenoid-derived terpenoid lactones. Recent studies have revealed the mechanisms by which endogenous SLs control numerous aspects of plant architecture formation and growth, including shoot branching, root architecture development, and the responses to various environmental stresses such as drought, cold, and low-phosphorus and low-nitrogen levels. As a relatively newly discovered type of plant hormone, SLs are gaining attention for their exceptional development value and application potential in the fields of agricultural science and forestry biology. In this review, we initially summarize the discovery history and structural characteristics of SLs. Then, we introduce the physiological regulatory patterns and molecular regulatory mechanisms of SLs in plant growth and development regulation, abiotic stress adaptation, and the interactions of SLs with other hormones. Finally, we discuss recent advances in SL research in forest trees and outline future research frontiers and emerging techniques involving SLs. Thus, this review provides theoretical guidance and technical support for the physiological and molecular basis of SL use in regulating plant growth and development and offers new perspectives for investigating the genetic regulatory networks of important traits for improved plant breeding via molecular design.

The protein kinase (PK) superfamily holds paramount importance in the plant kingdom, orchestrating myriad cellular processes integral to plant growth, development, as well as resilience against environmental stresses. Nonetheless, the extant biological data about the PK superfamily in perennial woody species remains sparse. In this study, our comprehensive genomic survey of Populus tomentosa unveiled an expansive repertoire of 1543 PK genes, meticulously classified into nine discrete groups and 125 specialized subfamilies. A collinearity analysis revealed that whole genome duplication events served as the preeminent driving forces for PK superfamily expansion. Utilizing cluster analysis of expression profiling in different tissues, we identified 248 PK genes distributed across two clusters implicated in secondary cell wall (SCW) biosynthesis and lignification. Additionally, an association mapping study conducted within a natural population of P. tomentosa pinpointed 176 significant associations, corresponding to 67 PK genes, which were linked to SCW compositional attributes. These findings underscore the genetic determinants shaping PK-mediated modulation of SCW biosynthesis in Populus species. Of particular note, the causative PK gene PtoCMGC48 emerged as a linchpin, exhibiting a pronounced signal associated with lignin content and was found to interact with and phosphorylate the SCW-related transcription factor PtoVND6-C2. This discovery posits a novel molecular paradigm wherein PKs orchestrate SCW biosynthesis via targeted phosphorylation events in P. tomentosa. Overall, our findings lay the groundwork for a deeper appreciation of PK gene functionalities embedded within the complex network governing SCW formation.

Secondary cell walls (SCWs) are critical for providing mechanical strength to the vascular tissues of plants, and the composition of SCWs is a key determinant of wood properties in trees. However, the genetic regulatory networks that dictate the function of basic helix-loop-helix (bHLH) transcription factors in governing SCW formation in perennial woody plants remain largely unexplored. Here, we deciphered the genetic basis of bHLH members, a major transcription factor family in plants, for wood chemical composition in a natural population of Populus tomentosa, using association mapping and preferential expression pattern analysis. We identified the genetic variant in PtobHLH125, preferentially expressed in mature xylem, which showed a peak signal (P = 5.66E-07) for lignin content in P. tomentosa. Knockdown plants of PtobHLH125 gene in poplar showed a decrease in xylem cell size (similar to 26.23 %) and lignin content (similar to 17.92 %) compared to wild-type plants, indicating the beneficial function of PtobHLH125 in lignin biosynthesis. Through transcriptome analysis and molecular experiments, we uncovered the genetic regulatory network of PtobHLH125 and demonstrated that PtobHLH125 regulates lignin content by influencing the expression of PtoCCoAOMT1 (caffeoyl-CoA O-methyltransferase), a key gene in lignin biosynthesis, in P. tomentosa. Strikingly, the long non-coding RNA TCONS_00088706 was predicted to be involved in regulating the PtobHLH125-PtoCCoAOMT1 module for lignin content, as revealed by expression correlations and epistatic analysis. Our findings propose a potential molecular cascade, TCONS_00088706-PtobHLH125-PtoCCoAOMT1, for SCW formation by regulating lignin content, exploiting the allelic combination that could be used in the molecular genetic improvement of wood property traits in Populus.

Background: Neuregulin-1 (NRG1) levels were elevated in prostate cancer patients receiving androgen deprivation therapy (ADT). However, it remains unclear whether NRG1 levels could predict castration-resistant prostate cancer (CRPC) progression. Methods: Prostate cancer patients were divided into CRPC and NCRPC (non-CRPC) groups. Baseline clinicopathological characteristics and prostate-specific antigen (PSA) levels were compared among two groups. Subsequently, the levels of NRG1 in blood and tumor tissue were detected using enzyme-linked immunosorbent assay, western blotting, and qPCR. The predictive value of NRG1 was evaluated using receiver operating characteristic (ROC) analysis. Meanwhile, the correlation of NRG1 with Gleason score and PSA levels was analyzed using Spearman analysis. Results: The comparison analysis showed that TNM classification, Gleason scores, and PSA levels were significantly correlated to CRPC. Moreover, the serum NRG1 and the protein and mRNA levels of NRG1 were higher in CRPC patients than in NCRPC patients. ROC analysis unveiled that NRG1 levels in the patients before ADT could predict CRPC progression. Moreover, Spearman analysis also showed that NRG1 was correlated to Gleason scores and PSA levels. Conclusions: Serum NRG1, NRG1 protein, and NRG1 mRNA in tumor tissue from prostate cancer patients before ADT could predict the incidence of CRPC in patients receiving ADT.

Plants have evolved complex signal transduction networks to regulate metabolism and adapt to their environment. Calcium ions serve as key messengers in these pathways, playing central roles in a wide range of signal transduction processes. In plants, multiple classes of calcium-binding proteins can detect transient calcium signal fluctuations triggered by various stimuli, and initiate downstream responses. Calcium-dependent protein kinases (CDPKs), single-molecule Ca2+ sensor and effector proteins in plants, detect calcium signals and convert them into phosphorylation events, making them ideal tools for signal transduction. In this review, we provide a comprehensive review of the structural characteristics and functional features of CDPKs. We present a systematic analysis of the modular architecture of CDPKs, which determines their biological functions in plants. To elucidate how CDPKs localization to specific organelles shapes their regulatory roles in development and stress signaling, we discuss the regulatory roles of CDPKs in plant developmental processes through environmental signal transduction and hormonal signaling cascades. We further delineate the functional specialization of CDPKs in responses to abiotic stress (e.g., drought, salinity, cold, and heat) and biotic stress (e.g., pathogen defense). Three priority directions for future CDPK research are also proposed: elucidation of the molecular mechanisms underlying growth plasticity; determination of multi-signal homeostasis maintenance mechanisms; and exploring allele-specific variations for crop adaptability improvement. This review establishes a foundation for the relation between CDPK-mediated signaling networks and plant phenotypic plasticity in fluctuating environments.

Paclitaxel resistance of castration-resistant prostate cancer (CRPC) remains a substantial challenge in clinical oncology. Our investigation aimed to explore the potential of stachydrine hydrochloride in addressing paclitaxel resistance within CRPC. Parental prostate cancer cells, PC3 and DU145, and the corresponding paclitaxel-resistant counterparts, PC3-TxR and DU145-TxR, were subjected to indicated concentrations to determine the IC50 of paclitaxel and stachydrine hydrochloride. Cell viability and proliferation were evaluated by MTT and colony formation assays. Stachydrine hydrochloride–mediated apoptosis, senescence, and ferroptosis were detected using Western blot, SA-β-gal staining, and QuantiChrom iron assay. We found a pronounced reduction in paclitaxel resistance in both PC3-TxR and DU145-TxR cells following exposure to stachydrine hydrochloride. The considerable decrease in paclitaxel’s IC50 values in these cells highlights the potential of stachydrine hydrochloride in sensitizing CRPC cells to paclitaxel-based therapies. Mechanistically, stachydrine hydrochloride treatment significantly upregulated the expression of estrogen receptor beta (ERβ) and promoted cell apoptosis, senescence, and ferroptosis pathways in CRPC. Our study provides promising insights into the potential of stachydrine hydrochloride as a novel therapeutic adjunct in overcoming paclitaxel resistance in CRPC.

Alternative splicing plays a crucial role in enhancing the protein diversity of eukaryotic genomes. However, alternative splicing has not been extensively studied in Botrytis cinerea. In this study, we examined the distribution and regulation of alternative splicing in the filamentous plant pathogenic fungus B. cinerea through strand-specific RNA sequencing at various stages of infection of Solanum lycopersicum. During infection (pre-penetration and biotrophic stage), most spliceosome genes had upregulated expression levels, indicating that splicing is altered at this stage. A total of 3308 genes underwent alternative splicing in B. cinerea, resulting in 7466 alternative splicing events, most of which were stage-specific. Transcripts generated through alternative splicing typically exhibit lower expression levels, coding potential, and functional domains, which are more prevalent during the hyphal phase compared to the infestation phase. To conclude, our research offers an extensive analysis of the genome-wide alternative splicing in B. cinerea throughout the infection process, acting as a significant resource for further clarifying the pathogenic mechanisms associated with B. cinerea.

Photosynthesis directly determines plant biomass accumulation by controlling carbon flow and energy input. Thus, increasing photosynthetic efficiency is a promising approach for boosting plant growth and yield. However, the genetic basis of photosynthesis in perennial woody plants remains largely unknown, and the causative alleles warrant comprehensive investigation. Here, we performed a genome-wide association study (GWAS) on photosynthetic traits in a natural population of Chinese white poplar (Populus tomentosa). We identified inflorescence and root apices receptor-like kinase-interacting protein (IRKI) as a causative gene of photosynthesis that is significantly associated with the activity of rubisco activase (Rca). The seventh leaves of PtoIRKI-overexpression (OE) plants exhibited a 27.77% increase in net photosynthetic rate (Pn), a 31.42% rise in starch content, and a 16.83% expansion in leaf area compared to wild-type plants, whereas ptoirki-knockdown (KD) plants displayed the opposite phenotypes. Further analyses indicated that PtoIRKI interacted with PtoRca to enhance Rca activity, leading to increases in the activation state of ribulose bisphosphate carboxylase oxygenase (rubisco) and photosynthetic efficiency. Importantly, we identified an elite haplotype, PtoIRKIhap2, which exhibited higher PtoIRKI expression and Pn than PtoIRKIhap1. Finally, we found that homeodomain-leucine zipper protein 1 (PtoHB1) specifically bound to the PtoIRKIhap2 promoter, thereby promoting PtoIRKI expression and photosynthetic efficiency, as validated by integrating machine learning models and molecular experiments. Our results shed light on the molecular mechanism through which PtoIRKI modulates photosynthetic efficiency. We also provide an excellent haplotype module, PtoHB1-PtoIRKIhap2-PtoRca, that can be used to improve the photosynthesis of woody plants via molecular breeding.

The CRISPR-Cas technology has sparked a new technological revolution, significantly enhancing our ability to understand and engineer organisms. The nuclease that underpins this technology is evolving from the “One Cas9 for all” model to a diverse CRISPR toolbox. Identifying PAM sequences is a critical bottleneck in developing novel Cas proteins. Given the limitations of experimental methods, bioinformatics approaches are essential for predicting PAM sequences of Cas proteins in advance. To date, there are only a few PAM sequence prediction programs, and their accuracy is relatively low due to the limited number of spacers in CRISPR-Cas systems. To overcome this challenge, we have developed a pipeline named PAMPHLET, which innovatively utilizes homology searches of Cas proteins to identify additional spacers. PAMPHLET was tested on 20 CRISPR-Cas systems with known PAMs, increasing the number of spacers by up to 18-fold compared to the original datasets and successfully predicting 18 PAM sequences for protospacers. For rigorous and high-quality wet-lab validation of the predictions made by PAMPHLET, we employed the published DocMF platform. This platform leverages next-generation sequencing chips to profile protein-DNA interactions and can simultaneously screen both 5’ and 3’ PAMs with high throughput. The PAMPHLET predictions showed high consistency with the DocMF results for four novel Cas proteins. We expect that PAMPHLET will overcome the current limitations in PAM sequence prediction, expedite the discovery of PAM sequences, and help to shorten the development cycle for CRISPR tools. Remarkably, PAMPHLET has revealed an intriguing genomic phenomenon: the C2c9 and C2c10 systems, which lack the canonical adaptation module, possess identical PAM sequences to those found in co-occurring type I systems, suggesting potential shared spacer acquisition mechanisms. This finding highlights the complex evolutionary relationships of CRISPR-Cas systems and propels us toward a deeper understanding of their mechanistic diversity and adaptability. ### Competing Interest Statement The authors have declared no competing interest.

Background and aim: Prostate cancer is a leading malignant tumor in men, associated with a high rate of mortality. Androgen deprivation therapy is commonly used to treat prostate cancer, which contributes to the progression of castration-resistant prostate cancer (CRPC). The current therapy has a low survival rate in patients with CRPC. Our study aims to develop a novel effective approach for CRPC treatment and improve survival benefits. Experimental procedure: CRPC cell line PC-3-Luc expressing luciferase and the CRPC cell line PC-3-IL6-Luc stably overexpressing IL-6 were used to establish the xenograft tumor mouse model. The tumor was monitored weekly using Bioluminescence imaging. Infiltrated macrophages were quantified by fluorescence-activated cell sorting using flow cytometry. IL6 mRNA level was determined using quantitative real-time PCR. The protein levels of total STAT3 and phosphorylated STAT3 were determined using Western blot. Results and conclusion: Zhoushi Qi Ling decoction (ZQD) treatment significantly reduced PC3 the xenograft tumor progression and the number of infiltrated macrophages when compared with saline treatment. IL6 mRNA level was remarkedly suppressed by ZQD treatment. Notably, the protein level of phosphorylated STAT3 was significantly decreased in PC3 the xenograft tumor treated with ZQD compared to saline treatment. Our findings demonstrated that ZQD treatment significantly reduced the progression of prostate cancer, evidenced by the reduced population of infiltrated macrophages and the inhibition of the IL6/STAT3 pathway.

Leaf development is a multifaceted and dynamic process orchestrated by a myriad of genes to shape the proper size and morphology. The dynamic genetic network underlying leaf development remains largely unknown. Utilizing a synergistic genetic approach encompassing dynamic genome-wide association study (GWAS), time-ordered gene co-expression network (TO-GCN) analyses and gene manipulation, we explored the temporal genetic architecture and regulatory network governing leaf development in Populus. We identified 42 time-specific and 18 consecutive genes that displayed different patterns of expression at various time points. We then constructed eight TO-GCNs that covered the cell proliferation, transition, and cell expansion stages of leaf development. Integrating GWAS and TO-GCN, we postulated the functions of 27 causative genes for GWAS and identified PtoGRF9 as a key player in leaf development. Genetic manipulation via overexpression and suppression of PtoGRF9 revealed its primary influence on leaf development by modulating cell proliferation. Furthermore, we elucidated that PtoGRF9 governs leaf development by activating PtoHB21 during the cell proliferation stage and attenuating PtoLD during the transition stage. Our study provides insights into the dynamic genetic underpinnings of leaf development and understanding the regulatory mechanism of PtoGRF9 in this dynamic process.