Single-Cell Transcriptomic Analysis Reveals Dynamic Cellular Processes in Corneal Epithelium During Wound Healing in Cynomolgus Monkeys.

Purpose: Corneal wounding healing is critical for maintaining clear vision, however, a complete understanding of its dynamic regulatory mechanisms remains elusive. Here, we used single-cell RNA sequencing (scRNA-seq) to analyze the cellular activities and transcriptional changes of corneal limbal epithelial cells at different stages after wound healing in cynomolgus monkeys, which exhibit a closer transcriptomic similarity to humans. Methods: Corneal limbal tissues were collected during uninjured, 1-day and 3-day healing stages, dissociated into single cells, and subjected to scRNA-seq using the 10× Genomics platform. Cell types were clustered by graph-based visualization methods and unbiased computational analysis. Additionally, cell migration assays and immunofluorescent staining were performed on cultured human corneal epithelial cells. Results: We characterized nine cell clusters by scRNA-seq analysis of the cynomolgus monkey corneal epithelium. By comparing heterogeneous transcriptional changes in major cell types during corneal healing, we highlighted the importance of limbal epithelial cells (LEPCs) and basal epithelial cells (BEPCs) in extracellular matrix (ECM) formation and wound healing, as well as suprabasal epithelial cells (SEPCs) in epithelial differentiation during the healing processes. We further identified five different sub-clusters in LEPC, including the transit amplifying cell (TAC) sub-cluster that promotes early healing through the activation of thrombospondin-1 (THBS1) expression. Conclusions: Our study represents the first comprehensive exploration of the detailed transcriptome profile of individual corneal cells during the wound healing process in nonhuman primates. We demonstrate the intricate mechanisms involved in corneal healing and provide a promising avenue for potential therapies in corneal wound healing.

Comprehensive review of the traditional uses and the potential benefits of epimedium folium.

Epimedium Folium has been extensively utilized for medicinal purposes in China for a significant period. This review undertakes a comprehensive examination of literature pertaining to Epimedium and its metabolites over the past decade, drawing from databases such as PubMed. Through meticulous organization and synthesis of pertinent research findings, including disease models, pharmacological effects, and related aspects, this narrative review sheds light on the principal pharmacological activities and associated mechanisms of Epimedium in safeguarding the reproductive system, promoting bone health, mitigating inflammation, and combating tumors and viral infections. Consequently, this review contributes to a more profound comprehension of the recent advances in Epimedium research.

Sustainable Natural Resources for Aphid Management: ß-Ionone and Its Derivatives as Promising Ecofriendly Botanical-Based Products.

ß-Ionone, sustainably derived from Petunia hybrida as a natural bioresource, was identified as a lead compound for integrated aphid management. A series of ß-ionone derivatives containing ester groups were designed and synthesized for the purpose of discovering renewable botanical-based products. The odorant-binding protein (OBP) binding test indicated that ß-ionone and its derivatives displayed binding affinities with Acyrthosiphon pisum OBP9 (ApisOBP9) and Harmonia axyridis OBP15 (HaxyOBP15). Bioactivity assays revealed that most ß-ionone derivatives exhibited a higher repellent activity than that of ß-ionone. ß-Ionone and derivatives 4g and 4l displayed attractiveness to H. axyridis. Specifically, 4g was a highly promising derivative, possessing good repellent activity against A. pisum and attractiveness to H. axyridis. Molecular dynamics simulations revealed that integrating the hydrophobic ester group into the ß-ionone framework strengthened the van der Waals interactions of 4g with ApisOBP9/HaxyOBP15, improving the binding affinity with OBPs and producing higher push-pull activity than ß-ionone; 4g also had low toxicity toward nontarget organisms. Thus, 4g is a potential ecofriendly, botanical-based option for aphid management.

[Action mechanisms of Qianlie Jindan Tablets on chronic nonbcterial prostatitis in rats: An exploration based on non-targeted urine metabolomics].

OBJECTIVE: To explore the mechanisms of Qianlie Jindan Tablets (QLJD) acting on chronic nonbacterial prostatitis (CNP) in rats based on non-targeted urine metabolomics. METHODS: According to the body mass index, we equally randomized 30 eight-week-old male SD rats into a blank control, a CNP model control and a QLJD medication group. We established the CNP model in the latter groups and, from the 4th day of modeling, treated the rats in the blank and model control groups intragastrically with normal saline and those in the QLJD medication group with QLJD suspension, qd, for 30 successive days. Then we detected the changes in the metabolites of the rats by ultra-high-performance liquid chromatography-tandem mass spectrometry, and identified the differential metabolites in different groups by multivariate statistical analysis, followed by functional annotation of the differential metabolites. RESULTS: Eight common metabolites were identified by metabolomics analysis, of which 5 were decreased in the CNP model controls and increased in the QLJD medication group, while the other 3 increased in the former and decreased in the latter group. Creatinine and genistein were important differential metabolites, and the arginine and proline metabolic pathways and isoflavone biosynthesis pathways were the main ones for QLJD acting on CNP. Compared with the blank controls, the model controls showed up-regulated arginine and proline metabolic pathways, increased production of creatinine, down-regulated isoflavone biosynthetic pathway and decreased production of genistein. The above changes in the model controls were all reversed in the QLJD medication group. CONCLUSION: QLJD acts effectively on CNP in male rats by regulating L-arginine and proline metabolic pathways, as well as the isoflavone biosynthesis pathway and naringenin metabolism.

High-throughput single-molecule long-read RNA sequencing analysis of tissue-specific genes and isoforms in lettuce (Lactuca sativa L.).

Lettuce is one of the most widely cultivated and consumed dicotyledonous vegetables globally. Despite the availability of its reference genome sequence, lettuce gene annotation remains incomplete, impeding comprehensive research and the broad application of genomic resources. Long-read RNA isoform sequencing (Iso-Seq) offers substantial advantages for analyzing RNA alternative splicing and aiding gene annotation, yet it faces throughput limitations. We present the HIT-ISOseq method tailored for bulk sample analysis, significantly enhancing RNA sequencing throughput on the PacBio platform by concatenating cDNA. Here we show, HIT-ISOseq generates 3-4 cDNA molecules per CCS read in lettuce, yielding 15.7 million long reads per PacBio Sequel II SMRT Cell 8 M. We validate its effectiveness in analyzing six lettuce tissue samples, including roots, stems, and leaves, revealing tissue-specific gene expression patterns and RNA isoforms. Leveraging diverse tissue long-read RNA sequencing, we refine the transcript annotation of the lettuce reference genome, expanding its GO and KEGG annotation repertoire. Collectively, this study serves as a foundational reference for genome annotation and the analysis of multi-sample isoform expression, utilizing high-throughput long-read transcriptome sequencing.

Liver injury protection of Artemisia stechmanniana besser through PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia stechmanniana Besser, one of the most prevalent botanical medicines in Chinese, has been traditionally used for hepatitis treatment. However, the bioactive components and pharmacological mechanism on alcohol-induced liver injury remains unclear. AIM OF THE STUDY: To investigate the effect of A. stechmanniana on alcohol-induced liver damage, and further explore its mechanism. MATERIALS AND METHODS: Phytochemical isolation and structural identification were used to determine the chemical constituents of A. stechmanniana. Then, the alcohol-induced liver damage animal and cell model were established to evaluate its hepato-protective potential. Network pharmacology, molecular docking and bioinformatics were integrated to explore the mechanism and then the prediction was further supported by experiments. Moreover, both compounds were subjected to ADMET prediction through relevant databases. RESULTS: 28 compounds were isolated from the most bioactive fraction, ethyl acetate extract A. stechmanniana, in which five compounds (abietic acid, oplopanone, oplodiol, hydroxydavanone, linoleic acid) could attenuate mice livers damage caused by alcohol intragastration, reduce the degree of oxidative stress, and serum AST and ALT, respectively. Furthermore, abietic acid and hydroxydavanone exhibited best protective effect against alcohol-stimulated L-O2 cells injury among five bioactive compounds. Network pharmacology and bioinformatics analysis suggested that abietic acid and hydroxydavanone exhibiting drug likeliness characteristics, were the principal active compounds acting on liver injury treatment, primarily impacting to cell proliferation, oxidative stress and inflammation-related PI3K-AKT signaling pathways. Both of them displayed strong binding energies with five target proteins (HRAS, HSP90AA1, AKT1, CDK2, NF-κB p65) via molecular docking. Western blotting results further supported the predication with up-regulation of protein expressions of CDK2, and down-regulation of HRAS, HSP90AA1, AKT1, NF-κB p65 by abietic acid and hydroxydavanone. CONCLUSION: Alcohol-induced liver injury protection by A. stechmanniana was verified in vivo and in vitro expanded its traditional use, and its two major bioactive compounds, abietic acid and hydroxydavanone exerted hepatoprotective effect through the regulation of PI3K-AKT signaling pathway.

Unconventional hcp/fcc Nickel Heteronanocrystal with Asymmetric Convex Sites Boosts Hydrogen Oxidation.

Developing non-platinum group metal catalysts for the sluggish hydrogen oxidation reaction (HOR) is critical for alkaline fuel cells. To date, Ni-based materials are the most promising candidates but still suffer from insufficient performance. Herein, we report an unconventional hcp/fcc Ni (u-hcp/fcc Ni) heteronanocrystal with multiple epitaxial hcp/fcc heterointerfaces and coherent twin boundaries, generating rugged surfaces with plenty of asymmetric convex sites. Systematic analyses discover that such convex sites enable the adsorption of *H in unusual bridge positions with weakened binding energy, circumventing the over-strong *H adsorption on traditional hollow positions, and simultaneously stabilizing interfacial *H2O. It thus synergistically optimizes the HOR thermodynamic process as well as reduces the kinetic barrier of the rate-determining Volmer step. Consequently, the developed u-hcp/fcc Ni exhibits the top-rank alkaline HOR activity with a mass activity of 40.6 mA mgNi -1 (6.3 times higher than fcc Ni control) together with superior stability and high CO-tolerance. These results provide a paradigm for designing high-performance catalysts by shifting the adsorption state of intermediates through configuring surface sites.

Chlorogenic acid can improve spermatogenic dysfunction in rats with varicocele by regulating mitochondrial homeostasis and inhibiting the activation of NLRP3 inflammasomes by oxidative mitochondrial DNA and cGAS/STING pathway.

In recent years, Varicocele (VC) has been recognized as a common cause of male infertility that can be treated by surgery or drugs. How to reduce the damage of VC to testicular spermatogenic function has attracted extensive attention in recent years. Among them, overexpressed ROS and high levels of inflammation may play a key role in VC-induced testicular damage. As the key mediated innate immune pathways, cGAS-STING shaft under pathological conditions, such as in cell and tissue damage stress can be cytoplasmic DNA activation, induce the activation of NLRP3 inflammatory corpuscle, triggering downstream of the inflammatory cascade reaction. Chlorogenic acid (CGA), as a natural compound from a wide range of sources, has strong anti-inflammatory and antioxidant activities, and is a potential effective drug for the treatment of varicocele infertility. The aim of this study is to investigate the role of CGA in the spermatogenic dysfunction of the rat testis induced by VC and the potential mechanisms. The results of this study have shown that CGA gavage treatment ameliorated the pathological damage of seminiferous tubules, increased the number of sperm in the lumen, and increased the expression levels of Occludin and ZO-1, which indicated the therapeutic effect of CGA on spermatogenic dysfunction in the testis of VC rats. Meanwhile, the damage of mitochondrial structure was alleviated and the expression levels of ROS, NLRP3 and pro-inflammatory cytokines (IL-1ß, IL-6, IL-18) were significantly reduced in the testicular tissues of model rats after CGA treatment. In addition, we demonstrated for the first time the high expression status of cGAS and STING in testicular tissues of VC model rats, and this was ameliorated to varying degrees after CGA treatment. In conclusion, this study suggests that CGA can improve the spermatogenic function of the testis by reducing mitochondrial damage and inhibiting the activation of the cGAS-STING axis, inhibiting the activation of the NLRP3 inflammasome, and improving the inflammatory damage of the testis, highlighting the potential of CGA as a therapeutic agent for varicocele infertility.

Electrochemical biosensor based on RNA aptamer and ferrocenecarboxylic acid signal probe for C-reactive protein detection.

Monitoring health-related biomarkers using fast and facile detection techniques provides key physicochemical information for disease diagnosis or reflects body health status. Among them, electrochemical detection of various bio-macromolecules, e.g., the C-reactive protein (CRP), is of great interest in offering potential diagnosis for acute inflammation caused by infections, heart diseases, etc. Herein, a novel electrochemical aptamer biosensor was constructed from Ti3C2Tx MXene and in-situ reduced Au NPs for thiolated-RNA aptamer immobilization and CRP protein detection using Fc(COOH) as the signal probe. The sensory performances for CRP detection were optimized based on working conditions, including the incubation times and the pH. The large surface area offered by Ti3C2Tx MXene and high electrical conductivity originating from Au NPs endowed the as-fabricated aptamer biosensor with a decent sensitivity for CRP in a wide linear range of 0.05-80.0 ng/mL, good selectivity over interfering substances, and a low detection limit of 0.026 ng/mL. Such aptamer biosensors also detected CRP in serum samples using the spike & recovery method with reasonable recovery rates. The results demonstrated the potential of the as-fabricated electrochemical aptamer biosensor for fast and facile CRP detection in practical applications.

An innovative label-free electrochemical aptamer sensor: utilizing Ti3C2Tx/MoS2/Au NPs for accurate interleukin-6 detection.

In the medical field, changes in interleukin-6 (IL-6) concentration serve as essential biomarkers for monitoring and diagnosing various conditions, including acute inflammatory responses such as those seen in trauma and burns, and chronic illnesses like cancer. This paper detailed a label-free electrochemical aptamer sensor designed for IL-6 quantification. A composite material consisting of Ti3C2Tx and MoS2 was successfully synthesized to fabricate this sensor. The synergistic effect of MoS2's catalytic action on hydrogen peroxide (H2O2), used as a signalling marker, when combined with the exceptional conductivity and large specific surface area of Ti3C2Tx, not only enables an increased loading of MoS2 but also significantly boosts the electrochemical response. The in situ-reduced Au NPs provided stable immobilization sites for DNA aptamers (DNAapt) and facilitated electron transfer, ensuring accurate IL-6 recognition. Under optimal conditions, the aptamer sensor exhibited a wide linear range (5 pg/mL to 100 ng/mL) and a low limit of detection (LOD) of 2.9 pg/mL. Its sensing performance in human serum samples highlights its potential as a promising clinical analysis tool.

Unilateral biportal endoscopy vs. open decompression for lumbar epidural lipomatosis-cohort study using a prospective registry.

Objective: This study aimed to compare the outcomes of unilateral biportal endoscopy, unilateral laminectomy bilateral decompression (UBE-ULBD), and open lumbar decompression (OLD) in patients with lumbar epidural lipomatosis (LEL). Methods: This prospective observational study was conducted from March 2019 to May 2022 and encompassed 33 patients with LEL who underwent lumbar decompression. The study included 15 cases of UBE-ULBD decompression and 18 cases of open decompression, which were followed up for 1 year. The baseline characteristics, initial clinical manifestations, and surgical details [including estimated blood loss (EBL) and preoperative complications] of all patients were recorded. Radiographic evaluation included the cross-sectional area (CSA) of the thecal sac and paraspinal muscles on MRI. Clinical results were analyzed using the Short Form-36 Score (SF-36), the Numeric Pain Rating Scale (NRS) for lumbar and leg pain, creatine kinase, the Roland and Morris Disability Questionnaire (RMDQ), and the Oswestry Disability Index (ODI). Results: The dural sac CSA increased considerably at the 1-year postoperative follow-up in both groups (p < 0.001). The operative duration in the OLD group (48.2 ± 7.2 min) was shorter than that in the UBE-ULBD group (67.7 ± 6.3 min, p < 0.001). The OLD group (97.2 ± 19.8 mL) was associated with more EBL than the UBE-ULBD group (40.6 ± 13.6 mL, p < 0.001). The duration of hospitalization in the OLD group (5.4 ± 1.3 days) was significantly longer compared with the UBE-ULBD group (3.5 ± 1.2 days, p < 0.01). The SF-36, NRS, RMDQ, and ODI scores improved in both groups postoperatively (p < 0.001). Serum creatine kinase values in the UBE-ULBD group (101.7 ± 15.5) were significantly lower than those in the OLD group (330.8 ± 28.1 U/L) 1 day after surgery (p < 0.001). The degree of paraspinal muscle atrophy in the UBE-ULBD group (4.81 ± 1.94) was significantly lower than that in the OLD group (12.15 ± 6.99) at 1 year (p < 0.001). Conclusion: UBE-ULBD and OLD demonstrated comparable clinical outcomes in treating LEL. However, UBE-ULBD surgery was associated with shorter hospital stays, lower rates of incision infection, lighter paravertebral muscle injury, and lower EBL than OLD surgery. Consequently, UBE-ULBD can be recommended in patients with LEL if conservative treatment fails.

Assessing the Feasibility of Simplifying the Scanning Protocol for Spinal Metastases With Vertebral Compression Fractures Using Only the Dixon T2-Weighted Sequence.

OBJECTIVE: Conventional imaging protocols, including sagittal T1-weighted imaging (T1WI) and water-only T2-weighted imaging (T2WI), are time consuming when screening for spinal metastases with vertebral compression fractures (VCFs). In this study, we aimed to assess the accuracy of using only the Dixon T2-weighted sequence in the diagnosis of spinal metastases with VCFs to determine its suitability as a simplified protocol for this task. METHODS: This retrospective study included 27 patients diagnosed with spinal metastases and VCFs. Qualitative analysis was performed separately by two musculoskeletal radiologists, who independently performed diagnostic evaluations of each vertebra using both conventional and simplified protocols. McNemar's test was then used to compare the differences in diagnostic results, and Cohen's kappa coefficient was used to assess interobserver and interprotocol agreement. Diagnostic performance values for both protocols, including sensitivity, specificity, and area under the curve, were then determined based on the reference standard. Quantitative image analysis was performed randomly for 30 metastases on T1WI and fat-only T2WI to measure the signal intensity, signal-to-noise ratio, and contrast-to-noise ratio. RESULTS: The diagnosis of VCFs by both radiologists was in full agreement with the reference standard. The classification of spinal metastases and diagnostic performance values determined by both radiologists were not significantly different between the two protocols (all P > 0.05), and the consistency between observers and protocols was excellent (κ = 0.973-0.991). The contrast-to-noise ratio of fat-only T2WI was significantly higher than that of T1WI ( P < 0.001). CONCLUSIONS: The Dixon T2-weighted sequence alone performed well in diagnosing spinal metastases with VCFs, performing no worse than the conventional protocol (T1WI and water-only T2WI). This suggests that the Dixon T2-weighted sequence alone can serve as a simplified protocol for the diagnosis of spinal metastases with VCFs, thereby avoiding the need for more intricate scanning procedures.

Trends in non-focal neurological chief complaints and CT angiography utilization among adults in the emergency department.

Prudent imaging use is essential for cost reduction and efficient patient triage. Recent efforts have focused on head and neck CTA in patients with emergent concerns for non-focal neurological complaints, but have failed to demonstrate whether increases in utilization have resulted in better care. The objective of this study was to examine trends in head and neck CTA ordering and determine whether a correlation exists between imaging utilization and positivity rates. This is a single-center retrospective observational study at a quaternary referral center. This study includes patients presenting with headache and/or dizziness to the emergency department between January 2017 and December 2021. Patients who received a head and neck CTA were compared to those who did not. The main outcomes included annual head and neck CTA utilization and positivity rates, defined as the percent of scans with attributable acute pathologies. Among 24,892 emergency department visits, 2264 (9.1%) underwent head and neck CTA imaging. The percentage of patients who received a scan over the study period increased from 7.89% (422/5351) in 2017 to 13.24% (662/5001) in 2021, representing a 67.4% increase from baseline (OR, 1.14; 95% CI 1.11-1.18; P < .001). The positivity rate, or the percentage of scans ordered that revealed attributable acute pathology, dropped from 16.8% (71/422) in 2017 to 10.4% (69/662) in 2021 (OR, 0.86; 95% CI 0.79-0.94; P = .001), a 38% reduction in positive examinations. Throughout the study period, there was a 67.4% increase in head and neck CTA ordering with a concomitant 38.1% decrease in positivity rate.

Positive benefit-risk ratio of Psoraleae Fructus: Comprehensive safety assessment and osteogenic effects in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (PF), the dried fruit of Psoralea corylifolia L., is a commonly used traditional medicine that has contributed to the treatment of orthopedic diseases for thousands of years in China. However, recent PF-related liver injury reports have drawn widespread attention regarding its potential hepatotoxicity risks. AIM OF THE STUDY: This study was aimed to evaluate the long-term efficacy and chronic toxicity of PF using a 26-week administration experiment on rats in order to simulate the clinical usage situation. MATERIALS AND METHODS: The PF aqueous extract was consecutively administrated to rats daily at dosages of 0.7, 2.0, and 5.6 g/kg (equivalent to 1-8 times the clinical doses for humans) for as long as 26 weeks. Samples were collected after 13, 26, and 32 weeks (withdrawal for 6 weeks) since the first administration. The chronic toxicity of PF was evaluated by conventional toxicological methods, and the efficacy of PF was evaluated by osteogenic effects in the natural growth process. RESULTS: In our experiments, only the H group (5.6 g/kg) for 26-week PF treatment demonstrated liver or kidney injury, which the injuries were reversible after 6 weeks of withdrawal. Notably, the PF treatment beyond 13 weeks showed significant benefits for bone growth and development in rats, with a higher benefit-risk ratio in female rats. CONCLUSIONS: PF displayed a promising benefit-risk ratio in the treatment and prevention of osteoporosis, a disease that lacks effective medicine so far. This is the first study to elucidate the benefit-risk balance associated with clinical dosage and long-term use of PF, thereby providing valuable insights for rational clinical use and risk control of PF.

A Patient Similarity Network (CHDmap) to Predict Outcomes After Congenital Heart Surgery: Development and Validation Study.

Background: Although evidence-based medicine proposes personalized care that considers the best evidence, it still fails to address personal treatment in many real clinical scenarios where the complexity of the situation makes none of the available evidence applicable. "Medicine-based evidence" (MBE), in which big data and machine learning techniques are embraced to derive treatment responses from appropriately matched patients in real-world clinical practice, was proposed. However, many challenges remain in translating this conceptual framework into practice. Objective: This study aimed to technically translate the MBE conceptual framework into practice and evaluate its performance in providing general decision support services for outcomes after congenital heart disease (CHD) surgery. Methods: Data from 4774 CHD surgeries were collected. A total of 66 indicators and all diagnoses were extracted from each echocardiographic report using natural language processing technology. Combined with some basic clinical and surgical information, the distances between each patient were measured by a series of calculation formulas. Inspired by structure-mapping theory, the fusion of distances between different dimensions can be modulated by clinical experts. In addition to supporting direct analogical reasoning, a machine learning model can be constructed based on similar patients to provide personalized prediction. A user-operable patient similarity network (PSN) of CHD called CHDmap was proposed and developed to provide general decision support services based on the MBE approach. Results: Using 256 CHD cases, CHDmap was evaluated on 2 different types of postoperative prognostic prediction tasks: a binary classification task to predict postoperative complications and a multiple classification task to predict mechanical ventilation duration. A simple poll of the k-most similar patients provided by the PSN can achieve better prediction results than the average performance of 3 clinicians. Constructing logistic regression models for prediction using similar patients obtained from the PSN can further improve the performance of the 2 tasks (best area under the receiver operating characteristic curve=0.810 and 0.926, respectively). With the support of CHDmap, clinicians substantially improved their predictive capabilities. Conclusions: Without individual optimization, CHDmap demonstrates competitive performance compared to clinical experts. In addition, CHDmap has the advantage of enabling clinicians to use their superior cognitive abilities in conjunction with it to make decisions that are sometimes even superior to those made using artificial intelligence models. The MBE approach can be embraced in clinical practice, and its full potential can be realized.

Rational design, synthesis and binding mechanisms of novel benzyl geranate derivatives as potential eco-friendly aphid repellents.

BACKGROUND: The push-pull strategy is considered as a promising eco-friendly method for pest management. Plant volatile organic compounds (PVOCs) act as semiochemicals constitute the key factor in implementing this strategy. Benzyl alcohol and geraniol, as functional PVOCs, were reported to regulate insect behavior, showing the potential application in pest control. Using geraniol as lead, a geraniol derivative 5i with fine repellent activity was discovered in our previous work. In order to explore novel, eco-friendly aphid control agents, a series of benzyl geranate derivatives was designed and synthesized using 5i as the lead and benzyl alcohol as the active fragment. RESULTS: Benzyl alcohol was firstly evaluated to have repellent activity to Acyrthosiphon pisum. Based on this repellent fragment, a series of novel benzyl geranate derivatives was rationally designed and synthesized using a scaffold-hopping strategy. Among them, compound T9, with a binding affinity (Kd = 0.43 µm) and a substantial repellency of 64.7% against A. pisum, is the most promising compound. Molecule docking showed that hydrophobic and hydrogen-bonding interactions substantially influenced the binding affinity of compounds with ApisOBP9. Additionally, T9 exhibited low-toxicity to honeybees and ladybugs. CONCLUSION: Using a simple scaffold-hopping strategy combined with active fragment benzyl alcohol, a new derivative T9, with high aphid-repellency and low-toxicity to nontarget organisms, can be considered as a novel potential eco-friendly aphid control agent for sustainable agriculture. © 2023 Society of Chemical Industry.

Splicing activates transcription from weak promoters upstream of alternative exons.

Transcription and splicing are intrinsically coupled. Alternative splicing of internal exons can fine-tune gene expression through a recently described phenomenon called exon-mediated activation of transcription starts (EMATS). However, the association of this phenomenon with human diseases remains unknown. Here, we develop a strategy to activate gene expression through EMATS and demonstrate its potential for treatment of genetic diseases caused by loss of expression of essential genes. We first identified a catalog of human EMATS genes and provide a list of their pathological variants. To test if EMATS can be used to activate gene expression, we constructed stable cell lines expressing a splicing reporter based on the alternative splicing of motor neuron 2 (SMN2) gene. Using small molecules and antisense oligonucleotides (ASOs) currently used for treatment of spinal muscular atrophy, we demonstrated that increase of inclusion of alternative exons can trigger an activation of gene expression up to 45-fold by enhancing transcription in EMATS-like genes. We observed the strongest effects in genes under the regulation of weak human promoters located proximal to highly included skipped exons.

High-throughput and high-accuracy single-cell RNA isoform analysis using PacBio circular consensus sequencing.

Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mouse Corneal Epithelial Cells.

Purpose: Corneal epithelial homeostasis is maintained by coordinated gene expression across distinct cell populations, but the gene regulatory programs underlying this cellular diversity remain to be characterized. Here we applied single-cell multi-omics analysis to delineate the gene regulatory profile of mouse corneal epithelial cells under normal homeostasis. Methods: Single cells isolated from the cornea epithelium (with marginal conjunctiva) of adult mice were subjected to scRNA-seq and scATAC-seq using the 10×Genomics platform. Cell types were clustered by the graph-based visualization method uniform manifold approximation and projection and unbiased computational informatics analysis. The scRNA-seq and scATAC-seq datasets were integrated following the integration pipeline described in ArchR and Seurat. Results: We characterized diverse corneal epithelial cell types based on gene expression signatures and chromatin accessibility. We found that cell type-specific accessibility regions were mainly located at distal regions, suggesting essential roles of distal regulatory elements in determining corneal epithelial cell diversity. Trajectory analyses revealed a continuum of cell state transition and higher coordination between transcription factor (TF) motif accessibility and gene expression during corneal epithelial cell differentiation. By integrating transcriptomic and chromatin accessibility analysis, we identified cell type-specific and shared gene regulation programs. We also uncovered critical TFs driving corneal epithelial cell differentiation, such as nuclear factor I (NFI) family members, Rarg, Elf3. We found that nuclear factor-κB (NF-κB) family members were positive TFs in limbal cells and some superficial cells, but they were involved in regulating distinct biological processes. Conclusions: Our study presents a comprehensive gene regulatory landscape of mouse cornea epithelial cells, and provides valuable foundations for future investigation of corneal epithelial homeostasis in the context of cornea pathologies and regenerative medicine.