Regulatory Pathways Research Articles

Cbf11 and Mga2 function together to activate transcription of lipid metabolism genes and promote mitotic fidelity in fission yeast.

Within a eukaryotic cell, both lipid homeostasis and faithful cell cycle progression are meticulously orchestrated. The fission yeast Schizosaccharomyces pombe provides a powerful platform to study the intricate regulatory mechanisms governing these fundamental processes. In S. pombe, the Cbf11 and Mga2 proteins are transcriptional activators of non-sterol lipid metabolism genes, with Cbf11 also known as a cell cycle regulator. Despite sharing a common set of target genes, little was known about their functional relationship. This study reveals that Cbf11 and Mga2 function together in the same regulatory pathway, critical for both lipid metabolism and mitotic fidelity. Deletion of either gene results in a similar array of defects, including slow growth, dysregulated lipid homeostasis, impaired cell cycle progression (cut phenotype), abnormal cell morphology, perturbed transcriptomic and proteomic profiles, and compromised response to the stressors camptothecin and thiabendazole. Remarkably, the double deletion mutant does not exhibit a more severe phenotype compared to the single mutants. In addition, ChIP-nexus analysis reveals that both Cbf11 and Mga2 bind to nearly identical positions within the promoter regions of target genes. Interestingly, Mga2 binding appears to be dependent on the presence of Cbf11 and Cbf11 likely acts as a tether to DNA, while Mga2 is needed to activate the target genes. In addition, the study explores the distribution of Cbf11 and Mga2 homologs across fungi. The presence of both Cbf11 and Mga2 homologs in Basidiomycota contrasts with Ascomycota, which mostly lack Cbf11 but retain Mga2. This suggests an evolutionary rewiring of the regulatory circuitry governing lipid metabolism and mitotic fidelity. In conclusion, this study offers compelling support for Cbf11 and Mga2 functioning jointly to regulate lipid metabolism and mitotic fidelity in fission yeast.

Abstract A015: Design and deciphering of precision peptide inhibitors for cancer stemness using generative deep learning and molecular dynamics simulations

Abstract The use of artificial intelligence (AI) and machine learning (ML) for exploring vast amino acid sequence spaces has recently gained traction in the discovery of antibiotics and the design of biomaterials with a wide array of algorithms. However, despite some advancements, designing peptide inhibitors to specifically modulate protein-protein interactions remains a significant challenge. In this contribution, we explore the use of a Long Short-Term Memory (LSTM) network - a type of recurrent neural network - to model peptide sequences, given its ability to process sequential data and capture long-term dependencies, critical for peptide design. Our research focuses on developing precision peptides that target the interaction between the Notch intracellular domain (NICD) and CBF1/RBPJ transcription factors, key regulators of the Notch signaling pathway implicated in breast and pancreatic cancer stemness. We inferred hydrophobic, hydrophilic, van der Waals, and salt bridge interactions from experimentally determined three-dimensional protein complex structures, which were used for feature engineering via one-hot encoding. Peptides, each 20 amino acids in length, were generated using temperature scaling in the LSTM model. These peptides were then structurally optimized and subjected to molecular dynamics (MD) simulations, followed by molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) analysis to assess their interactions and binding affinities with the Notch receptor. The MD simulations provide valuable molecular-level insights into the peptide-Notch interactions, helping to evaluate their binding strength. Further biological testing is underway to validate the efficacy of these lead peptide inhibitors and elucidate their molecular mechanisms in targeting cancer stem cells associated with breast cancer. Citation Format: Gurudeeban Selvaraj, Satyavani Kaliamurthi, Gilles H. Peslherbe. Design and deciphering of precision peptide inhibitors for cancer stemness using generative deep learning and molecular dynamics simulations. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Optimizing Therapeutic Efficacy and Tolerability through Cancer Chemistry; 2024 Dec 9-11; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(12_Suppl):Abstract nr A015

Scale-Up and Postapproval Changes in Orally Inhaled Drug Products: Scientific and Regulatory Considerations.

Approved drug products may be subject to change(s) for a variety of reasons. The changes may include, but are not limited to, increase in batch size, alteration of the drug product constituent(s), improvement in the manufacturing process, and shift in manufacturing sites. The extent of pharmaceutical testing and the regulatory pathway for timely implementation of any change in the approved product and/or process depends upon the nature and extent of change. The U.S. Food and Drug Administration (FDA) has published guidelines that outline its expectations for the Scale-Up and Postapproval Changes (SUPAC) in the solid oral immediate and modified release (MR) products, and semisolid formulations. However, to date, no such guidelines have been issued to address SUPAC in the orally inhaled drug products (OIDPs), and this article represents a seminal contribution in this direction. It is hoped that it will inspire contributions from the relevant multidisciplinary experts from the pharmaceutical industry and the agency in accomplishing formal regulatory guidelines relevant to the OIDP SUPAC. The OIDPs are complex drug-device combination products. Therefore, a conceptualization of SUPAC guidelines for these products warrants consideration of contributions of effect of change(s) in individual components (drug substance, formulation, device) as well as a compound effect that a single or multiple changes may have on product performance, and its safety and efficacy. This article provides a discussion of scientific aspects and regulatory bases relevant to the development of SUPAC for OIDPs, and it attempts to outline considerations that may be applicable in addressing issues related to the OIDP SUPAC in the context of human drugs. The authors' statements should not be viewed as recommendations from any regulatory agency, as the applicable guidelines would be determined on case-by-case evaluation by the relevant authorities.

Reaction Pathway Regulation for Gaseous and Liquid Products of Electrocatalytic CO2 Reduction under Adsorbate Interactions

Halide anion adsorption on transition metals can improve the performance of electrochemical CO2 reduction reaction (CO2RR), while the specific reaction mechanisms governing selective CO2RR pathways remain unclear. In this study, we demonstrate for the first time the distinct pathways for gaseous (CO) and liquid products (formate and ethanol) on the well‐defined Ag‐Cu nanostructures with controlled chlorination, respectively. We show that CO2 conversion to CO on Ag/AgCl can be tuned by adjusting the thickness of AgCl layer, achieving a Faradaic efficiency (FE) near 100% over a broad potential range in a 0.5 M KHCO3 using flow cell. In contrast, the optimized Cl‐Ag/Cu system enables the conversion of CO2 into liquid products including formate and ethanol with a total FE nearing 100%, delivering high current density under similar conditions. In situ infrared experiments and theoretical calculations reveal that the lateral adsorbate of *OCHO intermediate facilitates the thermodynamics of both the CO pathway on Cl‐Ag(111) and the formate pathway on Cl‐Ag/Cu(111) by reducing Gibbs free energy barriers of each potential‐limit step. This work uncovers the role of chlorination in the tuning of C‐bound or O‐bound intermediates during CO2RR on Ag‐Cu catalysts, determining the reaction pathway under lateral adsorbate effects.

Reaction Pathway Regulation for Gaseous and Liquid Products of Electrocatalytic CO2 Reduction under Adsorbate Interactions.

Halide anion adsorption on transition metals can improve the performance of electrochemical CO2 reduction reaction (CO2RR), while the specific reaction mechanisms governing selective CO2RR pathways remain unclear. In this study, we demonstrate for the first time the distinct pathways for gaseous (CO) and liquid products (formate and ethanol) on the well-defined Ag-Cu nanostructures with controlled chlorination, respectively. We show that CO2 conversion to CO on Ag/AgCl can be tuned by adjusting the thickness of AgCl layer, achieving a Faradaic efficiency (FE) near 100% over a broad potential range in a 0.5 M KHCO3 using flow cell. In contrast, the optimized Cl-Ag/Cu system enables the conversion of CO2 into liquid products including formate and ethanol with a total FE nearing 100%, delivering high current density under similar conditions. In situ infrared experiments and theoretical calculations reveal that the lateral adsorbate of *OCHO intermediate facilitates the thermodynamics of both the CO pathway on Cl-Ag(111) and the formate pathway on Cl-Ag/Cu(111) by reducing Gibbs free energy barriers of each potential-limit step. This work uncovers the role of chlorination in the tuning of C-bound or O-bound intermediates during CO2RR on Ag-Cu catalysts, determining the reaction pathway under lateral adsorbate effects.

Identification of differentially expressed miRNAs involved in vascular aging reveals pathways associated with the endocrine hormone regulation

Vascular aging refers to a series of processes where the elasticity of blood vessels diminishes, leading to stiffening, and deposition of fat components on the vessel walls, causing inflammation. Cardiovascular diseases, such as stroke and hypertension, play significant roles in morbidity and mortality rates among the elderly population. In this study, the Reactive Hyperemia Index (RHI) was measured to assess vascular endothelial function and aging-induced pathogenesis of vascular diseases in Korean subjects. We aimed to identify extracellular vesicle microRNAs (EV-miRNAs) with differential abundance between groups of individuals at the ends of a continuum in vascular aging acceleration, revealing miRNAs regulating genes in endocrine hormone regulation and tumor-related pathways. We also discovered that the principal component characterizing the global miRNA expression profile is significantly associated with clinical traits including cholesterol levels. Together, these data provide a foundation for understanding the role of miRNAs as modulators of longevity and for developing age-specific epigenetic biomarkers.

The Effect of Atorvastatin on Oncogenic miRNAs in Hematological Malignancies: A Central Study

The efficacy of statins as anti-cancer drugs has been demonstrated in several malignancies but has been poorly investigated in hematological malignancies (HM). By studying its effect on oncogenic miRNAs, we investigated the effect of statin therapy on HM patients. The data were used to identify enriched pathways that were altered due to statin treatment. The main aim of this study was to identify significantly differentially expressed miRNAs and involved regulatory pathways post-atorvastatin treatment in HM patients. A panel of 95 plasma circulating miRNAs involved in tumorigenesis, apoptosis, and differentiation were relatively quantified using qPCR for blood samples obtained from 12 HM patients, 4 with Chronic Myeloid Leukemia (CML), 4 with Non-Hodgkin Lymphoma (NHL), and 4 with Essential Thrombocythemia. Pre- and post-administration of a 6-week atorvastatin course miRNA expression levels were measured. Significantly differentially expressed miRNAs were those with a fold change >2 or <0.5 using the Livak method with a two-sided p-value < 0.05. To further understand the underlying mechanism of statin regulated miRNA, GO and KEGG pathway enrichment analyses were conducted for identified target genes using the DAVID 6.8 bioinformatics tool. Out of 95 miRNAs, 14 exhibited significant fold changes post-treatment with statins including miR-198, miR-29a+b+c, miR-204, miR-222, miR-224, miR-155, miR-128b, miR-296, miR-199a+b, miR-194, miR-125a, miR-200a, and the let-7-family that were upregulated and miR-150 that was downregulated post-statin treatment. Higher mir-222, mir-194, mir-128b, and mir-199b expressions were significantly associated with better overall survival using the Cancer Genomic Atlas leukemia and lymphoma patient cohorts. Enrichment analysis identified the PI3k-Akt pathway as well as other pathways involved in the epithelial–mesenchymal transition. Atorvastatin upregulated several tumor suppressor genes involved in mediating better prognosis. The data can be used to enhance personalized treatments for patients with hematological malignancies by helping to predict the different pathways that may be targeted and, therefore, result in better survival outcomes in these patients.

LINC01614 Accelerates CRC Progression via STAT1/LINC01614/miR-4443/PFKFB3-Mediated Aerobic Glycolysis.

Colorectal cancer (CRC) is an aggressive malignancy among malignant tumours, with a high incidence globally. LINC01614, a long non-coding RNA, has been identified as an essential regulator in multiple cancer types. However, its biological functions and underlying molecular mechanisms in CRC remain largely unknown. In this study, we employed RT-qPCR to assess the expression levels of LINC01614 in CRC samples. In vitro, glucose metabolism experiments were conducted to evaluate glucose metabolism in cells. The binding relationship between miR-4443, PFKFB3, and LINC01614 was confirmed through fluorescence reporter gene detection. The subcellular localization of LINC01614 in CRC cells was determined using FISH and subcellular fractionation experiments. Additionally, a mouse subcutaneous tumor model was established for in vivo experiments. Our findings reveal that LINC01614 is upregulated in CRC tissues. Silencing of LINC01614 suppresses the malignant behaviors of CRC cells, including cell proliferation, invasion, migration, and aerobic glycolysis. Furthermore, we discovered that LINC01614 promotes the expression of PFKFB3. Additional experiments demonstrated that LINC01614 binds to miR-4443, leading to the upregulation of PFKFB3 expression. Further experiments confirmed that the LINC01614/miR-4443/PFKFB3 axis promotes CRC cell malignancy by enhancing aerobic glycolysis. Additionally, we found that STAT1 promotes the transcription of LINC01614. These findings uncover a novel regulatory pathway wherein STAT1-induced LINC01614 enhances PFKFB3 expression by sponging miR-4443, thereby accelerating CRC development. This understanding may lead to novel therapeutic strategies for CRC treatment.

Revealing the core active pharmaceutical ingredients and targets of Jie-gu capsules for fracture treatment through network pharmacology and mendelian randomization.

Jie-gu capsules are widely used for the treatment of fractures in China. However, the core active pharmaceutical ingredients of Jie-gu capsules and the potential mechanisms for treating fractures remain unclear. This study aims to preliminarily elucidate the potential mechanisms of Jie-gu capsules in the treatment of fractures through network pharmacology and mendelian randomization methods. Data of fracture patients were obtained from the GEO database (GSE93215), and the active pharmaceutical ingredients and therapeutic targets of Jie-gu capsules were retrieved from the TCMSP and TCMID databases to identify the intersection genes. Subsequently, a protein-protein interaction network of the intersection genes was constructed using the STRING database. Then, GO and KEGG analyses were conducted on the intersection genes. In addition, mendelian randomization was employed to identify core targets. Finally, molecular docking techniques were used to perform molecular docking of the core active pharmaceutical ingredients and core targets for Jie-gu capsules in the treatment of fractures. In this study, a total of 65 intersection genes involved in Jie-gu capsule treatment of fractures were identified. GO and KEGG results indicated that these 65 intersection genes were primarily associated with biological processes such as response to tumor necrosis factor and are involved in signaling pathways, especially the regulation of the MAPK signaling pathway. We identified 5 core active ingredients of Jie-gu capsules (quercetin, baicalein, kaempferol, luteolin, and succinic acid). Mendelian randomization confirmed 2 core targets (ALOX12 and EGF). Molecular docking results demonstrated that the core active pharmaceutical ingredients (quercetin, baicalein, kaempferol, luteolin, and succinic acid) exhibit high affinities with the core targets (ALOX12 and EGF). This study has unveiled the core active pharmaceutical ingredients and potential action targets of the Jie-gu capsules in treating fractures, offering valuable insights for subsequent foundational research and the development of new medications.

F56F11.4 Extends Healthspan by Regulation of TOR-Related Pathway in C. elegans

Aging has emerged as a significant issue in human society, warranting intensified research efforts. Despite this, there is a noticeable dearth of research on genetic factors that could potentially extend lifespan. The genetic role of TOR/let-363 and its interactome was delved into in the model organism C. elegans. Utilizing RNA interference (RNAi), the impact of predicted interactors within the TOR pathway were explored on aging, with a particular emphasis on healthspan. This was achieved through comprehensive lifespan and pumping rate assays. Of the 20 genes examined, only the knockdown of F56F11.4 resulted in a reduction in both lifespan and early-stage pumping rate. To elucidate the interplay between F56F11.4 and TOR/let-363, double knockdown and RT-qPCR assays were employed to reveal their intricate regulatory roles in the aging process. Additionally, the conserved nature of F56F11.4 and let-363 across five well-studied organisms (C. elegans, H. sapiens, M. musculus, R. norvegicus, and D. melanogaster) was identified using bioinformatics analyses. The discovery of the influence of F56F11.4 on healthspan through the modulation of TOR/let-363 signaling suggests a promising target for the development of novel therapeutic strategies aimed at enhancing healthy aging.

A Comparative Transcriptomic and Proteomic Analysis of the Pileus of Agaricus bisporus During Its Different Developmental Phases

The analysis of the developmental stages of Agaricus bisporus, a major edible and medicinal mushroom, has always been an important focus in this research area. The process of the growth and development of edible mushrooms is complex and involves the regulation of multiple genes and metabolic pathways. Less data exist on the mechanism of their growth and development at the overall level. In this study, RNA sequencing analyses (RNA-Seq) and data-independent acquisition (DIA) proteomic analyses were carried out at the button phase (BP), harvesting phase (HP), and opening phase (OP) stages of Agaricus bisporus ‘Shuangbao 106’ to reveal the changes in gene expression during the different growth periods of its substrates. The authors screened and explored 3351 differentially expressed genes (DEGs) with a difference factor of ≥2.0, including 2080 up-regulated and 1271 down-regulated genes. After proteome sequencing, 1156 differentially expressed proteins (DEPs) were screened, including 524 up-regulated and 632 down-regulated genes. The expression in TPM of glycoside hydrolase, catalytic core, and chitinase II decreased during both the HP and OP compared with the BP. This may be because mushrooms require higher levels of glycoside hydrolase, catalytic core, and chitinase II activity during the BP to cope with external threats and the need for cell wall remodeling. Conversely, the growth of mushrooms slowed down and the need for cell wall remodeling decreased during the HP and OP, leading to a decrease in the expression of glycoside hydrolase, catalytic core, and chitinase II. This change is related to the need for environmental adaptation, immune defense, and cell wall remodeling, and may regulate the post-growth process of A. bisporus via the hydrolysis of cell wall chitin and glycoside hydrolase. It may also inhibit the growth of mushroom pilei.

Targeting Nudix Hydrolase 5 with Bioactive Flavonoids: Molecular Dynamics and Docking Studies for Breast Cancer Therapy.

Breast cancer (BC) is the most prevalent malignancy among women globally and the leading cause of cancer-related mortality. Consequently, there is an urgent need for new, effective treatment strategies for breast cancer. Research has shown that the enzyme nudix hydrolase 5 (NUDT5) plays a critical role in promoting breast cancer aggressiveness and serves as a key regulator of oncogenic pathways. The development of NUDT5 inhibitors presents a viable strategy for enhancing treatment results in managing BC. The ability of the flavonoids to modulate key biochemical pathways and improve therapeutic outcomes highlights their promise in developing novel breast cancer treatments. Hence, the main objective of the present investigation is to identify the potential interaction of structurally diverse bioactive flavonoids with the active site of the target NUDT5. Our docking analysis revealed that the flavonoids such as naringin and genistein have shown a significant binding association with residues Arg51, Asp60, Gln82, Arg84, Ala96, Leu98, Glu112, Glu116, Met132, Cys139, Ile141, and Glu166 of NUDT5, suggesting its potential as a potent inhibitor. The stabilizing effects of these leads (naringin and genistein) were further validated using molecular dynamics investigations, including RMSD, RMRF, Rg, SASA, PCA, and FEL. The results of the MD simulation studies evidenced a more significant interaction between genistein and NUDT5, indicating a steady and robust affinity, making genistein a more promising inhibitor. In conclusion, the flavonoid genistein has a strong potential as a therapeutic agent for targeting NUDT5 in breast cancer treatment making it viable candidates for further preclinical and clinical investigations.

The PpPep2-Triggered PTI-like Response in Peach Trees Is Mediated by miRNAs

Plant diseases diminish crop yields and put the world’s food supply at risk. Plant elicitor peptides (Peps) are innate danger signals inducing defense responses both naturally and after external application onto plants. Pep-triggered defense networks are compatible with pattern-triggered immunity (PTI). Nevertheless, in complex regulatory pathways, there is crosstalk among different signaling pathways, involving noncoding RNAs in the natural response to pathogen attack. Here, we used Prunus persica, PpPep2 and a miRNA-Seq approach to show for the first time that Peps regulate, in parallel with a set of protein-coding genes, a set of plant miRNAs (~15%). Some PpPep2-regulated miRNAs have been described to participate in the response to pathogens in various plant–pathogen systems. In addition, numerous predicted target mRNAs of PpPep2-regulated miRNAs are themselves regulated by PpPep2 in peach trees. As an example, peach miRNA156 and miRNA390 probably have a role in plant development regulation under stress conditions, while others, such as miRNA482 and miRNA395, would be involved in the regulation of resistance (R) genes and sulfate-mediated protection against oxygen free radicals, respectively. This adds to the established role of Peps in triggering plant defense systems by incorporating the miRNA regulatory network and to the possible use of Peps as sustainable phytosanitary products.

Edridge Green Lecture 2022-demystifying clinical trials and regulatory approvals in drug development.

This article provides a comprehensive overview of clinical trial design and regulatory pathways essential for drug development, specifically in the context of retinal diseases. Key concepts include trial structure, efficacy and safety endpoints, and regulatory expectations from agencies like the FDA. It delves into recent regulatory advancements, such as the inclusion of low-luminance vision as a secondary endpoint and analyses case studies from age-related macular degeneration (AMD) trials. Approvals for key retinal drugs, such as ranibizumab and aflibercept, treatments for AMD and diabetic macular oedema, are discussed highlighting criteria like the 15-letter gain/loss in visual acuity as approvable/clinical meaningful efficacy endpoints. Insights into geographic atrophy (GA) and diabetic retinopathy trials showcase the evolving landscape, where anatomical endpoints and new drugs bring fresh challenges and opportunities. It also emphasizes the importance of academic-industry collaboration, citing instances of gene therapy development and innovative endpoint measures like the Multi-Luminance Mobility Test for retinal dystrophies. The overarching aim of this lecture was to demystify the process that spans the design of clinical trials to regulatory approval of drugs so that clinicians understand these complexities. In particular, it is important to understand the reasons behind selection of trial design, inclusion and exclusion criteria, primary and secondary efficacy endpoints and safety endpoints. Since this lecture, there have been important changes in this field including new guidance from the Food and Drug Administration (FDA) as well as lessons learnt from recent drug approvals that are included in this manuscript.

The current socioeconomic and regulatory landscape of immune effector cell therapies

Immune cell effector therapies, including chimeric antigen receptor (CAR)-T cells, T-cell receptor (TCR) T cells, natural killer (NK) cells, and macrophage-based therapies, represent a transformative approach to cancer treatment, harnessing the immune system to target and eradicate malignant cells. CAR-T cell therapy, the most established among these, involves engineering T cells to express CARs specific to cancer cell antigens, showing remarkable efficacy in hematologic malignancies like leukemias, B-cell lymphomas, and multiple myeloma. Similarly, TCR-modified therapies, which reprogram T cells to recognize intracellular tumor antigens presented by major histocompatibility complex (MHC) molecules, offer promise for a range of solid tumors. NK-cell therapies leverage NK cells’ innate cytotoxicity, providing an allogeneic approach that avoids some of the immune-related complications associated with T-cell-based therapies. Macrophage-based therapies, still in early stages of the development, focus on reprogramming macrophages to stimulate an immune response against cancer cells in the tumor microenvironment. Despite their promise, socioeconomic and regulatory challenges hinder the accessibility and scalability of immune cell effector therapies. These treatments are costly, with CAR-T therapies currently exceeding $400,000 per patient, creating significant disparities in access based on socioeconomic status and geographic location. The high manufacturing costs stem from the personalized, labor-intensive processes of harvesting, modifying, and expanding patients’ cells. Moreover, complex logistics for manufacturing and delivering these therapies limit their reach, particularly in low-resource settings. Regulatory pathways further complicate the landscape. In the United States., the Food and Drug Administrations’ (FDA) accelerated approval processes for cell-based therapies facilitate innovation but do not address cost-related barriers. In Europe, the European Medicines Agency (EMA) offers adaptive pathways, yet decentralized reimbursement systems create uneven access across member states. Additionally, differing regulatory standards for manufacturing and quality control worldwide pose hurdles for global harmonization and access. To expand the reach of immune effector cell therapies, a multipronged approach is needed—streamlined regulatory frameworks, policies to reduce treatment costs, and international collaborations to standardize manufacturing. Addressing these socioeconomic and regulatory obstacles is essential to make these life-saving therapies accessible to a broader patient population worldwide. We present a literature review on the current landscape of immune effector cell therapies and barriers of access to currently approved standard of care therapy at various levels.

Identification of miRNA-TF Regulatory Pathways Related to Diseases from a Neuroendocrine-Immune Perspective

The neuroendocrine-immune (NEI) network is fundamental for maintaining body's homeostasis and health. While the roles of microRNAs (miRNAs) and transcription factors (TFs) in disease processes are well-established, their synergistic regulation within the NEI network has yet to be elucidated. In this study, we constructed a background NEI-related miRNA-TF regulatory network (NEI-miRTF-N) by integrating NEI signaling molecules (including miRNAs, genes, and TFs) and identifying miRNA-TF feed-forward loops. Our analysis reveals that the number of immune signaling molecules is the highest and suggests potential directions for signal transduction, primarily from the nervous system to both the endocrine and immune systems, as well as from the endocrine system to the immune system. Furthermore, disease-specific NEI-miRTF-Ns for depression, Alzheimer’s disease (AD) and dilated cardiomyopathy (DCM) were constructed based on the known disease molecules and significantly differentially expressed (SDE) molecules. Additionally, we proposed a novel method using depth-first-search algorithm for identifying significantly dysregulated NEI-related miRNA-TF regulatory pathways (NEI-miRTF-Ps) and verified their reliability from multiple perspectives. Our study provides an effective approach for identifying disease-specific NEI-miRTF-Ps and offers new insights into the synergistic regulation of miRNAs and TFs within the NEI network. Our findings provide information for new therapeutic strategies targeting these regulatory pathways.

Calcium sensing receptor regulate claudin-14 via PKA-STAT3 pathway in rat model of nephrolithiasis

BackgroundThe calcium-sensitive receptor (CaSR) has been identified as a key factor in the formation of kidney stones. A substantial body of research has illuminated the function of CaSR in stone formation with respect to oxidative stress, epithelial injury, crystal adhesion, and stone-associated proteins. Nevertheless, as a pivotal molecule in renal calcium excretion, its pathway that contributes to stone formation by regulating calcium supersaturation remains underexplored.MethodsAn in vitro rat calcium oxalate kidney stone model was established through the co-cultivation of calcium oxalate monohydrate (COM) with NRK-52E cells, while an in vivo model was constructed using the ethylene glycol method. Subsequently, the level of the CaSR-claudin-14 pathway was determined. To further elucidate the molecular pathway of CaSR-mediated regulation of claudin-14, drugs were selectively added to the in vitro and ex vivo kidney stone models, and the expression of claudin-14 and the levels of stone formation were detected. Moreover, the direct regulation of claudin-14 by CaSR with STAT3 serving as a transcription factor was examined via the dual luciferase assay. Eventually, a Cldn-14 knockout rat model and a model of kidney stone induction by ethylene glycol were generated using CRISPR-Cas9 technology to further clarify the role of claudin-14 in the CaSR-regulated formation of kidney stones.ResultsIn vitro and in vivo observations revealed that calcium oxalate induces high expression of CaSR-claudin-14. Specifically, CaSR regulates claudin-14 expression through phosphorylation modification of STAT3 via protein kinase A (PKA). In vitro, the intervention of PKA and STAT3 reversed the elevated claudin-14 levels and stone formation induced by CaSR. Finally, we generated cldn-14 knockout rats using CRISPR-Cas9 technology and observed that ethylene glycol still induced stone formation in these animals. Nevertheless, the specific activation or inhibition of CaSR demonstrated no notable impact on stone formation.ConclusionThe results of our study indicate that calcium oxalate crystals induce the activation of the pro-stone pathway of CaSR. That is, activated CaSR regulates claudin-14 levels via the PKA-STAT3 pathway, which further promotes calcium salt stone formation. The role of CaSR in the regulation of stone homeostasis is further enriched.

Genetic Foundation of Leaf Senescence: Insights from Natural and Cultivated Plant Diversity

Leaf senescence, the final stage of leaf development, is crucial for plant fitness as it enhances nutrient reutilization, supporting reproductive success and overall plant adaptation. Understanding its molecular and genetic regulation is essential to improve crop resilience and productivity, particularly in the face of global climate change. This review explores the significant contributions of natural genetic diversity to our understanding of leaf senescence, focusing on insights from model plants and major crops. We discuss the physiological and adaptive significance of senescence in plant development, environmental adaptation, and agricultural productivity. The review emphasizes the importance of natural genetic variation, including studies on natural accessions, landraces, cultivars, and artificial recombinant lines to unravel the genetic basis of senescence. Various approaches, from quantitative trait loci mapping to genome-wide association analysis and in planta functional analysis, have advanced our knowledge of senescence regulation. Current studies focusing on key regulatory genes and pathways underlying natural senescence, identified from natural or recombinant accession and cultivar populations, are highlighted. We also address the adaptive implications of abiotic and biotic stress factors triggering senescence and the genetic mechanisms underlying these responses. Finally, we discuss the challenges in translating these genetic insights into crop improvement. We propose future research directions, such as expanding studies on under-researched crops, investigating multiple stress combinations, and utilizing advanced technologies, including multiomics and gene editing, to harness natural genetic diversity for crop resilience.

The Impact of Resident Adipose Tissue Macrophages on Adipocyte Homeostasis and Dedifferentiation

Obesity is concurrent with immunological dysregulation, resulting in chronic low-grade inflammation and cellular dysfunction. In pancreatic islets, this loss of function has been correlated with mature β-cells dedifferentiating into a precursor-like state through constant exposure to inflammatory stressors. As mature adipocytes likewise have the capability to dedifferentiate in vitro and in vivo, we wanted to analyze this cellular change in relation to adipose tissue (AT) inflammation and adipose tissue macrophage (ATM) activity. Using our organotypic AT explant culture method combined with a double-reporter mouse model for labeling ATMs and mature adipocytes, we were able to visualize and quantify dedifferentiated fat (DFAT) cells in AT explants. Preliminary testing showed increased dedifferentiation after tamoxifen (TAM) stimulation, making TAM-dependent lineage-tracing models unsuitable for quantification of naturally occurring DFAT cells. The regulatory role of ATMs in adipocyte dedifferentiation was shown through macrophage depletion using Plexxicon 5622 or clodronate liposomes, which significantly increased DFAT cell levels. Subsequent bulk RNA sequencing of macrophage-depleted explants revealed enrichment of the tumor necrosis factor α (TNFα) signaling pathway as well as downregulation of associated genes. Direct stimulation with TNFα decreased adipocyte dedifferentiation, while application of a TNFα-neutralizing antibody did not significantly alter DFAT cell levels. Our findings suggest a regulatory role of resident ATMs in maintaining the mature adipocyte phenotype and preventing excessive adipocyte dedifferentiation. The specific regulatory pathways as well as the impact that DFAT cells might have on ATMs, and vice versa, are subject to further investigation.

Metabolic Engineering for Squalene Production: Advances and Perspectives.

Squalene is a linear polyunsaturated triterpene which has multiple physiological functions including anticancer, antioxidant, and skin-care. It has been widely used in the food, medicine, and cosmetics sectors and also serves as a precursor of triterpenes and steroids. Recently, the production of squalene by microbial cell hosts has drawn much attention due to its sustainability, environmental friendliness, and great efficiency. In this review, we first introduce the recent developments in the production of squalene by employing microbial cell factories, especially yeasts. Next, we underscore the primary metabolic strategies, including the biosynthetic pathway engineering, precursor manipulation, cofactor engineering, and organelle engineering. In addition to traditional metabolic engineering strategies, we also discuss some prospective metabolic regulation approaches, including regulation of lipid synthesis, identifying and manipulating related transcription factors, dynamic regulation of the metabolic pathway, and secretion engineering of membrane-impermeable terpenoids. These approaches provide insights for further metabolic engineering of squalene and related terpenoids.