Regulation Of Development Research Articles

Abstract 1543: 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers

Abstract Introduction: High-dimensional immunoprofiling is essential for studying host response to cancer immunotherapy. Synergistic agents often induce systemic changes in leukocyte abundance/function due to targeting, dosage, or delivery, necessitating multi-tissue analyses to decipher immune response mechanisms. We set out to dissect leukocyte composition in lymphoid tissues and tumors by using spectral cytometry, a multiplexed, high-throughput technology. Methods: We engineered a 40-color spectral flow cytometry panel to immunophenotype all major leukocytes and their functional subsets. Naïve and tumor-burdened C57BL/6 mice treated with combinations of agonist CD40 (aCD40) and checkpoint inhibitors were sacrificed, and tissues were processed. Stained cells were acquired on a 5-laser CyTEK Aurora. Unmixing accuracy and resolution was validated via 780 NxN plots, and high-dimensional analysis was performed. Results: Spectral cytometry affords resolution of 43+ parameters compared to 26 or less in conventional systems. We first designed a 25-marker backbone panel to identify all immune lineages. Additional channels were assigned to functional markers, allowing for profiling of migration, maturation, and activation in tumor-infiltrating leukocytes (TILs). In naïve secondary lymphoid tissues (SLTs), 35 distinct CD45+ populations were resolved. Germinal center B cells (17% vs. ~0.3% of CD45+ in other SLTs) and T follicular cells (31% vs. ~1-5% of CD4+ T cells in other SLTs) were mostly found in the Peyer’s patches, representing a hypothesized model tissue for studying tertiary immune structures in tumors. Peripheral blood contained the highest frequency of NK cells (4%), monocytes (6%), basophils (0.3%), eosinophils (4%), and neutrophils (7%) as a percentage of CD45+ cells. The spleen held the largest reservoir of CD4/8 central/effector memory cells (5% of CD45+) while ~80% of all T cells in the lymph nodes were naïve. In primary lymphoid tissues (PLTs), 12 distinct thymocyte and 7 B cell populations were resolved. Wanderlust trajectory analysis revealed CD24 and CD127 as key developmental regulators in lymphocyte differentiation. Autofluorescence extraction was essential for myeloid phenotyping. With this approach, we found monocytes comprised ~60% of TILs following aCD40 treatment (~8% in non-treatment and checkpoint only controls) and had altered macrophage differentiation fates. aCD40-treated tumors also contained higher frequencies of activated CD40/80/86+ DCs (~80/86/60% vs. ~60/51/50% of DCs in NTCs). Conclusion: This work describes a novel tool that efficiently resolves leukocytes on the axes of time, tissue, and treatment. Applications range from serial monitoring of circulating blood, endpoint analysis, validation of multiplexed-IHC, and cell sorting. PLT and SLT data has been made publicly available to serve as a training tool and reference proteomic atlas in future work. Citation Format: Aris John Kare, Lisa Nichols, Ricardo Zermeno, Marina N. Raie, Spencer K. Tumbale, Katherine W. Ferrara. 40-color spectral flow cytometry for comprehensive immunophenotyping of murine cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1543.

EjGASA6 promotes flowering and root elongation by enhancing gibberellin biosynthesis

The Gibberellic Acid-stimulated Arabidopsis (GASA) gene family is involved in the regulation of gene expression and plant growth, development, and stress responses. To investigate the function of loquat GASA genes in the growth and developmental regulation of plants, a loquat EjGASA6 gene homologous to Arabidopsis AtGASA6 was cloned. EjGASA6 expression was induced by gibberellin, and ectopic transgenic plants containing this gene exhibited earlier bloom and longer primary roots since these phenotypic characteristics are related to higher gibberellin content. Transcriptome analysis and qRT-PCR results showed that the expression levels of GA3ox1 and GA20ox1, which encode key enzymes in gibberellin biosynthesis, were significantly increased. Furthermore, we confirmed that EjGASA6 could promote the expression of GA20ox1 via the luciferase reporter system. Overall, our results suggest that EjGASA6 promotes blooming and main-root elongation by positively regulating gibberellin biosynthesis. These findings broaden our understanding of the role of GASAs in plant development and growth, and lay the groundwork for future research into the functions of EjGASA6 in regulating loquat growth and development.

When it’s cute but also dark: critical analysis of FarmVille 3’s game design in the digital economy

ABSTRACT ‘Cute design’ or ‘cuteness’ is a known design and development trend in the digital economy, with a proven advertising and marketing appeal to both children and adults. However, we postulate that it may also be an effective dark pattern. The following analysis uses the popular online game FarmVille 3 to illustrate the potential dark side of cuteness in digital game design. Through an explorative game analysis, the cute aesthetic can be identified as pervasive and multi-factored. At the same time, the history and economic steer behind the game shows a clear interest in user data ownership and control. Here, we flag cute design as a risk to human autonomy, and therefore a significant regulatory concern. This original conceptualisation of ‘cute but dark’ design is significant in its potential to manipulate the consumer autonomy of both children and adults. Current regulatory frameworks should empower the end user against the manipulate steer of cute but dark design, considering cultural nuances of what might be deemed cute user interfaces, and the vulnerabilities of certain consumer groups. Regulatory development should constitute a more streamlined approach to dark patterns, that enables a thriving digital economy while maintaining rigorous consumer protection.

Developing generic templates to shape the future for conducting integrated research platform trials

BackgroundInterventional clinical studies conducted in the regulated drug research environment are designed using International Council for Harmonisation (ICH) regulatory guidance documents: ICH E6 (R2) Good Clinical Practice—scientific guideline, first published in 2002 and last updated in 2016. This document provides an international ethical and scientific quality standard for designing and conducting trials that involve the participation of human subjects. Recently, there has been heightened awareness of the importance of integrated research platform trials (IRPs) designed to evaluate multiple therapies simultaneously. The use of a single master protocol as a key source document to fulfill trial conduct obligations has resulted in a re-examination of the templates used to fulfill the dynamic regulatory and modern drug development environment challenges.MethodsRegulatory medical writing, biostatistical, and other members of EU Patient-cEntric clinicAl tRial pLatforms (EU-PEARL) developed the suite of templates for IRPs over a 3.5-year period. Stakeholders contributing expertise included academic hospitals, pharmaceutical companies, non-governmental organizations, patient representative groups, and small and medium-sized enterprises (SMEs).ResultsThe suite of templates for IRPs based on TransCelerate’s Common Protocol Template (CPT) and statistical analysis plan (SAP) should help authors navigate relevant guidelines as they create study design content relevant for today’s IRP studies. It offers practical suggestions for adaptive platform designs which offer flexible features such as dropping treatments for futility or adding new treatments to be tested during a trial. The EU-PEARL suite of templates for IRPs comprises a preface, followed by the actual resource. The preface clarifies the intended use and underlying principles that inform resource utility. The preface lists references contributing to the development of the resource. The resource includes TransCelerate CPT guidance text, and EU-PEARL-derived guidance text, distinguished from one another using shading. Rationale comments are used throughout for clarification purposes. In addition, a user-friendly, functional, and informative Platform Trials Best Practices tool to support the setup, design, planning, implementation, and conduct of complex and innovative trials to support multi-sourced/multi-company platform trials is also provided. Together, the EU-PEARL suite of templates and the Platform Trials Best Practices tool constitute the reference user manual.ConclusionsThis publication is intended to enhance the use, understanding, and dissemination of the EU-PEARL suite of templates for designing IRPs. The reference user manual and the associated website (http://www.eu-pearl) should facilitate the designing of IRP trials.

Tanshinone IIA Regulates Synaptic Plasticity in Mg2+-Free-Induced Epileptic Hippocampal Neurons via the PI3K/Akt Signaling Pathway.

Tanshinone IIA (TSIIA) is an element of the effective ingredients of Salvia miltiorrhiza Bunge (Labiatae), exhibits a significant therapeutic effect in brain neuroprotection. The focus of this study was the examination of synaptic plasticity of in Mg2+-free-induced epileptic hippocampus neurons and how TSIIA protects against it. The purity of the primary hippocampal neurons extracted from Sprague Dawley rats was assessed within 24 hours by microtubule-associated protein (MAP2) immunofluorescence staining. A hippocampal neuron model for Mg2+-free-induced spontaneous recurrent epileptiform discharge was developed, five experimental groups were then randomized: blank (Blank), model (Model), TSIIA (TSIIA, 20 µM), LY294002 (LY294002, 25 µM), and TSIIA+LY294002 (TSIIA+LY294002, 20 µM+25 µM). FIJI software was used to examine variations of neurite complexity, total length of hippocampal neurons, number of primary dendrites and density of dendritic spines. Developmental regulation brain protein (Drebrin) and brain-derived neurotrophic factor (BDNF) expression was evaluated using immunofluorescence staining and the relative expression of phospho-protein kinase B (p-Akt)/Akt, BDNF, synaptophysin (SYN) and postsynaptic density 95 (PSD-95) determined by Western blot. In contrast to the model group, TSIIA drastically reduced damage to synaptic plasticity of hippocampal neurons caused by epilepsy (p < 0.05). The TSIIA group showed a significant increase in the relative expression of PSD-95, SYN, BDNF, and p-Akt/Akt (p < 0.01). TSIIA was effective in reducing harm to the synaptic plasticity of hippocampal neurons induced by persistent status epilepticus, with the possible mechanism being regulation of the phosphatidylinositol 3-kinase 56 (PI3K)/Akt signaling pathway.

COVID-19 self-testing: Countries accelerating policies ahead of WHO guidelines during pandemics, a global consultation.

The widespread use of antigen-detection rapid diagnostic tests (Ag-RDTs) has revolutionized SARS-CoV-2 (COVID-19) testing, particularly through the option of self-testing. The full extent of Ag-RDT utilization for self-testing, however, remains largely unexplored. To inform the development of WHO guidance on COVID-19 self-testing, we conducted a global consultation to gather the views and experiences of policy makers, researchers, and implementers worldwide. The consultation was conducted by disseminating a WHO questionnaire through professional networks via email and social media, encouraging onward sharing. We used a cross-sectional design with both closed and open-ended questions related to policy and program information concerning the regulation, availability, target population, indications, implementation, benefits, and challenges of COVID-19 self-testing (C19ST). We defined self-testing as tests performed and interpreted by an untrained individual, often at home. Descriptive summaries, cross-tabulations, and proportions were used to calculate outcomes at the global level and by WHO region and World Bank income classifications. All information was collated and reported according to WHO guideline development standards and practice for global consultations. Between 01 and 11 February 2022, 844 individuals from 139 countries responded to the survey, with 45% reporting affiliation with governments and 47% operating at the national level. 504 respondents from 101 countries reported policies supporting C19ST for a range of use cases, including symptomatic and asymptomatic populations. More respondents from low-and-middle-income countries (LMICs) than high-income countries (HICs) reported a lack of an C19ST policy (61 vs 11 countries) and low population-level reach of C19ST. Respondents with C19ST experience perceived that the tests were mostly acceptable to target populations, provided significant benefits, and highlighted several key challenges to be addressed for increased success. Reported costs varied widely, ranging from specific programmes enabling free access to certain users and others with high costs via the private sector. Based on this consultation, systems for the regulatory review, policy development and implementation of C19ST appeared to be much more common in HIC when compared to LIC in early 2022, though most respondents indicated self-testing was available to some extent (101 out of 139 countries) in their country. Addressing such global inequities is critical for ensuring access to innovative and impactful interventions in the context of a public health emergency of international concern. The challenges and opportunities highlighted by key stakeholders could be valuable to consider as future testing strategies are being set for outbreak-prone diseases.

A single base pair substitution in zebrafish distinguishes between innate and acute startle behavior regulation.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate independently from those regulating acute threshold regulation.

40 years of the homeobox: either it is wrong or it is quite interesting.

Many genes that regulate development share a 180 bp DNA sequence, called the homeobox, encoding a 60 amino acid DNA-binding domain ( McGinnis et al., 1984c; Scott and Weiner, 1984). Because the homeobox is long enough to hybridize to related, but different, genes, it has been a powerful tool for discovering developmental regulators. This year is the 40th anniversary of the first homeobox report. Here, I describe work carried out at Indiana University that led to the discovery of the homeobox. The accompanying Perspective from McGinnis and Levine describes the independent discovery made at the Biozentrum in Basel ( McGinnis and Levine, 2024). At the time, the competition was lively but, as we all met each other - and realized that no one cares more about your work than competitors - we fortunately became friends and have enjoyed many years of following and respecting each other's work.

MYRF: A unique transmembrane transcription factor- from proteolytic self-processing to its multifaceted roles in animal development.

The Myelin Regulator Factor (MYRF) is a master regulator governing myelin formation and maintenance in the central nervous system. The conservation of MYRF across metazoans and its broad tissue expression suggest it has functions extending beyond the well-established role in myelination. Loss of MYRF results in developmental lethality in both invertebrates and vertebrates, and MYRF haploinsufficiency in humans causes MYRF-related Cardiac Urogenital Syndrome, underscoring its importance in animal development; however, these mechanisms are largely unexplored. MYRF, an unconventional transcription factor, begins embedded in the membrane and undergoes intramolecular chaperone mediated trimerization, which triggers self-cleavage, allowing its N-terminal segment with an Ig-fold DNA-binding domain to enter the nucleus for transcriptional regulation. Recent research suggests developmental regulation of cleavage, yet the mechanisms remain enigmatic. While some parts of MYRF's structure have been elucidated, others remain obscure, leaving questions about how these motifs are linked to its intricate processing and function.

Innovations and Future Trends in Storage Pest Management

Current innovations and future trends in storage pest management, with a specific focus on the Indian context. The critical role of effective pest management in agriculture and food security is underscored, considering its impact on economic stability and public health. It begins by detailing the challenges faced in storage pest management, including the variety of pests like insects and rodents, and the damage they inflict on stored agricultural products. It critiques the limitations of traditional pest management methods, particularly chemical control, and highlights emerging issues such as climate change effects and pesticide resistance. The core examines the latest innovations in storage pest management. Advanced chemical approaches like novel pesticides and controlled release formulations are discussed, alongside the rise of nanotechnology applications in pest control, including nano-pesticides and smart delivery systems. The emergence of biological control innovations, particularly new biocontrol agents, and genetic control strategies like the sterile insect technique, marks a significant shift towards more sustainable pest management methods. Additionally, It explores developments in physical and mechanical control methods, emphasizing improved storage facilities and environmental control techniques. Integrated Pest Management (IPM) approaches are identified as a key future trend, promoting holistic and sustainable strategies, with case studies underscoring their success and practical application. It also addresses the vital role of policy and regulatory developments, considering the impact of global regulations and the importance of international cooperation and standards in shaping pest management practices in India. Ethical and environmental considerations form a crucial part of the discourse, focusing on the ecological impact of pest management and the balance between control measures and conservation efforts. It concludes with an examination of practical applications and case studies, providing insights into real-world challenges and the strategies employed to overcome them. Overall, It offers a thorough analysis of the state-of-the-art in storage pest management, highlighting the intersection of innovation, sustainability, and practicality. It aims to provide valuable guidance for researchers, policymakers, and practitioners in the field, contributing to the advancement of more effective, environmentally responsible, and sustainable pest management strategies in India.

Evolution of ride-hailing platforms regulations in India: a multi-level perspective

PurposeDigital platforms (DP) are transforming service delivery and affecting associated actors. The position of DPs is impacted by the regulations. However, emerging economies often lack the regulatory environment to support DPs. This paper aims to explore the regulatory developments for DPs using the multi-level perspective (MLP).Design/methodology/approachThe paper explores regulatory developments of ride-hailing platforms (RHPs) in India and their impacts. This study uses qualitative interview data from platform representatives, bureaucrats, drivers, experts and policy documents.FindingsRegulatory developments in the ride-hailing space cannot be explained as a linear progression. The static institutional assumptions, especially without considering the multi-actors and multi-levels in policy formulation, do not serve associated actors adequately in different times and spaces. The RHPs regulations must consider the perspective of new RHPs and the support available to them. Non-consideration of short- and long-term perspectives of RHPs may have unequal outcomes for established and new RHPs.Research limitations/implicationsThis research has implications for the digital economy regulatory ecosystem, DPs and implications for policymakers. Though the data from legal documents and qualitative interviews is adequate, transactional data from the RHPs and interviews with judiciary actors would have been insightful.Practical implicationsThe study provides insights into critical aspects of regulatory evolution, governance and regulatory impact on the DPs’ ecosystem. The right balance of regulations according to the business models of DPs allows DPs to have space for growth and development of the platform ecosystem.Social implicationsThis research shows the interactions in the digital space and how regulations can impact various actors. A balanced policy can guide the paths of DPs to have equal opportunities.Originality/valueDP regulations have a complex structure. The paper studies regulatory developments of DPs and the impacts of governance and controls on associated players and platform ecosystems.

Developmental regulation of zinc homeostasis in differentiating oligodendrocytes

Oligodendrocytes develop through sequential stages and understanding pathways regulating their differentiation remains an important area of investigation. Zinc is required for the function of enzymes, proteins and transcription factors, including those important in myelination and mitosis. Our previous studies using the ratiometric zinc sensor chromis-1 demonstrated a reduction in intracellular free zinc concentrations in mature MBP+ oligodendrocytes compared with earlier stages (Bourassa et al., 2018). We performed a more detailed developmental study to better understand the temporal course of zinc homeostasis across the oligodendrocyte lineage. Using chromis-1, we found a transient increase in free zinc after O4+,O1− pre-oligodendrocytes were switched from proliferation medium into terminal differentiation medium. To gather other evidence for dynamic regulation of free zinc during oligodendrocyte development, qPCR was used to evaluate mRNA expression of major zinc storage proteins metallothioneins (MTs) and metal regulatory transcription factor 1 (MTF1), which controls expression of MTs. MT1, MT2 and MTF1 mRNAs were increased several fold in mature oligodendrocytes compared to oligodendrocytes in proliferation medium. To assess the depth of the zinc buffer, we assayed zinc release from intracellular stores using the oxidizing thiol reagent 2,2′-dithiodipyridine (DTDP). Exposure to DTDP resulted in ∼ 100% increase in free zinc in pre-oligodendrocytes but, paradoxically more modest ∼ 60% increase in mature oligodendrocytes despite increased expression of MTs. These results suggest that zinc homeostasis is regulated during oligodendrocyte development, that oligodendrocytes are a useful model for studying zinc homeostasis in the central nervous system, and that regulation of zinc homeostasis may be important in oligodendrocyte differentiation.

BIOSURFACTANTS: SUSTAINABLE ALTERNATIVE TO SYNTHETIC SURFACTANTS AND THEIR APPLICATIONS

Biosurfactants are surface active agents produced by microorganisms, which help reduce surface or interfacial tension between two immiscible liquids like oil and water. In recent years, Due to their environmentally friendly nature and wide range of applications in various industries, they can act as a sustainable alternative to synthetic surfactants. This review article provides an overview of biosurfactants, emphasizing their need for biosurfactants, the production process, and their classification based on molecular weight, charge, and the microorganism they derived. The advantages include biodegradability, biocompatibility, low toxicity, surface activity, and specificity, and various areas where the biosurfactant used are emulsification, thermal stability, pH stability, wetting ability, foaming ability, and spreadability. Research on using biosurfactants in various formulations like nanoparticles, liposomes, transdermal application, nanoemulsion, and nanocapsules is also highlighted in this review to support its application in the medical field. Biosurfactants are also utilized in various fields like the pharmaceuticals, cosmetics, food, and oil industries. However, they have their drawbacks, which include high production costs, variability in production yield, sensitivity to the environment, lack of standardization, hurdles in regulatory approval, and research and development limitations. Despite certain drawbacks, biosurfactant offers a sustainable alternative to synthetic surfactants.

Di-n-butyl phthalate promotes the neural differentiation of mouse embryonic stem cells through neurogenic differentiation 1

An understanding of the risk of gene deletion and mutation posed by endocrine-disrupting chemicals (EDCs) is necessary for the identification of etiological reagents for many human diseases. Therefore, the characterization of the genetic traits caused by developmental exposure to EDCs is an important research subject. A new regenerative approach using embryonic stem cells (ESCs) holds promise for the development of stem-cell-based therapies and the identification of novel therapeutic agents against human diseases. Here, we focused on the characterization of the genetic traits and alterations in pluripotency/stemness triggered by phthalate ester derivatives. Regarding their in vitro effects, we reported the abilities of ESCs regarding proliferation, cell-cycle control, and neural ectoderm differentiation. The expression of their stemness-related genes and their genetic changes toward neural differentiation were examined, which led to the observation that the tumor suppressor gene product p53/retinoblastoma protein 1 and its related cascades play critical functions in cell-cycle progression, cell death, and neural differentiation. In addition, the expression of neurogenic differentiation 1 was affected by exposure to di-n-butyl phthalate in the context of cell differentiation into neural lineages. The nervous system is one of the most sensitive tissues to exposure to phthalate ester derivatives. The present screening system provides a good tool for studying the mechanisms underlying the effects of EDCs on the developmental regulation of humans and rodents, especially on the neuronal development of ESCs.

Using Social Media as a Source of Real-World Data for Pharmaceutical Drug Development and Regulatory Decision Making.

While pharmaceutical companies aim to leverage real-world data (RWD) to bridge the gap between clinical drug development and real-world patient outcomes, extant research has mainly focused on the use of social media in a post-approval safety-surveillance setting. Recent regulatory and technological developments indicate that social media may serve as a rich source to expand the evidence base to pre-approval and drug development activities. However, use cases related to drug development have been largely omitted, thereby missing some of the benefits of RWD. In addition, an applied end-to-end understanding of RWD rooted in both industry and regulations is lacking. We aimed to investigate how social media can be used as a source of RWD to support regulatory decision making and drug development in the pharmaceutical industry. We aimed to specifically explore the data pipeline and examine how social-media derived RWD can align with regulatory guidance from the US Food and Drug Administration and industry needs. A machine learning pipeline was developed to extract patient insights related to anticoagulants from X (Twitter) data. These findings were then analysed from an industry perspective, and complemented by interviews with professionals from a pharmaceutical company. The analysis reveals several use cases where RWD derived from social media can be beneficial, particularly in generating hypotheses around patient and therapeutic area needs. We also note certain limitations of social media data, particularly around inferring causality. Social media display considerable potential as a source of RWD for guiding efforts in pharmaceutical drug development and pre-approval settings. Although further regulatory guidance on the use of social media for RWD is needed to encourage its use, regulatory and technological developments are suggested to warrant at least exploratory uses for drug development.

Insights into the developmental trajectories of zygotic embryo, embryogenic callus and somatic embryo in coconut by single-cell transcriptomic analysis

Comprehensive utilization of coconut tissue culture in propagation and transformation is limited by the low efficiency of somatic embryogenesis. Understanding the potential mechanisms and developmental regulators (DRs) will facilitate the process. Here, we performed single-cell RNA sequencing (scRNA-seq) using protoplasts isolated from the zygotic embryo (ZE), embryogenic callus (EC) and somatic embryo (SE) of ‘Hainan Tall’ coconut. Cell types were identified with the assistance of known cell marker genes and further validated by RNA in situ hybridization. Differentiation trajectories from the ZE to SE transition were then constructed and identified potential DRs in the process. Notably, developmental initiation cells for both ZE and SE were identified as quiescent center-like cells (QC-L), while it was the dividing cell (DC) in EC. CnGRF12 (Growth Regulating Factor) was preferentially expressed in these developmental initiation cells, and its co-expression with CnGIF1 (GRF-interacting Factor) by transient transformation suggested that CnGRF12 has potential as a DR to accelerate transformation. Our comprehensive cell atlas provides single-cell-level insights into the developmental trajectories of ZE, EC and SE cells in coconut, and potential DRs investigated in this study could be useful for the future genotype-independent transformation and gene editing of coconut.

Harmonising cryptocurrency regulation in Europe: opportunities for preventing illicit transactions

This study focuses on the analysis of cryptocurrency regulatory developments in Europe. The aim is to review national crypto-legislation in Europe and the EU's latest initiative to create designated regulatory instruments for the crypto-economy. This study assessed whether the European Union's Regulation on Markets in Crypto-Assets (MiCA) would have the intended effect. Drawing on the results of a survey of crypto experts from five European countries, this study evaluated the effectiveness of current regulation across Europe and how it can be improved to reduce financial crimes. The findings show that a unified national legal framework for regulating transactions with crypto assets does not exist in European countries. Current crypto regulations are dictated by anti-money laundering recommendations. This study provides suggestions for improving MiCA regulation. The article offers recommendations for an international regulatory standard for crypto assets and insights for increasing efficiency in regulating DeFi, NFTs, and smart contracts.

Genomic Resources and Annotations for a Colonial Ascidian, the Light-Bulb Sea Squirt Clavelina lepadiformis.

Ascidian embryos have been studied since the birth of experimental embryology at the end of the 19th century. They represent textbook examples of mosaic development characterized by a fast development with very few cells and invariant cleavage patterns and lineages. Ascidians belong to tunicates, the vertebrate sister group, and their study is essential to shed light on the emergence of vertebrates. Importantly, deciphering developmental gene regulatory networks has been carried out mostly in two of the three ascidian orders, Phlebobranchia and Stolidobranchia. To infer ancestral developmental programs in ascidians, it is thus essential to carry out molecular embryology in the third ascidian order, the Aplousobranchia. Here, we present genomic resources for the colonial aplousobranch Clavelina lepadiformis: a transcriptome produced from various embryonic stages, and an annotated genome. The assembly consists of 184 contigs making a total of 233.6 Mb with a N50 of 8.5 Mb and a L50 of 11. The 32,318 predicted genes capture 96.3% of BUSCO orthologs. We further show that these resources are suitable to study developmental gene expression and regulation in a comparative framework within ascidians. Additionally, they will prove valuable for evolutionary and ecological studies.

Retinoic acid-loaded liposomes induce human mucosal CD103+ dendritic cells that inhibit Th17 cells and drive regulatory T-cell development in vitro.

The active vitamin A metabolite, all-trans-retinoic acid (RA), primes precursor dendritic cells (DCs) into a mucosal phenotype with tolerogenic properties characterized by the expression of integrin CD103. CD103+ DCs can counteract pathogenic Th1 and Th17 in inflammatory bowel disease (IBD) or celiac disease (CD). Tolerogenic manipulation of DCs using nanoparticles carrying tolerogenic adjuvants and disease-specific antigens is a valuable treatment strategy to induce antigen-specific mucosal tolerance in vivo. Here, we investigated the effects of RA-loaded liposomes on human DC phenotype and function, including DC-driven T-cell development, both during the generation of monocyte-derived DCs (moDCs) as well as by priming immature moDCs. RA liposomes drove CD103+ DC differentiation as well as ALDH1A2 expression in DCs. Neutrophil-dependent Th17 cell development was reduced by RA-liposome-differentiated and RA-liposome-primed DCs. Moreover, RA liposome treatment shifted T-cell development toward a Th2 cell profile. Importantly, RA liposomes induced the development of IL-10-producing and FoxP3+ regulatory T cells (Tregs) of various Treg subsets, including ICOS+ Tregs, that were potent inhibitors of bystander memory T-cell proliferation. Taken together, RA-loaded liposomes could be a novel treatment avenue for IBD or CD patients.

Comparative proteomic analysis across the developmental stages of the Eimeria tenella

Eimeria tenella is the main pathogen responsible for coccidiosis in chickens. The life cycle of E. tenella is, arguably, the least complex of all Coccidia, with only one host. However, it presents different developmental stages, either in the environment or in the host and either intracellular or extracellular. Its signaling and metabolic pathways change with its different developmental stages. Until now, little is known about the developmental regulation and transformation mechanisms of its life cycle. In this study, protein profiles from the five developmental stages, including unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), sporozoites (S) and second-generation merozoites (M2), were harvested using the label-free quantitative proteomics approach. Then the differentially expressed proteins (DEPs) for these stages were identified. A total of 314, 432, 689, and 665 DEPs were identified from the comparison of SO7h vs USO, SO vs SO7h, S vs SO, and M2 vs S, respectively. By conducting weighted gene coexpression network analysis (WGCNA), six modules were dissected. Proteins in blue and brown modules were calculated to be significantly positively correlated with the E. tenella developmental stages of sporozoites (S) and second-generation merozoites (M2), respectively. In addition, hub proteins with high intra-module degree were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analyses revealed that hub proteins in blue modules were involved in electron transport chain and oxidative phosphorylation. Hub proteins in the brown module were involved in RNA splicing. These findings provide new clues and ideas to enhance our fundamental understanding of the molecular mechanisms underlying parasite development.