MERISTEM-DEFECTIVE regulates the balance between stemness and differentiation in the root meristem through RNA splicing control.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner.

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease.

Photoreception and transcriptomic response to light during early development of a teleost with a life cycle tightly controlled by seasonal changes in photoperiod.

Light cues vary along the axis of periodicity, intensity and spectrum and perception of light is dependent on the photoreceptive capacity encoded within the genome and the opsins expressed. A global approach was taken to analyze the photoreceptive capacity and the effect of differing light conditions on a developing teleost prior to first feeding. The transcriptomes of embryos and alevins of Atlantic salmon (Salmo salar) exposed to different light conditions were analyzed, including a developmental series and a circadian profile. The results showed that genes mediating nonvisual photoreception are present prior to hatching when the retina is poorly differentiated. The clock genes were expressed early, but the circadian profile showed that only two clock genes were significantly cycling before first feeding. Few genes were differentially expressed between day and night within a light condition; however, many genes were significantly different between light conditions, indicating that light environment has an impact on the transcriptome during early development. Comparing the transcriptome data from constant conditions to periodicity of white light or different colors revealed overrepresentation of genes related to photoreception, eye development, muscle contraction, degradation of metabolites and cell cycle among others, and in constant light, several clock genes were upregulated. In constant white light and periodicity of green light, genes associated with DNA replication, chromatin remodeling, cell division and DNA repair were downregulated. The study implies a direct influence of light conditions on the transcriptome profile at early developmental stages, by a complex photoreceptive system where few clock genes are cycling.

Evaluation of the EMA log kow trigger for fish BCF testing based on data for several human pharmaceuticals.

In the European Medicines Agency (EMA) "Guideline for Environmental Risk Assessment of Medicinal Products for Human Use," a fish bioconcentration factor (BCF) study is triggered in Phase I for pharmaceuticals having log Kow >4.5, to support Persistence, Bioaccumulation and Toxicity (PBT) screening, and in Phase II to assess secondary poisoning and bioaccumulation ('B') potential when log Kow ≥3. The standard sampling schedule outlined in OECD Test Guideline 305 (TG305) may require assessment of approximately 200 fish following exposure to low- and high-test concentrations and a negative control. We report experimental log Kow and BCF values for 64 human pharmaceuticals that were used to evaluate the current BCF testing trigger of log Kow ≥3, and whether a single BCF exposure concentration allows accurate classification of bioaccumulation potential. Our data support raising the BCF testing trigger to log Kow ≥4, and use of a single test concentration. The resulting reduction in the use of fish is consistent with the 3 R s principle and did not adversely affect classification accuracy. An assessment of potential risk of secondary poisoning was also conducted for three drugs classified as either B or vB, and no risks were identified.

An integrated transcriptional analysis of the developing human retina.

The scarcity of embryonic/foetal material as a resource for direct study means that there is still limited understanding of human retina development. Here, we present an integrated transcriptome analysis combined with immunohistochemistry in human eye and retinal samples from 4 to 19 post-conception weeks. This analysis reveals three developmental windows with specific gene expression patterns that informed the sequential emergence of retinal cell types and enabled identification of stage-specific cellular and biological processes, and transcriptional regulators. Each stage is characterised by a specific set of alternatively spliced transcripts that code for proteins involved in the formation of the photoreceptor connecting cilium, pre-mRNA splicing and epigenetic modifiers. Importantly, our data show that the transition from foetal to adult retina is characterised by a large increase in the percentage of mutually exclusive exons that code for proteins involved in photoreceptor maintenance. The circular RNA population is also defined and shown to increase during retinal development. Collectively, these data increase our understanding of human retinal development and the pre-mRNA splicing process, and help to identify new candidate disease genes.

An Evaluation of the Occupational Health Hazards of Peptide Couplers.

Peptide couplers (also known as amide bond-forming reagents or coupling reagents) are broadly used in organic chemical syntheses, especially in the pharmaceutical industry. Yet, occupational health hazards associated with this chemical class are largely unexplored, which is disconcerting given the intrinsic reactivity of these compounds. Several case studies involving occupational exposures reported adverse respiratory and dermal health effects, providing initial evidence of chemical sensitization. To address the paucity of toxicological data, a pharmaceutical cross-industry task force was formed to evaluate and assess the potential of these compounds to cause eye and dermal irritation as well as corrosivity and dermal sensitization. The goal of our work was to inform health and safety professionals as well as pharmaceutical and organic chemists of the occupational health hazards associated with this chemical class. To that end, 25 of the most commonly used peptide couplers and five hydrolysis products were selected for in vivo, in vitro, and in silico testing. Our findings confirmed that dermal sensitization is a concern for this chemical class with 21/25 peptide couplers testing positive for dermal sensitization and 15 of these being strong/extreme sensitizers. We also found that dermal corrosion and irritation (8/25) as well as eye irritation (9/25) were health hazards associated with peptide couplers and their hydrolysis products (4/5 were dermal irritants or corrosive and 4/5 were eye irritants). Resulting outcomes were synthesized to inform decision making in peptide coupler selection and enable data-driven hazard communication to workers. The latter includes harmonized hazard classifications, appropriate handling recommendations, and accurate safety data sheets, which support the industrial hygiene hierarchy of control strategies and risk assessment. Our study demonstrates the merits of an integrated, in vivo -in silico analysis, applied here to the skin sensitization endpoint using the Computer-Aided Discovery and REdesign (CADRE) and Derek Nexus programs. We show that experimental data can improve predictive models by filling existing data gaps while, concurrently, providing computational insights into key initiating events and elucidating the chemical structural features contributing to adverse health effects. This interactive, interdisciplinary approach is consistent with Green Chemistry principles that seek to improve the selection and design of less hazardous reagents in industrial processes and applications.

Setting impurity limits for endogenous substances: Recommendations for a harmonized procedure and an example using fatty acids.

Endogenous substances, such as fatty, amino, and nucleic acids, are often purposefully used in parenterally pharmaceuticals, but may be present as impurities. Currently, no consensus guidance exists on setting impurity limits for these substances. Specific procedures are needed, as the amount and types of toxicity data available for endogenous substances are typically far less than those for other chemical impurities. Additionally, the parenteral route of administration of these substances is inherently non-physiological, resulting in potentially different or increased severity of toxicity. Risk Assessment Process Maps (RAPMAPs) are proposed as a model to facilitate the development of health-based exposure limits (HBELs) for endogenous substances. This yielded a framework that was applied to derive HBELs for several fatty acids commonly used in parenteral pharmaceuticals. This approach was used to derive HBELs with further vetting based on anticipated perturbations in physiological serum levels, impacts of dose-rate, and consideration of intermittent dosing. Parenteral HBELs of 100-500 mg/day were generated for several fatty acids, and a proposed class-based limit of 50 mg/day to be used in the absence of chemical-specific data. This default limit is consistent with the low toxicity of this chemical class and ICH Q3C value for Class 3 solvents.

Deriving harmonised permitted daily exposures (PDEs) for paracetamol (acetaminophen) CAS #: 103-90-2.

In the pharmaceutical industry, cleaning criteria are required for multipurpose manufacturing facilities. These Health Based Exposure Limits (HBELs), also called permitted daily exposures (PDEs) values, are derived from toxicological and pharmacological evaluation of the active pharmaceutical ingredients (APIs). The purpose of this publication is to show an example of how authors from different companies evaluate a generic drug, paracetamol, and discuss different approaches and relevance of the nonclinical studies for deriving PDEs. PDE limits of 25 mg/day for the oral route, and 20 mg/day for the intravenous (i.v.) and inhalation (inhal.) routes, respectively, were established herein. However, it has been already recognised that there are acceptable differences in the PDE calculations, which may be based on data accessibility, company-specific science-policy decisions or expert judgments. These differences can cause up to a 3-fold lower or higher values. If unnecessarily high factors are applied, this would result in a very conservative PDE value and unneeded additional cleaning and higher manufacturing costs. The PDE values presented are considered to be protective against adverse and pharmacological effects observed in clinical trials and in this case, a very long postmarketing period of paracetamol.

Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies.

Leachables from pharmaceutical container closure systems are a subset of impurities that present in drug products and may pose a risk to patients or compromise product quality. Extractable studies can identify potential leachables, and extractables and leachables (E&Ls) should be evaluated during development of the impurity control strategy. Currently, there is a lack of specific regulatory guidance on how to risk assess E&Ls; this may lead to inconsistency across the industry. This manuscript is a cross-industry Extractables and Leachables Safety Information Exchange (ELSIE) consortium collaboration and follow-up to Broschard et al. (2016), which aims to provide further clarity and detail on the conduct of E&L risk assessments. Where sufficient data are available, a health-based exposure limit termed Permitted Daily Exposure (PDE) may be calculated and to exemplify this, case studies of four common E&Ls are described herein, namely bisphenol-A, butylated hydroxytoluene, Irgafos® 168, and Irganox® 1010. Relevant discussion points are further explored, including the value of extractable data, how to perform route-to-route extrapolations and considerations around degradation products. By presenting PDEs for common E&L substances, the aim is to encourage consistency and harmony in approaches for deriving compound-specific limits.

Proteomic analysis highlights the role of detoxification pathways in increased tolerance to Huanglongbing disease.

BACKGROUND: Huanglongbing (HLB) disease is still the greatest threat to citriculture worldwide. Although there is not any resistance source in the Citrus germplasm, a certain level of moderated tolerance is present. A large-scale analysis of proteomic responses of Citrus may help: 1) clarifying physiological and molecular effects of disease progression, 2) validating previous data at transcriptomic level, and 3) identifying biomarkers for development of early diagnostics, short-term therapeutics and long-term genetic resistance. RESULTS: In this work we have conducted a proteomic analysis of mature leaves of two Citrus genotypes with well-known differing tolerances to HLB: Navel orange (highly susceptible) and Volkameriana (moderately tolerant). Pathway enrichment analysis showed that amino acid degradation processes occurred to a larger degree in the Navel orange. No clear differences between the two genotypes were observed for primary metabolic pathways. The most important finding was that four glutathione-S-transferases were upregulated in Volkameriana and not in Navel orange. These proteins are involved in radical ion detoxification. CONCLUSIONS: Upregulation of proteins involved in radical ion detoxification should be considered as an important mechanism of increased tolerance to HLB.

Integrated Stochastic Model of DNA Damage Repair by Non-homologous End Joining and p53/p21-Mediated Early Senescence Signalling.

Unrepaired or inaccurately repaired DNA damage can lead to a range of cell fates, such as apoptosis, cellular senescence or cancer, depending on the efficiency and accuracy of DNA damage repair and on the downstream DNA damage signalling. DNA damage repair and signalling have been studied and modelled in detail separately, but it is not yet clear how they integrate with one another to control cell fate. In this study, we have created an integrated stochastic model of DNA damage repair by non-homologous end joining and of gamma irradiation-induced cellular senescence in human cells that are not apoptosis-prone. The integrated model successfully explains the changes that occur in the dynamics of DNA damage repair after irradiation. Simulations of p53/p21 dynamics after irradiation agree well with previously published experimental studies, further validating the model. Additionally, the model predicts, and we offer some experimental support, that low-dose fractionated irradiation of cells leads to temporal patterns in p53/p21 that lead to significant cellular senescence. The integrated model is valuable for studying the processes of DNA damage induced cell fate and predicting the effectiveness of DNA damage related medical interventions at the cellular level.

Special endpoint and product specific considerations in pharmaceutical acceptable daily exposure derivation.

Recently, a guideline has been published by the European Medicines Agency (EMA) on setting safe limits, permitted daily exposures (PDE) [also called acceptable daily exposures (ADE)], for medicines manufactured in multi-product facilities. The ADE provides a safe exposure limit for inadvertent exposure of a drug due to cross-contamination in manufacturing. The ADE determination encompasses a standard risk assessment, requiring an understanding of the toxicological and pharmacological effects, the mechanism of action, drug compound class, and the dose-response as well as the pharmacokinetic properties of the compound. While the ADE concept has broad application in pharmaceutical safety there are also nuances and specific challenges associated with some toxicological endpoints or drug product categories. In this manuscript we discuss considerations for setting ADEs when the following specific adverse health endpoints may constitute the critical effect: genotoxicity, developmental and reproductive toxicity (DART), and immune system modulation (immunostimulation or immunosuppression), and for specific drug classes, including antibody drug conjugates (ADCs), emerging medicinal therapeutic compounds, and compounds with limited datasets. These are challenging toxicological scenarios that require a careful evaluation of all of the available information in order to establish a health-based safe level.

Using default methodologies to derive an acceptable daily exposure (ADE).

This manuscript discusses the different historical and more recent default approaches that have been used to derive an acceptable daily exposure (ADE). While it is preferable to derive a health-based ADE based on a complete nonclinical and clinical data package, this is not always possible. For instance, for drug candidates in early development there may be no or limited nonclinical or clinical trial data. Alternative approaches that can support decision making with less complete data packages represent a variety of methods that rely on default assumptions or data inputs where chemical-specific data on health effects are lacking. A variety of default approaches are used including those based on certain toxicity estimates, a fraction of the therapeutic dose, cleaning-based limits, the threshold of toxicological concern (TTC), and application of hazard banding tools such as occupational exposure banding (OEB). Each of these default approaches is discussed in this manuscript, including their derivation, application, strengths, and limitations. In order to ensure patient safety when faced with toxicological and clinical data-gaps, default ADE methods should be purposefully as or more protective than ADEs derived from full data packages. Reliance on the subset of default approaches (e.g., TTC or OEB) that are based on toxicological data is preferred over other methods for establishing ADEs in early development while toxicology and clinical data are still being collected.

Considerations in civil commitment of individuals with substance use disorders.

Several states currently have enacted laws that allow for civil commitment for individuals diagnosed with severe substance use disorders. Civil commitment or involuntary commitment refers to the legal process by which individuals with mental illness are court-ordered into inpatient and/or outpatient treatment programs. Although initially civil commitment laws were intended for individuals with severe mental illness, these statutes have been extended to cover individuals with severe substance use disorders. Much of the recent legislation allowing for civil commitment of individuals with substance use disorders has come about in response to the heroin epidemic and is designed to provide an alternative to the unrelenting progression of opioid use disorders. Civil commitment also provides an opportunity for individuals with opioid use disorders to make informed decisions regarding ongoing or continued treatment. However, civil commitment also raises concerns regarding the potential violation of 14th Amendment rights, specifically pertaining to abuses of deprivation of liberty or freedom, which are guaranteed under the 14th Amendment to the United States Constitution. This commentary examines these issues while supporting the need for effective brief civil commitment legislation in all states.

Macrophage migration inhibitory factor acts as a neurotrophin in the developing inner ear.

This study is the first to demonstrate that macrophage migration inhibitory factor (MIF), an immune system 'inflammatory' cytokine that is released by the developing otocyst, plays a role in regulating early innervation of the mouse and chick inner ear. We demonstrate that MIF is a major bioactive component of the previously uncharacterized otocyst-derived factor, which directs initial neurite outgrowth from the statoacoustic ganglion (SAG) to the developing inner ear. Recombinant MIF acts as a neurotrophin in promoting both SAG directional neurite outgrowth and neuronal survival and is expressed in both the developing and mature inner ear of chick and mouse. A MIF receptor, CD74, is found on both embryonic SAG neurons and adult mouse spiral ganglion neurons. Mif knockout mice are hearing impaired and demonstrate altered innervation to the organ of Corti, as well as fewer sensory hair cells. Furthermore, mouse embryonic stem cells become neuron-like when exposed to picomolar levels of MIF, suggesting the general importance of this cytokine in neural development.

Comparison of senescence-associated miRNAs in primary skin and lung fibroblasts.

MicroRNAs are non-coding RNAs with roles in many cellular processes. Tissue-specific miRNA profiles associated with senescence have been described for several cell and tissue types. We aimed to characterise miRNAs involved in core, rather than tissue-specific, senescence pathways by assessment of common miRNA expression differences in two different cell types, with follow-up of predicted targets in human peripheral blood. MicroRNAs were profiled in early and late passage primary lung and skin fibroblasts to identify commonly-deregulated miRNAs. Expression changes of their bioinformatically-predicted mRNA targets were then assessed in both cell types and in human peripheral blood from elderly participants in the InCHIANTI study. 57/178 and 26/492 microRNAs were altered in late passage skin and lung cells respectively. Three miRNAs (miR-92a, miR-15b and miR-125a-3p) were altered in both tissues. 14 mRNA targets of the common miRNAs were expressed in lung and skin fibroblasts, of which two demonstrated up-regulation in late passage skin and lung cells (LYST; p = 0.02 [skin] and 0.02 [lung] INMT; p = 0.03 [skin] and 0.04 [lung]). ZMPSTE24 and LHFPL2 demonstrated altered expression in late passage skin cells only (p = 0.01 and 0.05 respectively). LHFPL2 was also positively correlated with age in peripheral blood (p value = 6.6 × 10(-5)). We find that the majority of senescence-associated miRNAs demonstrate tissue-specific effects. However, miRNAs showing common effects across tissue types may represent those associated with core, rather than tissue-specific senescence processes.

Conditioning the cochlea to facilitate survival and integration of exogenous cells into the auditory epithelium.

The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media.

Threshold of toxicological concern (TTC) for developmental and reproductive toxicity of anticancer compounds.

Pharmaceutical companies develop specialized therapies to treat late stage cancer. In order to accelerate life-saving treatments and reduce animal testing, compounds to treat life-threatening malignancies are allowed modified requirements for preclinical toxicology testing. Limited data packages in early drug development can present product quality challenges at multi-product manufacturing facilities. The present analysis established an endpoint-specific threshold of toxicological concern (TTC) for developmental and reproductive toxicity (DART) for anticancer compounds. A comprehensive database was created consisting of over 300 no-observed adverse effect levels (NOAELs) for DART of 108 anticancer compounds. The 5th percentile NOAEL for developmental and reproductive toxicity was 0.005 mg/kg/day (300 µg/day), resulting in a human exposure threshold of 3 µg/day assuming standard uncertainty factors and a 60 kg human bodyweight. The analysis shows this threshold is protective for developmental and reproductive toxicity of highly potent groups of anticancer compounds. There were similar TTC values calculated for direct-acting and indirect-acting anticancer compounds.

Bioavailability of therapeutic proteins by inhalation--worker safety aspects.

A literature review and analysis of inhalation bioavailability data for large therapeutic proteins was conducted in order to develop a practical estimate of the inhalation bioavailability of these drugs. This value is incorporated into equations used to derive occupational exposure limits(OELs) to protect biopharmaceutical manufacturing workers from systemic effects. Descriptive statistics implies that a value of 0.05, or 5% is an accurate estimate for large therapeutic proteins (molecular weight ≥ 40kDa). This estimate is confirmed by pharmacokinetic modeling of data from a human daily repeat-dose inhalation study of immunoglobulin G. In conclusion, we recommend using 5% bioavailability by inhalation when developing OELs for large therapeutic proteins.

Corrigendum: Transcriptome-wide analyses of early immune responses in lumpfish leukocytes upon stimulation with poly(I:C).

[This corrects the article DOI: 10.3389/fimmu.2023.1198211.].