Cellular Outcomes Research Articles

Epstein-Barr virus reactivation induces divergent abortive, reprogrammed, and host shutoff states by lytic progression.

Viral infection leads to heterogeneous cellular outcomes ranging from refractory to abortive and fully productive states. Single cell transcriptomics enables a high resolution view of these distinct post-infection states. Here, we have interrogated the host-pathogen dynamics following reactivation of Epstein-Barr virus (EBV). While benign in most people, EBV is responsible for infectious mononucleosis, up to 2% of human cancers, and is a trigger for the development of multiple sclerosis. Following latency establishment in B cells, EBV reactivates and is shed in saliva to enable infection of new hosts. Beyond its importance for transmission, the lytic cycle is also implicated in EBV-associated oncogenesis. Conversely, induction of lytic reactivation in latent EBV-positive tumors presents a novel therapeutic opportunity. Therefore, defining the dynamics and heterogeneity of EBV lytic reactivation is a high priority to better understand pathogenesis and therapeutic potential. In this study, we applied single-cell techniques to analyze diverse fate trajectories during lytic reactivation in three B cell models. Consistent with prior work, we find that cell cycle and MYC expression correlate with cells refractory to lytic reactivation. We further found that lytic induction yields a continuum from abortive to complete reactivation. Abortive lytic cells upregulate NFκB and IRF3 pathway target genes, while cells that proceed through the full lytic cycle exhibit unexpected expression of genes associated with cellular reprogramming. Distinct subpopulations of lytic cells further displayed variable profiles for transcripts known to escape virus-mediated host shutoff. These data reveal previously unknown and promiscuous outcomes of lytic reactivation with broad implications for viral replication and EBV-associated oncogenesis.

DCas9-HDAC8-EGFP fusion enables epigenetic editing of breast cancer cells by H3K9 deacetylation

Epigenetic editing is thriving as a robust tool for manipulating transcriptional regulation and cell fate. Despite its regulatory role in gene downregulation, epigenetic editing with histone deacetylation has been sparsely studied, especially in the context of cancer. In this current study, we have reconstructed a dCas9-HDAC8-EGFP fusion to perform histone deacetylation on the promoter of the ESR1, TERT and CDKN1C genes for the first time in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in HEK293T cells. Our results demonstrated that dCas9-HDAC8-EGFP in combination with appropriate gRNAs were able to downregulate the expression of the ESR1, TERT and CDKN1C genes transcriptionally by specifically depleting the H3K9ac level on the recruitment loci. The addition of histone deacetylase inhibitors was found to neutralize the outcomes of dCas9-HDAC8-EGFP-induced epigenetic editing. Furthermore, we observed a significant downregulation of full length ERα expression in epigenetically edited MCF-7 cells with consequential alteration in cellular response toward estradiol and tamoxifen treatment due to dCas9-HDAC8-EGFP mediated epigenetic editing of the ESR1 gene. Overall, dCas9-HDAC8-EGFP is a novel circuit that enabled downregulation of crucial genes with cellular outcome in breast cancer cells by preferentially inducing H3K9 deacetylation of specific promoter regions.

Mitochondrial genome-derived circRNAs: Orphan epigenetic regulators in molecular biology

Mitochondria are vital for cellular activities, influencing ATP production, Ca2+ signaling, and reactive oxygen species generation. It has been proposed that nuclear genome-derived circular RNAs (circRNAs) play a role in biological processes. For the first time, this study aims to comprehensively explore experimentally confirmed human mitochondrial genome-derived circRNAs (mt-circRNAs) via in-silico analysis. We utilized wide-ranging bioinformatics tools to anticipate their roles in molecular biology, involving miRNA sponging, protein antagonism, and peptide translation. Among five well-characterized mt-circRNAs, SCAR/mc-COX2 stands out as particularly significant with the potential to sponge around 41 different miRNAs, which target several genes mostly involved in endocytosis, MAP kinase, and PI3K-Akt pathways. Interestingly, circMNTND5 and mecciND1 specifically interact with miRNAs through their unique back-splice junction sequence. These exclusively targeted miRNAs (has-miR-5186, 6888-5p, 8081, 924, 672-5p) are predominantly associated with insulin secretion, proteoglycans in cancer, and MAPK signaling pathways. Moreover, all mt-circRNAs intricately affect the P53 pathway through miRNA sequestration. Remarkably, mc-COX2 and circMNTND5 appear to be involved in the RNA’s biogenesis by antagonizing AGO1/2, EIF4A3, and DGCR8. All mt-circRNAs engaged with IGF2BP proteins crucial in redox signaling, and except mecciND1, they all potentially generate at least one protein resembling the immunoglobulin heavy chain protein. Given P53′s function as a redox-sensitive transcription factor, and insulin’s role as a crucial regulator of energy metabolism, their indirect interplay with mt-circRNAs could influence cellular outcomes. However, due to limited attention and infrequent data availability, it is advisable to conduct more thorough investigations to gain a deeper understanding of the functions of mt-circRNA.

Drug-induced oxidative stress actively prevents caspase activation and hepatocyte apoptosis

Cell death is a fundamental process in health and disease. Emerging research shows the existence of numerous distinct cell death modalities with similar and intertwined signaling pathways, but resulting in different cellular outcomes, raising the need to understand the decision-making steps during cell death signaling. Paracetamol (Acetaminophen, APAP)-induced hepatocyte death includes several apoptotic processes but eventually is executed by oncotic necrosis without any caspase activation. Here, we studied this paradoxical form of cell death and revealed that APAP not only fails to activate caspases but also strongly impedes their activation upon classical apoptosis induction, thereby shifting apoptosis to necrosis. While APAP intoxication results in massive drop in mitochondrial respiration, low cellular ATP levels could be excluded as an underlying cause of missing apoptosome formation and caspase activation. In contrast, we identified oxidative stress as a key factor in APAP-induced caspase inhibition. Importantly, caspase inhibition and the associated switch from apoptotic to necrotic cell death was reversible through the administration of antioxidants. Thus, exemplified by APAP-induced cell death, our study stresses that cellular redox status is a critical component in the decision-making between apoptotic and necrotic cell death, as it directly affects caspase activity.

Poly-ADP-ribosylation dynamics, signaling, and analysis.

ADP-ribosylation is a reversible post-translational modification that plays a role as a signaling mechanism in various cellular processes. This modification is characterized by its structural diversity, highly dynamic nature, and short half-life. Hence, it is tightly regulated at many levels by cellular factors that fine-tune its formation, downstream signaling, and degradation that together impacts cellular outcomes. Poly-ADP-ribosylation is an essential signaling mechanism in the DNA damage response that mediates the recruitment of DNA repair factors to sites of DNA damage via their poly-ADP-ribose (PAR)-binding domains (PBDs). PAR readers, encoding PBDs, convey the PAR signal to mediate cellular outcomes that in some cases can be dictated by PAR structural diversity. Several PBD families have been identified, each with variable PAR-binding affinity and specificity, that also recognize and bind to distinct parts of the PAR chain. PARylation signaling has emerged as an attractive target for the treatment of specific cancer types, as the inhibition of PAR formation or degradation can selectively eliminate cancer cells with specific DNA repair defects and can enhance radiation or chemotherapy response. In this review, we summarize the key players of poly-ADP-ribosylation and its regulation and highlight PBDs as tools for studying PARylation dynamics and the expanding potential to target PARylation signaling in cancer treatment.

3-N-Butylphthalide Confers Antiarrhythmic Features in Ischemia/Reperfusion Injury of Diabetic Heart by Targeting Mitochondria-Endoplasmic Reticulum Network and Inhibiting Oxidative Stress and Inflammation

While 3-N-butylphthalide (NBP) has demonstrated notable cardioprotective effects, its precise role in mitigating myocardial arrhythmia following ischemia/reperfusion (IR) injury in diabetes remains unclear. This study aimed to explore the potential mechanisms through which NBP mitigates reperfusion-induced myocardial arrhythmia in diabetic rats, with a particular focus on mitochondrial function and biogenesis, endoplasmic reticulum (ER) stress, and oxidative/inflammatory responses. Sixty Sprague-Dawley rats were divided into non-diabetic and diabetic groups, subjected to in-vivo myocardial IR injury, and treated with NBP (100 mg/kg, intraperitoneally) through different modalities: preconditioning, postconditioning, or a combination of both. Electrocardiography (ECG) was employed to assess the incidence and severity of arrhythmia. Fluorometric, Western blotting and ELISA analyses were utilized to measure the mitochondrial, ER stress, and cellular outcomes. Treatment of non-diabetic rats with NBP in preconditioned, postconditioned, and combined approaches significantly reduced cardiotroponin-I and the frequency and severity of arrhythmias induced by IR injury. However, only the combined preconditioning plus postconditioning approach of NBP had protective and antiarrhythmic effects in diabetic rats, in an additive manner. Moreover, the NBP combined approach improved mitochondrial function and upregulated the expression of PGC-1α, Sirt1, and glutathione while concurrently downregulating ER stress and oxidative and pro-inflammatory-related proteins in diabetic rats. In conclusion, the combined approach of NBP treatment was effective in mitigating myocardial arrhythmia in diabetic rats. This approach coordinates interactions within the mitochondria-endoplasmic reticulum network and inhibits oxidative and inflammatory mediators, offering a promising strategy for managing myocardial arrhythmia in diabetic patients.

Selective optogenetic inhibition of Gαq or Gαi signaling by minimal RGS domains disrupts circuit functionality and circuit formation

Optogenetic techniques provide genetically targeted, spatially and temporally precise approaches to correlate cellular activities and physiological outcomes. In the nervous system, G protein-coupled receptors (GPCRs) have essential neuromodulatory functions through binding extracellular ligands to induce intracellular signaling cascades. In this work, we develop and validate an optogenetic tool that disrupts Gαq signaling through membrane recruitment of a minimal regulator of G protein signaling (RGS) domain. This approach, Photo-induced Gα Modulator-Inhibition of Gαq (PiGM-Iq), exhibited potent and selective inhibition of Gαq signaling. Using PiGM-Iq we alter the behavior of Caenorhabditis elegans and Drosophila with outcomes consistent with GPCR-Gαq disruption. PiGM-Iq changes axon guidance in cultured dorsal root ganglia neurons in response to serotonin. PiGM-Iq activation leads to developmental deficits in zebrafish embryos and larvae resulting in altered neuronal wiring and behavior. Furthermore, by altering the minimal RGS domain, we show that this approach is amenable to Gαi signaling. Our unique and robust optogenetic Gα inhibiting approaches complement existing neurobiological tools and can be used to investigate the functional effects neuromodulators that signal through GPCR and trimeric G proteins.

A robust evaluation of TDP-43, poly GP, cellular pathology and behavior in a AAV-C9ORF72 (G4C2)66 mouse model.

The G4C2 hexanucleotide repeat expansion in C9ORF72 is the major genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Despite considerable efforts, the development of mouse models of C9-ALS/FTD useful for therapeutic development has proven challenging due to the intricate interplay of genetic and molecular factors underlying this neurodegenerative disorder, in addition to species differences. This study presents a robust investigation of the cellular pathophysiology and behavioral outcomes in a previously described AAV mouse model of C9-ALS expressing 66 G4C2 hexanucleotide repeats. Despite displaying key molecular ALS pathological markers including RNA foci, dipeptide repeat (DPR) protein aggregation, p62 positive stress granule formation as well as mild gliosis, the AAV-(G4C2)66 mouse model in this study exhibits negligible neuronal loss, no motor deficits, and functionally unimpaired TAR DNA-binding protein-43 (TDP-43). While our findings indicate and support that this is a robust and pharmacologically tractable model for investigating the molecular mechanisms and cellular consequences of (G4C2) repeat driven DPR pathology, it is not suitable for investigating the development of disease associated neurodegeneration, TDP-43 dysfunction, gliosis, and motor performance. Our findings underscore the complexity of ALS pathogenesis involving genetic mutations and protein dysregulation and highlight the need for more comprehensive model systems that reliably replicate the multifaceted cellular and behavioral aspects of C9-ALS.

PP2A phosphatase regulatory subunit PPP2R3C is a new positive regulator of the hedgehog signaling pathway

Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored.This study unveils a novel role for the B″gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity as measured by reduced expression of Gli1/2 and Hh target genes upon Hh signaling activation, and reduced growth of a Hh signaling-dependent medulloblastoma cell line. Moreover, we establish an antagonistic connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.

Ligand Shell Thickness of PEGylated Gold Nanoparticles Controls Cellular Uptake and Radiation Enhancement.

The drive to improve the safety and efficacy of radiotherapies for cancers has prompted the development of nanomaterials that can locally amplify the radiation dose at a tumor without damaging the surrounding healthy tissue. Gold nanoparticles (Au NPs), in particular, exhibit promising radiosensitizing properties under kilovolt X-ray exposure, although the precise mechanism behind this enhancement is not fully understood. While most studies recognize the involvement of factors such as core composition, size, shape, and ligand chemistry in the effectiveness of Au NPs for radiation-induced cancer treatment, there is a scarcity of direct assessments that connect the photophysical properties of the nanomaterial with the observed cellular or biological outcomes. Despite previous evidence of low-energy electron (LEE) emission from Au NPs and their potential to initiate biological damage, to our knowledge, no studies directly correlate the secondary LEE emission with radiation-induced cell death. In this study we assessed Au NPs functionalized with polyethylene glycol (PEG) ligands of varying molecular weights and lengths (1, 5, and 20 kDa PEG) as potential radiosensitizers of A549 lung cancer cells using kilovolt X-ray source potentials (33-130 kVp). We assessed NP internalization using mass cytometry, radiation dose enhancement using clonogenic survival assays, and secondary LEE emission using a retarding field analyzer. Results reveal a statistically significant difference in cellular uptake and radiation dose enhancement for 5 kDa PEG-Au NPs compared to formulations using 1 and 20 kDa PEG, while analysis of secondary LEE emission spectra demonstrated that differences in the length of the PEG ligand did not cause statistically significant attenuation of secondary LEE flux. Consequently, we inferred increased cellular uptake of NPs to be the cause for the observed enhancement in radiosensitivity for 5 kDa PEGylated Au NPs. The approach used in this study establishes a more complete workflow for designing and characterizing the performance of nanomaterial radiosensitizers, allowing for quantification of secondary LEEs and cellular uptake, and ultimately correlation with localized dose enhancement that leads to cell death.

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 µm intervals, covering a depth of 90 µm in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Artificial mitochondrial transplantation (AMT) reverses aging of mesenchymal stromal cells and improves their immunomodulatory properties in LPS-induced synoviocytes inflammation

Nowadays, regenerative medicine techniques are usually based on the application of mesenchymal stromal cells (MSCs) for the repair or restoration of injured damaged tissues. However, the effectiveness of autologous therapy is limited as therapeutic potential of MSCs declines due to patient's age, health condition and prolonged in vitro cultivation as a result of decreased growth rate. For that reason, there is an urgent need to develop strategies enabling the in vitro rejuvenation of MSCs prior transplantation in order to enhance their in vivo therapeutic efficiency.In presented study, we attempted to mimic the naturally occurring mitochondrial transfer (MT) between neighbouring cells and verify whether artificial MT (AMT) could reverse MSCs aging and improve their biological properties. For that reason, mitochondria were isolated from healthy donor equine adipose-derived stromal cells (ASCs) and transferred into metabolically impaired recipient ASCs derived from equine metabolic syndrome (EMS) affected horses, which were subsequently subjected to various analytical methods in order to verify the cellular and molecular outcomes of the applied AMT.Mitochondria recipient cells were characterized by decreased apoptosis, senescence and endoplasmic reticulum stress while insulin sensitivity was enhanced. Furthermore, we observed increased mitochondrial fragmentation and associated PARKIN protein accumulation, which indicates on the elimination of dysfunctional organelles via mitophagy. AMT further promoted physioxia and regulated autophagy fluxes. Additionally, rejuvenated ASCs displayed an improved anti-inflammatory activity toward LPS-stimulated synoviocytes. The presented findings highlight AMT as a promising alternative and effective method for MSCs rejuvenation, for potential application in autologous therapies in which MSCs properties are being strongly deteriorated due to patients' condition.

A primer for junior trainees: Recognition of RNA modifications by RNA-binding proteins.

The complexity of RNA cannot be fully expressed with the canonical A, C, G, and U alphabet. To date, over 170 distinct chemical modifications to RNA have been discovered in living systems. RNA modifications can profoundly impact the cellular outcomes of messenger RNAs (mRNAs), transfer and ribosomal RNAs, and noncoding RNAs. Additionally, aberrant RNA modifications are associated with human disease. RNA modifications are a rising topic within the fields of biochemistry and molecular biology. The role of RNA modifications in gene regulation, disease pathogenesis, and therapeutic applications increasingly captures the attention of the scientific community. This review aims to provide undergraduates, junior trainees, and educators with an appreciation for the significance of RNA modifications in eukaryotic organisms, alongside the skills required to identify and analyze fundamental RNA-protein interactions. The pumilio RNA-binding protein and YT521-B homology (YTH) family of modified RNA-binding proteins serve as examples to highlight the fundamental biochemical interactions that underlie the specific recognition of both unmodified and modified ribonucleotides, respectively. By instilling these foundational, textbook concepts through practical examples, this review contributes an analytical toolkit that facilitates engagement with RNA modifications research at large.

Generation of an induced pluripotent stem cell line (BIORTCi001-A) from a healthy adult indigenous Nigerian participant

Genetic backgrounds influence cellular phenotypes, drug responses, and health outcomes, yet most human iPSC lines are derived from individuals of European descent, with lines from indigenous Africans particularly scarce. Addressing this gap, we generated iPSCs from dermal fibroblasts of a healthy 60-year-old indigenous Nigerian male of the Babur ethnic group using Sendai virus. The iPSC line displayed a normal karyotype, was characterized for pluripotency markers and differentiated into neural progenitor cells and astrocytes. To enhance African representation in research, this iPSC line will be available to the scientific community, with ongoing efforts focused on creating an open-access African iPSC biobank.Resource Table:

Systemic Effects of Photoactivated 5,10,15,20-tetrakis(N-methylpyridinium-3-yl) Porphyrin on Healthy Drosophila melanogaster.

Porphyrins are frequently employed in photodynamic therapy (PDT), a non-invasive technique primarily utilized to treat subcutaneous cancers, as photosensitizing agents (PAs). The development of a new PA with improved tissue selectivity and efficacy is crucial for expanding the application of PDT for the management of diverse cancers. We investigated the systemic effects of 5,10,15,20-tetrakis(N-methylpyridinium-3-yl)-porphyrin (TMPyP3) using Drosophila melanogaster adult males. We established the oral administration schedule and demonstrated that TMPyP3 was absorbed and stored higher in neuronal than in non-neuronal extracts. Twenty-four hours after oral TMPyP3 photoactivation, the quantity of hydrogen peroxide (H2O2) increased, but exclusively in the head extracts. Regardless of photoactivation, TMPyP3 resulted in a reduced concentration of H2O2 after 7 days, and this was linked with a decreased capacity to climb, as indicated by negative geotaxis. The findings imply that systemic TMPyP3 therapy may disrupt redox regulation, impairing cellular signaling and behavioral outcomes in the process. To determine the disruptive effect of porphyrins on redox homeostasis, its duration, and the mechanistic variations in retention across various tissues, more research is required.

BET activity plays an essential role in control of stem cell attributes in Xenopus.

Neural crest cells are a stem cell population unique to vertebrate embryos that retains broad multi-germ layer developmental potential through neurulation. Much remains to be learned about the genetic and epigenetic mechanisms that control the potency of neural crest cells. Here, we examine the role that epigenetic readers of the BET (bromodomain and extra terminal) family play in controlling the potential of pluripotent blastula and neural crest cells. We find that inhibiting BET activity leads to loss of pluripotency at blastula stages and a loss of neural crest at neurula stages. We compare the effects of HDAC (an eraser of acetylation marks) and BET (a reader of acetylation) inhibition and find that they lead to similar cellular outcomes through distinct effects on the transcriptome. Interestingly, loss of BET activity in cells undergoing lineage restriction is coupled to increased expression of genes linked to pluripotency and prolongs the competence of initially pluripotent cells to transit to a neural progenitor state. Together these findings advance our understanding of the epigenetic control of pluripotency and the formation of the vertebrate neural crest.

Transfer learning of condition-specific perturbation in gene interactions improves drug response prediction.

Drug response is conventionally measured at the cell level, often quantified by metrics like IC50. However, to gain a deeper understanding of drug response, cellular outcomes need to be understood in terms of pathway perturbation. This perspective leads us to recognize a challenge posed by the gap between two widely used large-scale databases, LINCS L1000 and GDSC, measuring drug response at different levels-L1000 captures information at the gene expression level, while GDSC operates at the cell line level. Our study aims to bridge this gap by integrating the two databases through transfer learning, focusing on condition-specific perturbations in gene interactions from L1000 to interpret drug response integrating both gene and cell levels in GDSC. This transfer learning strategy involves pretraining on the transcriptomic-level L1000 dataset, with parameter-frozen fine-tuning to cell line-level drug response. Our novel condition-specific gene-gene attention (CSG2A) mechanism dynamically learns gene interactions specific to input conditions, guided by both data and biological network priors. The CSG2A network, equipped with transfer learning strategy, achieves state-of-the-art performance in cell line-level drug response prediction. In two case studies, well-known mechanisms of drugs are well represented in both the learned gene-gene attention and the predicted transcriptomic profiles. This alignment supports the modeling power in terms of interpretability and biological relevance. Furthermore, our model's unique capacity to capture drug response in terms of both pathway perturbation and cell viability extends predictions to the patient level using TCGA data, demonstrating its expressive power obtained from both gene and cell levels. The source code for the CSG2A network is available at https://github.com/eugenebang/CSG2A.

Prodrug Approach to Exploit (S)-Alanine Amide as Arginine Mimic Moiety in the Development of Protein Arginine Methyltransferase 4 Inhibitors.

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as an emerging target class in oncology and other diseases. A successful strategy to identify PRMT substrate-competitive inhibitors has been to exploit chemical scaffolds able to mimic the arginine substrate. (S)-Alanine amide moiety is a valuable arginine mimic for the development of potent and selective PRMT4 inhibitors; however, its high hydrophilicity led to derivatives with poor cellular outcomes. Here, we describe the development of PRMT4 inhibitors featuring a central pyrrole core and an alanine amide moiety. Rounds of optimization, aimed to increase lipophilicity and simultaneously preserve the inhibitory activity, produced derivatives that, despite good potency and physicochemical properties, did not achieve on-target effects in cells. On the other hand, masking the amino group with a NAD(P)H:quinone oxidoreductase 1 (NQO1)-responsive trigger group, led to prodrugs able to reduce arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155). These results indicate that prodrug strategies can be successfully applied to alanine-amide containing PRMT4 inhibitors and provide an option to enable such compounds to achieve sufficiently high exposures in vivo.

Patient engagement in hematopoietic stem cell transplantation and cell therapy: a survey by the EBMT patient engagement task force & transplantation complications working party.

The EBMT (European Blood and Marrow Transplantation Society) aims to connect patients, the scientific community, and other stakeholders to improve hematopoietic stem cell transplantation and cellular therapy outcomes. We performed a cross-sectional online survey to understand the perceptions regarding Patient Reported Outcomes (PROs) and Patient Active Involvement in Research (PAIR) in over 800 stakeholders (n = 813). Patients (n = 278) and health care professionals (HCPs) (n = 351) were compared. We observed high openness for EBMT PRO collection (n = 680, 84.5% across stakeholders' groups; patients n = 256, 93.1% versus HCPs n = 273, 78.4% [p < 0.001]) and PAIR (n = 702, 87.3% across stakeholder groups; patients n = 256, 92.4% versus HCPs n = 296, 85.8% [p = 0.009]), with a significantly higher proportion of patients expressing interest compared to HCPs. Priority domains for PROs data-collection identified were the assessment of symptom experience, psychosocial and cognitive functioning. The most important issues for patients specifically were the data-collection of PROs reflecting cognitive function, the option of reporting data at home, the importance of identifying actionable targets to improve their recovery, and receiving feedback on their input when participating in research projects. Our multistakeholder approach suggests an added value to embracing patient engagement in the development of meaningful research and service design within the transplantation and cellular therapy community.

Physicochemical metamorphosis of re-aerosolized urban PM2.5

The toxicity of particulate matter (PM) is dependent on particle physical and chemical properties and is commonly studied using in vivo and in vitro approaches. PM to be used for in vivo and in vitro studies is often collected on filters and then extracted from the filter surface using a solvent. During extraction and further PM sample handling, particle properties change, but this is often neglected in toxicology studies, with possible implications for health effect assessment. To address the current lack of knowledge and investigate changes in particle properties further, ambient PM with diameter less than 2.5 μm (PM2.5) was collected on filters at an urban site and extracted using a standard methanol protocol. After extraction, the PM was dried, dispersed in water and subsequently nebulized. The resulting aerosol properties were then compared to those of the ambient PM2.5. The number size distribution for the nebulized aerosol resembled the ambient in terms of the main mode diameter, and >90 % of particle mass in the nebulized size distribution was still in the PM2.5 range. Black carbon made up a similar fraction of PM mass in nebulized as in ambient aerosol. The sulfate content in the nebulized aerosol seemed depleted and the chemical composition of the organic fraction was altered, but it remains unclear to what extent other non-refractory components were affected by the extraction process. Trace elements were not distributed equally across size fractions, neither in ambient nor nebulized PM. Change in chemical form was studied for zinc, copper and iron. The form did not appear to be different between the ambient and nebulized PM for iron and copper, but seemed altered for zinc. Although many of the studied properties were reasonably well preserved, it is clear that the PM2.5 collection and re-aerosolization process affects particles, and thus potentially also their health effects. Because of this, the effect of the particle collection and extraction process must be considered when evaluating cellular and physiological outcomes upon PM2.5 exposure.