mRNA Research Articles

Abstract B008: Exploring the Dual Roles of RNAs as Oncogenic Drivers and Therapeutic Targets in Cancer: A Cross-Sectional Study

Abstract RNAs have emerged as pivotal molecules in the complex landscape of cancer biology, acting as both drivers of oncogenesis and therapeutic targets. This cross-sectional experimental design research explores the dual roles of RNAs, particularly focusing on the interplay between non-coding RNAs (ncRNAs), messenger RNAs (mRNAs), and their regulatory networks in various cancer types. The study systematically analyzes RNA expression profiles across a broad spectrum of cancer samples, employing high-throughput sequencing technologies combined with bioinformatics tools to identify key RNA species that contribute to tumorigenesis. The experimental design includes the collection of tumor and adjacent normal tissues from patients diagnosed with breast, lung, and colorectal cancers. RNA extraction and sequencing are performed to generate comprehensive expression profiles. Differential expression analysis is conducted to pinpoint RNAs that are significantly dysregulated in cancerous tissues compared to their normal counterparts. Subsequently, functional assays, including RNA interference (RNAi) and CRISPR/Cas9-mediated gene editing, are utilized to validate the oncogenic or tumor-suppressive roles of these RNAs in cancer cell lines. To further elucidate the mechanistic pathways, the study integrates data from RNA-binding protein immunoprecipitation (RIP) and crosslinking immunoprecipitation (CLIP) assays, providing insights into the RNA-protein interactions that influence cancer progression. Additionally, the research evaluates the therapeutic potential of targeting these RNAs, using small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs) in preclinical cancer models. Preliminary findings highlight the significance of long non-coding RNAs (lncRNAs) and micro RNAs (miRNAs) in modulating key signaling pathways involved in cell proliferation, apoptosis, and metastasis. This study underscores the importance of RNA-based diagnostics and therapeutics, paving the way for innovative treatment strategies in cancer management. Citation Format: Fathelrahman M Gameel. Exploring the Dual Roles of RNAs as Oncogenic Drivers and Therapeutic Targets in Cancer: A Cross-Sectional Study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B008.

Effect of soy protein isolate supplementation on the estradiol biosynthesis pathway in a female rat model

Background Soy protein isolate (SPI) has gained popularity as an alternative to animal proteins. Soy consumption may have a link to rising serum estrogen in humans. This concern is attributed to phytoestrogens, specifically isoflavones present in soy protein, acting as estrogen substitutes and modulators. Objective The objective of this research was to assess the influence of SPI supplementation on estradiol hormone levels and the expression of genes associated with estrogen synthesis in female rats. Materials and methods Female Wistar rats (n=18) were evenly divided into three groups: group 1 (normal control) received oral administration of a saline vehicle. Group 2 (low dose) received 450 mg/kg body weight of SPI for 30 days. Group 3 (high dose) received 900 mg/kg body weight of SPI for 30 days. All administrations were conducted intragastrically. Results and conclusion The results of our study indicate that there was a significant increase in the levels of estradiol hormone in groups receiving low and high doses of SPI when compared with the control group. There was an upregulation in the messenger ribonucleic acid expression of the Cyp19 gene in low-dose and high-dose groups. Moreover, the low-dose group showed upregulation in the expression of the HSD3B gene. These genes are involved in the biosynthesis pathway of estrogen hormone in females. Therefore, the use of SPI should be cautious because it contains phytoestrogens (isoflavones), which have structural similarities to endogenous female estrogen and may cause hormonal disturbance in females.

Abstract A022: Patho-DBiT: Spatially exploring RNA biology in archival formalin-fixed paraffin- embedded tissues

Abstract Despite recent breakthroughs in spatial biology, either imaging-based or sequencing-based spatial transcriptomics is largely equivalent to spatial messenger RNA (mRNA) expression quantification. An RNA molecule experiences a complex life cycle involving transcription, splicing, maturation, translation, and degradation . It is highly desirable to profile all these RNA species spanning their life cycle to explore the biology of RNA at genome scale and cellular level. Furthermore, formalin-fixed paraffin-embedded (FFPE) tissues are essential in clinical practice, being the backbone of human disease histopathological diagnoses . Pathology departments have accrued vast collections of FFPE blocks over time, creating a rich, yet underutilized compendium of materials that, accompanied by clinical data, stands as a treasure trove for human biology and translational research . In this evolving landscape, we present “Pathology-compatible Deterministic Barcoding in Tissue” (Patho-DBiT) not only enabling spatial full-coverage base-by-base whole transcriptome sequencing but also crafted to address the challenges of clinically archived FFPE tissues. Patho-DBiT integrates in situ polyadenylation, microfluidic in tissue barcoding, and computational innovations to decode rich RNA biology inherent in FFPE samples. The platform capitalizes on RNA fragmentation naturally occurring in FFPE specimens and appends poly(A) tails to a broad spectrum of RNA species, thereby overcoming traditional barriers associated with FFPE samples and even outperforming the assays conducted with frozen tissues. Patho-DBiT permits spatial co-profiling of gene expression and alternative splicing, unveiling region-specific isoforms in the mouse brain. High-sensitivity transcriptomics is constructed from 5-year archived T-cell lymphoma tissues, with cross-validation conducted using super-resolution spatial phenotyping technology (CODEX). Furthermore, genome-scale single nucleotide RNA variants (SNVs) are captured to distinguish malignant subclones from non-malignant cells in human B-cell lymphomas, dissecting spatial clonal architectures based on both SNVs and copy number variation (CNV) profiles. Patho-DBiT also enables spatially resolved co-profiling of large and small RNAs, facilitating the analysis of a microRNA-mRNA regulatory network and tRNA utilization within clinical biopsies and elucidating their roles in tumorigenesis. With superior intronic read capture efficiency, Patho-DBiT spatially mapped RNA splicing dynamics associated with the developmental trajectory of tumor B cells. High resolution Patho-DBiT with a 10-μm spot size reveals the heterogeneities of human lymphomas within a spatial neighborhood and traces the spatiotemporal molecular kinetics driving tumorprogression at the cellular level. Patho-DBiT represents a first-of-its-kind technology, enabling the spatial exploration of rich RNA biology in FFPE tissues to aid in clinical pathology research. Citation Format: Zhiliang Bai, Dingyao Zhang, Yan Gao, Bo Tao, Mingyao Li, Yi Xing, Jun Lu, Mina Xu, Rong Fan. Patho-DBiT: Spatially exploring RNA biology in archival formalin-fixed paraffin- embedded tissues [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A022.

Low Molecular Weight Alginate Oligosaccharides as Alternatives to PEG for Enhancement of the Diffusion of Cationic Nanoparticles Through Cystic Fibrosis Mucus

AbstractAirway mucus is a major barrier to the delivery of lipid‐based nanoparticles in chronic airway diseases such as cystic fibrosis (CF). Receptor‐Targeted Nanocomplexes (RTN), comprise mixtures of cationic lipids and bifunctional peptides with receptor‐targeting and nucleic acid packaging properties. The aim of this study is to improve the mucus‐penetrating properties of cationic siRNA and mRNA RTNs by combining them with low molecular weight alginate oligosaccharides, OligoG and OligoM. Cationic RTNs formulated with either alginate become strongly anionic, while PEGylated messenger RNA (mRNA) and short interfering RNA (siRNA) RTNs remain cationic. Both alginates enhance mucus diffusion rates of cationic siRNA and mRNA RTNs in a static mucus barrier diffusion model, with OligoG particularly effective. PEGylation also enhance mucus diffusion rates of siRNA RTNs but not mRNA RTNs. Electron microscopy shows that RTNs remained intact after mucosal transit. The transfection efficiency of OligoM‐coated mRNA RTNs is better than those coated with OligoG or PEG, and similar to cationic RTNs. In siRNA RTN transfections, OligoM is better than OligoG although 1% PEG is slightly better than both. The combination of cationic RTNs and alginate oligosaccharides represents a promising alternative to PEGylation for epithelial delivery of genetic therapies across the mucus barrier while retaining transfection efficiency.

Prophylactic and therapeutic vaccine development: advancements and challenges

AbstractBiomedical research is fundamental in developing preventive and therapeutic vaccines, serving as a cornerstone of global public health. This review explores the key concepts, methodologies, tools, and challenges in the vaccine development landscape, focusing on transitioning from basic biomedical sciences to clinical applications. Foundational disciplines such as virology, immunology, and molecular biology lay the groundwork for vaccine creation, while recent innovations like messenger RNA (mRNA) technology and reverse vaccinology have transformed the field. Additionally, it highlights the role of pharmaceutical advancements in translating lab discoveries into clinical solutions. Techniques like CRISPR-Cas9, genome sequencing, monoclonal antibodies, and computational modeling have significantly enhanced vaccine precision and efficacy, expediting the development of vaccines against infectious diseases. The review also discusses challenges that continue to hinder progress, including stringent regulatory pathways, vaccine hesitancy, and the rapid emergence of new pathogens. These obstacles underscore the need for interdisciplinary collaboration and the adoption of innovative strategies. Integrating personalized medicine, nanotechnology, and artificial intelligence is expected to revolutionize vaccine science further. By embracing these advancements, biomedical research has the potential to overcome existing challenges and usher in a new era of therapeutic and prophylactic vaccines, ultimately improving global health outcomes. This review emphasizes the critical role of vaccines in combating current and future health threats, advocating for continued investment in biomedical science and technology.

IMMU-63. MESSENGER RNA CHALLENGE PREDICTS CANCER IMMUNOGENICITY AND RESPONSE TO CHECKPOINT INHIBITORS

Abstract BACKGROUND Checkpoint inhibition has emerged as a promising cancer therapeutic approach. Checkpoint inhibitors (CIs) function by overcoming the adaptive T cell exhaustion induced by solid tumor microenvironment including brain tumors, particularly in the neoadjuvant setting. Unfortunately, not every patient who receives CIs will unlock effective antitumor immunity. Objectives: Microsatellite instability and high mutational burden are considered key criteria to predict response to CIs. However, only a minority of patients will fulfill these criteria suggesting the need to develop innovative technologies to identify CI responsive patients. METHODS To determine whether glioma tumors will respond to CIs, we have developed a novel multilamellar mRNA-lipid particle tool to predict response ex vivo. Messenger RNA is complexed into multilamellar lipid particles to form aggregates (RNA-LPAs) to induce pathogen recognition receptor signaling in tumor cells to define an inflammatory profile as an indirect predictor to CI response. RESULTS In naïve mice, we found that multilamellar RNA-LPAs induce activation of Type I IFN system as supported by decreased secretion of IFN-α in MyD88 KO mice compared with wild type mice. We next selected murine glioma cell lines with known response profile to CIs (responders – GL261 and SMA560 vs non-responders – KR158b and CT2A). Prior to evaluation of cytokine profiling, successful uptake of RNA-LPAs was achieved in all cell lines. In 2D cell lines cultures and their corresponding 3D tumor spheroids, we demonstrated that treatment of CI responsive murine glioma models showed a differential cytokine signature (increased secretion of IFN-β/IL-6/CCL4 versus non-responsive models). CONCLUSION We identified a unique inflammatory signature after ex vivo RNA-LPAs challenge that correlates with checkpoint blockade activity. These findings allow for creation of a diagnostic assay to quickly assess tumor immunogenicity, allowing for informed immunotherapeutic treatment.

Mining the secreted and membrane transcriptome of Hyalomma dromedarii ticks for identification of potential protective antigens

Abstract Background Members belonging to the tick genus Hyalomma function as a multi-host reservoir for several pathogens and important parasites infesting large animals, such as camels, goats, cattle and sheep. In Egypt, there is a high risk of pathogen transmission as camels and cattle are imported from Sudan and Ethiopia and shipped to slaughterhouses and animal markets located in populated areas. Hyalomma dromedarii ticks are semi-desert vectors and, similar to other members of the genus Hyalomma, characterized by long-term feeding. During this process, different physiological, biochemical and immunological interactions occur within both the feeding ticks and their hosts. These biological changes affect the different tick developmental phases. The aim of this study was to explore the transcriptome of mixed messenger RNAs (mRNAs) collected from H. dromedarii eggs, larvae, nymphs and fed and unfed adults, using the Gateway cDNA library prepared in pCMV sport6.1 vector Methods The clones were sequenced and searched for potential secreted, membrane-associated or transmembrane (SMaT) sequences. The identified SMaT sequences were compared to the National Center for Biotechnology Information (NCBI) non-redundant protein sequence database using Blastx. Annotation and functional classification were achieved by comparison to sequences in the UniProtKB/Swiss-Prot and VectorBase databases and to the publicly available annotated proteomes of six hard tick species (H. asiaticum, Rhipicephalus sanguineus sensu lato, Dermacentor silvarum, Rhipicephalus microplus, Ixodes scapularis and Haemaphysalis longicornis) in addition to the published H. dromedarii sialotranscriptome. For the common sequences, we predicted the physicochemical properties, secondary structures and antigenicity of the fragments similar to matched sequences in the UniProtKB/Swiss-Prot database using three different methods. Results The quality-trimmed sequences from the cDNA library revealed 319 SMaT transcripts among 1248 sequenced clones. Annotation of the SMaT sequences using the UniProtKB/Swiss-Prot database revealed only 232 non-redundant sequences with at least one match. According to the UniProtKB/Swiss-Prot and Vectorbase databases, the SMaT sequences were either secreted (extracellular) (29 sequences) or cellular (transmembrane and membrane-associated) (203 sequences). These were classified into 10 functional classes: biogenesis (49 sequences), defense (9 sequences), development (36 sequences), signal transduction (28 sequences), transport (15 sequences), protein modification (33 sequences), homeostasis (6 sequences), metabolism (45 sequences) and miscellaneous/uncharacterized (11 sequences). A total of 60 sequences were shared between H. dromedarii SMaT, the sialotransciptome and six other hard tick species. The peptide fragments of these sequences that aligned to proteins from the UniProtKB/Swiss-Prot database were predicted to be promising epitopes and mapped to 10 functional classes at different ratios. Conclusions Our immuno-informatics analysis identified 60 sequences common among hard tick species and encoded by H. dromedarii salivary glands. These annotated SMaT sequences of H. dromedarii will pave the way for the identification and discovery of novel potential protective antigens that are either secreted, membrane-associated or transmembrane. Graphical abstract

Replenishing co‐downregulated miR‐100‐5p and miR‐125b‐5p in malignant germ cell tumors causes growth inhibition through cell cycle disruption

MicroRNAs (miRNAs) are short, nonprotein‐coding RNAs, and their expression is dysregulated in malignant germ cell tumors (GCTs). Here, we investigated the causes and consequences of downregulated miR‐99a‐5p/miR‐100‐5p (functionally identical) and miR‐125b‐5p levels in malignant GCTs regardless of age, site, or subtype. Quantitative RT‐PCR was used to assess miR‐99a‐5p/miR‐100‐5p, miR‐125b‐5p, and associated gene expression in malignant GCT tissues/cell lines [seminoma (Sem), yolk sac tumor (YST), embryonal carcinoma (EC)]. Cells were treated with demethylating 5‐azacytidine and pyrosequencing was performed. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment was used to treat malignant GCT cells. Global messenger RNA (mRNA) targets of the replenished miRNAs were identified and Metascape used to study pathway effects. We found that expression levels of miR‐99a‐5p/miR‐100‐5p and miR‐125b‐5p, their respective pri‐miRNAs, and associated genes from chromosomes 11 and 21 (chr11/chr21) were downregulated and highly correlated in malignant GCT cells. Treatment with 5‐azacytidine caused upregulation of these miRNAs, with pyrosequencing revealing hypermethylation of their chr11/chr21 loci, likely contributing to miR‐100‐5p/miR‐125b‐5p downregulation. Combination miR‐100‐5p/miR‐125b‐5p mimic replenishment resulted in growth inhibition in Sem/YST cells, with miR‐100‐5p/miR‐125b‐5p mRNA targets enriched in downregulated genes, which were involved in cell cycle (confirmed by flow cytometry) and signaling pathways. Knockdown of the miR‐100‐5p/miR‐125b‐5p target tripartite motif containing 71 (TRIM71kd) recapitulated miR‐100‐5p/miR‐125b‐5p replenishment, with growth inhibition and cell cycle disruption of Sem/YST/EC cells. Further, replenishment led to reduced lin‐28 homolog A (LIN28A) levels and concomitant increases in let‐7 (MIRLET7B) tumor suppressor miRNAs, creating a sustained reversion of cell phenotype. In summary, combination miR‐100‐5p/miR‐125b‐5p mimic replenishment or TRIM71kd caused growth inhibition in malignant GCT cells via cell cycle disruption. Further studies are now warranted, including mimic treatment alongside conventional platinum‐based chemotherapy.

Exploring the ceRNA network involving AGAP2-AS1 as a novel biomarker for preeclampsia

Preeclampsia (PE) is an important research subject in obstetrics. Nevertheless, the underlying mechanisms of PE remain elusive. PE-related expression datasets (GSE96983, GSE96984 and GSE24129) were downloaded from the Gene Expression Omnibus (GEO) database. Firstly, the differentially expressed messenger RNAs (DE-mRNAs), DE-microRNA (DE-miRNAs) and DE-long non-coding RNA (DE-lncRNAs) between PE and control cohorts were identified, and the ceRNA network was constructed. Then candidate hub genes were obtained through five algorithms by the protein-protein intersection (PPI) network of the mRNAs. Further, five hub genes were identified by receiver operating characteristic (ROC) curve and gene expression profiles: DAXX, EFNB1, NCOR2, RBBP4 and SOCS1. The function of 5 hub genes was analyzed and the interaction between drugs and hub genes was predicted. A total of 5 small molecule drugs were predicted, namely benzbromarone, 9,10-phenanthrenequinone, chembl312032, insulin and aldesleukin. AGAP2-AS1 was mainly located in exosome and cytoplasm. Agap2-as1-related regulatory subnetworks were extracted from ceRNA networks which included 41 mRNAs, 2 miRNAs and 1 lncRNA, including the regulated relationship pairs AGAP2-AS1-hsa-miR-497-5p-SRPRB, and AGAP2-AS1-hsa-miR-195-5p-RPL36. In summary, we constructed a competitive endogenous RNA (ceRNA) network to identify five potential biomarkers (DAXX, EFNB1, NCOR2, SOCS1 and RBBP4) of PE. The in-depth analysis of the AGAP2-AS1 regulatory network will help to uncover more important molecules closely related to PE and provide a scientific Reference.

Co-regulated ceRNA network mediated by circRNA and lncRNA in patients with gouty arthritis

Numerous studies have demonstrated the involvement of messenger RNAs (mRNAs) and non-coding RNAs, including long non-coding RNAs (lncRNA), circular RNAs (circRNAs) and microRNA (miRNAs), in gouty arthritis onset; however, the regulatory mechanism has not yet been elucidated. Here, we applied whole-transcriptome sequencing to identify the differentially expressed circRNAs, lncRNAs, miRNAs and mRNAs between the gout patients and normal people, and constructed co-regulated networks of circRNAs and lncRNAs according to the competitive endogenous RNA (ceRNA) theory for gouty arthritis onset to improve our understanding of the pathogenesis of this disease. The most significant finding of this study is the co-regulated ceRNA network of circRNAs and lncRNAs in gouty arthritis. The circRNA novel_circ_0030384 and the lncRNAs AAMP, TRIM16, PKN1, XLOC_184579 and XLOC_189826 were upstream genes in the co-regulated network. These upstream genes upregulated miR550a-5p and miR550a-3-5p, which downregulated PSME1 and FERMT3 expression. These mRNAs participated in proteasome dynamics, antigen processing and presentation, and platelet activation, which are associated with inflammation in gouty arthritis. In addition, the circRNA and lncRNAs upregulated miR550a-5p, which downregulated GRK2 and OS9 expression. Also, it proved that the down-regulated of PSME1, FERMT3, GRK2 and OS9 can aggravate gouty arthritis in vitro. In summary, these genes mediate inflammation in gouty arthritis through chemokine signaling to regulate neutrophil function.

Upregulated miR-10b-5p as a potential miRNA signature in amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset disease marked by a progressive degeneration of motor neurons (MNs) present in the spinal cord, brain stem and motor cortex. Death in most patients usually occurs within 2–4 years after symptoms onset. Despite promising progress in delineating underlying mechanisms, such as disturbed proteostasis, DNA/RNA metabolism, splicing or proper nucleocytoplasmic shuttling, there are no effective therapies for the vast majority of cases. A reason for this might be the disease heterogeneity and lack of substantial clinical and molecular biomarkers. The identification and validation of such pathophysiology driven biomarkers could be useful for early diagnosis and treatment stratification. Recent advances in next generation RNA-sequencing approaches have provided important insights to identify key changes of non-coding RNAs (ncRNAs) implicated with ALS disease. Especially, microRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression to target several genes/pathways by degrading messenger RNAs (mRNAs) or repressing levels of gene expression. In this study, we expand our previous work to identify top-regulated differentially expressed (DE)-miRNAs by combining different normalizations to search for important and generalisable pathomechanistic dysregulations in ALS as putative novel biomarkers of the disease. For this we performed a consensus pipeline of existing datasets to investigate the transcriptomic profile (mRNAs and miRNAs) of MN cell lines from iPSC-derived SOD1- and TARDBP (TDP-43 protein)-mutant-ALS patients and healthy controls to identify potential signatures and their related pathways associated with neurodegeneration. Transcriptional profiling of miRNA–mRNA interactions from MN cell lines in ALS patients revealed differential expression of genes showed greater vulnerability to KEAP1-NRF2 stress response pathway, sharing a common molecular denominator linked to both disease conditions. We also reported that mutations in above genes led to significant upregulation of the top candidate miR-10b-5p, which we could validate in immortalized lymphoblast cell lines (LCLs) derived from sporadic and familial ALS patients and postmortem tissues of familial ALS patients. Collectively, our findings suggest that miRNA analysis simultaneously performed in various human biological samples may reveal shared miRNA profiles potentially useful as a biomarker of the disease.

Safety of Simultaneous vs Sequential mRNA COVID-19 and Inactivated Influenza Vaccines

Limited randomized clinical trial data exist on the safety of simultaneous administration of COVID-19 and influenza vaccines. To compare the reactogenicity, safety, and changes in health-related quality of life (HRQOL) after simultaneous vs sequential receipt of messenger RNA (mRNA) COVID-19 vaccine and quadrivalent inactivated influenza vaccine (IIV4). This randomized, placebo-controlled clinical trial was conducted between October 8, 2021, and June 14, 2023, at 3 US sites. Participants were nonpregnant persons aged 5 years or older with the intention of receiving both influenza and mRNA COVID-19 vaccines. Intramuscular administration in opposite arms of either IIV4 or saline placebo simultaneously with mRNA COVID-19 vaccine at visit 1. Those who received placebo at visit 1 received IIV4 and those who received IIV4 at visit 1 received placebo 1 to 2 weeks later at visit 2. The primary composite reactogenicity outcome was the proportion of participants with fever, chills, myalgia, and/or arthralgia of moderate or greater severity within 7 days after vaccination visits 1 and/or 2, using a 10% noninferiority margin. Secondary outcomes were solicited reactogenicity events and unsolicited adverse events (AEs) for 7 days after each visit separately and HRQOL after visit 1, assessed by the EuroQol 5-Dimension 5-Level (EQ-5D-5L) Index. Serious AEs (SAEs) and AEs of special interest (AESIs) were assessed for 121 days. Outcomes were compared between groups. A total of 335 persons (mean [SD] age, 33.4 [15.1] years) were randomized (169 to the simultaneous group and 166 to the sequential group); 211 (63.0%) were female, and 255 (76.1%) received bivalent BNT162b2 mRNA COVID-19 vaccine. The proportion with the primary composite reactogenicity outcome in the simultaneous group (25.6% [n = 43]) was noninferior to the proportion in the sequential group (31.3% [n = 52]) (site-adjusted difference, -5.6 percentage points [pp]; 95% CI, -15.2 to 4.0 pp). Respective proportions in each group were similar after each visit separately (visit 1, 40 [23.8%] vs 47 [28.3%]; visit 2, 5 [3.0%] vs 9 [5.4%]). No significant group differences in participants with AEs (21 [12.4%] vs 16 [9.6%]), SAEs (1 [0.6%] vs 1 [0.6%]), and AESIs (19 [11.2%] vs 9 [5.4%]) were observed in the simultaneous vs sequential groups, respectively. Among participants with severe reactogenicity, the mean (SD) EQ-5D-5L Index score decreased from 0.92 (0.08) to 0.92 (0.09) prevaccination to 0.81 (0.09) to 0.82 (0.12) postvaccination. In this randomized clinical trial assessing simultaneous vs sequential administration of mRNA COVID-19 and IIV4 vaccines, reactogenicity was comparable in both groups. These findings support the option of simultaneous administration of these vaccines. ClinicalTrials.gov Identifier: NCT05028361.

Extracellular Vesicle lncRNAs as Key Biomolecules for Cell-to-Cell Communication and Circulating Cancer Biomarkers

Extracellular vesicles (EVs) are secreted by almost every cell type and are considered carriers of active biomolecules, such as nucleic acids, proteins, and lipids. Their content can be uptaken and released into the cytoplasm of recipient cells, thereby inducing gene reprogramming and phenotypic changes in the acceptor cells. Whether the effects of EVs on the physiology of recipient cells are mediated by individual biomolecules or the collective outcome of the total transferred EV content is still under debate. The EV RNA content consists of several types of RNA, such as messenger RNA (mRNA), microRNA (miRNA), and long non-coding RNA (lncRNA), the latter defined as transcripts longer than 200 nucleotides that do not code for proteins but have important established biological functions. This review aims to update our insights on the functional roles of EV and their cargo non-coding RNA during cancer progression, to highlight the utility of EV RNA as novel diagnostic or prognostic biomarkers in cancer, and to tackle the technological advances and limitations for EV RNA identification, integrity assessment, and preservation of its functionality.

Delayed inflammatory reaction to hyaluronic acid filler following COVID-19 vaccination: A case report and review of the literature

Delayed inflammatory reactions (DIRs) to coronavirus disease 2019 (COVID-19) vaccines were rarely reported in patients receiving hyaluronic acid (HA) soft-tissue fillers. We present a case of a DIR to HA filler that occurred within days after receiving the second dose of the Pfizer-BioNTech COVID-19 messenger ribonucleic acid (RNA) vaccine and provide a comprehensive summary of its clinical presentation and treatment.

Dynamic multilayered control of m6A RNA demethylase activity

Similar to DNA and histone, RNA can also be methylated. In its most common form, a N6-methyladenosine (m6A) chemical modification is introduced into nascent messenger ribonucleic acid (mRNA) by a specialized methyltransferase complex and removed by the RNA demethylases, Fat mass and obesity-associated (FTO), and ALKBH5. The fate of m6A-marked mRNA is uniquely diverse, ranging from degradation to stabilization/translation, which has been suggested to be largely dependent on its interaction with the family of YT521-B homology (YTH) domain-containing proteins. Here, we highlight a series of control levers that impinge on the RNA demethylases. We present evidence to indicate that intermediary metabolism and various posttranslation modifications modulate the activity, stability, and the subcellular localization of FTO and ALKBH5, further dispelling the notion that m6A methylation is not a dynamic process. We also discuss how examination of these underappreciated regulatory nodes adds a more nuanced view of the role of FTO and ALKBH5 and should guide their study in cancer and nonmalignant conditions alike.

ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules.

Stress granules (SG) are part of a cellular protection mechanism where untranslated messenger RNAs and RNA-binding proteins are stored upon conditions of cellular stress. Compositional variations due to qualitative or quantitative protein changes can disrupt their functionality and alter their structure. This is the case of different forms of amyotrophic lateral sclerosis (ALS) where a causative link has been proposed between the cytoplasmic de-localization of mutant proteins, such as FUS (Fused in Sarcoma), and the formation of cytotoxic inclusions. Here, we describe the SG transcriptome in neuroblastoma cells and define several features for RNA recruitment in these condensates. We demonstrate that SG dynamics and RNA content are strongly modified by the incorporation of mutant FUS, switching to a more unstructured, AU-rich SG transcriptome. Moreover, we show that mutant FUS, together with its protein interactors and their target RNAs, are responsible for the reshaping of the mutant SG transcriptome with alterations that can be linked to neurodegeneration. Our data describe the molecular differences between physiological and pathological SG in ALS-FUS conditions, showing how FUS mutations impact the RNA and protein composition of these condensates.

Applications and Developments of Gene Therapy Drug Delivery Systems for Neurological Disorders

AbstractNeurological diseases (NDs) such as Alzheimer's disease, Parkinson's disease, ischemic strokes, spinal cord injuries, and other similar conditions that continue to pose a substantial health and economic burden on a global scale. It is crucial to tackle the difficulties provided by current medications due to the adverse effects and its immunological reactions to develop improved treatments for neurodegenerative illnesses. Gene therapy is currently being extensively used in preclinical and clinical studies for various diseases because of its ability to enhance the delivery and effectiveness of treatments. Various gene delivery techniques, including messenger RNA, small interfering RNA, antisense oligonucleotides, microRNA, CRISPR/Cas9 system, and plasmid DNA, have been created to address these difficulties. The goal of this study is to provide a clear overview of the pathophysiological underpinnings of NDs illnesses while also illuminating recent developments in gene delivery vector technologies. It goes over the main classifications of these vectors, their individual benefits and drawbacks, and their specific applications in the delivery of gene therapy.

Organophosphorus Flame Retardants and Metabolic Disruption: An in Silico, in Vitro, and in Vivo Study Focusing on Adiponectin Receptors.

Environmental chemical exposures have been associated with metabolic outcomes, and typically, their binding to nuclear hormone receptors is considered the molecular initiating event (MIE) for a number of outcomes. However, more studies are needed to understand the influence of such exposures on cell membrane-bound adiponectin receptors (AdipoRs), which are critical metabolic regulators. We aimed to clarify the potential interactions between AdipoRs and environmental chemicals, specifically organophosphorus flame retardants (OPFRs), and the resultant effects. Employing in silico simulation, cell thermal shift, and noncompetitive binding assays, we screened eight OPFRs for interactions with AdipoR1 and AdipoR2. We tested two key events underlying AdipoR modulation upon OPFR exposure in a liver cell model. The Toxicological Prioritization Index (ToxPi)scoring scheme was used to rank OPFRs according to their potential to disrupt AdipoR-associated metabolism. We further examined the inhibitory effect of OPFRs on AdipoR signaling activation in mouse models. Analyses identified pi-pi stacking and pi-sulfur interactions between the aryl-OPFRs 2-ethylhexyl diphenyl phosphate (EHDPP), triphenyl phosphate (TPhP), and tricresyl phosphate (TCP) and the transmembrane cavities of AdipoR1 and AdipoR2. Cell thermal shift assays showed a rightward shift in the AdipoR proteins' melting curves upon exposure to these three compounds. Although the binding sites differed from adiponectin, results suggest that aryl-OPFRs noncompetitively inhibited the binding of the endogenous peptide ligand ADP355 to the receptors. Analyses of key events underlying AdipoR modulation revealed that glucose uptake was notably lower, whereas lipid content was higher in cells exposed to aryl-OPFRs. EHDPP, TCP, and TPhP were ranked as the top three disruptors according to the ToxPi scores. A noncompetitive binding between these aryl-OPFRs and AdipoRs was also observed in wild-type (WT) mice. In db/db mice, the finding of lower blood glucose levels after ADP355 injection was diminished in the presence of a typical aryl-OPFR (TCP). WT mice exposed to TCP demonstrated lower AdipoR1 signaling, which was marked by lower phosphorylated AMP-activated protein kinase (pAMPK) and a higher expression of gluconeogenesis-related genes. Moreover, WT mice exposed to ADP355 demonstrated higher levels of pAMPK protein and peroxisome proliferator-activated receptor- messenger RNA. This was accompanied by higher glucose disposal and by lower levels of long-chain fatty acids and hepatic triglycerides; these metabolic improvements were negated upon TCP co-treatment. In silico, in vitro, and invivo assays suggest that aryl-OPFRs act as noncompetitive inhibitors of AdipoRs, preventing their activation by adiponectin, and thus function as antagonists to these receptors. Our study describes a novel MIE for chemical-induced metabolic disturbances and highlights a new pathway for environmental impact on metabolic health. https://doi.org/10.1289/EHP14634.

N6-methyladenosine modification of circDcbld2 in Kupffer cells promotes hepatic fibrosis via targeting miR-144-3p/Et-1 axis

Kupffer cells (KCs), as residents and sentinels of the liver, are involved in the formation of hepatic fibrosis (HF). However, the biological functions of circular RNAs (circRNAs) in KCs to HF have not been determined. In this study, the expression levels of circRNAs, microRNAs, and messenger RNAs (mRNAs) in KCs from a mouse model of HF mice were investigated using microarray and circRNA-Seq analyses. circDcbld2 was identified as a candidate circRNA in HF, as evidenced by its up-regulation in KCs. Silver staining and mass spectrometry showed that Wtap and Igf2bp2 bind to cirDcbld2. The suppression of circDcbld2 expression decreased the KC inflammatory response and oxidative stress and inhibited hepatic stellate cell (HSCs) activation, attenuating mouse liver fibrogenesis. Mechanistically, Wtap mediated the N6-methyladenosine (m6A) methylation of circDcbld2, and Igf2bp2 recognized m6A-modified circDcbld2 and increased its stability. circDcbld2 contributes to the occurrence of HF by binding miR-144-3p/Et-1 to regulate the inflammatory response and oxidative stress. These findings indicate that circDcbld2 functions via the m6A/circDcbld2/miR-144-3p/Et-1 axis and may act as a potential biomarker for HF treatment.

Bridging One Health: Computational design of a multi-epitope messenger RNA vaccine for cross-species immunization against Nipah virus

Background and Aim: Nipah virus (NiV) poses a threat to human and animal health, particularly swine, which serve as primary vectors for human transmission. Despite its severe risks, no NiV vaccine currently exists for humans or animal hosts; thus, innovative vaccine development approaches that address cross-species transmission are required. This study was computationally designed to evaluate a multi-epitope messenger RNA (mRNA) vaccine targeting NiV for human and swine immunization. Materials and Methods: B and T lymphocyte epitopes were identified from NiV structural proteins using multiple epitope prediction tools. All epitopes were linked to form a multi-epitope construct, and various adjuvant combinations were analyzed for physicochemical properties and immune simulation. Molecular docking and dynamics were employed to visualize the construct’s interaction with a host immune receptor. Signal peptides were added to the construct, and mRNA sequences were generated using LinearDesign. The minimum free energies (MFEs) and codon adaptation indices (CAI) were used to select the final mRNA sequence of the vaccine construct. Results: Computational tools predicted 10 epitopes within NiV structural proteins that can be recognized by human and swine immune receptors. The construct with β-defensin 2 adjuvant was selected as the final immunogenic region after showing favorable immunogenicity profiles and physicochemical properties. The final vaccine sequence had higher MFE and CAI compared to the BioNTech/Pfizer BNT162b2 and Moderna mRNA-1273 vaccines. Conclusion: The multi-epitope mRNA vaccine designed in this study shows promising results as a potential NiV vaccine candidate. Further in vivo and in vitro studies are required to confirm the efficacy. Keywords: computational design, cross-species immunization, messenger RNA vaccine, multi-epitope, Nipah virus.