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This article discusses the use of nanopore sequencing to detect chemical modifications in mRNA, specifically N6-methyladenosine (m6A), which can affect gene regulation. The study focused on the role of the enzyme METTL3 in regulating cardiomyocyte mitochondrial metabolism. Through nanopore sequencing, the researchers identified 394 m6A-modified mRNAs, with the modifications most frequently found in the stop codon and 3' untranslated region. The study also found that the location of the m6A mark and differential binding of m6A recognition proteins can control the fate of the mRNA. The altered proteins in METTL3-deficient hearts were linked to mitochondrial function, suggesting that mitochondrial dysfunction may be a primary driver of cardiomyopathy. This study provides valuable insights into the role of mRNA modifications in cardiac homeostasis. [Extracted from the article]

RNA therapeutics hold significant promise in the treatment of cardiovascular diseases. RNAs are biologically diverse and functionally specific and can be used for gain- or loss-of-function purposes. The effectiveness of mRNA-based vaccines in the recent COVID-19 pandemic has undoubtedly proven the benefits of an RNA-based approach. RNA-based therapies are becoming more common as a treatment modality for cardiovascular disease. This is most evident in hypertension where several small interfering RNA–based drugs have proven to be effective in managing high blood pressure in several clinical trials. As befits a rapidly burgeoning field, there is significant interest in other classes of RNA. Revascularization of the infarcted heart through an mRNA drug is under clinical investigation. mRNA technology may provide the platform for the expression of paracrine factors for myocardial protection and regeneration. Emergent technologies on the basis of microRNAs and gene editing are tackling complex diseases in a novel fashion. RNA-based gene editing offers hope of permanent cures for monogenic cardiovascular diseases, and long-term control of complex diseases such as essential hypertension, as well. Likewise, microRNAs are proving effective in regenerating cardiac muscle. The aim of this review is to provide an overview of the current landscape of RNA-based therapies for the treatment of cardiovascular disease. The review describes the large number of RNA molecules that exist with a discussion of the clinical development of each RNA type. In addition, the review also presents a number of avenues for future development. [ABSTRACT FROM AUTHOR]

Objective To investigate the change in intestinal flora and its association with liver CYP2E1 in mice with li-ver injury caused by high-intensity atorvastatin (ATO). Methods According to the treatment method, 40 C57BL/6 mice were divided into low-intensity ATO group (ATO-L group), medium-intensity ATO group (ATO-M group), high-intensity ATO group (ATO-H group), and control group (CON group), with 10 rats in each group. The mice in the ATO-L group, the ATO-M group, and the ATO-H group were given 1 mL normal saline containing ATO at doses of 10, 20, and 30 mg/kg, respectively, by gavage, and those in the CON group were given an equal volume of normal saline by gavage, for 4 consecutive weeks. HE staining and transmission electron microscopy were used to observe liver pathological state. Related methods were used to measure the se-rum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-10, the activity of superoxide dismutase (SOD) in liver tissue, and the mRNA expression level of CYP2E1 in liver tissue. 16SrDNA sequencing was used to analyze the distribution of intestinal flora in fresh feces of mice, and a correlation analysis was conducted to investigate the correlation of the relative abundance of bacterial genera with significant diffe-rences with the mRNA expression level of CYP2E1 and SOD activity in liver tissue. Results HE staining and transmission electron microscopy showed varying degrees of liver injury in the ATO-M and ATO-H groups. There were significant differences in the serum levels of ALT, AST, IL-6, and TNF-α and SOD activity in liver tissue between groups (F=4.57-7.74,P

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Purpose To detect elements governing the pathogenesis of diabetic cystopathy (DC), mRNA sequencing was carried out for bladder tissues from normal rats and those with induced diabetes mellitus (DM). This research therefore offers possible underlying molecular pathways for the advancement of DC in relation to differential mRNA expression, together with visceral functional and architectural alterations noted in individuals with this condition. Methods An intraperitoneal injection of streptozotocin (STZ) was utilized to provoke DM in male Sprague-Dawley rats. Dysregulation and significant variations between normal rats and those with induced DM were then identified by a fold change of ≥ 1.5 with a false discovery rate P < 0.05. Hierarchical clustering/heat map and Gene Ontology/DAVID reference sources were generated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction analysis were then performed. Results The diabetic rodent group exhibited a greater residual urine volume (4.0 ± 0.4 mL) than their control counterparts (0.7 ± 0.2 mL, P < 0.01) at 12 weeks after diagnosis of diabetes. Expression analysis revealed 16 upregulated and 4 downregulated genes in STZDM bladder samples. A notable increase in expression was seen in PTHLH, TNFAIP6, PRC1, MAPK10, LOC686120, CASQ2, ACTG2, PDLIM3, FCHSD1, DBN1, NKD2, PDLIM7, ATF4, RBPMS2, ITGB1 and HSPB8. A notable decrease in expression was seen in SREBLF1, PBGFR1, PBLD1 and CELF1. Major genetic themes associated with mRNA upregulation and downregulation ware identified via Gene Ontology analysis and KEGG pathways. Protein to protein interaction analysis detected PDLIM3, PDLIM7, ITGB1, ACTG2 as core high frequency nodes within the network. Conclusions Changes in mRNA expression together with biological process and pathways that contribute to the etiologies underlying visceral impairment of the bladder in DM are evident. Our strategy is promising for recognizing mRNAs exclusive to the bladder in DM that might offer useful targets for diagnosis and treatment.

Simple Summary: This manuscript aims to investigate the features of patients with metastatic sentinel lymph node (SLN), evaluated by OSNA, and to predict which patients have a high risk of positive ALND. The finding of the present study suggests a new cut-off of CK19 mRNA copy number in the sentinel lymph node useful to personalize surgical treatments and avoid unnecessary axillary surgical treatments. (1) Background: The main discriminant in breast cancer prognosis is axillary lymph node status. In a select cohort of patients, axillary lymph node dissection (ALND) may be safely spared. This study aimed to determine a new possible cut-off of cytokeratin (CK) 19 mRNA copy number in the SLN to predict cases at high risk of positive ALND. (2) Methods: Clinical records of 1339 patients were retrospectively reviewed and were separated into two groups according to the axillary status (negative: ALNs− and positive ALNs+). Receiver operative characteristic (ROC) curves were used to identify a new optimal cut-off of CK19 mRNA copy number in SLN; (3) Results: Large tumor size and high grade were found mostly in ALNs+. Results from the ROC analyses, with an AUC of 82.1%, identified a new cut-off (9150 CK19 mRNA copies) showing 94% sensitivity, 67.3% specificity, 61.2% positive, and 95.3% negative predictive values; (4) OSNA remains the most-important intra-operative tool to identify patients who can benefit from ALND but with the traditional cut-off, many patients undergo needless ALND. The results of the present study suggest a new cut-off helpful to personalize surgical treatment and avoid unnecessary invasive procedures. [ABSTRACT FROM AUTHOR]

Neurological monogenic loss-of-function diseases are hereditary disorders resulting from gene mutations that decrease or abolish the normal function of the encoded protein. These conditions pose significant therapeutic challenges, which may be resolved through the development of innovative therapeutic strategies. RNA-based technologies, such as mRNA replacement therapy, have emerged as promising and increasingly viable treatments. Notably, mRNA therapy exhibits significant potential as a mutation-agnostic approach that can address virtually any monogenic loss-of-function disease. Therapeutic mRNA carries the information for a healthy copy of the defective protein, bypassing the problem of targeting specific genetic variants. Moreover, unlike conventional gene therapy, mRNA-based drugs are delivered through a simplified process that requires only transfer to the cytoplasm, thereby reducing the mutagenic risks related to DNA integration. Additionally, mRNA therapy exerts a transient effect on target cells, minimizing the risk of long-term unintended consequences. The remarkable success of mRNA technology for developing coronavirus disease 2019 vaccines has rekindled interest in mRNA as a cost-effective method for delivering therapeutic proteins. However, further optimization is required to enhance mRNA delivery, particularly to the CNS, while minimizing adverse drug reactions and toxicity. In this comprehensive review, we delve into past, present and ongoing applications of mRNA therapy for neurological monogenic loss-of-function diseases. We also discuss the promises and potential challenges presented by mRNA therapeutics in this rapidly advancing field. Ultimately, we underscore the full potential of mRNA therapy as a game-changing therapeutic approach for neurological disorders., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The messenger RNA (mRNA) platform emerged at the forefront of vaccine development during the COVID-19 pandemic, with two mRNA COVID-19 vaccines being among the first authorized globally. These vaccines were developed rapidly. Informed by decades of laboratory research, and proved to be safe and efficacious tools for mitigating the global impact of the COVID-19 pandemic. The mRNA platform holds promise for a broader medical application beyond COVID-19. Herein, we provide an overview of this platform and describe lessons learned from the COVID-19 pandemic to help formulate strategies toward enhancing uptake of future mRNA-based interventions. We identify several strategies as vital for acceptance of an expanding array of mRNA-based vaccines and therapeutics, including education, accurate and transparent information sharing, targeted engagement campaigns, continued investment in vaccine safety surveillance, inclusion of diverse participant pools in clinical trials, and addressing deep-rooted inequalities in access to healthcare. We present findings from the Global Listening Project (GLP) initiative, which draws on quantitative and qualitative approaches to capture perceptions and experiences during the COVID-19 pandemic to help design concrete action plans for improving societal preparedness for future emergencies. The GLP survey (>70,000 respondents in 70 countries) revealed tremendous disparities across countries and sociodemographic groups regarding willingness to accept novel mRNA vaccines and medicines. The comfort in innovations in mRNA medicines was generally low (35%) and was marginally lower among women (33%). The GLP survey and lessons learnt from the COVID-19 pandemic provide actionable insights into designing effective strategies to enhance uptake of future mRNA-based medicines., Competing Interests: SMI, AMR, DE, MS, MR, AC, and FC are employees of Moderna, Inc., and hold stock/stock options in the company. HJL received research grants from GSK and Moderna related to the Global Listening Project. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Moderna, Inc. and GSK. Moderna, Inc. had the following involvement in the study: study design and analysis, preparation of the manuscript, and decision to publish. GSK had the following involvement in the study: study design., (Copyright © 2024 Iqbal, Rosen, Edwards, Bolio, Larson, Servin, Rudowitz, Carfi and Ceddia.)

We have used pulsed field gradient (PFG)-NMR diffusion experiments, also known as DOSY, in combination with small angle X-ray scattering measurements to investigate structure and molecular exchange dynamics between pharmaceutical lipid nanoparticles and the bulk phase. Using liposomes and lipoplexes formed after complexation of the liposomes with messenger mRNA as test systems, information on dynamics of encapsulated water molecules, lipids and excipients was obtained. The encapsulated fraction, having a diffusivity similar to that of the liposomes, could be clearly identified and quantified by the NMR diffusion measurements. The unilamellar liposome membranes allowed a fast exchange of water molecules, while sucrose, used as an osmolyte and model solute, showed very slow exchange. Upon interactions with mRNA a topological transition from a vesicular to a lamellar organization took place, where the mRNA was inserted in repeating lipid bilayer stacks. In the lipoplexes, a small fraction of tightly bound water molecules was present, with a diffusivity that was influenced by the additional presence of sucrose. This extended information on dynamic coherencies inside pharmaceutical nanoparticle products, provided by the combined application of SAXS and PFG-NMR diffusion measurements, can be valuable for evaluation of quality and comparability of nanoscaled pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Cancer genomics and transcriptomics studies have provided a large volume of data that enables to test of hypotheses based on real data from cancer patients. Ezrin (encoded by the EZR gene) is a highly expressed protein in cancer that contributes to linking the actin cytoskeleton to the cell membrane and signal transduction pathways involved in oncogenesis and disease progression. NSC305787 is a pharmacological ezrin inhibitor with potential antineoplastic effects. In the present study, the authors prospected EZR mRNA levels in a pan-cancer analysis and identified potential cancers that could benefit from anti-EZR therapies., Methods: This study analyzed TCGA data for 32 cancer types, emphasizing cervical squamous cell carcinoma and stomach adenocarcinoma. It investigated the impact of EZR transcript levels on clinical outcomes and identified differentially expressed genes. Cell lines were treated with NSC305787, and its effects were assessed through various cellular and molecular assays., Results: EZR mRNA levels are highly expressed, and their expression is associated with biologically relevant molecular processes in cervical squamous carcinoma and stomach adenocarcinoma. In cellular models of cervical and gastric cancer, NSC305787 reduces cell viability and clonal growth (p < 0.05). Molecular analyses indicate that the pharmacological inhibition of EZR induces molecular markers of cell death and DNA damage, in addition, to promoting the expression of genes associated with apoptosis and inhibiting the expression of genes related to survival and proliferation., Conclusion: The present findings provide promising evidence that ezrin may be a molecular target in the treatment of cervical and gastric carcinoma., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

In sexual assault cases, it is crucial to discriminate between peripheral blood and menstrual blood to provide evidence for vaginal intercourse with traumatic injury. In this study, the menstrual blood mRNA markers progestagen-associated endometrial protein (PAEP), matrix metallopeptidase 7 (MMP7), and left-right determination factor 2 (LEFTY2) were evaluated by quantitative RT-PCR (RT-qPCR) for the discrimination of menstrual blood from peripheral blood and vaginal fluid. As a result, all markers with cutoff delta cycle quantification (ΔCq) values were specifically determined in menstrual blood among forensically relevant body fluids. Even though the changes in the expression levels of each marker differed during the menstrual cycle, all markers were determined to be positive in most of the randomly collected menstrual blood samples that were analyzed. Additionally, the markers with proposed cutoff ΔCq values could discriminate between menstrual blood and peripheral blood-mixed vaginal fluid samples. The determination of positive markers was less affected by storage temperature under dry conditions than under wet conditions, while PAEP was detectable in samples stored below room temperature under wet conditions. The detectability of PAEP was considered to be the result of its higher expression level compared with MMP7 and LEFTY2. In conclusion, menstrual blood markers for the RT-qPCR procedure evaluated in this study were highly specific for menstrual blood. The proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract. In particular, PAEP is expected to be applicable to forensic casework samples because of its high specificity and robustness., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Personalized liposome-formulated mRNA vaccines (RNA-LPX) are a powerful new tool in cancer immunotherapy. In preclinical tumor models, RNA-LPX vaccines are known to achieve potent results when combined with conventional X-ray radiation therapy (XRT). Densely ionizing radiation used in carbon ion radiation therapy (CIRT) may induce distinct effects in combination with immunotherapy compared with sparsely ionizing X-rays., Methods and Materials: Within this study, we investigate the potential of CIRT and isoeffective doses of XRT to mediate tumor growth inhibition and survival in murine colon adenocarcinoma models in conjunction with neoantigen (neoAg)-specific RNA-LPX vaccines encoding both major histocompatibility complex (MHC) class I- and class II-restricted tumor-specific neoantigens. We characterize tumor immune infiltrates and antigen-specific T cell responses by flow cytometry and interferon-γ enzyme-linked immunosorbent spot (ELISpot) analyses, respectively., Results: NeoAg RNA-LPX vaccines significantly potentiate radiation therapy-mediated tumor growth inhibition. CIRT and XRT alone marginally prime neoAg-specific T cell responses detected in the tumors but not in the blood or spleens of mice. Infiltration and cytotoxicity of neoAg-specific T cells is strongly driven by RNA-LPX vaccines and is accompanied by reduced expression of the inhibitory markers PD-1 and Tim-3 on these cells. The neoAg RNA-LPX vaccine shows similar overall therapeutic efficacy in combination with both CIRT and XRT, even if the physical radiation dose is lower for carbon ions than for X-rays., Conclusions: We hence conclude that the combination of CIRT and neoAg RNA-LPX vaccines is a promising strategy for the treatment of radioresistant tumors., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Keratin 80 (KRT80) is a filament protein that makes up one of the major structural fibers of epithelial cells, and involved in cell differentiation and epithelial barrier integrity. Here, KRT80 mRNA expression was found to be higher in esophageal cancer than normal epithelium by RT-PCR and bioinformatics analysis ( p  < .05), opposite to KRT80 methylation ( p  < .05). There was a negative relationship between promoter methylation and expression level of KRT80 gene in esophageal cancer ( p  < .05). KRT80 mRNA expression was positively correlated with the differentiation, infiltration of immune cells, and poor prognosis of esophageal cancer ( p  < .05). KRT80 mRNA expression was positively linked to no infiltration of immune cells, the short survival time of esophageal cancers ( p  < .05). The differential genes of KRT80 mRNA were involved in fat digestion and metabolism, peptidase inhibitor, and intermediate filament, desosome, keratinocyte differentiation, epidermis development, keratinization, ECM regulator, complement cascade, metabolism of vitamins and co-factor ( p  < .05). KRT-80-related genes were classified into endocytosis, cell adhesion molecule binding, cadherin binding, cell-cell junction, cell leading edge, epidermal cell differentiation and development, T cell differentiation and receptor complex, plasma membrane receptor complex, external side of plasma membrane, metabolism of amino acids and catabolism of small molecules, and so forth ( p  < .05). KRT80 knockdown suppressed anti-apoptosis, anti-pyroptosis, migration, invasion, chemoresistance, and lipogenesis in esophageal cancer cells ( p  < .05), while ACC1 and ACLY overexpression reversed the inhibitory effects of KRT80 on lipogenesis and chemoresistance. These findings indicated that up-regulated expression of KRT80 might be involved in esophageal carcinogenesis and subsequent progression, aggravate aggressive phenotypes, and induced chemoresistance by lipid droplet assembly and ACC1- and ACLY-mediated lipogenesis.

Basophil activation test (BAT) with COVID-19 mRNA vaccine seems particularly suitable for detecting sensitization to polyethylene glycol (PEG) in patients with PEG allergy. It was the aim of this study to determine the cutoffs for BAT using BNT162B2 (Comirnaty®) in a larger group of PEG allergic patients and controls. 10 PEG allergic patients and 10 controls were studied. BAT was performed using anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation. Incubations with BNT162B2 at four different concentrations were performed. Basophil activation was significantly higher in PEG allergic patients compared to controls at the higher concentrations used. ROC curves showed best results with a sensitivity of 60% and specificity of 100% with a cutoff of 5% CD63 + basophils at a concentration of 4.5 µg/ml. Controls showed no positive results. In our group of PEG allergic patients, a concentration of 4.5 µg/ml BNT162B2 with a cutoff of 5% CD63 + basophils was the most suitable condition for identifying patients with a sensitization to PEG. Allergological work-up of PEG allergic patients including BAT with PEGylated lipid nanoparticles might play a role in the future when these substances will be used for other vaccines and cancer immunotherapies.

Breast cancer (BC), characterized by its diverse molecular profiles and clinical outcomes, presents a significant challenge in the development of effective therapeutic strategies. Metabolic reprogramming, a defining characteristic of cancer, has emerged as a promising target for novel therapies. SLC7A11, an amino acid transporter that facilitates cysteine uptake in exchange for glutamate, plays a crucial role in sustaining the altered metabolism of cancer cells. This study delves into the comprehensive analysis of SLC7A11 at the genomic, transcriptomic, and protein levels in extensive BC datasets to elucidate its potential role in different BC subtypes. SLC7A11 gene copy number and mRNA expression were evaluated using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort ( n  = 1,980) and Breast Cancer Gene Expression Miner ( n  = 4,712). SLC7A11 protein was assessed using immunohistochemistry in a large BC cohort ( n  = 1,981). Additionally, The Cancer Genome Atlas (TCGA) dataset was used to explore SLC7A11 DNA methylation patterns using MethSurv ( n  = 782) and association of SLC7A11 mRNA expression with immune infiltrates using TIMER ( n  = 1,100). High SLC7A11 mRNA and SLC7A11 protein expression were significantly associated with high tumor grade ( p  ≤ .02), indicating a potential role in cancer progression. Interestingly, SLC7A11 copy number gain was observed in HER2+ tumors ( p  = .01), suggesting a subtype-specific association. In contrast, SLC7A11 mRNA expression was higher in the basal-like/triple-negative (TN; p  < .001) and luminal B tumors ( p  = .02), highlighting its differential expression across BC subtypes. Notably, high SLC7A11 protein expression was predominantly observed in Estrogen Receptor (ER)-negative and Triple Negative (TN) BC, suggesting a role in these aggressive subtypes. Further analysis revealed that SLC7A11 was positively correlated with other amino acid transporters and enzymes associated with glutamine metabolism, implying a coordinated role in metabolic regulation. Additionally, SLC7A11 gene expression was positively associated with neutrophil and macrophage infiltration, suggesting a potential link between SLC7A11 and tumor immunity. Our findings suggest that SLC7A11 plays a significant role in BC metabolism, demonstrating differential expression across subtypes and associations with poor patient outcomes. Further functional studies are warranted to elucidate the precise mechanisms by which SLC7A11 contributes to BC progression and to explore its potential as a therapeutic target.

Objective: To study whether and, if so, how honokiol overcome dexamethasone resistance in DEX-resistant CEM-C1 cells. Methods: We investigated the effect of honokiol (0-20 µM) on cell proliferation, cell cycle, cell apoptosis and autophagy in DEX-resistant CEM-C1 cells and DEX-sensitive CEM-C7 cells. We also determined the role of c-Myc protein and mRNA in the occurrence of T-ALL associated dexamethasone resistance western blot and reverse transcription-qPCR (RT-qPCR) analysis. Results: Cell Counting Kit (CCK)-8 assay shows that DEX-resistant CEM-C1 cell lines were highly resistant to dexamethasone with IC50 of 364.1 ± 29.5 µM for 48 h treatment. However, upon treatment with dexamethasone in combination with 1.5 µM of honokiol for 48 h, the IC50 of CEM-C1 cells significantly decreased to 126.2 ± 12.3 µM, and the reversal fold was 2.88. Conversely, the IC50 of CEM-C7 cells was not changed combination of dexamethasone and honokiol as compared to that of CEM-C7 cells treated with dexamethasone alone. It has been shown that honokiol induced T-ALL cell growth inhibition by apoptosis and autophagy via downregulating cell cycle-regulated proteins (Cyclin E, CDK4, and Cyclin D1) and anti-apoptotic proteins BCL-2 and upregulating pro-apoptotic proteins Bax and led to PARP cleavage. Honokiol may overcome dexamethasone resistance in DEX-resistant CEM-C1 cell lines via the suppression of c-Myc mRNA expression. Conclusion: The combination of honokiol and DEX were better than DEX alone in DEX-resistant CEM-C1 cell lines. Honokiol may regulate T-ALL-related dexamethasone resistance by affecting c-Myc.

Background: The chemotherapy resistance often leads to chemotherapy failure. This study aims to explore the molecular mechanism by which MUC1 regulates paclitaxel resistance in lung adenocarcinoma (LUAD), providing scientific basis for future target selection., Methods: The bioinformatics method was used to analyse the mRNA and protein expression characteristics of MUC1 in LUAD. RT-qPCR and ELISA were used to detect the mRNA and protein expression, flow cytometry was used to detect CD133 + cells, and cell viability was detected by CCK-8 assay. The mRNA-seq was performed to analyse the changes in expression profile, GO and KEGG analysis were used to explore the potential biological functions., Results: MUC1 is highly expressed in LUAD patients and is associated with a higher tumour infiltration. In paclitaxel resistance LUAD cells (A549/TAX cells), the expression of MUC1, EGFR/p-EGFR and IL-6 were higher than that of A549 cells, the proportion of CD133 + cells was significantly increased, and the expression of cancer stem cell (CSCs) transcription factors (NANOG, OCT4 and SOX2) were significantly up-regulated. After knocking down MUC1 in A549/Tax cells, the activity of A549/Tax cells was significantly decreased. Correspondingly, the expression of EGFR, IL-6, OCT4, NANOG, and SOX2 were significantly down-regulated. The mRNA-seq showed that knocking down MUC1 affected the gene expression, DEGs mainly enriched in NF-κB and MAPK signalling pathway., Conclusion: MUC1 was highly expressed in A549/TAX cells, and MUC1-EGFR crosstalk with IL-6 may be due to the activation of NF-κB and MAPK pathways, which promote the enrichment of CSCs and lead to paclitaxel resistance.

This review has focused on the development of mRNA nano-vaccine and the biochemical interactions of anti-COVID-19 mRNA vaccines with various disease conditions and age groups. It studied five major groups of individuals with different disease conditions and ages, including allergic background, infarction background, adolescent, and adult (youngsters), pregnant women, and elderly. All five groups had been reported to have background-related adverse effects. Allergic background individuals were observed to have higher chances of experiencing allergic reactions and even anaphylaxis. Individuals with an infarction background had a higher risk of vaccine-induced diseases, e.g. pneumonitis and interstitial lung diseases. Pregnant women were seen to suffer from obstetric and gynecological adverse effects after receiving vaccinations. However, interestingly, the elderly individuals (> 65 years old) had experienced milder and less frequent adverse effects compared to the adolescent (<19 and >9 years old) and young adulthood (19-39 years old), or middle adulthood (40-59 years old) age groups, while middle to late adolescent (14-17 years old) was the riskiest age group to vaccine-induced cardiovascular manifestations.

In vitro transcription (IVT) using T7 RNA polymerase (RNAP) is integral to RNA research, yet producing this enzyme in E. coli presents challenges regarding endotoxins and animal-sourced toxins. This study demonstrates the viable production and characterization of T7 RNAP using ClearColi BL21(DE3) (an endotoxin-free E. coli strain) and animal-free media. Compared to BL21(DE3) with animal-free medium, soluble T7 RNAP expression is ~50% lower in ClearColi BL21(DE3). Optimal soluble T7 RNAP expression in flask fermentation is achieved through the design of experiments (DoE). Specification and functional testing showed that the endotoxin-free T7 RNAP has comparable activity to conventional T7 RNAP. After Ni-NTA purification, endotoxin levels were approximately 109-fold lower than T7 RNAP from BL21(DE3) with animal-free medium. Furthermore, a full factorial DoE created an optimal IVT system that maximized mRNA yield from the endotoxin-free and animal-free T7 RNAP. This work addresses critical challenges in recombinant T7 RNAP production through innovative host and medium combinations, avoided endotoxin risks and animal-derived toxins. Together with an optimized IVT reaction system, this study represents a significant advance for safe and reliable reagent manufacturing and RNA therapeutics. KEY POINTS: • Optimized IVT system maximizes mRNA yields, enabling the synthesis of long RNAs. • Novel production method yields endotoxin-free and animal-free T7 RNAP. • The T7 RNAP has equivalent specifications and function to conventional T7 RNAP., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: This study aims to detect the serum levels of IGF-1, bFGF, and PLGF and their expressions in placental bed tissues of patients with placenta previa complicated with PAS disorders., Methods: This case and control study included 40 multiparous pregnant women with complete placenta previa between 34 weeks and 38 weeks of gestation and they were divided into two groups: 25 patients with PAS (case group) and 15 patients without PAS (control group). The venous blood samples were collected 2 h before the cesarean section, and the placental bed tissues were taken intraoperatively at the placental implantation site and then were histologically examined to evaluate the gravity of the myometrial invasion of the placenta. According to FIGO PAS increasing grading, the 25 patients were also divided into three groups: PAS grade I group, PAS grade II group, and PAS grade III group. The concentrations of IGF-1, bFGF, and PLGF in serum were measured using ELISA, and the mean ratio of the relative mRNA expression of each biomarker in placental bed tissues was calculated using qRT-PCR. The staining intensity and the positive cells were quantitatively measured and expressed as means by using Image J software for IHC analysis., Results: IGF-1 had low serum levels and high placental bed expression in placenta previa patients with PAS disorders compared to those without PAS (all p  < 0.0001). PLGF had high serum levels ( p  = 0.0200) and high placental bed expression ( p  < 0.0001) in placenta previa patients with PAS disorders compared to those without PAS. IGF-1 serum levels decreased up to PAS grade II (means were 24.3 ± 4.03, 21.98 ± 3.29, and 22.03 ± 7.31, respectively for PAS grade I, PAS grade II, PAS grade III groups, p  = 0.0006). PLGF serum levels increased up to PAS grade II (means were 12.96 ± 2.74, 14.97 ± 2.56, and 14.89 ± 2.14, respectively for the three groups, p  = 0.0392). However, IGF-1 and PLGF mRNA placental bed expression increased up to PAS grade III. The relative expression of mRNA means for the three groups was 3.194 ± 1.40, 3.509 ± 0.63, and 3.872 ± 0.70, respectively for IGF-1; and 2.784 ± 1.14, 2.810 ± 0.71, and 2.869 ± 0.48, respectively for PLGF (all p  < 0.0001). Their IHC (immunohistochemical) staining also had increasing trends, but p  > 0.05. bFGF was not significantly expressed in placenta previa with PAS disorders in most of the analysis sections ( p  > 0.05)., Conclusions: Low serum levels and high expression in placental bed tissues of IGF-1, or high serum levels and high expression in placental bed tissues of PLGF, may differentiate placenta previa patients with FIGO PAS grade I and PAS grade II from those without PAS disorders. However, they could not significantly predict the degree of placental invasiveness in FIGO PAS grades II and III.

Objective: Extremely preterm infants have low Nuclear Receptor (NR) expression in their developing hepatobiliary systems, as they rely on the placenta and maternal liver for compensation. NRs play a crucial role in detoxification and the elimination of both endogenous and xenobiotic substances by regulating key genes encoding specific proteins. In this study, we utilized an Artificial Placenta Therapy (APT) platform to examine the liver tissue expression of NRs of extremely preterm ovine fetuses. This fetal model, resembling a "knockout placenta," lacks placental and maternal support, while maintaining a healthy extrauterine survival., Methods: Six ovine fetuses at 95 ± 1 d gestational age (GA; term = ∼150 d)/∼600 g delivery weight were maintained on an APT platform for a period of 120 h (APT Group). Six age-matched, in utero control fetuses were delivered at 99-100 d GA (Control Group). Fetal liver tissue samples and blood samples were collected at delivery from both groups and assessed mRNA expression of NRs and target transporters involved in the hepatobiliary transport system using quantitative PCR. Data were tested for group differences with ANOVA ( p  < .05 deemed significant)., Results: mRNA expression of NRs was identified in both the placenta and the extremely preterm ovine fetal liver. The expression of HNF4α , LRH1 , LXR , ESR1 , PXR , CAR , and PPARα/γ were significantly elevated in the liver of the APT Group compared to the Control Group. Moreover, target transporters NTCP , OATP1B3 , BSEP , and MRP4 were upregulated, whereas MRP2 and MRP3 were unchanged. Although there was no evidence of liver necrosis or apoptotic changes histologically, there was an impact in the fetal liver of the ATP group at the tissue level with a significant increase in TNFα mRNA, a cytokine involved in liver inflammation, and blood elevation of transaminases., Conclusion: A number of NRs in the fetal liver were significantly upregulated after loss of placental-maternal support. However, the expression of target transporter genes appeared to be insufficient to compensate role of the placenta and maternal liver and avoid fetal liver damage, potentially due to insufficient excretion of organic anions.

Influenza A virus (IAV) poses a threat to patients receiving immunosuppressive medications since they are more susceptible to infection with severe symptoms, and even death. Understanding the direct effects of immunosuppressants on IAV infection is critical for optimizing immunosuppression in these patients who are infected or at risk of influenza virus infection. We profiled the effects of 10 immunosuppressants, explored the antiviral mechanisms of immunosuppressants, and demonstrated the combined effects of immunosuppressants with the antiviral drug oseltamivir in IAV-infected cell models. We found that mycophenolic acid (MPA) strongly inhibits viral RNA replication via depleting cellular guanosine pool. Treatment with 6-Thioguanine (6-TG) promoted viral protein degradation through a proteasomal pathway. Filgotinib blocked mRNA splicing of matrix protein 2, resulting in decreased viral particle assembly. Furthermore, combined treatment with immunosuppressants and oseltamivir inhibits IAV viral particle production in an additive or synergic manner. Our results suggest that MPA, 6-TG, and filgotinib could be the preferential choices for patients who must take immunosuppressants but are at risk of influenza virus infection.

With the large number of atypical cases in the mpox outbreak, which was classified as a global health emergency by the World Health Organization (WHO) on 23 July 2022, rapid diagnosis of mpox and diseases with similar symptoms to mpox such as chickenpox and respiratory infectious diseases in the early stages of viral infection is key to controlling the spread of the outbreak. In this study, antibodies against the monkeypox virus A29L protein were efficiently and rapidly identified by combining rapid mRNA immunization with high-throughput sequencing of individual B cells. We obtained eight antibodies with a high affinity for A29L validated by ELISA, which were was used as the basis for developing an ultrasensitive fluorescent immunochromatographic assay based on multilayer quantum dot nanobeads (SiTQD-ICA). The SiTQD-ICA biosensor utilizing M53 and M78 antibodies showed high sensitivity and stability of detection: A29L was detected within 20 min, with a minimum detection limit of 5 pg/mL. A specificity test showed that the method was non-cross-reactive with chickenpox or common respiratory pathogens and can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. This antibody identification method can also be used for rapid acquisition of monoclonal antibodies in early outbreaks of other infectious diseases for various studies.

Objective: To explore the expression of microRNA (miRNA) -messenger RNA (mRNA) and predict its prognostic significance in cervical squamous carcinoma (CESC), analyze its significance in predicting the prognosis of CESC. Methods: TCGA database was used to screen the differential expression profiles of miRNA and mRNA in CESC and normal cervical tissue. The interaction between miRNA and targeted mRNA was verified by miRTarBase, TargetScan and miRDB databases. The prognostic markers of CESC were determined by Kaplan-Meier survival analysis and visualized by Cytoscape software. Construction the miRNA -mRNA regulatory network related to the prognosis of CESC, the selected mRNAs were analyzed by gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Results: Four miRNAs and nine mRNAs differentially expressed in CESC and normal tissues were detected. Four miRNAmRNA axes closely related to the prognosis of CESC were obtained. The target mRNA of hsa-miR-505-5p was CBX8, and hsamiR- 142-3p targeted ADAMTS3, PTPRB and SEC23A respectively. Biological function and pathway analysis showed that the differentially expressed mRNAs were mainly enriched in biological process and PI3K-Akt pathway, HPV infection pathway. Conclusions: Above miRNA-mRNA axes obtained in this study are related to the prognosis of CESC, which can be used as a new perspective for the study of the pathogenesis and an effective indicator for monitoring the prognosis of CESC. [ABSTRACT FROM AUTHOR]

Background/Aim. In vitro and in vivo studies show that CX3CL1 and CXCL16 chemokines and their specific receptors, CX3CR1 and CXCR6, respectively, mediate mechanism of neuroinflammation during the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate relative messenger ribonucleic acid (mRNA) levels of CX3CL1, CXCL16, CX3CR1 and CXCR6 in peripheral blood mononuclear cells, as potential molecular markers of relapsing-remitting (RR) MS. Methods. The study included 43 unrelated RR MS patients, 20 of them with clinically active disease (relapse) and 23 with clinically stable disease (remission), and 28 unrelated healthy subjects as controls. Real-time polymerase chain reactions (PCR) were performed using TaqMan® gene expression assays. Relative expression (mRNA) level of each target gene in each sample of peripheral blood mononuclear cells was calculated as the mean normalized expression. Results. The levels of CX3CR1 mRNA were significantly higher in clinically active RR MS patients compared to controls [fold change = 1.38, p (Mann-Whitney U test) = 0.009], and significantly lower in clinically stable vs active RR MS patients [fold change = - 1.43, p (t-test) = 0.03]. Stable RR MS patients had significantly higher CXCL16 mRNA levels than controls [fold change = 1.33, p (Mann-Whitney U test) = 0.006]. A trend of increased CXCR6 gene expression was found in active RR MS patients compared to controls [fold change = 1.23, p (Mann-Whitney U test) = 0.08]. In either active or stable RR MS patients there were no significant correlations of the clinical parameters with expression levels of the target genes. Conclusion. The current results show that increased CX3CR1 mRNA levels in peripheral blood mononuclear cells could represent a proinflammatory molecular marker of clinically active RR MS.

Background: The impact of Schistosoma mansoni infection over the immune response and the mechanisms involved in pathogenesis are not yet completely understood., Objectives: This study aimed to evaluate the expression of innate immune receptors in three distinct mouse lineages (BALB/c, C57BL/6 and Swiss) during experimental S. mansoni infection with LE strain., Methods: The parasite burden, intestinal tissue oogram and presence of hepatic granulomas were evaluated at 7- and 12-weeks post infection (wpi). The mRNA expression for innate Toll-like receptors, Nod-like receptors, their adaptor molecules, and cytokines were determined at 2, 7 and 12 wpi in the hepatic tissue by real-time quantitative polymerase chain reaction (qPCR)., Findings: Swiss mice showed 100% of survival, had lower parasite burden and intestinal eggs, while infected BALB/c and C57BL/6 presented 80% and 90% of survival, respectively, higher parasite burden and intestinal eggs. The three mouse lineages displayed distinct patterns in the expression of innate immune receptors, their adaptor molecules and cytokines, at 2 and 7 wpi., Main Conclusions: Our results suggest that the pathogenesis of S. mansoni infection is related to a dynamic early activation of innate immunity receptors and cytokines important for the control of developing worms.

Ethnopharmacological Relevance: Rujifang (RJF) constitutes a traditional Chinese medicinal compound extensively employed in the management of triple-negative breast cancer (TNBC). However, information regarding its potential active ingredients, antitumor effects, safety, and mechanism of action remains unreported., Aim of the Study: To investigate the efficacy and safety of RJF in the context of TNBC., Materials and Methods: We employed the ultra high-performance liquid chromatography-electrospray four-pole time-of-flight mass spectrometry technique (UPLC/Q-TOF-MS/MS) to scrutinize the chemical constituents of RJF. Subcutaneously transplanted tumor models were utilized to assess the impact of RJF on TNBC in vivo. Thirty female BLAB/c mice were randomly divided into five groups: the model group, cyclophosphamide group, and RJF high-dose, medium-dose, and low-dose groups. A total of 1 × 10 6 4T1 cells were subcutaneously injected into the right shoulder of mice, and they were administered treatments for a span of 28 days. We conducted evaluations on blood parameters, encompassing white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), platelet count (PLT), neutrophils, lymphocytes, and monocytes, as well as hepatorenal indicators including alkaline phosphatase (ALP), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), albumin, and creatinine (CRE) to gauge the safety of RJF. Ki67 and TUNEL were detected via immunohistochemistry and immunofluorescence, respectively. We prepared RJF drug-containing serum for TNBC cell lines and assessed the in vitro inhibitory effect of RJF on tumor cell growth through the CCK8 assay and cell cycle analysis. RT-PCR was employed to detect the mRNA expression of cyclin-dependent kinase and cyclin-dependent kinase inhibitors in tumor tissues, and Western blot was carried out to ascertain the expression of cyclin and pathway-related proteins., Results: 100 compounds were identified in RJF, which consisted of 3 flavonoids, 24 glycosides, 18 alkaloids, 3 amino acids, 8 phenylpropanoids, 6 terpenes, 20 organic acids, and 18 other compounds. In animal experiments, both CTX and RJF exhibited substantial antitumor effects. RJF led to an increase in the number of neutrophils in peripheral blood, with no significant impact on other hematological indices. In contrast, CTX reduced red blood cell count, hemoglobin levels, and white blood cell count, while increasing platelet count. RJF exhibited no discernible influence on hepatorenal function, whereas Cyclophosphamide (CTX) decreased ALP, GOT, and GPT levels. Both CTX and RJF reduced the expression of Ki67 and heightened the occurrence of apoptosis in tumor tissue. RJF drug-containing serum hindered the viability of 4T1 and MD-MBA-231 cells in a time and concentration-dependent manner. In cell cycle experiments, RJF diminished the proportion of G2 phase cells and arrested the cell cycle at the S phase. RT-PCR analysis indicated that RJF down-regulated the mRNA expression of CDK2 and CDK4, while up-regulating that of P21 and P27 in tumor tissue. The trends in CDKs and CDKIs protein expression mirrored those of mRNA expression. Moreover, the PI3K/AKT pathway displayed downregulation in the tumor tissue of mice treated with RJF., Conclusion: RJF demonstrates effectiveness and safety in the context of TNBC. It exerts anti-tumor effects by arresting the cell cycle at the S phase through the PI3K-AKT pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: Human metapneumovirus (hMPV) and parainfluenza virus type 3 (PIV3) are common respiratory illnesses in children. The safety and immunogenicity of an investigational mRNA-based vaccine, mRNA-1653, encoding membrane-anchored fusion proteins of hMPV and PIV3, was evaluated in hMPV/PIV3-seropositive children., Methods: In this phase 1b randomized, observer-blind, placebo-controlled, dose-ranging study, hMPV/PIV3-seropositive children were enrolled sequentially into 2 dose levels of mRNA-1653 administered 2 months apart; children aged 12 to 36 months were randomized (1:1) to receive 10-μg of mRNA-1653 or placebo and children aged 12 to 59 months were randomized (3:1) to receive 30-μg of mRNA-1653 or placebo., Results: Overall, 27 participants aged 18 to 55 months were randomized; 15 participants received 10-μg of mRNA-1653 (n = 8) or placebo (n = 7), whereas 12 participants received 30-μg of mRNA-1653 (n = 9) or placebo (n = 3). mRNA-1653 was well-tolerated at both dose levels. The only reported solicited local adverse reaction was tenderness at injection site; solicited systemic adverse reactions included grade 1 or 2 chills, irritability, loss of appetite, and sleepiness. A single 10-μg or 30-μg mRNA-1653 injection increased hMPV and PIV3 neutralizing antibody titers (geometric mean fold-rise ratio over baseline: hMPV-A = 2.9-6.1; hMPV-B = 6.2-13.2; PIV3 = 2.8-3.0) and preF and postF binding antibody concentrations (geometric mean fold-rise ratio: hMPV preF = 5.3-6.1; postF = 4.6-6.5 and PIV3 preF = 13.9-14.2; postF = 11.0-12.1); a second injection did not further increase antibody levels in these seropositive children. Binding antibody responses were generally preF biased., Conclusions: mRNA-1653 was well-tolerated and boosted hMPV and PIV3 antibody levels in seropositive children aged 12 to 59 months, supporting the continued development of mRNA-1653 or its components for the prevention of hMPV and PIV3., (Copyright © 2024 by the American Academy of Pediatrics.)

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry., (© 2024 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Histone deacetylase 6 (HDAC6) is a key therapeutic target in neurodegenerative diseases such as Alzheimer's disease (AD), which has been demonstrated to play an essential role in memory function and microtubule-associated tau physiology. In this study, W5 was used to treat AD model rats induced by Aβ/Cu 2+ to study the improving effect of W5 on learning and memory impairment in AD rats and its related mechanism, to provide the basis for the subsequent development of W5 as an anti-AD drug. Results showed that W5 could decrease the expression of Aβ, Tau, and p-Tau proteins in the hippocampus of AD rats to inhibit the formation of senile plaques and neurofibrillary tangles, down-regulate the expression of Bax mRNA and Caspase-3 mRNA, and up-regulate the expression of Bcl-2 mRNA to reduce the apoptosis of neuron cells, reverse the expression of TNF-α, IL-1β and IL-6 mRNA to regulate neuroinflammatory response in AD rat brain. W5 also could regulate the oxidative stress state of AD rats, and balance the neurotransmitter disorder in AD rats' brain tissue. Overall, W5 could recover the morphology of hippocampal neurons and improve the learning and memory dysfunction in AD rats by regulating multiple targets in AD rats, providing a promising therapeutic avenue for the treatment of AD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Dysregulated circular RNAs (circRNAs) are significantly related with tumor initiation and progression. However, biological activity and potential molecular mechanism of circRNAs in gastric cancer (GC) deserve further exploration. We carried out total RNA sequencing and acquired the expression profiles of circRNAs. Quantitative real-time PCR as well as RNA in situ hybridization helped to validate circ&#95;0000119 dysregulation. Various in vitro experiments were utilized to investigate the biological activities of circ&#95;0000119 in GC, and the clinical relation of circ&#95;0000119 in vivo was identified through nude mouse xenograft models. Finally, the molecular mechanism of circ&#95;0000119 was clarified via luciferase assays, western blot, and rescue experiments. Compared with adjacent normal tissues, the study found an increase in the expression of circ&#95;0000119 as well as its host linear gene MAN1A2 in GC tissues. Circ&#95;0000119 overexpression promoted proliferation and migration of GC cells in vitro and in vivo, whereas circ&#95;0000119 suppression had the opposite effect. Mechanistically, circ&#95;0000119 sponged miR-502-5p which played an inhibitory role in tumors. Furthermore, we found that miR-502-5p alleviated GC progression through targeting MTBP and downregulating its expression at mRNA and protein levels. In conclusion, our findings reveal a new regulatory mechanism for circ&#95;0000119, which sponges the miR-502-5p, suppresses MTBP expression, and finally promotes GC progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Ethnopharmacological Relevance: Baizhu (BZ) is the dried rhizome of Atractylodes macrocephala Koidz (Compositae), which invigorates the spleen, improves vital energy, stabilizes the fetus, and is widely used for treating spleen deficiency syndrome. However, the impact of BZ on gastrointestinal function during pregnancy remains unexplored., Aim of the Study: This study elucidated the ameliorative effects of BZ on gastrointestinal health and pregnancy outcomes in pregnant mice with spleen deficiency diarrhea (SDD)., Methods: To simulate an irregular human diet and overconsumption of cold and bitter foods leading to SDD, a model of pregnant mice with SDD was established using an alternate-day fasting and high-fat diet combined with oral administration of Sennae Folium. During the experiment, general indicators and diarrhea-related parameters were measured. Gastric and intestinal motility (small intestinal propulsion and gastric emptying rates) were evaluated. Serum motilin (MTL), ghrelin, growth hormone (GH), gastrin (Gas), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), chorionic gonadotropin β (β-CG), progesterone (P), and estradiol (E2) were quantified using an enzyme-linked immunosorbent assay. Pathological changes were examined by hematoxylin and eosin staining (H&E) and alcian blue periodic acid Schiff staining (AB-PAS). Immunohistochemistry and immunofluorescence were used to measure the expression levels of the intestinal barrier and water metabolism-related proteins in colonic tissues. The pregnancy rate, ovarian organ coefficient, uterus with fetus organ coefficient, small size, average fetal weight, and body length of fetal mice were calculated., Results: The results showed that BZ significantly improved general indicators and diarrhea in pregnant mice with SDD, increased gastric emptying rate and small intestinal propulsion rate, elevated the levels of gastrointestinal hormones (AMS, ghrelin, GH, and Gas) in the serum, and reduced lipid levels (TC and LDL-c). It also improved colonic tissue morphology, increased the number of goblet cells, and promoted the mRNA and protein expression of occludin, claudin-1, ZO-1, AQP3, AQP4, and AQP8 in colonic tissues, downregulating the mRNA and protein expression levels of claudin-2, thereby alleviating intestinal barrier damage and regulating the balance of water and fluid metabolism. BZ also held the levels of pregnancy hormones (β-CG, P, and E2) in the serum of pregnant mice with SDD. Moreover, it increased the pregnancy rate, ovarian organ coefficient, uterus with fetus organ coefficient, litter size, average fetal weight, and body length of fetal mice. These findings indicate that BZ can improve spleen deficiency-related symptoms in pregnant mice before and during pregnancy, regulate pregnancy-related hormones, and improve pregnancy outcomes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wang Chenxing: Investigation, Data curation, Writing – original draft, preparation. Su Jie: Data curation, Writing – original draft. Tian Yajuan: Investigation, Data curation. Li Ting: Conceptualization, Methodology. Zhong Yuying: Investigation. Chen Suhong: Writing – review & editing. Lv Guiyuan: Methodology, Validation, Writing – review & editing., (Copyright © 2024 Elsevier B.V. All rights reserved.)

mRNA vaccines have emerged as an optimistic technological platform for vaccine innovation in this new scientific era. mRNA vaccines have dramatically altered the domain of vaccinology by offering a versatile and rapid approach to combating infectious diseases and virus-induced cancers. Clinical trials have demonstrated efficacy rates of 94-95% in preventing COVID-19, and mRNA vaccines have been increasingly recognized as a powerful vaccine platform. Although mRNA vaccines have played an essential role in the COVID-19 pandemic, they still have several limitations; their instability and degradation affect their storage, delivery, and over-all efficiency. mRNA is typically enclosed in a transport mechanism to facilitate its entry into the target cell because it is an unstable and negatively charged molecule. For instance, mRNA that is given using lipid-nanoparticle-based vaccine delivery systems (LNPs) solely enters cells through endocytosis, establishing an endosome without damaging the cell membrane. The COVID-19 pandemic has accelerated the development of mRNA vaccine platforms used to treat and prevent several infectious diseases. This technology has the potential to change the future course of the disease by providing a safe and effective way to combat infectious diseases and cancer. A single-stranded genetic sequence found in mRNA vaccines instructs host cells to produce proteins inside ribosomes to elicit immunological responses and prepare the immune system to fight infections or cancer cells. The potential applications of mRNA vaccine technology are vast and can lead to the development of a preferred vaccine pattern. As a result, a new generation of vaccinations has gradually gained popularity and access to the general population. To adapt the design of an antigen, and even combine sequences from different variations in response to new changes in the viral genome, mRNA vaccines may be used. Current mRNA vaccines provide adequate safety and protection, but the duration of that protection can only be determined if further clinical research is conducted.

Exposure to Cr and/or Ni can have widespread implications on the environment and health. However, the specific toxic effects of chronic Cr and Ni co-exposure on mice liver have not been reported. To ascertain the combined toxic effects of chronic Cr and Ni co-exposure on liver damage in mice, 80 6-week-old female C57BL/6 J mice were randomly divided into 4 groups: the Con group, Cr group (Cr +6 50 mg/L), Ni group (Ni +2 110 mg/L), and Cr + Ni group (Cr +6 50 mg/L + Ni +2 110 mg/L). The trial period lasted for 16 weeks. The results showed that Cr +6 and/or Ni +2 increased liver weight and liver index (P < 0.05) in mice, caused histological abnormality and ultrastructural damage, and micronutrients imbalance in mice liver. These findings serve as the basis for subsequent experiments. Compared with the individual exposure group, chronic Cr and Ni co-exposure resulted in decreased levels and activities of ALT, AST, MDA, T-AOC, and T-SOD (P < 0.05) in liver tissue, and decreased the mRNA expression levels of the TLR4/mTOR pathway related factors (TLR4, TRAM, TRIF, TBK-1, IRF-3, MyD88, IRAK-4, TRAF6, TAK-1, IKKβ, NF-κB, IL-1β, IL-6, TNFα, ULK1, Beclin 1, LC3) (P < 0.05) and decreased the protein expression levels of the factors (TLR4, MyD88, TRAF6, NF-κB p50, IL-6, TNFα, ULK1, LC3II/LC3I) (P < 0.05). Moreover, factorial analysis revealed the interaction between Cr and Ni, which was manifested as antagonistic effects on Cr concentration, Ni concentration, and TLR4, MyD88, NF-κB, mTOR, LC3, and p62 mRNA expression levels. In conclusion, the TLR4/mTOR pathway as a mechanism through which chronic Cr and Ni co-exposure induce liver inflammation and autophagy in mice, and there was an antagonistic effect between Cr and Ni. The above results provided a theoretical basis for understanding the underlying processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sugar bingeing induces maladaptive neuroadaptations to decrease dietary control and promote withdrawal symptoms. This study investigated sex differences in sucrose bingeing, sucrose withdrawal-induced negative mood effects and underlying neuroimmune response in the prefrontal cortex (PFC) and nucleus accumbens (NAc) of C57BL/6J male and female mice. Two-bottle sucrose choice paradigm was used to develop sucrose dependence in mice. Female mice consumed more sucrose than male mice when given free access to water and 10% sucrose for four weeks. A significant increase in the mRNA expression of neuroinflammatory markers (Il1β, Tnfα) was found in the PFC of males exposed to sucrose withdrawal. Sucrose bingeing and subsequent sucrose withdrawal showed elevated protein levels of pro-inflammatory cytokines/chemokines/growth factors in the PFC (IL-1β, IL-6, TNFα, IFN-γ, IL-10, CCL5, VEGF) and NAc (IL-1β, IL-6, IL-10, VEGF) of male mice as compared to their water controls. These effects were concurrent with reduced mRNA expression of neuronal activation marker (cFos) in the PFC of sucrose withdrawal males. One week of sucrose withdrawal after prolonged sucrose consumption showed anxiety-like behavior in male mice, not in females. In conclusion, this study demonstrates that repeated access to sucrose induces anxiety-like behavior when the sugar is no longer available in the diet and these effects are male-specific. Elevated neuroinflammation in reward neurocircuitry may underlie these sex-specific effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A novel electrical impedance spectroscopy-based method for non-destructive sensing of gene expression in living cells is presented. The approach used takes advantage of the robustness and responsiveness of electrical impedance spectroscopy and the highly specific and selective nature of DNA hybridization. The technique uses electrical impedance spectroscopy and gold nanoparticles functionalized with single-stranded DNA complementary to an mRNA of interest to provide reliable, real-time, and quantifiable data on gene expression in live cells. The system was validated by demonstrating specific detection of the uidA mRNA, which codes for the β-glucuronidase (GUS) enzyme, in Solanum lycopersicum MsK8 cells. Gold nanoparticles were functionalized with single-stranded DNA oligonucleotides consisting of either a sequence complementary to uidA mRNA or an arbitrary sequence. The DNA-functionalized gold nanoparticles were mixed with cell suspensions, allowing the gold nanoparticles to penetrate into the cells. The impedance spectra of suspensions of cells with gold nanoparticles inserted within them were then studied. In suspensions of uidA-expressing cells and gold nanoparticles functionalized with the complementary single-stranded DNA oligonucleotide, the impedance magnitude in the frequency range of interest was significantly higher (146 %) in comparison to all other controls. Due to its highly selective nature, the methodology has the potential to be used as a precision agricultural sensing system for accurate and real-time detection of markers of stress, viral infection, disease, and normal physiological activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Heightened interest in messenger RNA (mRNA) therapeutics has accelerated the need for analytical methodologies that facilitate the production of supplies for clinical trials. Forced degradation studies are routinely conducted to provide an understanding of potential weak spots in the molecule that are exploited by stresses encountered during bulk purification, production, shipment, and storage. Consequently, temperature fluctuations and excursions are often experienced during these unit operations and may accelerate mRNA degradation. Here, we present a concise panel of chromatography-based stability-indicating assays for evaluating thermally stressed in vitro transcribed (IVT) mRNA as part of a forced degradation study. We found that addition of EDTA to the mRNAs prior to heat exposure reduced the extent of degradation, suggesting that transcripts may be fragmenting via a divalent metal-ion mediated pathway. Trace divalent metal contamination that can accelerate RNA instability is likely carried over from upstream steps. We demonstrate the application of these methods to evaluate the critical quality attributes (CQAs) of mRNAs as well as to detect intrinsic process- and product-related impurities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Diabetic ulcers represent a chronic condition characterized by prolonged hyperglycemia and delayed wound healing, accompanied by endocrine disorders, inflammatory responses, and microvascular damage in the epidermal tissue, demanding effective clinical treatment approaches. For thousands of years, ancient Chinese ethnopharmacological studies have documented the use of Poria cocos (Schw.) Wolf in treating diabetic ulcers. Recent research has substantiated the diverse pharmacological effects of Poria cocos (Schw.) Wolf, including its potential to alleviate hyperglycemia and exhibit anti-inflammatory, antioxidant, and immune regulatory properties, which could effectively mitigate diabetic ulcer symptoms. Furthermore, being a natural medicine, Poria cocos (Schw.) Wolf has demonstrated promising therapeutic effects and safety in the management of diabetic ulcers, holding significant clinical value. Despite its potential clinical efficacy and applications in diabetic ulcer treatment, the primary active components and underlying pharmacological mechanisms of Poria cocos (Schw.) Wolf remains unclear. Further investigations are imperative to establish a solid foundation for drug development in this domain., Aim of the Study and Materials and Methods: In this study, we aimed to identify the active compounds and potential targets of Poria cocos (Schw.) Wolf using UHPLC-Q-TOF-MS and TCMSP databases. Additionally, we attempt to identify targets related to diabetic ulcers. Following enrichment analysis, a network of protein-protein interactions was constructed to identify hub genes based on the common elements between the two datasets. To gain insights into the binding activities of the hub genes and active ingredients, molecular docking analysis was employed. Furthermore, to further validate the therapeutic effect of Poria cocos (Schw.) Wolf, we exerted in vitro experiments using human umbilical vein vascular endothelial cells and human myeloid leukemia monocytes (THP-1). The active ingredient of Poria cocos (Schw.) Wolf was applied in these experiments. Our investigations included various assays, such as CCK-8, scratch test, immunofluorescence, western blotting, RT-PCR, and flow cytometry, to explore the potential of Poria cocos (Schw.) Wolf triterpenoid extract (PTE) in treating diabetic ulcers., Results: The findings here highlighted PTE as the primary active ingredient in Poria cocos (Schw.) Wolf. Utilizing network pharmacology, we identified 74 potential targets associated with diabetic ulcer treatment for Poria cocos (Schw.) Wolf, with five hub genes (JUN, MAPK1, STAT3, AKT1, and CTNNB1). Enrichment analysis revealed the involvement of multiple pathways in the therapeutic process, with the PI3K-AKT signaling pathway showing significant enrichment. Through molecular docking, we discovered that relevant targets within this pathway exhibited strong binding with the active components of Poria cocos (Schw.) Wolf. In vitro experiments unveiled that PTE (10 mg/L) facilitated the migration of human umbilical vein vascular endothelial cells (P < 0.05). PTE also increased the expression of CD31 and VEGF mRNA (P < 0.05) while activating the expressions of p-PI3K and p-AKT (P < 0.05). Moreover, PTE demonstrated its potential by reducing the expression of IL-1β, IL-6, TNF-α, and NF-κB mRNA in THP-1 (P < 0.05) and fostering M2 macrophage polarization. These results signify the potential therapeutic effects of PTE in treating diabetic ulcers, with its beneficial actions mediated through the PI3K-AKT signaling pathway., Conclusions: PTE is the main active ingredient in Poria cocos (Schw.) Wolf that exerts therapeutic effects. Through PI3K-AKT signaling pathway activation and inflammatory response reduction, PTE promotes angiogenesis, thereby healing diabetic ulcers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To investigate the efficacy of epigallocatechin gallate (EGCG) and its underlying mechanism in preventing bisphenol-A-induced metabolic disorders, in this study, a mice model of metabolic disorders induced by BPA was developed to investigate the efficacy and mechanism of EGCG using microbiomes and metabolomics. The results showed that EGCG reduced body weight, liver weight ratio, and triglyceride and total cholesterol levels in mice by decreasing the mRNA expression of genes related to fatty acid synthesis (Elov16) and cholesterol synthesis (CYP4A14) and increasing the mRNA expression of genes related to fatty acid oxidation (Lss) and cholesterol metabolism (Cyp7a1). In addition, EGCG normalized BPA-induced intestinal microbial dysbiosis. Metabolic pathway analysis showed that low-dose EGCG was more effective than high-dose EGCG at affecting the biosynthesis of L-cysteine, glycerophosphorylcholine, and palmitoleic acid. These results provide specific data and a theoretical basis for the risk assessment of BPA and the utilization of EGCG., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) are noncoding small nucleic acids that contain ~22 nucleotides and are considered to promote the degradation or inhibit the translation of mRNA by targeting its 3'‑untranslated region. However, growing evidence has revealed that nuclear miRNAs, combined with gene promoters or enhancers, are able to directly mediate gene transcription. These miRNAs exert a critical influence on cancer progression by affecting cell growth, migration and invasion. In this review, the direct regulation of gene expression by nuclear miRNAs at the transcriptional level was discussed and summarized, and their mechanisms of action in cancers were highlighted with reference to the various body systems.

Background: Dengue is a rapidly emerging pandemic-prone disease, whose manifestations range from asymptomatic infection to life-threatening complications like Dengue Hemorrhagic Fever and Dengue Shock Syndrome. This study investigates and compares the immune response in clinically defined cohorts of Dengue with and without warning signs, with the aim of identifying immunological correlates of clinical disease and potential markers of disease severity., Methods: Blood samples, collected from study participants fulfilling the WHO definition of Dengue with and without warning signs and healthy volunteers, were analyzed using flow cell-based fluorometric methods for cytokines and chemokines. Gene expression analysis, using RT-PCR, was conducted on T helper cell subset-specific transcription factors and cytokines. Demographic details, virological markers, serotype distribution, and hematological parameters were also investigated in all the subjects., Results: The 35 participants recruited in the study, included 11 healthy volunteers and 12 patients each fulfilling the WHO criteria of Dengue with and without warning signs. While the demographic characteristics and serotype distribution was similar in Dengue with and without warning signs cohorts of the disease, platelet counts and Aspartate Aminotransferase (AST) levels changed significantly between Dengue with and without warning signs patients. Plasma cytokine analysis showed up-regulation of IL-4, IL-10, IP-10, and MCP-1 in Dengue patients compared to healthy volunteers. Disease severity was associated with elevated levels of IL-10, IP-10, IL-4, MCP-1, and MIP-1α. IL-8 and MIP-1α were significantly up-regulated in Dengue with warning sign compared to Dengue without warning signs cases. Transcription factor analysis indicated increased expression of RORα, FoxP3, and GATA3 in Dengue patients. mRNA expression of TGFβ and IL-4 was also elevated in Dengue patients. A positive correlation between mRNA expression of IL-4 and plasma IL-4 was observed., Conclusion: The study reveals a Th2-predominant immune response in all Dengue patients, regardless of disease severity, with overexpression of IL-8 and MIP-1α being observed in patients with warning signs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Iodine excess (IE) intake leads to lymphocyte dysfunction and contributes to autoimmune thyroiditis (AIT). Abnormal thyroid function is associated with adverse cardiovascular events, endothelial dysfunction is often an early pathophysiological feature in most cardiovascular disease. However, the relationship between iodine and the cardiovascular system is currently unclear. Therefore, the aim of this study was to investigate the effects of IE on endothelial function in mouse model., Methods: A total of 24 NOD.H-2 h4 mice were randomly divided into different groups. A sodium iodide (NaI) group supplied with 0.05% NaI water for 8 weeks. Serum levels of tumor necrosis factors α (TNFα), interleukin-6 (IL-6) and C-reactive Protein (CRP), as well as endothelin-1 (ET-1), von Willebrand factor (VWF) and thrombomodulin (THBD) were detected by Elisa. In addition, the mRNA and protein expression of these genes were measured by RT-PCR and Western blotting., Results: Here, we found the urinary iodine concentration (UIC) was higher in the NaI group compared to the control group. Serum levels of ET-1, VWF, and THBD were also significantly lower in the NaI group, however, CRP serum levels are significantly increased. In aorta, the mRNA and protein expression of ET-1, VWF, THBD were downregulated, however, the expression of IL-6, CRP and TNFα mRNA and protein were upregulated in the NaI group. A correlation analysis showed negative correlation between UIC with ET-1, VWF, and THBD, similarly, negative correlation between CRP with THBD was observed. In addition, positive correlations between UIC with CRP., Conclusion: Collectively, in the NOD.H-2 h4 mice, IE supplementation had a suppressive effect on endothelial function, and this inhibition maybe due to the increase expression of inflammatory cytokines., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Objective: Although antipsychotics constitute the best treatment for patients with schizophrenia, this treatment class carries a high risk of metabolic disarrangements thus developing metabolic syndrome (MetS). Altered fatty acid (FA) composition and desaturase indices have been associated with several metabolic diseases, including MetS. Herein, we determined fatty acid desaturase 1 (FADS1) and FADS2 gene expressions, serum delta-5 desaturase (D5D) and D6D indices in female adults with first-episode schizophrenia after olanzapine medication, as well as their relationship with the incidence of MetS., Methods: This study prospectively recruited 120 female patients with first-episode schizophrenia who completed 6-month olanzapine medication. Among these female patients, 31 patients developed MetS and 89 patients did not., Results: The mRNA expression levels of FADS1 and FADS2 in patients were analyzed according to the presence of MetS and evaluation times with results of two-way ANOVAs (FADS1: P MetS = 0.0006, P time = 0.004, P interaction = 0.010; FADS2: P MetS = 0.012, P time < 0.0001, P interaction = 0.001). The D5D and D6D indices in patients were analyzed according to the presence of MetS and evaluation times with results of two-way ANOVAs (D5D: P MetS = 0.002, P time = 0.009, P interaction = 0.014; D6D: P MetS = 0.011, P time = 0.006, P interaction = 0.0001). The SCD-16 and SCD-18 indices in patients were analyzed according to the presence of MetS and evaluation times (SCD-16: P MetS = 0.005, P time = 0.009, P interaction = 0.016; SCD-18: P MetS = 0.037, P time = 0.382, P interaction = 0.163). The following multiple comparisons test showed the MetS exhibited reduced FADS1 mRNA expression and D5D index, increased FADS2 mRNA expression and D6D index, concomitant with an enhanced SCD-16 index, compared to the non-MetS did not after 6-month olanzapine medication., Conclusion: The study suggests changes of FADS1, FADS2 expressions, and fatty acid desaturase indices including D5D, D6D, and SCD-16 may be associated with the development of MetS in female adults with first-episode schizophrenia after olanzapine medication., Competing Interests: Declaration of Competing Interest No conflict of interest is declared by the authors., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis, and its heterogeneity affects the response to clinical treatments. Glycolysis is highly associated with HCC therapy and prognosis. The present study aimed to identify a novel biomarker for HCC by exploring the heterogeneity of glycolysis in HCC. The intersection of both marker genes of glycolysis‑related cell clusters from single‑cell RNA sequencing analysis and mRNA data of liver HCC from The Cancer Genome Atlas were used to construct a prognostic model through Cox proportional hazard regression and the least absolute shrinkage and selection operator Cox regression. Data from the International Cancer Genome Consortium were used to validate the results of the analysis. Immune status analysis was then conducted. A significant gene in the prognostic model was identified as a potential biomarker and was verified through in vitro experiments. The results revealed that the glycolysis‑related prognostic model divided patients with HCC into high‑ and low‑risk groups. A nomogram combining the model and clinical features exhibited accurate predictive ability, with an area under the curve of 0.763 at 3 years. The high‑risk group exhibited a higher expression of checkpoint genes and lower tumor immune dysfunction and exclusion scores, suggesting that this group may be more likely to benefit from immunotherapy. The tumor tissues had a higher zinc finger protein (ZFP)41 mRNA and protein expression compared with the adjacent tissues. In vitro analyses revealed that ZFP41 played a crucial role in cell viability, proliferation, migration, invasion and glycolysis. On the whole, the present study demonstrates that the glycolysis‑related prognostic gene, ZFP41, is a potential prognostic biomarker and therapeutic target, and may play a crucial role in glycolysis and malignancy in HCC.

In an era of head and neck oncology where HPV status will soon dictate patient management, reliable HPV detection is critical. P16 immunohistochemistry (IHC) is currently recommended as the test of choice for oropharyngeal squamous cell carcinomas (OPSCCs). The purpose of this study was to determine the performance characteristics of p16 IHC based on a large clinical experience of squamous cell carcinomas (SCC) arising from HPV hot-spot regions of the head and neck. Consecutive OPSCCs, sinonasal SCCs, and metastatic SCCs of unknown primary sites were evaluated for the presence of HPV by p16 IHC and PCR-based HPV DNA testing as part of clinical care. For discrepant cases, high-risk HPV E6/E7 mRNA in situ hybridization (ISH) and, when possible, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MassArray) genotyping were performed. 746 cancers underwent HPV testing by p16 IHC and DNA PCR genotyping. There was a 95.6% concordance between the 2 assays. Of the 33 discrepant cases, 32 cases (4.3%) were p16 positive but HPV DNA negative. In these cases, 68% were positive for mRNA ISH, invariably related to a non-16 HPV genotype. P16 IHC had an overall accuracy of 98.8%, a sensitivity of 99.8%, and a specificity of 92.1%. P16 IHC is a sensitive and specific assay for determining HPV status. HPV DNA PCR appears vulnerable to HPV genotype diversity and is prone to missing rare non-16 genotypes. HPV mRNA ISH is a practical and reliable direct measure of HPV that may help eliminate the small number of false-positive p16 cases and avoid potential patient harm related to erroneous HPV classification., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: The COVID-19 pandemic required rapid development of vaccines within a short period of time which did not allow to assess vaccine effectiveness (VE) in the long-term., Methods: A computerized literature search was undertaken to identify eligible studies, with no language restrictions, published between 1 December 2020 and 30 June 2023., Results: Out of a total of 27,597 publications, 761 studies were included. Early VE of 87.2% decreased to 55.1% after 9 months among populations fully immunized not only with mRNA (proxy mRNA) vaccines, and 66.3% decreased to 23.5% in populations immunized exclusively with non-mRNA vaccines. Protection against severe COVID-19 declined to 80.9% for proxy mRNA vaccines and 67.2% for non-mRNA vaccines. Omicron variants significantly diminished VE. Within 6-8 months of receiving a single booster of an mRNA vaccine, VE declined to 14.0% and 67.7% for any and severe COVID-19, respectively. Multiple mRNA booster doses restored protection that declined to 29.5% and 70.6% for any and severe COVID-19, respectively, within 5-7 months., Conclusion: Outcomes of this meta-regression underscore the evolving nature of COVID-19 in response to vaccination, dosing schedules, and emerging variants, and provide crucial insights for public health interventions and vaccination strategies., Competing Interests: Declarations of competing interest We declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Monoclonal antibody-based therapeutics have achieved remarkable success in treating a wide range of human diseases. However, conventional systemic delivery methods have limitations in insufficient target tissue permeability, high costs, repeated administrations, etc. Novel technologies have been developed to address these limitations and further enhance antibody therapy. Local antibody delivery via respiratory tract, gastrointestinal tract, eye and blood-brain barrier have shown promising results in increasing local concentrations and overcoming barriers. Nucleic acid-encoded antibodies expressed from plasmid DNA, viral vectors or mRNA delivery platforms also offer advantages over recombinant proteins such as sustained expression, rapid onset, and lower costs. This review summarizes recent advances in antibody delivery methods and highlights innovative technologies that have potential to expand therapeutic applications of antibodies., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material., (© 2024 Sartorius BIA Separations. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Background: Non-nutritive sweeteners (NNS) are commonly used in sweetened foods and beverages; however their role in metabolic regulation is still not clear. In this experiment, we used guinea pigs as an animal model to study the effect of NNS on body growth and intestinal health by modifying gut microbiota and hypothalamus-related proteins., Results: For a 28-day feeding experiment a total of 40 guinea pigs were randomly divided into four groups, one control (CN) group and three treatments, in which three NNS were added to the diet: rebaudioside A (RA, 330 mg kg -1 ), sodium saccharin (SS, 800 mg kg -1 ), and sucralose (TGS, 167 mg kg -1 ), respectively. The TGS group exhibited significantly reduced food consumption in comparison with the CN group (P < 0.05) whereas the RA group showed increased food consumption in comparison with the CN group (P < 0.05). Notably, Taste receptor type 1 subunit 2 (T1R2) expression in the hypothalamus was significantly higher in the RA group than in the CN group (P < 0.05). The mRNA expressions of appetite-stimulated genes arouti-related neuropeptide (AGRP), neuropeptide Y (NPY), and thyroid stimulating hormone (TSHB) were significantly higher than those in the CN group (P < 0.05) but mRNA expressions of appetite-suppressed genes tryptophan hydroxylase 2(THP2) were significantly lower in the TGS group (P < 0.05). Furthermore, NNS in the guinea pig diets (RA, SS, TGS) significantly increased the relative abundance of Muribaculaceae but decreased the relative abundance of Clostridia&#95;vadin BB60 in comparison with the CN group (P < 0.05). We also found that dietary supplementation with RA also significantly altered the relative abundance of Lactobacillus., Conclusion: Our finding confirmed that dietary supplementation with RA and TGS affected body growth and intestinal health by modulating hypothalamic RNA profiles and ileum microbiota, suggesting that NNS should be included in guinea-pig feeding. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)