Abstract: While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Gene ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.

Abstract: Spatial transcriptomics enables the in situ mapping of gene expression, revolutionizing our ability to study tissue organization and cellular interactions. However, as this technology is increasingly adopted across biological and clinical research, many groups struggle with practical barriers to implementation—including platform selection, sample quality, and experimental scalability.Here, we provide a comprehensive, practical guide to spatial transcriptomics, informed by the processing and analysis of over 1,000 spatial samples across Visium, Visium HD, and Xenium platforms. We outline best practices for experimental design, tissue handling, sequencing, and computational analysis, with special attention to clinical samples and high-throughput settings.Our goal is to translate hands-on experience into actionable recommendations that support robust, reproducible spatial workflows. This guide is designed to assist researchers at all levels—from those designing their first spatial experiment to groups aiming to integrate ST into translational pipelines and large-scale studies.

Abstract: Long noncoding RNAs (lncRNAs), non-protein-coding transcripts exceeding 200 nucleotides, are critical regulators of gene expression through chromatin remodeling, transcriptional modulation, and post-transcriptional modifications. While ionizing radiation (IR) induces cellular damage through direct DNA breaks, reactive oxygen species (ROS)-mediated oxidative stress, and bystander effects, the functional involvement of lncRNAs in radiation response remains incompletely characterized. Here, through genome-wide CRISPR activation (CRISPRa) screening in non-small cell lung cancer (NSCLC) cells, we identified LOC401312 as a novel radiosensitizing lncRNA, the stable overexpression of which significantly enhanced IR sensitivity. Transcriptomic profiling revealed that LOC401312 transcriptionally upregulates carbamoyl-phosphate synthase 1 (CPS1), a mitochondrial enzyme involved in pyrimidine biosynthesis. Notably, CPS1 overexpression recapitulated the radiosensitization phenotype observed with LOC401312 activation. Mechanistic investigations revealed that CPS1 suppresses the phosphorylation of ATM kinase (Ser1981) and XRCC1 protein levels, which are key mediators of DNA damage checkpoint activation and base excision repair, respectively. This study establishes the LOC401312-CPS1-ATM/XRCC1 axis as a previously unrecognized regulatory network governing radiation sensitivity, highlighting the potential of lncRNA-directed metabolic rewiring to impair DNA repair fidelity. Our findings not only expand the functional landscape of lncRNAs in DNA damage response but also provide a therapeutic rationale for targeting the LOC401312-CPS1 axis to improve radiotherapy efficacy in NSCLC.

Abstract: Translation initiation in mitochondria involves unique mechanisms distinct from those in the cytosol or in bacteria. The Schizosaccharomyces pombe mitochondrial translation initiation factor 2 (Mti2) is the ortholog of human MTIF2, which plays a vital role in synthesizing proteins in mitochondria. Here, we investigate the insertion domain of Mti2, which stabilizes its interaction with the ribosome and is crucial for efficient translation initiation. Our results show that the insertion domain is critical for the proper folding and function of Mti2. The absence of the insertion domain disrupts cell growth and affects the expression of genes encoded by mitochondrial DNA. Additionally, we show that Mti2 physically interacts with the small subunits of mitoribosomes (mtSSU), and deletion of the insertion domain dissociates mitochondrial initiation factors from the mitoribosome, reducing the efficiency of mitochondrial translation. Altogether, these findings highlight the conserved role of the insertion domain in facilitating translation initiation in fission yeast and thus reveal shared principles of mitochondrial translation initiation in both fission yeast and humans.

Abstract: The ongoing demand for reliable, cost-effective, and scalable diagnostic solutions during the SARS-CoV-2 pandemic has highlighted the need for innovative production platforms. Here, we present a plant-based molecular farming (PMF) strategy for the production of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein fused with Fc (RBDw-Fc). The RBDw-Fc antigen was transiently expressed in Nicotiana benthamiana plant, achieving high yields and purity. Antigen-antibody binding assays were used to assess its functionality. The purified antigen was used to develop a rapid diagnostic blot assay to screen both pre and post vaccinated and pre and post infected plasma EDTA samples with high sensitivity and specificity. Our results demonstrate that the RBDw-Fc-based assay is effective for SARS-CoV-2 detection, and offers significant advantages in terms of production speed, scalability, and cost-efficiency compared to traditional systems, such as cell-culture-based production. This kind of diagnostic test deliver accurate results in just few minutes, enhancing its applicability in clinical and resource-limited settings. This study highlights the versatility of PMF as a platform for producing high-quality reagents, with potential applications beyond SARS-CoV-2 diagnostics. The RBDw-Fc antigen-based method offers a model for the development of rapid, economical, and adaptable screening tools for emerging infectious diseases and future pandemics.

Abstract: Cellular senescence is a well-established biological phenomenon in eukaryotes. It involves DNA damage, telomere shortening, a senescence-associated secretory phenotype (SASP), and the inability of cells to replicate. It is associated with ageing, and also with oxidative stress. Given the importance of oxidative stress in pre-eclampsia, there is considerable evidence, that we review, that senescence plays an important role in both normal placental development and in the development of both early- and late-term pre-eclampsia. Autophagy is capable of delaying or even reversing the development of senescence, and certain small molecules such as sulforaphane and spermidine can stimulate autophagy, including via the redox-sensitive transcription factor Nrf2. Ergothioneine is a thiohistidine antioxidant that is protective against a variety of cardiovascular and other diseases. Ergothioneine also interacts with Nrf2, and pre-eclampsia occurs far less frequently in individuals with higher plasma ergothioneine levels. Together, these elements provides a self-consistent, molecular and systems biology explanation for at least one mechanism by which ergothioneine may be protective against pre-eclampsia.

Abstract: Bruton's tyrosine kinase (BTK) is a key signaling molecule involved in both hematological malignancies and solid tumors. In B-cell malignancies such as Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL), BTK mediates B-cell receptor signaling, promoting tumor survival and proliferation, leading to the development of BTK inhibitors like ibrutinib that improve patient outcomes. In solid tumors, BTK isoforms, particularly p65BTK, contribute to tumor growth and therapy resistance, with inhibition showing promise in cancers like colorectal, ovarian, and non-small cell lung cancer. BTK also influences the tumor microenvironment by modulating immune cells such as myeloid-derived suppressor cells and tumor-associated macrophages, aiding immune evasion. BTK inhibition can enhance anti-tumor immunity and reduce inflammation-driven tumor progression. Additionally, BTK contributes to tumor angiogenesis, with inhibitors like ibrutinib showing anti-angiogenic effects. Beyond cancer, BTK is linked to aging, where its modulation may reduce senescent cell accumulation and preserve cognitive function. This review explores BTK's dual role, focusing on its oncogenic effects and potential impact on aging processes. We also discuss the use of BTK inhibitors in cancer treatment and their potential to address age-related concerns, providing a deeper understanding of BTK as a therapeutic target and mediator in the complex relationship between cancer and aging.

Abstract: Heparan-sulfate (HS) and chondroitin-sulfate (CS) proteoglycans (PGs) are essential regulators of many biological processes including cell differentiation, signalisation and proliferation. PGs interact, mainly via their glycosaminoglycan (GAG) chains, with a large number of ligands including growth factors, enzymes and extracellular matrix components, thereby modulating their biological activities. HSPGs and CSPGs share a common tetrasaccharide linker region, which undergoes modifications particularly phosphorylation of the xylose residue by the kinase Fam20B. Here, we demonstrated that Fam20B gain-of-function decreased, in a dose dependent manner, the synthesis of both CS- and HS-attached PGs. In addition, we showed that blockage of GAG chain synthesis by Fam20B was suppressed by mutation of aspartic acid residues D289 and D309 of the catalytic domain. Interestingly, we bring evidence that, in contrast to Fam20B, expression of the 2-phosphoxylose phosphatase XYLP increased, in a dose dependent manner, GAG chain synthesis and rescue the blockage of GAG chains synthesis induced by Fam20B. In line with previous reports, we found that Fam20B loss-of-function reduced GAG chain synthesis. Finaly, we found that Fam20B inhibits proliferation and migration of glioblastoma cells, thus revealing the critical role of GAG chains of PGs in glioblastoma cell tumorigenesis. This study revealed that both gain- and loss-of-function of Fam20B led to decreased GAG chain synthesis, therefore suggesting that a balance between phosphorylation and dephosphorylation of the xylose by Fam20B and XYLP, respectively is probably an essential factor of the regulation of the rate of PG synthesis.

Abstract: Background/Objectives: Colon cancer remains a significant health challenge world-wide, with inflammatory pathways such as tumor necrosis factor-alpha (TNF-α) playing a central role in its progression. TNF-α, a key proinflammatory cytokine, is implicated in various stages of colon cancer development, including inflammation, tumor growth, and metastasis. This review provides a comprehensive overview of the molecular mechanisms through which TNF-α contributes to colon cancer progression, with a focus on its interaction with signaling pathways like NF-κB, oxidative stress, and Wnt/β-catenin. Methods: A comprehensive review of the literature was conducted to elucidate the role of tumor necrosis factor-alpha in the pathogenesis of colon cancer. Peer-reviewed clin-ical and preclinical studies involving human subjects were identified through electronic databases, including PubMed, Scopus, and Web of Science. Selection criteria focused on studies examining the molecular mechanisms of TNF-α signaling, its interaction with key oncogenic pathways and therapeutic interventions targeting TNF-α. Data were synthesized to highlight both mechanistic insights and therapeutic applications. Results: TNF-α’s involvement in promoting tumorigenesis and its complex role in the tumor microenvironment highlight its potential as both a therapeutic target and a challenge for effective treatment. The review also delves into the potential of an-ti-TNF-α therapies and the emerging role of combination strategies with immune checkpoint inhibitors. Despite promising preclinical findings, clinical application faces challenges due to the dual role of TNF-α in both promoting and inhibiting tumor pro-gression. Conclusion: Future research should focus on overcoming resistance mechanisms, de-veloping personalized therapeutic strategies, and balancing the effects of TNF-α in cancer therapy.

Abstract: Modulating the enzymatic activity of amylase holds significant therapeutic promising in diabetes mellitus, primarily due to its ability to catalyze the hydrolysis of starch into simpler sugars. This study employs computational models utilizing experimental datasets, focusing on designing inhibitors of α-amylase. Despite limited information regarding in silico predictive models’ capability related to α-amylase, we collected various data and applied multiple linear regression-based machine learning technique (MLR-ML) to forecast the inhibitory activity of α-amylase inhibitors as antidiabetic agents. The model was developed using a dataset comprising compounds relevant to α-amylase's preventive action and the model exhibited R2 correlation values of 0.887 and 0.887 for training and prediction sets, respectively. These findings underscore the efficacy of an in silico approach employing machine learning (ML) techniques in identifying potential antidiabetic compounds. Collectively, our study demonstrates that this approach is a viable strategy for regulating postprandial hyperglycemia and mitigating diabetes risk.

Abstract: Immunoassays for cardiac troponin, such as the Elecsys® hs-TnT, have become the gold standard for myocardial infarction diagnostics. While various protein/chemical factors affecting the troponin complex and, thus, its diagnostic accuracy have been investigated, the role of coding single nucleotide polymorphisms remains underexplored. To evaluate potential cSNP-induced interference with antibody binding in the Elecsys® hs-TnT immunoassay, we applied ITEM-FOUR, a mass spectrometry-based method that quantifies changes in antibody binding upon amino acid substitutions in epitope peptides. Candidate cSNPs were selected from the dbSNP database and were mapped to human cardiac troponin T by molecular modeling. Consuming micromolar antibody concentrations and microliter sample volumes, two wild-type and 17 cSNP-derived variant epitope peptides—six for monoclonal antibody M7 and eleven for monoclonal antibody M11.7—were investigated to reveal the binding motifs 'V131-K134-E138-A142' for M7 and 'E146-I150-R154-E157' for M11.7. Loss of binding to M11.7 was observed for substitutions Q148R (rs730880232), R154W (rs483352832), and R154Q (rs745632066), whereas the E138K (rs730881100) exchange disrupted binding of M7. Except for cSNP Q148R they are associated with cardiomyopathies, placing affected individuals at risk for both, underlying heart disease and false-negative hs-TnT assay results in case of myocardial infarction. Our results highlight the need to account for cSNP-related interferences in antibody-based diagnostics. ITEM-FOUR offers a powerful approach for tackling this challenge, fostering next-generation assay development.

Abstract: Background/Objectives: Bacteria rarely live in well-defined environment. Even in the biotechnological reactors cells are surrounded by hundreds of byproducts and their derivatives, let alone complex microbial communities in the gut or soil. Yet all modern genome-scale metabolic (GEM) models were developed with minimal media in mind.Methods: In this study, we propose the use of a novel FBA-PRCC (Flux Balance Analysis – Partial Rank Correlation Coefficient) approach for the analysis of GEM models under nutrient-rich conditions. This method combines flux space sampling with global sensitivity analysis (GSA), enabling a more comprehensive understanding of the metabolic behavior of organisms beyond traditional constraint-based modeling techniques. Results: Using FBA-PRCC, we identify two novel modes of species–metabolite interaction: attraction and avoidance. These concepts offer a framework to quantify and utilize interspecies metabolic dependencies, potentially transforming how we understand community function in microbiomes. Our results show that sensitivity coefficients provide complementary insights to standard knockout analysis, Flux Variability Analysis (FVA), and CoPE-FBA. However, analysis of auxotrophic mutants reveals that sensitivity coefficients are highly non-robust in the presence of alternative pathways: even weakly active bypasses can suppress signals from key metabolic routes.Conclusions: While FBA models are usually developed for well-characterized laboratory strains in controlled conditions, they fail when applied to bacteria in the complex environments like the human gut or skin. Better description of the metabolite transport and new modelling approaches are required to overcome this problem.

Abstract: The prostaglandin E2 receptor EP3 is emerging as a promising therapeutic target in cardiovascular disease due to its involvement in vascular inflammation, platelet aggregation, and vasoconstriction. However, selective EP3 ligands with validated biological activity remain scarce. Here, we combine computational and experimental strategies to discover and validate novel EP3 receptor ligands with therapeutic potential. We implemented a high-throughput, structure- and ligand- based virtual screening pipeline, enabling the efficient exploration of approved drugs and natural compounds from DrugBank and FooDB libraries. Top-scoring candidates were prioritized based on binding energy, and pharmacophoric similarity. Selected hits underwent in silico ADME/Tox profiling using QikProp, identifying molecules with favorable pharmacokinetic and safety parameters. TUCA, masoprocol, and pravastatin sodium emerged as lead candidates and were validated in vitro using endothelial migration and platelet aggregation assays. TUCA exhibited the most consistent inhibitory effect on endothelial migration, while masoprocol, and hydrocortisone significantly reduced platelet aggregation. These findings establish a multidimensional workflow for the rational identification of EP3 ligands and support their potential in cardiovascular therapeutics.

Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology has revolutionized molecular biology and therapeutic research. In this review, we highlight a focused analysis of CRISPR-Cas applications in the treatment and study of five major human diseases based on select peer-reviewed articles. While not exhaustive, our investigation provides insight into trends, mutation targets, and experimental outcomes, highlighting the evolving landscape of CRISPR as a therapeutic tool. We also discuss challenges, emerging tools like Cas12, Cas13, and Cas15, and future directions.

Abstract: Actinobacterial natural ecological niches are characterized by variations in the availability of nutrients, resulting in a complex metabolism. Their remarkable ability to adapt to fluctuating nutrient conditions is possible through the utilization of large amounts of substrates. Recent discoveries in bacterial metabolism suggested the importance of polyamine metabolism in bacteria, particularly in those of the order Actinomycetales, to survive in their natural habitats. This makes such enzymes promising targets to inhibit their growth. Since the polyamine metabolism of soil bacteria of the genus Streptomyces and the human pathogenic Mycobacteria is surprisingly similar, a target-based drug development in Streptomyces and Mycobacterium spp. is an alternative approach to the classical search of antibiotics. The recent development of drugs to treat epidemic diseases like tuberculosis (TB) has gained attention due to the occurrence of multi-drug-resistant strains. In addition, drug repurposing plays a crucial role in the treatment of various complex diseases, such as malaria. With that notion, the treatment of TB could also benefit from this approach. For example, molecular chaperones, proteins that help other proteins to fold properly are found in almost all living organisms including the causative agents of TB. Therefore, targeting these molecules could help in the treatment of TB. We aim to summarize the knowledge of nitrogen and carbon metabolism of the two closely related actinobacterial genera, Streptomyces and Mycobacterium, and of the identification of new potential drug targets.

Abstract: The protective effect of isorhamnetin on myocardial injury induced by isoproterenol (ISO) was investigated to identify key targets and pathways involved, offering potential therapeutic insights for cardiovascular diseases. The myocardial injury model was established through intraperitoneal ISO injection, and the effects of isorhamnetin on apoptosis and oxidative stress in ISO-induced myocardial injury rats were assessed. Additionally, an ISO-induced H9c2 cell injury model was established to evaluate the impact of isorhamnetin on cellular damage. Transcriptomic sequencing of H9c2 cells was conducted to identify differentially expressed genes, followed by gene enrichment analysis. Intracellular glucose, lactate, and ATP levels were quantified, and protein expression of key pathway targets ENO1, PPARα, and PGC-1α was analyzed via immunoblotting. Isorhamnetin improved cardiac dysfunction and morphological damage, reduced serum markers of cardiac injury, and exerted cardioprotective effects by regulating oxidative stress and inhibiting apoptosis. Compared to the ISO group, the glycolytic process, with ENO1 as a key target and the PPAR signaling pathway as the core regulator, was significantly suppressed in the isorhamnetin-pretreated group. Furthermore, isorhamnetin pretreatment reduced intracellular glucose and lactate levels while increasing ATP content in a concentration-dependent manner. These findings suggest that isorhamnetin protects the heart by inhibiting ENO1, activating the PPARα/PGC-1α signaling axis, reversing isoprenaline-induced metabolic shifts in H9c2 cells, suppressing glycolysis, and enhancing ATP release, thereby mitigating apoptosis and oxidative stress.

Abstract: Cellular gene expression varies in different physiological and pathological conditions. Analysis of differential gene expression enables researchers to understand the cellular changes associated with physiological or pathological conditions. During gene expression analyses, researchers calculate the transcripts expressed from a gene under that gene's name, assuming only a single mRNA is expressed by that gene. However, this assumption is biologically inaccurate, but it is often preferred to avoid the complex analyses of isoforms. Most mammalian genes express more than three mRNAs, which encode various proteins or act as noncoding RNAs. We have previously addressed the molecular basis mRNA isoform formation and detection strategies. In this review article, we have discussed the physiological and pathological roles of mRNA isoforms. Any specific cell may express different isoforms of mRNA from a specific gene depending on its differentiation state. Different isoforms can be expressed from a single gene at various stages of development and during aging. The same cell may also express mRNA isoforms related to pathological conditions. Summarized findings highlight the importance of detecting mRNA isoforms over conventional gene identification.

Abstract: (1) Background: Previous theoretical studies have provided arguments for the existence of a ring or hairpin RNA that could have served as a primitive informational and functional molecule at the origin of life. The present article consists of searching in current genomes for RNAs closest to this ring in terms of occurrence of similar nucleotide motifs. (2) Methods: In searching for the smallest possible ring/hairpin RNA capable of interacting with amino acids in the construction of the peptides of the primitive living world, we found a circular docosamer RNA molecule (length 22), which we called AL ring (for Alpha or Archetypal Loop). Then, we started to systematically track AL relics in current genomes in the form of motifs like pentamers or pairs of consecutive codons in common with AL. (3) Results: The sequence correspondence between AL and RNA sequences of organisms from different kingdoms of life (Archaea, Bacteria and Eukarya) was found with high statistical significance with a frequency gradient depending on both the antiquity of the species and the functional necessity of the genes. (4) Conclusions: Considering the suitability of AL as a candidate for being a primitive sequence, and the evolution of the different species considered, we can consider the AL RNA ring as a possible actor that favored the appearance of life on Earth.

Abstract: Dairy propionibacteria are commonly ingested through the consumption of raw milk cheeses or Swiss type cheeses in which they are added as starter cultures. Some strains of the species Propionibacterium freudenreichii or their culture media have been commercialized in multi-strain probiotic preparations or to supply bioactive substances such as short chain fatty acids, bifidogenic molecules and vitamins, respectively. In recent years, many more mechanisms of action of dairy propionibacteria as probiotics for different novel applications were discovered and are summarized in this descriptive review. Strains of P. freudenreichii mitigated inflammatory bowel diseases (IBDs), mucositis and prevented necrotizing enterocolitis (NEC) in preterm newborns. Moreover, these bacteria exerted immunomodulation, particularly in food allergy, anti-obesity, anti-diabetic, anti-cancerogenic effects, inhibition of osteoclastogenesis in rheumatoid arthritis, and infection mitigation in animal models. Most of the observed effects were mediated by cell surface proteins or extracellular vesicle (EV) proteins such as the surface layer (S-layer) protein SlpB, DlaT and GroEL. Based on the available information, these bacteria do not present safety issues but investigations on the presence of transferable antibiotic resistance traits should be specifically assessed both phenotypically and genotypically. In most possible applications the confirmation of beneficial effects in clinical trials still need to be carried out to allow their use as health promoting agents.

Abstract: Unpredictable emerging and re-emerging of Mpox virus (MPXV) outbreaks account for urgent interventions. While no drug has been approved for Mpox treatment, repurposing of nucleoside analog drugs such as tecovirimat and cidofovir is the current focus of many intervention initiatives. Intrigued by their molecular structural similarities, we hypothesized that acyclovir, allopurinol, and B vitamins, which structurally resemble tecovirimat and cidofovir, could be competitive inhibitors of the MPXV DNA polymerase, abrogating DNA synthesis viral multiplication. We applied computational molecular docking experiments to investigate this hypothesis. Our results revealed that acyclovir fits exactly into the binding cavity for cidofovir and interacts with the same MPXV DNA polymerase amino acid residues with comparatively similar binding energy. We also found that oxipurinol (a metabolite of allopurinol) expresses higher affinity for MPXV DNA polymerase than cidofovir. Interestingly, the B vitamins riboflavin and folic acid outcompeted cidofovir, acyclovir and deoxyribonucleoside triphosphates (dNTPs), the natural substrates for DNA polymerase. From these ligand-receptor interactions we infer that acyclovir, oxipurinol and B vitamins are competitive inhibitors, targeting the active rather than the allosteric site of MPXV DNA polymerase. Our findings provide a novel insight into the B vitamin’s mode of action, emphasizing their antiviral potential via direct hijacking of the viral DNA replication machinery. While B vitamins are easy to find and cheaper, wet laboratory studies equipped with bioinformatics capacity could validate our findings to drive clinical trials. Once well established, B vitamins can be a cheap and reliable venture for pandemic preparedness for viral outbreaks.