mRNA Vaccines: Design Principles, Mechanisms, and Manufacturing-Insights From COVID-19 as a Model for Combating Infectious Diseases.

The full approval of two SARS-CoV-2 mRNA vaccines, Comirnaty and Spikevax, has greatly accelerated the development of numerous mRNA vaccine candidates targeting infectious diseases and cancer. mRNA vaccines provide a rapid, safe, and versatile manufacturing process while eliciting strong humoral and cellular immune responses, making them particularly beneficial for addressing emerging pandemics. Recent advancements in modified nucleotides and lipid nanoparticle delivery systems have further emphasized the potential of this vaccine platform.

KRAS mutations detection methodology: from RFLP to CRISPR/Cas based methods.

In personalized cancer medicine, the identification of KRAS mutations is essential for making treatment decisions and improving patient outcomes. This work presents a comprehensive review of the current approaches for detection of KRAS mutations in different cancers. We highlight the value of fast and reliable KRAS mutations discovery and the effectiveness of molecular testing for selecting individuals who might benefit from targeted therapy.

AI-driven covalent drug design strategies targeting main protease (m) against SARS-CoV-2: structural insights and molecular mechanisms.

The emergence of new SARS-CoV-2 variants has raised concerns about the effectiveness of COVID-19 vaccines. To address this challenge, small-molecule antivirals have been proposed as a crucial therapeutic option. Among potential targets for anti-COVID-19 therapy, the main protease (M) of SARS-CoV-2 is important due to its essential role in the virus's life cycle and high conservation.

The effect of modification of DNA interference on myostatin gene expression in mice.

Myostatin is a known negative regulator of muscle tissue growth. Thus, an inhibitor of myostatin may be therapeutically useful as an anabolic agent for the muscle tissue. A promising gene-silencing approach for gene therapy is DNA interference (DNAi), a sequence that is complementary to the promoter region of a target gene.

Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.

Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy.

An Update on the Application of CRISPR Technology in Clinical Practice.

The CRISPR/Cas system, an innovative gene-editing tool, is emerging as a promising technique for genome modifications. This straightforward technique was created based on the prokaryotic adaptive immune defense mechanism and employed in the studies on human diseases that proved enormous therapeutic potential. A genetically unique patient mutation in the process of gene therapy can be corrected by the CRISPR method to treat diseases that traditional methods were unable to cure.

Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus.

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

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood.

An overview of the vaccine platforms to combat COVID-19 with a focus on the subunit vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that has caused the recent coronavirus disease (COVID-19) global pandemic. The current approved COVID-19 vaccines have shown considerable efficiency against hospitalization and death. However, the continuation of the pandemic for more than two years and the likelihood of new strain emergence despite the global rollout of vaccination highlight the immediate need for the development and improvement of vaccines.

TWIST1 activates cancer stem cell marker genes to promote epithelial-mesenchymal transition and tumorigenesis in esophageal squamous cell carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is one of the deadliest cancers worldwide. Overexpression of EMT master transcription factors can promote differentiated cells to undergo cancer reprogramming processes and acquire a stem cell-like status.

Methods: The KYSE-30 and YM-1 ESCC cell lines were transduced with retroviruses expressing TWIST1 or GFP and analyzed by quantitative reverse transcription PCR (qRT-PCR), chromatin immunoprecipitation (ChIP), and immunostaining to investigate the correlation between TWIST1 and stemness markers expression.

Developing novel liquid biopsy by selective capture of viral RNA on magnetic beads to detect COVID-19.

Objectives: Early, specific, and sensitive detection methods of COVID-19 are essential for force stopping its worldwide infection. Although CT images of the lung and/or viral RNA extraction followed by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) are widely used; they have some limitations. Here, we developed a highly sensitive magnetic bead-based viral RNA extraction assay followed by rRT-PCR.

Insights into the structural peculiarities of the N-terminal and receptor binding domains of the spike protein from the SARS-CoV-2 Omicron variant.

Since the new variant of SARS-CoV-2, Omicron (BA.1) has raised serious concerns, it is important to investigate the effects of mutations in the NTD and RBD domains of the spike protein for the development of COVID-19 vaccines. In this study, computational analysis of the Wuhan and Omicron NTDs and RBDs in their unbound and bound states to mAb 4A8 and ACE2 were performed.

Guidelines for pre-analytical conditions for assessing the methylation of circulating cell-free DNA.

Methylation analysis of circulating cell-free DNA (cirDNA), as a liquid biopsy, has a significant potential to advance the detection, prognosis, and treatment of cancer, as well as many genetic disorders. The role of epigenetics in disease development has been reported in several hereditary disorders, and epigenetic modifications are regarded as one of the earliest and most significant genomic aberrations that arise during carcinogenesis. Liquid biopsy can be employed for the detection of these epigenetic biomarkers.

Pre-mRNA Processing Factors and Retinitis Pigmentosa: RNA Splicing and Beyond.

Retinitis pigmentosa (RP) is the most common inherited retinal disease characterized by progressive degeneration of photoreceptors and/or retinal pigment epithelium that eventually results in blindness. Mutations in pre-mRNA processing factors () have been linked to 15-20% of autosomal dominant RP (adRP) cases. Current evidence indicates that mutations cause retinal specific global spliceosome dysregulation, leading to mis-splicing of numerous genes that are involved in a variety of retina-specific functions and/or general biological processes, including phototransduction, retinol metabolism, photoreceptor disk morphogenesis, retinal cell polarity, ciliogenesis, cytoskeleton and tight junction organization, waste disposal, inflammation, and apoptosis.

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes.

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome-the most common cause of deaf-blindness.

Selective capture of plasma cell-free tumor DNA on magnetic beads: a sensitive and versatile tool for liquid biopsy.

Purpose: Recently, 'solid tumor biopsies' have been challenged by the emergence of 'liquid biopsies', which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC).

Methods: Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations.

Hydroxyapatite as a biomaterial - a gift that keeps on giving.

The synthetic analogue to biogenic apatite, hydroxyapatite (HA) has a number of physicochemical properties that make it an attractive candidate for diagnosis, treatment of disease and augmentation of biological tissues. Here we describe some of the recent studies on HA, which may provide bases for a number of new medical applications. The content of this review is divided to different medical application modes utilizing HA, including tissue engineering, medical implants, controlled drug delivery, gene therapies, cancer therapies and bioimaging.

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31 mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31 mouse retinae and RPE.

Prp19/Pso4 Is an Autoinhibited Ubiquitin Ligase Activated by Stepwise Assembly of Three Splicing Factors.

Human nineteen complex (NTC) acts as a multimeric E3 ubiquitin ligase in DNA repair and splicing. The transfer of ubiquitin is mediated by Prp19-a homotetrameric component of NTC whose elongated coiled coils serve as an assembly axis for two other proteins called SPF27 and CDC5L. We find that Prp19 is inactive on its own and have elucidated the structural basis of its autoinhibition by crystallography and mutational analysis.

The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes.

A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing.

Prp3 is an essential U4/U6 di-snRNP-associated protein whose functions and molecular mechanisms in pre-mRNA splicing are presently poorly understood. We show by structural and biochemical analyses that Prp3 contains a bipartite U4/U6 di-snRNA-binding region comprising an expanded ferredoxin-like fold, which recognizes a 3'-overhang of U6 snRNA, and a preceding peptide, which binds U4/U6 stem II. Phylogenetic analyses revealed that the single-stranded RNA-binding domain is exclusively found in Prp3 orthologs, thus qualifying as a spliceosome-specific RNA interaction module.

The G-patch protein Spp2 couples the spliceosome-stimulated ATPase activity of the DEAH-box protein Prp2 to catalytic activation of the spliceosome.

Structural rearrangement of the activated spliceosome (B(act)) to yield a catalytically active complex (B*) is mediated by the DEAH-box NTPase Prp2 in cooperation with the G-patch protein Spp2. However, how the energy of ATP hydrolysis by Prp2 is coupled to mechanical work and what role Spp2 plays in this process are unclear. Using a purified splicing system, we demonstrate that Spp2 is not required to recruit Prp2 to its bona fide binding site in the B(act) spliceosome.