The concept of preconditioning mesenchymal stem cells (MSCs) under different stress conditions or with bioactive molecules is introduced to optimize their therapeutic potential. This study investigates the physicochemical effect of hierarchical TiO 2 nanotube arrays, a versatile and easy-to-prepare nanosurface, on MSC behaviour. By precisely controlling the nanotopography through anodization, we demonstrate the significant influence of surface properties on MSC adhesion, proliferation and differentiation. Electrostatic interactions between surface charge and proteins play a crucial role in these cellular responses. In addition, preconditioning MSCs under specific conditions enhances their therapeutic potential by optimizing paracrine signalling and homing properties. Higher surface charges and increasing spiky character of surface roughness of titania samples after anodization at 60 V significantly upregulated chemokine receptor type 4 (CXCR4) and vascular endothelial growth factor A (VEGFA), indicating the enhanced migratory and angiogenic potential of MSCs. The study reveals the mechanotransductive effects of nanotopography on MSC differentiation, suggesting that tailored surface features can direct cellular fate. These findings highlight the potential of hierarchical TiO 2 nanotube arrays as a promising platform for regenerative medicine, offering a novel approach to improve tissue engineering and therapeutic outcomes.

BACKGROUND:La-related protein 7 (LARP7) is a key regulator of RNA metabolism and is thought to play a role in various cellular processes. LARP7 gene autosomal recessive mutations are the cause of Alazami syndrome, which presents with skeletal abnormalities, intellectual disabilities, and facial dysmorphisms. This study aimed to determine the role of LARP7 in modulating gene expression dynamics during osteogenesis. METHODS AND RESULTS:First, the temporal expression profile of the LARP7 gene during various stages of osteogenesis was examined. Then, RNA interference-mediated knockdown of LARP7 was implemented and high-throughput RNA-seq analysis was performed in order to identify global gene expression changes associated with knockdown of LARP7. The findings show there were significant alterations in the overall gene expression profile. The observed down-regulation in extracellular matrix (ECM) component genes suggests that it might lead to impairments in the structure and function of the bone matrix. Additionally, modulation of alternative splicing events were observed, especially in the RUNX2 and SPP1, indicating the potential contribution of LARP7 to the phenotypic features observed in Alazami syndrome. CONCLUSION:Overall, the findings clarify the regulatory mechanisms of LARP7 in osteogenic differentiation and illuminate potential avenues for therapeutic interventions in patients with skeletal disorders.

Introduction:Ectodermal dysplasias (EDs) represent a heterogeneous group of genetic disorders marked by impaired development of multiple tissue derivatives originating from the human ectoderm, including teeth, hair, nails, and sweat glands. Advances in next-generation sequencing technology have facilitated the identification of novel genes, such as TSPEAR, contributing to the emergence of the ectodermal dysplasia-14 of the hair/tooth type (ECTD14) phenotype, primarily characterized by hypotrichosis, hypodontia, and dysmorphic features. Methods:Five individuals from the same family were included in the study, three of whom were heterozygous and two homozygous for a novel frameshift TSPEAR variant. All displayed ED and/or tooth loss. Exome sequencing was performed in the index case, and Sanger sequence analysis was carried out to detect the carrier status in parents and relatives. Results:We identified a novel biallelic frameshift TSPEAR variant [NM_144991.2, c.1594_1595insA, p.(Phe532TyrfsTer26)] in two siblings who displayed oligodontia, sparse hair, and facial dysmorphism. The remaining heterozygous carriers manifested early tooth loss with non-syndromic isolated oligodontia. Conclusion:This study has identified individuals carrying biallelic and heterozygous TSPEAR variants, where heterozygous carriers often exhibit non-syndromic tooth agenesis. Moreover, the presence of inter- and intrafamilial variability emerges as a notable feature of the disease. This understanding underscores the complexity of the disease and the importance of considering genetic variability when diagnosing and managing affected individuals.

OBJECTIVES:Familial Mediterranean fever (FMF) is traditionally viewed as an autosomal recessive autoinflammatory disorder. However, a significant subset of patients harbouring a single pathogenic MEFV mutation exhibit a clinical phenotype indistinguishable from that of homozygous patients. We aimed to compare the transcriptomic profiles of patients carrying a single pathogenic mutation who exhibit the classical FMF phenotype with those of healthy carriers (with 1 pathogenic mutation), as well as with homozygous or compound heterozygous patients (with 2 pathogenic mutations), to identify differential molecular signatures and potential diagnostic pathways. METHODS:Peripheral blood mononuclear cells (PBMCs) from 10 patients with FMF (phenotypic carriers/homozygotes) and 5 healthy MEFV mutation carriers were isolated during asymptomatic, treatment-naïve phases. Transcriptome profiling employed globin mRNA-depleted, strand-specific Qiaseq libraries sequenced on Illumina NextSeq 500/550 (paired-end). Differential expression analysis applied TMM (trimmed mean of M values) based-normalised negative binomial models (|log₂FC| > 1, adjusted *P* < .01), with Reactome pathway enrichment. For immunoblotting, interferon (IFN)-α-stimulated monocytes/PBMCs of healthy individuals were lysed, denatured, and probed with antibodies targeting key proteins (IRF-3, IFN-stimulated gene 15 [ISG15], Pyrin, STAT1, AIM2, caspase-5, β-actin). CXCL10 levels were quantified using Luminex. RESULTS:PBMC profiling revealed 147 differentially expressed genes. Pathway analyses highlighted enrichment in type I IFN signalling and inflammasome-related pathways, with marked upregulation of type I ISGs: ISG15, IFIT2, STAT1, and the inflammasome sensor Pyrin encoded by the MEFV gene, mutated in FMF. Moreover, functional assays demonstrated that type I IFN stimulation increases Pyrin protein levels in PBMCs and isolated monocytes, revealing cross-talk between IFN responses and inflammasome signalling. CONCLUSIONS:These findings suggest that type I IFN signalling acts as a critical 'second hit', amplifying Pyrin expression in heterozygous individuals and enabling disease manifestation despite a single MEFV mutation. This study offers an explanation for the much-debated issue of the carriers expressing disease phenotypes in diseases such as FMF and presents novel insights for precision diagnosis and therapeutic intervention.

Skeletal dysplasias, characterized by bone, cartilage, and connective tissue abnormalities, often arise due to disruptions in extracellular matrix (ECM) dynamics and growth factor-dependent signaling pathways. RSPRY1, a secreted protein with RING and SPRY domains, has been implicated in bone development, yet its exact role remains to be determined. RSPRY1 gene mutations are associated with spondyloepimetaphyseal dysplasia (SEMD), a rare skeletal disorder characterized by severe epiphyseal and metaphyseal deformities. This study aimed to determine the molecular and cellular mechanisms by which RSPRY1 deficiency affects skeletal homeostasis. Transcriptome analysis of fibroblasts from patients with homozygous RSPRY1 mutations showed there was significant enrichment of transforming growth factor beta (TGF-β) signaling and ECM-related pathways. Functional wound healing assays showed that RSPRY1 knockout fibroblasts exhibited enhanced motility, a phenotype that was abrogated in RSPRY1 + SMAD3 double knockout fibroblasts, highlighting the SMAD3-dependence of RSPRY1′s effects. The observed limited response to exogenous TGF-β in RSPRY1-deficient cells indicated that there was constitutive pathway activation. These findings show that RSPRY1 is a critical regulator of TGF-β signaling in ECM dynamics and cell motility, contributing to the pathophysiology of SEMD. An improvement in our understanding of the molecular roles of RSPRY1 might yield novel therapeutic strategies that target TGF-β signaling in patients with SEMD and other skeletal dysplasias.

Retinal dystrophies are a common health problem worldwide that are currently incurable due to the inability of retinal cells to regenerate. Inherited retinal diseases (IRDs) are a diverse group of disorders characterized by progressive vision loss caused by photoreceptor cell dysfunction. The eye has always been an attractive organ for the development of novel therapies due to its independent access to the systemic pathway. Moreover, anti-sense oligonucleotides (ASOs), which facilitate manipulation of unwanted mRNAs via degradation or splicing, are undergoing rapid development and have been clinically deployed for the treatment of several diseases. The primary aim of this study was to establish a reliable in vitro model utilizing induced photoreceptor-like cells (PRCs) for assessing the efficacy and safety of ASOs targeting the BEST1 gene. Despite advances in gene therapy, effective treatments for a broad range of IRDs remain limited. An additional aim was to develop an in vitro model for evaluating RNA-based therapeutics, specifically ASOs, for the treatment in IRDs.Firstly, a cell culture model was established by induction of PRCs from dermal fibroblasts via direct programming. The induced PRCs were characterized at both the transcriptomic and protein level. Then, a common single nucleotide polymorphism (SNP) was identified in the BEST1 gene (rs1800007) for targeting with ASOs. ASOs were designed using the GapmeR strategy to target multiple alleles of this SNP, which is potentially suitable for a large proportion of the population. The efficacy and possible off-target effects of these ASOs were also analyzed in the induced PRC model.The findings show that the selected ASOs achieved allele-specific mRNA degradation with virtually no off-target effects on the global transcriptome profile, indicating their potential as safe and effective therapeutic agents. The presented in vitro model is a valuable platform for testing personalized IRD treatments and should inspire further research on RNA-based therapeutics. To the best of our knowledge this study is the first to test RNA-based therapeutics involving the use of ASOs in an induced PRC model. Based on the present findings, it will be possible to establish an ex vivo disease model using dermal fibroblast samples from affected individuals. In other words, the disease model and the ASOs that were successfully designed in this study can serve as a useful platform for the testing of personalized treatments for IRDs.

BackgroundRett syndrome (RTT) is a rare neurodevelopmental disorder that primarily affects females and is characterized by a period of normal development followed by severe cognitive, motor, and communication impairment. The syndrome is predominantly caused by mutations in the MECP2. This study aimed to use comprehensive multi-omic analysis to identify the molecular and metabolic alterations associated with Rett syndrome.Methods and resultsTranscriptomic and metabolomic profiling was performed using neuron-like cells derived from the fibroblasts of 3 Rett syndrome patients with different MECP2 mutations (R168X, P152R, and R133C) and 1 healthy control. Differential gene expression, alternative splicing events, and metabolite changes were analyzed to identify the key pathways and processes affected in patients with Rett syndrome. Transcriptomic analysis showed there was significant down-regulation of genes associated with the extracellular matrix (ECM) and cytoskeletal components, which was particularly notable in patient P3 (R133C mutation), who had non-random X chromosome inactivation. Additionally, significant changes in microtubule-related gene expression and alternative splicing events were observed, especially in patient P2 (P152R mutation). Metabolomic profiling showed that there were alterations in metabolic pathways, particularly up-regulation of ketone body synthesis and degradation pathways, in addition to an increase in free fatty acid levels. Integrated analysis highlighted the interplay between structural gene down-regulation and metabolic shifts, underscoring the adaptive responses to cellular stress in Rett neurons.ConclusionThe present findings provide valuable insights into the molecular and metabolic landscape of Rett syndrome, emphasizing the importance of combining omic data to enlighten the molecular pathophysiology of this syndrome.

Skeletal ciliopathies constitute a subgroup of ciliopathies characterized by various skeletal anomalies arising from mutations in genes impacting cilia, ciliogenesis, intraflagellar transport process, or various signaling pathways. Short-rib thoracic dysplasias, previously known as Jeune asphyxiating thoracic dysplasia (ATD), stand out as the most prevalent and prototypical form of skeletal ciliopathies, often associated with semilethality. Recently, pathogenic variants in GRK2, a subfamily of mammalian G protein-coupled receptor kinases, have been identified as one of the underlying causes of Jeune ATD. In this study, we report a new patient with Jeune ATD, in whom exome sequencing revealed a novel homozygous GRK2 variant, and we review the clinical features and radiographic findings. In addition, our findings introduce Morgagni hernia and an organoaxial-type rotation anomaly of the stomach and midgut malrotation for the first time in the context of this recently characterized GRK2-related skeletal ciliopathy.

The combination of gelatin and hydroxyapatite (HA) has emerged as a promising strategy in dental tissue engineering due to its favorable biocompatibility, mechanical properties, and ability to support cellular activities essential for tissue regeneration, rendering them ideal components for hard tissue applications. Besides, precise control over interconnecting porosity is of paramount importance for tissue engineering materials. Conventional methods for creating porous scaffolds frequently encounter difficulties in regulating pore size distribution. This study demonstrates the fabrication of gelatin-nano HA scaffolds with uniform porosity using a T-type junction microfluidic device in a single-step process. Significant improvements in control over the pore size distribution are achieved by regulating the flow parameters, resulting in effective and time-efficient manufacturing comparable in quality to the innovative 3D bioprinting techniques. The overall porosity of the scaffolds exceeded 60%, with a remarkably narrow size distribution. The incorporation of nano-HAinto 3D porous gelatin scaffolds successfully induced osteogenic differentiation in stem cells at both the protein and gene levels, as evidenced by the significant increase in osteocalcin (OCN), an important marker of osteogenic differentiation. The OCN levels are 26 and 43 times higher for gelatin and gelatin-HA scaffolds, respectively, compared to the control group.

Objective: Chondrocyte inflammation is a critical factor in degenerative joint diseases, such as osteoarthritis (OA), significantly impairing quality of life through chronic pain and limited mobility. Genetic predisposition is recognized as a critical factor in the progression of chondrocyte inflammation and OA, with particular focus on the role of genetic variants in the expression and regulation of inflammatory mediators. This study aimed to obtain information about the potential roles of a few genes containing ELF3-associated SNPs in the pathogenesis of chondrocyte inflammation. Materials and Methods: GVAT Database was used to select top candidate SNPs associated with ELF3, a cardinal transcription factor in chondrocyte inflammation. Inflammation was induced by IL-1β treatment in differentiated chondrocytes to analyze gene expression patterns. Transcriptome analysis was done by RNA sequencing. Results: The most important SNPs that could potentially affect the binding affinity of ELF3 transcription factor were analyzed. As a result of the analysis, 52% of ELF3-associated SNPs were found in protein-coding regions, 40% in gene-free intergenic regions, and 8% in non-coding RNA sequences. Some of these SNPs are located in regulatory regions (enhancers). A significant increase in expression levels in the ELF3 gene was detected after IL-1β administration, indicating that IL-1β promotes the activity of this transcription factor. mRNA expressions of TLN2, BABAM2, PEPD, and NUDT5 were also increased after IL-1β stimulation. Conclusion: The presence of ELF3-associated SNPs in the enhancer sequences of TLN2 and BABAM2 genes, in addition to the increased expression of these two genes upon IL-1β stimulation, suggested that TLN2 and BABAM2 genes may be associated with the severity of chondrocyte inflammation and OA.

Introduction: Gorlin syndrome is a rare, autosomal dominant multi-systemic disorder with a predisposition to the development of cancers such as medulloblastoma and nevoid basal cell carcinoma. Heterozygous pathogenic variants in PTCH1 are responsible for 90% of Gorlin syndrome cases. Pathogenic variants in PTCH1 cause overstimulation of the sonic hedgehog signaling pathway, which plays a role in the development of embryonic structures and tumorigenesis. Clinical major and minor diagnostic criteria for Gorlin syndrome have been determined. Odontogenic keratocyst (OKC) is the most common reason for medical admission in Gorlin syndrome. In this article, it is aimed to draw attention to the fact that patients with Gorlin syndrome are not very rare in our country and the variability in phenotypic and dysmorphic findings may be a clue for the diagnosis. Methods: Exome sequencing was performed on the Illumina NextSeq550 System platform by using the Ion Ampliseq exome RDY kit for Illumina. Sanger sequencing was performed accordingly for the other affected individuals in both families. Results: In this study, the clinical and molecular findings of 9 Gorlin syndrome patients from three unrelated families are presented. Macrocephaly, calcification of falx cerebri, palmar-plantar pits, rib anomalies, and OKC were detected in decreasing order in more than half of the patients. A novel heterozygous frameshift PTCH1 variant in family 1, a nonsense previously reported PTCH1 variant in family 2, and a novel heterozygous splice-site PTCH1 variant in family 3 were detected. Conclusion: Gorlin syndrome should be kept in mind in patients presenting with macrocephaly, palmoplantar pits, and OKC history. Careful examination of all family members is essential in the timely diagnosis of other affected individuals with minor phenotypic findings.

The differentiation of mesenchymal stem cells (MSCs) into chondrocytes, known as chondrogenesis, is a complex process that plays a fundamental role in cartilage formation and skeletal development. This study elucidates the transcriptional dynamics and phenotypic correlations at various stages of chondrogenesis (early, mid, and late) using RNA-seq data. We focused on the differential expression of transcription factors (TFs) and RNA-binding proteins (RBPs). We identified critical genes during their highest expression periods and generated heatmaps to visualize these temporal patterns. Additionally, we conducted a comprehensive analysis of skeletal dysplasia nosology genes, determining their highest expression periods and phenotypic implications using the DisGeNET database. Our findings reveal that early-stage (D1) gene expression is linked to craniofacial development and limb formation anomalies, primarily involving genes responsible for extracellular matrix (ECM) organization and signal transduction. Mid-stage (D7) genes are associated with cartilage matrix composition and skeletal growth, highlighting roles in chondrocyte proliferation and matrix deposition. Late-stage (D21) genes are implicated in bone mineral density, cartilage integrity, and joint formation, ensuring the maturation and functionality of cartilage tissue. This study provides a detailed analysis of gene expression regulators and their phenotypic correlations during chondrogenesis, offering insights into the molecular mechanisms driving cartilage development and skeletal dysplasias. Understanding these temporal gene expression patterns enhances our knowledge of chondrogenesis and aids in developing targeted therapies for cartilage-related diseases. These findings underscore the significance of time-point analyses in capturing the dynamic regulation of gene expression throughout the differentiation process.

AbstractThis work explores the application of Allium sativum (Garlic) extract, in the creation of novel polymeric core‐sheath fibers for wound therapy applications. The core‐sheath pressurized gyration (CS PG) technology is utilized to mass‐produce fibers with a polycaprolactone (PCL) core and a polyethylene oxide (PEO) sheath, loaded with garlic extract. The produced fibers maintain structural integrity, long‐term stability and provide a cell‐friendly surface with rapid antibacterial activity. The physical properties, morphology, therapeutic delivery, cytotoxicity, thermal and chemical stability of PCL, PEO, PEO/Garlic, Core‐Sheath (CS) PEO/PCL and PEO/Garlic/PCL fibers are analyzed. Findings show that the addition of garlic extract greatly increases the fibers’ thermal durability, while decreasing their diameter, thus improving cell adhesion and proliferation. In‐vitro release tests reveal a rapid release of garlic extract, which has significant antibacterial action against both Gram‐negative Escherichia coli (E. coli) and Gram‐positive Staphylococcus aureus (S. aureus) bacteria species. Cell viability experiments validate the fiber samples' biocompatibility and nontoxicity, making them appropriate for integrative medicine applications. These core‐sheath structures emphasize the potential of combining natural therapeutic agents with advanced material technologies to develop cost‐effective, sustainable and highly effective wound dressings, offering a promising solution to the growing concerns associated with conventional synthetic antibacterial agents.

Placental transmogrification of the lung (PTL) is a rare pulmonary condition characterized by the presence of immature placental villous structures. The etiology and molecular mechanisms underlying this disease remain largely unknown. This functional study aimed to identify the molecular signatures in the pathogenesis of PTL via comprehensive transcriptome analysis. Comparative transcriptomic assessment of PTL tissue and stromal cells showed differential expression of 257 genes in PTL tissue and 189 genes in stromal cells. Notably, several transcription factors and regulators, including FOSB, FOS, JUN, and ATF3, were upregulated in PTL tissue. Additionally, genes associated with the extracellular matrix and connective tissue, such as COL1A1, MMP2, and SPARC, were significantly altered, indicating possible fibrotic changes. Gene set enrichment analysis highlighted the role of vascular development and extracellular matrix organization, and the Activator Protein-1 (AP-1) transcription factor was significantly activated in PTL tissue. Furthermore, the analysis highlighted an overlap of 25 genes between PTL tissue and stromal cells, underscoring the importance of shared molecular pathways in the pathogenesis of PTL. Among the shared genes, JUND, COL4A2, COL6A2, IGFBP5, and IGFBP7 were consistently upregulated, highlighting the possible involvement of AP-1-mediated signaling and fibrotic changes in the pathogenesis of PTL. The present findings pave the way for further research into the molecular mechanisms underlying PTL and offer novel insights for therapeutic interventions. Given the rarity of PTL, these molecular findings represent a significant step forward in our understanding this enigmatic disease.

Resistance to immunity is associated with the selection of cancer cells with superior capacities to survive inflammatory reactions. Here, we tailored an ex vivo immune selection model for acute myeloid leukemia (AML) and isolated the residual subpopulations as “immune-experienced” AML (ieAML) cells. We confirmed that upon surviving the immune reactions, the malignant blasts frequently decelerated proliferation, displayed features of myeloid differentiation and activation, and lost immunogenicity. Transcriptomic analyses revealed a limited number of commonly altered pathways and differentially expressed genes in all ieAML cells derived from distinct parental cell lines. Molecular signatures predominantly associated with interferon and inflammatory cytokine signaling were enriched in the AML cells resisting the T-cell-mediated immune reactions. Moreover, the expression and nuclear localization of the transcription factors c-MYB and KLF6 were noted as the putative markers for immune resistance and identified in subpopulations of AML blasts in the patients’ bone marrow aspirates. The immune modulatory capacities of ieAML cells lasted for a restricted period when the immune selection pressure was omitted. In conclusion, myeloid leukemia cells harbor subpopulations that can adapt to the harsh conditions established by immune reactions, and a previous “immune experience” is marked with IFN signature and may pave the way for susceptibility to immune intervention therapies.

Ion channels gated selectively by mechanical stimulus are the key elements of mechanosensation. Several genes have been associated with putative mechanosensitive ion channels or mechanosensitive channel complexes. Transmembrane channel (TMC)-like protein is one of those candidate proteins that have been explored in mammals and several invertebrates. The presence and possible function of TMC related genes has not been investigated yet in crustaceans. In the present work an mRNA coding TMC-like protein was firstly cloned in Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae) and expressed in HEK293T cells. Three-dimensional structural calculations of the protein predicted a channel. Functional studies, however, indicated that the mechanosensitivity of the transfected HEK293T cells is similar to that in the control cells. It was concluded that a TMC-like protein is present in the crayfish but future studies are necessary to define its function.

Abstract Ion channels gated selectively by mechanical stimulus are the key elements of mechanosensation. Several genes have been associated with putative mechanosensitive ion channels or mechanosensitive channel complexes. Transmembrane channel (TMC)-like protein is one of those candidate proteins that have been explored in mammals and several invertebrates. The presence and possible function of TMC related genes has not been investigated yet in crustaceans. In the present work an mRNA coding TMC-like protein was firstly cloned in Astacus leptodactylus (Eschscholtz, 1823) (Decapoda: Astacidea: Astacidae) and expressed in HEK293T cells. Three-dimensional structural calculations of the protein predicted a channel. Functional studies, however, indicated that the mechanosensitivity of the transfected HEK293T cells is similar to that in the control cells. It was concluded that a TMC-like protein is present in the crayfish but future studies are necessary to define its function.