Genomic sequencing for newborn screening: current perspectives and challenges.

Traditional newborn screening (NBS) serves as a critical tool in identifying conditions that may impact a child's health from an early stage. Newborn sequencing (NBSeq), the comprehensive analysis of an infant's genome, holds immense promise for revolutionizing health care throughout the lifespan. NBSeq allows for early detection of genetic disease risk and precision personalized medicine. The rapid evolution of DNA sequencing technologies and increasing affordability have spurred numerous endeavors to explore the potential of whole-genome sequencing in newborn screening. However, this transformative potential cannot be realized without challenges. Ethical aspects must be carefully navigated to safeguard individual rights and maintain public trust. Moreover, genomic data interpretation poses complex challenges due to its amount, the presence of variants of uncertain significance, and the dynamic nature of our understanding of genetics. Implementation hurdles, including cost, infrastructure, and specialized expertise, also present barriers to the widespread adoption of NBSeq. Addressing these challenges requires collaboration among clinicians, researchers, policymakers, ethicists, and stakeholders across various sectors. Robust frameworks for informed consent, data protection, and governance are essential. Advances in bioinformatics, machine learning, and genomic interpretation are crucial for translation into actionable clinical insights. Scalability and improving downstream health care access are vital for equitability, particularly in underserved communities. By fostering interdisciplinary collaboration, advancing technology and infrastructure, and upholding ethical principles, we can unlock the full potential of NBSeq as a tool for precision medicine and pave the way toward a future where every child has the opportunity for a healthier, genomics-informed start to life.

Tissue engineering and future directions in regenerative medicine for knee cartilage repair: a comprehensive review.

This review evaluates the current landscape and future directions of regenerative medicine for knee cartilage repair, with a particular focus on tissue engineering strategies. In this context, scaffold-based approaches have emerged as promising solutions for cartilage regeneration. Synthetic scaffolds, while offering superior mechanical properties, often lack the biological cues necessary for effective tissue integration. Natural scaffolds, though biocompatible and biodegradable, frequently suffer from inadequate mechanical strength. Hybrid scaffolds, combining elements of both synthetic and natural materials, present a balanced approach, enhancing both mechanical support and biological functionality. Advances in decellularized extracellular matrix scaffolds have shown potential in promoting cell infiltration and integration with native tissues. Additionally, bioprinting technologies have enabled the creation of complex, bioactive scaffolds that closely mimic the zonal organization of native cartilage, providing an optimal environment for cell growth and differentiation. The review also explores the potential of gene therapy and gene editing techniques, including CRISPR-Cas9, to enhance cartilage repair by targeting specific genetic pathways involved in tissue regeneration. The integration of these advanced therapies with tissue engineering approaches holds promise for developing personalized and durable treatments for knee cartilage injuries and osteoarthritis. In conclusion, this review underscores the importance of continued multidisciplinary collaboration to advance these innovative therapies from bench to bedside and improve outcomes for patients with knee cartilage damage.

Implementing Whole Genome Sequencing (WGS) in Clinical Practice: Advantages, Challenges, and Future Perspectives.

The integration of whole genome sequencing (WGS) into all aspects of modern medicine represents the next step in the evolution of healthcare. Using this technology, scientists and physicians can observe the entire human genome comprehensively, generating a plethora of new sequencing data. Modern computational analysis entails advanced algorithms for variant detection, as well as complex models for classification. Data science and machine learning play a crucial role in the processing and interpretation of results, using enormous databases and statistics to discover new and support current genotype-phenotype correlations. In clinical practice, this technology has greatly enabled the development of personalized medicine, approaching each patient individually and in accordance with their genetic and biochemical profile. The most propulsive areas include rare disease genomics, oncogenomics, pharmacogenomics, neonatal screening, and infectious disease genomics. Another crucial application of WGS lies in the field of multi-omics, working towards the complete integration of human biomolecular data. Further technological development of sequencing technologies has led to the birth of third and fourth-generation sequencing, which include long-read sequencing, single-cell genomics, and nanopore sequencing. These technologies, alongside their continued implementation into medical research and practice, show great promise for the future of the field of medicine.

Population Pharmacogenomics in Croatia: Evaluating the PGx Allele Frequency and the Impact of Treatment Efficiency.

BACKGROUND: Adverse drug reactions (ADRs) are a significant cause of mortality, and pharmacogenomics (PGx) offers the potential to optimize therapeutic efficacy while minimizing ADRs. However, there is a lack of data on the Croatian population, highlighting the need for investigating the most common alleles, genotypes, and phenotypes to establish national guidelines for drug use. METHODS: A single-center retrospective cross-sectional study was performed to examine the allele, genotype, and phenotype frequencies of drug-metabolizing enzymes, receptors, and other proteins in a random sample of 522 patients from Croatia using a 28-gene PGx panel. RESULTS: Allele frequencies, genotypes, and phenotypes for the investigated genes were determined. No statistically significant differences were found between the Croatian and European populations for most analyzed genes. The most common genotypes observed in the patients resulted in normal metabolism rates. However, some genes showed higher frequencies of altered metabolism rates. CONCLUSIONS: This study provides insights into the allele, genotype, and phenotype frequencies of drug-metabolizing enzymes, receptors, and other associated proteins in the Croatian population. The findings contribute to optimizing drug use guidelines, potentially reducing ADRs, and improving therapeutic efficacy. Further research is needed to tailor population-specific interventions based on these findings and their long-term benefits.

Novel Approach in Rectovaginal Fistula Treatment: Combination of Modified Martius Flap and Autologous Micro-Fragmented Adipose Tissue.

In this paper, we introduce an innovative therapeutic approach for managing rectovaginal fistulas (RVF), by combining the modified Martius flap and micro-fragmented adipose tissue (MFAT) enriched with mesenchymal stem cells (MSC). This novel approach aims to deal with the difficulties associated with RVF, a medically complex condition with a lack of effective treatment options. We present the case of a 45-year-old female patient with a 15-year history of Crohn's disease (CD). During the preceding eight years, she had encountered substantial difficulties resulting from a rectovaginal fistula (RVF) that was active and considerable in size (measuring 3.5 cm in length and 1 cm in width). Her condition was accompanied by tissue alterations at both the vaginal and rectal openings. Following her admission to our hospital, the patient's case was discussed during both surgical and multidisciplinary hospital team (IRB) meetings. The team decided to combine a modified Martius flap with autologous MFAT containing MSCs. The results were remarkable, leading to comprehensive anatomical and clinical resolution of the RVF. Equally significant was the improvement in the patient's overall quality of life and sexual satisfaction during the one-year follow-up period. The integration of the modified Martius flap with MFAT emerges as a highly promising approach for addressing CD-related RVFs that had historically been, and still are, difficult to treat, given their often refractory nature and low healing success rates.

Case report: State-of-the-art risk-modifying treatment of sudden cardiac death in an asymptomatic patient with a mutation in the SCN5A gene and a review of the literature.

Brugada syndrome is a rare hereditary disorder characterized by distinct ECG findings, complex genetics, and a high risk of sudden cardiac death. Recognition of the syndrome is crucial as it represents a paradigm of sudden death tragedy in individuals at the peak of their lives. Notably, Brugada syndrome accounts for more than 20% of sudden cardiac deaths in individuals with structurally normal hearts. Although this syndrome follows an autosomal dominant inheritance pattern, it is more prevalent and severe in males. Diagnosis is primarily based on the characteristic ECG pattern observed in the right precordial leads. Mutations in the SCN5A gene, resulting in loss of function, are the most common genetic cause. We presented a 36-year-old proband with a family history of sudden cardiac death. Although the patient was asymptomatic for Brugada syndrome, his father had experienced sudden death at the age of 36. The proband was admitted to St. Catherine's Specialty Hospital where blood was taken and subjected to next-generation sequencing (NGS) using a "Sudden cardiac death" panel. The analysis identified a pathogenic variant in the SCN5A gene [c.4222G > A(p.Gly1408Arg)], which is associated with autosomal dominant Brugada syndrome. Based on the positive genetic test result, the patient was referred for further examination. ECG with modified precordial lead positioning confirmed the presence of the Brugada phenotype, displaying the type-2 and type-1 ECG patterns. Therefore, we made the diagnosis and decided to implant an implantable cardioverter-defibrillator (ICD) based on the results of broad genetic NGS testing, diagnostic criteria (ECG), and considering the high burden of sudden cardiac death in the patient's family, as well as his concerns that limited his everyday activities. This case shows that genetics and personalized medicine hold immense potential in the primary prevention, diagnosis, and treatment of Brugada syndrome and sudden cardiac death.

Unlocking the Potential of Mesenchymal Stem Cells in Gynecology: Where Are We Now?

Stem cells, with their remarkable capacity for differentiation into diverse cell types, are vital for the development as well as maintenance of health and homeostasis. Two unique abilities set them apart from other cells: self-renewal and the capacity for differentiation. They play important roles in embryogenesis, development, regeneration, and various other processes. Over the last decade, there has been increased interest in their potential use in the treatment of numerous diseases and disorders across multiple fields of medicine in acute, chronic, innate, and acquired diseases. Stem cells are key to maintaining the body's homeostasis and regulating growth and tissue functions. There are several types of stem cells-embryonic, adult, and human-induced pluripotent cells. Currently, mesenchymal stem cells are of great interest due to their regenerative, immunomodulatory, analgesic, and antimicrobial (anti-inflammatory) effects. Recent studies have shown the potent regenerative effect of stem cell therapy in gynecologic diseases such as infertility, Asherman syndrome, lichen sclerosus, polycystic ovary syndrome, premature ovarian insufficiency, genitourinary syndrome of menopause, and rectovaginal fistulas. Moreover, the successful isolation of oogonial stem cells could lead to a revolution in the field of gynecology and the potential treatment of the conditions discussed. This review aims to provide a better understanding of the latest therapeutic options involving stem cells and raise awareness of this promising yet not widely known topic in gynecology and medicine in general.

Successful Treatment of a Rare Cholesterol Homeostasis Disorder Due to CYP27A1 Gene Mutation with Chenodeoxycholic Acid Therapy.

Cerebrotendinous xanthomatosis (CTX) is a genetic disorder of the cholesterol metabolic pathway, most often associated with variants in the CYP27A1 gene. The dysregulation of cholesterol metabolism results in the accumulation of metabolites such as cholestanol, which has a predilection for neuronal tissue and tendons. The condition is treatable with chenodeoxycholic acid (CDCA), which halts the production of these metabolites. We present two adult brothers, without diagnosis, suffering from ataxia, general muscle weakness and cognitive deficits. Both brothers suffered from early onset cataracts, watery stools and thoracic kyphoscoliosis. Magnetic resonance imaging revealed hyperintense alterations in the central nervous system and intratendinous xanthomas in the Achilles tendons. A biochemical analysis showed elevated levels of cholestanol, lathosterol and 7-dehydrocholesterol. Their family history was negative for neurological and metabolic disorders. Genetic testing revealed a pathogenic CYP27A1 variant (c.1184+1G>A) in both brothers, confirming the diagnosis. The patients were started on CDCA therapy and have shown significant improvement at their follow-up examinations. Early diagnosis and treatment initiation in CTX patients is of great importance, as the significant reversal of disease progression can be achieved. For this reason, clinical genetic testing is necessary when it comes to patients with an onset of cataracts, chronic diarrhea, and neurological symptoms in early childhood.

Importance of Cellular Immunity and IFN-γ Concentration in Preventing SARS-CoV-2 Infection and Reinfection: A Cohort Study.

Recent studies have highlighted the underestimated importance of the cellular immune response after the emergence of variants of concern (VOCs) of SARS-CoV-2, and the significantly reduced neutralizing power of antibody titers in individuals with previous SARS-CoV-2 infection or vaccination. Our study included 303 participants who were tested at St. Catherine Specialty Hospital using the Quan-T-Cell SARS-CoV-2 in combination with the Quan-T-Cell ELISA (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany) for the analysis of IFN-γ concentration, and with Anti-SARS-CoV-2 QuantiVac ELISA IgG (Euroimmun Medizinische Labordiagnostika, Lübeck, Germany) for the detection of human antibodies of the immunoglobulin class IgG against the S1 domain of the SARS-CoV-2 spike protein. The statistical analysis showed a significant difference in the concentration of IFN-γ between reinfected participants and those without infection (p = 0.012). Participants who were not infected or reinfected with SARS-CoV-2 after vaccination and/or previous SARS-CoV-2 infection had a significantly higher level of cellular immunity. Furthermore, in individuals without additional vaccination, those who experienced infection/reinfection had significantly lower levels of IFN-γ compared to uninfected participants (p = 0.016). Our findings suggest a long-lasting effect of cellular immunity, measured by IFN-γ concentrations, which plays a key role in preventing infections and reinfections after the emergence of SARS-CoV-2 variants of concern.

Autologous Platelet-Rich Plasma (PRP) for Treating Androgenetic Alopecia: A Novel Treatment Protocol Standardized on 2 Cases.

Platelet-rich plasma (PRP) treatment has emerged in recent years as a valuable, effective, and affordable treatment for androgenetic alopecia. Androgenetic alopecia is the most common type of alopecia, affecting both men and women, and is characterized by diminished hair follicles mainly pronounced in the frontal region and vertex. A considerable variety of PRP treatment regimens have been described so far, but there is no consensus on the standardization of PRP preparation or administration protocol. Our study was conducted on two patients to test the efficacy of a new PRP application protocol of only two treatments by using a combination of a PRP collecting device and a conventional kit. Efficacy of treatment was assessed after a 6-month follow-up by artificial intelligence (AI)-driven software on microscopic images of treated regions. An average number of hairs, cumulative hair thickness, and the number of follicular units increased in the vertex region of both patients by 30/59%, 35/53%, and 14/48%, respectively. The novel treatment regimen showed significant effectiveness in only six months.

Mesenchymal Stem Cell Mechanisms of Action and Clinical Effects in Osteoarthritis: A Narrative Review.

With the insufficient satisfaction rates and high cost of operative treatment for osteoarthritis (OA), alternatives have been sought. Furthermore, the inability of current medications to arrest disease progression has led to rapidly growing clinical research relating to mesenchymal stem cells (MSCs). The availability and function of MSCs vary according to tissue source. The three primary sources include the placenta, bone marrow, and adipose tissue, all of which offer excellent safety profiles. The primary mechanisms of action are trophic and immunomodulatory effects, which prevent the further degradation of joints. However, the function and degree to which benefits are observed vary significantly based on the exosomes secreted by MSCs. Paracrine and autocrine mechanisms prevent cell apoptosis and tissue fibrosis, initiate angiogenesis, and stimulate mitosis via growth factors. MSCs have even been shown to exhibit antimicrobial effects. Clinical results incorporating clinical scores and objective radiological imaging have been promising, but a lack of standardization in isolating MSCs prevents their incorporation in current guidelines.

Experience with comprehensive pharmacogenomic multi-gene panel in clinical practice: a retrospective single-center study.

AIM: To assess the prevalence of actionable pharmacogenetic interventions in patients who underwent pharmacogenetic testing with a multi-gene panel. METHODS: We retrospectively reviewed single-center electronic health records. A total of 319 patients were enrolled who underwent pharmacogenomic testing with the RightMed test panel using TaqMan quantitative real-time PCR method and copy number variation analysis to determine the SNPs in the 27 target genes. RESULTS: Actionable drug-gene pairs were found in 235 (73.7%) patients. Relevant guidelines on genotype-based prescribing were available for 133 (56.7%) patients at the time of testing. Based on the patients' genotype, 139 (43.6%) patients were using at least one drug with significant pharmacogenetic interactions. CONCLUSION: Two out of three patients had at least one drug-gene pair in their therapy. Further studies should assess the clinical effectiveness of integrating pharmacogenomic data into patients' electronic health records.

Medicinal signaling cells niche in stromal vascular fraction from lipoaspirate and microfragmented counterpart.

AIM: To expand our previous findings by increasing the number of patients in a study characterizing medicinal signaling cells (MSC) of stromal vascular fraction from lipoaspirate (SVF-LA) and from microfragmented lipoaspirate (SVF-MLA) applied for the treatment of osteoarthritis (OA). METHODS: Twenty OA patients, including 8 new patients, acquiring autologous microfragmented adipose tissue were enrolled. In-parallel immunophenotyping of SVF-LA and SVF-MLA was performed. The samples were incubated in a DuraClone SC prototype tube targeting the CD31, CD34, CD45, CD73, CD90, CD105, and CD146 surface markers, stained with the DRAQ7 cell nuclear dye and Live/Dead Yellow Fixable Stain, and analyzed by flow cytometry. RESULTS: The population phenotypes in SVF-LA and SVF-MLA samples included CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells. Compared with the autologous SVF-LA samples, the prevailing cell type in SVF-MLA were EP, which outnumbered leukocytes and supra-adventitial-adipose stromal cells (SA-ASC). The ratio of progenitor cells in SVF-MLA samples differed between female and male patients, showing a higher EP-pericyte and pericyte-SA-ASC ratio in men. CONCLUSION: Our results, hallmarked by EP-enriched anti-inflammatory features and indicating a possible sex-specific impact, contribute to defining the cellular composition of the clinically applied MSC serving as a regenerative cell therapy in OA.

Adaptive Immune Responses and Immunity to SARS-CoV-2.

Since the onset of the COVID-19 pandemic, the medical field has been forced to apply the basic knowledge of immunology with the most up-to-date SARS-CoV-2 findings and translate it to the population of the whole world in record time. Following the infection with the viral antigen, adaptive immune responses are activated mainly by viral particle encounters with the antigen-presenting cells or B cell receptors, which induce further biological interactions to defend the host against the virus. After the infection has been warded off, the immunological memory is developed. The SARS-CoV cellular immunity has been shown to persist even 17 years after the infection, despite the undetectable humoral component. Similar has been demonstrated for the SARS-CoV-2 T cell memory in a shorter period by assessing interferon-gamma levels when heparinized blood is stimulated with the virus-specific peptides. T cells also play an irreplaceable part in a humoral immune reaction as the backbone of a cellular immune response. They both provide the signals for B cell activation and the maturation, competence, and memory of the humoral response. B cell production of IgA was shown to be of significant influence in mediating mucosal immunity as the first part of the defense mechanism and in the development of nasal vaccines. Here, we interpret the recent SARS-CoV-2 available research, which encompasses the significance and the current understanding of adaptive immune activity, and compare it among naive, exposed, and vaccinated blood donors. Our recent data showed that those who recovered from COVID-19 and those who are vaccinated with EMA-approved vaccines had a long-lasting cellular immunity. Additionally, we analyze the humoral responses in immunocompromised patients and memory mediated by cellular immunity and the impact of clonality in the SARS-CoV-2 pandemic regarding breakthrough infections and variants of concern, both B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants.

Cellular Immunity-The Key to Long-Term Protection in Individuals Recovered from SARS-CoV-2 and after Vaccination.

Previous clinical and epidemiological studies have shown that over time antibody titers decrease, and they do not provide long-term mucosa protection against SARS-CoV-2 infection. Additionally, the increase in breakthrough infections that occur more frequently in the vaccinated than in the study participants with previous SARS-CoV-2 infection has recently become a priority public health concern. We measured the amount of interferon-gamma (Quan-T-Cell ELISA) and the level of antibodies (Anti-SARS-CoV-2 QuantiVac ELISA IgG) in the blood of the same patients simultaneously to compare cellular and humoral immunity. A total of 200 study participants (before Omicron variant appearance) were divided into four groups whose levels of cellular and humoral immunity we compared: study participants previously infected with SARS-CoV-2 (group 1); study participants vaccinated with EMA-approved vaccines (group 2); study participants previously infected with SARS-CoV-2, and vaccination history (group 3); and study participants without a history of SARS-CoV-2 infection or vaccination (group 4). Our results showed that study participants who received one of the EMA-approved vaccines and who recovered from COVID-19 (group 3) had significantly higher levels of cellular immunity and antibody titers in comparison with groups 1 and 2. Additionally, we have noticed that the study participants previously infected with SARS-CoV-2 and the study participants vaccinated with EMA-approved vaccines had a long-lasting cellular immunity. Furthermore, antibody levels showed a negative correlation with time since the last contact with a viral antigen, while cellular immunity within 20 months showed as long-term protection. Moreover, out of 200 study participants, only 1 study participant who recovered from COVID-19 (0.5%) was re-infected, while a total of 6 study participants (3%) were infected with SARS-CoV-2 after receiving the vaccine. This study suggests that cellular immunity-unlike humoral immunity, thanks to memory T cells-represents long-term protection in individuals recovered from SARS-CoV-2 and after vaccination.

Results of Treating Mild to Moderate Knee Osteoarthritis with Autologous Conditioned Adipose Tissue and Leukocyte-Poor Platelet-Rich Plasma.

Knee osteoarthritis (KOA) is one of the most common musculoskeletal disorders. Much progress has been made in regenerative medicine for the symptomatic treatment of KOA, including products containing stromal vascular fraction (SVF) and platelet-rich plasma (PRP). The aim of this study was to evaluate clinical and radiological findings after the application of autologous conditioned adipose tissue (ACA) and leukocyte-poor PRP (LP-PRP) in patients with mild to moderate KOA. A total of 16 patients (eight male and eight female) with changes related to KOA on the magnetic resonance imaging (MRI), but without severe osteophytosis, full-thickness cartilage loss, or subchondral bone involvement were included in this study. Patients received an intraarticular, ultrasound-guided injection of ACA and LP-PRP. Clinical scores, including a visual analog scale for pain (VAS), Knee Injury and Osteoarthritis Outcome Score (KOOS), and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) were evaluated at baseline and at the three and six month follow-ups showing a statistically significant improvements at three and six months post-intervention. Furthermore, the delayed gadolinium-enhanced MRI of the cartilage (dGEMRIC) indices were evaluated at baseline and at the three and six month follow-ups showing no significant changes after treatment with ACA and LP-PRP, which were actually equal to the dGEMRIC indices measured in the control group (hyaluronic acid applied in contralateral knees without osteoarthritis). ACA with LP-PRP presents a viable minimally invasive therapeutic option for the clinical improvement of mild to moderate KOA. However, MFAT produced by different systems is likely to differ in cellular content, which can directly affect the paracrine effect (cytokine secretion) of mesenchymal stem cells and consequently the regeneration process.

X-Linked Osteogenesis Imperfecta Possibly Caused by a Novel Variant in PLS3.

Osteogenesis imperfecta (OI) represents a complex spectrum of genetic bone diseases that occur primarily due to mutations and deletions of the COL1A1 and COL1A2 genes. Recent molecular studies of the network of signaling pathways have contributed to a better understanding of bone remodeling and the pathogenesis of OI caused by mutations in many other genes associated with normal bone mineralization. In this paper, a case of a rare X-linked variant of OI with a change in the gene encoding plastin 3-a protein important for the regulation of the actin cytoskeleton, is presented. A 16-year-old patient developed ten bone fractures caused by minor trauma or injury, including a compression fracture of the second lumbar vertebra during his lifetime. Next-generation sequencing analysis did not show pathologically relevant deviations in the COL1A1 and COL1A2 genes. Targeted gene analyses (Skeletal disorder panel) of the patient, his father, mother and sister were then performed, detecting variants of uncertain significance (VUS) for genes PLS3, FN1 and COL11A2. A variant in the PLS3 gene were identified in the patient, his mother and sister. Since the PLS3 gene is located on the X chromosome, the mother and sister showed no signs of the disease. Although the variant in the PLS3 gene (c.685G>A (p.Gly229Arg)) has not yet been described in the literature, nor is its pathogenicity known, clinical findings combined with genetic testing showed that this variant may explain the cause of X-linked OI in our patient. This rare case of the PLS3 variant of X-linked OI might point to a novel target for personalized therapy in patients with this severe disease.

Polychromatic Flow Cytometric Analysis of Stromal Vascular Fraction from Lipoaspirate and Microfragmented Counterparts Reveals Sex-Related Immunophenotype Differences.

Mesenchymal stem/stromal cells or medicinal signaling cells (MSC)-based therapy holds promise as a beneficial strategy for treating knee OA (osteoarthritis), but there is no standardized protocols nor mechanistic understanding. In order to gain a better insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of the stromal vascular fraction from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry and investigated the cellular components considered responsible for cartilage regeneration. We found an enrichment of the regenerative cellular niche of the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate, which, in female patients, contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31highCD34high endothelial progenitors as well as of CD73 on supra-adventitialadipose stromal cells. Some of these MSC-expression differences were present after microfragmentation and indicated a differential phenotype pattern of the applied MSC mixture in female and male patients. Our results provide a better insight into the heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, with an emphasis on interesting differences between women and men.

Integrative cBioPortal Analysis Revealed Molecular Mechanisms That Regulate EGFR-PI3K-AKT-mTOR Pathway in Diffuse Gliomas of the Brain.

Diffuse gliomas are a heterogeneous group of tumors with aggressive biological behavior and a lack of effective treatment methods. Despite new molecular findings, the differences between pathohistological types still require better understanding. In this in silico analysis, we investigated AKT1, AKT2, AKT3, CHUK, GSK3ß, EGFR, PTEN, and PIK3AP1 as participants of EGFR-PI3K-AKT-mTOR signaling using data from the publicly available cBioPortal platform. Integrative large-scale analyses investigated changes in copy number aberrations (CNA), methylation, mRNA transcription and protein expression within 751 samples of diffuse astrocytomas, anaplastic astrocytomas and glioblastomas. The study showed a significant percentage of CNA in PTEN (76%), PIK3AP1 and CHUK (75% each), EGFR (74%), AKT2 (39%), AKT1 (32%), AKT3 (19%) and GSK3ß (18%) in the total sample. Comprehensive statistical analyses show how genomics and epigenomics affect the expression of examined genes differently across various pathohistological types and grades, suggesting that genes AKT3, CHUK and PTEN behave like tumor suppressors, while AKT1, AKT2, EGFR, and PIK3AP1 show oncogenic behavior and are involved in enhanced activity of the EGFR-PI3K-AKT-mTOR signaling pathway. Our findings contribute to the knowledge of the molecular differences between pathohistological types and ultimately offer the possibility of new treatment targets and personalized therapies in patients with diffuse gliomas.

Methylation Patterns of DKK1, DKK3 and GSK3ß Are Accompanied with Different Expression Levels in Human Astrocytoma.

In the present study, we investigated genetic and epigenetic changes and protein expression levels of negative regulators of Wnt signaling, DKK1, DKK3, and APC as well as glycogen synthase kinase 3 (GSK3ß) and ß-catenin in 64 human astrocytomas of grades II-IV. Methylation-specific PCR revealed promoter methylation of DKK1, DKK3, and GSK3ß in 38%, 43%, and 18% of samples, respectively. Grade IV comprised the lowest number of methylated GSK3ß cases and highest of DKK3. Evaluation of the immunostaining using H-score was performed for ß-catenin, both total and unphosphorylated (active) forms. Additionally, active (pY216) and inactive (pS9) forms of GSK3ß protein were also analyzed. Spearman's correlation confirmed the prevalence of ß-catenin's active form (rs = 0.634, p < 0.001) in astrocytoma tumor cells. The Wilcoxon test revealed that astrocytoma with higher levels of the active pGSK3ß-Y216 form had lower expression levels of its inactive form (p < 0.0001, Z = -5.332). Changes in APC's exon 11 were observed in 44.44% of samples by PCR/RFLP. Astrocytomas with changes of APC had higher H-score values of total ß-catenin compared to the group without genetic changes (t = -2.264, p = 0.038). Furthermore, a positive correlation between samples with methylated DKK3 promoter and the expression of active pGSK3ß-Y216 (rs = 0.356, p = 0.011) was established. Our results emphasize the importance of methylation for the regulation of Wnt signaling. Large deletions of the APC gene associated with increased ß-catenin levels, together with oncogenic effects of both ß-catenin and GSK3ß, are clearly involved in astrocytoma evolution. Our findings contribute to a better understanding of the etiology of gliomas. Further studies should elucidate the clinical and therapeutic relevance of the observed molecular alterations.