Evaluation of Vitamin D-enriched Bone Graft in Surgically-induced Critical-sized Bone Defects: An Experimental Study.

BACKGROUND: Restoration of bone defects in the craniac vault may require the use of autografts, allografts, xenografts, or synthetic grafts. There are promising data that vitamin D may play a positive role in graft incorporation. The purpose of the present study is the evaluation of the impact of vitamin D addition to human-derived bone grafts in the healing of critical-sized bone defects in porcine skulls. MATERIALS AND METHODS: Four identical critical-sized defects were created in the calvaria of 8 adult Landrace Large White pigs. The first defect was left blank as control, the second defect was filled with human-derived bone graft, the third defect was filled with human-derived bone graft enriched with a low concentration of vitamin D (2 mg/mL), and the fourth defect was filled with human-derived bone graft enriched with a high concentration of vitamin D (10 mg/mL). The animals were sacrificed after 12 weeks. Harvested tissue specimens were qualitatively evaluated by histology. New bone formation (bone volume/tissue volume) was quantitatively measured by histomorphometry. RESULTS: Signs of bone formation were evident in all bone sockets. Mean values of the bone volume/tissue volume of the 4 defects were 10.91%, 11.05%, 10.40% and 10.87% respectively, at 12 weeks. In 5 animals, high concentration of vitamin D caused a significant improvement in bone formation in relation to controls. In 3 animals, a high concentration of vitamin D was associated with decreased bone formation compared with controls. No statistical difference was observed in the graft healing among the 4 graft sites ( P > 0.05). CONCLUSIONS: The results of this study have shown that the addition of vitamin D to human-derived bone grafts does not have a significant effect on bone formation and graft incorporation in critical-sized bone defects of the porcine calvaria. Further high-quality studies are needed to fully elucidate the role of vitamin D in bone formation and bone graft union.

Cancer Associated PRDM9: Implications for Linking Genomic Instability and Meiotic Recombination.

The PR domain-containing 9 or PRDM9 is a gene recognized for its fundamental role in meiosis, a process essential for forming reproductive cells. Recent findings have implicated alterations in the PRDM9, particularly its zinc finger motifs, in the onset and progression of cancer. This association is manifested through genomic instability and the misregulation of genes critical to cell growth, proliferation, and differentiation. In our comprehensive study, we harnessed advanced bioinformatic mining tools to delve deep into the intricate relationship between PRDM9F and cancer. We analyzed 136,752 breakpoints and found an undeniable association between specific PRDM9 motifs and the occurrence of double-strand breaks, a phenomenon evidenced in every cancer profile examined. Utilizing R statistical querying and the Regioner package, 55 unique sequence variations of PRDM9 were statistically correlated with cancer, from a pool of 1024 variations. A robust analysis using the Enrichr tool revealed prominent associations with various cancer types. Moreover, connections were noted with specific phenotypic conditions and molecular functions, underlining the pervasive influence of PRDM9 variations in the biological spectrum. The Reactome tool identified 25 significant pathways associated with cancer, offering insights into the mechanistic underpinnings linking PRDM9 to cancer progression. This detailed analysis not only confirms the pivotal role of PRDM9 in cancer development, but also unveils a complex network of biological processes influenced by its variations. The insights gained lay a solid foundation for future research aimed at deciphering the mechanistic pathways of PRDM9, offering prospects for targeted interventions and innovative therapeutic approaches in cancer management.

Impact of ZBTB7A hypomethylation and expression patterns on treatment response to hydroxyurea.

BACKGROUND: We aimed to clarify the emerging epigenetic landscape in a group of genes classified as "modifier genes" of the ß-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/ß-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented. RESULTS: We examined the CpG islands' DNA methylation profile of BCL11A, KLF1, MYB, MAP3K5, SIN3A, ZBTB7A, and GATA2, along with γ-globin and LRF/ZBTB7A expression levels. In vitro treatment of hematopoietic stem cells (HSCs) with HU induced a significant DNA hypomethylation pattern in ZBTB7A (p*, 0.04) and GATA2 (p*, 0.03) CpGs exclusively in the HU non-responders. Also, this group of patients exhibited significantly elevated baseline methylation patterns in ZBTB7A, before the HU treatment, compared to HU responders (p*, 0.019) and to control group of healthy individuals (p*, 0.021), which resembles a potential epigenetic barrier for the γ-globin expression. γ-Globin expression in vitro matched with detected HbF levels during patients' monitoring tests (in vivo) under HU treatment, implying a good reproducibility of the in vitro HU epigenetic effect. LRF/ZBTB7A expression was elevated only in the HU non-responders under the influence of HU. CONCLUSIONS: This is one of the very first pharmacoepigenomic studies indicating that the hypomethylation of ZBTB7A during HU treatment enhances the LRF expression, which by its turn suppresses the HbF resumption in the HU non-responders. Its role as an epigenetic regulator of hemoglobin switching is also supported by the wide distribution of ZBTB7A-binding sites within the 5' CpG sequences of all studied human HBB cluster "modifier genes." Also, the baseline methylation level of selective CpGs in ZBTB7A and GATA2 could be an indicator of the negative HU response among the ß-type hemoglobinopathy patients.

Mechanical stress affects methylation pattern of GNAS isoforms and osteogenic differentiation of hAT-MSCs.

Mechanical stress exerts a substantial role on skeletal-cell renewal systems, whereas accumulating evidence suggests that epigenetic mechanisms induce changes and differential gene expression. Although the underlying mechanisms remain to be fully elucidated, our study suggests that the influence of the long term mechanical stimulation elicits epigenetic modifications controlling osteogenic differentiation of human adipose tissue multipotential stromal cells (hAT-MSCs) and contributes to an accelerating in vitro osteogenesis. GNAS imprinting gene acts as a critical regulator of osteoblast differentiation and is implicated in human genetic disorders with pathological formation of ectopic-skeletal bone. Investigating a wide variety of stimuli, we showed that daily mechanical stretch on hAT-MSCs of 7th and 15th days' intervals induced a significant down-regulation in DNA methylation status of critical CpG sites of NESP and GNASXL isoforms, accompanied by up-regulation of the corresponding gene transcripts, and osteogenic differentiation earlier in culture. Importantly, methylation analysis of differentiating bone marrow-derived MSCs revealed similar methylation patterns. Bioinformatic analysis further showed that all CpG islands exhibiting significant methylation alterations encompassed transcriptional repressor CTCF binding sites. We hereby emphasize the need to investigate the epigenetic alterations on hAT-MSCs during environmental mechanical forces and to consider how the knowledge gained through these studies may foster new means of symptoms prevention and management of ectopic bone formation in the clinic.

A novel splicing isoform of protein arginine methyltransferase 1 (PRMT1) that lacks the dimerization arm and correlates with cellular malignancy.

Methylation of arginine residues is an important modulator of protein function that is involved in epigenetic gene regulation, DNA damage response and RNA maturation, as well as in cellular signaling. The enzymes that catalyze this post-translational modification are called protein arginine methyltransferases (PRMTs), of which PRMT1 is the predominant enzyme. Human PRMT1 has previously been shown to occur in seven splicing isoforms, which are differentially abundant in different tissues, and have distinct substrate specificity and intracellular localization. Here we characterize a novel splicing isoform which does not affect the amino-terminus of the protein like the seven known isoforms, but rather lacks exons 8 and 9 which encode the dimerization arm of the enzyme that is essential for enzymatic activity. Consequently, the isoform does not form catalytically active oligomers with the other endogenous PRMT1 isoforms. Photobleaching experiments reveal an immobile fraction of the enzyme in the nucleus, in accordance with earlier results from our laboratory that had shown a tight association of inhibited or inactivated PRMT1 with chromatin and the nuclear scaffold. Thus, it apparently is able to bind to the same substrates as catalytically active PRMT1. This isoform is found in a variety of cell lines, but is increased in those of cancer origin or after expression of the EMT-inducing transcriptional repressor Snail1. We discuss that the novel isoform could act as a modulator of PRMT1 activity in cancer cells by acting as a competitive inhibitor that shields substrates from access to active PRMT1 oligomers.

Antiproliferative and cytotoxic action of N-(p-coumaroyl) serotonin in lung cancer cells.

PURPOSE: Lung cancer is among the leading causes of cancer-related cases and cancer-associated deaths. Tumor cells frequently acquire chemoresistance and, due to that, new therapies are always needed in the fight against cancer. Pharmaceutical plants continue to offer novel compounds as anticancer therapies. METHODS: We studied the action of N-p-coumaroyl-serotonin (CS), a natural compound from Centaurea seed and safflower on a lung adenocarcinoma cell line. Cytotoxic or antiproliferative effect was studied using the MTT assay. Cell cycle, caspase-8 activation, mitochondrial membrane potential (MMP) and expression of CD15/CD56/CD24/CD44/CD58/CD71 were studied by flow cytometry. RESULTS: CS exterted antiproliferative and cytotoxic activity, independent of mitochondrial membrane disruption. This compound caused S phase arrest and a decrease in the expression of CD24/CD44/CD58/CD71. CONCLUSION: This is the first report on the in vitro action of CS against lung cancer, necessitating further studies towards its use as a potential anticancer agent.

N-(p-coumaroyl) serotonin induces cell cycle arrest and apoptosis in breast cancer cells.

PURPOSE: Breast cancer is the most commonly diagnosed malignancy among women. Breast cancer cells may develop resistance to current chemotherapy, thus new chemotherapeutic agents are urgently needed. METHODS: A major number of drugs with anticancer activity have been isolated from plants. Herewith, we investigated for the first time the effect of N-(p-coumaroyl) serotonin (CS), isolated from Centaurea seed on a drug-resistant breast carcinoma (MCF-7) cells. Viability and proliferation of the cells were examined with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Caspace-8, cell cycle, and CD24/CD44/CD56/ CD58/CD71/CD15 expression were tested with flow cytometry. RESULTS: Treatment with CS significantly reduced cell viability. Induction of cell death and cell cycle arrest was confirmed with flow cytometry. After treatment with CS, there was a dose-dependent decrease in CD24/CD44/CD58/CD71 expression, whereas there was no change in CD56 and CD15 expression. CONCLUSION: The treatment of breast cancer cells with CS may represent a novel therapeutic strategy and requires further investigation.

Thermally Activated Delayed Fluorescence and Aggregation Induced Emission with Through-Space Charge Transfer.

Emissive molecules comprising a donor and an acceptor bridged by 9,9-dimethylxanthene, were studied (XPT, XCT, and XtBuCT). The structures position the donor and acceptor with cofacial alignment at distances of 3.3-3.5 Å wherein efficient spatial charge transfer can occur. The quantum yields were enhanced by excluding molecular oxygen and thermally activated delayed fluorescence with lifetimes on the order of microseconds was observed. Although the molecules displayed low quantum yields in solution, higher quantum yields were observed in the solid state. Crystal structures revealed π-π intramolecular interactions between a donor and an acceptor, however, the dominant intermolecular interactions were C-H···π, which likely restrict the molecular dynamics to create aggregation-induced enhanced emission. Organic light emitting devices using XPT and XtBuCT as dopants displayed electroluminescence external quantum efficiencies as high as 10%.

Design of efficient molecular organic light-emitting diodes by a high-throughput virtual screening and experimental approach.

Virtual screening is becoming a ground-breaking tool for molecular discovery due to the exponential growth of available computer time and constant improvement of simulation and machine learning techniques. We report an integrated organic functional material design process that incorporates theoretical insight, quantum chemistry, cheminformatics, machine learning, industrial expertise, organic synthesis, molecular characterization, device fabrication and optoelectronic testing. After exploring a search space of 1.6 million molecules and screening over 400,000 of them using time-dependent density functional theory, we identified thousands of promising novel organic light-emitting diode molecules across the visible spectrum. Our team collaboratively selected the best candidates from this set. The experimentally determined external quantum efficiencies for these synthesized candidates were as large as 22%.

Moschamine inhibits proliferation of glioblastoma cells via cell cycle arrest and apoptosis.

Glioblastoma is the most common and most malignant primary brain tumor with a median survival of 15 months. Moschamine is an indole alkaloid that has a serotoninergic and cyclooxygenase inhibitory effect. In this study, we sought to determine whether moschamine could exert cytotoxic and cytostatic effects on glioma cells in vitro. Moschamine was tested for toxicity in zebrafish. We investigated the effect of moschamine on U251MG and T98G glioblastoma cell lines. Viability and proliferation of the cells were examined with trypan blue exclusion assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the xCELLigence system. Apoptosis (annexin-propidium iodide), cell cycle, and CD24/CD44/CD56/CD15 expression were tested with flow cytometry. Treatment with moschamine significantly reduced cell viability in both cell lines tested. Induction of cell death and cell cycle arrest was confirmed with flow cytometry in both cell lines. After treatment with moschamine, there was a dose-dependent decrease in CD24 and CD44 expression, whereas there was no change in CD56 and CD15 expression in T98G cell line. The zebrafish mortality on the fifth post-fertilization day was zero even for 1 mM of moschamine concentration. The treatment of glioblastoma cell lines with moschamine may represent a novel strategy for targeting glioblastoma.

N-(p-coumaroyl) serotonin inhibits glioblastoma cells growth through triggering S-phase arrest and apoptosis.

Glioblastoma is the most common and most malignant primary brain tumor with a median survival of 15 months. N-(p-coumaroyl) serotonin (CS) is an indole alkaloid with antioxidant, cardioprotective effects after ischemia and antitumor activity. In the present study we sought to determine whether could exert cytotoxic and cytostatic effects in glioma cells in vitro. CS was tested for toxicity in zebrafish. We investigated the effect of CS in U251MG and T98G glioblastoma cell lines. Viability and proliferation of the cells were examined with trypan blue exclusion assay and the xCELLigence system. Cell cycle, activation of caspase-8, mitochondrial membrane potential and CD24/CD44/CD56/CD15/CD71 expression were tested with flow cytometry. Treatment with CS significantly reduced cell viability in both cell lines tested. Induction of cell death and cell cycle arrest at G2/M and S-phase was confirmed with flow cytometry in both cell lines. CS produced significant higher activity of caspase-8 compared to control. After treatment with CS there was a dose-dependent increase in CD15 and CD71 expression, whereas there was no change in CD24/CD44/CD56 expression in both cell lines. The zebrafish mortality on the fifth post fertilization day was zero for even 1 mM of CS concentration. The treatment of glioblastoma cell lines with CS may represent a novel strategy for targeting glioblastoma. Further studies are obviously needed to elucidate the complete mechanism of its antitumor activity.

¹9F NMR fingerprints: identification of neutral organic compounds in a molecular container.

Improved methods for quickly identifying neutral organic compounds and differentiation of analytes with similar chemical structures are widely needed. We report a new approach to effectively "fingerprint" neutral organic molecules by using (19)F NMR and molecular containers. The encapsulation of analytes induces characteristic up- or downfield shifts of (19)F resonances that can be used as multidimensional parameters to fingerprint each analyte. The strategy can be achieved either with an array of fluorinated receptors or by incorporating multiple nonequivalent fluorine atoms in a single receptor. Spatial proximity of the analyte to the (19)F is important to induce the most pronounced NMR shifts and is crucial in the differentiation of analytes with similar structures. This new scheme allows for the precise and simultaneous identification of multiple analytes in a complex mixture.

An interstitial 4q31.21q31.22 microdeletion associated with developmental delay: case report and literature review.

The 4q deletion syndrome phenotype consists of growth failure and developmental delay, minor craniofacial dysmorphism, digital anomalies, and cardiac and skeletal defects. We have identified an inversion (inv(1)(q25.2q31.1)) and an interstitial deletion in a boy with developmental delay using array-comparative genomic hybridization. This de novo deletion is located at 4q31.21q31.22 (145,963,820- 147,044,764), its size is 0.9-1.1 Mb, and it contains 7 genes (ABCE1, OTUD4, SMAD1, MMAA, C4orf51, ZNF827, and ANAPC10) as well as 5 retrotransposon-derived pseudogenes. Bioinformatic analysis revealed that while small copy number variations seem to have no impact on the phenotype, larger deletions or duplications in the deleted region are associated with developmental delay. Additionally, we found a higher coverage in transposable element sequences in the 4q31.21q31.22 region compared to that of the expected repeat density when regarding any random genome region. Transposable elements might have contributed to the reshaping of the genome architecture and, most importantly, we identified 3 L1PA family members in the breakpoint regions, suggesting their possible contribution in the mechanism underlying the appearance of this deletion. In conclusion, this is one of the smallest deletions reported associated with developmental delay, and we discuss the possible role of genomic features having an impact on the phenotype.

Intraoperative Flow Cytometry for the Rapid Diagnosis and Validation of Surgical Clearance of Non-Melanoma Skin Cancer: A Prospective Clinical Feasibility Study.

Non-melanoma skin cancer (NMSC) is the most prevalent cancer in humans, with a high global incidence. We present a prospective clinical feasibility study on the use of intraoperative flow cytometry (iFC) for the instant diagnosis of NMSC and its complete surgical clearance. Flow cytometry, a laser-based technique, quantifies cell features, which has applications in cancer research. This study aim is to explore the potential applicability of iFC in detecting and characterizing NMSC and its surgical margins. In total, 30 patients who underwent diagnosis for NMSC were recruited. The method demonstrated high sensitivity (95.2%) and specificity (87.1%), with an accuracy of 91.1%, as confirmed with a receiver operating characteristic curve analysis. The results also indicated that most tumors were diploid, with two cases being hypoploid. The average G0/G1 fractions for normal and tumor tissue samples were 96.03 ± 0.30% and 88.03 ± 1.29%, respectively, with the tumor index escalating from 3.89 ± 0.30% to 11.95 ± 1.29% in cancerous cells. These findings underscore iFC's capability for precise intraoperative NMSC characterization and margin evaluation, promising enhanced complete tumor excision rates. Given the technique's successful application in various other malignancies, its implementation in NMSC diagnosis and treatment holds significant promise and warrants further research in clinical trials.

Imaging Flow Cytometry: Development, Present Applications, and Future Challenges.

Imaging flow cytometry (ImFC) represents a significant technological advancement in the field of cytometry, effectively merging the high-throughput capabilities of flow analysis with the detailed imaging characteristics of microscopy. In our comprehensive review, we adopt a historical perspective to chart the development of ImFC, highlighting its origins and current state of the art and forecasting potential future advancements. The genesis of ImFC stemmed from merging the hydraulic system of a flow cytometer with advanced camera technology. This synergistic coupling facilitates the morphological analysis of cell populations at a high-throughput scale, effectively evolving the landscape of cytometry. Nevertheless, ImFC's implementation has encountered hurdles, particularly in developing software capable of managing its sophisticated data acquisition and analysis needs. The scale and complexity of the data generated by ImFC necessitate the creation of novel analytical tools that can effectively manage and interpret these data, thus allowing us to unlock the full potential of ImFC. Notably, artificial intelligence (AI) algorithms have begun to be applied to ImFC, offering promise for enhancing its analytical capabilities. The adaptability and learning capacity of AI may prove to be essential in knowledge mining from the high-dimensional data produced by ImFC, potentially enabling more accurate analyses. Looking forward, we project that ImFC may become an indispensable tool, not only in research laboratories, but also in clinical settings. Given the unique combination of high-throughput cytometry and detailed imaging offered by ImFC, we foresee a critical role for this technology in the next generation of scientific research and diagnostics. As such, we encourage both current and future scientists to consider the integration of ImFC as an addition to their research toolkit and clinical diagnostic routine.

Association of left ventricular diastolic dysfunction with inflammatory activity, renal dysfunction, and liver-related mortality in patients with cirrhosis and ascites.

Left ventricular diastolic dysfunction (LVDD) is the predominant cardiac abnormality in cirrhosis. We investigated the association of LVDD with systemic inflammation and its impact on renal function, occurrence of hepatorenal syndrome (HRS) and survival in patients with cirrhosis and ascites. We prospectively enrolled 215 patients with cirrhosis and ascites. We evaluated the diagnosis and grading of LVDD by Doppler echocardiography, inflammatory markers, systemic hemodynamics, vasoactive factors, radioisotope-assessed renal function and blood flow, HRS development and liver-related mortality. LVDD was diagnosed in 142 (66%) patients [grade 2/3: n  = 61 (43%)]. Serum lipopolysaccharide-binding protein (LBP), plasma renin activity (PRA) and glomerular filtration rate (GFR) were independently associated with the presence of grade 2/3 LVDD and the severity of diastolic dysfunction. Serum tumor necrosis factor-α, cardiac output and plasma noradrenaline were also independently associated with the presence of grade 2/3 LVDD. The diastolic function marker E / e ' was strongly correlated with serum LBP ( r  = 0.731; P  < 0.001), PRA ( r  = 0.714; P  < 0.001) and GFR ( r  = -0.609; P  < 0.001) among patients with LVDD. The 5-year risk of HRS development and death was significantly higher in patients with grade 2/3 LVDD compared to those with grade 1 (35.5 vs. 14.4%; P  = 0.01 and 53.3 vs. 28.2%; P  = 0.03, respectively). The occurrence and severity of LVDD in patients with cirrhosis and ascites is closely related to inflammatory activity. Advanced LVDD is associated with baseline circulatory and renal dysfunction, favoring HRS development, and increased mortality.