Clinical signs and genetic evaluation of patients with neurofibromatosis type 1 with and without optic pathway gliomas in a center in Turkey.

PURPOSE: Optic pathway gliomas (OPGs) occur in 15% of patients with neurofibromatosis type 1 (NF1). Their location renders biopsy or surgical resection difficult because of the risk of vision loss. Therefore, only a few NF1-OPGs have been used for tissue diagnosis, and only a few analyses have been published on the molecular changes that drive tumorigenesis. METHODS: Due to this reason, we evaluated 305 NF1 patients, 34 with OPG and 271 without OPG for germ line mutations. All subjects underwent clinical examination and DNA analysis of NF1, confirming the diagnosis of NF1. RESULTS: Clinically, the group with OPG had a significantly higher incidence of bone dysplasia (P < 0.001) and more café-au-lait spots (P = 0.001) compared to those in the group without OPG. The frequency of Lisch nodules was on the borderline of statistical significance (P = 0.058), whereas the frequency of neurofibromas did not differ significantly (cutaneous, P = 0.64; plexiform, P = 0.44). Individuals with OPG mostly had mutations in the first one-third of the NF1 gene compared with that in patients who did not have OPG. Some identical mutations were detected in unrelated families with NF1-OPG. CONCLUSION: The observation of certain phenotypic features and the correlation between genotype and phenotype might help to determine the risk of developing OPG with NF1.

Combined effects of bone morphogenetic protein-7 and mineral trioxide aggregate on the proliferation, migration, and differentiation of human dental pulp stem cells.

BACKGROUND: Bioactive molecules present the potential to be used along with biomaterials in vital pulp therapy and regenerative endodontic treatment. OBJECTIVE: The aim of this study was to assess the effects of the combined use of bone morphogenetic protein-7 (BMP-7) and mineral trioxide aggregate (MTA) on the proliferation, migration, and differentiation of human dental pulp stem cells (DPSCs). METHODOLOGY: For the proliferation analysis, DPSCs were incubated with a growth medium and treated with MTA and/or BMP-7 at different concentrations. For the following analyses, DPSCs were incubated with a differentiation medium and treated with MTA and/or BMP-7. Moreover, there were groups in which DPSCs were incubated with the growth medium (control), the differentiation medium, or DMEM/F12 containing fetal bovine serum, and not treated with MTA or BMP-7. Cell proliferation was analyzed using the WST-1 assay. The odontogenic/osteogenic differentiation was evaluated by immunocytochemistry, alkaline phosphatase (ALP) activity assay, alizarin red staining, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was evaluated using a wound-healing assay. Data were analyzed using analysis of variance and Tukey test (p=0.05). RESULTS: The use of BMP-7 with MTA presented no significant effect on cell proliferation in comparison with the treatment with MTA alone (p>0.05), but showed higher ALP activity, increased mineralization, and higher expression of DMP1 and DSPP when compared with other groups (p<0.05). Nestin expression was higher in the control group than in groups treated with MTA and/or BMP-7 (p<0.05). The cell migration rate increased after treatment with MTA when compared with other groups in all periods of time (p<0.05). At 72 hours, the wound area was smaller in groups treated with MTA and/or BMP-7 than in the control group (p<0.05). CONCLUSION: The use of BMP-7 with MTA increased odontogenic/osteogenic differentiation without adversely affecting proliferation and migration of DPSCs. The use of BMP-7 with MTA may improve treatment outcomes by increasing repair and regeneration capacity of DPSCs.

Combined effects of bone morphogenetic protein-7 and mineral trioxide aggregate on the proliferation, migration, and differentiation of human dental pulp stem cells

Abstract Bioactive molecules present the potential to be used along with biomaterials in vital pulp therapy and regenerative endodontic treatment. Objective The aim of this study was to assess the effects of the combined use of bone morphogenetic protein-7 (BMP-7) and mineral trioxide aggregate (MTA) on the proliferation, migration, and differentiation of human dental pulp stem cells (DPSCs). Methodology For the proliferation analysis, DPSCs were incubated with a growth medium and treated with MTA and/or BMP-7 at different concentrations. For the following analyses, DPSCs were incubated with a differentiation medium and treated with MTA and/or BMP-7. Moreover, there were groups in which DPSCs were incubated with the growth medium (control), the differentiation medium, or DMEM/F12 containing fetal bovine serum, and not treated with MTA or BMP-7. Cell proliferation was analyzed using the WST-1 assay. The odontogenic/osteogenic differentiation was evaluated by immunocytochemistry, alkaline phosphatase (ALP) activity assay, alizarin red staining, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was evaluated using a wound-healing assay. Data were analyzed using analysis of variance and Tukey test (p=0.05). Results The use of BMP-7 with MTA presented no significant effect on cell proliferation in comparison with the treatment with MTA alone (p>0.05), but showed higher ALP activity, increased mineralization, and higher expression of DMP1 and DSPP when compared with other groups (p<0.05). Nestin expression was higher in the control group than in groups treated with MTA and/or BMP-7 (p<0.05). The cell migration rate increased after treatment with MTA when compared with other groups in all periods of time (p<0.05). At 72 hours, the wound area was smaller in groups treated with MTA and/or BMP-7 than in the control group (p<0.05). Conclusion The use of BMP-7 with MTA increased odontogenic/osteogenic differentiation without adversely affecting proliferation and migration of DPSCs. The use of BMP-7 with MTA may improve treatment outcomes by increasing repair and regeneration capacity of DPSCs.

Possible modifier genes in the variation of neurofibromatosis type 1 clinical phenotypes.

Neurofibromatosis type 1 (NF1) is the most common neurogenetic disorder worldwide, caused by mutations in the (NF1) gene. Although NF1 is a single-gene disorder with autosomal-dominant inheritance, its clinical expression is highly variable and unpredictable. NF1 patients have the highest known mutation rate among all human disorders, with no clear genotype-phenotype correlations. Therefore, variations in NF1 mutations may not correlate with the variations in clinical phenotype. Indeed, for the same mutation, some NF1 patients may develop severe clinical symptoms whereas others will develop a mild phenotype. Variations in the mutant NF1 allele itself cannot account for all of the disease variability, indicating a contribution of modifier genes, environmental factors, or their combination. Considering the gene structure and the interaction of neurofibromin protein with cellular components, there are many possible candidate modifier genes. This review aims to provide an overview of the potential modifier genes contributing to NF1 clinical variability.

Application of Fourier transform infrared spectroscopy to biomolecular profiling of cultured fibroblast cells from Gaucher disease patients: A preliminary investigation.

BACKGROUND: Gaucher disease (GD) is defined as an autosomal recessive disorder resulting from the deficiency of glucocerebrosidase (E.C. 3.2.1.45). Glucocerebrosidase is responsible for the degradation of glucosylceramide into ceramide and glucose. The deficiency of this enzyme results in the accumulation of undegraded glucosylceramide, almost exclusively in macrophages. With Fourier transform infrared (FTIR) spectroscopy, the complete molecular diversity of the samples can be studied comparatively and the amount of the particular materials can be determined. Also, the secondary structure ratios of proteins can be determined by analysing the amide peaks. OBJECTIVES: The primary aim of this study is to introduce FTIR-ATR spectroscopy technique to GD research for the first time in the literature and to assess its potential as a new molecular method. MATERIAL AND METHODS: Primary fibroblast cell cultures obtained from biopsy samples were used, since this material is widely used for the diagnosis of GD. Intact cells were placed onto a FTIR-ATR crystal and dried by purging nitrogen gas. Spectra were recorded in the mid-infrared region between 4500-850 cm-1 wavenumbers. Each peak in the spectra was assigned to as organic biomolecules according to their chemical bond information. A quantitative analysis was performed using peak areas and we also used a hierarchical cluster analysis as a multivariate spectral analysis. RESULTS: We obtained FTIR spectra of fibroblast samples and assigned the biomolecule origins of the peaks. We observed individual heterogeneity in FTIR spectra of GD fibroblast samples, confirming the well-known phenotypic heterogeneity in GD at the molecular level. Significant alterations in protein, lipid and carbohydrate levels related to the enzyme replacement therapy were also observed, which is also supported by cluster analysis. CONCLUSIONS: Our results showed that the application of FTIR spectroscopy to GD research deserves more attention and detailed studies with an increased sample size in order to evaluate its potential in the diagnosis and follow-up of GD patients.

In Vitro Comparison of Adipokine Export Signals.

Mammalian cells are widely used for recombinant protein production in research and biotechnology. Utilization of export signals significantly facilitates production and purification processes. 35 years after the discovery of the mammalian export machinery, there still are obscurities regarding the efficiency of the export signals. The aim of this study was the comparative evaluation of the efficiency of selected export signals using adipocytes as a cell model. Adipocytes have a large capacity for protein secretion including several enzymes, adipokines, and other signaling molecules, providing a valid system for a quantitative evaluation. Constructs that expressed N-terminal fusion export signals were generated to express Enhanced Green Fluorescence Protein (EGFP) as a reporter for quantitative and qualitative evaluation. Furthermore, fluorescent microscopy was used to trace the intracellular traffic of the reporter. The export efficiency of six selected proteins secreted from adipocytes was evaluated. Quantitative comparison of intracellular and exported fractions of the recombinant constructs demonstrated a similar efficiency among the studied sequences with minor variations. The export signal of Retinol Binding Protein (RBP4) exhibited the highest efficiency. This study presents the first quantitative data showing variations among export signals, in adipocytes which will help optimization of recombinant protein distribution.

Periostin is temporally expressed as an extracellular matrix component in skeletal muscle regeneration and differentiation.

The transcriptional events and pathways responsible for the acquisition of the myogenic phenotype during regeneration and myogenesis have been studied extensively. The modulators that shape the extracellular matrix in health and disease, however, are less understood. Understanding the components and pathways of this remodeling will aid the restoration of the architecture and prevent deterioration under pathological conditions such as fibrosis. Periostin, a matricellular protein associated with remodeling of the extracellular matrix and connective tissue architecture, is emerging in pathological conditions associated with fibrosis in adult life. Periostin also complicates fibrosis in degenerative skeletal muscle conditions such as dystrophies. This study primarily addresses the spatial and temporal involvement of periostin along skeletal muscle regeneration. In the acute skeletal muscle injury model that shows recovery without fibrosis, we show that periostin is rapidly disrupted along with the extensive necrosis and periostin mRNA is transiently upregulated during the myotube maturation. This expression is stringently initiated from the newly regenerating fibers. However, this observation is contrasting to a model that displays extensive fibrosis where upregulation of periostin expression is stable and confined to the fibrotic compartments of endomysial and perimysial space. In vitro myoblast differentiation further supports the claim that upregulation of periostin expression is a function of extracellular matrix remodeling during myofiber differentiation and maturation. We further seek to identify the expression kinetics of various periostin isoforms during the differentiation of rat and mouse myoblasts. Results depict that a singular periostin isoform dominated the rat muscle, contrasting to multiple isoforms in C2C12 myoblast cells. This study shows that periostin, a mediator with deleterious impact on conditions exhibiting fibrosis, is also produced and secreted by myoblasts and regenerating myofibers during architectural remodeling in the course of development and regeneration.

Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: a novel gene related to nuclear envelopathies.

We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patient's muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle.