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Driven by the acknowledged health and functional properties of milk fat globules (MFGs), there is a growing interest to develop gentle methodologies for separation of fat from milk. In this study, separation of fat from raw milk and fractionation in streams containing MFGs of different size was achieved using a series of two silicon carbide ceramic membranes. A first step consisting of a 1.4 µm membrane aimed to concentrate the bulk of the fat, i.e. the larger MFGs (D[4,3] â¼ 4 µm) followed by a 0.5 µm fractionation aimed to concentrate the residual milk fat in the permeate, i.e. fraction with the smaller MFGs (D[4,3] â¼ 1.8-2.4 µm. The fat separation performance showed a yield of 92 % for the 1.4 µm membrane and 97 % for the 0.5 µm membrane. Both fat enriched retentates showed, by the confocal laser scanning microscopy, intact MFGs with limited damage in the MFG membrane. The fatty acid profile analysis and SAXS showed minor differences in fat acid composition and the crystallization behavior was related to differences in the fat content. The 0.5 µm permeate containing the smallest MFGs however showed larger aggregates and a trinomial particle size distribution, due to probably pore pressure induced coalescences. The series of silicon carbide membranes showed potential to concentrate some of MFGM proteins such as Periodic Schiff base 3/4 and cluster of differentiation 36 especially in the 0.5 µm retentates. A shift in casein to whey protein ratio from 80:20 (milk) to 50:50 was obtained in the final 0.5 µm permeate, which opens new opportunities for product development.
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Compuestos Inorgánicos de Carbono , Glucolípidos , Glicoproteínas , Gotas Lipídicas , Leche , Compuestos de Silicona , Gotas Lipídicas/química , Compuestos de Silicona/química , Glucolípidos/química , Compuestos Inorgánicos de Carbono/química , Glicoproteínas/química , Glicoproteínas/análisis , Animales , Leche/química , Membranas Artificiales , Tamaño de la Partícula , Ácidos Grasos/análisis , Ácidos Grasos/química , Difracción de Rayos X , Sialoglicoproteínas , Dispersión del Ángulo Pequeño , Fraccionamiento Químico/métodosRESUMEN
AIMS: This study aims to assess the synergistic effect of utilizing a bioceramic sealer, NeoPutty, with photobiomodulation (PBM) on dental pulp stem cells (DPSCs) for odontogenesis. MATERIALS AND METHODS: Dental pulp stem cells were collected from 10 premolars extracted from healthy individuals. Dental pulp stem cells were characterized using an inverted-phase microscope to detect cell shape and flow cytometry to detect stem cell-specific surface antigens. Three experimental groups were examined: the NP group, the PBM group, and the combined NP and PBM group. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) experiment was conducted to assess the viability of DPSCs. The odontogenic differentiation potential was analyzed using Alizarin red staining, RT-qPCR analysis of odontogenic genes DMP-1, DSPP, and alkaline phosphatase (ALP), and western blot analysis for detecting BMP-2 and RUNX-2 protein expression. An analysis of variance (ANOVA) followed by a post hoc t-test was employed to examine and compare the mean values of the results. RESULTS: The study showed a notable rise in cell viability when NP and PBM were used together. Odontogenic gene expression and the protein expression of BMP-2 and RUNX-2 were notably increased in the combined group. The combined effect of NeoPutty and PBM was significant in enhancing the odontogenic differentiation capability of DPSCs. CONCLUSION: The synergistic effect of NeoPutty and PBM produced the most positive effect on the cytocompatibility and odontogenic differentiation potential of DPSCs. CLINICAL SIGNIFICANCE: Creating innovative regenerative treatments to efficiently and durably repair injured dental tissues. How to cite this article: Alshawkani HA, Mansy M, Al Ankily M, et al. Regenerative Potential of Dental Pulp Stem Cells in Response to a Bioceramic Dental Sealer and Photobiomodulation: An In Vitro Study. J Contemp Dent Pract 2024;25(4):313-319.
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Proteína Morfogenética Ósea 2 , Diferenciación Celular , Pulpa Dental , Terapia por Luz de Baja Intensidad , Odontogénesis , Células Madre , Pulpa Dental/citología , Humanos , Células Madre/efectos de los fármacos , Terapia por Luz de Baja Intensidad/métodos , Diferenciación Celular/efectos de los fármacos , Odontogénesis/efectos de los fármacos , Materiales de Obturación del Conducto Radicular/farmacología , Fosfatasa Alcalina/metabolismo , Técnicas In Vitro , Supervivencia Celular/efectos de los fármacos , Regeneración/efectos de los fármacos , Cerámica , Proteínas de la Matriz Extracelular , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Sialoglicoproteínas , FosfoproteínasRESUMEN
SummaryWe previously demonstrated that among various histological types of human testicular germinal cell tumors (GCTs), embryonal carcinoma (EC) preferentially expresses low-sulfated keratan sulfate (KS) consisting of repeating N-acetyllactosamine (LacNAc) disaccharide units composed of galactose and 6-O-sulfated N-acetylglucosamine (GlcNAc), which is recognized by the R-10G antibody. Recently, we generated another anti-low-sulfated KS monoclonal antibody, 294-1B1. Immunohistochemical analysis of testicular GCTs (n=83) revealed that the low-sulfated KS recognized by 294-1B1 is also preferentially expressed in EC but minimally in other GCT histological types. Moreover, immunolabeling with R-10G and 294-1B1 antibodies was resistant to peptide-N-glycosidase F digestion, and EC was not stained with the MECA-79 antibody, indicating that low-sulfated KS expressed in EC contains mucin-type core 2 O-glycans carrying GlcNAc-6-O-sulfated oligo-LacNAc. Double immunofluorescence staining showed that R-10G and 294-1B1 antibody signals colocalized with those for podocalyxin (PODXL). Furthermore, western blot analysis of recombinant human PODXLâ¢IgG fusion proteins secreted from low-sulfated KS-expressing human embryonic kidney 293T cells revealed that PODXL functions as a core protein for low-sulfated KS. Taken together, these findings strongly suggest that the PODXL glycoform decorated with low-sulfated KS is preferentially expressed in human testicular EC and may therefore serve as a diagnostic marker for this malignancy.
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Carcinoma Embrionario , Sulfato de Queratano , Sialoglicoproteínas , Neoplasias Testiculares , Humanos , Masculino , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patología , Neoplasias Testiculares/diagnóstico , Sialoglicoproteínas/análisis , Sialoglicoproteínas/metabolismo , Carcinoma Embrionario/patología , Carcinoma Embrionario/metabolismo , Sulfato de Queratano/metabolismo , Sulfato de Queratano/análisis , Inmunohistoquímica , Línea Celular Tumoral , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias de Células Germinales y Embrionarias/patología , Neoplasias de Células Germinales y Embrionarias/diagnósticoRESUMEN
In India, fish roes are generally considered worthless garbage and disposed of without recovering the valuable molecules, creating environmental and disposal problems. The present investigation aimed to optimize the extraction conditions, partial purification, and characterization of sialoglycoproteins (RRSGP) from Labeo rohita (rohu) roes. RSM generated optimum conditions for maximum RRSGP (70.49 %) extraction, which were 1.25 M NaCl, 1:32.5(w/v) solid-to-liquid ratio, 47.5 °C temperature, and 3 h time. Further, sialoglycoproteins from RRSGPs were partially purified, and result revealed that obtained peak-1 (PRRSGP) using QFF anion exchange chromatography exhibited higher glycoprotein and sialic acid content (p < 0.05). SDS-PAGE pattern of PRRSGP presented dominant bands of 97 kDa and 27 kDa glycoproteins. FTIR spectrum of PRRSGP confirmed the presence of glycated proteins. HPLC analysis revealed that PRRSGP consists of Neu5Ac. Furthermore, ß-elimination reaction elucidated that PRRSGP contained N-glycosidic linkage. PRRSGP exhibited tyrosine and glutamate as primary amino acids. Glycan part of PRRSGP presented mannose and N-acetyl galactosamine as dominant neutral and amino sugar, respectively. Furthermore, PRRSGP exhibited antioxidant activity with EC50 value for DPPH (8.79 mg/ml) and ABTS (2.21 mg/ml). Besides, RRSGP displayed better protein solubility, foaming, and emulsion properties. Therefore, rohu roes are potential source of sialoglycoproteins that can be recovered and used as bio-functional ingredients in food and nutraceutical applications.
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Sialoglicoproteínas , Animales , Sialoglicoproteínas/aislamiento & purificación , Sialoglicoproteínas/química , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Cyprinidae , Glicoproteínas/química , Glicoproteínas/aislamiento & purificaciónRESUMEN
BACKGROUND: Extracellular matrix (ECM) protein malfunction or defect may lead to temporomandibular joint osteoarthritis (TMJ OA). Dentin sialophophoprotein (DSPP) is a mandibular condylar cartilage ECM protein, and its deletion impacted cell proliferation and other extracellular matrix alterations of postnatal condylar cartilage. However, it remains unclear if long-term loss of function of DSPP leads to TMJ OA. The study aimed to test the hypothesis that long-term haploinsufficiency of DSPP causes TMJ OA. MATERIALS AND METHODS: To determine whether Dspp+/- mice exhibit TMJ OA but no severe tooth defects, mandibles of wild-type (WT), Dspp+/-, and Dspp homozygous (Dspp-/-) mice were analyzed by Micro-computed tomography (micro-CT). To characterize the progression and possible mechanisms of osteoarthritic degeneration over time in Dspp+/- mice over time, condyles of Dspp+/- and WT mice were analyzed radiologically, histologically, and immunohistochemically. RESULTS: Micro-CT and histomorphometric analyses revealed that Dspp+/- and Dspp-/- mice had significantly lower subchondral bone mass, bone volume fraction, bone mineral density, and trabecular thickness compared to WT mice at 12 months. Interestingly, in contrast to Dspp-/- mice which exhibited tooth loss, Dspp+/- mice had minor tooth defects. RNA sequencing data showed that haplodeficency of DSPP affects the biological process of ossification and osteoclast differentiation. Additionally, histological analysis showed that Dspp+/- mice had condylar cartilage fissures, reduced cartilage thickness, decreased articular cell numbers and severe subchondral bone cavities, and with signs that were exaggerated with age. Radiographic data showed an increase in subchondral osteoporosis up to 18 months and osteophyte formation at 21 months. Moreover, Dspp+/- mice showed increased distribution of osteoclasts in the subchondral bone and increased expression of MMP2, IL-6, FN-1, and TLR4 in the mandibular condylar cartilage. CONCLUSIONS: Dspp+/- mice exhibit TMJ OA in a time-dependent manner, with lesions in the mandibular condyle attributed to hypomineralization of subchondral bone and breakdown of the mandibular condylar cartilage, accompanied by upregulation of inflammatory markers.
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Proteínas de la Matriz Extracelular , Osteoartritis , Fosfoproteínas , Sialoglicoproteínas , Trastornos de la Articulación Temporomandibular , Animales , Ratones , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Cóndilo Mandibular/patología , Cóndilo Mandibular/diagnóstico por imagen , Osteoartritis/patología , Osteoartritis/diagnóstico por imagen , Osteoartritis/genética , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Articulación Temporomandibular/patología , Articulación Temporomandibular/diagnóstico por imagen , Trastornos de la Articulación Temporomandibular/patología , Trastornos de la Articulación Temporomandibular/diagnóstico por imagen , Trastornos de la Articulación Temporomandibular/etiología , Trastornos de la Articulación Temporomandibular/genética , Microtomografía por Rayos XRESUMEN
OBJECTIVES: To determine the efficacy of a newly developed kit in dentine sialophosphoprotein (DSPP) detection and compare it with enzyme-linked immunosorbent assay (ELISA). User acceptance was also determined. MATERIALS AND METHODS: This cross-sectional study consisted of 45 subjects who were divided into 3 groups based on the severity of root resorption using radiographs: normal (RO), mild (RM), and severe (RS). DSPP in GCF samples was analyzed using both methods. Questionnaires were distributed to 30 orthodontists to evaluate future user acceptance. RESULTS: The sensitivity and specificity of the kit were 0.98 and 0.8 respectively. The DSPP concentrations measured using ELISA were the highest in the RS group (6.33 ± 0.85 ng/mL) followed by RM group (3.77 ± 0.36 ng/mL) and the RO group had the lowest concentration (2.23 ± 0.55 ng/mL). The new kit portrayed similar results as the ELISA, the optical density (OD) values were the highest in the RS group (0.62 ± 0.10) followed by RM group (0.33 ± 0.03) and the RO group (0.19 ± 0.06). The differences among all the groups were statistically significant (p < 0.05) for both methods. The Pearson correlation coefficient showed a statistically significant (p < 0.001) strong and positive correlation between DSPP concentrations and OD values. CONCLUSIONS: The new kit was validated to detect the colour intensities of different severity of root resorptions. Most of the responses to the survey were positive towards the new kit for being a safer and simpler method to detect apical root resorption.
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Proteínas de la Matriz Extracelular , Resorción Radicular , Humanos , Resorción Radicular/diagnóstico por imagen , Estudios Transversales , Sialoglicoproteínas , Líquido del Surco Gingival/química , Fosfoproteínas , Biomarcadores/análisisRESUMEN
High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)-spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.
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Carcinoma , Neoplasias Ováricas , Femenino , Humanos , Carboplatino/farmacología , Carboplatino/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismoRESUMEN
The dentine sialophosphoprotein (DSPP) gene is the only identified causative gene for dentinogenesis imperfecta type 2 (DGI-II), dentinogenesis imperfecta type 3 (DGI-III) and dentine dysplasia type 2 (DD-II). These three disorders may have similar molecular mechanisms involved in bridging the DSPP mutations and the resulting abnormal dentine mineralisation. The DSPP encoding proteins DSP (dentine sialoprotein) and DPP (dentine phosphoprotein) are positive regulators of dentine formation and perform a function during dentinogenesis. The present review focused on the recent findings and viewpoints regarding the relationship between DSPP and dentinogenesis as well as mineralisation from multiple perspectives, involving studies relating to spatial structure and tissue localisation of DSPP, DSP and DPP, the biochemical characteristics and biological function of these molecules, and the causative role of the proteins in phenotypes of the knockout mouse model and in hereditary dentine defects.
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Calcinosis , Dentinogénesis Imperfecta , Fosfoproteínas , Sialoglicoproteínas , Animales , Ratones , Calcificación Fisiológica , Dentina , Dentinogénesis Imperfecta/genética , Modelos Animales de Enfermedad , Ratones Noqueados , Humanos , Sialoglicoproteínas/genética , Fosfoproteínas/genéticaRESUMEN
An overexpression of sialic acid is an indicator of metastatic cancer, and selective detection of sialic acid shows potential for cancer diagnosis. Boronic acid is a promising candidate for this purpose because of its ability to specifically bind to sialic acid under acidic conditions. Notably, the binding strength can be easily modulated by adjusting the pH, which allows for a simple dissociation of the bound sialic acid. In this study, we developed 5-boronopicolinic acid (5-BPA)-modified magnetic particles (BMPs) to selectively capture sialic acid biomolecules. We successfully captured fetuin, a well-known sialoglycoprotein, on BMPs at >104 molecules/particle using an acetate buffer (pH 5.0). Facile dissociation then occurred when the system was changed to a pH 7.6 phosphate buffer. This capture-and-release process could be repeated at least five times. Moreover, this system could enrich fetuin by more than 20 times. In summary, BMPs are functional particles for facile purification and concentration through the selective capture of sialic acid proteins and can improve detection sensitivity compared with conventional methods. This technology shows potential for the detection of sialic acid overexpression by biological particles.
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Ácido N-Acetilneuramínico , Neoplasias , Humanos , Ácido N-Acetilneuramínico/química , Sialoglicoproteínas/metabolismo , Ácidos Borónicos/química , FetuínasRESUMEN
OBJECTIVES: The aim of this study was to investigate the molecular mechanism underlying odontoblast damage repair in dentin hypersensitivity (DH) and the role of Yes-associated protein (YAP) in this process. METHODS: The DH model was constructed in Sprague-Dawley (SD) rats, and the in vivo expression of Piezo1, Integrin αvß3, YAP, and dentin sialophosphoprotein (DSPP) was detected by immunohistochemistry. COMSOL Multiphysics software was used to simulate the dentinal tubule fluid flow velocity and corresponding fluid shear stress (FSS) on the odontoblast processes. MDPC-23 cells were cultured in vitro and loaded with a peristaltic pump for 1 hour at FSS values of 0.1, 0.3, 0.5, and 0.7 dyne/cm2. The expression of Piezo1, Integrin αvß3, and YAP was detected by immunofluorescence. Verteporfin (a YAP-specific inhibitor) was utilised to confirm the effect of YAP on the expression of dentineogenesis-related protein under FSS. RESULTS: The level and duration of external mechanical stimuli have an effect on the functional expression of odontoblasts. In DH, the harder the food that is chewed, the faster the flow of the dentinal tubule fluid and the greater the FSS on the odontoblast processes. The expression of Piezo1, Integrin αvß3, and YAP can be promoted when the FSS is less than 0.3 dyne/cm2. After YAP inhibition, the DSPP protein expression level was reduced at 0.3 dyne/cm2 FSS. CONCLUSIONS: These results suggest that appropriate FSS can enhance the expression of odontoblast-related factors in odontoblasts via the Piezo1-Integrin αvß3-YAP mechanotransduction pathway and the YAP appears to play an essential role in the response of odontoblasts to external mechanical stimuli.
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Sensibilidad de la Dentina , Odontoblastos , Proteínas Señalizadoras YAP , Animales , Ratas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sensibilidad de la Dentina/genética , Sensibilidad de la Dentina/metabolismo , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Inmunohistoquímica , Integrina alfaVbeta3/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana , Odontoblastos/metabolismo , Fosfoproteínas/metabolismo , Ratas Sprague-Dawley , Sialoglicoproteínas/metabolismo , Estrés Mecánico , Verteporfina/farmacología , Verteporfina/uso terapéuticoRESUMEN
Determination of the relative expression levels of the α2,3/α2,6-sialic acid linkage isomers on glycoproteins is critical to the analysis of various human diseases such as cancer, inflammation, and viral infection. However, it remains a challenge to separate and differentiate site-specific linkage isomers at the glycopeptide level. Some derivatization methods on the carboxyl group of sialic acid have been developed to generate mass differences between linkage isomers. In this study, we utilized chemical derivatization that occurred on the vicinal diol of sialic acid to separate linkage isomers on a reverse-phase column using a relatively short time. 2-Aminobenzamide (2AB) labeling derivatization, including periodate oxidation and reductive amination, took only â¼3 h and achieved high labeling efficiency (>90%). Within a 66 min gradient, the sialic acid linkage isomers of 2AB-labeled glycopeptides from model glycoproteins can be efficiently resolved compared to native glycopeptides. Two different methods, neuraminidase digestion and higher-energy collision dissociation tandem mass spectrometry (HCD-MS2) fragmentation, were utilized to differentiate those isomeric peaks. By calculating the diagnostic oxonium ion ratio of Gal2ABNeuAc and 2ABNeuAc fragments, significant differences in chromatographic retention times and in mass spectral peak abundances were observed between linkage isomers. Their corresponding MS2 PCA plots also helped to elucidate the linkage information. This method was successfully applied to human blood serum. A total of 514 2AB-labeled glycopeptide structures, including 152 sets of isomers, were identified, proving the applicability of this method in linkage-specific structural characterization and relative quantification of sialic acid isomers.
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Ácido N-Acetilneuramínico , Espectrometría de Masas en Tándem , Humanos , Ácido N-Acetilneuramínico/química , Espectrometría de Masas en Tándem/métodos , Sialoglicoproteínas , Cromatografía Líquida con Espectrometría de Masas , Cromatografía Liquida , Glicoproteínas , Glicopéptidos/análisis , Polisacáridos/químicaRESUMEN
Several sialoglycopeptides were isolated from several fish eggs and exerted anti-osteoporosis effects. However, few papers have explored sialoglycopeptide from tuna eggs (T-ES). Here, a novel T-ES was prepared through extraction with KCl solution and subsequent enzymolysis. Pure T-ES was obtained through DEAE-Sepharose ion exchange chromatography and sephacryl S-300 gel filtration chromatography. The T-ES was composed of 14.07% protein, 73.54% hexose, and 8.28% Neu5Ac, with a molecular weight of 9481 Da. The backbone carbohydrate in the T-ES was â4)-ß-D-GlcN-(1â3)-α-D-GalN-(1â3)-ß-D-Glc-(1â2)-α-D-Gal-(1â2)-α-D-Gal-(1â3)-α-D-Man-(1â, with two branches of ß-D-GlcN-(1â and α-D-GalN-(1â linking at o-4 in â2,4)-α-D-Gal-(1â. Neu5Ac in the T-ES was linked to the branch of α-D-GlcN-(1â. A peptide chain, Ala-Asp-Asn-Lys-Ser*-Met-Ile that was connected to the carbohydrate chain through O-glycosylation at the -OH of serine. Furthermore, in vitro data revealed that T-ES could remarkably enhance bone density, bone biomechanical properties, and bone microstructure in SAMP mice. The T-ES elevated serum osteogenesis-related markers and reduced bone resorption-related markers in serum and urine. The present study's results demonstrated that T-ES, a novel sialoglycopeptide, showed significant anti-osteoporosis effects, which will accelerate the utilization of T-ES as an alternative marine drug or functional food for anti-osteoporosis.
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Sialoglicoproteínas , Atún , Humanos , Ratones , Animales , Secuencia de Carbohidratos , Carbohidratos , HexosasRESUMEN
Dentin dysplasia type â ¡ (DD-â ¡) is a subtype of hereditary dentin disorders. The dentin sialophosphoprotein (DSPP) gene has been revealed to be the causative gene, whose mutations could affect the normal tooth development process. The lesions involve both deciduous and permanent dentition, mainly manifested as tooth discoloration, attrition and even the subsequent malocclusion. If not treated in time, it will significantly affect the physical and psychological health of patients. The disease is difficult to be diagnosed in clinic accurately as its low incidence and hidden manifestations. The present article aims to discuss the clinical and radiographic characteristics, diagnosis, treatment of DD-â ¡, in order to improve the overall understanding on DD-â ¡ for clinicians.
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Displasia de la Dentina , Dentinogénesis Imperfecta , Diente , Humanos , Displasia de la Dentina/diagnóstico , Displasia de la Dentina/genética , Displasia de la Dentina/patología , Dentinogénesis Imperfecta/diagnóstico , Dentinogénesis Imperfecta/genética , Dentinogénesis Imperfecta/patología , Sialoglicoproteínas/genética , Diente/patología , Mutación , Proteínas de la Matriz Extracelular/genética , Fosfoproteínas/genética , Dentina/patologíaRESUMEN
Sialic acid, a monosaccharide containing nine carbon atoms, is widely distributed in eukaryotic cells. The bound sialic acids are mainly present at the glycan ends of glycoconjugates via α2-3 or α2-6 glycosidic bonds, and alterations in their expression levels and linkage types are associated with the progress of many diseases and tumors. The present study provides a new strategy for quantification of α2,3- and α2,6-linked sialic acids in sialylated glycoproteins. In fact, quantification of α2,3-linked sialic acids were based on the difference of the bound sialic acids in the sample before and after treatment with α2-3 neuraminidase, whereas the α2,6-linked sialic acids were equal to the bound sialic acids in the α2-3 neuraminidase-treated sample. Subsequently, α2,3/6-linked sialic acids in salivary glycoproteins from healthy volunteers and diabetic patients were quantified in accordance with this method. This work provides an accurate method for the quantification of α2,3- and α2,6-linked sialic acids in the sialoglycoproteins, which is more instructive for understanding the biological roles of α2,3/6-linked sialic acid in sialoglycoproteins.
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Ácido N-Acetilneuramínico , Ácidos Siálicos , Humanos , Ácidos Siálicos/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Glicoproteínas/metabolismo , SialoglicoproteínasRESUMEN
Metamorphic, or fold-switching, proteins feature different folds that are physiologically relevant. The human chemokine XCL1 (or Lymphotactin) is a metamorphic protein that features two native states, an [Formula: see text] and an all[Formula: see text] fold, which have similar stability at physiological condition. Here, extended molecular dynamics (MD) simulations, principal component analysis of atomic fluctuations and thermodynamic modeling based on both the configurational volume and free energy landscape, are used to obtain a detailed characterization of the conformational thermodynamics of human Lymphotactin and of one of its ancestors (as was previously obtained by genetic reconstruction). Comparison of our computational results with the available experimental data show that the MD-based thermodynamics can explain the experimentally observed variation of the conformational equilibrium between the two proteins. In particular, our computational data provide an interpretation of the thermodynamic evolution in this protein, revealing the relevance of the configurational entropy and of the shape of the free energy landscape within the essential space (i.e., the space defined by the generalized internal coordinates providing the largest, typically non-Gaussian, structural fluctuations).
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Linfocinas , Sialoglicoproteínas , Humanos , Termodinámica , Linfocinas/química , Linfocinas/metabolismo , Sialoglicoproteínas/química , Sialoglicoproteínas/metabolismo , Simulación de Dinámica MolecularRESUMEN
Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' - 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp-1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp-1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp-1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp-1fs mice may be valuable for the study of autophagy and ER-phagy.
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Proteínas de la Matriz Extracelular , Mutación del Sistema de Lectura , Ratones , Humanos , Animales , Proteínas de la Matriz Extracelular/genética , Odontoblastos , Mutación , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Dentina , Autofagia/genéticaRESUMEN
OBJECTIVE: This study aimed to identify candidate genes for inheritable dentin defects in three Chinese pedigrees and characterize the property of affected teeth. DESIGN: Clinical and radiological features were recorded for the affected individuals. Genomic DNA obtained from peripheral venous blood or saliva were analyzed by whole-exome sequencing. The density and microhardness of affected dentin was measured. Scanning electron microscopy (SEM) was also performed to obtain the microstructure phenotype. RESULTS: 1) General appearance: the affected dentitions shared yellowish-brown or milky color. Radiographs showed that the pulp cavity and root canals were obliterated in varying degrees or exhibited a pulp aspect in the 'thistle tube'. Some patients exhibited periapical infections without pulpal exposure, and some affected individuals showed shortened, abnormally thin roots accompanied by severe alveolar bone loss. 2) Genomic analysis: three new frameshift mutations (NM_014208.3: c.2833delA, c.2852delGand c.3239delA) were identified in exon 5 of dentin sialophosphoprotein (DSPP) gene, altering dentin phosphoprotein (DPP) as result. In vitro studies showed that the density and microhardness of affected dentin were decreased, the dentinal tubules were sparse and arranged disorderly, and the dentinal-enamel-junction (DEJ) was abnormal. CONCLUSIONS: In this study, we identified three novel frameshift mutations of dentin sialophosphoprotein gene related to inherited dentin defects. These mutations are speculated to cause abnormal coding of dentin phosphoprotein C-terminus, which affect dentin mineralization. These results expand the spectrum of dentin sialophosphoprotein gene mutations causing inheritable dentin defects and broaden our understanding of the biological mechanisms by which dentin forms.
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Dentinogénesis Imperfecta , Mutación del Sistema de Lectura , Humanos , Dentinogénesis Imperfecta/genética , Fosfoproteínas/genética , Proteínas de la Matriz Extracelular/genética , Sialoglicoproteínas/genética , DentinaRESUMEN
Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.
Asunto(s)
Neoplasias Óseas , Neoplasias Pulmonares , Ratones , Animales , Sialoproteína de Unión a Integrina/genética , Sialoproteína de Unión a Integrina/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Metaloproteinasa 14 de la Matriz , Fosfatidilinositol 3-Quinasas , Línea Celular Tumoral , Neoplasias Óseas/metabolismoRESUMEN
Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses an Escherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azido-sialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and copper-catalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.
Asunto(s)
Glicoproteínas , Sialoglicoproteínas , Glicosilación , Lectinas/metabolismo , Polisacáridos/metabolismo , Sialoglicoproteínas/metabolismo , Sistema Libre de CélulasRESUMEN
Dentin is a major type of hard tissue of teeth and plays essential roles for normal tooth function. Odontoblasts are responsible for dentin formation. Mutations or deficiency in various genes affect the differentiation of odontoblasts, leading to irreversible dentin developmental defects in animals and humans. Whether such dentin defects can be reversed by gene therapy for odontoblasts remains unknown. In this study, we compare the infection efficiencies of six commonly used adeno-associated virus (AAV) serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVDJ) in cultured mouse odontoblast-like cells (OLCs). We show that AAV6 serotype infects OLCs with the highest efficiency among the six AAVs. Two cellular receptors, which are able to recognize AAV6, AAV receptor (AAVR), and epidermal growth factor receptor (EGFR), are strongly expressed in the odontoblast layer of mouse teeth. After local administration to mouse molars, AAV6 infects the odontoblast layer with high efficiency. Furthermore, AAV6-Mdm2 was successfully delivered to teeth and prevents the defects in odontoblast differentiation and dentin formation in Mdm2 conditional knockout mice (a mouse model of dentinogenesis imperfecta type â ¢). These results suggest that AAV6 can serve as a reliable and efficient vehicle for gene delivery to odontoblasts through local injection. In addition, human OLCs were also successfully infected by AAV6 with high efficiency, and both AAVR and EGFR are strongly expressed in the odontoblast layer of extracted human developing teeth. These findings suggest that AAV6-mediated gene therapy through local injection may be a promising treatment approach for hereditary dentin disorders in humans.