Non-endothelial expression of endomucin in the mouse and human choroid.

Endomucin (EMCN) is a 261 amino acid transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells where it plays a role in VEGF-mediated angiogenesis and regulation of immune cell recruitment. However, it is better known as a histological marker, where it has become widespread due to the commercial availability of high-quality antibodies that work under a wide range of conditions and in many tissues. The specificity of EMCN staining has been well-validated in retinal vessels, but while it has been used extensively as a marker in other tissues of the eye, including the choroid, the pattern of expression has not been described in detail. Here, in addition to endothelial expression in the choriocapillaris and deeper vascular layers, we characterize a population of EMCN-positive perivascular cells in the mouse choroid that did not co-localize with cells expressing other endothelial markers such as PECAM1 or PODXL. To confirm that these cells represented a new population of EMCN-expressing stromal cells, we then performed single cell RNA sequencing in choroids from adult wild-type mice. Analysis of this new dataset confirmed that, in addition to endothelial cells, Emcn mRNA expression was present in choroidal pericytes and a subset of fibroblasts, but not vascular smooth muscle cells. Besides Emcn, no known endothelial gene expression was detected in these cell populations, confirming that they did not represent endothelial-stromal doublets, a common technical artifact in single cell RNA seq datasets. Instead, choroidal Emcn-expressing fibroblasts exhibited high levels of chemokine and interferon signaling genes, while Emcn-negative fibroblasts were enriched in genes encoding extracellular matrix proteins. Emcn expressing fibroblasts were also detected in published datasets from mouse brain and human choroid, suggesting that stromal Emcn expression was not unique to the choroid and was evolutionarily conserved. Together, these findings highlight unique fibroblast and pericyte populations in the choroid and provide new context for the role of EMCN in the eye.

Structural and physical-chemical properties of milk fat globules fractionated by a series of silicon carbide membranes.

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] âˆ¼ 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] âˆ¼ 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.

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte.

INTRODUCTION: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. METHODS: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. RESULTS: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. CONCLUSION: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

RAC1 inhibition ameliorates IBSP-induced bone metastasis in lung adenocarcinoma.

Macrophage-to-osteoclast differentiation (osteoclastogenesis) plays an essential role in tumor osteolytic bone metastasis (BM), while its specific mechanisms remain largely uncertain in lung adenocarcinoma BM. In this study, we demonstrate that integrin-binding sialoprotein (IBSP), which is highly expressed in the cancer cells from bone metastatic and primary lesions of patients with lung adenocarcinoma, can facilitate BM and directly promote macrophage-to-osteoclast differentiation independent of RANKL/M-CSF. In vivo results further suggest that osteolytic BM in lung cancer specifically relies on IBSP-induced macrophage-to-osteoclast differentiation. Mechanistically, IBSP regulates the Rac family small GTPase 1 (Rac1)-NFAT signaling pathway and mediates the forward shift of macrophage-to-osteoclast differentiation, thereby leading to early osteolysis. Moreover, inhibition of Rac1 by EHT-1864 or azathioprine in mice models can remarkably alleviate IBSP-induced BM of lung cancer. Overall, our study suggests that tumor-secreted IBSP promotes BM by inducing macrophage-to-osteoclast differentiation, with potential as an early diagnostic maker for BM, and Rac1 can be the therapeutic target for IBSP-promoted BM in lung cancer.

Regenerative Potential of Dental Pulp Stem Cells in Response to a Bioceramic Dental Sealer and Photobiomodulation: An In Vitro Study.

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.

Expression of Podocalyxin Potentially Decorated With Low-sulfated Keratan Sulfate in Human Testicular Embryonal Carcinoma.

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 PODXL•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.

Extraction optimization, partial purification, and characterization of sialoglycoproteins from Labeo rohita roes.

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.

Long-term haplodeficency of DSPP causes temporomandibular joint osteoarthritis in mice.

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.

A newly developed kit for dental apical root resorption detection: efficacy and acceptability.

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.

The Role of DSPP in Dentine Formation and Hereditary Dentine Defects.

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.

Podocalyxin promotes the formation of compact and chemoresistant cancer spheroids in high grade serous carcinoma.

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.

Simple Binding and Dissociation of a Sialoglycoprotein Using Boronic Acid-Modified Functional Interfaces on Microparticles.

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.

Role of YAP in Odontoblast Damage Repair in a Dentin Hypersensitivity Model.

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.

Isomeric Separation of α2,3/α2,6-Linked 2-Aminobenzamide (2AB)-Labeled Sialoglycopeptides by C18-LC-MS/MS.

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 ∼3 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.

Structural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs.

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 →4)-ß-D-GlcN-(1→3)-α-D-GalN-(1→3)-ß-D-Glc-(1→2)-α-D-Gal-(1→2)-α-D-Gal-(1→3)-α-D-Man-(1→, with two branches of ß-D-GlcN-(1→ and α-D-GalN-(1→ linking at o-4 in →2,4)-α-D-Gal-(1→. Neu5Ac in the T-ES was linked to the branch of α-D-GlcN-(1→. 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.

[Recognition on dentin dysplasia type â ¡].

Dentin dysplasia type Ⅱ (DD-Ⅱ) 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-Ⅱ, in order to improve the overall understanding on DD-Ⅱ for clinicians.

A novel strategy for quantification of α2,3- and α2,6-linked sialic acids in sialylated glycoproteins.

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.

Thermodynamic Evolution of a Metamorphic Protein: A Theoretical-Computational Study of Human Lymphotactin.

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).

Novel dentin sialophosphoprotein gene frameshift mutations affect dentin mineralization.

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.

Dentin defects caused by a Dspp-1 frameshift mutation are associated with the activation of autophagy.

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.