A proteomics informed by transcriptomics insight into the proteome of Ornithodoros erraticus adult tick saliva.

BACKGROUND: The argasid tick Ornithodoros erraticus is the main vector of tick-borne human relapsing fever (TBRF) and African swine fever (ASF) in the Mediterranean Basin. The prevention and control of these diseases would greatly benefit from the elimination of O. erraticus populations, and anti-tick vaccines are envisaged as an effective and sustainable alternative to chemical acaricide usage for tick control. Ornithodoros erraticus saliva contains bioactive proteins that play essential functions in tick feeding and host defence modulation, which may contribute to host infection by tick-borne pathogens. Hence, these proteins could be candidate antigen targets for the development of vaccines aimed at the control and prevention of O. erraticus infestations and the diseases this tick transmits. The objective of the present work was to obtain and characterise the proteome of the saliva of O. erraticus adult ticks as a means to identify and select novel salivary antigen targets. METHODS: A proteomics informed by transcriptomics (PIT) approach was applied to analyse samples of female and male saliva separately using the previously obtained O. erraticus sialotranscriptome as a reference database and two different mass spectrometry techniques, namely liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-dependent acquisition mode and sequential window acquisition of all theoretical fragment ion spectra MS (SWATH-MS). RESULTS: Up to 264 and 263 proteins were identified by LC-MS/MS in the saliva of O. erraticus female and male ticks, respectively, totalling 387 non-redundant proteins. Of these, 224 were further quantified by SWATH-MS in the saliva of both male and female ticks. Quantified proteins were classified into 23 functional categories and their abundance compared between sexes. Heme/iron-binding proteins, protease inhibitors, proteases, lipocalins and immune-related proteins were the categories most abundantly expressed in females, while glycolytic enzymes, protease inhibitors and lipocalins were the most abundantly expressed in males. Ninety-seven proteins were differentially expressed between the sexes, of which 37 and 60 were overexpressed in females and males, respectively. CONCLUSIONS: The PIT approach demonstrated its usefulness for proteomics studies of O. erraticus, a non-model organism without genomic sequences available, allowing the publication of the first comprehensive proteome of the saliva of O. erraticus reported to date. These findings confirm important quantitative differences between sexes in the O. erraticus saliva proteome, unveil novel salivary proteins and functions at the tick-host feeding interface and improve our understanding of the physiology of feeding in O. erraticus ticks. The integration of O. erraticus sialoproteomic and sialotranscriptomic data will drive a more rational selection of salivary candidates as antigen targets for the development of vaccines aimed at the control of O. erraticus infestations and the diseases it transmits.

Impact of histological response after neoadjuvant therapy on podocalyxin as a prognostic marker in pancreatic cancer.

Podocalyxin overexpression associates with poor survival in pancreatic cancer (PDAC). We investigated whether podocalyxin expression correlates with treatment response or survival in neoadjuvant-treated PDAC. Through immunohistochemistry, we evaluated podocalyxin expression in 88 neoadjuvant and 143 upfront surgery patients using two antibodies. We developed a six-tier grading scheme for neoadjuvant responses evaluating the remaining tumor cells in surgical specimens. Strong podocalyxin immunopositivity associated with poor survival in the patients responding poorly to the neoadjuvant treatment (HR 4.16, 95% CI 1.56-11.01, p = 0.004), although neoadjuvant patients exhibited generally low podocalyxin expression (p = 0.017). Strong podocalyxin expression associated with perineural invasion (p = 0.003) and lack of radiation (p = 0.036). Two patients exhibited a complete neoadjuvant response, while a strong neoadjuvant response (≤ 5% of residual tumor cells) significantly associated with lower stage, pT-class and grade, less spread to the regional lymph nodes, less perineural invasion, and podocalyxin negativity (p < 0.05, respectively). A strong response predicted better survival (HR 0.28, 95% CI 0.09-0.94, p = 0.039). In conclusion, strong podocalyxin expression associates with poor survival among poorly responding neoadjuvant patients. A good response associates with podocalyxin negativity. A strong response associates with better outcome.

Podocyte Involvement in Renal Thrombotic Microangiopathy: A Clinicopathological Study.

BACKGROUND: The current study aimed to evaluate the associations between podocyte injury and clinicopathological features in renal thrombotic microangiopathy (TMA) based on a Chinese cohort, which might be underscored in this disease. METHODS: The clinical, laboratory, and renal histopathological data of patients with renal biopsy-proven TMA from 2000 to 2015 in our institute were collected. Foot process effacement (FPE) was quantified by foot process width (FPW) by electron microscopy. Podocytes in the renal specimens were also detected by stainings for podocyte-specific markers, including Wilms tumor 1 (WT-1), synaptopodin, and podocalyxin. The associations between FPW and clinico-histopathological data were further analyzed. A composite end-point was defined by all-cause death or end-stage renal disease to address the predictive value of FPW. RESULTS: Sixty-three patients with renal biopsy-proven TMA were enrolled. The FPW of renal TMA patients was 1,090 ± 637 nm (range, 572-4,748 nm), which was significantly higher than the normal range in our center (p = 0.005). By immunohistochemistry and immunofluorescence assays, we found decreased expressions of synaptopodin, podocalyxin, and WT-1 and continued stainings of WT-1 in some podocytes without detectable synaptopodin stainings in the areas of sclerotic tufts and cellular crescents. The FPW value was correlated with the serum albumin concentration (rs = -0.281, p = 0.026), proteinuria amount (rs = 0.255, p = 0.047), serum creatinine levels (rs = 0.339, p = 0.007), and eGFR (rs = -0.335, p = 0.007). According to ROC curve analysis, the optimal cutoff level of FPW for predicting the composite end-point was 869 nm. In patients with FPW ≥ 869 nm, FPW levels were further correlated with the severity of mesangiolysis (rs = 0.351, p = 0.033) and glomerulosclerosis (rs = 0.369, p = 0.025) in pathological evaluations. Patients without clinical remission also had higher FPW than those with remission (1,240 ± 793 vs. 925 ± 344 nm, p = 0.013). The multivariate Cox hazard model showed that FPW ≥ 869 nm was an independent risk factor for the composite end-point (hazard ratio: 3.64, 95% CI: 1.37-9.66, p = 0.009). CONCLUSION: The podocyte injury was prevalent and the FPW levels were closely associated with clinicopathological features, especially prognosis, in renal TMA patients.

The influence of alveolar bone healing degree on its potential as a source of human alveolar bone-derived cells.

BACKGROUND: The concept of bone tissue engineering has emerged as a novel alternative approach that comprises three essential components: osteogenic cells, osteoinductive signals and osteoconductive scaffolds. The low-speed drilling represents a useful and accessible autologous source for human alveolar bone-derived cells (hABCs). The aim of this study was to compare the efficacy of two donor sites (healing sites (HS) and non-augmented healed sites (NAHS)) as a source of hABCs. METHODS: Nineteen patients were enrolled in this study. The patients' demographic data were described. Bone type and dental implant location were also determined. The hABCs obtained were characterized. Apoptosis and sclerostin expression in the samples were also assessed with immunohistochemistry. RESULTS: The hABCs left earlier the tissue explants of the HS than the NAHS. The proliferation of the hABCs had reached the sub-confluence stage in both groups. Cellular efficacy was not statistically significant between the two groups. The hABCs exhibited osteogenic phenotype as they expressed bone sialoprotein (BSP), osteopontin (OP) and tissue non-specific alkaline phosphatase (TNAP). In both groups, the level and the distribution pattern of apoptotic cells and sclerostin expression were similar. CONCLUSIONS: Within the limitations of this study, both HS and NAHS were similarly effective to provide hABCs.

Identifying Sialylation Linkages at the Glycopeptide Level by Glycosyltransferase Labeling Assisted Mass Spectrometry (GLAMS).

Precise assignment of sialylation linkages at the glycopeptide level is of importance in bottom-up glycoproteomics and an indispensable step to understand the function of glycoproteins in pathogen-host interactions and cancer progression. Even though some efforts have been dedicated to the discrimination of α2,3/α2,6-sialylated isomers, unambiguous identification of sialoglycopeptide isomers is still needed. Herein, we developed an innovative glycosyltransferase labeling assisted mass spectrometry (GLAMS) strategy. After specific enzymatic labeling, oxonium ions from higher-energy C-trap dissociation (HCD) fragmentation of α2,3-sailoglycopeptides then generate unique reporters to distinctly differentiate those of α2,6-sailoglycopeptide isomers. With this strategy, a total of 1236 linkage-specific sialoglycopeptides were successfully identified from 161 glycoproteins in human serum.

Evaluation of dentinogenesis inducer biomaterials: an in vivo study.

When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. OBJECTIVE: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. METHODOLOGY: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). RESULTS: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. CONCLUSIONS: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications.

Evaluation of dentinogenesis inducer biomaterials: an in vivo study

Abstract When exposure of the pulp to external environment occurs, reparative dentinogenesis can be induced by direct pulp capping to maintain pulp tissue vitality and function. These clinical situations require the use of materials that induce dentin repair and, subsequently, formation of a mineralized tissue. Objective: This work aims to assess the effect of tricalcium silicate cements and mineral trioxide aggregate cements, including repairing dentin formation and inflammatory reactions over time after pulp exposure in Wistar rats. Methodology: These two biomaterials were compared with positive control groups (open cavity with pulp tissue exposure) and negative control groups (no intervention). The evaluations were performed in three stages; three, seven and twenty-one days, and consisted of an imaging (nuclear medicine) and histological evaluation (H&E staining, immunohistochemistry and Alizarin Red S). Results: The therapeutic effect of these biomaterials was confirmed. Nuclear medicine evaluation demonstrated that the uptake of 99mTc-Hydroxymethylene diphosphonate (HMDP) showed no significant differences between the different experimental groups and the control, revealing the non-occurrence of differences in the phosphocalcium metabolism. The histological study demonstrated that in mineral trioxide aggregate therapies, the presence of moderate inflammatory infiltration was found after three days, decreasing during follow-ups. The formation of mineralized tissue was only verified at 21 days of follow-up. The tricalcium silicate therapies demonstrated the presence of a slight inflammatory infiltration on the third day, increasing throughout the follow-up. The formation of mineralized tissue was observed in the seventh follow-up day, increasing over time. Conclusions: The mineral trioxide aggregate (WhiteProRoot®MTA) and tricalcium silicate (Biodentine™) present slight and reversible inflammatory signs in the pulp tissue, with the formation of mineralized tissue. However, the exacerbated induction of mineralized tissue formation with the tricalcium silicate biomaterial may lead to the formation of pulp calcifications

Isolation and characterization of a human cementocyte-like cell line, HCY-23.

Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

Isolation and characterization of a human cementocyte-like cell line, HCY-23

Abstract Cementum is the mineralized tissue covering the tooth root that functions in tooth attachment and post-eruptive adjustment of tooth position. It has been reported to be highly similar to bone in several respects but remains poorly understood in terms of development and regeneration. Here, we investigate whether cementocytes, the residing cells in cellular cementum, have the potential to be protagonist in cementum homeostasis, responding to endocrine signals and directing local cementum metabolism. Cells from healthy erupted human teeth were isolated using sequential collagenase/EDTA digestions, and maintained in standard cell culture conditions. A cementocyte-like cell line was cloned (HCY-23, for human cementocyte clone 23), which presented a cementocyte compatible gene expression signature, including the expression of dentin matrix protein 1 ( DMP1 ), sclerostin ( SOST ), and E11/gp38/podoplanin ( E11 ). In contrast, these cells did not express the odontoblast/dentin marker dentin sialoprotein ( DSPP ). HCY-23 cells produced mineral-like nodules in vitro under differentiation conditions, and were highly responsive to inorganic phosphate (Pi). Within the limits of the present study, it can be concluded that cementocytes are phosphate-responsive cells, and have the potential do play a key role in periodontal homeostasis and regeneration.

[Value of podocalyxin levels in urinary extracellular vesicles for diagnosis of diabetic nephropathy].

OBJECTIVE: To explore the value of detecting podocalyxin (PCX) level in urinary extracellular vesicles for the diagnosis of diabetic nephropathy. METHODS: This study was conducted among 57 diabetic patients admitted during the period from March to September, 2017, including 34 with uncomplicated diabetics and 23 with diabetic nephropathy; 21 patients with other types of nephropathy and 11 healthy individuals were also included to serve as the controls. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to verify the separation of urinary extracellular vesicles. The molecular markers of extracellular vesicles (TSG101 and podocalyxin [PCX]) were detected using Western blotting. PCX levels in extracellular vesicles were also detected using ELISA. RESULTS: TEM reveal the presence of numerous extracellular vesicles in the urine with intact morphology and different sizes, and most of them were below 300 nm in diameter as shown by NTA. TSG101 expression was detected in the samples from all the 4 groups. Positive expression of PCX was detected in the samples from patients with diabetic nephropathy but not in the other groups. In patients with diabetic nephropathy, the mean PCX levels (3.27±2.30 ng/µmol)was significantly higher than those in the healthy control group (1.22±0.36 ng/µmol), uncomplicated diabetes group (2.22±1.29 ng/µmol) and nephropathy group (1.24±0.45 ng/µmol). CONCLUSIONS: PCX level in urinary extracellular vesicles is significantly increased in patients with diabetic nephropathy, suggesting the value of PCX as a potential marker for clinical diagnosis of diabetic nephropathy.

Pre-B acute lymphoblastic leukemia expresses cell surface nucleolin as a 9-O-acetylated sialoglycoprotein.

Precursor B acute lymphoblastic leukemias (pre-B ALLs) abnormally express a specific glycan structure, 9-O-acetylated sialic acid (9-O-Ac-Sia), on their cell surface, but glycoproteins that carry this modification have not been identified. Using three different lectins that specifically recognize this structure, we establish that nucleolin (NCL), a protein implicated in cancer, contains 9-O-Ac-Sia. Surprisingly, antibodies against the glycolipid 9-O-Ac-Sia GD3 also detected 9-O-Ac-Sia NCL. NCL is present on the surface of pre-B ALL cells as a sialoglycoprotein that is partly 9-O-acetylated and conversely, 9-O-Ac-Sia-containing structures other than NCL are present on these cells as well. Interestingly, NCL and the 9-O-Ac-Sia signal had less co-localization on normal pre-B cells. We also investigated regulation of NCL on the cell surface and found that sialidase treatment increased the percentage of cells positive for cell surface NCL, suggesting that sialylation of NCL promotes internalization. Treatment of pre-B ALL cells with the chemotherapy drug vincristine also increased the percentage of cells with surface NCL and correlated with increased 9-O-Ac-Sia expression. All tested leukemia cells including primary samples expressed NCL, suggesting it as a possible therapeutic target. We confirmed this by showing inhibition of cell proliferation in some pre-B ALLs by exposure to a NCL-specific aptamer AS1411.

Effects of Er:YAG and Diode Laser Irradiation on Dental Pulp Cells and Tissues.

Vital pulp therapy (VPT) is to preserve the nerve and maintain healthy dental pulp tissue. Laser irradiation (LI) is beneficial for VPT. Understanding how LI affects dental pulp cells and tissues is necessary to elucidate the mechanism of reparative dentin and dentin regeneration. Here, we show how Er:YAG-LI and diode-LI modulated cell proliferation, apoptosis, gene expression, protease activation, and mineralization induction in dental pulp cells and tissues using cell culture, immunohistochemical, genetic, and protein analysis techniques. Both LIs promoted proliferation in porcine dental pulp-derived cell lines (PPU-7), although the cell growth rate between the LIs was different. In addition to proliferation, both LIs also caused apoptosis; however, the apoptotic index for Er:YAG-LI was higher than that for diode-LI. The mRNA level of odontoblastic gene markers-two dentin sialophosphoprotein splicing variants and matrix metalloprotease (MMP)20 were enhanced by diode-LI, whereas MMP2 was increased by Er:YAG-LI. Both LIs enhanced alkaline phosphatase activity, suggesting that they may help induce PPU-7 differentiation into odontoblast-like cells. In terms of mineralization induction, the LIs were not significantly different, although their cell reactivity was likely different. Both LIs activated four MMPs in porcine dental pulp tissues. We helped elucidate how reparative dentin is formed during laser treatments.

Endometrial L-selectin ligand is downregulated in the mid-secretory phase during the menstrual cycle in women with adenomyosis.

OBJECTIVE: Defects in L-selectin ligand (LSL) expression have been reported to cause implantation failure, but little is known about LSL expression in adenomyosis. This study evaluates LSL expression throughout the menstrual cycle in women with adenomyosis. MATERIALS AND METHODS: Endometrial samples were obtained from reproductive-aged women with adenomyosis who underwent hysterectomy. A total of 42 endometrial biopsies were included. There were 12 women in proliferative phase, 10 in early-secretory phase, 9 in mid-secretory phase, and 11 in late-secretory phase. Immunohistochemistry, western blotting, and RT-PCR were performed to evaluate LSL expression. A non-parametric Kruskal-Wallis one-way analysis of variance with multiple comparisons was performed to examine differences among menstrual phases. RESULTS: Immunohistochemistry analysis with MECA-79 shows that LSL is expressed with weak intensity in the endometrium in all phases. In the luminal epithelium, MECA-79 reactivity increased from the proliferative to the late-secretory phase but decreased in the mid-secretory phase. There were significant differences in the mean histological scores (HSCOREs) among the proliferative, early-secretory, and late-secretory phases (p < 0.05). Five LSL genes were detected in the adenomyotic endometria: PODXL, EMCN, CD300LG, GLYCAM1, and CD34. The mRNA expression of LSL genes occurred differentially among phases. Moreover, PODXL differed significantly among phases (p < 0.05). CONCLUSIONS: LSL expressions were downregulated in the luminal epithelium of adenomyotic endometria in the mid-secretory phase. The mRNA expressions of LSL genes also had differential expression patterns throughout the menstrual cycle, especially for PODXL. Our study showed that adenomyosis may cause abnormalities of LSL production in the mid-secretory phase, which may contribute to impaired endometrial receptivity and implantation failure.

Metabolic glycan labeling-assisted discovery of cell-surface markers for primary neural stem and progenitor cells.

A chemical approach was developed for identifying cell-surface markers for primary neural stem cells (NSCs). Using an in vitro coculture system of primary NSCs combined with metabolic labeling of sialoglycans with bioorthogonal functional groups, we selectively enriched and identified a list of cell-surface sialoglycoproteins that were more abundantly expressed in neural stem and progenitor cells.

Identification of Sialic Acid Linkages on Intact Glycopeptides via Differential Chemical Modification Using IntactGIG-HILIC.

Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration. Graphical Abstract ᅟ.

Value of immunohistochemical expression of podocalyxin in active lupus nephritis.

Podocalyxin is an electronegative sialoglycoprotein that prevents the podocyte foot process from collapsing. The aim of this study was to detect an association between the glomerular immunohistochemical (IHC) expression of podocalyxin and the degree of podocyte effacement detected by electron microscopy, and to evaluate the role of podocalyxin IHC expression as a novel marker for disease activity in lupus nephritis (LN). METHODS: Thirty-two renal biopsies of active lupus nephritis patients were studied. Clinical assessment by the systemic lupus activity measure (SLAM-R) score and laboratory data were included [serum creatinine, 24-h urinary protein, antinuclear antibodies (ANA), anti-double-strand DNA antibodies (anti-dsDNA), C3 and C4]. Light (L/M) and electron microscopic (E/M) examination was conducted. Podocyte loss was evaluated by immunohistochemistry with monoclonal anti-podocalyxin antibodies by means of a semiquantitative score that was graded from 0 to 4+ according to the percentage of glomerular involvement. RESULTS: 22 cases (68.8%) with LN class IV, 6 (18.8%) with class III and 4 (12.5%) with class V. The mean age was (25.41±10.13) years. There was a significant negative correlation between IHC podocalyxin score and LN class, and NIH activity parameters such as leukocyte infiltration, endocapillary proliferation, fibrinoid necrosis and cellular crescent and disease activity index but not chronicity index. There was a highly significant negative correlation between IHC podocalyxin and podocyte effacement by E/M (rs=-0.903, P=0.000), and E/M immune deposits (r=-0.53, P=0.001), and a significant association with degree of proteinuria, ANA and SLAM score (P<0.05). CONCLUSIONS: Podocyte loss indicated by podocalyxin immunohistochemical expression reflects the degree of activity and severity of LN and the degree of podocyte effacement by E/M.

Implanted Dental Pulp Cells Fail to Induce Regeneration in Partial Pulpotomies.

Cell-based partial pulp regeneration is one of the promising approaches to obtain newly formed functional dentin-pulp complex. It relies on the preservation of the healthy tissue while regenerating the damaged pulp. The aim of this study was to investigate whether this regenerative process could be achieved by implanting porcine dental pulp cells (pDPCs) in pulp defects in the minipig. By split-mouth model, self-assembling injectable nanopeptide hydrogel, with and without pDPCs, was implanted after cameral pulpotomy in premolars and molars. At day 21 after surgery, 3-dimensional morphometric characterization, Masson's trichrome staining, and immunolabeling for DSP and BSP (dentin sialoprotein and bone sialoprotein) were performed on treated teeth. This study demonstrated no pulp regeneration but systematic reparative dentinogenesis. In fact, regardless of the presence of pDPCs in the scaffold, an osteodentin bridge-the microarchitecture of which significantly differed from the native dentin-was systematically obtained. Furthermore, the presence of pDPCs significantly affected the microstructure of the dentin bridges. In the radicular area of each treated tooth, hyperemia in the remaining pulp and external root resorptions were observed. Under the conditions tested in this work, pulp regeneration was not achieved, which highlights the need of further investigations to develop favorable regenerative microenvironment.

Facile and Selective Enrichment of Intact Sialoglycopeptides Using Graphitic Carbon Nitride.

Combining powerful selectivity, high stability, convenient operation, mild condition, and eco-friendliness, a novel graphitic carbon nitride (g-C3N4)-based enrichment method of intact sialoglycopeptides (SGs) was developed. The intact SGs could be simply enriched and separated from protein tryptic digests by hydrogen bonding without damage of glycan structures due to the specific structure of g-C3N4. By optimizing the enrichment and elution conditions, 45 and 38 SGs were detected from the tryptic digests of bovine fetuin and transferrin, respectively. Under the synergistic effect of hydrogen bonding and electrostatic adsorption, the SGs could be enriched simply in less than 2 h with a detection limit of 50 fmol. The method is repeatable due to the high stability of g-C3N4 and the simple protocol of the method, indicating the potential application of g-C3N4 in efficient and selective enrichment of intact SGs.

Cancer Secretome May Influence BSP and DSP Expression in Human Salivary Gland Cells.

One of the biggest challenges in managing head and neck cancers, especially salivary gland cancers, is the identification of secreted biomarkers of the disease that can be evaluated noninvasively. A relevant source of enriched tumor markers could potentially be found in the tumor secretome. Although numerous studies have evaluated secretomes from various cancers, the influence of the cancer secretome derived from salivary gland cancers on the behavior of normal cells has not yet been elucidated. Our data indicate that secretome derived from salivary gland cancer cells can influence the expression of two potential biomarkers of oral cancer-namely, bone sialoprotein (BSP) and dentin sialoprotein (DSP)-in normal salivary gland cells. Using routine immunohistochemistry, immunofluorescence, and immunoblotting techniques, we demonstrate an enrichment of BSP and DSP in human salivary gland (HSG) cancer tissue, unique localizations of BSP and DSP in HSG cancer cells, and enriched expression of BSP and DSP in normal salivary gland cells exposed to a cancer secretome. The secretome domain of the cancer microenvironment could alter signaling cascades responsible for normal cell proliferation, migration, and invasion, thus enhancing cancer cell survival and the potential for cancer progression. The cancer secretome may be critical in maintaining and stimulating "cancer-ness," thus potentially promoting specific hallmarks of metastasis.

Expressão de DMP-1, DSPP, VEGF e FGF2 em células derivadas da polpa de dentes decíduos após o uso do laser de baixa intensidade / FGF2, VEGF, DSPP, and DMP-1 expression in cells of the pulp deciduous teeth with a low intensity laser

O objetivo do presente trabalho foi avaliar o efeito de diferentes densidades de energia do Laser de Baixa Intensidade na viabilidade e proliferação celular de fibroblastos derivados da polpa de dentes decíduos humanos e na expressão de RNAm para DMP- 1, DSPP, VEGF e FGF-2. Amostras de fibroblastos pulpares da polpa de dentes decíduos humanos foram obtidas de um Biorrepositório. Foram utilizadas células entre a 4ª e a 7ª passagem, irradiadas com Laser de Baixa Intensidade (InGaAlP) de acordo com os seguintes grupos experimentais: Grupo 1: 1,2 J/cm2 - 05 mW - 10s; Grupo 2: 2,5 J/cm2 - 05 mW - 20s; Grupo 3: 3,7 J/cm2 - 05 mW - 30s; Grupo 4: 5,0 J/cm2 - 05 mW - 40s; Grupo 5: 6,2 J/cm2 - 05 mW - 50s; Grupo 6: 2,5 J/cm2 - 10 mW - 10s; Grupo 7: 3,7 J/cm2 - 15 mW - 10s; Grupo 8: 5,0 J/cm2 - 20 mW - 10s; Grupo 9: 6,2 J/cm2 - 25 mW - 10s; Controle Negativo: DMEM 1% SFB ­ não irradiado; Controle Positivo: DMEM 10% SFB ­ não irradiado. As técnicas utilizadas para as análises de viabilidade e proliferação celular foram MTT e CV. A técnica utilizada para avaliação da expressão de RNAm para os alvos DMP-1, DSPP, VEGF e FGF-2 foi RT-PCR. Os resultados foram analisados pelo método ANOVA a dois critérios, seguido pelo teste de Tukey (p<0,05). Para o teste MTT, na comparação intragrupos observou-se que houve diferença estatisticamente significativa entre os períodos 6h, 12h e 24h, diminuindo a viabilidade com o passar do tempo, exceto para o Grupo 1. Na comparação intergrupos, o MTT mostrou menor viabilidade para o controle negativo em comparação com os outros grupos (p<0,05), exceto com grupo 5 (5mW/50 seg). Observou-se que os grupos com maiores potências (10mW, 15mW, 20mW e 25mW), menores tempos de aplicação (10 segundos) e densidades de energia entre 2,5 J/cm2 e 6,2 J/cm2, apresentaram estatisticamente maior viabilidade que o grupo com menor potência (5mW), maior tempo de aplicação (50 segundos) e densidade de energia de 6,2 J/cm2. Para o teste CV não houve diferença intragrupos, mas houve diferença intergrupos entre os controles positivo e negativo. Para a expressão de RNAm por RTPCR, os fatores de crescimento VEGF e FGF-2 foram expressos em grande quantidade no primeiro período experimental, enquanto que DMP-1 e DSPP não foram expressos de maneira significativa. De acordo com os resultados obtidos, frente as diferentes densidades de energia, sugere-se que a terapia a laser de baixa intensidade manteve os fibroblastos viáveis e aumentou a expressão de RNAm para VEGF e FGF-2.(AU)

This study aimed to evaluate the effect of different energy densities of Low Level Laser (LLL) on cell viability and proliferation of fibroblasts from the pulp of human primary teeth (DHPF) and on the RNAm expression of DMP-1, DSPP, VEGF and FGF-2. DHPF were obtained from a biorepository and used at passages 4th to 7th. The cells were irradiated with LLL (InGaAlP) according to the following experimental groups: Group 1: 1.2 J/cm2 - 05 mW - 10s; Group 2: 2.5 J/cm2 - 05 mW - 20s; Group 3: 3.7 J/cm2 - 05 mW - 30s; Group 4: 5.0 J/cm2 - 05 mW - 40s; Group 5: 6.2 J/cm2 - 05 mW - 50s; Group 6: 2.5 J/cm2 - 10 mW - 10s; Group 7: 3,7 J/cm2 - 15 mW - 10s; Group 8: 5.0 J/cm2 - 20 mW - 10s; Group 9: 6.2 J/cm2 - 25 mW - 10s; Negative Control: DMEM 1% SFB ­ not irradiated; Positive Control: DMEM 10% SFB ­ not irradiated. The techniques used to evaluate the cell viability/proliferation were MTT and Crystal Violet (CV) assays. RT-PCR was used to verify the RNAm expression of DMP-1, DSPP, VEGF, and FGF-2. Two-way ANOVA, followed by Tukey test (p<0.05) was used to analyze the results. In the intragroup comparison, MTT assay revealed statistically significant differences among the periods of 6h, 12h, and 24h, with viability reduction as time went by, except for Group 1. In the intergroup comparison, the MTT assay showed that the negative control had statistically lower viability than that of the other groups (p<0.05), except for Group 5 (5mW/50 s). The groups with higher powers (10mW, 15mW, 20mW, and 25mW), shortest application periods (10 s), and energy densities between 2.5 J/cm2 and 6.2 J/cm2 exhibited statistically higher viability than that of the group with small power (5mW), longer application period (50 s), and energy density of 6.2 J/cm2 . CV assay did not show intergroup statistically differences. In the intragroup comparison, CV assay revealed statistically significant differences between positive and negative controls (p<0.05). RT-PCR revealed increased RNAm expression of the growth factors VEGF and FGF-2 at the first experimental period, while DMP-1 and DSPP was not significant. Based on the results and different energy densities used, LLL maintained DHPF viability and increased the RNAm expression of VEGF and FGF-2.(AU)