Temperature effects on plasmalogen profile and quality characteristics in Pacific oyster (Crassostrea gigas) during depuration.

The quality of Pacific oyster (Crassostrea gigas) can be affected by many factors during depuration, in which temperature is the major element. In this study, we aim to determine the quality and plasmalogen changes in C. gigas depurated at different temperatures. The quality was significantly affected by temperature, represented by varying survival rate, glycogen content, total antioxidant capacity, alkaline phosphatase activity between control and stressed groups. Targeted MS analysis demonstrated that plasmalogen profile was significantly changed during depuration with PUFA-containing plasmalogen species being most affected by temperature. Proteomics analysis and gene expression assay further verified that plasmalogen metabolism is regulated by temperature, specifically, the plasmalogen synthesis enzyme EPT1 was significantly downregulated by high temperature and four plasmalogen-related genes (GPDH, PEDS, Pex11, and PLD1) were transcriptionally regulated. The positive correlations between the plasmalogen level and quality characteristics suggested plasmalogen could be regarded as a quality indicator of oysters during depuration.

A distinct, high-affinity, alkaline phosphatase facilitates occupation of P-depleted environments by marine picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus, the two most abundant phototrophs on Earth, thrive in oligotrophic oceanic regions. While it is well known that specific lineages are exquisitely adapted to prevailing in situ light and temperature regimes, much less is known of the molecular machinery required to facilitate occupancy of these low-nutrient environments. Here, we describe a hitherto unknown alkaline phosphatase, Psip1, that has a substantially higher affinity for phosphomonoesters than other well-known phosphatases like PhoA, PhoX, or PhoD and is restricted to clade III Synechococcus and a subset of high light I-adapted Prochlorococcus strains, suggesting niche specificity. We demonstrate that Psip1 has undergone convergent evolution with PhoX, requiring both iron and calcium for activity and likely possessing identical key residues around the active site, despite generally very low sequence homology. Interrogation of metagenomes and transcriptomes from TARA oceans and an Atlantic Meridional transect shows that psip1 is abundant and highly expressed in picocyanobacterial populations from the Mediterranean Sea and north Atlantic gyre, regions well recognized to be phosphorus (P)-deplete. Together, this identifies psip1 as an important oligotrophy-specific gene for P recycling in these organisms. Furthermore, psip1 is not restricted to picocyanobacteria and is abundant and highly transcribed in some α-proteobacteria and eukaryotic algae, suggesting that such a high-affinity phosphatase is important across the microbial taxonomic world to occupy low-P environments.

Selected osteointegration markers in different timeframes after dental implantation: findings and prognostic value.

The study aimed to determine the osteointegration markers after dental implantation and evaluate their predictive value. The study was performed on 60 practically healthy persons who needed teeth rehabilitation using dental implants. The conical-shaped implants (CI) and hexagonal implants (HI) were used. The content of Osteopontin (OPN), Osteocalcin (OC), Alkaline Phosphatase (ALP), Osteoprotegerin (OPG), and nitric oxide (NO) was determined in patients' gingival crevicular fluid (GCF) and peri-implant sulcular fluid (PISF), collected 1, 3, and 6 months after implantation. During the 3-6 months of observation level of OPN increased in patients with CIs (<50 years > 50 years) and HIs (<50 years) (CI: <50 years F = 36.457, p < 0.001; >50 years F = 30.104, p < 0.001; HI < 50 years F = 2.246, p < 0.001), ALP increased in patients with CIs (<50 years: F = 19.58, p < 0.001; >50 years: F = 12.01; p = 0.001) and HIs (<50 years) (F = 18.51, p < 0.001), OC increased in patients <50 years (CI: F = 33.72, p < 0.001; HI: F = 55.57, p < 0.001), but in patients >50 years - on the 3 days month (CI: F = 18.82, p < 0.001; HI: F = 26.26, p < 0.001), but sharply decreased at the end of sixth month. OPG increased during 1-3 months of the observation in patients <50 years (CI: F = 4.63, p = 0.037; HI: F = 2.8927, p = 0.046), but at the end of the sixth month returned to the initial level; NO content in PISF increased in patients with CI (>50 years) during 1-6 months of the observation (F = 27.657, p < 0.001). During the post-implantation period, age-related differences in osteointegration were observed. Patients <50 years old had relatively high levels of OPN, ALP, OC, and OPG in PISF, resulting in less alveolar bone destruction around dental implants and more intensive osteointegration. These indicators may be used as biological markers for monitoring implant healing. The process of osseointegration was more intense in CIs due to their comparatively high mechanical loading.

Hydrolyzed egg yolk peptide prevented osteoporosis by regulating Wnt/ß-catenin signaling pathway in ovariectomized rats.

Hydrolyzed egg yolk peptide (YPEP) was shown to increase bone mineral density in ovariectomized rats. However, the underlying mechanism of YPEP on osteoporosis has not been explored. Recent studies have shown that Wnt/ß-catenin signaling pathway and gut microbiota may be involved in the regulation of bone metabolism and the progression of osteoporosis. The present study aimed to explore the preventive effect of the YPEP supplementation on osteoporosis in ovariectomized (OVX) rats and to verify whether YPEP can improve osteoporosis by regulating Wnt/ß-catenin signaling pathway and gut microbiota. The experiment included five groups: sham surgery group (SHAM), ovariectomy group (OVX), 17-ß estradiol group (E2: 25 µg /kg/d 17ß-estradiol), OVX with low-dose YPEP group (LYPEP: 10 mg /kg/d YPEP) and OVX with high-dose YPEP group (HYPEP: 40 mg /kg/d YPEP). In this study, all the bone samples used were femurs. Micro-CT analysis revealed improvements in both bone mineral density (BMD) and microstructure by YPEP treatment. The three-point mechanical bending test indicated an enhancement in the biomechanical properties of the YPEP groups. The serum levels of bone alkaline phosphatase (BALP), bone gla protein (BGP), calcium (Ca), and phosphorus (P) were markedly higher in the YPEP groups than in the OVX group. The LYPEP group had markedly lower levels of alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide of type I collagen (CTX-I) than the OVX group. The YPEP groups had significantly higher protein levels of the Wnt3a, ß-catenin, LRP5, RUNX2 and OPG of the Wnt/ß-catenin signaling pathway compared with the OVX group. Compared to the OVX group, the ratio of OPG/RANKL was markedly higher in the LYPEP group. At the genus level, there was a significantly increase in relative abundance of Lachnospiraceae_NK4A136_group and a decrease in Escherichia_Shigella in YPEP groups, compared with the OVX group. However, in the correlation analysis, there was no correlation between these two bacteria and bone metabolism and microstructure indexes. These findings demonstrate that YPEP has the potential to improve osteoporosis, and the mechanism may be associated with its modulating effect on Wnt/ß-catenin signaling pathway.

[Zinc finger protein-36 deficiency inhibits osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells and preosteoblasts by activating the ERK/MAPK pathway].

OBJECTIVE: To explore the role of zinc finger protein 36(ZFP36) in regulating osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and preosteoblasts. METHODS: ZFP36 expression was observed in primary mouse BMSCs and mouse preosteoblasts (MC3T3-E1 cells) during induced osteogenic differentiation. Zfp36-deficient cell models were constructed in the two cells using RNA interference technique and the changes in differentiation capacities of the transfected cells into osteoblasts were observed. Transcriptome sequencing was used to investigate the potential mechanisms of ZFP36 for regulating osteoblast differentiation of the two cells. U0126, a ERK/MAPK signal suppressor, was used to verify the regulatory mechanism of Zfp36 in osteogenic differentiation of Zfp36-deficient cells. RESULTS: During the 14-day induction of osteogenic differentiation, both mouse BMSCs and MC3T3-E1 cells exhibited increased expression of ZFP36, and its mRNA expression reached the peak level on Day 7(P < 0.0001). The Zfp36-deficient cell models showed reduced intensity of alkaline phosphatase (ALP) staining and alizarin red staining with significantly lowered expressions of the osteogenic marker genes including Alpl, Sp7, Bglap and Ibsp (P < 0.01). Transcriptome sequencing verified the reduction of bone mineralization-related gene expressions in Zfp36-deficient cells and indicated the involvement of ERK signaling in the potential regulatory mechanism of Zfp36. Immunoblotting showed that pERK protein expression increased significantly in Zfp36-deficient cells compared with the control cells. In Zfp36-deficient MC3T3-E1 cells, inhibition of activated ERK/MAPK signaling with U0126 resulted in obviously enhanced ALP staining and significantly increased expressions of osteoblast differentiation markers Runx2 and Bglap (P < 0.05). CONCLUSIONS: ZFP36 is involved in the regulation of osteoblast differentiation of mouse BMSCs and preosteoblasts, and ZFP36 deficiency causes inhibition of osteoblast differentiation of the cells by activating the ERK/MAPK signaling pathway.

Detection of alkaline phosphatase activity with a CsPbBr3/Y6 heterojunction-based photoelectrochemical sensor.

An ultra-sensitive photoelectrochemical (PEC) sensor based on perovskite composite was developed for the determination of alkaline phosphatase (ALP) in human serum. In contrast to CsPbBr3 or Y6 that generated anodic current, the heterojunction of CsPbBr3/Y6 promoted photocarriers to separate and generated cathodic photocurrent. Ascorbic acid (AA) was produced by ALP hydrolyzing L-ascorbic acid 2-phosphate trisodium salt (AAP), which can combine with the holes on the photoelectrode surface, accelerating the transmission of photogenerated carriers, leading to enhanced photocurrent intensity. Thus, the enhancement of PEC current was linked to ALP activity. The PEC sensor exhibits good sensitivity for detection of ALP owing to the unique photoelectric properties of the CsPbBr3/Y6 heterojunction. The detection limit of the sensor was 0.012 U·L-1 with a linear dynamic range of 0.02-2000 U·L-1. Therefore, this PEC sensing platform shows great potential for the development of different PEC sensors.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

The utility of Hibiscus sabdariffa L. to prepare metal oxides NPs for clinical application on osteoporosis supported by theoretical study.

Green synthesis of metal oxides as a treatment for bone diseases is still exploring. Herein, MgO and Fe2O3 NPs were prepared from the extract of Hibiscus sabdariffa L. to study their effect on vit D3, Ca+2, and alkaline phosphatase enzyme ALP associated with osteoporosis. Computational chemistry was utilized to gain insight into the possible interactions. These oxides were characterized by X-ray diffraction, SEM, FTIR, and AFM. Results revealed that green synthesis of MgO and Fe2O3 NPs was successful with abundant. MgO NPs were in vitro applied on osteoporosis patients (n = 35) and showed a significant elevation of vit D3 and Ca+2 (0.0001 > p < 0.001) levels, compared to healthy volunteers (n = 25). Thus, Hibiscus sabdariffa L. is a good candidate to prepare MgO NPs, with a promising enhancing effect on vit D3 and Ca+2 in osteoporosis. In addition, interactions of Fe2O3 and MgO NPs with ALP were determined by molecular docking study.

Enzymatic fluorescent supramolecular hydrogel with aggregation-induced emission characteristics for sensing alkaline phosphatase.

Alkaline phosphatase is an important biomarker for medical diagnosis. An enzymatic fluorescence supramolecular hydrogel with AIE properties was developed and used for sensing alkaline phosphatase in vitro and in living cells. In the presence of ALP, K(TPE)EFYp was partially converted to the hydrogelator K(TPE)EFY and self-assembled into nanofibers to form Hydrogel. With the sol-gel transition and the AIE effect, the fluorescence emission was turned on. The linear concentration range of ALP activity in vitro quantified by this method was determined as 0-3 U/L with aLODat 0.02 U/L. In addition, cell imaging and serum experiment showed that K(TPE)EFYp could also be used to detect ALP activity in living cells and biological samples.

Metastatic Melanoma of the Common Bile Duct Presented with Dyspepsia.

Malignant melanoma (MM) is an aggressive tumor that can metastasize to any organ, but biliary tract metastasis is scarce. We describe a very rare case of MM metastasis to the common bile duct (CBD), presented with only dyspeptic symptoms. The patient had mildly elevated alkaline phosphatase and gamma-glutamyl transferase levels. Magnetic resonance cholangiopancreatography demonstrated a dilated common bile duct with a distal stricture. The MM diagnosis was established with the ampulla of Vater biopsy specimens obtained by endoscopic retrograde cholangiopancreatography (ERCP), and the patient's symptoms were resolved after biliary stenting. Both primary CBD cancer and other cancer types like MM that metastasize to CBD can cause obstruction and can be manifested only by dyspeptic symptoms. MM metastasis to CBD can cause obstruction manifested only by dyspeptic symptoms without obstructive jaundice. ERCP can be employed as a promising option for treatment and diagnosis. New-onset dyspeptic symptoms in patients with a history of MM should be investigated thoroughly, especially in the context of biliary metastasis.

[Chidamide Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells from Patients with Myelodysplastic Syndromes].

OBJECTIVE: To explore the effects and mechanisms of chidamide on the osteogenic differentiation of bone marrow mesenchymal stromal cells (MSC) from myelodysplastic syndromes (MDS). METHODS: MSC were isolated and cultured from bone marrow of MDS patients and healthy donors. CCK-8 assay was used to detect the effects of chidamide on the proliferation of MSC. The effects of chidamide on the activity of histone deacetylase (HDAC) in MSC was measured by a fluorescence assay kit and Western blot. Alkaline phosphatase (ALP) activity was detected on day 3 and calcium nodule formation was observed by Alizarin Red staining on day 21 after osteogenic differentiation. The expression of early and late osteogenic genes was detected on day 7 and day 21, respectively. RT-PCR and Western blot were used to detect the effects of chidamide on mRNA and protein expression of RUNX2 which is the key transcription factor during osteogenesis. RESULTS: As the concentration of chidamide increased, the proliferation of MSC was inhibited. However, at a low concentration (1 µmol/L), chidamide had no significant inhibitory effect on MSC proliferation but significantly inhibited HDAC activity. In MSC from both MDS patients and healthy donors, chidamide (1 µmol/L) significantly increased ALP activity, calcium nodule formation, thereby mRNA expression of osteogenic genes, and restored the reduced osteogenic differentiation ability of MDS-MSC compared to normal MSC. Mechanistic studies showed that the osteogenic-promoting effect of chidamide may be related to the upregulation of RUNX2 . CONCLUSION: Chidamide can inhibit HDAC activity in MSC, upregulate the expression of the osteogenic transcription factor RUNX2, and promote the osteogenic differentiation of MDS-MSC.

Probiotics from kefir: Evaluating their immunostimulant and antioxidant potential in the carpet shell clam (Ruditapesdecussatus).

This study aimed to investigate the impact of incorporating kefir into the diet on biometric parameters, as well as the immune and antioxidant responses of the carpet shell clam (Ruditapes decussatus) after an experimental infection by Vibrio alginolyticus. Clams were divided into a control group and a treated group. The control group was fed on spirulina (Arthrospira platensis) alone. While, the treated group was fed on spirulina supplemented with 10% dried kefir. After 21 days, clams were immersed in a suspension of V. alginolyticus 5 × 105 CFU mL -1 for 30 min. Seven days after experimental infection, survival was 100% in both groups. The obtained results showed a slight increase in weight and condition index in clams fed with kefir-supplemented diet for 21 days compared to control clams. Regarding antioxidant responses, the treated group showed higher superoxide dismutase activity compared to the control group. However, the malondialdehyde level was lower in the treated clams than in the control. In terms of immune parameters, the treated group showed slightly elevated activities of phenoloxidase, lysozyme and alkaline phosphatase, whereas a decreased lectin activity was observed compared to the control group. The obtained results suggest that kefir enhanced both the antioxidant and immune response of infected clams.

Effects of surface nanomorphology on the senescence of periodontal ligament stem cells.

OBJECTIVES: The effect of TiO2 nanotube morphology on the differentiation potency of senescent periodontal ligament stem cells was investigated. METHODS: Two types of titanium sheets with TiO2 nanotube morphology (20V-NT and 70V-NT) were prepared via anodic oxidation at 20 and 70 V separately, and their surface morphology was observed. Young periodontal ligament stem cells were cultivated in an osteogenic induction medium, and the most effective surface morphology in promoting osteogenic differentiation was selected. RO3306 and Nutlin-3a were used to induce the aging of young periodontal ligament stem cells, and senescent periodontal ligament stem cells were obtained. The osteogenic differentiation of senescent periodontal ligament stem cells was induced, and the effect of surface morphology on osteogenic differentiation was observed. RESULTS: Nanotube morphology was achieved on the surfaces of titanium sheets through anodic oxidation, and the diameters of the nanotubes increased with voltage. A significant difference in the effect of nanotube morphology was found among nanotubes with different diameters in the young periodontal ligament stem cells. The surface nanotube morphology of 20V-NT had a more significant effect that promoted osteogenic differentiation. Compared with a smooth titanium sheet, the surface nanotube morphology of 20V-NT increased the number of alkaline phosphatase-positive senescent periodontal ligament stem cells and promoted calcium deposition and the expression of osteogenic marker genes Runt-related transcription factor 2, osteopontin, and osteocalcin. CONCLUSIONS: A special nanotube morphology enhances the differentiation ability of senescent periodontal ligament stem cells, provides an effective method for periodontal regeneration, and further improves the performance of implants.

A bioetching-induced visualized-organic photoelectrochemical transistor dual-signal mode sensor for alkaline phosphatase detection.

A study of an integrated OPECT biosensor gate and the EC color-changing region on the same chip was carried out, achieving sensitive detection through bioetching-induced signal changes. Enzymatic bioetching enables specific alkaline phosphatase (ALP) detection by catalyzing the production of CdS, which modulates the channel current and generates a visual signal.

Enhanced osteogenic differentiation potential of Arnica montana and Bellis perennis in C3H10T1/2 multipotent mesenchymal stem cells.

BACKGROUND: Arnica montana and Bellis perennis are two medicinal plants that are thought to accelerate bone repair in homoeopathic literature. Mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate and regenerate bone or osteogenesis. Hence, we aimed to determine the role of Arnica montana and Bellis perennis on the osteogenic differentiation of the C3H10T1/2 stem cell line. METHODS AND RESULTS: The cell proliferation of Arnica montana and Bellis perennis was evaluated by MTT assay. Osteogenic differentiation of C3H10T1/2 was induced by the addition of ß-glycerophosphate, ascorbic acid and dexamethasone in the differentiation medium over 3 weeks. Cells were treated with Arnica montana and Bellis perennis individually as well as in combination. The osteogenic differentiation potential of Arnica montana and Bellis perennis to differentiate C3H10T1/2 into osteoblasts was measured by alkaline phosphatase activity, alizarin red staining and the expression of Osteocalcin using immunostaining and qRT-PCR. Arnica montana and Bellis perennis could enhance C3H10T1/2 cell proliferation at 1600 µg. Further, the compound showed the ability to augment osteogenesis as confirmed by increased expression of alkaline phosphatase and enhanced calcium accumulation as seen by the Alizarin Red staining and quantification. Enhanced osteogenesis was further supported by the increased expression of osteocalcin in the treated cells with individual and combined doses of Arnica montana and Bellis perennis. Therefore, the findings provide additional support for the positive impact of Arnica montana and Bellis perennis on bone formation. CONCLUSIONS: Our findings suggest that homoeopathic compounds Arnica montana and Bellis perennis can augment osteogenesis individually as well as in combination.

Repurposing the drug, amprolium as a novel molluscicide against the land snail (Eobania vermiculata).

Amprolium (AMP) is an organic compound used as a poultry anticoccidiostat. The aim of this work is to repurpose AMP to control the land snail, Eobania vermiculata in the laboratory and in the field. When snails treated with ½ LC50 of AMP, the levels of alkaline phosphatase (ALP), total lipids (TL), urea, creatinine, malondialdehyde (MDA), catalase (CAT), and nitric oxide (NO) were significantly increased, whereas the levels of acetylcholinesterase (AChE), total protein (TP), and glutathione (GSH) decreased. It also induced histopathological and ultrastructural changes in the digestive gland, hermaphrodite gland, kidney, mucus gland, and cerebral ganglion. Furthermore, scanning electron micrographs revealed various damages in the tegumental structures of the mantle-foot region of E. vermiculata snails. The field application demonstrated that the AMP spray caused reduced percentages in snail population of 75 and 84% after 7 and 14 days of treatment. In conclusion, because AMP disrupts the biology and physiology of the land snail, E. vermiculata, it can be used as an effective molluscicide.

The role of the Piezo1 channel in osteoblasts under cyclic stretching: A study on osteogenic and osteoclast factors.

OBJECTIVES: Orthodontic tooth movement is a mechanobiological reaction induced by appropriate forces, including bone remodeling. The mechanosensitive Piezo channels have been shown to contribute to bone remodeling. However, information about the pathways through which Piezo channels affects osteoblasts remains limited. Thus, we aimed to investigate the influence of Piezo1 on the osteogenic and osteoclast factors in osteoblasts under mechanical load. MATERIALS AND METHODS: Cyclic stretch (CS) experiments on MC3T3-E1 were conducted using a BioDynamic mechanical stretching device. The Piezo1 channel blocker GsMTx4 and the Piezo1 channel agonist Yoda1 were used 12 h before the application of CS. MC3T3-E1 cells were then subjected to 15% CS, and the expression of Piezo1, Piezo2, BMP-2, OCN, Runx2, RANKL, p-p65/p65, and ALP was measured using quantitative real-time polymerase chain reaction, western blot, alkaline phosphatase staining, and immunofluorescence staining. RESULTS: CS of 15% induced the highest expression of Piezo channel and osteoblast factors. Yoda1 significantly increased the CS-upregulated expression of Piezo1 and ALP activity but not Piezo2 and RANKL. GsMTx4 downregulated the CS-upregulated expression of Piezo1, Piezo2, Runx2, OCN, p-65/65, and ALP activity but could not completely reduce CS-upregulated BMP-2. CONCLUSIONS: The appropriate force is more suitable for promoting osteogenic differentiation in MC3T3-E1. The Piezo1 channel participates in osteogenic differentiation of osteoblasts through its influence on the expression of osteogenic factors like BMP-2, Runx2, and OCN and is involved in regulating osteoclasts by influencing phosphorylated p65. These results provide a foundation for further exploration of osteoblast function in orthodontic tooth movement.

[Correlation between CRAB Symptoms and Antioxidant Enzyme Activity in Patients with Multiple Myeloma].

OBJECTIVE: To investigate the relationship between clinical indicators of CRAB symptoms and antioxidant enzyme activity in patients with multiple myeloma (MM). METHODS: The activity of catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase (SOD) in the bone marrow supernatants of 44 patients with MM and 12 patients with non-malignant hematological diseases was detected by colorimetric assay, and then the differences in the activity of antioxidant enzymes between the two groups were compared. Furthermore, the relationship between the activity of antioxidant enzymes in the MM group and the levels of serum calcium, serum creatinine (Scr), hemoglobin (Hb), alkaline phosphatase (ALP) as well as bone lesions were analyzed. RESULTS: The antioxidant enzyme activity was lower in MM patients compared with the control group (P < 0.05). When the concentrations of serum calcium and ALP were higher than the normal levels, Hb was lower than 85 g/L, and there were multiple bone lesions, the activity of CAT, SOD and GPX was significantly declined (P < 0.05); When the concentration of Scr≥177 µmol/L, the activity of GPX was significantly declined (P < 0.05). Regression analyses showed that CAT, SOD and GPX were negatively correlated with serum calcium (r =-0.538, r =-0.456, r =-0.431), Scr (r =-0.342, r =-0.384, r =-0.463), and ALP (r =-0.551, r =-0.572, r =-0.482). CONCLUSION: The activity of antioxidant enzymes, including CAT, SOD and GPX, were decreased in patients with MM and they were negatively correlated with some clinical indicators of CRAB symptoms (such as serum calcium, Scr, and ALP), which suggests that promoting the activity of antioxidant enzymes may be beneficial to treat the CRAB symptoms of the patients with MM.

[Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway].

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Decellularized human amniotic membrane scaffolds: influence on the biological behavior of dental pulp stem cells.

OBJECTIVE: The objective of this study was to assess the characterization of human acellular amniotic membrane (HAAM) using various decellularization methods and their impact on the proliferation and differentiation of human dental pulp stem cells (DPSCs). The goal was to identify scaffold materials that are better suited for pulp regeneration. METHODS: Six different decellularization methods were used to generate the amniotic membranes. The characteristics of these scaffolds were examined through hematoxylin and eosin (H&E) staining, scanning electron microscopy (SEM), and immunohistofluorescence staining (IHF). The DPSCs were isolated, cultured, and their capacity for multidirectional differentiation was verified. The third generation (P3) DPSCs, were then combined with HAAM to form the decellularized amniotic scaffold-dental pulp stem cell complex (HAAM-DPSCs complex). Subsequently, the osteogenic capacity of the HAAM-DPSCs complex was evaluated using CCK8 assay, live-dead cell staining, alizarin red and alkaline phosphatase staining, and real-time quantitative PCR (RT-PCR). RESULTS: Out of the assessed decellularization methods, the freeze-thaw + DNase method and the use of ionic detergent (CHAPS) showed minimal changes in structure after decellularization, making it the most effective method. The HAAM-DPSCs complexes produced using this method demonstrated enhanced biological properties, as indicated by CCK8, alizarin red, alkaline phosphatase staining, and RT-PCR. CONCLUSION: The HAAM prepared using the freeze-thaw + DNase method and CHAPS methods exhibited improved surface characteristics and significantly enhanced the proliferation and differentiation capacity of DPSCs when applied to them. The findings, therefore demonstrate the capacity for enhanced pulp regeneration therapy.