OBJECTIVE: Ascorbic acid (AA) is a water-soluble vitamin that has antioxidant properties and regulates homeostasis of connective tissue through controlling various enzymatic activities. Two cell surface glycoproteins, sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2, are known as ascorbate transporters. The purpose of this study was to investigate the expression pattern and functions of SVCTs in periodontal ligament (PDL) and PDL fibroblast (PDLF).
METHODS: Gene expression was examined using real-time polymerase chain reaction (PCR) and reverse transcription PCR. SVCT2 expression was determined by immunofluorescence staining, western blot and flow cytometry. ALP activity and collagen production were examined using ALP staining and collagen staining. Short interfering RNA was used to knock down the gene level of SVCT2. Change of comprehensive gene expression under SVCT2 knockdown condition was examined by RNA-sequencing analysis.
RESULTS: Real-time PCR, fluorescent immunostaining, western blot and flowy cytometry showed that SVCT2 was expressed in PDLF and PDL. ALP activity, collagen production, and SVCT2 expression were enhanced upon AA stimulation in PDLF. The enhancement of ALP activity, collagen production, and SVCT2 expression by AA was abolished under SVCT2 knockdown condition. RNA-sequencing revealed that gene expression of CLDN4, Cyclin E2, CAMK4, MSH5, DMC1, and Nidgen2 were changed by SVCT2 knockdown. Among them, the expression of MSH5 and DMC1, which are related to DNA damage sensor activity, was enhanced by AA, suggesting the new molecular target of AA in PDLF.
CONCLUSIONS: Our study reveals the SVCT2 expression in PDL and the pivotal role of SVCT2 in mediating AA-induced enhancements of ALP activity and collagen production in PDLF. Additionally, we identify alterations in gene expression profiles, highlighting potential molecular targets influenced by AA through SVCT2. These findings deepen our understanding of periodontal tissue homeostasis mechanisms and suggest promising intervention targeting AA metabolism.

Hypophosphatasia (HPP) is characterized by low activity of tissue nonspecific alkaline phosphatase (TNSALP). The enzyme replacement therapy asfotase alfa has been approved for childhood-onset forms of HPP. MicroRNAs (miRNAs) have emerged as a novel disease biomarker, with potential application in therapy monitoring. Circulating miRNAs were analyzed at baseline, months 1, 2, 4, and 16 in a 49-yr-old woman with childhood-onset HPP, chronic musculoskeletal pain, and non-traumatic fractures prior to enzyme replacement therapy. Serum RNA was extracted and sequenced using miRNeasy Mini Kit (Qiagen, Germany), RealSeq Biosciences Kit (Santa Cruz, US) together with miND spike-in control kit (TAmiRNA, Austria) and Illumina NovaSeq 6000 SP1 flow cell (San Diego, US). Brief Pain Inventory Severity and Interference scores (BPI-S/BPI-I), fatigue severity scale (FSS), Patient Global Impression of Improvement (PGI-I), Western Ontario and McMaster university hip disability and osteoarthritis outcome score (WOMAC), fibromyalgia impact questionnaire (FIQ), 6-Minute Walking Test (6-MWT), chair-rise-test (CRT), and handgrip dynamometry (HD) were performed at baseline and different timepoints during the therapy. Out of >800 screened, 84 miRNAs were selected based on differences in expression profiles between 24 HPP patients and 24 healthy controls. Six miRNAs showed a clear graphic trend and were up- or downregulated by ≥50% reads per million (rpm). These included hsa-let-7i-5p (+50%), hsa-miR-1-3p (-66.66%), hsa-miR-1294 (+63.63%), hsa-miR-206 (-85.57%), hsa-miR-375-3p (-71.43%), and hsa-miR-624-5p (+69.44%). hsa-miR-1-3p and hsa-miR-206 were identified as muscle-specific miRNAs. hsa-mir-375-3p, which negatively regulates osteogenesis, was significantly downregulated. In terms of patient-reported outcomes, BPI-S, BPI-I, FSS, PGI-I, WOMAC, and FIQ showed a reduction by -58.62%, -68.29%, -33.33%, -75.00%, -63.29%, and -43.02%, respectively. 6-MWT improved by +33.89% and CRT by -44.46%. Mean hand grip strength of the right/left hand measured by HD improved by +12.50% and + 23.53%, respectively. miRNA profile changes during the therapy with asfotase alfa, accompanying improvements in functionality tests and quality of life scores.

Intermittent and continuous mechanical loads are known to influence osteogenic activity. The present study examines the effects of matched intermittent and continuous load in vitro on bone formation markers. MC3T3 (mouse pre-osteoblasts) were cultured and placed in a bioreactor to undergo continuous, intermittent, or unloading for 1, 3 and 12 days. Loading conditions were matched for magnitude, duration and frequency. Each time point was analysed for alkaline phosphatase (ALP) activity, procollagen 1 N-terminal propeptide (PINP) and alizarin red staining (ARS). Intermittent load caused an increase in ALP activity across all time points compared to continuous loading (↑30%-59%) and unloaded conditions (↑70%-90%). PINP concentrations from intermittent load were lower than continuous load (↓112%) on day 3. However, no differences were observed in PINP concentrations between loading conditions at other time points. No differences were observed for ARS between loading conditions. Intermittent load caused an increase in bone formation marker ALP, but not PINP, when compared to continuous loading and unloaded conditions. These findings further our knowledge in bone formation response and provide additional tools for the analysis of osteogenesis in vitro.

This study introduces an innovative approach for the real-time and efficient detection of alkaline phosphatase (ALP) activity, using a calcein fluorescence probe and leveraging the static quenching properties of calcein fluorescence by Ce3+ metal ions. In this method, calcein serves as the signal element, with its fluorescence effectively preserved through energy transfer or charge transfer when coordinated with Ce3+. Conversely, ALP catalyzes the phosphopeptide substrate to generate a substantial amount of Pi, preventing calcein fluorescence quenching due to the higher affinity between Pi and Ce3+ compared with that between calcein and Ce3+. The fluorescence intensity ratio (F-F0/F0) exhibited excellent linearity, facilitating sensitive ALP detection. The proposed ALP detection method covers a range from 0 to 1.4 mU/mL (R2 = 0.9942), with the limit of detection at 0.069 mU/mL (S/N = 3). Additionally, this method was successfully applied for detecting ALP in serum samples and studying its inhibitors. This research introduces a novel clinical diagnosis approach for ALP sensing while broadening the potential applications of calcein.

Ubiquitin-fold modifier 1 (UFM1) is covalently conjugated to protein substrates via a cascade of enzymatic reactions, a process known as UFMylation. UFMylation orchestrates an array of vital biological functions, including maintaining endoplasmic reticulum (ER) homeostasis, facilitating protein biogenesis, promoting cellular differentiation, regulating DNA damage response, and participating in cancer-associated signaling pathways. UFMylation has rapidly evolved into one of the forefront research areas within the last few years, yet much remains to be uncovered. In this review, first, UFMylation and its cellular functions associated with diseases are briefly introduced. Then, we summarize the proteomic approaches for identifying UFMylation substrates and explore the impact of UFMylation on gene transcription, protein translation, and maintenance of ER homeostasis. Next, we highlight the intricate regulation between UFMylation and two protein degradation pathways, the ubiquitin-proteasome system and the autophagy-lysosome pathway, and explore the potential of UFMylation system as a drug target. Finally, we discuss emerging perspectives in the UFMylation field. This review may provide valuable insights for drug discovery targeting the UFMylation system.

In this work, we propose a new technique involving the modification of commercial screen-printed carbon electrodes with electrochemically reduced graphene oxide to serve as the starting point of a future electrochemical biosensor for the detection of two osteogenic biomarkers: alkaline phosphatase (ALP) and Runt-related transcription factor 2 (RUNX2). The electrodes were characterized after each modification by cyclic voltammetry and electrochemical impedance spectroscopy, showing the appropriate electrochemical characteristics for each modification type. The results obtained from scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, and contact angle measurements are well correlated with each other, demonstrating the successful modification of the electrodes with graphene oxide and its subsequent reduction. The bioreceptors were immobilized on the electrodes by physical adsorption, which was confirmed by electrochemical methods, structural characterization, and contact angle measurements. Finally, the functionalized electrodes were incubated with the specific target analytes and the detection relied on monitoring the electrochemical changes occurring after the hybridization process. Our results indicated that the pilot platform has the ability to detect the two biomarkers up to 1 nM, with increased sensitivity observed for RUNX2, suggesting that after further optimizations, it has a high potential to be employed as a future biosensor.

Surface pre-reacted glass-ionomer (S-PRG) filler is a bioactive glass filler capable of releasing various ions. A culture medium to which was added an S-PRG filler eluate rich in boron was reported to enhance alkaline phosphatase (ALP) activity in human dental pulp-derived stem cells (hDPSC). To clarify the role of boron eluted from S-PRG fillers, the modified S-PRG filler eluate with different boron concentrations was prepared by using an anion exchange material. Therefore, elemental mapping analysis of anion exchange material, adsorption ratio, hDPSCs proliferation and ALP activity were evaluated. For statistical analysis, Kruskal-Wallis test was used, with statistical significance determined at p<0.05. ALP activity enhancement was not observed in hDPSC cultured in the medium that contained the S-PRG filler eluate from which boron had been removed. The result suggested the possibility that an S-PRG filler eluate with controlled boron release could be useful for the development of novel dental materials.

Observational studies have shown an association between liver dysfunction and hepatocellular carcinoma (HCC), but the causality relationship between them is unclear. We aimed to determine whether there is a bidirectional causal relationship between liver function indicators (alanine aminotransferase, ALT; aspartate aminotransferase, AST; alkaline phosphatase, ALP; γ-glutamyltransferase, GGT) and HCC. Our two-sample Mendelian randomization (MR) study acquired single nucleotide polymorphisms (SNPs) associated with liver function indicators (ALT, n = 134,182; AST, n = 134,154; GGT, n = 118,309; ALP, n = 105,030) and with HCC (n = 197,611) from publicly available genome-wide association studies (GWAS) of East Asian ancestry in Japan (BioBank Japan, BBJ). Univariable MR analyses were performed to identify whether the genetic evidence of exposure was significantly associated with outcome. Multivariable MR analysis was conducted to estimate the independent effects of exposures on outcome. Univariable MR analysis indicated that the level of ALT, AST, and GGT was the risk factor for HCC incidence. Meanwhile, multivariable MR analysis revealed that AST was an independent risk factor for HCC. The hazard ratio (HR) of the probability of HCC was 3.045 [95% confidence interval (95%CI), 1.697-5.463, p = 0.003] for AST. The results of reverse MR analyses showed that gene-predictive HCC incidence could increase the levels of AST (HR = 1.031, 95%CI: 1.009-1.054, p = 2.52 × 10-4) and ALT (HR = 1.040, 95%CI: 1.019-1.063, p = 0.005). Meanwhile, HCC may be negatively correlated with ALP levels (HR = 0.971, 95%CI: 0.947-0.995, p = 0.018). This study provides evidence to support that genetically predicted higher levels of AST are related to increased risk of HCC, with no strong evidence of a causal effect of genetically predicted ALP, ALP, and GGT on HCC. In addition, genetic predisposition to HCC could influence blood concentration of ALT, AST, and ALP. Thus, this may create a vicious cycle.

BACKGROUND: Vitamin D (Vit D) deficiency is common in patients with hyperparathyroidism, but the importance of replacement before surgery is controversial. It can be predicted that hypocalcemia risk will be higher in patients with high bone turnover.
OBJECTIVE: In this study, the effect of preoperative ALP/Vit D ratio on postoperative hypocalcemia was investigated.
METHODS: Among the primary hyperparathyroidism cases who were operated between 2015 and 2022, 158 patients with complete data were included in the study. Preoperative laboratory results, radiological images, and pathology reports of the patients were evaluated retrospectively. The cross-sectional value of the ALP/Vit D value predicting hypocalcemia was calculated. The effect of these parameters on postoperative hypocalcemia was investigated.
RESULTS: The mean age of our patients was 54 (21-81 years). When factors affecting postoperative hypocalcemia were evaluated by univariable analysis, Vit D deficiency and insufficiency (p < 0.001), ALP (p < 0.001), ALP/Vit D ratio (p < 0.001), and T score (p = 0.026) found to be factors affecting postoperative hypocalcemia. In multivariate analysis, the ALP/Vit D ratio was found to be an independent variable in predicting hypocalcemia. It was found that hypocalcemia was 45 times more common in patients with ALP/Vit D > 6.34 (p < 0.001). ALP/Vit D ratio predicts patients who will develop postoperative hypocalcemia with 87.2% sensitivity and 87.1% specificity.
CONCLUSIONS: Vit D deficiency increases the risk of postoperative hypocalcemia, but it is not sufficient alone to predict it. The risk increases more in patients with high bone turnover. The preoperative ALP/Vit D ratio is the strongest predictor of postoperative hypocalcemia risk.

Osseous disease accounts for over half of chronic pathologies, but there is a limited supply of autografts, the gold standard; hence, there is a demand for new synthetic biomaterials. Herein, we present the use of a promising, new dairy-derived biomaterial: whey protein isolate (WPI) in the form of hydrogels, modified with the addition of different concentrations of the biotechnologically produced protein-like polymeric substance poly-γ-glutamic acid (γ-PGA) as a potential scaffold for tissue regeneration. Raman spectroscopic analysis demonstrated the successful creation of WPI-γ-PGA hydrogels. A cytotoxicity assessment using preosteoblastic cells demonstrated that the hydrogels were noncytotoxic and supported cell proliferation from day 3 to 14. All γ-PGA-containing scaffold compositions strongly promoted cell attachment and the formation of dense interconnected cell layers. Cell viability was significantly increased on γ-PGA-containing scaffolds on day 14 compared to WPI control scaffolds. Significantly, the cells showed markers of osteogenic differentiation; they synthesised increasing amounts of collagen over time, and cells showed significantly enhanced alkaline phosphatase activity at day 7 and higher levels of calcium for matrix mineralization at days 14 and 21 on the γ-PGA-containing scaffolds. These results demonstrated the potential of WPI-γ-PGA hydrogels as scaffolds for bone regeneration.