High-Fidelity Spatiotemporal Recognition of Golgi ALP through an Initial-Accumulation and Postactivation Strategy.

Various signal molecules mediate complex physiological processes collectively in the Golgi. However, most currently accessible probes are questionable in illuminating the functions of these reactive species in Golgi because of the inability to irradiate these probes only at the desired Golgi location, which compromises specificity and accuracy. In this study, we rationally designed the first photocontrollable and Golgi-targeted fluorescent probe to in situ visualize the Golgi alkaline phosphatase (ALP). The designed probe with natural yellow fluorescence can provide access into Golgi and monitor the exact timing of accumulation in Golgi. On-demand photoactivation at only the desired Golgi location affords a significant emission response to ALP with illuminating red fluorescence at 710 nm. Through the photocontrollable fluorescence responsiveness to ALP, precise spatiotemporal recognition of Golgi ALP fluctuations is successfully performed. With this probe, for the first time, we revealed the Golgi ALP levels during cisplatin-induced acute kidney injury (AKI), which will further facilitate and complement the comprehensive exploration of ALP kinetics during physiological and pathological processes.

Alpha-synuclein aggregates are phosphatase resistant.

Alpha-synuclein (αsyn) is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of αsyn at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we investigated experimental conditions under which two distinct PSER129 pools, namely endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the mammalian brain. Results showed that in the wild-type (WT) mouse brain, perfusion fixation conditions greatly influenced the detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30-min and 1-h postmortem interval). Exposure to anesthetics (e.g., Ketamine or xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase calf alkaline phosphatase (CIAP) selectively dephosphorylated endogenous-PSER129 while αsyn preformed fibril (PFF)-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp-Lantos bodies in Parkinson's disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. The phosphatase resistance of aggregates was abolished by sample denaturation, and CIAP-resistant PSER129 was closely associated with proteinase K (PK)-resistant αsyn (i.e., a marker of aggregation). CIAP pretreatment allowed for highly specific detection of seeded αsyn aggregates in a mouse model that accumulates non-aggregated-PSER129. We conclude that αsyn aggregates are impervious to phosphatases, and CIAP pretreatment increases detection specificity for aggregated-PSER129, particularly in well-preserved biological samples (e.g., perfusion fixed or flash-frozen mammalian tissues) where there is a high probability of interference from endogenous-PSER129. Our findings have important implications for the mechanism of PSER129-accumulation in the synucleinopathy brain and provide a simple experimental method to differentiate endogenous-from aggregated PSER129.

Modified and alternative bone cements can improve the induced membrane: Critical size bone defect model in rat femur.

BACKGROUND: As a two-stage surgical procedure, Masquelet's technique has been used to care for critical-size bone defects (CSD). We aimed to determine the effects of modified and altered bone cement with biological or chemical enriching agents on the progression of Masquelet's induced membrane (IM) applied to a rat femur CSD model, and to compare the histopathological, biochemical, and immunohistochemical findings of these cements to enhance IM capacity. METHODS: Thirty-five male rats were included in five groups: plain polymethyl methacrylate (PMMA), estrogen-impregnated PMMA (E+PMMA), bone chip added PMMA (BC+PMMA), hydroxyapatite-coated PMMA (HA) and calcium phosphate cement (CPC). The levels of bone alkaline phosphatase (BALP), osteocalcin (OC), and tumor necrosis factor-alpha (TNF-α) were analyzed in intracardiac blood samples collected at the end of 4 weeks of the right femur CSD intervention. All IMs collected were fixed and prepared for histopathological scoring. The tissue levels of rat-specific Transforming Growth Factor-Beta (TGF-ß), Runt-related Transcription Factor 2 (Runx2), and Vascular Endothelial Growth Factor (VEGF) were analyzed immunohistochemically. RESULTS: Serum levels of BALP and OC were significantly higher in E+PMMA and BC+PMMA groups than those of other groups (P = 0.0061 and 0.0019, respectively). In contrast, TNF-α levels of all groups with alternative bone cement significantly decreased compared to bare PMMA (P = 0.0116). Histopathological scores of E+PMMA, BC+PMMA, and CPC groups were 6.86 ± 1.57, 4.71 ± 0.76, and 6.57 ± 1.51, respectively, which were considerably higher than those of PMMA and HA groups (3.14 ± 0.70 and 1.86 ± 0.69, respectively) (P < 0.0001). Significant increases in TGF-ß and VEGF expressions were observed in E+PMMA and CPC groups (P = 0.0001 and <0.0001, respectively) whereas Runx2 expression significantly increased only in the HA group compared to other groups (P < 0.0001). CONCLUSIONS: The modified PMMA with E and BC, and CPC as an alternative spacer resulted in a well-differentiated IM and increased IM progression by elevating BALP and OC levels in serum and by mediating expressions of TGF-ß and VEGF at the tissue level. Estrogen-supplemented cement spacer has yielded promising findings between modified and alternative bone cement.

Novel hydrogel pillar array based ratiometric multicolor fluorescence biosensor for visual detection of alkaline phosphatase activity.

Enzyme-induced in-situ fluorescence is crucial for the development of biosensing mechanisms and correlative spectroscopic analysis. Inspired by simple p-aminophenol (AP)-controlled synthesis and the specific catalytic reaction of 4-aminophenyl phosphate (APP) triggered by alkaline phosphatase (ALP), our research proposed a strategy to prepare carbon dots (CDs) as fluorescent signals for ALP detection using AP and 3-aminopropyltrimethoxysilane (APTMS) as the precursors. The further constructed ratiometric fluorescence sensor reduced the detection limit of ALP to 0.075 µU/mL by a significant margin. Considering the need for point-of-care testing (POCT), we chose agarose for the preparation of portable hydrogel sensors so that even untrained personnel can quickly achieve semi-quantitative visual detection of ALP using colorimetric cards. These results demonstrate the practical applicability of ratiometric fluorescence sensing hydrogel pillar arrays, which are important for high-sensitivity, visualization, and portable rapid enzyme activity assays.

[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.

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.

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.

A preliminary study on calcifying nanoparticles in dental plaque: Isolation, characterization, and potential mineralization mechanism.

OBJECTIVES: Calcifying nanoparticles (CNPs), referred to as nanobacteria (NB), are recognized to be associated with ectopic calcification. This study aims to isolate and culture CNPs from the dental plaque of patients with periodontal disease and investigate their possible role in unravelling the aetiology of periodontal disease. MATERIAL AND METHODS: Supragingival and subgingival plaques were sampled from 30 periodontitis patients for CNPs isolation and culture. Alkaline phosphatase (ALP) content changes were tracked over time. Positive samples underwent thorough morphological identification via hematoxylin and eosin (HE) staining, Alizarin red S (ARS), and transmission electron microscopy (TEM). The chemical composition of CNPs analysis involved calcium (Ca) and phosphorus (P) content determination, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD). RESULTS: The subgingival plaque dental group exhibited a higher CNPs isolation rate at 36.67% (11/30) compared to the supragingival dental plaque group at 66.67% (20/30). ALP activity varied among the positive, negative and control groups. Morphological observation characterized the CNPs as round, oval, and ellipsoid particles with Ca deposits. Chemical analysis revealed the Ca/P ratio was 0.6753. Hydroxyl, methyl, carbonate, phosphate, hydrogen phosphate, and dihydrogen phosphate were detected by FTIR; the main chemical components detected by XRD were hydroxyapatite and tricalcium phosphate. CONCLUSION: CNPs were found in periodontitis-related dental plaque and exhibited the potential to develop calcified structures resembling dental calculus. However, the potential involvement of ALP in CNPs formation requires deeper exploration, as does the precise nature of its role and the interrelation with periodontitis demand a further comprehensive investigation.

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.

Odontogenic and anti-inflammatory effects of magnesium-doped bioactive glass in vital pulp therapy.

This study aimed to investigate the effects of magnesium-doped bioactive glass (Mg-BG) on the mineralization, odontogenesis, and anti-inflammatory abilities of human dental pulp stem cells (hDPSCs). Mg-BG powders with different Mg concentrations were successfully synthesized via the sol-gel method and evaluated using x-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. Apatite formation was observed on the surfaces of the materials after soaking in simulated body fluid. hDPSCs were cultured with Mg-BG powder extracts in vitro, and no evident cytotoxicity was observed. Mg-BG induced alkaline phosphatase (ALP) expression and mineralization of hDPSCs and upregulated the expression of odontogenic genes, including those encoding dentin sialophosphoprotein, dentin matrix protein 1, ALP, osteocalcin, and runt-related transcription factor 2. Moreover, Mg-BG substantially suppressed the secretion of inflammatory cytokines (interleukin [IL]-4, IL-6, IL-8, and tumor necrosis factor-alpha). Collectively, the results of this study suggest that Mg-BG has excellent in vitro bioactivity and is a potential material for vital pulp therapy of inflamed pulps.

Assessing the effects of a commercial fungicide and an herbicide, alone and in combination, on Apis mellifera: Insights from biomarkers and cognitive analysis.

Agrochemicals play a vital role in protecting crops and enhancing agricultural production by reducing threats from pests, pathogens and weeds. The toxicological status of honey bees can be influenced by a number of factors, including pesticides. While extensive research has focused on the lethal and sublethal effects of insecticides on individual bees and colonies, it is important to recognise that fungicides and herbicides can also affect bees' health. Unfortunately, in the field, honey bees are exposed to mixtures of compounds rather than single substances. This study aimed to evaluate the effects of a commercial fungicide and a commercial herbicide, both individually and in combination, on honey bees. Mortality assays, biomarkers and learning and memory tests were performed, and the results were integrated to assess the toxicological status of honey bees. Neurotoxicity (acetylcholinesterase and carboxylesterase activities), detoxification and metabolic processes (glutathione S-transferase and alkaline phosphatase activities), immune system function (lysozyme activity and haemocytes count) and genotoxicity biomarkers (Nuclear Abnormalities assay) were assessed. The fungicide Sakura® was found to activate detoxification enzymes and affect alkaline phosphatase activity. The herbicide Elegant 2FD and the combination of both pesticides showed neurotoxic effects and induced detoxification processes. Exposure to the herbicide/fungicide mixture impaired learning and memory in honey bees. This study represents a significant advance in understanding the toxicological effects of commonly used commercial pesticides in agriculture and contributes to the development of effective strategies to mitigate their adverse effects on non-target insects.

Ascertaining variations in the activity of larval midgut enzymes of Helicoverpa armigera by dietary emamectin benzoate through biochemical and in silico docking study.

Helicoverpa armigera, a ubiquitous polyphagous pest, poses a significant threat to global agriculture, causing substantial economic losses and demonstrating resistance to synthetic pesticides. This study investigates the potential of emamectin benzoate (EMB), an avermectin derivative, as an effective control agent against H. armigera. The larvae of the NBII-MP-NOC-01 strain of H. armigera were reared on an artificial diet. The impact of dietary EMB was examined on four midgut enzymes; alanine aminotransferase (ALT), aspartate aminotransferase (AST), acid phosphatase (ACP), and alkaline phosphatase (ALP). Results showed a dose-dependent and time-dependent reduction in ALT and AST activity, while an initial increase and subsequent decline in ACP and ALP activity at higher EMB concentrations. Computational modelling of enzyme structures and molecular docking studies revealed differential binding of EMB with the midgut enzymes. The strongest interaction was observed between EMB and ALT residues, contrasting with weakest interactions observed with AST. The study also showed that decreased activity of transaminases in H. armigera caused by EMB may be because of stability-activity trade-off, while in phosphatases reverse may be the case. This research provides crucial insights into the biochemical responses and the intricate insecticide-enzyme interactions in H. armigera caused by EMB exposure. This study lays the foundation for further research aimed at developing environmentally friendly approaches for managing H. armigera, addressing the challenges associated with conventional pesticides.

Conformation-Switchable Polypeptides as Molecular Gates for Controllable Drug Release.

The control over secondary structure has been widely studied to regulate the properties of polypeptide materials, which is used to change their functions in situ for various biomedical applications. Herein, we designed and constructed enzyme-responsive polypeptides as gating materials for mesoporous silica nanoparticles (MSNs), which underwent a distorted structure-to-helix transition to promote the release of encapsulated drugs. The polypeptide conjugated on the MSN surface adopted a negatively charged, distorted, flexible conformation, covering the pores of MSN to prevent drug leakage. Upon triggering by alkaline phosphatase (ALP) overproduced by tumor cells, the polypeptide transformed into positively charged, α-helical, rigid conformation with potent membrane-penetrating capabilities, which protruded from the MSN surface to uncover the pores. Such a transition thus enabled cancer-selective drug release and cellular internalization to efficiently kill tumor cells. This study highlights the important role of chain flexibility in modulating the biological function of polypeptides and provides a new application paradigm for synthetic polypeptides with secondary-structure transition.

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.

Based ATP-gating mechanism for detection of alkaline phosphatase in single-glass micropipettes functionalized by three-dimensional DNA network.

The construction of gating system in artificial channels is a cutting-edge research direction in understanding biological process and application sensing. Here, by mimicking the gating system, we report a device that easily synthesized single-glass micropipettes functionalized by three-dimensional (3D) DNA network, which triggers the gating mechanism for the detection of biomolecules. Based on this strategy, the gating mechanism shows that single-glass micropipette assembled 3D DNA network is in the "OFF" state, and after collapsing in the presence of ATP, they are in the "ON" state, at which point they exhibit asymmetric response times. In the "ON" process of the gating mechanism, the ascorbic acid phosphate (AAP) can be encapsulated by a 3D DNA network and released in the presence of adenosine triphosphate (ATP), which initiates a catalyzed cascade reaction under the influence of alkaline phosphatase (ALP). Ultimately, the detection of ALP can be responded to form the fluorescence signal generated by terephthalic acid that has captured hydroxyl radicals, which has a detection range of 0-250 mU/mL and a limit of detection of 50 mU/mL. This work provides a brand-new way and application direction for research of gating mechanism.

Bone turnover prediction in patients with chronic kidney disease (CKD) undergoing hemodialysis using shortened dynamic 18F-NaF PET/CT Ki-Patlak.

This study investigated whether Ki-Patlak derived from a shortened scan time for dynamic 18F-NaF PET/CT in chronic kidney disease (CKD) patients undergoing hemodialysis can provide predictive accuracy comparable to that obtained from a longer scan. Twenty-seven patients on chronic hemodialysis, involving a total of 42 scans between December 2021 and August 2023 were recruited. Dynamic 18F-NaF PET/CT scans, lasting 60-90 min, were immediately acquired post-injection, covering the mid-twelfth thoracic vertebra to the pelvis region. Ki-Patlak analysis was performed on bone time-activity curves at 15, 30, 45, 60, and 90 min in the lumbar spine (L1-L4) and both anterior iliac crests. Spearman's rank correlation (rs) and interclass correlation coefficient were used to assess the correlation and agreement of Ki-Patlak between shortened and standard scan times. Bone-specific alkaline phosphatase (BsAP) and tartrate-resistant acid phosphatase isoform 5b (TRAP5b) were tested for their correlation with individual Ki-Patlak. Strong correlations and good agreement were observed between Ki-Patlak values from shortened 30-min scans and longer 60-90-min scans in both lumbar spine (rs = 0.858, p < 0.001) and anterior iliac crest regions (rs = 0.850, p < 0.001). The correlation between BsAP and Ki-Patlak in the anterior iliac crests was weak and statistically insignificant. This finding suggests that a proposed shortened dynamic 18F-NaF PET/CT scan is effective in assessing bone metabolic flux in CKD patients undergoing hemodialysis, offering a non-invasive alternative approach for bone turnover prediction.

Enzyme-responsive oncolytic polypeptide for tumor therapy.

Host defense peptide-mimicking cationic oncolytic polymers have attracted increasing attention for cancer treatment in recent years. However, polymers with large amounts of positive charge may cause rapid clearance and severe off-target toxicity. To facilitate in vivo application, an alkaline phosphatase (ALP)-responsive oncolytic polypeptide precursor (C12-PLL/PA) has been reported in this work. C12-PLL/PA could be hydrolyzed into the active form of the oncolytic polypeptide (C12-PLL) by the extracellular alkaline phosphatase within solid tumors, thereby resulting in the conversion of the negative charge to positive charge and restoring its membrane-lytic activity. Detailed mechanistic studies showed that C12-PLL/PA could effectively destroy cancer cell membranes and subsequently result in rapid necrosis of cancer cells. More importantly, C12-PLL/PA significantly inhibited the tumor growth in the 4T1 orthotopic breast tumor model with negligible side effects. In summary, these findings demonstrated that the shielding of the amino groups with phosphate groups represents a secure and effective strategy to develop cationic oncolytic polypeptide, which represents a valuable reference for the design of enzyme-activated oncolytic polymers. STATEMENT OF SIGNIFICANCE: Recently, there has been a growing interest in fabricating host defense peptide-mimicking cationic oncolytic polymers for cancer therapy. However, there remain concerns about the tumor selectivity and off-target toxicity of these cationic polymers. In this study, an alkaline phosphatase-responsive oncolytic polypeptide precursor (C12-PLL/PA) has been developed to selectively target cancer cells while sparing normal cells. Mechanistic investigations demonstrated that C12-PLL/PA effectively disrupted cancer cell membranes, leading to rapid necrosis. Both in vitro and in vivo experiments showed promising anticancer activity and reliable safety of C12-PLL/PA. The findings suggest that this synthetic enzyme-responsive polypeptide holds potential as a tumor-specific oncolytic polymer, paving the way for future applications in cancer therapy.

Osteogenic potential of adipose stem cells on hydroxyapatite-functionalized decellularized amniotic membrane.

Amniotic membrane (AM) is an attractive source for bone tissue engineering because of its low immunogenicity, contains biomolecules and proteins, and osteogenic differentiation properties. Hydroxyapatite is widely used as bone scaffolds due to its biocompatibility and bioactivity properties. The aim of this study is to design and fabricate scaffold based on hydroxyapatite-coated decellularized amniotic membrane (DAM-HA) for bone tissue engineering purpose. So human amniotic membranes were collected from healthy donors and decellularized (DAM). Then a hydroxyapatite-coating was created by immersion in 10X SBF, under variable parameters of pH and incubation time. Hydroxyapatite-coating was characterized and the optimal sample was selected. Human adipose-derived mesenchymal stem cell behaviors were assessed on control, amniotic membrane, and coated amniotic membrane. The results of the SEM, MTT assay, and Live-Dead staining showed that DAM and DAM-HA support cell adhesion, viability and proliferation. Osteogenic differentiation was evaluated by assessment of alkaline phosphatase activity and expression of osteogenic markers. Maximum gene expression values compared to control occurred in 14 days for alkalin phosphatase, while the highest values for osteocalcin and osteopontin in 21 days. These gene expression values in DAM and DAM-HA for alkalin phosphatase is 6.41 and 8.47, for osteocalcin is 3.95 and 5.94 and for osteopontin is 5.59 and 9.9 respectively. The results of this study indicated DAM supports the survival and growth of stem cells. Also, addition of hydroxyapatite component to DAM promotes osteogenic differentiation while maintaining viability. Therefore, hydroxyapatite-coated decellularized amniotic membrane can be a promising choice for bone tissue engineering applications.

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.