Non-specific Alkaline Phosphatase Research Articles

Differential glycosylation of tissue non-specific alkaline phosphatase in mesenchymal stromal cells differentiated into either an osteoblastic or adipocytic phenotype

It has long been known that tissue non-specific alkaline phosphatase (TNAP) is essential for the correct formation of bone, as altered expression or function of this enzyme results in hypophosphatasia, a disease characterised by compromised bone structure, density and strength. However, recent evidence strongly suggests that the enzyme also has a role in lipid accrual and adipogenesis, a function that seems far removed from bone formation. Given that mesenchymal stromal cells (MSCs) are progenitors of both osteoblasts and adipocytes, the question arises of how TNAP is regulated to potentially have a different function when MSCs undergo either osteogenesis or adipogenesis. As the primary protein sequence is unchanged for the enzyme during both types of differentiation, any differences in function must be attributed to post-translational modification and/or localisation. We therefore examined the location of TNAP in bone- or adipose-derived MSCs differentiated into an adipocytic phenotype and compared the glycosylation state of the enzyme in MSCs differentiated into either osteoblasts or adipocytes. TNAP was found to co-locate with perilipin around lipid droplets in MSCs from bone, subcutaneous- and visceral adipose tissue during adipocytic differentiation. Treatment of TNAP with wheat germ lectin followed by electrophoresis showed minor differences in glycosylation between the phosphatase isolated from cells from these tissues, whereas electrophoresis after neuraminidase digestion highlighted differential glycosylation between cell types and during adipogenesis and osteoblastogenesis. This infers that post-translational modification of TNAP is altered during differentiation and is dependent on the eventual phenotype of the cells.

Design, Synthesis, and Biological Evaluation of Novel Dihydropyridine and Pyridine Analogs as Potent Human Tissue Nonspecific Alkaline Phosphatase Inhibitors with Anticancer Activity: ROS and DNA Damage-Induced Apoptosis.

Small molecules with nitrogen-containing scaffolds have gained much attention due to their biological importance in the development of new anticancer agents. The present paper reports the synthesis of a library of new dihydropyridine and pyridine analogs with diverse pharmacophores. All compounds were tested against the human tissue nonspecific alkaline phosphatase (h-TNAP) enzyme. Most of the compounds showed excellent enzyme inhibition against h-TNAP, having IC50 values ranging from 0.49 ± 0.025 to 8.8 ± 0.53 µM, which is multi-fold higher than that of the standard inhibitor (levamisole = 22.65 ± 1.60 µM) of the h-TNAP enzyme. Furthermore, an MTT assay was carried out to evaluate cytotoxicity against the HeLa and MCF-7 cancer cell lines. Among the analogs, the most potent dihydropyridine-based compound 4d was selected to investigate pro-apoptotic behavior. The further analysis demonstrated that compound 4d played a significant role in inducing apoptosis through multiple mechanisms, including overproduction of reactive oxygen species, mitochondrial dysfunction, DNA damaging, and arrest of the cell cycle at the G1 phase by inhibiting CDK4/6. The apoptosis-inducing effect of compound 4d was studied through staining agents, microscopic, and flow cytometry techniques. Detailed structure–activity relationship (SAR) and molecular docking studies were carried out to identify the core structural features responsible for inhibiting the enzymatic activity of the h-TNAP enzyme. Moreover, fluorescence emission studies corroborated the binding interaction of compound 4d with DNA through a fluorescence titration experiment.

MiR-335-3p/miR-155-5p Involved in IGFBP7-AS1–Enhanced Odontogenic Differentiation

BackgroundThe differentiation of stem cells from exfoliated deciduous teeth (SHEDs) into odontoblasts determines the regeneration of dentin-pulp complex. Non-coding RNAs (ncRNAs), including microRNA (miRNA) and long non-coding RNA (lncRNA), participate in many multiple biological processes, but the specific miRNAs involved in odontogenesis are incompletely defined. It was confirmed that lncRNA IGFBP7-AS1 could positively regulate odontogenetic differentiation in SHEDs. To investigate the downstream mechanisms of this process, miR-335-3p and miR-155-5p were found to be closely related with SHED odontogenic differentiation through whole-genome sequencing. The aim of the current study was to determine the role of miR-335-3p/miR-155-5p in IGFBP7-AS1–enhanced SHED differentiation and explore the potential mechanism of IGFBP7-AS1–mediated odontogenesis. MethodsPutative miR-335-3p/miR-155-5p binding sites within IGFBP7-AS1 were identified by bioinformatics analysis, and the binding of miR-335-3p/miR-155-5p to these sites was confirmed by dual-luciferase reporter gene assays. The effects of miR-335-3p/miR-155-5p in odontogenesis were detected by tissue nonspecific alkaline phosphatase staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR) analyses, and western blot testing. The molecular mechanisms of miR-335-3p/miR-155-5p involved in IGFBP7-AS1–mediated odontogenesis were analysed by qRT-PCR and western blot testing. ResultsDual-luciferase reporter gene assays showed that miR-335-3p/miR-155-5p could directly bind to IGFBP7-AS1. MiR-335-3p and miR-155-5p both could down-regulate dentin sialophosphoprotein expression, and both miRNAs could inhibit IGFBP7-AS1–mediated SHED odontogenetic differentiation via suppression of the extracellular signal-regulated kinase (ERK) pathway. ConclusionsBoth miR-335-3p and miR-155-5p were negative regulators to IGFBP7-AS1-enhanced odontogenic differentiation of SHED through suppression of the ERK pathway.

Does the RGD region of certain proteins affect metabolic activity?

A better understanding of the role of mineralized tissues and their associated factors in governing whole-body metabolism should be of value toward informing clinical strategies to treat mineralized tissue and metabolic disorders, such as diabetes and obesity. This perspective provides evidence suggesting a role for the arginine-glycine-aspartic acid (RGD) region, a sequence identified in several proteins secreted by bone cells, as well as other cells, in modulating systemic metabolic activity. We focus on (a) two of the SIBLING (small integrin-binding ligand, N-linked glycoprotein) family genes/proteins, bone sialoprotein (BSP) and osteopontin (OPN), (b) insulin-like growth factor-binding protein-1 & 2 (IGFBP-1, IGFBP-2) and (c) developmental endothelial locus 1 (DEL1) and milk fat globule–EGF factor-8 (MFG-E8). In addition, for our readers to appreciate the mounting evidence that a multitude of bone secreted factors affect the activity of other tissues, we provide a brief overview of other proteins, to include fibroblast growth factor 23 (FGF23), phosphatase orphan 1 (PHOSPHO1), osteocalcin (OCN/BGLAP), tissue non-specific alkaline phosphatase (TNAP) and acidic serine aspartic-rich MEPE-associated motif (ASARM), along with known/suggested functions of these factors in influencing energy metabolism.

Increased PHOSPHO1 expression mediates cortical bone mineral density in renal osteodystrophy.

Patients with advanced chronic kidney disease (CKD) often present with skeletal abnormalities, a condition known as renal osteodystrophy (ROD). While tissue non-specific alkaline phosphatase (TNAP) and PHOSPHO1 are critical for bone mineralization, their role in the etiology of ROD is unclear. To address this, ROD was induced in both WT and Phospho1 knockout (P1KO) mice through dietary adenine supplementation. The mice presented with hyperphosphatemia, hyperparathyroidism, and elevated levels of FGF23 and bone turnover markers. In particular, we noted that in CKD mice, bone mineral density (BMD) was increased in cortical bone (P < 0.05) but decreased in trabecular bone (P < 0.05). These changes were accompanied by decreased TNAP (P < 0.01) and increased PHOSPHO1 (P < 0.001) expression in WT CKD bones. In P1KO CKD mice, the cortical BMD phenotype was rescued, suggesting that the increased cortical BMD of CKD mice was driven by increased PHOSPHO1 expression. Other structural parameters were also improved in P1KO CKD mice. We further investigated the driver of the mineralization defects, by studying the effects of FGF23, PTH, and phosphate administration on PHOSPHO1 and TNAP expression by primary murine osteoblasts. We found both PHOSPHO1 and TNAP expressions to be downregulated in response to phosphate and PTH. The in vitro data suggest that the TNAP reduction in CKD-MBD is driven by the hyperphosphatemia and/or hyperparathyroidism noted in these mice, while the higher PHOSPHO1 expression may be a compensatory mechanism. Increased PHOSPHO1 expression in ROD may contribute to the disordered skeletal mineralization characteristic of this progressive disorder.

Anabolic actions of parathyroid hormone in a hypophosphatasia mouse model

SummaryHypophosphatasia, the rare heritable disorder caused by TNAP enzyme mutations, presents wide-ranging severity of bone hypomineralization and skeletal abnormalities. Intermittent PTH (1-34) increased long bone volume in Alpl-/- mice but did not alter the skull phenotype. PTH may have therapeutic value for adults with TNAP deficiency–associated osteoporosis.IntroductionHypophosphatasia is the rare heritable disorder caused by mutations in the tissue non-specific alkaline phosphatase (TNAP) enzyme leading to TNAP deficiency. Individuals with hypophosphatasia commonly present with bone hypomineralization and skeletal abnormalities. The purpose of this study was to determine the impact of intermittent PTH on the skeletal phenotype of TNAP-deficient Alpl-/- mice.MethodsAlpl-/- and Alpl+/+ (wild-type; WT) littermate mice were administered PTH (1-34) (50 µg/kg) or vehicle control from days 4 to 12 and skeletal analyses were performed including gross measurements, micro-CT, histomorphometry, and serum biochemistry.ResultsAlpl-/- mice were smaller with shorter tibial length and skull length compared to WT mice. Tibial BV/TV was reduced in Alpl-/- mice and daily PTH (1-34) injections significantly increased BV/TV and BMD but not TMD in both WT and Alpl-/- tibiae. Trabecular spacing was not different between genotypes and was decreased by PTH in both genotypes. Serum P1NP was unchanged while TRAcP5b was significantly lower in Alpl-/- vs. WT mice, with no PTH effect, and no differences in osteoclast numbers. Skull height and width were increased in Alpl-/- vs. WT mice, and PTH increased skull width in WT but not Alpl-/- mice. Frontal skull bones in Alpl-/- mice had decreased BV/TV, BMD, and calvarial thickness vs. WT with no significant PTH effects. Lengths of cranial base bones (basioccipital, basisphenoid, presphenoid) and lengths of synchondroses (growth plates) between the cranial base bones, plus bone of the basioccipitus, were assessed. All parameters were reduced (except lengths of synchondroses, which were increased) in Alpl-/- vs. WT mice with no PTH effect.ConclusionPTH increased long bone volume in the Alpl-/- mice but did not alter the skull phenotype. These data suggest that PTH can have long bone anabolic activity in the absence of TNAP, and that PTH may have therapeutic value for individuals with hypophosphatasia-associated osteoporosis.

Dysregulation of the endomembrane homeostasis triggers vascular smooth muscle cell phenotype switching

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft Background Cardiovascular diseases are a leading cause of mortality globally, with cardiovascular calcification as a prominent predictor and contributor. Notably, microcalcifications might cause atherosclerotic plaque rupture, leading to myocardial infarction. Calcified plaque remodeling accompanies phenotypic modulation of vascular smooth muscle cells (SMCs). Previously, we showed that extracellular vesicles (EVs) released from calcifying SMCs are pivotal for microcalcification formation. EVs originate from the endolysosomal system. The lipid kinase FYVE-type zinc finger containing phosphoinositide kinase (PIKfyve) plays a role in the endolysosomal maturation. Here, we investigated how modulation of the endolysosomal system intersects with SMC phenotype regulation. Methods and Results In calcified arteries and SMCs, we observed increased PIKfyve expression. Inhibition of PIKfyve with the small molecule Apilimod inhibited osteogenic differentiation of SMCs in an in vitro calcification model. Apilimod reduced tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA (-79%, p&amp;lt;0.01), protein (-96%, p&amp;lt;0.01) and activity (-92%, p&amp;lt;0.01) levels. Additionally, matrix mineralization and collagen network formation are inhibited. SMC-secreted EVs exhibited a lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Transcriptome analyses revealed that the anti-calcifying effect is accompanied by a decreased expression of contractile SMC genes. Validation by RT-PCR supported that SMC-specific markers (ACTA2, SM22) and the key SMC transcription factor MYOCD were downregulated, while the expression of macrophage-like SMC markers (LGALS3, CD68) was induced. On the other hand, Apilimod increased the expression of adipogenic transcription factors (CEBPA, PPARG) and genes of the cholesterol and fatty acid metabolism pathways (SREBF2, HMGCR, FABP3, CD36) and induced the nuclear translocation of PPARγ, the main regulator of adipogenic differentiation. Fluorescence-based assays revealed increased fatty acid and disrupted low-density lipoprotein (LDL) uptake in Apilimod-treated SMCs. In Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks and treated with Apilimod for 5 weeks, we found differentially distributed plaque lipid depositions detected by Oil Red O staining. Conclusion Disrupting the endolysosomal maturation by PIKfyve inhibition ameliorates SMC calcification and induces a phenotypic adipogenic-like, pro-inflammatory adaption. Our data elucidates the importance of SMC phenotype monitoring during therapeutic interference. The exact lineage characteristics of these SMCs remain to be further investigated.

Loss of BMP2 and BMP4 Signaling in the Dental Epithelium Causes Defective Enamel Maturation and Aberrant Development of Ameloblasts.

BMP signaling is crucial for differentiation of secretory ameloblasts, the cells that secrete enamel matrix. However, whether BMP signaling is required for differentiation of maturation-stage ameloblasts (MA), which are instrumental for enamel maturation into hard tissue, is hitherto unknown. To address this, we used an in vivo genetic approach which revealed that combined deactivation of the Bmp2 and Bmp4 genes in the murine dental epithelium causes development of dysmorphic and dysfunctional MA. These fail to exhibit a ruffled apical plasma membrane and to reabsorb enamel matrix proteins, leading to enamel defects mimicking hypomaturation amelogenesis imperfecta. Furthermore, subsets of mutant MA underwent pathological single or collective cell migration away from the ameloblast layer, forming cysts and/or exuberant tumor-like and gland-like structures. Massive apoptosis in the adjacent stratum intermedium and the abnormal cell-cell contacts and cell-matrix adhesion of MA may contribute to this aberrant behavior. The mutant MA also exhibited severely diminished tissue non-specific alkaline phosphatase activity, revealing that this enzyme’s activity in MA crucially depends on BMP2 and BMP4 inputs. Our findings show that combined BMP2 and BMP4 signaling is crucial for survival of the stratum intermedium and for proper development and function of MA to ensure normal enamel maturation.

Single-molecule studies reveal method for tuning the heterogeneous activity of alkaline phosphatase

Single-molecule-enzymology (SME) methods have enabled the observation of heterogeneous catalytic activities within a single enzyme population. Heterogeneous activity is hypothesized to originate from conformational changes in the enzyme that result from changes in the local environment leading to catalytically active substates. Here, we use SME to investigate the mechanisms of heterogeneous activity exhibited by tissue nonspecific alkaline phosphatase (TNSALP), which reveals two subpopulations with different catalytic activities. We show the effect of pH and temperature on the distribution of TNSALP activity and confirm the presence of two subpopulations attributed to half- and fully active TNSALP substates. We provide mechanistic insight about protein structure using molecular dynamic simulations and show pH- and temperature-dependent conformational transitions that corroborate experimentally observed changes in TNSALP activity. These results show the utility of SME to understand heterogeneous enzyme activity and demonstrate a simple approach using pH and temperature to tune catalytic activity within an enzyme population.

Abstract #1151796: Hypophosphatasia and Microcephalic Osteodysplastic Primordial Dwarfism type II: Diagnosing patients with minor symptoms

Hypophosphatasia (HPP) and Microcephalic Osteodysplastic Primordial Dwarfism type II (MOPD II) are rare inherited diseases which arise from the ALPL gene and PCNT gene respectively. HPP has nonspecific alkaline phosphatase (TNSALP) deficiency which causes the manifestations of skeletal deformities, fractures, nephrocalcinosis, seizures, respiratory failure and poor dentition. While in MOPD typically presents with bone, vascular and hematological abnormalities. Both of these diseases have a variety of symptoms which can vary in severity which can make the diagnosis difficult but important to avoid future complications.

A new perspective on the function of Tissue Non-Specific Alkaline Phosphatase: from bone mineralization to intra-cellular lipid accumulation.

Tissue-nonspecific alkaline phosphatase (TNAP) is one of four isozymes, which include germ cell, placental and intestinal alkaline phosphatases. The TNAP isozyme has 3 isoforms (liver, bone and kidney) which differ by tissue expression and glycosylation pattern. Despite a long history of investigation, the exact function of TNAP in many tissues is largely unknown. Only the bone isoform has been well characterised during mineralization where the enzyme hydrolyses pyrophosphate to inorganic phosphate, which combines with calcium to form hydroxyapatite crystals deposited as new bone. The inorganic phosphate also increases gene expression of proteins that support tissue mineralization. Recent studies have shown that TNAP is expressed in preadipocytes from several species, and that inhibition of TNAP activity causes attenuation of intracellular lipid accumulation in these and other lipid-storing cells. The mechanism by which TNAP stimulates lipid accumulation is not known; however, proteins that are important for controlling phosphate levels in bone are also expressed in adipocytes. This review examines the evidence that inorganic phosphate generated by TNAP promotes transcription that enhances the expression of the regulators of lipid storage and consequently, that TNAP has a major function of lipid metabolism.

Altered Topographic Distribution and Enhanced Neuronal Expression of Adenosine-Metabolizing Enzymes in Rat Hippocampus and Cortex from Early to late Adulthood.

The present study demonstrates altered topographic distribution and enhanced neuronal expression of major adenosine-metabolizing enzymes, i.e. ecto-5'-nucleotidase (eN) and tissue non-specific alkaline phosphatase (TNAP), as well as adenosine receptor subtype A2A in the hippocampus and cortex of male rats from early to late adulthood (3, 6, 12 and 15 months old males). The significant effect of age was demonstrated for the increase in the activity and the protein expression of eN and TNAP. At 15-m, enzyme histochemistry demonstrated enhanced expression of eN in synapse-rich hippocampal and cortical layers, whereas the upsurge of TNAP was observed in the hippocampal and cortical neuropil, rather than in cells and layers where two enzymes mostly reside in 3-m old brain. Furthermore, a dichotomy in A1R and A2AR expression was demonstrated in the cortex and hippocampus from early to late adulthood. Specifically, a decrease in A1R and enhancement of A2AR expression were demonstrated by immunohistochemistry, the latter being almost exclusively localized in hippocampal pyramidal and cortical superficial cell layers. We did not observe any glial upregulation of A2AR, which was common for both advanced age and chronic neurodegeneration. Taken together, the results imply that the adaptative changes in adenosine signaling occurring in neuronal elements early in life may be responsible for the later prominent glial enhancement in A2AR-mediated adenosine signaling, and neuroinflammation and neurodegeneration, which are the hallmarks of both advanced age and age-associated neurodegenerative diseases.

Histone Deacetylase Inhibitors Downregulate Calcium Pyrophosphate Crystal Formation in Human Articular Chondrocytes.

Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-β1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5′-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-β1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.

A Japanese single-center experience of the efficacy and safety of asfotase alfa in pediatric-onset hypophosphatasia

BackgroundHypophosphatasia (HPP) is a rare inherited metabolic disorder caused by mutations in the ALPL gene, which encodes tissue nonspecific alkaline phosphatase. The severity of HPP is widely diverse from the perinatal form to the adult mild form. The former represents the most severe form and was earlier associated with high mortality due to pneumonia which was caused by severe hypomineralization of the bones—such as chest deformity and fractured ribs—and muscle weakness. Enzyme replacement therapy using asfotase alfa (AA) was approved in 2015 in Japan for treating patients with HPP and has improved their pulmonary function and life prognosis. There are several practical and ethical challenges related to using orphan drugs for a rare disorder in a publicly funded healthcare system. Sharing experiences about their application is essential towards formulating guidelines to assist clinicians with decisions about their initiation and withdrawal. We report the details of AA experience in ten cases of pediatric-onset HPP in nine families from January 2015 to November 2019 (median [interquartile range] age 11.0 [7.6–12.5] years; 60% male). This is a study of a single-center cohort describing the clinical course of patients with HPP, mainly consisting of the mild childhood form of HPP, treated with AA in Japan.ResultsOne case of perinatal form of HPP, two cases of benign prenatal form, and seven cases of childhood form were observed. The most common symptom at onset was pain. All patients had low serum alkaline phosphatase levels as compared to the age-matched reference range before the commencement of AA. All HPP patients seem to have responded to AA treatment, as evidenced by pain alleviation, increased height standard deviation, improvement in respiratory condition and 6-min walk test result improvement, disappearance of kidney calcification, alleviation of fatigue, and/or increases in bone mineralization. There were no serious adverse events, but all patients had an injection site reaction and skin changes at the injection sites. Genetic analysis showed that eight out of ten patients had compound heterozygosity.ConclusionsAA may be effective in patients with mild to severe pediatric-onset forms of HPP.

Hyperglycemic environment disrupts phosphate transporter function and promotes calcification processes in podocytes and isolated glomeruli.

Soft tissue calcification is a pathological phenomenon that often occurs in end-stage chronic kidney disease (CKD), which is caused by diabetic nephropathy, among other factors. Hyperphosphatemia present during course of CKD contributes to impairments in kidney function, particularly damages in the glomerular filtration barrier (GFB). Essential elements of the GFB include glomerular epithelial cells, called podocytes. In the present study, we found that human immortalized podocytes express messenger RNA and protein of phosphate transporters, including NaPi 2c (SLC34A3), Pit 1 (SLC20A1), and Pit 2 (SLC20A2), which are sodium-dependent and mediate intracellular phosphate (Pi) transport, and XPR1, which is responsible for extracellular Pi transport. We found that cells that were grown in a medium with a high glucose (HG) concentration (30 mM) expressed less Pit 1 and Pit 2 protein than podocytes that were cultured in a standard glucose medium (11 mM). We found that exposure of the analyzed transporters in the cell membrane of the podocyte is altered by HG conditions. We also found that the activity of tissue nonspecific alkaline phosphatase increased in HG, causing a rise in Pi generation. Additionally, HG led to a reduction of the amount of ectonucleotide pyrophosphatase/phosphodiesterase 1 in the cell membrane of podocytes. The extracellular concentration of pyrophosphate also decreased under HG conditions. These data suggest that a hyperglycemic environment enhances the production of Pi in podocytes and its retention in the extracellular space, which may induce glomerular calcification.

Oral immunizations with Bacillus subtilis spores expressing MCP protein provide protection against red-spotted grouper nervous necrosis virus (RGNNV) infection in juvenile grouper, Epinephelus coioides

Nervous necrosis virus (NNV) causes severe viral disease in grouper, resulting in major economic losses in the global grouper aquaculture industry as a result of the high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect grouper against NNV infection. To achieve effective oral and bath immune responses, spores of Bacillus subtilis (B. subtilis) WB600 were utilized as the vehicle to deliver MCP (major capsid protein of RGNNV) partnered with CotC, one of the coat proteins of B. subtilis, to the gastrointestinal tract. After routine culturing for 8–12 h in LB medium, B. subtilis containing CotC-MCP (B.s-CotC-MCP) was transferred into the sporulation culture medium. Western blot analysis and the growth curve indicated that the best sporulation time for recombinant WB600 was 24–30 h at 37 °C with continuous shaking (250 rpm). The grouper then received surface displaying B·S-CotC-MCP vaccine against RGNNV via either oral or bath immunization and their immune responses were monitored. The results revealed that some non-specific immune parameters (acid phosphatase (ACP), alkaline phosphatase (AKP) and total antioxidant capacity (T-AOC)) were strongly enhanced in juvenile grouper following oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). The levels of specific antibodies against RGNNV and the transcription of immune-related genes (TNF-α, IL-1β, MHCI and IgM) were also significantly enhanced in juvenile grouper flowing oral or bath immunizations (1 × 106, 1 × 107, and 1 × 108 CFU g−1). However, only the highest dose of bath vaccination induced the production of specific antibodies significantly and up-regulated transcriptions of several immune-related genes (IgM and MHCI). The lower cumulative mortality in vaccinated groups after RGNNV challenge clearly demonstrated that surface-displayed MCP vaccine could protect fish against RGNNV infection. The relative percentage survival (RPS) value in the orally vaccinated group (88.89%) was much higher than the bath group (18.89%), which was consistent with the production of specific serum antibodies, non-specific immune response and immune related gene expression. Therefore, this study demonstrated that the CotC-MCP spores can trigger high levels of mucosal and humoral immunity, and represent a promising candidate vaccine against NNV infection.

Azomethine-clubbed thiazoles as human tissue non-specific alkaline phosphatase (h-TNAP) and intestinal alkaline phosphatase (h-IAP) Inhibitors: kinetics and molecular docking studies.

Thiazole derivatives are known inhibitors of alkaline phosphatase, but various side effects have reduced their curative efficacy. Conversely, compounds bearing azomethine linkage display a broad spectrum of biological applications. Therefore, combining the two scaffolds in a single structural unit should result in joint beneficial effects of both. A new series of azomethine-clubbed thiazoles (3a-i) was synthesized and appraised for their inhibitory potential against human tissue non-specific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). Compounds 3c and 3f were found to be most potent compounds toward h-TNAP with IC50 values of 0.15 ± 0.01 and 0.50 ± 0.01µM, respectively, whereas 3a and 3f exhibited maximum potency for h-IAP with IC50 value of 2.59 ± 0.04 and 2.56 ± 0.02µM, respectively. Molecular docking studies were also performed to find the type of binding interaction between potential inhibitor and active sites of enzymes. The enzymes inhibition kinetics studies were carried out to define the mechanism of enzyme inhibition. The current study leads to discovery of some potent inhibitors of alkaline phosphatase that is promising toward identification of compounds with druggable properties.

Inhibitors of ectonucleotidases have paradoxical effects on synaptic transmission in the mouse cortex.

Extracellular adenosine plays prominent roles in the brain in both physiological and pathological conditions. Adenosine can be generated following the degradation of extracellular nucleotides by various types of ectonucleotidases. Several ectonucleotidases are present in the brain parenchyma: ecto-nucleotide triphosphate diphosphohydrolases 1 and 3 (NTPDase 1 and 3), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP 1), ecto-5'-nucleotidase (eN), and tissue non-specific alkaline phosphatase (TNAP, whose function in the brain has received little attention). Here we examined, in a living brain preparation, the role of these ectonucleotidases in generating extracellular adenosine. We recorded local field potentials evoked by electrical stimulation of the lateral olfactory tract in the mouse piriform cortex in vitro. Variations in adenosine level were evaluated by measuring changes in presynaptic inhibition generated by adenosine A1 receptors (A1Rs) activation. A1R-mediated presynaptic inhibition was present endogenously and was enhanced by bath-applied AMP and ATP. We hypothesized that inhibiting ectonucleotidases would reduce extracellular adenosine concentration, which would result in a weakening of presynaptic inhibition. However, inhibiting TNAP had no effect in controlling endogenous adenosine action and no effect on presynaptic inhibition induced by bath-applied AMP. Furthermore, contrary to our expectation, inhibiting TNAP reinforced, rather than reduced, presynaptic inhibition induced by bath-applied ATP. Similarly, inhibition of NTPDase 1 and 3, NPP1, and eN induced stronger, rather than weaker, presynaptic inhibition, both in endogenous condition and with bath-applied ATP and AMP. Consequently, attempts to suppress the functions of extracellular adenosine by blocking its extracellular synthesis in living brain tissue could have functional impacts opposite to those anticipated.

Deletion of the Pyrophosphate Generating Enzyme ENPP1 Rescues Craniofacial Abnormalities in the TNAP-/- Mouse Model of Hypophosphatasia and Reveals FGF23 as a Marker of Phenotype Severity.

Hypophosphatasia is a rare heritable metabolic disorder caused by deficient Tissue Non-specific Alkaline Phosphatase (TNAP) enzyme activity. A principal function of TNAP is to hydrolyze the tissue mineralization inhibitor pyrophosphate. ENPP1 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 1) is a primary enzymatic generator of pyrophosphate and prior results showed that elimination of ENPP1 rescued bone hypomineralization of skull, vertebral and long bones to different extents in TNAP null mice. Current TNAP enzyme replacement therapy alleviates skeletal, motor and cognitive defects but does not eliminate craniosynostosis in pediatric hypophosphatasia patients. To further understand mechanisms underlying craniosynostosis development in hypophosphatasia, here we sought to determine if craniofacial abnormalities including craniosynostosis and skull shape defects would be alleviated in TNAP null mice by genetic ablation of ENPP1. Results show that homozygous deletion of ENPP1 significantly diminishes the incidence of craniosynostosis and that skull shape abnormalities are rescued by hemi- or homozygous deletion of ENPP1 in TNAP null mice. Skull and long bone hypomineralization were also alleviated in TNAP−/−/ENPP1−/− compared to TNAP−/−/ENPP1+/+ mice, though loss of ENPP1 in combination with TNAP had different effects than loss of only TNAP on long bone trabeculae. Investigation of a relatively large cohort of mice revealed that the skeletal phenotypes of TNAP null mice were markedly variable. Because FGF23 circulating levels are known to be increased in ENPP1 null mice and because FGF23 influences bone, we measured serum intact FGF23 levels in the TNAP null mice and found that a subset of TNAP−/−/ENPP1+/+ mice exhibited markedly high serum FGF23. Serum FGF23 levels also correlated to mouse body measurements, the incidence of craniosynostosis, skull shape abnormalities and skull bone density and volume fraction. Together, our results demonstrate that balanced expression of TNAP and ENPP1 enzymes are essential for microstructure and mineralization of both skull and long bones, and for preventing craniosynostosis. The results also show that FGF23 rises in the TNAP−/− model of murine lethal hypophosphatasia. Future studies are required to determine if the rise in FGF23 is a cause, consequence, or marker of disease phenotype severity.

Heparin Enriched-WPI Coating on Ti6Al4V Increases Hydrophilicity and Improves Proliferation and Differentiation of Human Bone Marrow Stromal Cells.

Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure.