Alkaline Phosphatase Gene Research Articles

Transcriptomics Analysis of the Toxicological Impact of Enrofloxacin in an Aquatic Environment on the Chinese Mitten Crab (Eriocheir sinensis).

Enrofloxacin is an important antimicrobial drug that is widely used in aquaculture. Enrofloxacin residues can have negative effects on aquatic environments and animals. The toxicological effects of different concentrations of enrofloxacin residues in cultured water on Chinese mitten crabs (Eriocheir sinensis) were compared. A histological analysis of the E. sinensis hepatopancreas demonstrated that the hepatopancreas was damaged by the different enrofloxacin residue concentrations. The hepatopancreas transcriptome results revealed that 1245 genes were upregulated and that 1298 genes were downregulated in the low-concentration enrofloxacin residue group. In the high-concentration enrofloxacin residue group, 380 genes were upregulated, and 529 genes were downregulated. The enrofloxacin residues led to differentially expressed genes related to the immune system and metabolic processes in the hepatopancreas of the Chinese mitten crab, such as the genes for alkaline phosphatase, NF-kappa B inhibitor alpha, alpha-amylase, and beta-galactosidase-like. The gene ontology terms "biological process" and "molecular function" were enriched in the carboxylic acid metabolic process, DNA replication, the synthesis of RNA primers, the transmembrane transporter activity, the hydrolase activity, and the oxidoreductase activity. A Kyoto Encyclopedia of Genes and Genomes pathway analysis determined that the immune and metabolic signal transduction pathways were significantly enriched. Furthermore, the nonspecific immune enzyme (alkaline phosphatase) and the metabolic enzyme system played a role in the enrofloxacin metabolism in the E. sinensis hepatopancreas. These findings helped us to further understand the basis of the toxicological effects of enrofloxacin residues on river crabs and provided valuable information for the better utilization of enrofloxacin in aquatic water environments.

Growth hormone-releasing hormone agonist attenuates vascular calcification in diabetic db/db mice.

Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. VC increases mortality of all-causes. VC is one of most common cardiovascular complications in type II diabetes. So far, no therapy has been proven to be effective in treatment of clinical VC. The present study investigated the therapeutic effects of MR409, an agonistic analog of growth hormone-releasing hormone (GHRH-A), on VC in diabetic db/db mice. Diabetic mice were injected with MR409 subcutaneously every day for 8 weeks. Long-term treatment with MR409 improved serum lipid profile and endothelium-dependent relaxation to acetylcholine, and reduced vascular structural injury in diabetic mice without affecting serum growth hormone level. Echocardiography showed that calcium plaques present in heart valve of diabetic mice disappeared in diabetic mice after treatment with MR409. MR409 inhibited vascular calcium deposition associated with a marked reduction in the expressions of osteogenic-regulated alkaline phosphatase (ALP) and transcription osteogenic marker gene Runx2 in diabetic mice. MR409 also inhibited vascular reactive oxygen species (ROS) generation and upregulated the expressions of anti-calcifying protein Klotho in diabetic mice. Our results demonstrate that GHRH-A MR409 can effectively attenuate VC and heart valve calcification, and protect against endothelial dysfunction and vascular injury in diabetic mice without significantly affecting pituitary-growth hormone axis. The mechanisms may involve upregulation of anti-calcifying protein Klotho and reduction in vascular ROS and the expression of redox sensitive osteogenic genes Runx2 and ALP. GHRH-A may represent a new pharmacological strategy for treatment of VC and diabetics associated cardiovascular complications.

1,25-Dihydroxyvitamin D3: A Positive Factor for the Osteogenic Differentiation of hPDLSCs and for the Tissue Regenerative Activity of Cell Sheets

This study aims to investigate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2VitD3) on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the activity of hPDLSC sheets and the differences in the tissue regeneration activity of hPDLSC sheets on tooth root fragment treated by different methods. Healthy caries-free premolars were collected. The hPDLSCs were obtained by enzymatic digestion. Surface markers of stem cells were analyzed by flow cytometry and the multidirectional differentiation ability of hPDLSCs was detected. During the osteogenic differentiation of hPDLSCs, 1,25(OH)2VitD3 was added and the effect of 1,25(OH)2VitD3 on osteogenic differentiation of hPDLSCs was assessed using Western blotting, quantitative reverse transcription–polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, cell staining, and immunofluorescence. After hPDLSC sheets were prepared, histology and immunofluorescence analysis of the effect of 1,25(OH)2VitD3 on sheet activity were performed. In addition, root fragments were prepared and treated with scaling, 24% EDTA (ethylenediamide tetraacetic acid), and Er,Cr:YSGG lasers, respectively, and the tissue regeneration activity of hPDLSC sheets on different root fragments were observed. 1,25(OH)2VitD3 promoted the high gene and protein expressions of osteogenic markers ALP (alkaline phosphatase), Runx2, and OPN (osteopontin antibody) in hPDLSCs, along with enhanced ALP activity and staining, alizarin red staining, and immunofluorescence staining, indicating that the osteogenic differentiation ability of hPDLSCs was improved. Extracellular matrix secretion was increased in hPDLSC sheets, along with the positive expressions of the protein markers fibronectin and collagen I, suggesting that 1,25(OH)2VitD3 could enhance these effects. In addition, the root fragments treated by Er,Cr:YSGG laser were more suitable for the attachment and regeneration of hPDLSC sheets, demonstrating that 1,25(OH)2VitD3 could improve the tissue regeneration performance of these sheets. 1,25(OH)2VitD3 can promote osteogenic differentiation of hPDLSCs and thus plays an active role in hPDLSC sheet formation and tissue regeneration. In addition, the Er,Cr:YSGG laser can be used as the recommended treatment method for the root surface regenerated by hPDLSCs.

The effect of Cissus quadrangularis Salisb. extract on maturation of rat mesenchymal stem cells.

Degenerative diseases, such as osteoporosis, could be treated by stem cells. The aim of this study was to identify the gene expression of bone marrow mesenchymal stem cells (BM-MSC) derived from Sprague Dawley rats and to assess the effect of Cissus quadrangularis Salisb. extract on their maturation into bone cells. The BM-MSC were divided into three groups: (a) BM-MSCs + osteoblast cell growth basal medium as the positive control; (b) BM-MSCs + Dulbecco's modified eagle's medium (DMEM) + 0.3 mg/mL methanol extract of C. quadrangularis as methanol group; and (c) BM-MSC + DMEM + 0.3 mg/mL ethyl acetate extract of C. quadrangularis as ethyl acetate group. A relative quantification approach using was used to analyze the expression of the alp (alkaline phosphatase) gene, with the beta-actin gene was used to normalize the expression of the alp gene. The intra-assay variation was calculated to validate the RT-qPCR data. Our study found that the intra-assay variation value was acceptable, with most of the coefficients of variability (CV) value <5. Ethyl acetate solvent outperformed methanol solvent in extracting the active compound C. quadrangularis. In the ethyl acetate extract group, the expression of the alp gene increased three times compared to the positive control. In methanol extract group, the expression of alp gene was lower six times compared to positive control. This study suggests that C. quadrangularis extracts using ethyl acetate could induce the maturation of BM-MSCs. However, further studies are warrant to confirm this effect using different indicators.

Effect of Er: YAG Laser Irradiation on Bone Metabolism-Related Factors Using Cultured Human Osteoblasts.

Introduction: A variety of laser treatments have been applied in numerous medical fields. In dentistry, laser treatments are used for caries, root canals, and periodontal disease, as well as surgical resection. Numerous reports have recently been published on the use of lasers for bone regeneration. If laser irradiation is found to promote the activation of bone metabolism, it might also be effective for periodontal treatment, peri-implantitis, and bone regeneration. Therefore, the present in vitro study aimed to elucidate the mechanisms underlying the effects of erbium-doped yttrium aluminum garnet (Er: YAG) laser irradiation on the bone using osteoblast-like cells. Methods: Osteoblast-like Saos 2 cells (5.0×104 cells) were seeded in 24-well plates. 24 hours after being seeded, the cells were subjected to 0.3 W, 0.6 W, and 2.0 W Er: YAG laser irradiation and then allowed to recover for 48 hours. The expression levels of bone metabolism-related factors alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteoprotegerin (OPG) were then evaluated using reverse transcription-quantitative polymerase chain reaction and western blot analyses. Results: Saos 2 cells subjected to Er: YAG laser irradiation at 0.3 W, 0.6 W, and 2.0 W showed normal growth. When the Er: YAG laser irradiation and control groups were compared after 48 hours, increases were observed in ALP, BSP, and OPG gene and protein expression in the 2.0 W group. Similar results were obtained in the western blot analysis. Conclusion: These findings suggest that the Er: YAG laser irradiation of osteoblast-like cells is effective for activating bone metabolism factors.

Silver Nanoparticles Alone or in Combination with Calcium Hydroxide Modulate the Viability, Attachment, Migration, and Osteogenic Differentiation of Human Mesenchymal Stem Cells.

This study aimed to evaluate the effect of silver nanoparticles (AgNPs) alone or in combination with calcium hydroxide (Ca(OH)2) on the proliferation, viability, attachment, migration, and osteogenic differentiation of human mesenchymal stem cells (hMSCs). Different concentrations of AgNPs alone or mixed with Ca(OH)2 were prepared. Cell proliferation was measured using AlamarBlue, and hMSCs attachment to dentin disks was evaluated using scanning electron microscopy. Live-dead imaging was performed to assess apoptosis. Wound healing ability was determined using the scratch-migration assay. To evaluate osteogenic differentiation, the expression of Runt-related transcription factor (RUNX2), Transforming growth factor beta-1 (TGF-β1), Alkaline Phosphatase (ALP), and Osteocalcin (OCN) were measured using real-time reverse transcriptase polymerase chain reaction. ALP staining and activity were also performed as indicators of osteogenic differentiation. AgNPs alone seemed to favor cell attachment. Lower concentrations of AgNPs enhanced cell proliferation. AgNP groups showed markedly less apoptosis. None of the medicaments had adverse effects on wound closure. The expression of TGF-β1 was significantly upregulated in all groups, and OCN was highly expressed in the AgNP groups. AgNPs 0.06% showed the most enhanced ALP gene expression levels, activity, and marked cytochemical staining. In conclusion, AgNPs positively affect hMSCs, making them a potential biomaterial for various clinical applications.

Correlation of gene content in selected bifidobacteria with folate supplier or scavenger status during growth in laboratory media

Five different bifidobacterial cultures were screened to investigate if their de novo folate production ability could be predicated based on their folate biosynthetic gene content. As previously developed folate free media could not adequately support the growth of these five strains, even with added folate, an alternative medium (YNB+) was developed. Consistent with its genome prediction, Bifidobacterium animalis subsp. lactis strain Bb-12 required folate for its growth even in the presence of p-aminobenzoic acid (PABA). Although B. longum DJO10A and B. breve ATCC 15701 lack an alkaline phosphatase gene for the formation of one pathway intermediate (DHPPP), both were found to be able to grow independent from folate and PABA suggesting that lack of these genes in genome annotations cannot be used to predict folate dependency in bifidobacteria. Similarly, having all required genes cannot always predict folate production, as B. longum subsp. infantis RECb4 contained a full set of pathway genes, but required folate or PABA for growth. While many bifidobacteria can produce folate, this also cannot be used to predicate their ability to supply folate as some can act as scavengers when folate is readily available in the environment, such as B. breve ATCC 15701 in this study. As bifidobacterial cultures are sometimes considered for use in foods based on their folate production abilities, it is important to consider whether they function as folate scavengers or suppliers when growing in folate containing media.

Not just a carrier: Clinical presentation and management of patients with heterozygous disease-causing alkaline phosphatase (ALPL) variants identified through expanded carrier screening.

Hypophosphatasia (HPP) is an underrecognized, complex bone mineralization disorder with variable manifestations caused by one or two deleterious variants in the alkaline phosphatase (ALPL) gene. Expanded carrier screening (ECS), inclusive of ALPL, intends to inform reproductive risk but may incidentally reveal an HPP diagnosis with 50% familial risks. We sought to investigate at-risk individuals and develop a multidisciplinary referral and evaluation protocol for ECS-identified ALPL heterozygosity. A retrospective database query of ECS results from 8 years to 1 month for heterozygous pathogenic/likely pathogenic ALPLvariants was completed. We implemented a clinical protocol for diagnostic testing and imaging, counseling, and interdisciplinary care management for identified patients, and outcomes were documented. Heterozygous ALPL variants were identified in 12/2248 unrelated patients undergoing ECS (0.53%; heterozygote frequency 1/187). Of 10 individuals successfully contacted, all demonstrated symptomatology and/or alkaline phosphatase values consistent with HPP. ECS may reveal incidental health risks, including recognition of missed HPP diagnoses in ALPL heterozygotes. In our cohort, all ECS-identified ALPL heterozygotes with clinical and/or biochemical data available demonstrated features of HPP. Referral to a genetics professional familiar with HPP is indicated for family history assessment, genetic counseling, cascade testing, and long-term bone health management.

Donor age effects on in vitro chondrogenic and osteogenic differentiation performance of equine bone marrow- and adipose tissue-derived mesenchymal stromal cells

BackgroundBone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro.BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15–17 months), adult (5–8 years), middle-aged (12–18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers.ResultsChondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types.ConclusionsThe data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.

RF30 | PSAT137 Low Level of Alkaline Phosphatase as a Clue to Noncompliance in Patient with Hypophosphatasia

Abstract Introduction Hypophosphatasia is rare, inherited metabolic disorder caused by mutations in the tissue nonspecific alkaline phosphatase gene. This mutation leads to persistently low level of alkaline phosphatase (ALP) which is the hallmark of the hypophosphatasia but also elevated substrates mostly inorganic pyrophosphate (PPi) and pyridoxal-5-phospate (PLP). Enzyme replacement therapy (ERT) with asfotase alpha presents currently available medical treatment, in which administration of enzyme leads to increase in ALP activity up to several thousand units per liter. Measurement of ALP and other biochemical parameters are crucial during follow-up of treatment with ERT. Clinical case: 42-year-old female with medical history significant for early onset scoliosis secondary to hypophosphatasia diagnosed at age of 6 months presented at endocrinology clinic to establish care. In early childhood patient had 3 nontraumatic femur fractures and ankle fracture at age 25. Patient underwent fixation of bilateral proximal femurs and spinal surgery due to scoliosis. She has been started on weight base treatment with asfotase alpha 6 years ago with 2 mg/kg 3 times per week. During current visit patient continued to experience back pain despite surgery treatment and ongoing treatment with asfotase alpha. Her ALP level in blood was 234 u/l. This ALP level was low considering that patient is on 6 years long treatment and careful history taking revealed that patient is taking smaller dosage of asfotase alpha than recommended. Patient explained that due to side effects of medications: palpitations and nervousness she decreased her dose to 2 times per week which led to inappropriate self-medication and non-compliance. Conclusion Although there are many different causes of low level of ALP like osteogenesis imperfecta, vitamin D intoxication, multiple myeloma, vitamin C deficiency, hypothyroidism, Cushing's syndrome and bisphosphonate therapy recent studies found out that prevalence of mild form of hypophosphatasia can be much higher than expected even 1/508. Due to relative rarity of the disease and lack of the awareness of hypophosphatasia among physicians there are usually delay in the diagnosis and management especially in mild forms. Measurement of ALP activity is crucial for diagnosis of hypophosphatasia but is also essential minimum for monitoring of treatment. Relatively low level of ALP while on treatment with asfotase alpha as in our case should prompt for assessment of medication compliance or treatment failure. Further assessment with detailed information about medication injections, dosage, and frequency as well as measurements of biochemical parameters, radiography, pain assessment and mobility are needed in follow up of adults with hypophosphatasia. More awareness and education on diagnosis and follow up of patients with hypophosphatasia are needed so patients can be effectively treated with available enzyme replacement therapy. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 12:54 p.m. - 12:59 p.m.

Unsuspected functions of alkaline phosphatase PhoD in the diatom Phaeodactylum tricornutum

Alkaline phosphatase (AP) in algae is known as the indicator of phosphorus nutrient (P) starvation and an enzyme to hydrolyze dissolved organic phosphorus (DOP) as an alternative source of P when the primary source of P, dissolved inorganic phosphate, DIP, is limited in the marine environment. Here, we document the roles of an intracellular PhoD type AP gene in the diatom Phaeodactylum tricornutum other than DOP scavenging when P supply is abundant. CRISPR-mediated mutation of PhoD_45757 resulted in increased lipid content, pigment content, photosynthetic efficiency, and oxygen evolution. Meanwhile, PhoD_45757 mutation also altered physiological and transcriptomic performance under different P conditions. Mutant strains grown in the P-replete medium exhibited significantly lower growth rates than the wild type, apparently by inhibiting G1 phase in the cell cycle, and higher phosphate uptake rate, accompanied by increased particulate P and RNA contents per cell. In addition, mPhoD cells increased in cellular carbon (including lipid) and nitrogen contents, and reprogrammed their metabolic response to changes in the nutrient condition. Our physiological and transcriptomic results reveal the roles of a PhoD in pigment synthesis and photosynthesis, lipid accumulation, cell cycle regulation, and nutrient homeostasis in a diatom when DOP scavenging is not required.

Changes in rhizosphere phosphorus fractions and phosphate-mineralizing microbial populations in acid soil as influenced by organic acid exudation

Interactions were explored between organic acids, phosphate-mineralizing microorganisms, and phosphorus fractions in rhizosphere soil. Bulk soil, rhizosphere soil, and root samples were randomly collected from a mature lemon tree plantation. Composition of acid (phoC) and alkaline (phoD) phosphatase gene harboring bacterial populations in rhizosphere soil were different. Whereas only soil properties affected the phoC-harboring bacterial population, soil properties and organic acids shaped the phoD-harboring bacterial population. Organic acids, especially oxalic acid, rather than phosphatase significantly directly and indirectly affected phosphorus fractions. When oxalic acid concentration increased, abundances of phoD-harboring Pseudomonas and Bacillus also increased. Those genera then released alkaline phosphatase, which reduced the contents of calcium-bound organophosphorus, insoluble organophosphorus, and residual phosphorus. By contrast, when oxalic acid concentration decreased, abundance of phoC-harboring Xanthomonas increased. The genus released acid phosphatase, which reduced calcium-bound organophosphorus content. In addition, alkaline phosphatase released by phoD-harboring bacteria was more important than acid phosphatase in acid soil. In conclusion, oxalic acids have essential roles in directly and indirectly changing phosphorus fractions in lemon tree rhizosphere soil.

Moringa oleifera leaf extract induces osteogenic-like differentiation of human osteosarcoma SaOS2 cells

IntroductionMoringa oleifera is known as a ‘natural nutrition of the tropics’ because it provides vital nutritional supplements and a variety of pharmacological benefits. The focus of this study was to elucidate the dose dependent effects of Moringa oleifera leaf (MOL) extract on the growth of the human osteoblast-like osteosarcoma SaOS-2 cell line and primary osteoblast cells. MethodsTrypan blue & tetrazolium assay, intracellular ROS generation, chromatin condensation, cell cycle analysis, alkaline phosphatase (ALP), mineralization, and osteogenic gene expression were tested on both treated and untreated osteosarcoma SaOS-2 cells. ResultsAs revealed by cell viability assay, growth activity was observed at concentrations 25 and 50 μg/mL of MOL extract, whereas 100 and 200 μg/mL doses decreased the proliferation activity, resulting in ROS production and chromatin condensation. Cell cycle study revealed that MOL extract at 50 and 100 μg/mL concentrations arrested the cells in the G2/M phase. Low doses increased the ALP levels, mineralization, and expression of the bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (Runx2) genes in osteoblast-like SaOS-2 cells, however, high doses inhibited the proliferation properties of MOL extract. Through AutoDock Vina and iGEMDOCK 2.1, the interaction of active components of MOL, such as β-sitosterol, quercetin and kaempferol, with BMP2 and Runx2 proteins revealed a reasonable binding affinity. Moreover, these components did not show any Lipinski's rule of five violation and showed predictable pharmacokinetic properties. ConclusionThe results of the biphasic dose-response of MOL extract on the growth activity of osteoblast-like SaOS-2 cells and in silico binding interface, may provide a therapeutic and/or preventive implication in prospective drug development.

Vitamin B-related Gene Polymorphisms and Cardiovascular Disease.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis, even in early childhood. A mutation in genes that code homocysteine metabolism enzymes or deficiency of specific vitamin cofactors may cause hyperhomocysteinemia. Vitamin B complex has been correlated with serum homocysteine levels. Any abnormality in its metabolism or nutritional deficiency may lead to hyperhomocysteinemia. Both vitamin B complex and homocysteine levels are partly genetically determined. Specifically, the most studied polymorphism is 677T-C in exon 5 of the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene, which plays an important role in folate's metabolism. This polymorphism has been shown to be correlated with hypertension and cardiovascular disease. Polymorphisms in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L) gene have also been correlated with increased risk for coronary artery disease. Other common serious polymorphisms regard the area with high linkage disequilibrium, including the neuroblastoma breakpoint family, NBPF3 gene, and ~ 12-50 kb upstream of the tissue nonspecific alkaline phosphatase gene. Finally, the polymorphisms which have been mostly associated with vitamin B12 concentration are the rs11254363 polymorphism at intron 52 of the intrinsic factor vitamin B12 receptor of the CUBN and the rs526934 polymorphism at intron 8 of transcobalamin I. To sum up, several polymorphisms have already been associated with vitamin B complexes and therefore homocysteine level, highlighting the complex nature of vitamin B genetics.

Characterization and Structure of Alternatively Spliced Transcript Variant of Human Intestinal Alkaline Phosphatase (ALPI) Gene.

Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.

Effects of rearing systems on the eggshell quality, bone parameters and expression of genes related to bone remodeling in aged laying hens.

Public concerns regarding animal welfare are changing the selection of rearing systems in laying hens. This study investigated the effects of rearing systems on eggshell quality, bone parameters and relative expression levels of genes related to bone remodeling in aged laying hens. A total of 2,952 55-day-old Jing Tint Six pullets were randomly assigned to place in the conventional caging system (CCS) or aviary system (AVS) and kept until 95 weeks of age. The AVS group delayed the decrease of eggshell quality and alleviated the symptoms of osteoporosis in the humerus rather than in the femur. Eggshell breaking strength, thickness, weight, weight ratio, stiffness and fracture toughness were decreased linearly with age (from 55 to 95 weeks of age, p < 0.05). The AVS group had higher eggshell breaking strength, stiffness and fracture toughness than the CCS group (p < 0.05). Higher total calcium and phosphorus per egg were presented in the AVS group at 95 weeks of age (p < 0.05). At 95 weeks of age, the AVS group had a humerus with higher weight, volume, length, midpoint perimeter, cortical index, fat-free dry weight, ash content, total calcium per bone, total phosphorus per bone, average bone mineral density, strength, stiffness and work to fracture compared to the CCS group (p < 0.05). Such differences did not appear in the femur. The relative expression levels of alkaline phosphatase (ALP) and osteocalcin (OCN) genes in the femur and hormone receptors (vitamin D receptor (VDR), estrogen receptor alpha (ERα) and fibroblast growth factor 23 (FGF23)) genes in the humerus were significantly upregulated (p < 0.05) in the AVS group. The level of tartrate-resistant acid phosphatase (TRAP) transcripts was also increased (p < 0.05) in the femur of the AVS group. Overall, compared with the CCS, the AVS alleviated the deterioration of eggshell and bone qualities of aged laying hens, which may be related to the changes in the expression of genes associated with bone remodeling.

Stimulation phosphatidylinositol 3-kinase/protein kinase B signaling by Porphyromonas gingivalis lipopolysacch aride mediates interleukin-6 and interleukin-8 mRNA/protein expression in pulpal inflammation

The signaling mechanisms for Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammation in human dental pulp cells are not fully clarified. This invitro study aimed to evaluate the involvement of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in PgLPS-induced pulpal inflammation. Human dental pulp cells (HDPCs) were challenged with PgLPS with or without pretreatment and coincubation with a PI3K/Akt inhibitor (LY294002). The gene or protein levels of PI3K, Akt, interleukin (IL)-6, IL-8, alkaline phosphatase (ALP), osteocalcin and osteonectin were analyzed by reverse transcription polymerase chain reaction (PCR), real-time PCR, western blotting, and immunofluorescent staining. In addition, an enzyme-linked immunosorbent assay was used to analyze IL-6 and IL-8 levels in culture medium. In response to 5μg/ml PgLPS, IL-6, IL-8, and PI3K, but not Akt mRNA expression of HDPCs, was upregulated. IL-6, IL-8, PI3K, and p-Akt protein levels were stimulated by 10-50μg/ml of PgLPS in HDPCs. PgLPS also induced IL-6 and IL-8 secretion at concentrations higher than 5μg/ml. Pretreatment and co-incubation by LY294002 attenuated PgLPS-induced IL-6 and IL-8 mRNA expression in HDPCs. The mRNA expression of ALP, but not osteocalcin and osteonectin, was inhibited by higher concentrations of PgLPS in HDPCs. P. gingivalis contributes to pulpal inflammation in HDPCs by dysregulating PI3K/Akt signaling pathway to stimulate IL-6 and IL-8 mRNA/protein expression and secretion. These results are useful for understanding the pulpal inflammation and possible biomarkers of inflamed pulp diagnosis and treatment.

Effects of recombinant human growth hormone on proliferation and differentiation of cementoblast and ERK1 / 2, JNK / SAPK and p38MAPK signaling pathway

To investigate the effects of recombinant human growth hormone on the proliferation and differentiation of cementoblast and the signal pathways of ERK1 / 2, JNK / SAPK and p38MAPK, osteoblasts (OCCM-30) were cultured in vitro. The OCCM-30 was treated with different concentrations of recombinant human growth hormone (rhGH) (0, 10, 50 ng/mL) for 1 d and 2 d, respectively. MTT assay was used to test the proliferation of OCCM-30 cells by rhGH. The effect of BSP, OPN, OCN and ALP genes was detected by RT-PCR after one day. The activity of alkaline phosphatase (ALP) was detected after treatment with OCCM-30 for five days at different concentrations of rhGH. After treatment with OCCM-30 at 100 ng/mL rhGH for 0 min, 5 min, 10 min, 15 min, 30 min and 60 min, the phosphorylation levels of ERK1/2, JNK/SAPK and p38MAPK were detected by Western blot. Results showed that rhGH could promote the proliferation of OCCM-30 cells, and the proliferation of OCCM-30 cells increases with the increase of rhGH concentration. After one day of culture, the levels of the BSP and ALP genes increased with the increase of rhGH concentration (P<0.05); the OPN gene level in the 10 ng/mL group was significantly higher than that in the blank group, and the 50 ng/mL group was significantly lower than the blank group. (P<0.05); OCN gene levels in the 10 ng/mL group and 50 ng/mL group were not significantly different from those in the blank group (P>0.05). Compared with the blank group, the ERK 1/2 phosphorylation level increased at 5 min in the 100 ng/mL group, reached the maximum at 10 min, decreased significantly at 15 min, decreased to the original level at 30 min, and had no significant change in ERK 1/2 total protein level; 100 ng/mL rhGH had no significant effect on SAPK/JNK, p38MAPK phosphorylation and total protein levels in OCCM-30 cells (P>0.05). It was concluded that 10ng/mL and 50ng/mL rhGH could promote the proliferation of OCCM-30 cells and promote the expression of the BSP gene and ALP gene. Low-dose rhGH is beneficial to OPN gene expression, and high-dose rhGH inhibits OPN gene expression. 100 ng/mL rhGH promoted the ERK 1/2 pathway and had no effect on the SAPK/JNK and p38 MAPK pathways.

Aneuploidy Rate and Stemness in Low-Level Mosaic Human Embryonic Stem Cells in the Presence/Absence of Bortezomib, Paclitaxel, and Lapatinib

Human embryonic stem cells (hESCs) are predisposed to aneuploidy through continual passages. Some reports indicate more sensitivity of aneuploid hESCs cells to anticancer drugs. The present study was designed to investigate the cytotoxicity of three anticancer drugs (including bortezomib, paclitaxel, and lapatinib) and their effect on aneuploidy rate in hESCs. To create a low-level mosaic cell line, normal hESCs (80%) and trisomic hESCs for chromosomes 12 and 17 (20%) were mixed. The effect of the 3 mentioned anticancer drugs on the chromosomal status was assessed by metaphase spread analysis after selection of the nontoxic conditions. Expression of pluripotency genes was analyzed, and an alkaline phosphatase test was performed to assess pluripotency preservation. Our data showed that treatment with bortezomib, paclitaxel, and lapatinib was nontoxic at 0.01, 0.01, and 0.2 μ<sc>M</sc> concentrations, respectively. Alkaline phosphatase and pluripotency gene expression analyses revealed maintenance of pluripotency following treatment with above-noted nontoxic concentrations. Aneuploid cells were dominant in treated and control groups with a minimum abundance of 70%, with no significant differences between groups. Drug treatments had no negative effect on pluripotency. Insensitivity of aneuploid cells in treatment groups could be related to the specific characteristics of each cell line in response to the drug and the proliferative superiority of cells with trisomies 12 and 17.

Establishment of a new human iPSC cell line (UOMi007-A) from a patient with Hypophosphatasia

Hypophosphatasia (HPP) is a rare, inherited, metabolic, genetic disorder, which arises due to loss of function mutation in the alkaline phosphatase (ALPL) gene. We have created a new induced pluripotent stem cell line (UOMi007-A) from peripheral blood mononuclear cells (PBMCs) of an 18 yr. old male patient having compound heterozygous mutations in the ALPL gene c.571G>A (p.Glu191Lys) and c.1001G>A (p.Gly334Asp) respectively. This line can be used for exploration into the molecular mechanisms of disease pathophysiology, screen new potential drugs and design cell therapy studies that can be personalized or used for future patients.