Cell Alkaline Phosphatase Research Articles

Biomedical and histological evidence of Boswellia sp. Burseraceae on kidney and liver function in mice

Boswellia sp. is a species of Boswellia. Burseraceae is an essential conventional natural product that has received a lot of attention in the pharmaceutical industry in recent years. The aim of this study was to role of Boswellia sp. Burseraceae on kidney and liver of Balb/c mice. Different concentrations of Boswellia sp. Burseraceae solution was given to 5–7 weeks old wild-type Balb/c mice of both sexes (n = 24) for 21 days, with a control group given normal water to determine the physiological, biochemical, haematological, (0.5%, 1%), and histopathological effect of (0.5%, 1% and 5%) Boswellia sp..The results of this study showed that physiological parameters were changed with the increasing concentration of Boswellia sp. Burseraceae solution; like daily food consumption was reduced and fluid intake was more, decrease in body weight, urine output was low with less Na+ & K+ resulting into hypernatremia, hyperkalemia and hyperuricemia. Also elevated creatinine level in blood increase levels of bilirubin, Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP) and degeneration of cells, glomerular atrophy of kidney cells and degeneration of hepatocytes. In conclusion, all of the results support to dosage toxicity. Biochemical indicators such as white blood cells, glucose, and haemoglobin did not alter significantly. Based on the findings, it is concluded that the extract exhibits dosage toxicity, which is corroborated by histopathological indicators.

Clinical Outcome and 8-Year Follow-Up of Alveolar Bone Tissue Engineering for Severely Atrophic Alveolar Bone Using Autologous Bone Marrow Stromal Cells with Platelet-Rich Plasma and β-Tricalcium Phosphate Granules.

Background: Although bone tissue engineering for dentistry has been studied for many years, the clinical outcome for severe cases has not been established. Furthermore, there are limited numbers of studies that include long-term follow-up. In this study, the safety and efficacy of bone tissue engineering for patients with a severely atrophic alveolar bone were examined using autogenous bone marrow stromal cells (BMSCs), and the long-term stability was also evaluated. Methods: BMSCs from iliac bone marrow aspirate were cultured and expanded. Then, induced osteogenic cells were transplanted with autogenous platelet-rich plasma (PRP) and β-tricalcium phosphate granules (β-TCP) for maxillary sinus floor and alveolar ridge augmentation. Eight patients (two males and six females) with an average age of 54.2 years underwent cell transplantation. Safety was assessed by monitoring adverse events. Radiographic evaluation and bone biopsies were performed to evaluate the regenerated bone. Results: The major population of transplanted BMSCs belonged to the fraction of CD34−, CD45dim, and CD73+ cells, which was only 0.065% of the total bone marrow cells. Significant deviations were observed in cell growth and alkaline phosphatase activities among individuals. However, bone regeneration was observed in all patients and the average bone area in the biopsy samples was 41.9% 6 months following transplantation, although there were also significant deviations among each case. No adverse events related to the transplants were observed. In the regenerated bone, 27 out of 29 dental implants were integrated. Dental implants and regenerated bone were stable for an average follow-up period of 7 years and 10 months. Conclusions: Although individual variations were observed, the results showed that bone tissue engineering using BMSCs with PRP and β-TCP was feasible for patients with severe atrophic maxilla throughout a long-term follow-up period and was considered safe. However, further studies with a larger number of cases and controls to confirm the efficacy of BMSCs and the development of a protocol to establish a reproducible quality of stem cell-based graft material will be required.

Rejuvenated BCMA-Specific CD8 + Cytotoxic T Lymphocytes Derived from Antigen-Specific Induced Pluripotent Stem Cells : Immunotherapeutic Application in Multiple Myeloma

T cell regenerative medicine represents an emerging immunotherapeutic approach using antigen-specific Induced Pluripotent Stem Cells (iPSC) to rejuvenate CD8 + cytotoxic T lymphocytes (CTL). Here we report on an iPSC-derived therapeutic strategy targeting B-Cell Maturation Antigen (BCMA) against multiple myeloma (MM) via establishment of antigen-specific iPSC, followed by differentiation into highly functional BCMA-specific CD8 + CTL. The reprogrammed BCMA-specific iPSC displayed normal karyotypes and pluripotency potential as evidenced by expression of stem cell markers (SSEA-4, TRA1-60) and alkaline phosphatase, along with differentiation into three germ layers (Ectoderm, Mesoderm, Endoderm). During embryoid body formation, BCMA-specific iPSC further polarized into the mesoderm germ layer, evidenced by the activation of SNAI2, TBX3, PLVAP, HAND1 and CDX2 transcriptional regulators. Next, the BCMA-specific iPSC clones committed to CD8 + T cell differentiation were characterized by analyzing their hematopoietic progenitor cells (HPC; CD34 + CD43 +/CD14 - CD235a -) for specific transcriptional regulation. RNAseq analyses indicated a low variability and similar profiles of gene transcription within the iPSC clones committed to CD8 + CTL compared to increased transcriptional variability within iPSC clones committed to different cell types. The unique transcriptional profiles of the iPSC committed to CD8 + T cells included upregulation of transcriptional regulators controlling CD4/CD8 T cell differentiation ratio, memory CTL formation, NF-kappa-B/JNK pathway activation, and cytokine transporter/cytotoxic mediator development, as well as downregulation of regulators controlling B and T cell interactions, CD4 + Th cells, and inhibitory receptor development. Specifically, a major regulatory shift, indicated by upregulation of specific genes involved in immune function, was detected in HPC from the iPSC committed to CD8 + T cells. BCMA-specific T cells differentiated from the iPSC were characterized as displaying mature CTL phenotypes including high expression of CD3, CD8a, CD8b, TCRab, CD7 along with no CD4 expression (Fig. 1). In addition, the final BCMA iPSC-T cells were predominantly CD45RO + memory cells (central memory and effector memory cells) expressing high level of T cell activation (CD38, CD69) and costimulatory (CD28) molecules. Importantly, these BCMA iPSC-T cells lacked immune checkpoints (CTLA4, PD1, LAG3, Tim3) expression and regulatory T cells induction, distinct from other antigen-stimulated T cells. The rejuvenated BCMA iPSC-T cells demonstrated a high proliferative (1,000 folds increase) during the differentiation process as well as poly-functional anti-tumor activities and Th1 cytokine (IFN-g, IL-2, TNF-a) production triggered in response to MM patients' cells in HLA-A2-restricted manner (Fig. 2). Furthermore, the immune responses induced by these BCMA iPSC-T cells were specific to the parent heteroclitic BCMA 72-80 (YLMFLLRKI) peptide used to reprogram and establish the antigen-specific iPSC. Evaluation of 88 single cell Tetramer + CTL from the BCMA iPSC-T cells revealed a clonotype of unique T cell receptor (TCRa, TCRb) sequence. The BCMA-specific iPSC clones maintained their specific differentiation potential into the antigen-specific CD8 + memory T cells, following multiple subcloning in long-term cultures under feeder-free conditions or post-thaw after long-term (18 months) cryopreservation at -140 oC, which provides additional benefits to treat patients in a continuous manner. Taken together, rejuvenated CD8 + CTL differentiated from BCMA-specific iPSC were highly functional with significant (*p < 0.05) levels of anti-MM activities including proliferation, cytotoxic activity and Th-1 cytokine production. Therefore, the antigen-specific iPSC reprogramming and T cells rejuvenation process can provide an effective and long-term source of antigen-specific memory CTL lacking immune checkpoints and suppressors for clinical application in adoptive immunotherapy to improve patient outcome in MM. [Display omitted] DisclosuresMunshi: Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Legend: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Novartis: Consultancy; Pfizer: Consultancy. Ritz: Amgen: Research Funding; Equillium: Research Funding; Kite/Gilead: Research Funding; Avrobio: Membership on an entity's Board of Directors or advisory committees; Akron: Consultancy; Biotech: Consultancy; Blackstone Life Sciences Advisor: Consultancy; Clade Therapeutics, Garuda Therapeutics: Consultancy; Immunitas Therapeutic: Consultancy; LifeVault Bio: Consultancy; Novartis: Consultancy; Rheos Medicines: Consultancy; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy. Anderson: Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Effect of Azithromycin on Mineralized Nodule Formation in MC3T3-E1 Cells.

Azithromycin displays immunomodulatory and anti-inflammatory effects in addition to broad-spectrum antimicrobial activity and is used to treat inflammatory diseases, including respiratory and odontogenic infections. Few studies have reported the effect of azithromycin therapy on bone remodeling processes. The aim of this study was to examine the effects of azithromycin on the osteogenic function of osteoblasts using osteoblast-like MC3T3-E1 cells. Cells were cultured in the presence of 0, 0.1, 1, and 10 µg/mL azithromycin, and cell proliferation and alkaline phosphatase (ALPase) activity were determined. In vitro mineralized nodule formation was detected with alizarin red staining. The expression of collagenous and non-collagenous bone matrix protein was determined using real-time PCR or enzyme-linked immunosorbent assays. In cells cultured with 10 µg/mL azithromycin, the ALPase activity and mineralized nodule formation decreased, while the type I collagen, bone sialoprotein, osteocalcin, and osteopontin mRNA expression as well as osteopontin and phosphorylated osteopontin levels increased. These results suggest that a high azithromycin concentration (10 µg/mL) suppresses mineralized nodule formation by decreasing ALPase activity and increasing osteopontin production, whereas low concentrations (≤l.0 µg/mL) have no effect on osteogenic function in osteoblastic MC3T3-E1 cells.

The cytotoxic effects of various endodontic irrigants on the viability of dental mesenchymal stem cells.

This study aimed to evaluate cytotoxic effects of various irrigation solutions used in regenerative endodontic treatments (RETs) on mesenchymal stem cells, and further examine the long-term effect of hypochlorous acid (HOCl) on the cell viability and alkaline phosphatase (ALP) activity. Stem cells were exposed to various concentrations of NaOCl, EDTA, chlorhexidine (CHX), etidronic acid (HEDP)/NaOCl combination and HOCl. HOCl was tested for its effects on ALP activity up to 21 days. Additionally, cell viability was measured fluorescently using calcein AM. The most cytotoxic irrigant was CHX even with the lowest concentration. NaOCl and HEDP/NaOCl with 1:100 dilution decreased viability to around 40%. HOCl showed the lowest cytotoxicity among all tested irrigants. HOCl also showed no significant reduction in ALP activity compared with the controls. The cytotoxicity of endodontic irrigants was time and concentration dependent. HOCl demonstrated promising results regarding viability and ALP activity, since RETs require host stem cell survival.

Immunohistochemical insights into a hidden pathology: Canine cryptorchidism

Cryptorchidism is a common disorder in the canine population with some aspects still unclear. Although the bilateral condition is known to lead to fertility problems and predisposition to testicular cancer, the neoplastic risk for scrotal testis in unilateral cryptorchid dog is controversial. Therefore, the therapeutic approach to the canine unilateral cryptorchid is arbitrary so far.This study aimed to investigate precancerous testicular lesions, such as immaturity and atrophy, and compare them in scrotal and undescended testes using an in-depth diagnostic analysis based on immunophenotypic patterns. With this purpose, 26 adult male dogs of different ages and breeds, affected by unilateral or bilateral cryptorchidism were enrolled. After surgical removal, testes were examined immunohistochemically to assess their positivity for specific markers of the canine foetal/neonatal period, that is vimentin (VIM), cytokeratin (CK), desmin (DES), inhibin-α (INH), and anti-Müllerian hormone (AMH) in Sertoli cells, and placental alkaline phosphatase (PLAP) in germ cells. Except for the ubiquitous VIM, all the markers were more expressed in neoplastic gonads compared to healthy ones (P < 0.05). Similarly, testes detected with Sertoli cell-only tubules as well as with Sertoli cells hyperplasia showed higher expression than gonads without such alterations for CK, DES, AMH and PLAP, and for CK and DES, respectively (P < 0.05). The same trend was observed in undescended respect to scrotal testes even though their positivity was significant only for DES, INH and AMH (P < 0.05). Immunohistochemical positivity found in scrotal testes of unilateral cryptorchid dogs, even in absence of detectable anatomical abnormalities, was suggestive of precancerous lesions.Despite the limited sample size, this study could help to clarify the predisposition to neoplastic development in normally descended testes. These markers expression in adult life could allow identifying the early stages of the testicular carcinogenesis process besides suggesting a precautionary bilateral surgical approach in unilateral cryptorchid dogs.

Osteogenic Activity of Lupeol Isolated from Clinacanthus nutans Lindau: Activity and Mode of Action

Clinacanthus nutans Lindau has been traditionally used for healing of bone fragility, but the mechanism of actions has not been clarified yet. In this study, the bone regeneration activity of lupeol derived from C. nutans was assessed using an in vitro model of osteoblast cells MC3T3-E1. The finding revealed that the compound was not significantly toxic to osteoblast cells at concentration of ≤40 μg/mL. Lupeol demonstrated the osteogenic activity through enhancement of alkaline phosphatase (ALP) of osteoblast cells up to 31.2%, 21%, and 12% at concentrations of 5, 10, and 20 µg/mL, respectively ( p &lt; 0.05). Besides, the mineralization activity was increased up to 170, 230, 185, and 117% at concentration of 5, 10, 20, and 40 μg/mL, respectively ( p &lt; 0.05). The marker genes related to osteoblast differentiation evaluated on the expression level in the presence of lupeol, including collagen I (col 1), osteopontin (opn), osterix (osx), and runx2, showed upregulated expression in all the test genes ( p &lt; 0.05). The Western blot analysis demonstrated a clear effect of lupeol on expression of p38/p-p38, and ERK/p-ERK proteins involved in the MAPK signaling pathway. Thus, lupeol isolated from C. nutans exhibited the osteogenic activity by enhancing expression of important markers of osteogenesis, as well as affected the MAPK signaling pathway relating to osteoblast differentiation. This is the first report on the detailed mechanism of action of lupeol on bone regeneration and also explains for the traditional use of this medicinal plant for bone healing.

Antibacterial and Cellular Behaviors of Novel Zinc-Doped Hydroxyapatite/Graphene Nanocomposite for Bone Tissue Engineering.

Bacteria are one of the significant causes of infection in the body after scaffold implantation. Effective use of nanotechnology to overcome this problem is an exciting and practical solution. Nanoparticles can cause bacterial degradation by the electrostatic interaction with receptors and cell walls. Simultaneously, the incorporation of antibacterial materials such as zinc and graphene in nanoparticles can further enhance bacterial degradation. In the present study, zinc-doped hydroxyapatite/graphene was synthesized and characterized as a nanocomposite material possessing both antibacterial and bioactive properties for bone tissue engineering. After synthesizing the zinc-doped hydroxyapatite nanoparticles using a mechanochemical process, they were composited with reduced graphene oxide. The nanoparticles and nanocomposite samples were extensively investigated by transmission electron microscopy, X-ray diffraction, and Raman spectroscopy. Their antibacterial behaviors against Escherichia coli and Staphylococcus aureus were studied. The antibacterial properties of hydroxyapatite nanoparticles were found to be improved more than 2.7 and 3.4 times after zinc doping and further compositing with graphene, respectively. In vitro cell assessment was investigated by a cell viability test and alkaline phosphatase activity using mesenchymal stem cells, and the results showed that hydroxyapatite nanoparticles in the culture medium, in addition to non-toxicity, led to enhanced proliferation of bone marrow stem cells. Furthermore, zinc doping in combination with graphene significantly increased alkaline phosphatase activity and proliferation of mesenchymal stem cells. The antibacterial activity along with cell biocompatibility/bioactivity of zinc-doped hydroxyapatite/graphene nanocomposite are the highly desirable and suitable biological properties for bone tissue engineering successfully achieved in this work.

Mechnical Properties and Biocompatibility of Hydroxyapatite/Manganese Dioxide/Palladium Nanocomposite Scaffolds Filled by Natural Chitosan

The main objective of the present study is to evaluate the mechanical properties, biocompatibility, and bioactivity behavior of scaffolds made of hydroxyapatite (HA)-modified by MnO2 and Palladium (Pd) for biomedical applications. Throughout the research, HA, MnO2, and Pd were developed using sol-gel and precipitation methods, respectively. The properties of the scaffolds were determined using Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDX), atomic absorption, and Brunauer−EmmeS−Teller (BET) method. To investigate the in vitro cell proliferation and alkaline phosphatase (ALP) assays, cell culture was done. Furthermore, the mechanical properties of the scaffolds were investigated before and after immersion in Simulated Body Fluid (SBF), and the interaction of Dental Pulp Stem Cells (DPSCs) with the nanocomposite scaffolds was assessed. The obtained results showed that the HA/MnO2/Pd scaffolds were characterized by higher compressive strength (35.72%), toughness (35.68%), microhardness (80%), and density (0.44%) than HA/MnO2/Pd filled by chitosan (CS) binder scaffolds. The biocompatibility properties indicated higher cell proliferation and ALP assay on the HA/MnO2/Pd filled by CS scaffolds than those of HA/MnO2/Pd scaffolds.

Abstract P152: Downregulation Of Tissue-nonspecific Alkaline Phosphatase In Endothelial Cells Does Not Affect Calcification But Reduces Cardiac Function In Mice With Atherosclerosis

Introduction: We have previously demonstrated that overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells increases vascular calcification and accelerates atherosclerosis. Whether TNAP activity in vascular endothelium is essential for intimal calcification and the progression of atherosclerosis is unknown. Hypothesis: We hypothesized that elimination of TNAP expression in endothelial cells would be sufficient to reduce intimal calcification associated with atherosclerosis. Methods: Endothelial TNAP knockout (KO) mice were developed on the background of the low density lipoprotein receptor mutation. Endothelial TNAP KO (n = 8) and control (n = 9) mice were fed a Western diet starting from 8 weeks and until 52 weeks of age. Cardiac structure and function were assessed by echocardiography. Calcium volume was measured in the aortic root and aortic arch area by microcomputed tomography (microCT). Data were analyzed using a 2-way ANOVA to calculate the effects of the genotype adjusted for sex. Results: MicroCT data did not show any statistically significant decrease in calcification in endothelial TNAP KO mice compared to controls; however, other cardiac parameters were affected by genotype. The physiological examination of the mice demonstrated that ablation of TNAP expression in the endothelium resulted in significant reduction in ejection fraction, stroke volume, and cardiac output in the absence of cardiac hypertrophy. Conclusion: Contrary to our hypothesis, TNAP has no effect on natural progression of calcified atherosclerosis; however, our data indicate that TNAP is required for protection of cardiac function under conditions of atherosclerosis in mice

Fibrous dysplasia of thoracic and lumbar spine: A case report

IntroductionFibrous dysplasia (FD) is a rare genetic disease characterized by development of dysplastic bone tissue leading to pain, deformities and compressive complications. Spinal involvement is rare particularly for the polyostotic form. Case reportHerein, we present a case of 52-year-old man presented with isolated low back pain who received non-steroidal anti-inflammatory drugs (NSAIDs) with partial pain relief. On examination, the thoracic and lumbar spines were tender. Lumbar spine movements, attested by Shöber’s test and finger-to-floor test, were limited. There was no radicular pain and neurological examination was normal. The erythrocyte sedimentation rate was 20 mm/1st hour, C- reactive protein 2 mg/l and white blood cell count, serum calcium, phosphorus, alkaline phosphatase and parathormone were normal. Plain x-ray lumbar and thoracic spine and hips were normal. X-ray cervical spine and peripheral bone showed no lesions. Computed tomography (CT) and magnetic resonance imaging showed multiple osteolytic lesions with ground-glass opacity and sclerotic rims of thoracic and lumbar (L1 and L2) spine. Investigations to rule out malignancy or infections were negative. CT guided biopsy of a lumbar vertebra was performed and showed the presence of abnormal fibrous matrix surrounding an irregular shaped immature bone. The diagnosis of polyostotic FD of the spine was made. Zoledronic acid infusion was introduced and the patient felt better thereafter with no additional osteolytic lesions or new pain localization reported for 2 years on follow up. ConclusionThoracic and lumbar spine FD is a rare localization that should be considered in the differential diagnosis of back pain.

Biological activity of biomimetic dermal papilla spheres prepared by culture of dermal papilla cells of mice based on hanging drops of gelatin methacrylate and its hair-inducing function in nude mice

Biological activity of biomimetic dermal papilla spheres prepared by culture of dermal papilla cells of mice based on hanging drops of gelatin methacrylate and its hair-inducing function in nude mice

Machine learning approaches in predicting ambulatory same day discharge patients after total hip arthroplasty

BackgroundWith continuing financial and regulatory pressures, practice of ambulatory total hip arthroplasty is increasing. However, studies focusing on selection of optimal candidates are burdened by limitations related to traditional statistical...

Biological activity of titania coating prepared with zirconium oxychloride and titania on zirconia surface

Zirconia is recognized as a promising dental implant material because of its good biocompatibility, sufficient mechanical strength, minimal ion release and aesthetic effects similar to natural teeth. However, the limitations of inert surface of zirconia affect the long-term efficacy of zirconia implants. To enhance the osseointegration of zirconia implants, titania (TiO2) coating is prepared on the zirconia surface by immersion in a mixed zirconium oxychloride (ZrOCl2) and TiO2 suspension in a water bath. The surface and longitudinal section morphology are observed by scanning electron microscopy (SEM). The chemical composition is evaluated through energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD). The roughness and hydrophilicity of zirconia surface are also examined. A three-point bending test is conducted on the samples to explore the effect of this surface treatment on the mechanical strength of zirconia. Vickers hardness measurements are performed to evaluate the gradient change of the longitudinal section of the zirconia substrate. The MC3T3-E1 cells are seeded on zirconia discs, and a LIVE/DEAD double-staining test is conducted to detect the cytotoxicity of the TiO2 coating. The cell morphology is studied through fluorescence microscope. The degrees of cell proliferation, mineralization and alkaline phosphatase (ALP) activity are calculated and compared. Detection of the mRNA expression of osteogenic differentiation-related markers is performed by RT-PCR. A TiO2 coating is generated on the zirconia surface and significantly improves the surface roughness and hydrophilicity while not adversely affecting the mechanical strength of zirconia. The hardness of the zirconia substrate shows a gradient change. The TiO2 coating can promote proliferation, spreading and osteogenic differentiation of MC3T3-E1 cells. These findings suggest that modifying the surface of zirconia with a TiO2 coating may have a favourable osteogenic effect.

Femtosecond laser-induced periodic grooves and nanopore clusters make a synergistic effect on osteogenic differentiation

Polyether-ether-ketone (PEEK) materials have good biocompatibility, excellent corrosion resistance, chemical stability and an elastic modulus close to that of natural bone. However, due to its biological inertness, PEEK may affect osteogenic differentiation and leads to osseointegration failure, though PEEK is expected to improve osseointegration. In this work, by changing the power of femtosecond laser, micro-grooves are made on the PEEK surface. As observed by scanning electron microscopy, the trench has a periodic structure, the micro shape is neat, and the trench is also covered with nanometer-level pore clusters. In the in vitro culture experiments, through the proliferation experiment of mouse bone marrow mesenchymalstem cells (mBMSCs), cell viability analysis and alkaline phosphatase activity analysis, it is proven that after femtosecond laser treatment of the PEEK surface, the micro-grooves on the surface and the nanopore clusters due to laser energy ablation can produce a synergistic effect, enhancing the osteogenic differentiation ability of cells, and improving the bone integration ability of PEEK materials.

Expression of the developmental important candidate genes in oocytes, embryos, embryonic stem cells, cumulus cells, and fibroblast cells of buffalo (Bubalus bubalis)

The present study was undertaken to study the expression of the developmental important gene transcripts in immature oocytes, mature oocytes, different stages of IVF produced embryos, embryonic stem (ES), cumulus (BCC), fetal fibroblast (BFF), newborn fibroblast (NBF) and adult fibroblast (BAF) cells of buffalo by semi-quantitative RT-PCR. The expression of GLUT1, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts was found in immature oocytes, mature oocytes, 2-cell, 4-cell, 8–16 cell, morula, and the blastocyst. Interestingly, the CX43 expression was found in oocytes, embryos, and other cell types, but it was not detected in the blastocyst. However, the IFNT expression was found in the blastocyst only, but not in other cells. The buffalo ES cells showed the expression of intracellular and cell surface markers (NANOG, OCT4, SOX2, FOXD3, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and alkaline phosphatase activity. Two ES cell lines (S-line and M-line-II) were continued to survive up to 98th passages (~630 days) and 97th passages (~624 days), respectively. It was interesting to note that GLUT1, CX43, HSP70.1, POL A Polymerase, GDF9, BMP15, and SURVIVIN transcripts (except the IFNT) were expressed in buffalo ES, BCC, BFF, NBF and BAF cells. This is the first preliminary report that the buffalo ES, BCC, BFF, NBF, and BAF cells expressed the several developmental important candidate genes. It is concluded that the expression of the major developmental important genes was not only expressed in the oocytes and embryos but also expressed in the ES, BCC, BFF, NBF, and BAF cells of buffalo.

Ameliorative effect of the dietary Egyptian leek ( Allium ampeloprasum L. var. kurrat ) on zinc toxicity of the African catfish Clarias gariepinus (Burchell, 1822)

There is scant knowledge available concerning the protective role of the Allium ampeloprasum L. on African catfish (Clarias gariepinus) that is chronically exposed to zinc (Zn). Six fish groups were used: three were fed diets supplemented with leek extract (LEE 0%, 2% and 4%); and the three other groups received LEE and were exposed to a sublethal dose of zinc sulphate (7.8 mg L−1). After 45 days, fish exposed only to Zn exhibited the highest values of weight gain, specific growth rate, white blood cell count, alanine aminotransferase, alkaline phosphatase, proteins, albumin, globulin, glucose, creatinine and total antioxidant concentration significantly. Fish exposed to Zn-only also revealed insignificant changes in the condition coefficient, hepatosomatic index, gonadosomatic index and almost all haematological parameters. Moreover, exposure to only Zn resulted in a significant decrease in aspartate aminotransferase (AST). Feeding on LEE 2% increased growth, whereas LEE 4% had an insignificant effect on growth; both LEE doses caused insignificant changes in haematological and biochemical parameters. Feeding on LEE while being exposed to Zn showed a reduction in some elevated biochemical parameters compared with the Zn-only-exposed group, with LEE 2% displaying a greater protective role than LEE 4%. Histopathological investigations revealed many changes in the livers of fish fed with LEE, while it has more degenerative and necrotic changes with exposure to Zn. So, leek extract could be used at the lower dose for African catfish as a feed supplement to improve fish growth and alleviate the possible undesired effects of chronic exposure to Zn.

Epigenetic Effects of Blackberry Extract on Human Colorectal Cancer Cells

Fruit-derived polyphenolic compounds have been shown to exert anticancer effects via epigenetic mechanisms. In this study, we investigated the effect of blackberry extract on the expression of DNMTs (Dnmt1, Dnmt3a, and Dnmt3b) and HDACs (HDAC1-4 and SIRT1) and its influence on the cellular differentiation and promoter DNA methylation of tumor-related genes using a panel of six human CRC cell lines. Treatment with IC20 and IC50 concentrations of blackberry extract for 72 h significantly reduced Dnmt1 and Dnmt3b transcript levels in HCT116, SW480, HT29/219, SW742, and LS180 cells in a dose-dependent manner. Blackberry also induced promoter DNA demethylation of SFRP2 and p16 genes in four tested CRC cell lines. Berry treatment, however, upregulated Dnmt3a genes in SW480, SW742, and HT29/219 cell lines. A dose-dependent and cell-type-specific reduction of HDAC1, HDAC2, and HDAC4 expressions were observed in CRC-treated cells. Treatment with berry extract induced the expression of SIRT1 gene in HCT116 and HT29/219 cells and increased the expression of two colonic epithelial cell differentiation markers, carcinoembryonic antigen (CEA) and alkaline phosphatase in LS180 cells in a time-dependent manner. This study is the first to report the epigenetic effects of blackberry in cancer cells.

Abstract 1159: Hexamethylene bisacetamide (HMBA) inhibits the induction of alkaline phosphatase by butyrate in some bladder and colon cancer cell lines

Abstract Induction of alkaline phosphatase in colon cancer cells by inhibitors of histone deacetylase activity has been widely used to monitor the induction of differentiation. We have previously identified three human bladder cancer cell lines in which alkaline phosphatase activity can be induced by inhibitors of histone deacetylase (5637, HT1197 and HT1376 cells). In earlier work it was observed that some bromodomain inhibitors inhibit growth and induction of alkaline phosphatase in these cells. After a recent report that HMBA acts as a bromodomain inhibitor, the effects of HMBA on growth and induction of alkaline phosphatase were investigated in two colon cancer cell lines (Caco-2 and HT29) and in 5637, HT1197 and HT1376 bladder cancer cells. Growth was monitored by staining with sulforhodamine B. Incubation with 2 mM butyrate for 72 hours caused induction of alkaline phosphatase in the five cell lines. In some cell lines there was inhibition of growth and of the induction of alkaline phosphatase activity by 2 mM HMBA. Increasing the HMBA concentration to 5 mM caused significant growth inhibition in all five cell lines and significant inhibition of induction of alkaline phosphatase activity by 2 mM butyrate in the 5637, Caco-2 and HT1376 cells, whether expressed as total activity or activity per unit protein. Although the effect of HMBA on induction of alkaline phosphatase in HT29 cells was not statistically significant there is evidence in the literature for inhibition with longer incubation times. The most marked effects on enzyme induction were seen with the HT1376 bladder cancer cells and this distinction was in line with previous studies on the combined effects of butyrate and the well-established bromodomain inhibitor, JQ1. HMBA has been found by several investigators to be an effective inducer of differentiation in erythroleukemia cells as judged by hemoglobin production. The lack of induction of alkaline phosphatase by HMBA as a single agent in any of the cell lines investigated in the present work suggests that different mechanisms of induction of differentiation operate in erythroleukemia cells than in bladder and colon cancer cells. Citation Format: Michael A. Lea. Hexamethylene bisacetamide (HMBA) inhibits the induction of alkaline phosphatase by butyrate in some bladder and colon cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1159.

RNA analysis based on a small number of manually isolated fixed cells (RNA-snMIFxC) to profile stem cells from human deciduous tooth-derived dental pulp cells

BackgroundExpression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette.ResultsTwo or more colored cells (~ 10), after staining with a chromogen such a 3,3′-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells.ConclusionThese findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.