Alkaline Phosphatase Enzyme Activity Research Articles

Soil phosphorus transformation characteristics in response to molybdenum supply in leguminous crops

Phosphorus (P) is one of the most restrictive essential elements to crop growth and development due to less availability in the soil system. Previous studies have reported the synergistic effects between molybdenum (Mo) and P fertilizer on P uptake in various crops. However, an induced long term effect of Mo on soil P dynamics in the rhizosphere and non-rhizosphere has not been reported yet in leguminous crops. In this study, a long term field experiment was conducted to explore the P transformation characteristics and bioavailability in Mo-deficient (-Mo) and Mo-enriched (+Mo) soil under leguminous (broad bean-soybean) cropping system. The results indicated that long-term Mo application increased the plant dry matter accumulation (14.23%–35.27%, for broad bean; 24.40%–37.46%, for soybean) from March–September. In rhizosphere soil, the percent decrease in pH (8.10%) under +Mo treatment of the soybean crop was recorded more during September as compared to broad bean crop. Under Mo supply, H2O-Pi fraction increased up to 28.53% and 43.67% while for NaHCO3-Pi this increase was up to 5.61% and 11.98%, respectively in the rhizosphere soil of broad bean and soybean, whereas, residual-P exhibited the highest proportion of P fractions. Moreover, compared with -Mo, +Mo treatments significantly increased the soil acid phosphatase (broad bean = 17.43 μmol/d/g; soybean = 28.60 μmol/d/g), alkaline phosphatase (broad bean = 3.34 μmol/d/g; soybean 6.35 μmol/d/g) and phytase enzymes activities (broad bean = 2.45 μmol/min/g; soybean = 5.91 μmol/min/g), transcript abundance of phoN/phoC genes and microbial biomass P (MBP) in rhizosphere soil. In crux, the findings of this study suggest that long term Mo application enhanced P bioavailability through increased available P, MBP, P related enzymes activities and their genes expressions which may represent a strategy of Mo to encounter P deficiencies in the soil system.

Oxytocin and bone quality in the femoral neck of rats in periestropause

The objective of this study is to identify whether oxytocin (OT) contributes to the reduction of osteopenia in the femoral neck of rats in periestropause. Animals in irregular estrous cycles received two NaCl injections (0.15 mol/L) or OT (134 μg/kg) over a 12-h interval, and after thirty-five days without treatments, the biological sample collection was performed. The oxytocin group (Ot) demonstrated the highest enzymatic activity of alkaline phosphatase (p = 0.0138), lowest enzymatic activity of tartrate-resistant acid phosphatase (p = 0.0045), higher percentage of compact bone (p = 0.0359), cortical expression of runt-related transcription factor 2 (p = 0.0101), osterix (p = 0.0101), bone morphogenetic protein-2/4 (p = 0.0101) and periostin (p = 0.0455). Furthermore, the mineral-to-matrix ratio (ν1PO4/Proline) was higher and type-B carbonate substitution (CO3/ν1PO4) was lower (p = 0.0008 and 0.0303) in Ot group. The Ot showed higher areal bone mineral density (p = 0.0050), cortical bone area (p = 0.0416), polar moment of inertia, maximum, minimum (p = 0.0480, 0.0480, 0.0035), bone volume fraction (p = 0.0166), connectivity density (p < 0.0001), maximal load (p = 0.0003) and bone stiffness (p = 0.0145). In Ot percentage of cortical pores (p = 0.0102) and trabecular number (p = 0.0088) was lower. The results evidence action of OT in the reduction of osteopenia, suggesting that it is a promising anabolic strategy for the prevention of primary osteoporosis during the periestropause period.

Actividad de fosfatasa-alcalina y crecimiento del arroz con inoculación biológica y micronutrientes

La inoculación con bacterias promotoras del crecimiento en cultivos es una alternativa para reducir el uso de fertilizantes nitrogenados y los costos de producción. El objetivo de esta investigación fue evaluar el efecto de la inoculación de semillas de arroz Oryza sativa L. con la bacteria Azospirillum brasilense sobre la actividad enzimática de la fosfatasa-alcalina y su crecimiento. Se realizó en la Facultad de Ciencias Agrarias y la Facultad de Ciencias Químicas de la Universidad Nacional de Asunción, Paraguay. Se utilizó un diseño completamente al azar con cinco tratamientos y cinco repeticiones. Los tratamientos consistieron en T1 (Control abosoluto): semillas de arroz sin tratamiento, T2 (Co+Mo): semillas de arroz tratadas con cobalto más molibdeno, T3 (Az): semillas de arroz inoculadas con A. brasilense, T4 (Az+1F): semillas de arroz inoculadas con A. brasilense más una aplicación foliar del inoculante y T5 (Az+2F): semillas de arroz inoculadas más dos aplicaciones foliares. Se aplicó Anava y la prueba de Tukey (p&lt; 0.05) para el análisis de las variables agronómicas y la prueba de Kruskal-Wallis (p&lt; 0.05) para las evaluaciones enzimáticas. Las variables evaluadas fueron longitud de raíces (cm), masa seca de partes aéreas y raíces (g) y la actividad enzimática la fosfatasa-alcalina (μg de PNP g-1 suelo h-1). No se observaron diferencias significativas entre tratamientos con Azospirillum brasilense respecto a la masa seca. La actividad fosfatasa en los tratamientos con A. brasilense osciló entre 1 655.49 en el T3 y 7429,55 μg de PNP g-1 suelo h-1 en el T4. La utilización de micronutrientes a base de cobalto y molibdeno propició un mayor crecimiento de raíces.

Toxicological impact of green synthesized silver nanoparticles and protective role of different selenium type on Oreochromis niloticus: hematological and biochemical response

BackgroundThe present work aimed to detect the toxicological effects of green synthesized silver nanoparticles (Ag-NPs) by using Moringa Oleifera leaves extract on hematological and biochemical parameters of Oreochromis niloticus. MethodsAdult fish were exposed to two sublethal concentrations (1.95 and 3.9 ppm) of Ag-NPs against sodium selenite (0.1 ppm) and biosynthesized selenium nanoparticles (Se-NPs); 0.1 ppm; protection role for 2 and 4 weeks. Hematological parameters; erythrocyte count (RBCs), hemoglobin content (Hb), haematocrit value (Hct), mean corpuscular volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC), leucocytes (WBCs), with differential count Micronucleus (MN) and alerted cells and biochemical parameters; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) enzyme activities, serum protein (total protein, albumin and globulin) concentration, urea, creatinine, glucose, cholesterol (Cho) and triglyceride (Tg) were detected. ResultsThe present investigation showed that Ag-NPs in different doses led to a significant reduction (p < 0.05) in RBCs, Hb, Hct, MCV, WBCs, LYM and serum proteins concentration. However, MCHC, MN, alerted cells, NEUT, AST, ALT, ALP enzyme activities, urea, creatinine, glucose, Cho and Tg showed a significant increases (p < 0.05) when compared with control group. Sodium selenite (Se) and biosynthesized selenium nanoparticles (Se-NPs) play an optimistic role in detoxification of Ag-NPs toxicity. ConclusionThe results suggest the negative impact of Ag-NPs on hematology and biochemical parameters of fish. Moreover, Se-NPs showed a full improvement of hematological and biochemical parameters more than that of sodium selenite in elimination of Ag-NPs toxicity.

Extract of Adenopus breviflorus Fruit Alters the Expressions of Hepatic Cytochrome C, Caspase 3 and Sperm Parameters in Rats

Adenopus breviflorus fruit is used in traditional medicine against the outbreak of measles and the treatment of gonorrhoea in men. The fruit has been shown to have anti‐implantation activity. However, the safety of the aqueous extract of Adenopus breviflorus (AEAB) fruit on the function of the liver and male reproduction has not been studied. Therefore, this study investigated the effect of the oral administration of the AEAB fruit on rat liver function enzymes, cytochrome c release, caspase 3 expression and male reproductive functions. Adult male rats (15) were randomly divided into three groups of 5 animals per group. Animals in group 1 received distilled water while animals in groups 2 and 3 received an oral dose of 200 mg/kg and 400 mg/kg of AEAB fruit for 30 days, respectively. Alanine transaminase, alkaline phosphatase and aspartate transaminase enzyme activities were assayed spectrophotometrically. Cytochrome c and caspase 3 protein expressions in rat liver tissues were determined using immunohistochemistry. Sperm abnormality, sperm count and sperm motility were also determined. The result obtained showed that there was no significant (p &lt; 0.05) difference in serum alanine transaminase, alkaline phosphatase and aspartate transaminase between the treated groups and the control. Immunohistochemistry showed that cytochrome c release and caspase 3 proteins were significantly (p &lt; 0.05) expressed in the liver of animals in the treated groups when compared to the control. Furthermore, sperm abnormality significantly (p &lt; 0.05) increased while sperm count of rats orally exposed to AEAB fruit were significantly (p &lt; 0.05) reduced in the treated groups when compared to the control group. Specifically, sperm motility was significantly (p &lt; 0.05) reduced only in group 3 when compared with the control. This study show that the aqueous extract of Adenopus breviflorus fruit may not be hepatotoxic. However, this extract may have the potential to activate mitochondria cell death through cytochrome c release and activation of caspase 3 in rat liver. Furthermore, our study revealed that the extract may cause reproductive dysfunction in men. Therefore, the use of the extract in the treatment of gonorrhoea in traditional medicine should be carried out with utmost care.

The role of alkaline phosphatase (alp) and γ-glutamyltransferase (ggt) enzymes activities in monitoring the effect of enzyme replacement therapy in children with gaucher disease

To assess the role of γ-Glutamyltransferase (GGT) and alkaline phosphatase (ALP) in the diagnosis of Gaucher disease (GD) and monitoring the response of liver and bone to the treatment with enzyme replacement therapy (ERT).A case-control study was done on 67 children age range from 3-10 years who had Gaucher disease recruited from the Pediatric Department and Unit of rare disease at Al Imamain Al-Kathemeaain medical city,Gastroenterology and Hepatology Teaching Hospital, Children Welfare Hospital Consultation Clinic and Central Child’s Teaching Hospital. The activity of Alkaline Phosphatase (ALP) and γ- GlutamylTransferase (GGT) enzymes were measured in the samples of 67 Gaucher patients who were categorized as newly diagnosed untreated patients (n=9), patients receiving ERT for 3-5 months (n=18) 6-12 months (n=20) and patients receiving ERT for more than one year (n=20) and compared with twenty age-matched control subjects. The data indicated that the levels of both ALP and GGT in GD patients (216.82 ± 64.51, 35.94 ± 7.11; respectively) were significantly higher than those of controls (163.17 ± 49.34, 28.33 ± 3.36; respectively). Levels of ALP showed to return to levels comparable to those of controls after more than one year of treatment, while GGT levels become comparable to controls’ levels within 6-12 months. Negative significant (p&lt;0.05) correlations were obtained between the levels of ALP and GGT activities with the duration of receiving ERT. Receiver Operating Characteristic (ROC) curve results revealed that GGTshowed a higher area under the curve (AUC), sensitivity, and specificity than ALP activities in all studied groups and subgroups.The possibility of using ALP and GGT as supportive diagnostic markers and as powerful markers for the monitoring of the liver and bone improvement with the administration of ERT.

Heavy metal accumulation and changes in soil enzymes activities and bacterial functional diversity under long-term treated wastewater irrigation in East Central region of Tunisia (Monastir governorate)

Climate change and population growth generates a decrease in water availability around the world which can compromise the maintenance of sustainable agriculture. Thus, treated wastewater (TWW) became an alternative to minimize water shortage. However, this may indirectly affect the soil’s microbial properties. In this study different soils irrigated for 0, 1, 8 and 20 years with TWW were sampled and from the east central region of Tunisia. The objective was to investigate its effect on heavy metal accumulation and enzyme activities of β-glucosidase, arylsulfatase, acid phosphatase, alkaline phosphatase, urease, dehydrogenase and fluorescein diacetate (FDA). Moreover, carbon microbial biomass (Cmic) using Substrate induced respiration (SIR) technique and microbial functional diversity using Biolog EcoPlate™ were assessed. Our results had shown a crucial decrease of the enzymatic activities in soils that were newly irrigated with TWW, while a crucial increase was recorded in soils exposed to TWW for 20 years. In addition, chemical analysis revealed an important accumulation of heavy metals such as Cd, Cu, Zn and Ni along with the irrigation period, especially in 20 years-irrigated soils with TWW. The assessment of the bacterial functional diversity showed an important change in Average well color development (AWCD) and in diversity index values. Besides, substrate utilization profile revealed a significant variation along with the irrigation periods. In conclusion, long-term TWW irrigation has increased the metabolic activities of the soil’s microorganisms. Meanwhile, traces elements accumulation could pose a serious threat for biological processes in soils and a limiting factor for the reuse of TWW in agriculture.

Responses of soil biochemical properties and microbial community structure to short and long‐term no‐till systems

AbstractTillage activities play a crucial role in impacting soil physical and chemical properties, which in turn alter soil biochemical activities and microbial community structure. In this study, responses of soil biochemical activities and microbial community structure to short‐term (NTS, &lt;5 years) and long‐term (NTL, &gt;10 years) no‐till (NT) systems were studied in comparison with conventional till (CT) at four different locations (Garretson, Crooks, Beresford and Mitchell) in South Dakota, USA. Data showed that the NTL resulted in increased soil labile and microbial biomass carbon (C) and nitrogen (N) compared with the CT treatment at the Garretson, Crooks and Beresford sites. At the Crooks and Beresford sites, soils under NTL and NTS had significantly increased the β‐glucosidase, urease, arylamidase, acid and alkaline phosphatase and arylsulphatase enzyme activities compared with those under CT treatment. The β‐glucosidase, urease, acid phosphatase and arylsulphatase enzyme activities showed significant correlations with the microbial biomass content of the soils. Phospholipid fatty acid (PLFA) analysis showed increased PLFA content in NTL soils compared with the CT. The content of total bacterial, actinobacterial, Gram‐positive bacterial, total fungal, arbuscular mycorrhizal fungal (AMF) and saprophyte PLFAs was consistently increased in the NTL and NTS compared with the CT soils at the Garretson site. These differences were more pronounced between NTL and CT systems than between NTS and CT systems in most of the studied sites. The findings suggest that usage of NT for a longer duration is highly beneficial to soil labile C and N pool retention and enzyme activities, and provides less disturbance to soil microbial activities and their functions, which in turn regulates nutrient transformation. Our results also suggest that converting CT soils into NT can improve some soil health indicators even over a short duration, and hence adopting NT can enhance soil microbial and plant growth.Highlights NTS and NTL systems increased soil labile C and N and microbial biomass as compared to CT Total fungi biomass was significantly enhanced under NT compared with CT Short‐term NT significantly increased β‐glucosidase activity at Crooks and Beresford sites compared to CT Compared to the CT system, NT significantly increased soil biological health

Assessment of antidiabetic activity of combination of Murraya koenigii leaves extract and Vitis vinifera seeds extract in alloxan-induced diabetic rats

Background: Murraya koenigii is a well-known curry leaf tree. Its leaves are used as a spice in food recipe in India. Its related antidiabetic activity is attributed to alpha glucosidase activity of carbazole alkaloids. The proanthocyanidins present in Vitis vinifera contribute to its hyperglycemic activity through antioxidant effect and preservation of β-cell function. Aims and Objective: The aim of this study was to assess antidiabetic effect of the combination of M. koenigii leaves extract and V. vinifera seeds extract. Materials and Methods: A total of 36 animals were randomly selected for the study and were divided in six different groups: control group, alloxan (130 mg/kg; intraperitoneal [i.p.]) treated group, alloxan (130 mg/kg) treatment + M. koenigii leaves extract (300 mg/kg; per oral [p.o.]) treated group, alloxan (130 mg/kg) i.p. treatment + V. vinifera seeds extract (200 mg/kg; p.o.) treated group, alloxan (130 mg/kg) + M. koenigii leaves extract (150 mg/kg; p.o.) treatment + V. vinifera seeds extract (100 mg/kg; p.o.) treated group, and alloxan (130 mg/kg) treatment + glibenclamide (5 mg/kg; i.p.) treated group. Treatment was given for 21 days after induction of diabetes. Result: The combination treated group showed a significant reduction in serum glucose levels when compared to individual test extracts. It also attenuated the elevated activity of alkaline phosphatase enzyme in diabetic rats as compared with the healthy controls. The combination treatment showed reversal of the serum glutamic oxaloacetic transaminase (P < 0.001) and serum glutamic pyruvic transaminase (P < 0.001) levels. It also significantly decreased cholesterol level to near normalcy (P < 0.001). Conclusion: The findings of this study suggest additive antidiabetic effect of the combination of M. koenigii leaves extract and V. vinifera seeds extract.

Effects of β-sitosterol on growth, development and midgut enzymes of Helicoverpa armigera Hübner

Helicoverpa armigera is a global agricultural pest of serious concern. Continued use of chemical insecticides as control measures has raised grave health and environment concerns, necessitating a search for botanicals as safe alternatives. The current study investigates the effects of ?-sitosterol, a bioactive phytocomponent in Thevetia neriifolia, on the growth and development, as well as on midgut enzymes of H. armigera. Dietary ?-sitosterol produced dose-dependent systemic toxicity and growth inhibitory effects in H. armigera; the most significant effects were obtained with 10 ?g/mL dietary ?-sitosterol. Higher prepupal and pupal mortality in comparison to larval mortality and a comparatively greater reduction in average weight gained by later instars point to cumulative effects of ?-sitosterol. The delayed effects were ascertained by the 82.05%-57.89% reduction in adult emergence in comparison to 95.02% emergence in controls. Dose-dependent effects of ?-sitosterol were observed as significantly decreased enzyme activities of alanine aminotransaminase (ALT), aspartate aminotransaminase (AST) and alkaline phosphatase (ALP) in the larval midgut. Suppression of enzyme activity was obtained in the order ALT&gt;AST&gt;ALP. Impaired activity of gut enzymes possibly lowered the energy reserves and affected nutrient transport through the gut epithelium, affecting the growth and development of H. armigera. Our study points to a promising use of ?-sitosterol against H. armigera, although further examination and field studies are needed to ascertain its possible use in control programs.

Investigating the Effects of Tissue-Specific Extracellular Matrix on the Adipogenic and Osteogenic Differentiation of Human Adipose-Derived Stromal Cells Within Composite Hydrogel Scaffolds.

While it has been postulated that tissue-specific bioscaffolds derived from the extracellular matrix (ECM) can direct stem cell differentiation, systematic comparisons of multiple ECM sources are needed to more fully assess the benefits of incorporating tissue-specific ECM in stem cell culture and delivery platforms. To probe the effects of ECM sourced from decellularized adipose tissue (DAT) or decellularized trabecular bone (DTB) on the adipogenic and osteogenic differentiation of human adipose-derived stem/stromal cells (ASCs), a novel detergent-free decellularization protocol was developed for bovine trabecular bone that complemented our established detergent-free decellularization protocol for human adipose tissue and did not require specialized equipment or prolonged incubation times. Immunohistochemical and biochemical characterization revealed enhanced sulphated glycosaminoglycan content in the DTB, while the DAT contained higher levels of collagen IV, collagen VI and laminin. To generate platforms with similar structural and biomechanical properties to enable assessment of the compositional effects of the ECM on ASC differentiation, micronized DAT and DTB were encapsulated with human ASCs within methacrylated chondroitin sulfate (MCS) hydrogels through UV-initiated crosslinking. High ASC viability (>90%) was observed over 14 days in culture. Adipogenic differentiation was enhanced in the MCS+DAT composites relative to the MCS+DTB composites and MCS controls after 14 days of culture in adipogenic medium. Osteogenic differentiation studies revealed a peak in alkaline phosphatase (ALP) enzyme activity at 7 days in the MCS+DTB group cultured in osteogenic medium, suggesting that the DTB had bioactive effects on osteogenic protein expression. Overall, the current study suggests that tissue-specific ECM sourced from DAT or DTB can act synergistically with soluble differentiation factors to enhance the lineage-specific differentiation of human ASCs within 3-D hydrogel systems.

Loss of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis

Sepsis is a host response to systemic inflammation and infection that may lead to multi-organ dysfunction and eventual death. While acute brain dysfunction is common among all sepsis patients, chronic neurological impairment is prevalent among sepsis survivors. The brain microvasculature has emerged as a major determinant of sepsis-associated brain dysfunction, yet the mechanisms that underlie its associated neuroimmune perturbations and behavioral deficits are not well understood. An emerging body of data suggests that inhibition of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels may be associated with changes in endothelial cell barrier integrity. The objective of this study was to elucidate the connection between alterations in cerebrovascular TNAP enzyme activity and brain microvascular dysfunction in late sepsis. We hypothesized that the disruption of TNAP enzymatic activity in cerebral microvessels would be coupled to the sustained loss of brain microvascular integrity, elevated neuroinflammatory responses, and behavioral deficits. Male mice were subjected to cecal ligation and puncture (CLP), a model of experimental sepsis, and assessed up to seven days post-sepsis. All mice were observed daily for sickness behavior and underwent behavioral testing. Our results showed a significant decrease in brain microvascular TNAP enzyme activity in the somatosensory cortex and spinal cord of septic mice but not in the CA1 and CA3 hippocampal regions. Furthermore, we showed that loss of cerebrovascular TNAP enzyme activity was coupled to a loss of claudin-5 and increased perivascular IgG infiltration in the somatosensory cortex. Analyses of whole brain myeloid and T-lymphoid cell populations also revealed a persistent elevation of infiltrating leukocytes, which included both neutrophil and monocyte myeloid derived suppressor cells (MDSCs). Regional analyses of the somatosensory cortex, hippocampus, and spinal cord revealed significant astrogliosis and microgliosis in the cortex and spinal cord of septic mice that was accompanied by significant microgliosis in the CA1 and CA3 hippocampal regions. Assessment of behavioral deficits revealed no changes in learning and memory or evoked locomotion. However, the hot plate test uncovered a novel anti-nociceptive phenotype in our septic mice, and we speculate that this phenotype may be a consequence of sustained GFAP astrogliosis and loss of TNAP activity in the somatosensory cortex and spinal cord of septic mice. Taken together, these results demonstrate that the loss of TNAP enzyme activity in cerebral microvessels during late sepsis is coupled to sustained neuroimmune dysfunction which may underlie, in part, the chronic neurological impairments observed in sepsis survivors.

Raman spectroscopy links differentiating osteoblast matrix signatures to pro-angiogenic potential

Mineralization of bone is achieved by the sequential maturation of the immature amorphous calcium phase to mature hydroxyapatite (HA) and is central in the process of bone development and repair. To study normal and dysregulated mineralization in vitro, substrates are often coated with poly-l-lysine (PLL) which facilitates cell attachment. This study has used Raman spectroscopy to investigate the effect of PLL coating on osteoblast (OB) matrix composition during differentiation, with a focus on collagen specific proline and hydroxyproline and precursors of HA. Deconvolution analysis of murine derived long bone OB Raman spectra revealed collagen species were 4.01-fold higher in OBs grown on PLL. Further, an increase of 1.91-fold in immature mineral species (amorphous calcium phosphate) was coupled with a 9.32-fold reduction in mature mineral species (carbonated apatite) on PLL versus controls. These unique low mineral signatures identified in OBs were linked with reduced alkaline phosphatase enzymatic activity, reduced Alizarin Red staining and altered osteogenic gene expression. The promotion of immature mineral species and restriction of mature mineral species of OB grown on PLL were linked to increased cell viability and pro-angiogenic vascular endothelial growth factor (VEGF) production. These results demonstrate the utility of Raman spectroscopy to link distinct matrix signatures with OB maturation and VEGF release. Importantly, Raman spectroscopy could provide a label-free approach to clinically assess the angiogenic potential of bone during fracture repair or degenerative bone loss.

RANKL treatment of vascular endothelial cells leading to paracrine pro-calcific signaling involves ROS production.

Numerous studies have highlighted the causal link between over-production of reactive oxygen species (ROS) and cardiovascular complications such as vascular calcification (VC). Receptor-activator of nuclear factor-κB ligand (RANKL) has previously been shown to act on endothelial cells, eliciting the production/release of paracrine pro-calcific signals that act, in-turn, upon underlying vascular smooth muscle cells (VSMCs) to induce osteoblastic differentiation and VC. A role for endothelial ROS signaling in this process has not been established however. In the current paper, we investigate the possibility that RANKL leads to ROS signaling within the endothelial layer as part of the RANKL-driven VC signaling cascade. Human aortic endothelial cells (HAECs) were treated with RANKL (25ng/ml, 72h) and monitored for ROS production, in parallel with various pro-calcific signaling indices. Antioxidant co-treatments included TRAIL (5ng/ml), apocynin (10mM) and N-acetylcysteine (5mM). Treatment of HAECs with RANKL-induced robust ROS production. This surge could be partially attenuated by TRAIL and strongly attenuated by apocynin and N-acetylcysteine. RANKL also elicited a range of signaling events in HAECs that we have previously demonstrated are coupled to osteoblastic differentiation in underlying VSMCs. These include non-canonical NF-κB/p52 activation, elevated BMP-2 release and increased alkaline phosphatase (ALP) enzyme activity (cellular and extracellular). Importantly, these RANKL-induced signaling events could be completely prevented by co-treatment of HAECs with antioxidants. In summary, RANKL elicits ROS generation in HAECs with direct consequences for generation of paracrine pro-calcific signals known to effect calcification in underlying VSMCs.

Biochar addition to forest plantation soil enhances phosphorus availability and soil bacterial community diversity

Depletion of soil nutrients is a major cause of decline in productivity of forest plantations in successive rotations. Biochar amendment in agricultural systems has been shown to yield various beneficial effects, including increasing soil phosphorus (P) availability. However, the direct and indirect effects of biochar addition on forest soil P dynamics have largely been unexplored. The objective of this study was to examine how biochar produced from harvest residue (leaves and woodchips) affect the P dynamics in second rotation Cunninghamia lanceolata (Chinese fir) plantation soil. An incubation experiment which involved mixing of forest soil with 1% or 3% w/w leaf or woodchip biochar, pyrolyzed at 300 °C or 600 °C, was conducted for 80 days at 20 °C. After 7, 40 and 80 days of incubation, soil samples were analyzed for total and available P, inorganic and organic P pools, and soil phosphatase activity. At the end of the incubation period, bacterial community composition and diversity were analyzed by 16S rDNA sequencing. The leaf biochar produced at both pyrolysis temperatures was more alkaline and had significantly higher soluble P, nitrogen and calcium contents than the woodchip biochar. Soil total and available P increased significantly in all leaf biochar treatments after 80 days incubation compared to the untreated control soil, but the woodchip biochar treatments had no significant effects. At the end of the experiment, Al-P content was significantly lower and Ca10-P content higher in soil amended with both biochar types compared to the control soil, and Fe-P content was significantly higher in the leaf biochar treatments. Contrary to expectations, acid and alkaline phosphatase enzyme activities were significantly lower in some of the biochar treatments after 80 days incubation compared to the control soil. Nevertheless, the diversity of the bacterial community was higher in leaf biochar-amended forest soil than the woodchip biochar-amended and untreated soil at the end of the experiment. In particular, the abundance increased in the leaf biochar soil treatments of P-solubilizing bacteria, such as Burkholderia-Paraburkholderia, Planctomyces, Sphingomonas and Singulisphaera, which can indirectly improve P availability in soil. Thus, conversion of tree harvest residues, particularly leaves, into biochar and recycling back into the soil could be a viable option to boost P availability and help to conserve nutrients or reduce nutrient losses for the next rotation. Before recommending plantation management with biochar, long-term studies are required assessing the life cycle of biochar under field conditions and its promoting effect on growth of C. lanceolata.

Evaluation of human primary intestinal monolayers for drug metabolizing capabilities

BackgroundThe intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo.ResultsThe monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes.ConclusionsThe use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells.Graphical abstract

Arbuscular mycorrhizal fungi modulates dynamics tolerance expression to mitigate drought stress in Ephedra foliata Boiss.

Arbuscular mycorrhizal fungi (AMF) are one of the most important drivers of soil ecosystem dynamics. AMF have the potential to improve plant growth and development by modulating key hormonal pathways, which result in decreasing the adverse impact of abiotic stress, such as drought. Pot experiments were conducted in this study to investigate the ability of AMF to ameliorate the adverse impact of drought in Ephedra foliate. Non-inoculated AMF E. foliate (Ef) plants, exhibited reduced growth in response to drought stress with a concomitant lowering of chlorophyll pigments, relative to non-stressed and AMF inoculated plant. AMF inoculated E. foliate showed improved nitrogen metabolism by positively regulating nitrate and nitrite reductase activity which results in greater ammonium availability for the synthesis of amino acids. Inoculation with AMF also increased antioxidant enzyme activity, ascorbic acid contents, and reduction in glutathione level. This resulted in significant amelioration of oxidative damage to plant membranes by restricting the excess generation of reactive oxygen species (ROS), such as hydrogen peroxide. Greater content of proline, glucose, and total soluble protein in AMF-inoculated plants provided further benefit to E. foliate plants and their ability to withstand drought stress, and also evident by a greater level of sucrose phosphate synthase activity. AMF significantly enhanced the uptake of essential nutrients like K, Mg, and Ca. Importantly, higher concentrations of plant hormones, including indole acetic acid (IAA), indole butyric acid (IBA), gibberellic acid (GA), and abscisic acid (ABA), were maintained in AMF-inoculated Ef plants. AMF inoculation also boosted phosphorous metabolism by increasing alkaline and acid phosphatase enzyme activity. In summary, AMF-inoculation of Ef plants significantly reduced the deleterious effect of drought stress by up-regulating the antioxidant defense system, synthesis of osmolytes, and maintaining phytohormone levels.

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients.

Radioprotective Effect of Arbutin in Megavoltage Therapeutic X-irradiated Mice using Liver Enzymes Assessment.

Background:Medical use of ionizing radiation has direct/indirect undesirable effects on normal tissues. In this study, the radioprotective effect of arbutin in megavoltage therapeutic x-irradiated mice was investigated using serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), and asparate amniotransferase (AST) activity measurements. Material and Methods: In this analytical and experimental lab study, sixty mice (12 identical groups) were irradiated with 6 MV x-ray beam (2 and 4 Gy in one fraction). Arbutin concentrations were chosen 50, 100, and 200 mg/kg and injected intraperitoneal 2 hours before irradiation. Samples of peripheral blood cells were collected and serum was separated on the 1, 3, and 7 days post-x-radiation; in addition, the level of ALP, ALT, and AST were measured. Data were analyzed using one-way ANOVA, and Tukey HSD test. Results: X-radiation (2 and 4 Gy) increased the ALT and AST activity levels on the 1, 3, and 7 days post- irradiation, but the ALP level significantly increased on the 1 and 7 days and decreased on the third day compared to the control group (P< 0.001). ALP, ALT and AST activity levels in “2 and 4 Gy x irradiation + distilled water” groups were significantly higher than “2 and 4 Gy irradiation + 50, 100, and 200 mg/kg arbutin” groups on the first and seventh day post-irradiation (P< 0.001). Conclusion:Arbutin is a strong radioprotector for reducing the radiation effect on the whole-body tissues by measuring ALP, ALT and AST enzyme activity levels. Furthermore, the concentration of 50 mg/kg arbutin showed higher radioprotective effect.

Magnetic nanocomposite hydrogels and static magnetic field stimulate the osteoblastic and vasculogenic profile of adipose-derived cells

Exposure of cells to externally applied magnetic fields or to scaffolding materials with intrinsic magnetic properties (magnetic actuation) can regulate several biological responses. Here, we generated novel magnetized nanocomposite hydrogels by incorporation of magnetic nanoparticles (MNPs) into polyethylene glycol (PEG)-based hydrogels containing cells from the stromal vascular fraction (SVF) of human adipose tissue. We then investigated the effects of an external Static Magnetic Field (SMF) on the stimulation of osteoblastic and vasculogenic properties of the constructs, with MNPs or SMF alone used as controls. MNPs migrated freely through and out of the material following the magnetic gradient. Magnetically actuated cells displayed increased metabolic activity. After 1 week, the enzymatic activity of Alkaline Phosphatase (ALP), the expression of osteogenic markers (Runx2, Collagen I, Osterix), and the mineralized matrix deposition were all augmented as compared to controls. With magnetic actuation, strong activation of endothelial, pericytic and perivascular genes paralleled increased levels of VEGF and an enrichment in the CD31+ cells population. The stimulation of signaling pathways involved in the mechanotransduction, like MAPK8 or Erk, at gene and protein levels suggested an effect mediated through the mechanical stimulation. Upon subcutaneous implantation in mice, magnetically actuated constructs exhibited denser, more mineralized and faster vascularized tissues, as revealed by histological and micro-computed tomographic analyses. The present study suggests that magnetic actuation can stimulate both the osteoblastic and vasculogenic potentials of engineered bone tissue grafts, likely at least partially by mechanically stimulating the function of progenitor cells.