Alkaline Phosphatase Enzyme Activity Research Articles

Array of individually addressable two-electrode electrochemical cells sharing a single counter/reference electrode for multiplexed enzyme activity measurements

This work reports on the fabrication and performance of a new on-chip array of gold thin-film electrodes arranged into five individually addressable miniaturized electrochemical cells. Each cell shows a two-electrode configuration comprising a single working electrode and a counter/pseudo-reference electrode that is compartmentalized to be shared among all the cells of the array. Using this configuration, just six contact pads are required, which significantly reduces the chip overall surface area. Electrochemical characterization studies are carried out in solutions containing the two species of reversible redox pairs. The concentration of one redox species can reliably be measured at the working electrode by applying potentiostatic techniques to record the current due to the corresponding electrochemical reaction. The redox counterpart in turn undergoes an electrochemical process at the counter/pseudo-reference electrode, which, under optimized experimental conditions, injects current and keeps the applied potential in the electrochemical cell without limiting the current being recorded at the working electrode. Under these conditions, the electrode array shows an excellent performance in electrochemical detection studies without any chemical or electrical cross-talk between cells. The enzymatic activity of horseradish peroxidase, alkaline phosphatase and myeloperoxidase enzymes is analyzed using different redox mediators. Quasi-simultaneous measurements with the five electrochemical cells of the array are carried out within 1 s time frame. This array layout can be suitable for multiplexed electrochemical immunoassays and immunosensor approaches and implementation in simplified electrochemical ELISA platforms that make use of enzyme labels. Moreover, the array reduced dimensions facilitate the integration into compact fluidic devices.

Ramipril, an angiotensin-converting enzyme inhibitor, ameliorates oxidative stress, inflammation, and hepatic fibrosis in alloxan-induced diabetic rats

Angiotensin-II is considered as a peptide responsible for the vascular dysfunction and complications in various tissues including liver through inducing free radicle mediated oxidative stress. This study aimed to evaluate the effect of ramipril, an angiotensin-converting enzyme inhibitor (ACE inhibitor), on oxidative stress, inflammation, and fibrosis in the liver of alloxan-induced diabetic rats. In this investigation, rats were divided into four groups (six rats in each group): control, control+ramipril, alloxan, and alloxan+ ramipril. A single dose (90 mg/kg) of alloxan was given intra-peritoneally to induce type two diabetes. After the induction of diabetes, ramipril (10 mg/kg) was administered to each animal for 21 days. An oral glucose tolerance test (OGTT) was performed. All animals were sacrificed at the end of the study. Blood and liver tissues were collected from each animal and stored for further biochemical studies. Liver marker enzymes and oxidative stress parameters were also assayed followed by histological examination in the liver. Alloxan administration in rats showed oral glucose intolerance and increased fasting blood glucose levels. Ramipril (10 mg/kg) treatment in alloxan administered rats improved the OGTT and lowered fasting blood glucose level. This study also revealed the elevation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzymes activities in the alloxan administered rats which were attenuated by ramipril treatment. Oxidative stress parameters such as advanced protein oxidative products (APOP), nitric oxide (NO), and malondialdehyde (MDA) were also increased in alloxan administered rats which were diminished by the treatment of ramipril. Moreover, alloxan administration increased inflammation and fibrosis in the liver, which was further prevented by ramipril treatment. In conclusion, ramipril alleviated oxidative stress and fibrosis in the liver by suppressing oxidative stress. This investigation suggests that ACE inhibitors may be useful for treating diabetic complications and liver injury in alloxan-administered rats.

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

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

A Study to Analyze the Alkaline Phosphatase and Lactate Dehydrogenase Enzyme Activity in Gingival Crevicular Fluid During Orthodontic Tooth Movements.

Background:In orthodontic tooth development, bone turnover is described by bone testimony at locales of strain and bone resorption at areas of stress. There are metabolic disorders that may lead to tooth movement when the periodontal tissues are under mechanical stress. We needed to discover how the chemical movement of alkaline phosphatase and lactate dehydrogenase in gingival crevicular liquid (GCF) vacillated when tensions were coordinated during introductory arrangement (P1) and withdrawal (P2).Materials and Procedures:Fifty persons, ranging in age from 11 to 21 years, were enrolled in the trial, all of whom required first premolar extractions and were enduring fixed orthodontic treatment. Every subject's test and control teeth were the maxillary canine (TT) and mandibular second molar (CT). Two μL of GCF was taken from the mesial side of both TT and CT and tried for the action of ALP and LDH utilizing a spectrophotometer on the 14th day following the finish of each stage.Results:The findings were analyzed using Student's t-test. Enzyme activity changed when the teeth moved during orthodontic treatment. There was a statistically significant difference in the levels of ALP and LDH activity between P1 and P2 at TT (P = 0.005 and P = 0.001). Neither ALP nor LDH activity differed significantly between P1 and P2, with a statistical significance of 0.054 and 0.061, respectively.Conclusion:According to this research, GCF ALP and LDH activity, as well as periodontal biologic activity during orthodontic tooth movement, can be properly detected.

Metabolic blood parameters of pigs of different age groups in the pathogenesis of nonspecific bronchopneumonia

Despite the sufficient amount of antimicrobial drugs and specific therapy and prevention, the proportion of respiratory diseases in pigs is still quite high [2, 5, 6, 13]. In some farms, the incidence, as well as post-slaughter changes in the lungs, can reach up to 100%. At the same time, mortality is usually insignificant. The main damage among the pig population consists of an economic component - it is the culling of slaughter products, a decrease in weight gain, low conversion of feed, carrying out therapeutic and preventive measures. There is evidence of hypoxemia and hypoxia developing against this background, which can lead to the development of acidosis, a decrease in the barrier properties of organs and tissues and the development of organopathologies. The previously obtained data on immunological blood tests allow us to talk about the suppression of innate and adaptive immunity in nonspecific bronchopneumonia [2, 4, 5, 6, 12, 13].The purpose of this work was to conduct biochemical studies to study the state of metabolic parameters of the blood of pigs of different age groups, patients with nonspecific bronchopneumonia.The research was carried out at one of the large pig farms in the North-Western region of Russia and at the Department of Pathophysiology of the St. Petersburg State Medical University.As a result of studies in all experimental groups of animals, the effect of nonspecific bronchopneumonia on biochemical processes in piglets was established. In all sick animals, a noticeable decrease in the content of total protein, albumins and globulins was found, which can lead to inhibition of the function of protective mechanisms, and from all links of immunity (innate and adaptive). At the same time, the activity of alkaline phosphatase and lactate dehydrogenase enzymes was established in sick piglets, which indicates the generalization of the process with liver damage and the development of acidosis.

Evaluation of the biological and physicochemical properties of calcium silicate-based and epoxy resin-based root canal sealers.

Biocompatibility, dimensional stability, radiopacity, flow, and low solubility are the characteristics of an ideal endodontic sealer. This study evaluated and compared in vivo and in vitro biological and physicochemical properties of calcium silicate-based sealers: Sealer Plus BC (BC), MTA Fillapex (MF); and resin-based sealers: AH Plus (AHP) and Sealer Plus (SP). Apical papilla cells were exposed to sealer extracts and subjected to MTT, SRB, scratch, alkaline phosphatase enzyme activity (ALP) and Alizarin red staining (ALZ) assays. Sealers were histologically evaluated in connective tissue of Wistar rats in different periods. Radiopacity, film thickness, flow, setting time, pH and element analyses were investigated. BC had better results compared to AHP and MF at hour 72 for MTT assay (p < .05), and the highest cell viability under SRB (p < .05). All sealers presented ALP activity. BC presented the highest mineralized deposition under ALZ (p < .05). BC and MF promoted wound healing. All sealers induced an initial inflammation reaction that decreased over time. Eosinophils were observed at day 7 in MF (p < .05). Despite MF did not present final setting time, the sealers properties were in accordance to ISO 6876/2012 and ASTM C266-08. All sealers presented cell viability and biocompatibility. BC presented higher pH values and bioactivity. The materials tested showed physico-chemical properties in accordance with standards, except for MF setting time.

Interaction between ciliate and plant growth promoting bacteria influences the root structure of rice plants, soil PLFAs and respiration properties

Plant growth and productivity depend on the complex and dynamic interaction between the plant roots and soil microorganisms. At present, the research on rhizosphere associated microbes is largely focused on bacteria and fungi; whereas the interaction of soil protists with plants and other microbes remains unexplored. The present study aimed to investigate the interactive impact of a ciliate (Kreyellidae, C5) and two plant growth-promoting bacteria (PGPB) i.e., Pseudomonas sp. (Ps) and Enterobacter sp. (Ec), on the growth of rice plants, soil microbial community composition and soil chemical properties. It was observed that the protist-PGPB interaction significantly modified the root structure leading to an enhanced outgrowth of lateral roots (272.08%–380.41%) and seminal roots (190.40%–250.45%), in addition to an increase in the primary root length (p < 0.05). The phospholipid fatty acid (PLFA) analysis indicated a striking shift in the overall soil microbial communities, especially in the Gram negative bacterial community and fungi, which were found to be decreased in treatments due to the presence of a predator. The combined ciliate-bacterial treatments further increased the microbial carbon biomass to 223.59% and 310.57% as compared to control and PGPB treatments respectively. A similar enhancement of dehydrogenase enzyme activity was observed in soil samples of rice plants on combined treatments. In contrast, the alkaline phosphatase and fluorescein diacetate enzyme activities were recorded to be more in soil samples treated with PGPB. The combined treatment of rice plants also enhanced the uptake of N and P moderately, as compared to PGPB treated plants. The colony-forming unit and most probable number were found to be significantly higher in PGPB treated soil and combined treatments (Tukey's HSD, p < 0.05). In conclusion, this is the first study that demonstrates significant modification of root structure and increased nutrient uptake by rice plants through interaction between ciliate and PGPB.

ALPL Genotypes in Patients With Atypical Femur Fractures or Other Biochemical and Clinical Signs of Hypophosphatasia.

Hypophosphatasia (HPP) is a rare metabolic disorder caused by deficiency of alkaline phosphatase (ALP) enzyme activity, leading to defective mineralization, due to pathogenic variants of the ALPL gene, encoding the tissue nonspecific alkaline phosphatase (TNSALP) enzyme. Inheritance can be autosomal recessive or autosomal dominant. An abnormal ALPL genetic test enables accurate diagnosis, avoiding the administration of contraindicated antiresorptive drugs that, in patients with HPP, substantially increase the risk of atypical femur fractures (AFFs) and worsen the fracture healing process that is usually already compromised in these patients. Performing ALPL genetic testing to identify rare variants in suspected adult patients with HPP. Comparing frequencies of ALPL common variants in individuals with biochemical and/or clinical signs suggestive of adult HPP and non-HPP controls, and among different clinical subgroups of patients with a clinical suspicion of adult HPP. Patients with suspected adult HPP were retrospectively selected for the genetic testing of the ALPL gene. Patients included were from 3 main European Bone Units (Florence, Naples, and Geneva); 106 patients with biochemical and/or clinical signs suggestive of a mild form of HPP were included. Genetic testing led to the identification of a heterozygote rare variant in 2.8% of cases who were initially referred as suspected osteoporosis. The analysis of frequencies of ALPL common variants showed a high prevalence (30.8%) of homozygosity in subjects who developed an AFF, in association with normal serum total ALP activity. The results suggest homozygosity of common ALPL variants as a possible genetic mark of risk for these fractures.

Overexpression Effects of miR-424 and BMP2 on the Osteogenesis of Wharton's Jelly-Derived Stem Cells.

Recently, the translational application of noncoding RNAs is accelerated dramatically. In this regard, discovering therapeutic roles of microRNAs by developing synthetic RNA and vector-based RNA is attracting attention. Here, we studied the effect of BMP2 and miR-424 on the osteogenesis of Wharton's jelly-derived stem cells (WJSCs). For this purpose, human BMP2 and miR-424 DNA codes were cloned in the third generation of lentiviral vectors and then used for HEK-293T cell transfection. Lentiviral plasmids contained miR424, BMP-2, miR424-BMP2, green fluorescent protein (GFP) genes, and helper vectors. The recombinant lentiviral particles transduced the WJSCs, and the osteogenesis was evaluated by real-time PCR, Western blot, Alizarin Red staining, and alkaline phosphatase enzyme activity. According to the results, there was a significant increase in the expression of the BMP2 gene and secretion of Osteocalcin protein in the group of miR424-BMP2. Moreover, the amount of dye deposition in Alizarin Red staining and alkaline phosphatase activity was significantly higher in the mentioned group (p < 0.05). Thus, the current study results clarify the efficacy of gene therapy by miR424-BMP2 vectors for bone tissue engineering. These data could help guide the development of gene therapy-based protocols for bone tissue engineering.

Thymol as Molluscicidal Agent Against Biomphalaria alexandrina, Intermediate Host of Schistosoma mansoni

Biomphalaria alexandrina snails have received much concern due to their great medical importance as vectors for transmitting Schistosoma mansoni infection to humans. Sustainable control of B. alexandrina snails represents a logical approach to achieve its elimination targets. This work aimed to evaluate the efficacy of the monoterpenoid thymol as a plant molluscicide in the control of this schistosomiasis vector snail. The molluscicidal activity of thymol was determined according to the WHO method. The results showed that the lethal concentration LC50 and LC90 values of thymol were 21.90 and 34.06 ppm, respectively. Also, it was observed that the accumulative effect of sublethal concentration LC25 (15.94 ppm) of thymol for 4 successive weeks had destructive effects on the survival rate, egg-laying capacity, reproduction rate, and biochemical parameters of exposed snails. Our results demonstrated that thymol showed an oviposition inhibitory activity against B. alexandrina snails. Furthermore, the sublethal toxicity of thymol induced a significant increase in levels of total proteins and glucose as well as in enzyme activities of alkaline phosphatase and acid phosphatase of hemolymph of exposed snails, whereas a significant inhibition in aspartate aminotransferase and alanine aminotransferase activities was recorded. These significant biochemical disorders in turn explained noticeable changes in reproduction and other biological activities of treated snails. It was concluded that thymol can be used as a safe promising plant molluscicide for B. alexandrina snails’ control as it interferes with the snails’ biology and physiology. So thymol can be effectively used in the National S. mansoni control programs.

Responses of the Soil Microbial Community to Salinity Stress in Maize Fields.

Simple SummarySoil microorganisms are the core of maintaining soil ecological functions. Recognition of microbial community diversity in saline soil contributes to nutrient management and crop production. Meanwhile, microbial activity is easily affected by changes in soil properties. This study addressed how the composition and diversity of bacterial and fungal communities changed under different saline conditions to identify the sensitivity of bacteria or fungi to salinity. The primary objective was to evaluate the relationship between soil’s microbial community diversity and soil’s physicochemical factors, and to explore the vital microbial predictors in salinized soil. The results showed that Firmicutes and Bacteroidetes are pivotal in salinized soil, and this finding can provide guidance for the demand for plant rhizosphere growth-promoting bacteria as a bioindicator in saline soil.To investigate the diversity and structure of soil bacterial and fungal communities in saline soils, soil samples with three increasing salinity levels (S1, S2 and S3) were collected from a maize field in Yanqi, Xinjiang Province, China. The results showed that the K+, Na+, Ca2+ and Mg2+ values in the bulk soil were higher than those in the rhizosphere soil, with significant differences in S2 and S3 (p < 0.05). The enzyme activities of alkaline phosphatase (ALP), invertase, urease and catalase (CAT) were lower in the bulk soil than those in the rhizosphere. Principal coordinate analysis (PCoA) demonstrated that the soil microbial community structure exhibited significant differences between different salinized soils (p < 0.001). Data implied that the fungi were more susceptible to salinity stress than the bacteria based on the Shannon and Chao1 indexes. Mantel tests identified Ca2+, available phosphorus (AP), saturated electrical conductivity (ECe) and available kalium (AK) as the dominant environmental factors correlated with bacterial community structures (p < 0.001); and AP, urease, Ca2+ and ECe as the dominant factors correlated with fungal community structures (p < 0.001). The relative abundances of Firmicutes and Bacteroidetes showed positive correlations with the salinity gradient. Our findings regarding the bacteria having positive correlations with the level of salinization might be a useful biological indicator of microorganisms in saline soils.

Synergistic impact of platelet rich plasma-heparin sulfate with hydroxyapatite/zirconia on the osteoblast differentiation potential of adipose-derived mesenchymal stem cells.

3D porous hydroxyapatite (HA) has been reinforced by zirconia (ZrO2) coating and impregnation with a combination of platelet rich plasma (PRP) as a source of growth factors (GFs) and Heparin sulfate (HS) to sustain the release of GFs. Adipose mesenchymal stem cells (ADMSCs) were characterized by flow cytometry for CD (cluster of differentiation) 44, CD105, CD106, CD34 and CD144, along with checking the multipotency by differentiation into the adipocytes and osteoblasts. Then, they were cultured on the scaffold treated with and without osteogenic media on days 7, 14 and 21. Electron micrograph and PKH staining show that the ADMSCs have a fusiform phenotype in the absence of osteogenic induction. Cell viability assay shows a higher number of the viable cells on the PRP-containing scaffolds than PRP-free scaffolds on day 7. Colorimetric evaluation, quantitative RT-PCR and immunocytochemistry demonstrate that PRP and HS significantly elevate the alkaline phosphatase enzyme activity and also accelerate the production of both early and mid-osteogenic markers, including collagen I and osteopontin expression with and without osteogenic conditions. The PRP-HS also accelerates the expression of the late osteogenic marker, osteocalcin, in both mRNA and protein level expression with a peak on day 21. In conclusion, supplementation of HA/ZrO2 with PRP/HS has a synergistic impact on the ADMSCs, even in the absence of chemical induction. It seems that HA/ZrO2/PRP/HS scaffold provides a higher osteoconductive microenvironment for stem cell differentiation to osteoblasts.

O14 An atypical femoral fracture without a history of bisphosphonate use

Case report - IntroductionA 60-year-old female presented with an insufficiency fracture of the midshaft of the left femur and a persistently low serum alkaline phosphatase. A diagnosis of adult hypophosphatasia was made. We present this case alongside a series of other cases that we have identified locally and discuss the importance of identifying this condition in clinical practice.Case report - Case descriptionA 60-year-old female presented with left thigh pain and an 8-year history of polyarthralgia involving the medium-large sized joints. She had a traumatic fracture of the humerus 4 years previously. She had no history or examination findings suggestive of an inflammatory arthritis, connective tissue disease or systemic illness.An X-ray of left femur revealed a cortical-based lucency at the lateral cortex of midshaft. An MRI identified a focal area of cortical thickening but no evidence of bone marrow oedema suggestive of an osteoid osteoma. Finally, a CT scan confirmed the diagnosis of a healed stress fracture. The patient had never received any form of bisphosphonate treatment, no previous history of a non-traumatic fracture and no history of dental problems.Laboratory evaluation revealed persistently low levels of serum alkaline phosphatase (∼23 IU/L) with a normal vitamin D and PTH level. Bone density scan showed a T-score of -1.1 at the femoral neck and -2.6 at the lumbar spine. Serum pyridoxal phosphate level was elevated at 457 nmol/L (35-100). A diagnosis of adult hypophosphatasia was suspected and genetic study for ALPL gene confirmed the diagnosis.The following table summarises a number of other cases of adult hypophosphatasia, which we have identified locally. CaseAge / GenderPresentationAlkaline phospha tase (IU/L)Pyridoxal phosphate (nmol/L)Notes1 (Index case)62 / FWidespread pain, Atypical femoral fracture23457Genetic study confirmed278 / FWidespread pain, Osteomalacia22201Genetic study confirmed356 / MSon of Case 2, Osteoporosis, Diabetes mellitus22 – 29awaited Bisphosphonate stoppedGenetic study awaited 440 / FRecurrent metatarsal fractures, Low BMD24NormalBisphosphonate stopped548 / FPremature loss of teeth, fatigue, Widespread pain, Diabetes mellitus, hyperlipidaemia13-23327Genetic study confirmed675 / FSeronegative polyarthritis21-26120Genetic study awaitedCase report - DiscussionHypophosphatasia (HPP) is a rare inherited metabolic disease caused by mutations in the alkaline phosphatase liver type (ALPL) gene, localized on the chromosome region 1p36.1–34, encoding the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). The disease is related to a decrease in both level and activity of alkaline phosphatase enzyme (ALP). The clinical expression is highly variable, ranging from lethal forms to mild forms diagnosed in adults. Fractures, chondrocalcinosis, calcific periarthritis, and chronic muscle and bone pain are key features for the diagnosis of HPP in adults. Teriparatide has been used for treatment in some case reports with inconsistent results. Antiresorptive drugs are contraindicated as they can worsen underlying osteomalacia.Case report - Key learning pointsCheck for persistent low serum alkaline phosphatase levels in adults presenting with fragility fractures, crystal-related arthropathies and nonspecific generalised aches.Avoid bisphosphonates in patients with low alkaline phosphatase.

Interactions between Biochar and Compost Treatment and Mycorrhizal Fungi to Improve the Qualitative Properties of a Calcareous Soil under Rhizobox Conditions

Most calcareous soils have relatively low levels of organic matter. To evaluate the effect of pruning waste biochar (PWB) and pruning waste compost (PWC) combined with arbuscular mycorrhizal fungi (AMF) on the biological indices, a rhizobox study on wheat using a completely randomized design was conducted under greenhouse conditions. The studied factors included the source of organic material (PWB, PWC, and control), the microbial inoculation (+AMF or −AMF), and the zone (rhizosphere and non-rhizosphere soil). At the end of the plant growth period, organic carbon (OC), microbial biomass carbon (MBC), microbial biomass phosphorous (MBP), microbial respiration (BR), substrate-induced respiration (SIR), and alkaline (ALP) and acid (ACP) phosphatase enzyme activities in the rhizosphere and non-rhizosphere soils were determined. Simultaneous application of a source of organic matter and AMF inoculation significantly increased the OC and biological indices of soil relative to those observed when applying organic matter without AMF inoculation. Additionally, MBC, MBP, ACP, and ALP enzymes activities in the rhizosphere zone were significantly higher than in the non-rhizosphere. AMF increased BR and SIR levels in the rhizosphere by 13.06% and 7.95% compared to those in the non-rhizosphere, respectively. It can be concluded that PWC and PWB can improve soil biological properties by increasing microbial activity.

Calcium-Enriched Nanofibrillated Cellulose/Poloxamer in-situ Forming Hydrogel Scaffolds as a Controlled Delivery System of Raloxifene HCl for Bone Engineering.

PurposeTEMPO-oxidized nanofibrillated cellulose (TONFC) originating from an agricultural waste (sugar cane) was utilized to prepare injectable in-situ forming hydrogel scaffolds (IHS) for regenerative medicine.MethodsTONFC was prepared and characterized for its morphology and chemical structure using TEM and FT-IR, respectively. The cold method was applied to prepare hydrogels. Various concentrations of poloxamer 407 were added to the prepared TONFC (0.5%w/w). Different sources of calcium, Fujicalin® (DCP) or hydroxyapatite (TCP), were used to formulate the aimed calcium-enriched raloxifene hydrochloride-loaded IHS. Gelation temperature, drug content, injectability and in-vitro drug release were evaluated along with the morphological characters. Cytocompatibility studies and tissue regeneration properties were assessed on Saos-2 cells.ResultsTEM photograph of TONFC showed fibrous nanostructure. The selected formulation “Ca-IHS4” composed of TONFC+15% P407+10% TCP showed the most prolonged release pattern for 12 days with the least burst effect (about 25% within 24 h). SEM micro-photographs of the in-situ formed scaffolds showed a highly porous 3D structure. Cytocompatibility studies of formulation “Ca-IHS4” revealed the biocompatibility as well as improved cell adhesion, alkaline phosphatase enzyme activity and calcium ion deposition.ConclusionThe outcomes suggest that Ca-IHS4 presents a simple, safe-line and non-invasive strategy for bone regeneration.

Synthesis of New Boron Derived Compounds; Anticancer, Antioxidant and Antimicrobial Effect in Vitro Glioblastoma Tumor Model.

ObjectiveThe aim of our study is to investigate the cytotoxic, antioxidant, and antimicrobial effects of newly synthesized boron compounds in U87MG glioblastoma cell treatment. MethodsWe synthesized boron glycine monoester (BGM) and boron glycine diester (BGD) structures containing boron atoms and determined their cytotoxic activities on glioblastoma by the MTT method. The inhibitory concentration 50 (IC50) value was calculated with GraphPad Prism 5.0 program. The IC50 values were administered 48 hours on U87MG glioblastoma cell. Catalase (CAT), acid phosphatase (ACP) and alkaline phosphatase (ALP) enzyme activity, malondialdehyde (MDA), total glutathione (GSH), and total protein levels were detected using spectrophotometric methods. We determined the antimicrobial activities of BGM and BGD with the disc diffusion method.ResultsAfter 48 hours of BGM and BGD application to U87MG glioblastoma cells, we found the IC50 value as 6.6 mM and 26 mM, respectively. CAT and ACP enzyme activities were decreased in BGM and BGD groups. MDA which is a metabolite of lipid peroxidation was increased in both boron compounds groups. GSH level was reduced especially in BGD group. BGM and BGD have been found to be antimicrobial effects. ConclusionBoron compounds, especially the BGM, can provide a new therapeutic approach for the treatment of glioblastoma with their anticancer, antioxidant, and antimicrobial effects.

The Effect of the Repair of Induced Articular Cartilage Defects in Pigs Using Calcium Phosphate Cement with Aminoacids on the Concentrations of Selected Inflammatory Markers and Serum Enzyme Activities

The repair of articular cartilage defects is an intensively developing area of research. Severe inflammatory reactions after surgical interventions on bones or their structures may lead to changes of bone or joint asymmetry. In laboratory diagnostics, some inflammatory biomarkers and serum enzymes are available for the evaluation of these inflammatory processes. A general understanding of inflammatory responses following the repair of cartilage defects is still lacking. The aim of this study was to describe the alterations in the values of five inflammatory markers and the activities of several enzymes in seven 5-month-old pigs within the first 30 days following the restoration of induced articular cartilage defects using the tetracalcium phosphate/nanomoneite cement powder enriched with amino acids (CAL). The reconstruction of surgically induced defects was accompanied by a significant increase of serum amyloid A (SAA, p &lt; 0.05), haptoglobin (Hp, p &lt; 0.001), C-reactive protein (CRP, p &lt; 0.01), and pig major acute phase protein (pig-MAP, p &lt; 0.001). Their concentrations decreased gradually within one-month post-surgery, the values recorded at the end of this period were higher than those obtained prior to surgery. The concentrations of α1-acid glycoprotein (AGP) showed no significant changes during the evaluated period. Alterations were also found in the enzyme activities of creatine kinase (p &lt; 0.05), lactate-dehydrogenase and alkaline phosphatase (p &lt; 0.01). These findings suggest that SAA, Hp, CRP and pig-MAP might be applicable biomarkers of acute phase response for the monitoring of postoperative period.

Dorsomorphin attenuates Jagged1-induced mineralization in human dental pulp cells.

To investigate whether TGF-β/BMP signalling participates in Jagged1-induced osteogenic differentiation in human dental pulp cells (hDPs). Bioinformatic analysis of publicly available RNA sequencing data of Jagged1-treated hDPs was performed using NetworkAnalyst. The mRNA expression was validated using real-time polymerase chain reaction. hDPs were seeded on Jagged1 immobilized surfaces in the presence or absence of TGF-β or BMP inhibitor. Osteogenic differentiation was evaluated using alkaline phosphatase staining, osteogenic marker gene expression and mineralization assay. Statistical analyses were performed using a Kruskal-Wallis test, followed by a pairwise comparison for more than three group comparison. Mann-Whitney U-test was employed for two group comparison. The statistical significance was considered at p<.05. Jagged1 treatment in growth medium significantly promoted TGFB1, TGFB2 and TGFB3 whilst significantly inhibited BMP2, BMP4 and BMP6mRNA expression (p<.05). In osteogenic induction medium, Jagged1significantly up-regulated TGFB1, TGFB2 and TGFB3 at days 1 and 3 (p<.05). Pre-treatment with TGF-β1, TGF-β2 or TGF-β3 prior to osteogenic induction resulted in the significant increase of osteogenic marker gene expression, collagen type 1 protein expression, alkaline phosphatase enzymatic activity and mineral deposition (p<.05). However, TGF-β signalling inhibition with SB431542 (4μmolL-1 ) or SB505124 (47 and 129nmolL-1 ) failed to attenuate the effect of Jagged1-induced osteogenic differentiation in hDPs. Dorsomorphin (4 and 8μmolL-1 ) treatment significantly abolished the effect of Jagged1 on mineralization by hDPs (p<.05). Notch signalling activation by Jagged1modulated TGF-β and BMP ligand expression. Dorsomorphin, but not TGF-β receptor inhibitor, attenuated Jagged1-induced osteogenic differentiation in hDPs.

Effect of aqueous extract of Gangronema latifolium (G. latifolium) (Utazi) on n-acetyl-para-aminophenol (acetaminophen) - induced hepatic injury on Wistar albino rats

The present study investigated the effect of leaf extract of Gangronema latifolium (G. latifolium) on acetaminophen (APAP) - induced liver injury in Wistar albino rats. In this study, sixty (60) male Wistar albino rats were divided into five (5) groups of twelve (12) rats each. Animals in group 1 served as control group and received a placebo of 0.9% saline solution. Group 2 served as APAP control group, administered with 800 mg/kg body weight of APAP only. Groups 3, 4 and 5 served as the experimental groups and received oral dosage of 800 mg/kg body weight of APAP plus 150 mg/kg, 200 mg/kg and 250 mg/kg body weight of G. latifolium respectively. The results showed that the enzymatic activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) in the serum were decreased significantly (p ≤ 0.05) in the experimental groups dosed with 150 mg/kg, 200mg/kg and 250 mg/kg of G. latifolium respectively. For 150 mg/kg G. latifolium treated group, ALT decreased from 23.3 ± 7.31 to 9.00 ± 1.52 IU/L, while AST and ALP decreased from 17.6 ± 2.66 to 15.00 ± 1.00 IU/L and 92.8 ± 2.34 to 83.8 ± 7.94 IU/L respectively. In conclusion, the results showed that aqueous extract of G. latifolium has a protective effect on rat liver induced with APAP injury.

Reference haematology and blood biochemistry profiles of striped catfish (Pangasianodon hypophthalmus) in summer and winter seasons

Pangasianodon hypophthalmus is a major aquaculture species in South and Southeast Asia with a high level of production. Standard physiological indices of candidate species are of great value for proper health monitoring and better production. Since fish physiology is grossly influenced by temperature, the reference intervals (RIs) of haematology and blood biochemistry of the healthy reference population of this species are established here in winter and summer seasons following standard methodologies and non-parametric statistics. Haemoglobin, alkaline phosphatase enzyme activity, glucose, urea, and creatinine levels were significantly higher in summer, while leucocyte count, globulin concentration, alanine aminotransferase enzyme activity were higher in winter suggesting the need for use of season-specific RIs for correct interpretation of test results. The normometric database would be useful in season-specific stress assessment, monitoring of well-being, and pathological progressions in this catfish which contributes about 2% to the global aquaculture production.