Alkaline Phosphatase Activity Research Articles

High dietary Mucuna pruriens utilis seed meal compromises growth performance, carcass traits, haemato-biochemistry, and meat quality of broilers.

Usage of soyabean meal (SBM) in broiler diets is economically and environmentally unsustainable thus necessitating investigation of alternative protein sources. Therefore, this study investigated effects of incremental inclusion levels of Mucuna pruriens utilis seed meal (MSM) for partial substitution of SBM in broiler diets. In a completely randomized design (CRD), 400day-old Ross 308 chicks were allotted to 5 iso-caloric-nitrogenous MSM-containing (0, 5, 10, 15, and 20%) dietary treatments. Each treatment was replicated 8 times, with each pen having 10 birds, during starter (d1 - 14), grower (d15 - 28), and finisher (d29 - 42) phases. Results showed that dietary MSM decreased feed intake (FI: quadratic: P < 0.001), body weight gain (BWG: linear: P < 0.001), and feed conversion efficiency (FCE: linear: P < 0.001) as it linearly decreased slaughter weight (SW: P < 0.001), hot carcass weight (HCW: P < 0.001), cold carcass weight (CCW: P < 0.001), dressing percentage (P < 0.001), and breast weight (P < 0.05). In contrast, dietary MSM linearly increased the weights of the liver (P < 0.01), proventriculus (P < 0.001), gizzard (P < 0.001), duodenum (P = 0.01), jejunum (P < 0.001), ileum (P < 0.001), caecum (P < 0.01), and colon (P < 0.01). Also, dietary MSM quadratically increased blood heterophils (P < 0.05) and alkaline phosphatase activity (P < 0.05) of the chickens whilst linearly increasing their serum amylase (P = 0.001) and lipase (P = 0.001) activities and linearly decreasing their serum symmetric dimethylarginine (SDMA: P = 0.001) and cholesterol (P < 0.05). Further, dietary MSM linearly decreased chicken breast meat ultimate pH (P < 0.05) whilst linearly increasing its cooking loss (P < 0.01), drip loss (P < 0.05) and shear force (P < 0.01). In conclusion, dietary MSM compromised growth performance, carcass characteristics, and meat quality of broilers as it increased the weights of their digestive-metabolic organs.

Low frequency‑pulsed electromagnetic fields promote osteogenic differentiation of bone marrow‑derived mesenchymal stem cells by regulating connexin 43 expression.

The present study investigated the effect of connexin 43 (Cx43) on the regulation of osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) using low-frequency-pulsed electromagnetic fields (LPEMF). The BMSCs were isolated and cultured in vitro using adherent whole-bone marrow cultures. CCK-8 assay was used to detect the effects of LPEMF on the proliferation ability of BMSCs and alkaline phosphatase (ALP) activity and the levels of osteogenic marker genes were detected to evaluate the osteogenic ability change following LPEMF treatment. Lentiviral vector-mediated RNA interference was transfected into BMSCs to inhibit the expression of Cx43 and western blotting was used to detect Cx43 expression. The BMSCs showed the highest proliferation following LPEMF treatment at 80 Hz for 1 h. The results of ALP activity, osteogenic marker genes and Alizarin Red S staining showed that the osteogenic ability was notably increased following LPEMF treatment at 80 Hz for 1 h. Cx43 expression increased during the osteogenic differentiation of BMSCs following LPEMF treatment at 80 Hz. The enhanced osteogenic differentiation of the LPEMF-treated BMSCs were partially reversed when Cx43 expression was inhibited. LPEMF may promote the osteogenic differentiation of BMSCs by regulating Cx43 expression and enhancing osteogenic ability.

The Role of Natural Background Radiation in Maintaining Genomic Stability in the CGL1 Human Hybrid Model System.

Natural background ionizing radiation is present on the earth's surface; however, the biological role of this chronic low-dose-rate exposure remains unknown. The Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR) project is examining the impacts of sub-natural background radiation exposure through experiments conducted 2 km underground in SNOLAB. The rock overburden combined with experiment-specific shielding provides a background radiation dose rate 30 times lower than on the surface. We hypothesize that natural background radiation is essential for life and maintains genomic stability and that prolonged exposure to sub-background environments will be detrimental to biological systems. To evaluate this, human hybrid CGL1 cells were continuously cultured in SNOLAB and our surface control laboratory for 16 weeks. Cells were assayed every 4 weeks for growth rate, alkaline phosphatase (ALP) activity (a marker of cellular transformation in the CGL1 system), and the expression of genes related to DNA damage and cell cycle regulation. A subset of cells was also exposed to a challenge radiation dose (0.1 to 8 Gy of X rays) and assayed for clonogenic survival and DNA double-strand break induction to examine if prolonged sub-background exposure alters the cellular response to high-dose irradiation. At each 4-week time point, sub-background radiation exposure did not significantly alter cell growth rates, survival, DNA damage, or gene expression. However, cells cultured in SNOLAB showed significantly higher ALP activity, a marker of carcinogenesis in these cells, which increased with longer exposure to the sub-background environment, indicative of neoplastic progression. Overall, these data suggest that sub-background radiation exposure does not impact growth, survival, or DNA damage in CGL1 cells but may lead to increased rates of neoplastic transformation, highlighting a potentially important role for natural background radiation in maintaining normal cellular function and genomic stability.

Exploration of new thiazolidinones derived from natural verbenone: Design, synthesis, characterization, and in silico evaluation of alkaline phosphatase and carbonic anhydrase-II inhibition activity

Hybrid compounds featuring isoxazole and thiazolidinone nuclei, alongside verbenone, have become pivotal in pharmacological research due to their versatile properties and straightforward synthesis methods. In this study, the hybrid compounds 3–5 were thoroughly analyzed using high-resolution mass spectrometry (HRMS) and 1H- and 13C NMR spectroscopy, confirming the presence of the isoxazoline and thiazolidinone cores. Additionally, text of in vitro cytotoxicity, computational molecular docking and dynamics studies were performed to study the cytotoxic profile and the modes of possible interactions between the synthesized compounds and the biological targets. The cytotoxicity test indicated that product 5 exhibited the highest activity, with IC50 values ranging from 25.87±3.21 to 28.12±3.54 µM against MDA-MB-231 and A-549 cancer cell lines. From a theoretical point of view, the comparative analysis of compounds 3–5 against the references in the in silico study revealed significant interactions for these compounds with the isoenzymes of alkaline phosphatase and carbonic anhydrase.

Low-energy red light-emitting diode irradiation enhances osteogenic differentiation of periodontal ligament stem cells by regulating miR-146a-5p.

The study aimed to investigate the role of miR-146a-5p in osteogenesis of hPDLSCs irradiated with low-energy red LEDs. After irradiation with 5 J/cm2 red LED, miR-146a-5p expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and osteogenic markers expression was determined by RT-qPCR and Western blotting. Alkaline phosphatase (ALP) activity was assessed by ALP staining, and mineralization was assessed by Alizarin Red staining, respectively. Lentiviral vectors were designed to regulate miR-146a-5p expression. Dual-luciferase reporter assay was performed to confirm the targeted relationship between miR-146a-5p and MAPK1. Short hairpin RNA (shRNA) was used to regulate MAPK1 expression. RT-qPCR and western blotting revealed that 5 J/cm2 irradiation elevated the levels of the osteogenic markers osterix (OSX) and bone sialoprotein (BSP) in hPDLSCs. miR-146a-5p is downregulated in hPDLSCs under the low-energy red LED light irradiation. miR-146a-5p underexpression markedly promoted the osteogenic potential of hPDLSCs. miR-146a-5p targeted MAPK1. 5 J/cm2 red LED irradiation rescued the inhibitory effects of upregulated miR-146a-5p on osteogenic differentiation, and the positive influence of red LED irradiation could be reversed by downregulated MAPK1. These findings confirm that miR-146a-5p is involved in the effect of LED irradiation on the osteogenic differentiation of hPDLSCs by targeting MAPK1. Red LED irradiation may be a potential clinical adjunct therapy for periodontal regeneration.

Locally released dexamethasone and its effects on osteogenic activity at implant-tissue interface.

The osseointegration of titanium implants within the host tissue holds crucial importance. The introduction of functional coatings at tissue-implant interface enhances the bioactivity of titanium implants, improves their therapeutic outcomes, and enhances the effectiveness of treatments. In this study, we focused on enhancing the bioactivity of titanium-based implant materials by coating the titanium surfaces with chitosan microspheres, which are loaded with osseointegration-promoting agent dexamethasone (DEX). Initially, chitosan microspheres were successfully produced, followed by DEX loading through diffusion, resulting in a drug loading efficiency of around 50.2 (wt %). The subsequent drug release profile displayed a 24-hour duration, releasing approximately 32.6 (wt %) of the loaded DEX. In cell proliferation assays using human osteosarcoma (SAOS-2) cells, Ti surfaces coated with DEX-loaded chitosan microspheres initially exhibited lower cell numbers compared with DEX-free ones. This observation was attributed to transient osteogenic differentiation effects of DEX, since a notable increase in cell proliferation was observed on the 7th day. Von Kossa staining revealed mineralization beginning on the 14th day, particularly evident in DEX-loaded samples. Moreover, alkaline phosphatase (ALP) activity displayed a pattern of initial increase and subsequent decrease, with DEX release from chitosan microspheres showing a clear influence on the osteogenic differentiation, especially on the 7th day. These findings align with literature, highlighting DEX's potential to enhance osteogenic differentiation and cellular behavior on chitosan microsphere-coated titanium surfaces. This study emphasizes the promising implications for functionalizing surfaces of implant materials with DEX-loaded chitosan microspheres to improve their biocompatibility and bioactivity.

Interactive Effects of Dietary Probiotic and Succinic Acid on the Growth Performance, Digestive Enzyme Activities, Immunomodulation, Antioxidative Capacity, and Disease Resistance in Rainbow Trout (Oncorhynchus mykiss) Juveniles

Abstract The current study was carried out to explore the effects of lactofeed (LA) as a multi-strain probiotic and succinic acid (SA) on growth variables, gut lactic acid bacteria count, digestive enzymes, innate immune responses, antioxidant capacity, and resistance against yersinia ruckeri in rainbow trout juveniles (Oncorhynchus mykiss). Three hundred sixty healthy rainbow trout juveniles (13.21±0.41 g) were randomly divided into twelve tanks (300 L) as four experimental groups. They were fed with basal diet (Control; BD), FT1: BD + 1 g/kg LA, FT2: BD + 5 g/kg SA, and FT3: BD + 1 g/kg LA + 5 g/kg SA for eight weeks. According to the results, final weight (FW), weight gain (WG), protein efficiency rate (PER), and feed conversion rate (FCR) revealed a remarkable discrepancy compared to the control group. In addition, dietary inclusion of LA (FT1 and FT3) significantly increased the specific growth rate (SGR). Dietary supplementation of LA+SA (FT3) remarkably improved total bacteria count (TBC) and amylase activity compared to the unsupplemented group. Gut lactic acid bacteria (LAB) count and digestive protease activity in all supplemented fish were remarkably higher than in unsupplemented group. Blood immunological factors including white blood cell (WBC) count, total immunoglobulin content (Ig), and the activity of lysozyme (LYZ), alternative complement pathway (ACH50), and acid phosphatase (ACP) were significantly enhanced in the supplemented groups. Neutrophil (NEU) count, skin mucus Ig and hepatic glutathione peroxidase (GPX) increased in SA (FT2 and FT3) treatments. In addition, monocyte (MON) count and skin mucus LYZ activity were significantly elevated following feeding with the FT3 diet. Other immunological parameters of skin mucus including protease activity, alkaline phosphatase (ALP) activity, and ACH50 activity, as well as hepatic superoxide dismutase (SOD) and catalase (CAT) activities increased in fish fed with diets containing LA and/or SA. Malondialdehyde (MDA) value was remarkably decreased in all supplemented rainbow trout compared to the fish fed with BD. Disease resistance against Y. ruckeri in fish fed with supplemented diets significantly improved with respect to the results obtained in the control specimens. Overall, dietary LA+SA supplementation was beneficial to improve growth performance, gut LAB count, digestive enzyme activities, innate immune responses, antioxidant capacity, and disease resistance in rainbow trout. According to these findings, 1 g/kg LA + 5 g/kg SA is suggested for adding to rainbow trout diet.

A fluorescence biosensor for organophosphorus pesticide detection with a portable fluorescence device-based smartphone

An innovative fluorescence biosensor was successfully developed to detect organophosphorus pesticide (OPs) by utilizing smartphone technology. The assay relied on the enzymatic activity of alkaline phosphatase (ALP), which facilitated the conversion of L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AAP) into L-ascorbic acid (AA). The AA that generated was then reactedwith o-phenylenediamine (OPD) to yield a fluorescent marker identified as 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxalin-1(3H)-one (DFQ). A novel bandpass approach was specifically developed for a smartphone that was integrated with a customized portable fluorescence device to measure the fluorescence emission of DFQ. The device has a unique application that converts the fluorescence intensity into an RGB signal. In the presence of OPs, malathion was chosen as the representative of the OPs substance; the enzymatic activity of the ALP was inhibited, resulting in a decrease in fluorescence intensity, which was proportional to the concentration of malathion. Smartphones can be used to measure fluorescence emission, offering a calibration sensitivity more than 70 times higher than that of conventional spectrofluorometer. The recently developed methodology can be employed to identify malathion within the concentration range of 0.1–1 ppm, with a detection limit of 0.05 ppm. The practical applicability of the method was established using vegetable samples, and the acquired results were in good agreement with those obtained using the standard HPLC approach. This innovative method provides both portability and accuracy, while also exhibiting a notable degree of sensitivity in detecting traceamounts of OPs.

Food waste biochar for sustainable agricultural use: Effects on soil enzymes, microbial community, lettuce, and earthworms

This study investigates the effects of food waste biochar (FWB) on the biological properties of soil, including the microbial community structure, enzyme activities, lettuce growth, and earthworm ecotoxicity. This holistic assessment of various soil organisms was used to assess the potential of FWB as a soil amendment strategy. Pot experiments were carried out over a 28-d period using various FWB concentrations in soil (0–3% w/w). The presence of FWB enhanced the activity of alkaline phosphatase and beta-glucosidase in proportion to the FWB concentration. Similarly, the dehydrogenase activity after 28 d was positively correlated with the FWB concentration. Notably, the application of FWB improved the bacterial diversity in the soil, particularly among hydrocarbonoclastic bacteria, while also prompting a shift in the fungal community structure at the class level. Measures of lettuce growth, including total fresh weight, shoot length, and leaf number, also generally improved with the addition of FWB, particularly at higher concentrations. Importantly, FWB did not adversely affect the survival or weight of earthworms. Collectively, these findings suggest that FWB can enhance soil microbial enzyme activity and support plant growth-promoting rhizobacteria, potentially leading to increased crop yields. This highlights the potential of FWB as an eco-friendly soil amendment strategy.

Nanostructured lipid carriers decorated with polyphosphate coated linear and loop cell-penetrating peptides

AimThis study aimed to evaluate the cellular uptake of nanostructured lipid carriers (NLCs) decorated with polyphosphate coated linear and loop cell-penetrating peptides (CPPs). MethodsLinear-CPPs and loop-CPPs were synthesized via ring-opening polymerization and anchored on the surface NLCs, followed by coating with polyphosphate (PP). These nanocarriers (NCs) were characterized in terms of particle size, polydispersity index (PDI), and zeta potential. Cell viability and hemolysis, as well as enzyme-induced charge conversion via phosphate cleavage by free and membrane-bound intestinal alkaline phosphatase (IAP) were investigated. Cellular uptake studies by Caco-2 and HEK cells were quantitatively analyzed by flow cytometry and visualized by confocal microscopy. ResultsA shift in charge from positive to negative was obtained for both linear- and loop-CPPs-NLCs by coating with PP. PP-linear-CPPs-NLCs and PP-loop-CPPs-NLCs exhibited a particle size < 270 nm and a PDI of approximately 0.3. They had a minor effect on cell viability and caused in a concentration of 0.1 % (m/v) around 10 % hemolysis within 24 h. IAP triggered the cleavage and release of monophosphate from the surface of NLCs causing charge conversion from –22.2 mV to + 5.3 mV (Δ27.5 mV) for PP-linear-CPPs-NLCs and from −19.2 mV to + 11.9 mV (Δ31.1 mV) for PP-loop-CPPs-NLCs. Inhibition of alkaline phosphatase activity on Caco-2 and HEK cells confirmed the involvement of this enzyme in charge conversion. PP-linear-CPPs-NLCs showed on Caco-2 cells a higher uptake than PP-loop-CPPs-NLCs, whereas on HEK cells uptake of both types of NLCs was on the same level. The results of cellular uptake were confirmed visually by confocal microscopy. ConclusionCPPs-NLCs coated with polyphosphate are a promising approach to overcome the polycationic dilemma and to enhance cellular uptake.

Melanoma-inhibiting activity promotes the migration and odontoblastic differentiation of stem cells of apical papilla

ObjectiveMelanoma Inhibitory Activity (MIA) has been predominantly studied in the context of melanoma and cartilage development. However, its role in dental pulp development and stem cell behavior remains largely unexplored. This study investigates the expression pattern of MIA in dental pulp tissues and its potential role in the proliferation, migration, and odontoblastic differentiation of stem cells from the apical papilla (SCAPs). Design:MIA expression in human pulp tissue was demonstrated by immunohistochemistry. SCAPs were cultured in normal and mineralization induction media, with MIA levels monitored via RT-qPCR and Western blot. Cell proliferation was evaluated using the CCK8 assay, while transwell and cell scratch assays were conducted to examine cell migration. The effect of MIA on odontoblastic differentiation was examined by qRT-PCR, Alkaline phosphatase activity assay, and Western blot. siRNA was used to knock down MIA to investigate its effect. A mouse subcutaneous implantation model was used to assess whether MIA promotes odontoblastic differentiation in vivo. Results:MIA expression was observed in the papilla and odontoblasts layer of the developing pulp. In vitro, MIA expression increased during SCAPs differentiation and was found to significantly enhance migration, and odontoblastic differentiation but not proliferation. Gene knockdown experiments confirmed MIA’s pivotal role in promoting SCAPs migration and differentiation. In vivo, MIA facilitated the formation of dentin-like structures and enhanced pulp-dentin complex regeneration. Conclusion:MIA plays a crucial role in SCAPs’ migration and differentiation, suggesting its potential application in pulp-dentin regeneration therapies. Further studies are required to fully elucidate the underlying mechanisms.

Control of alkaline phosphatase activity and pH stability by taurine in liquid boar semen

Control of alkaline phosphatase activity and pH stability by taurine in liquid boar semen

Osteogenic effect of poly(lactic-co-glycolic acid) microcapsules with different molecular weights encapsulating bone morphogenetic protein 2.

This study aimed to explore the effects of bone morphogenetic protein 2 (BMP-2) encapsula-ted in poly(lactic-co-glycolic acid) (PLGA) microcapsules with different molecular weights on the osteogenic ability of osteoblasts. PLGA microcapsules with different molecular weights (12 000, 30 000) encapsulating BMP-2, were prepared using a dual-channel microinjection pump. The morphology and structure of the microcapsules were characterized by optical microscopy and scanning electron microscopy. The sustained-release performance of the microcapsules was characterized by phosphate buffered saline immersion method. The cell compatibility of the microcapsules was detected by the Calcein-AM/PI staining and CCK-8 method. The chemotactic effect of BMP-2-encapsulated microcapsules on MC3T3-E1 cells after 48 h of treatment was detected by the Transwell assay. The alkaline phosphatase activity assay and Alizarin Red S staining were used to characterize the effect of microcapsules on the osteogenic ability of MC3T3-E1 cells. Both types of microcapsules with different molecular weights exhibited smooth surfaces, as well as uniform and good cell compatibility. The chemotactic effect of the 12 000 microcapsules was outstanding. The 30 000 microcapsules had a longer sustained-release time, and the initial burst release was reduced by approximately 25% compared with the 12 000 microcapsules. In addition, 30 000 microcapsules performed better in long-term osteogenesis induction than 12 000 microcapsules. In this study, the release of BMP-2 is regulated by adjusting the molecular weight of PLGA, and the results indicate that 30 000 microcapsules can better induce the long-term osteogenic ability of MC3T3-E1 cells.

Osteoking prevents bone loss and enhances osteoblastic bone formation by modulating the AGEs/IGF-1/β-catenin/OPG pathway in type 2 diabetic db/db mice.

Type 2 diabetic osteoporosis (T2DOP) is a skeletal metabolic syndrome characterized by impaired bone remodeling due to type 2 diabetes mellitus, and there are drawbacks in the present treatment. Osteoking (OK) is widely used for treating fractures and femoral head necrosis. However, OK is seldom reported in the field of T2DOP, and its role and mechanism of action need to be elucidated. Consequently, this study investigated whether OK improves bone remodeling and the mechanisms of diabetes-induced injury. We used db/db mice as a T2DOP model and stimulated MC3T3-E1 cells (osteoblast cell line) with high glucose (HG, 50 mM) and advanced glycation end products (AGEs, 100 µg/mL), respectively. The effect of OK on T2DOP was assessed using a combined 3-point mechanical bending test, hematoxylin and eosin staining, and enzyme-linked immunosorbent assay. The effect of OK on enhancing MC3T3-E1 cell differentiation and mineralization under HG and AGEs conditions was assessed by an alkaline phosphatase activity assay and alizarin red S staining. The AGEs/insulin-like growth factor-1(IGF-1)/β-catenin/osteoprotegerin (OPG) pathway-associated protein levels were assayed by western blot analysis and immunohistochemical staining. We found that OK reduced hyperglycemia, attenuated bone damage, repaired bone remodeling, increased tibial and femoral IGF-1, β-catenin, and OPG expression, and decreased receptor activator of nuclear kappa B ligand and receptor activator of nuclear kappa B expression in db/db mice. Moreover, OK promoted the differentiation and mineralization of MC3T3-E1 cells under HG and AGEs conditions, respectively, and regulated the levels of AGEs/IGF-1/β-catenin/OPG pathway-associated proteins. In conclusion, our results suggest that OK may lower blood glucose, alleviate bone damage, and attenuate T2DOP, in part through activation of the AGEs/IGF-1/β-catenin/OPG pathway.

Optimizing mycorrhizal fungi application for improved nutrient uptake, growth, and disease resistance in cardamom seedlings (Elettaria cardamomum (L.) Maton)

This study evaluated the impact of various doses (5, 10, 15 g) and application sequences (1, 2, or 3 times at monthly intervals) of arbuscular mycorrhizal (AM) fungal inoculum on cardamom seedlings over two years (2020–2021 and 2021–2022). The results indicated that the dosage of AM inoculum had a more substantial effect on the seedlings than the application sequence. A 10 g dose significantly increased shoot length and dry weight, while three applications of 5 g each improved the number of fibrous roots. Although potassium uptake was not affected, phosphorus and calcium uptake were highest with the 10 g dose. Arbuscular mycorrhizal inoculation also enhanced phosphatase activity in the rhizosphere, with 5 g improving acid phosphatase and 10 g improving alkaline phosphatase activity. Disease incidence, including seedling rot was lower with the 10 g dose, and additional sequential applications did not further reduce disease. Structural equation modeling (SEM) revealed that AM colonization positively influenced dry weight through the number of fibrous roots, showing a strong relationship between AM dose, colonization, spore count, and mycorrhizal dependency. This study indicates that applying a 10 g dose of AM fungal inoculum can be particularly beneficial in agroecosystems for improving cardamom seedling growth, nutrient uptake, and disease resistance.

Low Testosterone Concentration Improves Colonisation and Viability in the Co-Cultured Goat Spermatogonial Stem Cell With Sertoli Cells.

Spermatogonial stem cells (SSCs) maintain spermatogenesis through self-renewal and differentiation. The proliferation of SSCs in culture systems can provide a valuable source of germ cells. Several studies have investigated new reproductive technologies, including the production of transgenic animals and recombinant proteins secreted from milk in goats. While studies in other species exist, research on goat SSC culture remains limited. We investigated the impact of different testosterone concentrations on the survival and colonisation of cocultured goat SSCs with Sertoli cells. Cells were isolated from immature goats using two-step enzymatic digestion and enriched by differential exclusion method. DMEM/F12 culture medium containing 1% antibiotic and 5% FBS, supplemented with GDNF (20 ng/mL), EGF, bFGF and LIF (10 ng/mL), was used with different testosterone concentrations (0, 60, 120 and 240 μg/mL) and cultured for 10 days. SC subpopulations were confirmed using PGP9.5 immunocytochemistry, and the expression of germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was evaluated through RT-PCR. Alkaline phosphatase activity provided additional SSC presence. The survival rate of SSCs after isolation and the number and area of colonies on Days 4, 7 and 10 were measured using an inverted microscope. The presence of PGP 9.5 antigens and germ cell markers (ID-4, UCHL-1, THY-1, β1-integrin, BCL6B, VASA, PLZF and OCT-4) was confirmed by immunocytochemistry and RT-PCR, respectively. According to the results, the group with 60 μg/mL testosterone had the highest number and area of colonies. The number of colonies in the 60 μg/mL testosterone group was significantly higher than the control group (p < 0.05), but no significant difference was observed compared to other groups (p ≥ 0.05). This study suggests that a low testosterone concentration (60 μg/mL) is optimal for goat SSC colonisation and viability in coculture with Sertoli cells, potentially leading to advancements in goat reproductive technologies.

Antibacterial activity and mechanism of combination natural essential oils from oregano and cinnamon against enterotoxigenic Escherichia coli K99

Essential oils from oregano (OEO) and cinnamon (CEO) have been reported an excellent antibacterial activity, but their synergistic antibacterial effect is rarely studied. In this study, the chemical compositions of OEO, CEO and mixed OEO with CEO (MEO) and their antibacterial activities were analyzed, the mode of MEO action against enterotoxigenic Escherichia coli K99 (ETEC K99) were investigated. Results showed that MEO had the synergistic antibacterial effect, which was proven by fractional inhibitory concentration index of 0.375. The inhibition zone diameter measured varying with volume ratio of OEO and CEO showed that the best synergistic effect was at a ratio 2:8. Meanwhile, the antibacterial mechanism of MEO demonstrated that the MEO changed the morphology of ETEC K99 by scanning and transmission electron microscopy, and disrupted the cell membrane integrity that were proved by the leakage of intracellular nucleic acid and lower alkaline phosphatase activity of ETEC K99. In addition, the MEO also inhibited DNA biosynthesis, biofilm formation and scavenged mature biofilm of ETEC K99. These results confirm the synergistic effect on improvement of antibacterial activity and elucidate a variety of mechanisms through which the ETEC K99 was inactivated, and support the application of MEO to against ETEC K99.

Impact of Polydeoxyribonucleotides on the Morphology, Viability, and Osteogenic Differentiation of Gingiva-Derived Stem Cell Spheroids

Background and Objectives: Polydeoxyribonucleotides (PDRN), composed of DNA fragments derived from salmon DNA, is widely recognized for its regenerative properties. It has been extensively used in medical applications, such as dermatology and wound healing, due to its ability to enhance cellular metabolic activity, stimulate angiogenesis, and promote tissue regeneration. In the field of dentistry, PDRN has shown potential in promoting periodontal healing and bone regeneration. This study aims to investigate the effects of PDRN on the morphology, survival, and osteogenic differentiation of gingiva-derived stem cell spheroids, with a focus on its potential applications in tissue engineering and regenerative dentistry. Materials and Methods: Gingiva-derived mesenchymal stem cells were cultured and formed into spheroids using microwells. The cells were treated with varying concentrations of PDRN (0, 25, 50, 75, and 100 μg/mL) and cultivated in osteogenic media. Cell morphology was observed over seven days using an inverted microscope, and viability was assessed with Live/Dead Kit assays and Cell Counting Kit-8. Osteogenic differentiation was evaluated by measuring alkaline phosphatase activity and calcium deposition. The expression levels of osteogenic markers RUNX2 and COL1A1 were quantified using real-time polymerase chain reaction. RNA sequencing was performed to assess the gene expression profiles related to osteogenesis. Results: The results demonstrated that PDRN treatment had no significant effect on spheroid diameter or cellular viability during the observation period. However, a PDRN concentration of 75 μg/mL significantly enhanced calcium deposition by Day 14, suggesting increased mineralization. RUNX2 and COL1A1 mRNA expression levels varied with PDRN concentration, with the highest RUNX2 expression observed at 25 μg/mL and the highest COL1A1 expression at 75 μg/mL. RNA sequencing further confirmed the upregulation of genes involved in osteogenic differentiation, with enhanced expression of RUNX2 and COL1A1 in PDRN-treated gingiva-derived stem cell spheroids. Conclusions: In summary, PDRN did not significantly affect the viability or morphology of gingiva-derived stem cell spheroids but influenced their osteogenic differentiation and mineralization in a concentration-dependent manner. These findings suggest that PDRN may play a role in promoting osteogenic processes in tissue engineering and regenerative dentistry applications, with specific effects observed at different concentrations.

Comparison of the Effects of Hesperidin Over 30- and 60-Day Intervals on Rainbow Trout: A Potential Biostimulant to Promote Growth, Immunological and Antioxidant Responses, and Disease Resistance

Abstract The current research evaluated the effects of dietary hesperidin (HSP) on growth parameters, digestive enzyme activities, innate immune markers, and antioxidant responses in rainbow trout, Oncorhynchus mykiss after the 30- and 60-day feeding trial. Then, specimens were subjected to Yersinia ruckeri infection for 14 days. For this, six hundred rainbow trout juveniles (initial weight; 25.49±0.40 g) were fed with different levels of HSP including 0 (HSP0; control), 50 (HSP50), 100 (HSP100), 150 (HSP150), and 200 (HSP200) mg/kg feed. After 60 days, dietary HSP100 supplementation significantly improved growth and feed efficiency indices. The optimal dose of HSP based on the regression test for WG and FCR detected 122 and 131 mg/kg, respectively. At the end of the 30th day, dietary HSP150 supplementation markedly boosted serum lysozyme (LYZ), myeloperoxidase (MPO), complement component C3, and immunoglobulin (Ig) levels, but decreased malondialdehyde (MDA) content. In the same period, dietary administration of HSP at the different concentrations markedly increased complement component C4 (HSP150 and HSP200), superoxide dismutase (SOD), skin mucus alkaline phosphatase (ALP) (HSP100–HSP200), skin mucus LYZ and catalase (CAT) (HSP100 and HSP 150), and skin mucus Ig level (HSP50–HSP200). After 60 days, all dietary HSP supplementation significantly improved lipase, serum C3, glutathione peroxidase (GPX), and skin mucus ALP, LYZ, and Ig levels, but decreased MDA, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) values. After 60 days, dietary administration of HSP150 induced a significant discrepancy in the activity of serum LYZ, MPO, respiratory burst (RB), and lactate dehydrogenase (LDH) compared to the control group. In the same period, dietary HSP supplementation at different levels induced a remarkable increase in digestive protease activity (HSP100), serum ACH50 and CAT activities (HSP100 and HSP150), skin mucus peroxidase and serum SOD levels (HSP100–HSP200), and serum C4 value (HSP50, HSP150, and HSP200), but markedly decreased ALP activity (HSP150 and HSP200). In addition, dietary HSP supplementation, especially HSP150, markedly boosted disease resistance against Y. ruckeri infection. The current data demonstrated that the oral administration of 100–150 mg/kg HSP has a high potential to promote growth performance, disease resistance, and faster induction of immune and antioxidant responses.

Effect of Humic Acids Extracted From Different Organic Sources and Inoculation of Bacillus Subtilis or Aspergillus Niger in Alkaline Phosphatase Activity in Calcareous Soil

Humic acids have received a large attention of researching and development as amendments for poor fertility conditions and deteriorating physical properties, since the process of extracting is an easy common process to implement, as well as dilution of these acids with water enables to be used at economically acceptable levels over large areas. It is necessary to notice that the properties of these acids depending on the organic source and the concentration added to the soil.A laboratory experiment was conducted at the department of soil Sciences and water resources, collage of Agriculture ,University of Basrah ,Iraq to demonstrate the effect of humic acids extracted from different sources and inoculation with Bacillus subtilis or Aspergillus niger on alkaline phosphatase activity in calcareous soil calcareous, southern Iraq. Humic acids extracted from wheat straw, alfalfa leaves, goat manure, and poultry manure which composted previously . Humic acid levels were 0, 25 and 50 L ha-1 .The soil also inoculated with Bacillus subtilis or Aspergillus niger, then samples were incubated at 28±2°C. After 7 or 30 days of incubation the activity of alkaline phosphatase was estimated . Enzyme phosphatase activity was increased in soils treated with humic acid as compared to control with highest activity at Poultry manure (391.54 µg p-nitrophenol gm-1 soil.1h-1).The results also indicated that increasing the level of humic acid significantly increased enzyme activity. For soil treated with Bacillus subtilis and Aspergillus niger , phosphatase activity increased-as compared to control with a higher activity at Bacilluas subtilis (373.1 µg p-nitrophenol gm-1 soil.1h-1).