Phosphatase Activity Research Articles

Age-dependent haemogram and sex-dependent serum biochemistry values in semi-feral Konik horses.

Semi-feral, free-roaming Konik polski horses are used in some European countries for preserving semi-open pasture landscapes. The estimation of their health status is still limited by insufficient data on various blood parameters. Therefore, our study aimed at the sex- and age-dependent analysis of haemogram and selected biochemistry parameters in healthy, semi-feral Koniks. In order to reach this aim, we took blood samples from 53 female and 18 male (8 uncastrated, 10 castrated) Koniks living in two Middle German nature reserves. They were of different age (9-266 months) and without signs of illness. Blood samples were analysed by an accredited laboratory. We identified age- but not sex-dependent changes in the white blood cell count (WBC). Higher age mainly caused a decrease in lymphocytes. Therefore, WBC correlated negatively and granulocyte-to-lymphocyte ratio positively with increasing age. Serum values of selected biochemical parameters did not depend on age but showed some sex-related differences. In this regard, serum total protein, triglyceride and the enzymatic activities of alkaline phosphatase, aspartate transaminase and g-glutamyltransferase were higher in males than females. However, the sex dependency of these enzymatic activities was restricted to uncastrated males. They also showed higher serum values for calcium and selenium than castrated males or all females. As far as the respective group sizes permitted, we then calculated age- or sex-dependent reference interval values for all parameters analysed. These values improve now the estimation of the health status of semi-feral, free-roaming Konik horses and provide a stable basis for future studies.

Straw return can increase maize yield by regulating soil bacteria and improving soil properties in arid and semi-arid areas

Straw return has been found to benefit soil fertility and crop yield, however, by which it affects microbial communities to mediate soil factors driving crop yields under maize continuous cropping systems in dryland areas is still unclear. To fill this gap, a 6-year field experiment was established with five straw return amounts (T0, T1, T2, T3, and T4, representing 0, 3,000, 6,000, 9,000, and 12,000kgha-1 of straw, respectively), and investigated the effects of on soil properties, enzymes, bacterial community composition and diversity, and crop yields. Our analysis showed that soil organic carbon (SOC), total nitrogen (TN), and total phosphorus (TP) contents significantly increased by 1-8%, 5-25%, and 2-9% under straw return treatments, respectively, compared to the T0, and soil catalase, urease, and alkaline phosphatase activities increased by at least 34.00%. Additionally, crop yield significantly increased by 4.23-12.00% under T1-T4 treatments, and showed highly significant relationships with SOC, TN, and TP. Importantly, we found straw return significantly altered the community of bacteria involved in the carbon and nitrogen cycle, and their abundance of strong responses depending on the amounts of straw return. For example, straw input increased the abundance of Proteobacteria (+2.64–5.57%), Acidobacteria (+3.82–13.83%), and Bacteroidetes (+15.37–30.49%). Similarly, the amount of straw application increased the bacterial diversity indexes (Shannon, 2.65–10.93%; Chao1, 13.47–18.50%), and had significant positive correlations with SOC, TN, and TP contents. Structural equation models (SEM) revealed that straw return management practice had positive and indirect effects on crop yields by influencing soil properties or the bacteria community. In conclusion, our findings revealed common associations and variations of bacterial community diversity with soil factors and crop yields at different straw return rates, and these findings provide insights and options for the development of better straw return strategies and sustainable agriculture in semi-arid regions.

Determinants of rhizospheric organic carbon fractions and accumulation in four different vegetations of coastal saline-alkali soils

Rhizosphere processes are key pathways through which plants influence the carbon cycling of diverse ecosystems and are closely associated with vegetation types. However, the environmental responses of rhizospheric soil organic carbon (SOC) mediated by the interaction between rhizospheric effect and vegetation type remain uncertain, severely limiting our assessment of the carbon sequestration potential of different coastal vegetation. Here, we identified the core factors driving changes in various soil carbon fractions in the rhizosphere of four coastal vegetation types by testing the contents of the reactive, dissolved, particulate, mineral-associated, heavy and light fractions of SOC and their relationships with physicochemical properties and enzyme activities. It was found that the rhizosphere effect significantly influenced SOC, with rhizospheric SOC contents varying significantly among vegetation types, ranging from 2.12 to 9.04 g/kg. Besides, rhizospheric soil pH was found to contribute significantly to SOC fractions (except for light organic carbon) and exert significant inhibitory effects on SOC, reactive organic carbon, mineral-associated organic carbon and heavy organic carbon. Despite the different responses of various SOC fractions to physicochemical properties and enzyme activities, soil available phosphorus and alkaline phosphatase activity emerged as core environmental factors influencing rhizospheric SOC accumulation across different coastal vegetation types. This study presents a promising framework for understanding the determinants of SOC fractions in rhizospheric soil, and their accumulation in the vegetation of coastal saline-alkali soils. In addition to alkalinity, phosphorus acquisition capacity can be a key predictor of coastal SOC fractions and their accumulation.

Protein phosphatase 1 suppresses PKR/EIF2α signaling during human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects the majority of the world's population. Lytic HCMV replication in immunocompromised individuals or neonates can lead to severe disease in multiple organ systems and even death. The establishment of lytic replication is driven by the first viral proteins expressed upon infection, the immediate early proteins, which play a key role in creating an intracellular environment conducive to virus replication. Two immediate early proteins, the functional orthologs pTRS1 and pIRS1, stimulate immediate early gene expression by suppressing antiviral PKR/eIF2α signaling and enhance the translation of viral mRNAs independent of PKR antagonism. To better understand the molecular functions of pTRS1, we used proximity labeling proteomics to identify proteins that interact with pTRS1 in infected cells. Multiple novel host and viral interactors were identified, including the catalytic subunits of the protein phosphatase 1 (PP1) holoenzyme. Mutations to a PP1 catalytic subunit known to disrupt binding to PP1 regulatory subunits decreased binding to pTRS1. pTRS1 immune complexes contained phosphatase activity, and inhibition of phosphatase activity in transfected or infected cells reversed the ability of pTRS1 to inhibit the antiviral kinase PKR. Depletion of individual PP1 catalytic subunits decreased virus replication and increased the phosphorylation of the PKR substrate eIF2α. Taken together, our data suggest potential novel functions for pTRS1 and define a novel role for PP1 as an antagonist of the antiviral PKR/eIF2α signaling axis during HCMV infection.IMPORTANCEThe human cytomegalovirus (HCMV) pTRS1 and pIRS1 proteins are critical regulators of HCMV replication, both during primary infection and during reactivation from viral latency. Thus, defining the molecular functions of pTRS1/pIRS1 is important for understanding the molecular events controlling HCMV replication and viral disease. These data provide new insights into potential pTRS1 functional roles, providing a starting point for others to understand new features of infected cell biology. Another important result of this study is the finding that specific protein phosphatase 1 (PP1) regulatory subunits are required to suppress PKR/eIF2α signaling, a critical cellular innate immune defense to viral infection. These data lay the groundwork for future efforts to discover therapeutics that disrupt pTRS1 interaction with PP1 allowing cellular defenses to limit HCMV replication and disease.

The uremic toxin indoxyl sulfate decreases osteocyte RANKL/OPG and increases wnt inhibitor RNA expression that is reversed by PTH

Abstract Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14 days (early) and 35 days (mature osteocytes) and incubated with 500 μM of indoxyl sulfate after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14 days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared to IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with PTH and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH, increased osteocyte mRNA Wnt inhibitor expression in both early and mature osteocytes and decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when elevated PTH, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes which would worsen bone resorption.

Arbuscular mycorrhizal fungi and intercropping Vicia villosa mediate plant biomass, soil properties, and rhizosphere metabolite profiles of walnuts

Intercropping is a prevalent soil management strategy within orchards, whereas it is unclear how inoculation with arbuscular mycorrhizal (AM) fungi and intercropping affect tree growth, soil properties, and rhizosphere metabolite profiles. This study investigated the effects of inoculation with Diversispora spurca and intercropping with hairy vetch (Vicia villosa) on biomass production, soil available nutrients, water-stable aggregate (WSA) distribution, phosphatase activity, and secondary metabolite profiles in walnuts (Juglans regia). The intercropping only elevated soil nitrate N levels and WSA distribution at the 0.5–2 mm size, and also triggered 2159 differential metabolites (1378 up-regulated and 781 down-regulated), with armillaramide as the most prominently up-regulated metabolite, followed by the substance diminished upon D. spurca inoculation. Conversely, D. spurca inoculation increased walnut biomass, WSA distribution across the 0.25 − 2 mm size, and acid and neutral phosphatase activities, as well as triggered 2489 differential metabolites (897 up-regulated and 1592 down-regulated), with pteroside D being highest up-regulated differential metabolite, allowing a competitive advantage to AM plants in combating soil pathogens. Despite significantly suppressing root AM fungal colonization and biomass production in AM walnuts, intercropping significantly increased soil ammonium and nitrate N levels in AM walnuts as well as WSAs at the 1–4 mm size, exhibiting a synergistic effect. Flavone and flavonol biosynthesis and pyruvate metabolism were simultaneously involved following AM inoculation or intercropping. Co-application of AM inoculation and intercropping triggered 1006 differential metabolites, with urocanic acid being the most up-regulated metabolite, although it decreased following AM inoculation, suggesting the involvement of mycorrhizal hyphae in soil histidine uptake. Under intercropping, AM inoculation elicited 418 differential metabolites, with the most up-regulated metabolite being implicated in flavonoid pathways. AM inoculation primarily triggered the biosynthesis of unsaturated fatty acids, regardless of intercropping or not, implying a potential increase in unsaturated fatty acid contents of walnut kernels. It concluded that AM inoculation and intercropping interactively affected walnut growth, soil attributes, and soil microenvironment.Graphical

Optimizing Irrigation Scheduling to Enhance Nutrient Uptake and Soil Microbial Activity in Linseed Cultivation (Linum usitatissimum L.)

Linseed crop cultivated under residual moisture and water scarcity conditions in rabi season. To improve the productivity of linseed irrigation water is applied at different stages of crop. So, to optimize the irrigation scheduling and enhance the nutrient uptake and soil microbial activity, this experiment was conducted at Main Agriculture Research Station, University of Agricultural Sciences, Raichur (which comes under North Eastern Dry zone of Karnataka) (India) during rabi 2023-24. The experiment was carried out with Randomized Complete Block Design (RCBD) with five treatments and four replications. The results revealed that, pre sowing irrigation fb three irrigations at vegetative stage (30-35 DAS), flowering stage (40-45 DAS) and capsule development stage (60-65 DAS) (T4) recorded significantly higher nutrient uptake by linseed crop (46.07, 19.87 and 39.83 NPK kg ha-1). The rainfed condition treatment (T5) recorded significantly lower nutrient uptake by the crop (19.35, 8.15 and 18.40 NPK kg ha-1). Among the different treatments, pre sowing irrigation fb three irrigations at vegetative stage (30-35 DAS), flowering stage (40-45 DAS) and capsule development stage (60-65 DAS) recorded significantly higher soil microbial population of bacteria (16.1 X 106 cfu g-1 of soil), fungi (13.8 X 104 cfu g-1 of soil) and actinomycetes (6.8 X 103 cfu g-1 of soil) as compared to other treatments. The soil enzymatic activity was significantly influenced by scheduling of irrigation at different growth stages in linseed. At harvest among the different treatments, pre sowing irrigation fb three irrigations at vegetative stage (30-35 DAS), flowering stage (40-45 DAS) and capsule development stage (60-65 DAS) recorded significantly higher dehydrogenase, urease and alkaline phosphatase activity (18.63 μg TPF g-1 soil day-1, 22.5 μg NH4-N g-1 soil hr-1 and 14.4 µg PNP g-1 soil hr-1, respectively).

A photo-triggered bioactive nanohydrogel loaded with niobium carbide for stem cell osteogenic differentiation

Photo-Cross-Linkable hydrogel has attracted immense interest in the regeneration of bone repair and regeneration strategies due to its superior biocompatibility and tunable mechanical properties. Recently, Nb was reported to strongly promote the bone regeneration process via an accelerated osteoblast-modulated alkaline phosphatase activity mechanism. In particular, Nb2C MXenes have drawn widespread attention due to their excellent biocompatibility and ability to induce bone formation. However, the easy agglomeration of Nb2C nanosheets and subsequent low cell endocytosis efficacy greatly suppressed the osteogenesis effect. In this study, a subtractive nanopore-engineered Nb2C MXene was prepared through a microwave combustion method, gelatin methacrylate was used as the carrier hydrogel, and the photo-triggered Porous-Nb2C@GelMA hydrogel was fabricated by a photo-triggered process. The pore-forming strategy not only successfully improved the distribution of Nb2C and formed more homogenous Porous-Nb2C@GelMA hydrogels but also guided bone marrow mesenchymal stem cells (BMSCs) toward osteoblast differentiation. Porous Nb2C provided convenient cellular grasping and endocytosis for BMSCs, which further created a favorable environment for differentiation and osteogenesis. This, in turn, leads to an increase in the expression of osteogenic markers, such as ALP and ARS, as well as osteogenic factors, such as BMP-2, COL-1, OCN, and OPN. Consequently, enhancing the regenerative microenvironment by incorporating porous Nb2C composite hydrogels shows promise for application in bone regeneration.

Ultraviolet laser induced periodic surface structures positively influence osteogenic activity on titanium alloys

Background/ObjectiveEndoprostheses might fail due to complications such as implant loosening or periprosthetic infections. The surface topography of implant materials is known to influence osseointegration and attachment of pathogenic bacteria. Laser-Induced Periodic Surface Structures (LIPSS) can improve the surface topography of orthopedic implant materials. In this preclinical in vitro study, laser pulses with a wavelength in the ultraviolet (UV) spectrum were applied for the generation of LIPSS to positively influence formation of extracellular matrix by primary human Osteoblasts (hOBs) and to reduce microbial biofilm formation in vitro.MethodsLaser machining was employed for generating UV-LIPSS on sample disks made of Ti6Al4V and Ti6Al7Nb alloys. Sample disks with polished surfaces were used as controls. Scanning electron microscopy was used for visualization of surface topography and adherent cells. Metal ion release and cellular metal levels were investigated by inductively coupled plasma mass spectrometry. Cell culture of hOBs on sample disks with and without UV-LIPSS surface treatments was performed. Cells were investigated for their viability, proliferation, osteogenic function and cytokine release. Biofilm formation was facilitated by seeding Staphylococcus aureus on sample disks and quantified by wheat germ agglutinin (WGA) staining.ResultsUV-LIPSS modification results in topographies with a periodicity of 223 nm ≤ λ ≤ 278 nm. The release of metal ions was found increased for UV-LIPSS on Ti6Al4V and decreased for UV-LIPSS on Ti6Al7Nb, while cellular metal levels remain unaffected. Cellular adherence was decreased for hOBs on UV-LIPSS Ti6Al4V when compared to controls while proliferation rate was unaffected. Metabolic activity was lower on UV-LIPSS Ti6Al7Nb when compared to the control. Alkaline phosphatase activity was upregulated for hOBs grown on UV-LIPSS on both alloys. Less pro-inflammatory cytokines were released for cells grown on UV-LIPSS Ti6Al7Nb when compared to polished surfaces. WGA signals were significantly lower on UV-LIPSS Ti6Al7Nb indicating reduced formation of a S. aureus biofilm.ConclusionOur results suggest that UV-LIPSS texturing of Ti6Al7Nb positively influence bone forming function and cytokine secretion profile of hOBs in vitro. In addition, our results indicate diminished biofilm formation on UV-LIPSS treated Ti6Al7Nb surfaces. These effects might prove beneficial in the context of long-term arthroplasty outcomes.

Accumulation of coumaric acid is a key factor in tobacco continuous cropping obstacles

IntroductionPhenolic acids are believed to play a significant role in tobacco continuous cropping obstacles, but the strength and potential mechanisms of different phenolic acids remain unclear.MethodsThis study evaluated the allelopathic effects of six phenolic acids that exhibited cumulative effects in our previous research. Different concentrations of phenolic acids with the strongest allelopathic effects were added to potting soil to explore their impacts on tobacco growth and physiological characteristics, as well as on soil chemical properties and microbial community structure. ResultsThe results showed that coumaric acid exhibited the strongest direct allelopathic effect. Exogenous coumaric acid significantly reduced soil pH and shifted the soil microbial community from bacteria-dominated to fungi-dominated. Simultaneously, the abundance of bacteria related to nutrient utilization (e.g., Flavisolibacter, Methylobacterium) and fungi related to disease resistance (e.g., Fusicolla, Clonostachys) gradually decreased, along with a reduction in soil catalase, urease, invertase, and acid phosphatase activities. Leaf MDA levels increased continuously with higher concentrations of coumaric acid, while the root resistance hormone (jasmonic acid and the jasmonate-isoleucine complex) levels show the opposite trend.DiscussionCoumaric acid may inhibit tobacco growth by influencing the physiological processes in tobacco plants directly and the broader soil microecological balance indirectly. This study provides theoretical guidance for precise mitigation of continuous cropping obstacles in future tobacco cultivation.

Investigating the Biological Efficacy of Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF): A Study on Cytokine Dynamics and Osteoblast Behavior

The development of effective biomaterials for tissue regeneration has led to the exploration of blood derivatives such as leucocyte- and platelet-rich fibrin (L-PRF). A novel variant, Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF), has been introduced to improve structural stability and bioactivity, making it a promising candidate for bone regeneration. This study aimed to evaluate Alb-PRF’s capacity for cytokine and growth factor release, along with its effects on the proliferation, differentiation, and mineralization of human osteoblasts in vitro. Alb-PRF membranes were analyzed using histological, scanning electron microscopy, and fluorescence microscopy techniques. Cytokine and growth factor release was quantified over seven days, and osteoinductive potential was evaluated with MG-63 osteoblast-like cells. Structural analysis showed Alb-PRF as a biphasic, highly cellularized material that releases lower levels of inflammatory cytokines and higher concentrations of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) compared to L-PRF. Alb-PRF exhibited higher early alkaline phosphatase activity and in vitro mineralization (p < 0.05) and significantly increased the OPG/RANKL mRNA ratio (p < 0.05). These results indicate that Alb-PRF has promising potential as a scaffold for bone repair, warranting further in vivo and clinical assessments to confirm its suitability for clinical applications.

The Impact of Acute Ammonia Nitrogen Stress on Serum Biochemical Indicators and Spleen Gene Expression in Juvenile Yellowfin Tuna (Thunnus albacares)

The presence of ammonia nitrogen in water has a significant impact on the serum and spleen of fish, potentially leading to changes in substances such as proteins in the serum while also causing damage to the immune function of the spleen. To investigate the effects of ammonia nitrogen (NH3-N) stress on juvenile yellowfin tuna (Thunnus albacares), this study established three NH3-N concentrations, 0, 5, and 10 mg/L, denoted as L0, L1, and L2, respectively. Serum and spleen samples were collected at 6, 24, and 36 h. The effects of different NH3-N concentrations and exposure times on the physiological status of juvenile fish were investigated by analyzing serum biochemical indices and splenic gene expression. The results indicate that in the L1 group, the serum high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), complement 3 (C3) and complement 4 (C4) levels, and acid phosphatase (ACP) activity showed a trend of initially increasing and then decreasing. In the L2 group, the serum low-density lipoprotein cholesterol (LDL-C), HDL-C, and C4 levels and ACP activity also displayed an initially rising and then declining trend, while TG, C3, and creatinine (CRE) levels and alkaline phosphatase (AKP) activity showed an upward trend. In the L1 group, glutathione peroxidase 1b (GPX1b), interleukin 10 (IL-10), interleukin 6 receptor (IL-6r), and tumor necrosis factor alpha (TNF-α) gene expression levels in the spleen exhibited a trend of first increasing and then decreasing. In the L2 group, IL-10, IL-6r, tumor necrosis factor beta (TNF-β), caspase 2 (casp2), and caspase 9 (casp9) gene expression levels in the spleen also showed an initial increase followed by a decrease. When NH3-N levels are below 5 mg/L, it is recommended to limit stress exposure to no more than 36 h for the juvenile fish. For concentrations ranging from 5 to 10 mg/L, stress should be strictly controlled to within 24 h. Exposure to high NH3-N levels may affect biochemical indicators such as serum lipid metabolism, immunity, and metabolism in juvenile fish, and may damage the expression of antioxidant, immune gene, and apoptosis factors in the spleen. This study aims to deepen our understanding of the effects of NH3-N on juvenile tuna, with the goal of establishing effective water quality monitoring and management strategies. This will ensure the quality of aquaculture water, reduce the harm caused by NH3-N to juvenile yellowfin tuna, and enhance aquaculture efficiency and product quality.

Orientation-dependent CD45 inhibition with viral and engineered ligands.

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as "beads on a string" that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses.

High keratinase and other types of hydrolase activity of the new strain of Bacillus paralicheniformis.

Keratinases, a subclass of proteases, are used to degrade keratin thereby forming peptones and free amino acids. Bacillus paralicheniformis strain T7 was isolated from soil and exhibited high keratinase, protease, collagenase, amylase, xylanase, lipase, and phosphatase activities. Keratinases of the strain showed maximum activity at 70°C and pH 9.0 as well as high thermal stability. A mass-spectrometric analysis identified seven peptidases with molecular masses of 26.8-154.8 kDa in the secretory proteome. These peptidases are members of S8 and S41 serine peptidase families and of M14, M42, and M55 metallopeptidase families. Additionally, α-amylase (55.2 kDa), alkaline phosphatase (59.8 kDa), and esterase (26.8 kDa) were detected. The strong keratinolytic properties of the strain were confirmed by degradation of chicken and goose feathers, which got completely hydrolyzed within 4 days. Submerged fermentation by strain B. paralicheniformis T7 was carried out in a pilot bioreactor, where the highest keratinase production was noted after 19 h of cultivation. After the fermentation, in the culture fluid, the keratinase activity toward keratin azure was 63.6 ± 5.8 U/mL. The protease activity against azocasein was 715.7 ± 40.2 U/mL. The possibility of obtaining enzyme preparations in liquid and powder form was demonstrated, and their comparative characteristics are given. In the concentrate, the keratinase, protease, α-amylase, phosphatase, and esterase/lipase activities were 2,656.7 ± 170.4, 29,886.7 ± 642.9, 176.1 ± 16.3, 23.9 ± 1.8, and 510.9 ± 12.2 U/mL, respectively. In the lyophilizate, these activities were 57,733.3 ± 8,911.4, 567,066.7 ± 4,822.2, 2,823.0 ± 266.8, 364.2 ± 74.8, and 17,618.0 ± 610.3 U/g, respectively. In the preparation obtained by air flow drying at 55°C, these activities were 53,466.7 ± 757.2, 585,333.3 ± 4,277.1, 2,395.8 ± 893.7, 416.7 ± 52.4, and 15,328.1 ± 528.6 U/g, respectively. The results show high potential of B. paralicheniformis strain T7 as a producer of keratinases and other enzymes for applications in agricultural raw materials and technologies for processing of keratin-containing animal waste.

Innovative 3D-Printed Titanium Specimens Favor a Modulation of Inflammation in Dental Pulp Stem Cells During Liposome-Triggered Mineralization.

developing customized titanium specimens, with innovative surfaces, is a suitable strategy to overcome implant failure. Additionally, a faster and efficient osteogenic commitment assists tissue regeneration. To investigate the interplay between inflammation and differentiation upon implantation, Dental Pulp Stem Cells (DPSCs) were cultured on 3D-printed titanium owning an internal open cell form, administering osteogenic factors by a liposomal formulation (LipoMix) compared to traditional delivery of differentiation medium (DM). osteogenic differentiation was evaluated by western blot, by measuring β1 integrin expression, and by and real-time RT-PCR, by measuring SP7 and Collagen I gene expression; while.angiogenesis was characterized by measuring VEGF secretion levels. Matrix mineralization was assessed by means of Alizarin Red Staining, cell adhesion and inflammation responses through western blot, enzymatic and ELISA assays evaluating Nrf2 expression, catalase activity and Prostaglandin E2 secretion, respectively. LipoMix enhances cell proliferation and adhesion, as revealed by increased integrin β1 expression. Mineralized matrix deposition, SP7 gene expression, Collagen I release and Alkaline Phosphatase activity appear increased in LipoMix condition. Additionally, the redox-sensitive transcription factor Nrf2 is overexpressed at the earliest experimental times, triggering the catalase activity. data reported confirm that internal topography and post-production treatments on titanium surfaces dynamically and positively condition the DPSC progress towards the osteogenic phenotype, moreover, the combination with LipoMix fastens the positive modulation of inflammation under osteogenic conditions. Therefore, the development of customized surfaces along with the administration of differentiating factors enclosed in a liposomal delivery system, could represent a promising and innovative tool in regenerative dentistry.

Vancomycin-Loaded Silk Fibroin/Calcium Phosphate/Methylcellulose-Based In Situ Thermosensitive Hydrogel: A Potential Function for Bone Regeneration

This study explores the efficacy of a vancomycin-loaded silk fibroin/calcium phosphate/methylcellulose-based in situ thermosensitive hydrogel (VM-SF/CaP/MC) in promoting the osteogenic differentiation of preosteoblast cells. Three VM-SF/CaP/MC formulations with varying low (L) and high (H) concentrations of silk fibroin (SF) and calcium phosphate (CaP) were prepared: VM-HSF/LCaP/MC, VM-LSF/HCaP/MC, and VM-HSF/HCaP/MC. These hydrogels significantly enhanced MC3T3-E1 cell migration and proliferation in a dose- and time-dependent manner, achieving complete cell migration within 48 h. In addition, they significantly promoted alkaline phosphatase activity, collagen content, and mineralization in MC3T3-E1 cells, indicating their potential for osteogenesis. Among the hydrogel formulations, the VM-HSF/HCaP/MC hydrogel, with high SF and CaP content, demonstrated superior potential in promoting the osteogenic differentiation of MC3T3-E1 cells. It exhibited the highest ALP activity (11.13 ± 0.91 U/mg protein) over 14 days, along with increased collagen content (54.00 ± 1.71 µg/mg protein) and mineralization (15.79 ± 1.48 mM) over 35 days. Therefore, this formulation showed a promising candidate for clinical application in localized bone regeneration, particularly in treating osteomyelitis.

The de-sulfinylation enzyme sulfiredoxin-1 attenuates hepatic stellate cell activation and liver fibrosis by modulating the PTPN12-NLRP3 axis.

Liver fibrosis is characterized by the progressive scarring of liver tissue. Oxidative stress is a critical causal factor of hepatic stellate cell (HSC) activation and the subsequent liver fibrogenesis, but the mechanism is not fully understood. Cysteine sulfinic acid (Cys-SO2H), a modification of reactive cysteine residues, is a unique form of oxidative response that alters the structure and function of proteins. Sulfiredoxin 1 (SRXN1) is responsible for ATP-dependent reduction of the Cys-SO2H to sulfenic acid (Cys-SOH). We found that the expression of SRXN1 was increased in activated HSCs and in human and mouse fibrotic livers. HSC-specific ablation of Srxn1 or pharmacological inhibition of Srxn1 exacerbated HSC activation and sensitized mice to liver fibrosis. Mechanistically, SRXN1 inhibited HSC activation by de-sulfinylating the phosphatase protein tyrosine phosphatase non-receptor type 12 (PTPN12), which enhanced its phosphatase activity and protein stability, leading to decreased tyrosine phosphorylation and reduced activation of the pro-fibrotic inflammasome protein NLRP3. The anti-fibrotic effect of SRXN1 was abolished when NLRP3 was inhibited. In contrast, overexpression of PTPN12 attenuated NLRP3 activation, and this effect was further amplified by the C164A S-sulfinylation resistant mutant of PTPN12. Our findings have uncovered an important role of SRXN1 and protein S-sulfinylation in HSC activation and liver fibrosis. The SRXN1-PTPN12-NLRP3 axis represents potential therapeutic targets for liver fibrosis.

Potential of Cranberry to Stimulate Osteogenesis: An In Vitro Study

This study investigated the potential of Cranberry extract to stimulate osteogenesis in vitro. The total phenolic and monomeric anthocyanin contents in the Cranberry were determined. Liquid chromatography coupled with mass spectrometry was used to identify the Cranberry’s constituents. To assess the Cranberry’s cytotoxicity, a thiazolyl blue tetrazolium bromide assay was employed. Concerning the osteogenesis potential of Cranberry, alkaline phosphatase (ALP) activity, bone morphogenetic protein 2 (BMP-2) expression, and extracellular matrix mineralization were evaluated. The total phenolic content was 522.72 ± 9.80 mg GAE g-1 ES and 364.95 ± 12.49 mg GAE/g detected by the Fast Blue BB and Folin–Ciocalteu method, respectively. For monomeric anthocyanin, the content was 460 ± 30 mg ECG g-1 ES. Moreover, Cranberry concentrations ranged from 62.5 to 500 mg/mL and were found to be biocompatible with osteoblasts and mesenchymal stromal cells. Regarding osteogenesis, 20 mg/mL of Cranberry promoted 2-fold more ALP activity and almost 1.5-fold more BMP-2 than compared to the positive control group. Additionally, 200 mg/mL of Cranberry stimulated a 1.7-fold increase in extracellular matrix mineralization compared to the positive control group. In conclusion, Cranberry displayed potential in stimulating early and late markers of osteogenesis. Its ability to promote osteogenesis and its biocompatibility at higher concentrations hold promise for future application into biomaterials for bone regeneration.

Real-Time Fluorescence Visualization of the Dynamic Distribution of Zn2+ Ions during Osteoblast Differentiation.

The differentiation and maturation of osteoblasts are essential for bone formation. Zn2+ plays a crucial role in cell differentiation and is involved in osteogenic differentiation. The concentration and distribution of Zn2+ in the nucleus and cytoplasm indicate the differentiation states of osteoblasts. However, there is an absence of a real-time method for monitoring the dynamic fluctuations of endogenous Zn2+ within the nucleus. Here, a novel Zn2+ fluorescent probe (NTAD-N1) with nuclear membrane permeability was designed and developed, allowing for distribution throughout the entire cell, including the nucleus. The NTAD-N1 probe successfully showed the dynamic distribution and concentration changes of Zn2+ in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during the 21-day differentiation period. The results showed that free Zn2+ increased significantly during differentiation of osteoblasts (2-21 days). Importantly, after 4 days of differentiation, osteoblasts are mainly distributed in the nucleus, which is confirmed by metallothionein expression. Subsequently, the level of free Zn2+ in the cytoplasm remained at a high level, which promoted the increase in alkaline phosphatase activity and inhibited the activity of cis-aconitase in the tricarboxylic acid cycle, resulting in the accumulation of citric acid. This series of events promotes the formation of mineralized nodules. In the process of osteoblast differentiation, the detection time of Zn2+ (≤7 days) is ahead of the late marker of alkaline phosphatase (14 days) and mineralized nodules (14-21 days). This indicates that Zn2+ can be used as a biomarker and an intervention point for early differentiation of osteoblasts.

Constitutive activation of two-component systems reveals regulatory network interactions in Streptococcus agalactiae

Bacterial two-component systems (TCSs) are signaling modules that control physiology, adaptation, and host interactions. A typical TCS consists of a histidine kinase (HK) that activates a response regulator via phosphorylation in response to environmental signals. Here, we systematically test the effect of inactivating the conserved phosphatase activity of HKs to activate TCS signaling pathways. Transcriptome analyses of 14 HK mutants in Streptococcus agalactiae, the leading cause of neonatal meningitis, validate the conserved HK phosphatase mechanism and its role in the inhibition of TCS activity in vivo. Constitutive TCS activation, independent of environmental signals, enables high-resolution mapping of the regulons for several TCSs (e.g., SaeRS, BceRS, VncRS, DltRS, HK11030, HK02290) and reveals the functional diversity of TCS signaling pathways, ranging from highly specialized to interconnected global regulatory networks. Targeted analysis shows that the SaeRS-regulated PbsP adhesin acts as a signaling molecule to activate CovRS signaling, thereby linking the major regulators of host-pathogen interactions. Furthermore, constitutive BceRS activation reveals drug-independent activity, suggesting a role in cell envelope homeostasis beyond antimicrobial resistance. This study highlights the versatility of constitutive TCS activation, via phosphatase-deficient HKs, to uncover regulatory networks and biological processes.