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ABSTRACT Parascedosporium putredinis NO1 is a plant biomass-degrading ascomycete with a propensity to target the most recalcitrant components of lignocellulose. Here we applied proteomics and activity-based protein profiling (ABPP) to investigate the ability of P. putredinis NO1 to tailor its secretome for growth on different lignocellulosic substrates. Proteomic analysis of soluble and insoluble culture fractions following the growth of P. putredinis NO1 on six lignocellulosic substrates highlights the adaptability of the response of the P. putredinis NO1 secretome to different substrates. Differences in protein abundance profiles were maintained and observed across substrates after bioinformatic filtering of the data to remove intracellular protein contamination to identify the components of the secretome more accurately. These differences across substrates extended to carbohydrate-active enzymes (CAZymes) at both class and family levels. Investigation of abundant activities in the secretomes for each substrate revealed similar variation but also a high abundance of “unknown” proteins in all conditions investigated. Fluorescence-based and chemical proteomic ABPP of secreted cellulases, xylanases, and β-glucosidases applied to secretomes from multiple growth substrates for the first time confirmed highly adaptive time- and substrate-dependent glycoside hydrolase production by this fungus. P. putredinis NO1 is a promising new candidate for the identification of enzymes suited to the degradation of recalcitrant lignocellulosic feedstocks. The investigation of proteomes from the biomass bound and culture supernatant fractions provides a more complete picture of a fungal lignocellulose-degrading response. An in-depth understanding of this varied response will enhance efforts toward the development of tailored enzyme systems for use in biorefining.IMPORTANCEThe ability of the lignocellulose-degrading fungus Parascedosporium putredinis NO1 to tailor its secreted enzymes to different sources of plant biomass was revealed here. Through a combination of proteomic, bioinformatic, and fluorescent labeling techniques, remarkable variation was demonstrated in the secreted enzyme response for this ascomycete when grown on multiple lignocellulosic substrates. The maintenance of this variation over time when exploring hydrolytic polysaccharide-active enzymes through fluorescent labeling, suggests that this variation results from an actively tailored secretome response based on substrate. Understanding the tailored secretomes of wood-degrading fungi, especially from underexplored and poorly represented families, will be important for the development of effective substrate-tailored treatments for the conversion and valorization of lignocellulose.

This research investigates the relationship between the returns of selected Indonesian and US stock market indexes and their risks so as to guide new investors on how to choose their investments wisely. A quantitative descriptive method was used using performance data from three Indonesian and three US stock indexes over ten years to calculate an average return. The Sharpe Index was used to measure each index's risk. The results show that the average stock return for each index in the US is higher than the Indonesia indexes, while the level of risk in the US, on average, is lower. Investors are advised to invest in index categories with higher returns and low risk to increase the chance of gaining better returns while managing their risk to be as low as possible.

In the life order of military soldiers, there are rules of soldier discipline that are useful for enforcing discipline in order to minimize soldier violations. The problem discussed in this study is how a soldier can be subject to disciplinary punishment as mandated by law number 25 of 2014 concerning Military Discipline Law. The purpose of this study is to describe and analyze the implementation of disciplinary enforcement in order to minimize the level of disciplinary violations by soldiers and to describe and analyze the factors that encourage and hinder disciplinary enforcement. The method used in this study is a normative juridical approach to reveal problems in the field related to military disciplinary law against violations by family members. The results of the study show that the settlement of Military Discipline Law Violations is to educate and prevent the recurrence of TNI Soldier Discipline Law Violations, Policy Law number 25 of 2014 concerning military discipline law requires that the imposition of punishment be handed over to superiors who have the right to punish, Implementation of military discipline punishment for soldiers the military for violations/criminal acts committed by members of their families should not be subject to articles 8 and 9 of Law number 25 of 2014 concerning Military Discipline Law, this is because the legal subject imposed is a Military Soldier, while the family is a civilian.

Improving national defense can be done by strengthening the national defense industry. A strong defense industry will produce modern and sophisticated military equipment according to user needs. The large number of military equipment imported from abroad requires efforts to increase the domestic defense industry to meet the needs of military equipment to increase defense capabilities. The purpose of this study is to analyze strategies and programs for the development of the defense industry and to analyze several factors that play a role in the development of the national defense industry. The collection technique in this research is through Focus Group Discussion, the results of which are input for SWOT and AHP analysis. The results of the SWOT analysis show that the defense industry has a strong category of competitiveness, and has a high opportunity to be developed because it has its own market. The three main factors for the sustainability of the defense industry are: the capability of the defense industry, the quality of its human resources, and the accuracy of delivery.

Activity-based protein profiling is used to screen lignocellulose-degrading enzymes from the white rot fungus Phanerochaete chrysosporium to identify those specifically active in the presence of wood substrate.

Diabetic foot ulcers (DFUs) are considered one of the most severe chronic complications of diabetes and can lead to amputation in severe cases. In addition, bacterial infections in diabetic chronic wounds aggravate this scenario by threatening human health. Wound dressings made of polymer matrices with embedded metal nanoparticles can inhibit microorganism growth and promote wound healing, although the current clinical treatments for diabetic chronic wounds remain unsatisfactory. In this view, this research reports the synthesis and characterization of innovative hybrid hydrogels made of carboxymethyl cellulose (CMC) and poly(vinyl alcohol) (PVA) chemically crosslinked by citric acid (CA) functionalized with silver nanoparticles (AgNPs) generated in situ using an eco-friendly aqueous process. The results assessed through comprehensive in vitro and in vivo assays demonstrated that these hybrid polymer hydrogels functionalized with AgNPs possess physicochemical properties, cytocompatibility, hemocompatibility, bioadhesion, antibacterial activity, and biocompatibility suitable for wound dressings to support chronic wound healing process as well as preventing and treating bacterial infections. Hence, it can be envisioned that, with further research and development, these polymer-based hybrid nanoplatforms hold great potential as an important tool for creating a new generation of smart dressings for treating chronic diabetic wounds and opportunistic bacterial infections.

Optimal carfilzomib dosing is a matter of debate. We analyzed the inhibition profiles of proteolytic proteasome subunits β5, β2 and β1 after low-dose (20/27 mg/m2) versus high-dose (≥36 mg/m2) carfilzomib in 103 pairs of peripheral blood mononuclear cells from patients with relapsed/refractory (RR) multiple myeloma (MM). β5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas β2 and β1 were co-inhibited only by 36 and 56 mg/m2, respectively. Coinhibition of β2 (P=0.0001) and β1 activity (P=0.0005) differed significantly between high-dose and low-dose carfilzomib. Subsequently, high-dose carfilzomib showed significantly more effective proteasome inhibition than low-dose carfilzomib in vivo (P=0.0003). We investigated the clinical data of 114 patients treated with carfilzomib combinations. High-dose carfilzomib demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose carfilzomib. Therefore, we escalated the carfilzomib dose to ≥36 mg/m2 in 16 patients who progressed during low-dose carfilzomib-containing therapies. High-dose carfilzomib recaptured response (≥ partial remission) in nine (56%) patients with a median PFS of 4.4 months. Altogether, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose carfilzomib differs from that of low-dose carfilzomib by coinhibition of β2 and β1 proteasome subunits and, consequently, high-dose carfilzomib achieves a superior anti-MM effect than low-dose carfilzomib and recaptures the response in RRMM resistant to low-dose carfilzomib. The optimal carfilzomib dose should be ≥36 mg/m2 to reach a sufficient anti-tumor activity, while the balance between efficacy and tolerability should be considered in each patient.

Molecularly thin films are important in material sciences but their use in a wide range of applications requires control over their chemical functionalities, which is difficult to achieve. Here, the authors use decacyclene to form such freestanding and mechanically stable molecular films held together by supramolecular interactions without requiring covalent crosslinking of any kind

A commercial niobium oxide was used to form a gelled material, which was lyophilized producing a novel polyoxoniobate catalyst (L-AmNbO) containing peroxo groups. The activity of this new catalyst was assessed through oxidation reactions of the cyclohexene. In the presence of L-AmNbO catalyst, the oxidation of cyclohexene with H2O2 reached 90% of conversion after 12 h at 75 °C, with selectivity for 1,2-cyclohexanediol (∼ 50%). The catalytic results and the surface properties of this new material indicated that it can be a promising catalyst for niobium compounds applications in the selective oxidation of cyclohexene.

Glioblastoma (GBM) is the most common primary brain cancer in adults. Despite the remarkable advancements in recent years in the realm of cancer diagnosis and therapy, regrettably, GBM remains the most lethal form of brain cancer. In this view, the fascinating area of nanotechnology has emerged as an innovative strategy for developing novel nanomaterials for cancer nanomedicine, such as artificial enzymes, termed nanozymes, with intrinsic enzyme-like activities. Therefore, this study reports for the first time the design, synthesis, and extensive characterization of innovative colloidal nanostructures made of cobalt-doped iron oxide nanoparticles chemically stabilized by a carboxymethylcellulose capping ligand (i.e., Co-MION), creating a peroxidase-like (POD) nanozyme for biocatalytically killing GBM cancer cells. These nanoconjugates were produced using a strictly green aqueous process under mild conditions to create non-toxic bioengineered nanotherapeutics against GBM cells. The nanozyme (Co-MION) showed a magnetite inorganic crystalline core with a uniform spherical morphology (diameter, 2R = 6–7 nm) stabilized by the CMC biopolymer, producing a hydrodynamic diameter (HD) of 41–52 nm and a negatively charged surface (ZP~−50 mV). Thus, we created supramolecular water-dispersible colloidal nanostructures composed of an inorganic core (Cox-MION) and a surrounding biopolymer shell (CMC). The nanozymes confirmed the cytotoxicity evaluated by an MTT bioassay using a 2D culture in vitro of U87 brain cancer cells, which was concentration-dependent and boosted by increasing the cobalt-doping content in the nanosystems. Additionally, the results confirmed that the lethality of U87 brain cancer cells was predominantly caused by the production of toxic cell-damaging reactive oxygen species (ROS) through the in situ generation of hydroxyl radicals (·OH) by the peroxidase-like activity displayed by nanozymes. Thus, the nanozymes induced apoptosis (i.e., programmed cell death) and ferroptosis (i.e., lipid peroxidation) pathways by intracellular biocatalytic enzyme-like activity. More importantly, based on the 3D spheroids model, these nanozymes inhibited tumor growth and remarkably reduced the malignant tumor volume after the nanotherapeutic treatment (ΔV~40%). The kinetics of the anticancer activity of these novel nanotherapeutic agents decreased with the time of incubation of the GBM 3D models, indicating a similar trend commonly observed in tumor microenvironments (TMEs). Furthermore, the results demonstrated that the 2D in vitro model overestimated the relative efficiency of the anticancer agents (i.e., nanozymes and the DOX drug) compared to the 3D spheroid models. These findings are notable as they evidenced that the 3D spheroid model resembles more precisely the TME of “real” brain cancer tumors in patients than 2D cell cultures. Thus, based on our groundwork, 3D tumor spheroid models might be able to offer transitional systems between conventional 2D cell cultures and complex biological in vivo models for evaluating anticancer agents more precisely. These nanotherapeutics offer a wide avenue of opportunities to develop innovative nanomedicines for fighting against cancerous tumors and reducing the frequency of severe side effects in conventionally applied chemotherapy-based treatments.

Benchmarking is a method that can be used to measure progress and create awareness about the performance of organisations. Benchmarking the housing stock energy performance of Dutch housing associations can be used to measure and assess progress towards the decarbonisation of the housing stock. A new national climate agreement was signed in 2019, and in 2021 a new method to determine the theoretical energy performance of dwellings came into force in the Netherlands. To benchmark energy performance, a set of indicators is created that adequately represents the performance of Dutch housing associations according to the changed policies. A process involving key stakeholders is presented here to identify, assess and combine possible indicators. These were then integrated into four integrated models, which led to a final benchmark model. A model was chosen that consists of three indicators covering the energy performance of Dutch housing associations. The process and arguments that led to this final model are presented. While applicable within the Dutch context, the method and research results provide generalisable insights for the creation of energy performance benchmarks for building stocks. 'Practice relevance' This paper provides both researchers and policymakers with a practical approach to monitor and benchmark the energy performance of dwellings owned by organisations. An analysis of the Dutch policy context is presented. Examples of possible benchmark indicators are described and evaluated. A method is created to assess indicators and it is shown how to integrate indicators in different benchmark models. The final model consists of three indicators: (1) the average theoretical primary fossil energy consumption (energy label value); (2) the difference between the theoretical heating demand (quality building envelope) and the theoretical maximum heating demand of dwellings; and (3) the average actual CO2 emissions from gas consumption. Researchers and policymakers from other countries can adapt both the process and the final benchmark model to create similar benchmark models across housing stocks.

Abstract Background Preoperative antiseptic skin solutions are used prior to most surgical procedures; however, there is no definitive research comparing infection-related outcomes following use of the various solutions available to orthopedic trauma surgeons. The objective of this pilot study was to test the feasibility of a cluster randomized crossover trial that assesses the comparative effectiveness of a 10% povidone-iodine solution versus a 4% chlorhexidine gluconate solution for the management of open fractures. Methods Two orthopedic trauma centers participated in this pilot study. Each of these clinical sites was randomized to a starting solution (povidone-iodine solution or chlorhexidine gluconate) then subsequently crossed over to the other treatment after 2 months. During the 4-month enrollment phase, we assessed compliance, enrollment rates, participant follow-up, and accurate documentation of the primary clinical outcome. Feasibility outcomes included (1) the implementation of the interventions during a run-in period; (2) enrollment of participants during two 2-month enrollment phases; (3) application of the trial interventions as per the cluster randomization crossover scheme; (4) participant follow-up; and (5) accurate documentation of the primary outcome (surgical site infection). Feasibility outcomes were summarized using descriptive statistics reported as means (standard deviation) or medians (first quartile, third quartile) for continuous variables depending on their distribution and counts (percentage) for categorical variables. Corresponding 95% confidence intervals (CIs) were also reported. Results All five of the criteria for feasibility were met. During the run-in phase, all 18 of the eligible patients identified at the two clinical sites received the correct cluster-assigned treatment. A total of 135 patients were enrolled across both sites during the 4-month recruitment phase, which equates to 92% (95% CI 85.9 to 96.4%) of eligible patients being enrolled. Compliance with the assigned treatment in the pilot study was 98% (95% CI 93.5 to 99.8%). Ninety-eight percent (95% CI 93.5 to 99.8%) of participants completed the 90-day post-surgery follow-up and the primary outcome (SSI) was accurately documented for 100% (95% CI 96.6 to 100.0%) of the participants. Conclusions These results confirm the feasibility of a definitive study comparing antiseptic solutions using a cluster randomized crossover trial design. Building upon the infrastructure established during the pilot phase, a definitive study has been successfully initiated. Trial registration ClincialTrials.gov , number NCT03385304 . Registered December 28, 2017.

Individuals with Netherton syndrome (NTS) have increased serine protease activity, which strongly impacts the barrier function of the skin epidermis and leads to skin inflammation. Here, we investigated how serine protease activity in NTS correlates with changes in the stratum corneum (SC) ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, β-glucocerebrosidase (GBA) and acid-sphingomyelinase (ASM). We compared in situ expression levels and activities of GBA and ASM between NTS patients and controls and correlated the expression and activities with i) SC ceramide profiles, ii) in situ serine protease activity, and iii) clinical presentation of patients. Using activity-based probe labeling, we visualized and localized active epidermal GBA, and a newly developed in situ zymography method enabled us to visualize and localize active ASM. Reduction in active GBA in NTS patients coincided with increased ASM activity, particularly in areas with increased serine protease activity. NTS patients with scaly erythroderma exhibited more pronounced anomalies in GBA and ASM activities than patients with ichthyosis linearis circumflexa. They also displayed a stronger increase in SC ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered SC ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS.

Chemical tools to monitor drug-target engagement of endogenous enzymes are essential for preclinical target validation. Here, the authors present a chemical genetics strategy to study target engagement of endogenous kinases, achieving specific labeling and inactivation of FES kinase to provide insights into FES’ role in neutrophil phagocytosis.

Activity-based protein profiling (ABPP) has so far scarcely been applied in Archaea in general and, especially, in extremophilic organisms. We herein isolated a novel Thermococcus strain designated sp. strain 2319x1E derived from the same enrichment culture as the recently reported Thermococcus sp. strain 2319x1. Both strains are able to grow with xylan as the sole carbon and energy source, and for Thermococcus sp. strain 2319x1E (optimal growth at 85°C, pH 6–7), the induction of xylanolytic activity in the presence of xylan was demonstrated. Since the solely sequence-based identification of xylanolytic enzymes is hardly possible, we established a complementary approach by conducting comparative full proteome analysis in combination with ABPP using α- or β-glycosidase selective probes and subsequent mass spectrometry (MS)-based analysis. This complementary proteomics approach in combination with recombinant protein expression and classical enzyme characterization enabled the identification of a novel bifunctional maltose-forming α-amylase and deacetylase (EGDIFPOO_00674) belonging to the GH57 family and a promiscuous β-glycosidase (EGIDFPOO_00532) with β-xylosidase activity. We thereby further substantiated the general applicability of ABPP in archaea and expanded the ABPP repertoire for the identification of glycoside hydrolases in hyperthermophiles.

Pseudomonas aeruginosa, a pathogenic Gram-negative bacterium for which currently antibiotic resistance is posing a significant problem and for which no vaccines are available, protects itself by the formation of a biofilm. The Pel polysaccharide, a cationic polymer composed of cis-linked galactosamine (GalN), N-acetyl galactosamine (GalNAc), glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) monosaccharides, is an important constituent of the biofilm. Well-defined Pel oligosaccharides will be valuable tools to probe the biosynthesis machinery of this polysaccharide and may serve as diagnostic tools or be used as components of glycoconjugate vaccines. We here, report on the development of synthetic chemistry to access well-defined Pel-oligosaccharides. The chemistry hinges on the use of di-tert-butylsilylidene protected GalN and GlcN building blocks, which allow for completely cis-selective glycosylation reactions. We show the applicability of the chemistry by the assembly of a matrix of 3 × 6 Pel heptasaccharides, which has been generated from a single set of suitably protected Pel heptasaccharides, in which a single glucosamine residue is incorporated and positioned at different places along the Pel oligo-galactosamine chain.

μ-glucosidases [GBA1 (glucocerebrosidase) and GBA2] are ubiquitous essential enzymes. Lysosomal GBA1 and cytosol-facing GBA2 degrade glucosylceramide (GlcCer); GBA1 deficiency causes Gaucher disease, a lysosomal storage disorder characterized by lysosomal accumulation of GlcCer, which is partly converted to glucosylsphingosine (GlcSph). GBA1 and GBA2 also may transfer glucose from GlcCer to cholesterol, yielding glucosylated cholesterol (GlcChol). Here, we aimed to clarify the role of zebrafish Gba2 in glycosphingolipid metabolism during Gba1 deficiency in zebrafish (Danio rerio), which are able to survive total Gba1 deficiency. We developed Gba1 (gba1−/−), Gba2 (gba2−/−), and double (gba1−/−:gba2−/−) zebrafish knockouts using CRISPR/Cas9 and explored the effects of both genetic and pharmacological interventions on GlcCer metabolism in individual larvae. Activity-based probes and quantification of relevant glycolipid metabolites confirmed enzyme deficiency. GlcSph increased in gba1−/− larvae (0.09 pmol/fish) but did not increase more in gba1−/−:gba2−/− larvae. GlcCer was comparable in gba1−/− and WT larvae but increased in gba2−/− and gba1−/−:gba2−/− larvae. Independent of Gba1 status, GlcChol was low in all gba2−/− larvae (0.05 vs. 0.18 pmol/fish in WT). Pharmacologic inactivation of zebrafish Gba1 comparably increased GlcSph. Inhibition of GlcCer synthase (GCS) in Gba1-deficient larvae reduced GlcCer and GlcSph, and concomitant inhibition of GCS and Gba2 with iminosugars also reduced excessive GlcChol. Finally, overexpression of human GBA1 and injection of recombinant GBA1 both decreased GlcSph. We determined that zebrafish larvae offer an attractive model to study glucosidase actions in glycosphingolipid metabolism in vivo, and we identified distinguishing characteristics of zebrafish Gba2 deficiency.

Background. The increased usage of marginal grafts has triggered interest in perfused kidney preservation to minimize graft injury. We used a donation after circulatory death (DCD) porcine kidney autotransplantation model to compare 3 of the most frequently used ex vivo kidney perfusion techniques: nonoxygenated hypothermic machine perfusion (non-oxHMP), oxygenated hypothermic machine perfusion (oxHMP), and normothermic ex vivo kidney perfusion (NEVKP). Methods. Following 30 min of warm ischemia, grafts were retrieved and preserved with either 16 h of non-oxHMP, oxHMP, or NEVKP (n = 5 per group). After contralateral nephrectomy, grafts were autotransplanted and animals were followed for 8 d. Kidney function and injury markers were compared between groups. Results. NEVKP demonstrated a significant reduction in preservation injury compared with either cold preservation method. Grafts preserved by NEVKP showed superior function with lower peak serum creatinine (NEVKP versus non-oxHMP versus oxHMP: 3.66 ± 1.33 mg/dL, 8.82 ± 3.17 mg/dL, and 9.02 ± 5.5 mg/dL) and more rapid recovery. The NEVKP group demonstrated significantly increased creatinine clearance on postoperative day 3 compared with the cold perfused groups. Tubular injury scores on postoperative day 8 were similar in all groups. Conclusions. Addition of oxygen during HMP did not reduce preservation injury of DCD kidney grafts. Grafts preserved with prolonged NEVKP demonstrated superior initial graft function compared with grafts preserved with non-oxHMP or oxHMP in a model of pig DCD kidney transplantation.

The Energy Performance of Buildings Directive (EPBD) enhanced the sustainable improvement of dwellings in the European Union. Member states formulated measurable goals to improve the housing stock, and monitoring systems were developed to give insights into the improvements. In the Netherlands, non-profit housing associations agreed to improve the quality of their housing stock to an average Dutch energy label B (energy index (EI NV) = 1.40) by 2020. Research assessing this progress over time is presented using an annual monitoring system based on 2.0 million energy performance calculations of 264 Dutch non-profit housing associations between 2017 and 2020. The assessment includes: a detailed description of the development of the state of the stock over time; the effect of changes to the stock (construction and demolition) and changes within the stock (different types of retrofit measures); and the different characteristics of non-profit housing associations. Insights from this research show which specific retrofit and other measures are adopted and have substantial impact over time. This provides a useful frame of reference for building stock analysis and accelerating the improvement of the building stock. It also creates a baseline of information for the future sustainable development of this particular stock. 'Practice relevance' This research reveals which energy saving measures are most and least employed over time in Dutch non-profit housing associations sector. Large urban housing associations own a large share of the Dutch non-profit housing stock, and their dwellings have on average a lower energy rating. However, the improvement of their dwellings between 2017 and 2020 is higher than for smaller housing associations, which already have on average a higher energy rating. While the construction and demolition of dwellings contribute to 15.6% of the annual improvement, most of the improvement of the energy performance depends on retrofitting the existing stock. The trends are found to rely most on traditional measures ('e.g'. the installation of high-efficiency gas boilers and improved insulation). However, the rate of adding photovoltaic (PV) solar systems has increased rapidly in recent years, while futureproof systems ('e.g'. heat pumps and district heating) only have a steady adoption rate in this sector.

Glioblastoma remains the most lethal form of brain cancer, where hybrid nanomaterials biofunctionalized with polysaccharide peptides offer disruptive strategies relying on passive/active targeting and multimodal therapy for killing cancer cells. Thus, in this research, we report for the first time the rational design and synthesis of novel hybrid colloidal nanostructures composed of gold nanoparticles stabilized by trisodium citrate (AuNP@TSC) as the oxidase-like nanozyme, coupled with cobalt-doped superparamagnetic iron oxide nanoparticles stabilized by carboxymethylcellulose ligands (Co-MION@CMC) as the peroxidase-like nanozyme. They formed inorganic–inorganic dual-nanozyme systems functionalized by a carboxymethylcellulose biopolymer organic shell, which can trigger a biocatalytic cascade reaction in the cancer tumor microenvironment for the combination of magnetothermal–chemodynamic therapy. These nanoassemblies were produced through a green aqueous process under mild conditions and chemically biofunctionalized with integrin-targeting peptide (iRDG), creating bioengineered nanocarriers. The results demonstrated that the oxidase-like nanozyme (AuNP) was produced with a crystalline face-centered cubic nanostructure, spherical morphology (diameter = 16 ± 3 nm), zeta potential (ZP) of −50 ± 5 mV, and hydrodynamic diameter (DH) of 15 ± 1 nm. The peroxide-like nanostructure (POD, Co-MION@CMC) contained an inorganic crystalline core of magnetite and had a uniform spherical shape (2R = 7 ± 1 nm) which, summed to the contribution of the CMC shell, rendered a hydrodynamic diameter of 45 ± 4 nm and a negative surface charge (ZP = −41 ± 5 mV). Upon coupling both nanozymes, water-dispersible colloidal supramolecular vesicle-like organic–inorganic nanostructures were produced (AuNP//Co-MION@CMC, ZP = −45 ± 4 mV and DH = 28 ± 3 nm). They confirmed dual-nanozyme cascade biocatalytic activity targeted by polymer–peptide conjugates (AuNP//Co-MION@CMC_iRGD, ZP = −29 ± 3 mV and DH = 60 ± 4 nm) to kill brain cancer cells (i.e., bioenergy “starvation” by glucose deprivation and oxidative stress through reactive oxygen species generation), which was boosted by the magneto-hyperthermotherapy effect when submitted to the alternating magnetic field (i.e., induced local thermal stress by “nanoheaters”). This groundwork offers a wide avenue of opportunities to develop innovative theranostic nanoplatforms with multiple integrated functionalities for fighting cancer and reducing the harsh side effects of conventional chemotherapy.

Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific β-glycosidic bond in glycoconjugate substrates; β-glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (β/α)8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (α/α)6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic β-glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the functional variety of these enzymes and their therapeutic potential.

Abstract: Deficiency of glucocerebrosidase (GBA), a lysosomal β-glucosidase, causes Gaucher disease. The enzyme hydrolyzes β-glucosidic substrates and transglucosylates cholesterol to cholesterol-β-glucoside. Here we show that recombinant human GBA also cleaves β-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced β-glucosidase activity were similarly impaired in β-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous β-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing β-glucosidase GBA2. We later sought an endogenous β-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived β-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

Targeting proteasome with proteasome inhibitors (PIs) is an approved treatment strategy in multiple myeloma that has also been explored pre-clinically and clinically in other hematological malignancies. The approved PIs target both the constitutive and the immunoproteasome, the latter being present predominantly in cells of lymphoid origin. Therapeutic targeting of the immunoproteasome in cells with sole immunoproteasome activity may be selectively cytotoxic in malignant cells, while sparing the non-lymphoid tissues from the on-target PIs toxicity. Using activity-based probes to assess the proteasome activity profile and correlating it with the cytotoxicity assays, we identified B-cell chronic lymphocytic leukemia (B-CLL) to express predominantly immunoproteasome activity, which is associated with high sensitivity to approved proteasome inhibitors and, more importantly, to the immunoproteasome selective inhibitors LU005i and LU035i, targeting all immunoproteasome active subunits or only the immunoproteasome β5i, respectively. At the same time, LU102, a proteasome β2 inhibitor, sensitized B-CLL or immunoproteasome inhibitor-inherently resistant primary cells of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, multiple myeloma and plasma cell leukemia to low doses of LU035i. The immunoproteasome thus represents a novel therapeutic target, which warrants further testing with clinical stage immunoproteasome inhibitors in monotherapy or in combinations.

Plants contain numerous glycoconjugates that are metabolized by specific glucosyltransferases and hydrolyzed by specific glycosidases, some also catalyzing synthetic transglycosylation reactions. The documented value of plant-derived glycoconjugates to beneficially modulate metabolism is first addressed. Next, focus is given to glycosidases, the central theme of the review. The therapeutic value of plant glycosidases is discussed as well as the present production in plant platforms of therapeutic human glycosidases used in enzyme replacement therapies. The increasing knowledge on glycosidases, including structure and catalytic mechanism, is described. The novel insights have allowed the design of functionalized highly specific suicide inhibitors of glycosidases. These so-called activity-based probes allow unprecedented visualization of glycosidases cross-species. Here, special attention is paid on the use of such probes in plant science that promote the discovery of novel enzymes and the identification of potential therapeutic inhibitors and chaperones.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is involved in neuronal differentiation. This study aimed to identify the biosynthetic enzymes responsible for 2-AG production during retinoic acid (RA)-induced neurite outgrowth of Neuro-2a cells. First, we confirmed that RA stimulation of Neuro-2a cells increases 2-AG production and neurite outgrowth. The diacylglycerol lipase (DAGL) inhibitor DH376 blocked 2-AG production and reduced neuronal differentiation. Surprisingly, CRISPR/Cas9-mediated knockdown of DAGLα and DAGLβ in Neuro-2a cells did not reduce 2-AG levels, suggesting another enzyme capable of producing 2-AG in this cell line. Chemical proteomics revealed DAGLβ and α,β-hydrolase domain containing protein (ABHD6) as the only targets of DH376 in Neuro-2a cells. Biochemical, genetic and lipidomic studies demonstrated that ABHD6 possesses DAGL activity in conjunction with its previously reported monoacylglycerol lipase activity. RA treatment of Neuro-2a cells increased by three-fold the amount of active ABHD6. Our study shows that ABHD6 exhibits significant DAG lipase activity in Neuro-2a cells in addition to its known MAG lipase activity and suggest it is involved in neuronal differentiation.

Epidermal β-glucocerebrosidase (GBA1), an acid β-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis: i) an optimized zymography method that is less labor intensive and visualizes enzymatic activity with higher resolution than currently reported methods using either substrate 4-methylumbelliferyl-β-D-glucopyranoside or resorufin-β-D-glucopyranoside; and ii) a novel technique to visualize active GBA1 molecules by their specific labeling with a fluorescent activity-based probe (ABP), MDW941. The latter method pro­ved to be more robust and sensitive, provided higher resolution microscopic images, and was less prone to sample preparation effects. Moreover, in contrast to the zymography substrates that react with various β-glucosidases, MDW941 specifically labeled GBA1. We demonstrate that active GBA1 in the epidermis is primarily located in the extracellular lipid matrix at the interface of the viable epidermis and the lower layers of the SC. With ABP-labeling, we observed reduced GBA1 activity in 3D-cultured skin models when supplemented with the reversible inhibitor, isofagomine, irrespective of GBA expression. This inhibition affected the SC ceramide composition: MS analysis revealed an inhibitor-dependent increase in the glucosylceramide:ceramide ratio.

Abstract Background Retinal degeneration (RD) is a leading cause of irreversible blindness, affecting millions of people worldwide. Stem cell transplantation has been considered a promising therapy for retinal degenerative diseases. This study aimed to investigate the therapeutic potential of human periodontal ligament-derived stem cells (hPDLSCs) for intervention in the progress of this degeneration in the Royal College Surgeons (RCS) rat. Methods hPDLSCs were injected into the subretinal space of 3-week-old RCS rats. Control animals received a phosphate-buffered saline injection or were untreated. Retinal function was assessed by electroretinography recording. Eyes were collected afterward for histology and molecular studies. Results Retinal function maintenance was observed at 2 weeks and persisted for up to 8 weeks following hPDLSC transplantation. Retinal structure preservation was demonstrated in hPDLSC-transplanted eyes at 4 and 8 weeks following transplantation, as reflected in the preservation of outer nuclear layer thickness and gene expression of Rho, Crx, and Opsin. The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic photoreceptors was significantly lower in the hPDLSC-injected retinas than in those of the control groups. hPDLSCs were also found to express multiple neurotrophic factors, including vascular endothelial growth factor, bioactive basic fibroblast growth factor, brain-derived neurotrophic factor, neurotrophin-3, insulin-like growth factor 1, nerve growth factor, and glial cell line-derived neurotrophic factor. Conclusions Our findings suggest that hPDLSC transplantation is effective in delaying photoreceptor loss and provides significant preservation of retinal function in RCS rats. This study supports further exploration of hPDLSCs for treating RD.

Proteasome inhibitors (PIs) are approved backbone treatments in multiple myeloma. More recently, inhibition of proteasome activity with the PI bortezomib has been clinically evaluated as a novel treatment strategy in pediatric acute lymphoblastic leukemia (ALL). However, we lack a marker that could identify ALL patients responding to PI-based therapy. By using a set of activity-based proteasome probes in conjunction with cytotoxicity assays, we show that B-cell precursor ALL (BCP-ALL), in contrast to T-ALL, demonstrates an increased activity of immunoproteasome over constitutive proteasome, which correlates with high ex vivo sensitivity to the PIs bortezomib and ixazomib. The novel selective PI LU015i-targeting immunoproteasome β5i induces cytotoxicity in BCP-ALL containing high β5i activity, confirming immunoproteasome activity as a novel therapeutic target in BCP-ALL. At the same time, cotreatment with β2-selective proteasome inhibitors can sensitize T-ALL to currently available PIs, as well as to β5i selective PI. In addition, levels of total and spliced forms of XBP1 differ between BCP-ALL and T-ALL, and only in BCP-ALL does high-spliced XBP1 correlate with sensitivity to bortezomib. Thus, in BCP-ALL, high immunoproteasome activity may serve as a predictive marker for PI-based treatment options, potentially combined with XBP1 analyses.

Herein, we validated novel functionalized hybrid semiconductor bioconjugates made of fluorescent quantum dots (QD) with the surface capped by chitosan (polysaccharide) and chemically modified with O-phospho-L-serine (OPS) that are biocompatible with different human cell sources. The conjugation with a directing signaling molecule (OPS) allows preferential accumulation in human bone mesenchymal stromal cells (HBMSC). The chitosan (Chi) shell with the fluorescent CdS core was characterized by spectroscopical (UV spectrophotometry and photoluminescence), by morphological techniques (Transmission Electron Microscopy (TEM)) and showed small size (ø 2.3 nm) and a stable photoluminescence emission band. The in vitro biocompatibility results were not dependent on the polysaccharide chain length (Chi with higher and lower molecular weight) but were remarkably affected by the surface modification (Chi or Chi-OPS). In addition, the efficiency of nanoparticles uptake by the cells was dependent on cells nature (human primary cells or cell lines) and tissue source (bone or skin) in the presence or absence of the OPS modification. The complex cellular uptake pathways involved in the cell labeling with the nanoparticles do not interfere on the normal cellular biology (adhesion and proliferation), osteogenic differentiation, and gene expression. The bone cells particles uptake evaluation showed a possible pathway by Caveolin-1 that regulates cell transduction in the membrane’s Caveolae. Caveolae mediates non-specific endocytosis, and it is upregulated in HBMSC. The OPS-modified nanoparticles promoted an intense intracellular trafficking by the HBMSCs that showed late-osteoblast phenotype with an increase of extracellular matrix (ECM) mineralization (Alizarin red and Von Kossa staining for calcium phosphate crystals). In this work, the OPS modified bioconjugated QD proved to be a reliable and stable fluorescent bioprobe for cell imaging and targeting research that could also help in clarifying some cellular mechanisms of particles intracellular traffic through the cytoplasmic membrane and osteogenic differentiation induction. The in vitro HBMSC’s biocompatibility responses indicated that the OPS-modified chitosan QDs have a prospective future in laboratory and pre-clinical applications such as bioimaging analysis and for ex-vivo cellular evaluation of biomedical implants.

Abstract Quantum dots (QDs) are colloidal semiconductor nanocrystals with unique properties that can be engineered by controlling the nanoparticle size and chemical composition by doping and alloying strategies. However, due to their potential toxicity, augmenting their biocompatibility is yet a challenge for expanding to several biomedical and environmentally friendly applications. Thus, the main goal of this study was to develop composition-tunable and biocompatible Zn x Cd1 − x S QDs using carboxymethylcellulose polysaccharide as direct capping ligand via green colloidal aqueous route at neutral pH and at room temperature for potential biomedical and environmental applications. The ternary alloyed QDs were extensively characterized using UV–vis spectroscopy, photoluminescence spectroscopy (PL), transmission electron microscopy (TEM), X-ray diffraction (XRD), electron energy loss spectroscopy (EELS), and X-ray photoelectrons spectroscopy (XPS). The results indicated that Zn x Cd(1 − x)S QDs were surface stabilized by carboxymethylcellulose biopolymer with spherical morphology for all composition of alloys and narrow sizes distributions ranging from 4 to 5 nm. The XRD results indicated that monophasic ternary alloyed Zn x Cd1 − x S nanocrystals were produced with homogenous composition of the core as evidenced by EELS and XPS analyses. In addition, the absorption and emission optical properties of Zn x Cd1 − x S QDs were red shifted with increasing the amount of Cd2+ in the alloyed nanocrystals, which have also increased the quantum yield compared to pure CdS and ZnS nanoparticles. These properties of alloyed nanomaterials were interpreted based on empirical model of Vegard’s law and chemical bond model (CBM). As a proof of concept, these alloyed-QD conjugates were tested for biomedical and environmental applications. The results demonstrated that they were non-toxic and effective fluorophores for bioimaging live HEK293T cells (human embryonic kidney cells) using confocal laser scanning fluorescence microscopy. Moreover, these conjugates presented photocatalytic activity for photodegradation of methylene blue used as model organic industrial pollutant in water. Hence, composition-tunable optical properties of ternary Zn x Cd1 − x S (x = 0–1) fluorescent alloyed QDs was achieved using a facile eco-friendly aqueous processing route, which can offer promising alternatives for developing innovative nanomaterials for applications in nanomedicine and environmental science and technology.

The processes of cellular differentiation and phenotypic maintenance can be influenced by stimuli from a variety of different factors. One commonly overlooked factor is the mechanical properties of the growth substrate in which stem cells are maintained or differentiated down various lineages. Here we explored the effect that growth on an elastic silicone substrate had on the myogenic expression and cytoskeletal morphology of periodontal ligament derived stem cells. Cells were grown on either collagen I coated tissue culture polystyrene plates or collagen I coated elastic silicone membranes for a period of 4 days without further induction from soluble factors in the culture media. Following the 4-day growth, gene expression and immunohistochemical analysis for key cardiomyogenic markers was performed along with a morphological assessment of cytoskeletal organization. Results show that cells grown on the elastic substrate significantly upregulate key markers associated with contractile activity in muscle tissues. Namely, the myosin light chain polypeptides 2 and 7, as well as the myosin heavy chain polypeptide 7 genes underwent a statistically significant upregulation in the cells grown on elastic silicone membranes. Similarly, the cells on the softer elastic substrate stained positive for both sarcomeric actin and cardiac troponin t proteins following just 4 days of growth on the softer material. Cytoskeletal analysis showed that substrate stiffness had a marked effect on the organization and distribution of filamentous actin fibers within the cell body. Growth on silicone membranes produced flatter and shorter cellular morphologies with filamentous actin fibers projecting anisotropically throughout the cell body. These results demonstrate how crucial the mechanical properties of the growth substrate of cells can be on the ultimate cellular phenotype. These observations highlight the need to further optimize differentiation protocols to enhance the application of cellular products in the fields of regenerative medicine and stem cell therapies.

Gaucher disease (GD), the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of β-glucocerebrosidase (GCase). Recently it was reported that progranulin (PGRN) insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD.

Dendritic cells (DCs) are important initiators of adaptive immunity, and they possess a multitude of Pattern Recognition Receptors (PRR) to generate an adequate T cell mediated immunity against invading pathogens. PRR ligands are frequently conjugated to tumor-associated antigens in a vaccination strategy to enhance the immune response toward such antigens. One of these PPRs, DC-SIGN, a member of the C-type lectin receptor (CLR) family, has been extensively targeted with Lewis structures and mannose glycans, often presented in multivalent fashion. We synthesized a library of well-defined mannosides (mono-, di-, and tri-mannosides), based on known “high mannose” structures, that we presented in a systematically increasing number of copies (n = 1, 2, 3, or 6), allowing us to simultaneously study the effect of mannoside configuration and multivalency on DC-SIGN binding via Surface Plasmon Resonance (SPR) and flow cytometry. Hexavalent presentation of the clusters showed the highest binding affinity, with the hexa-α1,2-di-mannoside being the most potent ligand. The four highest binding hexavalent mannoside structures were conjugated to a model melanoma gp100-peptide antigen and further equipped with a Toll-like receptor 7 (TLR7)-agonist as adjuvant for DC maturation, creating a trifunctional vaccine conjugate. Interestingly, DC-SIGN affinity of the mannoside clusters did not directly correlate with antigen presentation enhancing properties and the α1,2-di-mannoside cluster with the highest binding affinity in our library even hampered T cell activation. Overall, this systematic study has demonstrated that multivalent glycan presentation can improve DC-SIGN binding but enhanced binding cannot be directly translated into enhanced antigen presentation and the sole assessment of binding affinity is thus insufficient to determine further functional biological activity. Furthermore, we show that well-defined antigen conjugates combining two different PRR ligands can be generated in a modular fashion to increase the effectiveness of vaccine constructs.

Background: Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of β-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. Methods: Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. Findings: Serum PGRN levels were significantly lower in GD patients (96.65 ± 53.45 ng/ml) than those in healthy controls of the general population (164.99 ± 43.16 ng/ml, p

Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

Stem cells from human exfoliated deciduous teeth (SHED) are a unique source of stem cells because they are relatively easy to obtain and harvest from discarded teeth. While they could differentiate into neurons, adipocytes, osteoblasts, and myocytes, little is known whether they can differentiate into cardiomyocytes. The potential use of a biomaterial scaffold to deliver cardiomyocytes to the site of damaged heart tissue for cellular therapy is an attractive concept. Gelatin-B and poly-(lactic-co-glycolic acid) (PLGA) were selected for the present study. The gelatin-B and PLGA scaffolds were constructed using an electro-spinning technique. SHED cells proliferate in both PLGA scaffolds and gelatin-B scaffolds and maintain adequate viability as determined with calcein-AM staining and DNA quantification. SHED cells were treated with a predetermined optimized cardio-treatment protocol. qPCR analysis of the cardiomyotic genes, MEF2.C, Cx-43, TNNT2.C, Nkx2.5, and GATA-4, showed that SHED cells differentiated on PLGA significantly up-regulated these cardiogenic markers compared to SHED cells cultured in control media . In summary, we demonstrate the growth and cardiomyogenesis of SHED cells on electrospun gelatin and PLGA scaffolds. Further development of our research concepts for cardiovascular regeneration using in vivo research and clinical trials, could allow the development of therapies involving the delivery of cardiomyocytes differentiated from SHED using electrospun scaffolds to the site of damaged heart tissue.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading β-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-β-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and 13C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through β-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.

The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies.

Doping calcium phosphates with ionic species can play an important role in biological responses promoting alkaline phosphatase activity, and, therefore inducing the generation of new bone. Thus, in this study, the synthesis of niobium-doped hydroxyapatite (Nb-HA) nanosize particles obtained by the precipitation process in aqueous media followed by thermal treatment is presented. The bioceramics were extensively characterized by X-ray diffraction, wavelength dispersive X-ray fluorescence spectrometry, Fourier transform infrared spectroscopy, scanning electron microscopy/energy dispersive X-ray spectroscopy analysis, transmission electron microscopy, atomic force microscopy and thermal analysis regarding their chemical composition, structure and morphology. The results showed that the precipitate dried at 110 °C was composed of amorphous calcium phosphate and HA, with polidisperse particles ranging from micro to nano dimensions. After the thermal treatment at 900 °C, the bioceramic system evolved predominantly to HA crystalline phase, with evident features of particle sintering and reduction of surface area. Moreover, the addition of 10 mol% of niobium salt precursor during the synthesis indicated the complete incorporation of the Nb(V) species in the HA crystals with detectable changes in the original lattice parameters. Furthermore, the incorporation of Nb ions caused a significant refinement on the average particle size of HA. Finally, the preliminary cytocompatibility response of the biomaterials was accessed by human osteoblast cell culture using MTT and resazurin assays, which demonstrated no cytotoxicity of the Nb-alloyed hydroxyapatite. Thus, these findings seem promising for developing innovative Nb-doped calcium phosphates as artificial biomaterials for potential use in bone replacements and repair.