Search

An array of new nanomaterials have been designed to mimic the catalyst characteristics of natural enzymes (termed "nanozymes"), which connects an essential bridge between nanotechnology and biomedical science. Thus, the research on nanozymes has increased dramatically and a new multidisciplinary field has emerged in recent years named nanozymology. Magnetic iron oxide nanoparticles (e.g., Fe2O3 and Fe3O4, MIONs) have revealed to perform an enzyme-like activity with pH-dependent behavior. These nanozymes can catalytically decompose H2O2 into products (e.g., H2O and O2) under mild pH conditions mimicking enzyme-like bioactivity. More importantly, under mildly acidic conditions, they can use H2O2 as the substrate for producing highly toxic reactive oxygen species (ROS) through the generation of hydroxyl radicals ( OH), displaying peroxidase-like activity. Therefore, here we designed and developed a novel class of hybrid nanocatalysts based on the conjugation of GOx (natural enzyme) and MIONs (inorganic nanozyme) stabilized by a biocompatible polymer shell of carboxymethyl cellulose. They created supramolecular vesicle-like nanostructures that were tested for killing brain cancer cells (U-87 MG) in vitro, where they proved anticancer activity attributed to ferroptosis-induced cell death. These hybrid nanostructures performed as a cascade of integrated nanocatalysts: a) GOx worked as the starting catalyst, where the glucose in the medium generated H2O2; b) next, H2O2 was catalyzed by the downstream MION nanozymes (Fe3O4) via Fenton-like reactions releasing toxic species (ROS), which caused the cancer cell death. Remarkably, this nanozyme-induced cell death was more pronounced in brain cancer cells ascribed to the more acidic microenvironment than on healthy cells.

BACKGROUND It is hypothesized that leukocyte-poor (LP) platelet-rich plasma (PRP) is preferred over leukocyte-rich (LR) PRP for the treatment of knee osteoarthritis (OA). METHODS The MEDLINE, Embase, and Cochrane databases were reviewed for all English-language studies comparing LP-PRP or LR-PRP with relevant controls or each other. The follow-up periods were 6 months and 12 months. The primary outcome measure was the change in the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score between baseline and follow-up. The secondary outcome measures were changes in the WOMAC pain subscale, visual analog scale (VAS) for pain, and International Knee Documentation Committee (IKDC) subjective score between baseline and follow-up, and the incidence of local adverse reactions. Treatment outcomes were analyzed using the mean difference between treatments for continuous outcomes and the odds ratio for binary outcomes, with 95% credibility intervals. Treatment modalities were ranked using the surface under the cumulative ranking (SUCRA) probabilities. Risk of bias was assessed using the relevant Cochrane tools, RoB 2 (version 2 of the Cochrane risk-of-bias tools) for randomized controlled trials (RCTs) and ROBINS-I (Risk of Bias in Non-Randomized Studies - of Interventions) for prospective comparative studies (PCSs). RESULTS This network meta-analysis included 23 studies: 20 RCTs and 3 PCSs, with a total of 2,260 patients and a mean follow-up period of 9.9 months. The overall risk-of-bias assessment of the RCTs revealed that 9 studies had low risk, 7 had some concerns, and 4 had high risk. The overall risk-of-bias assessment of the PCSs revealed that 1 study had low risk and 2 had moderate risk. We found no significant (p < 0.05) difference in all outcome measures and local adverse reactions between LP-PRP and LR-PRP. SUCRA rankings revealed that, for all outcome measures, LP-PRP is preferred to LR-PRP across follow-up periods. CONCLUSIONS Leukocyte concentration of PRP does not play a significant role in patient-reported outcome measures for knee OA. LP-PRP is preferred to LR-PRP according to SUCRA rankings, but this preference may not be important in clinical practice. LEVEL OF EVIDENCE Therapeutic Level II. See Instructions for Authors for a complete description of levels of evidence.

Venous thromboembolism from a “thrombotic storm”–like syndrome is a major cause of morbidity and mortality in patients with active or “recovered” COVID-19. Patients should be risk-stratified, optimally by a pulmonary embolism (PE) response team (PERT), and considered for escalation of care if found with intermediate or high-risk PE. We present a series of patients with COVID-19-associated PE and thrombotic storm with D-dimer >10 000 ng/mL who underwent successful mechanical thrombectomy for intermediate to high-risk PE. All patients had immediate improvement in hemodynamics and large amounts of thrombi were retrieved.

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR- analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody

The HIV-protease inhibitor nelfinavir has shown broad anticancer activity in various preclinical and clinical contexts. In patients with advanced, proteasome inhibitor (PI)–refractory multiple myeloma, nelfinavir-based therapy resulted in 65% partial response or better, suggesting that this may be a highly active chemotherapeutic option in this setting. The broad anticancer mechanism of action of nelfinavir implies that it interferes with fundamental aspects of cancer cell biology. We combined proteome-wide affinity-purification of nelfinavir-interacting proteins with genome-wide CRISPR/Cas9–based screening to identify protein partners that interact with nelfinavir in an activity-dependent manner alongside candidate genetic contributors affecting nelfinavir cytotoxicity. Nelfinavir had multiple activity-specific binding partners embedded in lipid bilayers of mitochondria and the endoplasmic reticulum. Nelfinavir affected the fluidity and composition of lipid-rich membranes, disrupted mitochondrial respiration, blocked vesicular transport, and affected the function of membrane-embedded drug efflux transporter ABCB1, triggering the integrated stress response. Sensitivity to nelfinavir was dependent on ADIPOR2, which maintains membrane fluidity by promoting fatty acid desaturation and incorporation into phospholipids. Supplementation with fatty acids prevented the nelfinavir-induced effect on mitochondrial metabolism, drug-efflux transporters, and stress-response activation. Conversely, depletion of fatty acids/cholesterol pools by the FDA-approved drug ezetimibe showed a synergistic anticancer activity with nelfinavir in vitro. These results identify the modification of lipid-rich membranes by nelfinavir as a novel mechanism of action to achieve broad anticancer activity, which may be suitable for the treatment of PI–refractory multiple myeloma. Significance: Nelfinavir induces lipid bilayer stress in cellular organelles that disrupts mitochondrial respiration and transmembrane protein transport, resulting in broad anticancer activity via metabolic rewiring and activation of the unfolded protein response.

Introduction Quality in medical care must be measured in order to be improved. Trauma management is part of health care, and by definition, it must be checked constantly. The only way to measure quality and outcomes is to systematically accrue data and analyze them. Material and methods A systematic revision of the literature about quality indicators in trauma associated to an international consensus conference Results An internationally approved base core set of 82 trauma quality indicators was obtained: Indicators were divided into 6 fields: prevention, structure, process, outcome, post-traumatic management, and society integrational effects. Conclusion Present trauma quality indicator core set represents the result of an international effort aiming to provide a useful tool in quality evaluation and improvement. Further improvement may only be possible through international trauma registry development. This will allow for huge international data accrual permitting to evaluate results and compare outcomes.

Anthracyclines are effective drugs in the treatment of various cancers, but their use comes with severe side effects. The archetypal anthracycline drug, doxorubicin, displays two molecular modes of action: DNA double-strand break formation (through topoisomerase II alpha poisoning) and chromatin damage (via eviction of histones). These biological activities can be modulated and toxic side effects can be reduced by separating these two modes of action through alteration of the aminoglycoside moiety of doxorubicin. We herein report on the design, synthesis, and evaluation of a coherent set of configurational doxorubicin analogues featuring all possible stereoisomers of the 1,2-amino-alcohol characteristic for the doxorubicin 3-amino-2,3-dideoxyfucoside, each in nonsubstituted and N,N-dimethylated forms. The set of doxorubicin analogues was synthesized using appropriately protected 2,3,6-dideoxy-3-amino glycosyl donors, equipped with an alkynylbenzoate anomeric leaving group, and the doxorubicin aglycon acceptor. The majority of these glycosylations proceeded in a highly stereoselective manner to provide the desired axial alpha-linkage. We show that both stereochemistry of the 3-amine carbon and N-substitution state are critical for anthracycline cytotoxicity and generally improve cellular uptake. N,N-Dimethylepirubicin is identified as the most potent anthracycline that does not induce DNA damage while remaining cytotoxic.

In this work, bionanocomposite membranes of hydroxyapatite nanoparticles (nHA) embedded in chitosan (CHI) or carboxymethyl-chitosan (CMC) matrices were chemically crosslinked with epichlorohydrin (...

Purpose: Disturbances that affect the inside of the eyeball tend to be highly harmful since they compromise the homeostasis of this organ. Alongside this, the eyeball has several anatomical barriers that prevent the entry of substances. This way, diseases that affect the retina are among those that present greater difficulty in the treatment. In many cases, abnormal proliferation of blood vessels (neovascularization) occurs from the lower layers of the retina. This process damages its structure physiologically and anatomically, causing the rapid and irreversible loss of visual capacity. This work aims to develop nanosuspensions of quantum dots (QDs) conjugated to bevacizumab. Methods: Two types of QDs were produced by aqueous route, stabilized with chitosan conjugated to bevacizumab. The antiangiogenic activity was evaluated in the chorioallantoic membrane model, in which results indicated discrete activity at the doses tested. Samples were assessed for their biosafety in animals, after intravitreal administration, by means of electroretinography (ERG), intraocular pressure (IOP) measurement, histological, morphometric, and immunohistochemical evaluation. Results: No significant alterations were detected in ERG that suggests damage to retinal function by the samples. No significant changes in IOP were also detected. The histological sections did not show signs of acute inflammation, although there was evidence of late retinal damage. The immunohistochemical analysis did not detect any apoptotic bodies. Conclusion: Preliminary results suggest that QDs present potential applicability in ocular therapy, and it is necessary to better characterize their in vivo behavior and to optimize their dosage.

Periodontal ligament (PDL) stem cell properties are critical in the periodontal tissue regeneration for periodontitis. Previously, we have demonstrated that cigarette smoking attenuates PDL-derived stem cell (PDLSC) regenerative properties. Here, we report the findings on the regenerative properties of human PDLSCs with different donor ages and the underlying mechanisms. Human PDLSCs from 18 independent donors were divided into different age groups (≤ 20, 20–40, and > 40 years old). The proliferation of PDLSCs with donor age of ≤ 20 years old was significantly higher than that of the 20–40- and > 40-years-old groups, whereas the migration of PDLSCs with donor age of ≤ 20 and 20–40 years old was significantly higher than that of the > 40-years-old group. Moreover, the mesodermal lineage differentiation capabilities of PDLSCs were also higher in the donor age group of ≤ 20 years old than the donor age of > 40 years old. In addition, shorter telomere length and lower expression of SSEA4 were found in PDLSCs with donor age of > 40 years old, compared with those with donor age of ≤ 20-years-old group. Besides, PDLSCs with donor age of 20–40 and > 40 years old had higher IL6 and CXCL8 gene expressions. In summary, results from this study revealed the attenuated proliferation, migration, and mesodermal lineage differentiation properties in human PDLSCs with older donor ages. Donor age of PDLSCs should be considered as the selection criteria for the periodontal tissue regeneration treatment.

Fluorescent cell surface receptor agonists allow visualization of processes that are set in motion by receptor activation. This study describes the synthesis of two fluorescent, low molecular weight ligands for the follicle-stimulating hormone receptor (FSHR), based on a dihydropyridine (DHP) agonist. We show that both BODIPY- and Cy5-conjugated DHP (m-DHP-BDP and m-DHP-Cy5) are potent FSHR agonists, able to activate receptor signalling with nanomolar potencies and to effect receptor internalisation at higher concentrations. FSHR-dependent uptake of m-DHP-Cy5 is in stark contrast to the cellular uptake of m-DHP-BDP which was efficiently internalised also in the absence of FSHR. Our results comprise a first-in-class fluorescent low molecular weight ligand for in situ FSHR imaging and pertain the potential means for targeted delivery of drugs into the endolysosomal pathway of FSHR-expressing cells., Discovery of a potent, small-molecule, fluorescent agonist of the follicle-stimulating hormone receptor (FSHR) for selective staining of FSHR-expressing cells.

Despite the undeniable advances in recent decades, cancer remains one of the deadliest diseases of the current millennium, where the triple-negative breast cancer (TNBC) is very aggressive, extremely metastatic, and resistant to conventional chemotherapy. The nanotheranostic approach focusing on targeting membrane receptors often expressed at abnormal levels by cancer cells can be a strategic weapon for fighting malignant tumors. Herein, we introduced a novel "all-in-one nanosoldier" made of colloidal hybrid nanostructures, which were designed for simultaneously targeting, imaging, and killing TNBC cells. These nanohybrids comprised four distinct components: (a) superparamagnetic iron oxide nanoparticles, as bi-functional nanomaterials for inducing ferroptosis via inorganic nanozyme-mediated catalysis and magnetotherapy by hyperthermia treatment; (b) carboxymethyl cellulose biopolymer, as a water-soluble capping macromolecule; (c) folic acid, as the membranotopic vector for targeting folate receptors; (d) and doxorubicin (DOX) drug for chemotherapy. The results demonstrated that this novel strategy was highly effective for targeting and killing TNBC cells in vitro, expressing high levels of folate membrane-receptors. The results evidenced that three integrated mechanisms triggered the deaths of the cancer cells in vitro: (a) ferroptosis, by magnetite nanoparticles inducing a Fenton-like reaction; (b) magneto-hyperthermia effect by generating heat under an alternate magnetic field; and (c) chemotherapy, through the DOX intracellular release causing DNA dysfunction. This "all-in-one nanosoldier" strategy offers a vast realm of prospective alternatives for attacking cancer cells, combining multimodal therapy and the delivery of therapeutic agents to diseased sites and preserving healthy cells, which is one of the most critical clinical challenges faced in fighting drug-resistant breast cancers.

Glycosphingolipids are important building blocks of the outer leaflet of the cell membrane. They are continuously recycled, involving fragmentation inside lysosomes by glycosidases. Inherited defects in degradation cause lysosomal glycosphingolipid storage disorders. The relatively common glycosphingolipidosis Gaucher disease is highlighted here to discuss new insights in the molecular basis and pathophysiology of glycosphingolipidoses reached by fundamental research increasingly using chemical biology tools. We discuss improvements in the detection of glycosphingolipid metabolites by mass spectrometry and review new developments in laboratory diagnosis and disease monitoring as well as therapeutic interventions.

In addition to their hemostatic role, platelets play a significant role in immunity. Once activated, platelets release extracellular vesicles (EVs) formed by the budding of their cytoplasmic membranes. Because of their heterogeneity, platelet EVs (PEVs) are thought to perform diverse functions. It is unknown, however, whether the proteasome is transferred from platelets to PEVs or whether its function is retained. We hypothesized that functional protein processing and antigen presentation machinery are transferred to PEVs by activated platelets. Using molecular and functional assays, we found that the active 20S proteasome was enriched in PEVs, along with major histocompatibility complex class I (MHC-I) and lymphocyte costimulatory molecules (CD40L and OX40L). Proteasome-containing PEVs were identified in healthy donor blood, but did not increase in platelet concentrates that caused adverse transfusion reactions. They were augmented, however, after immune complex injections in mice. The complete biodistribution of murine PEVs after injection into mice revealed that they principally reached lymphoid organs, such as spleen and lymph nodes, in addition to the bone marrow, and to a lesser extent, liver and lungs. The PEV proteasome processed exogenous ovalbumin (OVA) and loaded its antigenic peptide onto MHC-I molecules, which promoted OVA-specific CD8+ T-lymphocyte proliferation. These results suggest that PEVs contribute to adaptive immunity through cross-presentation of antigens and have privileged access to immune cells through the lymphatic system, a tissue location that is inaccessible to platelets.

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.

Wall teichoic acids (WTAs) are important components of the cell wall of the opportunistic Gram‐positive bacterium Staphylococcus aureus. WTAs are composed of repeating ribitol phosphate (RboP) residues that are decorated with d‐alanine and N‐acetyl‐d‐glucosamine (GlcNAc) modifications, in a seemingly random manner. These WTA‐modifications play an important role in shaping the interactions of WTA with the host immune system. Due to the structural heterogeneity of WTAs, it is impossible to isolate pure and well‐defined WTA molecules from bacterial sources. Therefore, here synthetic chemistry to assemble a broad library of WTA‐fragments, incorporating all possible glycosylation modifications (α‐GlcNAc at the RboP C4; β‐GlcNAc at the RboP C4; β‐GlcNAc at the RboP C3) described for S. aureus WTAs, is reported. DNA‐type chemistry, employing ribitol phosphoramidite building blocks, protected with a dimethoxy trityl group, was used to efficiently generate a library of WTA‐hexamers. Automated solid phase syntheses were used to assemble a WTA‐dodecamer and glycosylated WTA‐hexamer. The synthetic fragments have been fully characterized and diagnostic signals were identified to discriminate the different glycosylation patterns. The different glycosylated WTA‐fragments were used to probe binding of monoclonal antibodies using WTA‐functionalized magnetic beads, revealing the binding specificity of these WTA‐specific antibodies and the importance of the specific location of the GlcNAc modifications on the WTA‐chains., By using tailor‐made teichoic acid building blocks, a library of glycosylated ribitol phosphate Staphylococcus aureus wall teichoic acids (WTA) has been assembled, ranging in length, and varying in N‐acetyl glucosamine substitution pattern. The library of well‐defined WTAs has been used to develop a magnetic bead binding assay to interrogate monoclonal antibody binding, revealing the preference of the various antibodies for different glycosylation patterns.

Malignant melanoma is the utmost severe form of skin cancer, causing the vast majority of deaths related to skin cancer in humans. Although melanoma is treatable at its early stages at localized fo...

The most common primary brain tumors in adults are gliomas, where glioblastoma multiforme is one of the deadliest in humans. In this study, we engineered novel nanoconjugates composed of Ag-In-S/ZnS quantum dots directly stabilized by chitosan polysaccharide ligand in aqueous medium and biofunctionalized with monoclonal antibody against vascular endothelial growth factor (VEGF) receptors. These nano-immunoconjugates demonstrated bi-functional capabilities combining fluorescent emission for bioimaging and killing activity towards brain cancer cells with very promising perspectives in nanotheranostic applications.

Multiple myeloma (MM) is a haematological malignancy being characterized by clonal plasma cell proliferation in the bone marrow. Targeting the proteasome with specific inhibitors (PIs) has been proven a promising therapeutic strategy and PIs have been approved for the treatment of MM and mantle‐cell lymphoma; yet, while outcome has improved, most patients inevitably relapse. As relapse refers to MM cells that survive therapy, we sought to identify the molecular responses induced in MM cells after non‐lethal proteasome inhibition. By using bortezomib (BTZ), epoxomicin (EPOX; a carfilzomib‐like PI) and three PIs, namely Rub999, PR671A and Rub1024 that target each of the three proteasome peptidases, we found that only BTZ and EPOX are toxic in MM cells at low concentrations. Phosphoproteomic profiling after treatment of MM cells with non‐lethal (IC10) doses of the PIs revealed inhibitor‐ and cell type‐specific readouts, being marked by the activation of tumorigenic STAT3 and STAT6. Consistently, cytokine/chemokine profiling revealed the increased secretion of immunosuppressive pro‐tumorigenic cytokines (IL6 and IL8), along with the inhibition of potent T cell chemoattractant chemokines (CXCL10). These findings indicate that MM cells that survive treatment with therapeutic PIs shape a pro‐tumorigenic immunosuppressive cellular and secretory bone marrow microenvironment that enables malignancy to relapse.

It is estimated that over 100 million people have been infected with human immunodeficiency virus (HIV-1) resulting in approximately 30 million deaths globally. Herein, we designed and developed novel nano-immunoconjugates using gold nanoparticles (AuNPs) and carboxymethylcellulose (CMC) biopolymer, which performed simultaneously as an eco-friendly in situ reducing agent and surface stabilizing ligand for the aqueous colloidal process. These AuNPs-CMC nanocolloids were biofunctionalized with the gp41 glycoprotein receptor (AuNPs-CMC-gp41) or HIV monoclonal antibodies (AuNPs-CMC_PolyArg-abHIV) for detection using the laser light scattering immunoassay (LIA). These AuNPs-CMC bioengineered nanoconjugates were extensively characterized by morphological and physicochemical methods, which demonstrated the formation of spherical nanocrystalline colloidal AuNPs with the average size from 12 to 20 nm and surface plasmon resonance peak at 520 nm. Thus, stable nanocolloids were formed with core-shell nanostructures composed of AuNPs and biomacromolecules of CMC-gp41, which were cytocompatible based on in vitro cell viability results. The AuNPs-CMC-gp41 nanoconjugates were tested against HIV monoclonal antibodies conjugates (AuNPs-CMC_PolyArg-abHIV) using the light scattering immunoassay (LIA) where they behaved as active nanoprobes for the detection at nM level of HIV-1 antigenic proteins. This strategy offers a novel nanoplatform for creating bioprobes using green nanotechnology for the detection of HIV-1 and other virus-related diseases.

Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.

Glucocerebrosidase (GBA) is a lysosomal β‐glucosidase‐degrading glucosylceramide. Its deficiency causes Gaucher disease (GD), a common lysosomal storage disorder. Carrying a genetic abnormality in GBA constitutes at present the largest genetic risk factor for Parkinson's disease (PD). Conduritol B epoxide (CBE), a mechanism‐based irreversible inhibitor of GBA, is used to generate cell and animal models for investigations on GD and PD. However, CBE may have additional glycosidase targets besides GBA. Here, we present the first in vivo target engagement study for CBE, employing a suite of activity‐based probes to visualize catalytic pocket occupancy of candidate off‐target glycosidases. Only at significantly higher CBE concentrations, nonlysosomal glucosylceramidase (GBA2) and lysosomal α‐glucosidase were identified as major off‐targets in cells and zebrafish larvae. A tight, but acceptable window for selective inhibition of GBA in the brain of mice was observed. On the other hand, cyclophellitol, a closer glucose mimic, was found to inactivate with equal affinity GBA and GBA2 and therefore is not suitable to generate genuine GD‐like models. Enzymes Glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html), nonlysosomal β‐glucocerebrosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/45.html); cytosolic β‐glucosidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/21.html); α‐glucosidases (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/20.html); β‐glucuronidase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/31.html)., In vivo target engagement of mechanism‐based glucocerebrosidase (GBA) inhibitors—conduritol B epoxide (CBE) and cyclophellitol (CP)—were examined in cultured cells, zebrafish larvae and mice by competitive activity‐based protein profiling (ABPP). This method utilizes suicide fluorescent enzyme reporter molecules to assess active site occupancy of target glycosidases by inhibitors. The in vivo targets of CBE and CP and their selectivity towards GBA were revealed.

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.

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation., Click handle-containing antigens can be used to study uptake, processing and presentation by immune cells.

Synthetic vaccines, based on antigenic peptides that comprise MHC−I and MHC‐II T‐cell epitopes expressed by tumors, show great promise for the immunotherapy of cancer. For optimal immunogenicity, the synthetic peptides (SPs) should be adjuvanted with suitable immunostimulatory additives. Previously, we have shown that improved immunogenicity in vivo is obtained with vaccine modalities in which an SP is covalently connected to an adjuvanting moiety, typically a ligand to Toll‐like receptor 2 (TLR2). SPs were covalently attached to UPam, which is a derivative of the classic TLR2 ligand Pam3CysSK4. A disadvantage of the triply palmitoylated UPam is its high lipophilicity, which precludes universal adoption of this adjuvant for covalent modification of various antigenic peptides as it renders the synthetic vaccine insoluble in several cases. Here, we report a novel conjugatable TLR2 ligand, mini‐UPam, which contains only one palmitoyl chain, rather than three, and therefore has less impact on the solubility and other physicochemical properties of a synthetic peptide. In this study, we used SPs that contain the clinically relevant neoepitopes identified in a melanoma patient who completely recovered after T‐cell therapy. Homogeneous mini‐UPam‐SP conjugates have been prepared in good yields by stepwise solid‐phase synthesis that employed a mini‐UPam building block pre‐prepared in solution and the standard set of Fmoc‐amino acids. The immunogenicity of the novel mini‐UPam‐SP conjugates was demonstrated by using the cancer patient's T‐cells., Less lipophilic ligands: A novel TLR2 ligand has been designed and synthesized to be subsequently conjugated with synthetic peptides containing clinically relevant neoepitopes. By treating HEK‐TLR2 cells, human moDCs and antigen specific human T‐cells with these new constructs, the immunogenic potential of the new TLR2 ligand and the conjugation of TLR ligands with neoepitopes was assessed.

Anthracycline anticancer drugs doxorubicin and aclarubicin have been used in the clinic for several decades to treat various cancers. Although closely related structures, their molecular mode of action diverges, which is reflected in their biological activity profile. For a better understanding of the structure-function relationship of these drugs, we synthesized ten doxorubicin/aclarubicin hybrids varying in three distinct features: aglycon, glycan, and amine substitution pattern. We continued to evaluate their capacity to induce DNA breaks, histone eviction, and relocated topoisomerase II alpha in living cells. Furthermore, we assessed their cytotoxicity in various human tumor cell lines. Our findings underscore that histone eviction alone, rather than DNA breaks, contributes strongly to the overall cytotoxicity of anthracyclines, and structures containing N,N-dimethylamine at the reducing sugar prove that are more cytotoxic than their nonmethylated counterparts. This structural information will support further development of novel anthracycline variants with improved anticancer activity.

Self-adjuvanting vaccines, wherein an antigenic peptide is covalently bound to an immunostimulating agent, have been shown to be promising tools for immunotherapy. Synthetic Toll-like receptor (TLR) ligands are ideal adjuvants for covalent linking to peptides or proteins. We here introduce a conjugation-ready TLR4 ligand, CRX-527, a potent powerful lipid A analogue, in the generation of novel conjugate-vaccine modalities. Effective chemistry has been developed for the synthesis of the conjugation-ready ligand as well as the connection of it to the peptide antigen. Different linker systems and connection modes to a model peptide were explored, and in vitro evaluation of the conjugates showed them to be powerful immune-activating agents, significantly more effective than the separate components. Mounting the CRX-527 ligand at the N-terminus of the model peptide antigen delivered a vaccine modality that proved to be potent in activation of dendritic cells, in facilitating antigen presentation, and in initiating specific CD8+ T-cell-mediated killing of antigen-loaded target cells in vivo. Synthetic TLR4 ligands thus show great promise in potentiating the conjugate vaccine platform for application in cancer vaccination.

Platinum-based antineoplastic agents play a major role in the treatment of numerous types of cancer. A new bulky, lipophilic, and chiral ligand based on 1,2-diaminodiamantane in both of its enantiomeric forms was employed for the preparation of new platinum(ii) complexes with chloride and oxalate ligands. The dichloride complexes have a higher solubility and were evaluated as anti-proliferation agents for human ovarian cancer cell lines A2780 and cisplatin-resistant A2780cis. Its R,R-enantiomer showed increased efficacy compared to cisplatin for both cancer cell lines. A chromatographic approach was used to estimate the solvent partition coefficient of the dichloride complex. The binding of diamondoid-based platinum complexes to nucleotides was tested for both enantiomers with guanosine monophosphate (GMP) and deoxyguanosine monophosphate (dGMP) and occurs at a similar or faster rate for both isomers compared to cisplatin despite greatly increased steric demand. These findings highlight the potential in 1,2-diaminodiamantane as a viable pharmacophore.

Overview: Malignant brain tumors remain one of the greatest challenges faced by health professionals and scientists among the utmost lethal forms of cancer. Nanotheranostics can play a pivotal role in developing revolutionary nanoarchitectures with multifunctional and multimodal capabilities to fight cancer. Mitochondria are vital organelles to eukaryotic cells, which have been recognized as a significant target in cancer therapy where, by damaging the mitochondria, it will cause irreparable cell death or apoptosis. Methods: We designed and produced novel hybrid nanostructures comprising a fluorescent semiconductor core (AgInS2, AIS) and cysteine-modified carboxymethylcellulose (termed thiomer, CMC_Cys) conjugated with mitochondria-targeting peptides (KLA) forming a macromolecular shell for combining bioimaging and for inducing brain cancer cell (U-87 MG) death. Results: The optical and physicochemical properties of the nanoconjugates demonstrated suitability as photoluminescent nanostructures for cell bioimaging and intracellular tracking. Additionally, the results proved a remarkable killing activity towards glioblastoma cells of cysteine-bearing CMC conjugates coupled with KLA peptides through the half-maximal effective concentration values, approximately 70-fold higher compared to the conjugate analogs without Cys residues. Moreover, these thiomer-based pro-apoptotic drug nanoconjugates displayed higher lethality against U-87 MG cancer cells than doxorubicin, a model drug in chemotherapy, although extremely toxic. Remarkably, these peptidomimetic nanohybrids demonstrated a relative "protective effect" regarding healthy cells while maintaining high killing activity towards malignant brain cells. Conclusion: These findings pave the way for developing hybrid nanoarchitectures applied as targeted multifunctional platforms for simultaneous imaging and therapy against cancer while minimizing the high systemic toxicity and side-effects of conventional drugs in anticancer chemotherapy.

The interplay between host and pathogen relies heavily on rapid protein synthesis and accurate protein targeting to ensure pathogen destruction. To gain insight into this dynamic interface, we combined Click chemistry with pulsed stable isotope labelling of amino acids in cell culture to quantify the host proteome response during macrophage infection with the intracellular bacterial pathogen Salmonella enterica Typhimurium. We monitored newly synthesized proteins across different host cell compartments and infection stages. Within this rich resource, we detected aberrant trafficking of lysosomal proteases to the extracellular space and the nucleus. We verified that active cathepsins re-traffic to the nucleus and that these are linked to cell death. Pharmacological cathepsin inhibition and nuclear targeting of a cellular cathepsin inhibitor (stefin B) suppressed S. enterica Typhimurium-induced cell death. We demonstrate that cathepsin activity is required for pyroptotic cell death via the non-canonical inflammasome, and that lipopolysaccharide transfection into the host cytoplasm is sufficient to trigger active cathepsin accumulation in the host nucleus and cathepsin-dependent cell death. Finally, cathepsin inhibition reduced gasdermin D expression, thus revealing an unexpected role for cathepsin activity in non-canonical inflammasome regulation. Overall, our study illustrates how resolution of host proteome dynamics during infection can drive the discovery of biological mechanisms at the host-microbe interface.

Proteasome inhibitors are established therapeutic agents for the treatment of hematological cancers, as are anthracyclines such as doxorubicin. We here present a new drug targeting approach that combines both drug classes into a single molecule. Doxorubicin was conjugated to an immunoproteasome-selective inhibitor via light-cleavable linkers, yielding peptide epoxyketone-doxorubicin prodrugs that remained selective and active toward immunoproteasomes. Upon cellular uptake and immunoproteasome inhibition, doxorubicin is released from the immunoproteasome inhibitor through photoirradiation. Multiple myeloma cells in this way take a double hit: immunoproteasome inhibition and doxorubicin-induced toxicity. Our strategy, which entails targeting of a cytotoxic agent, through a covalent enzyme inhibitor that is detrimental to tumor tissue in its own right, may find use in the search for improved anticancer drugs.

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 ceramides, which are crucial components of the skin barrier. We examined two key enzymes involved in epidermal ceramide biosynthesis, 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) stratum corneum 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 stratum corneum ceramides processed via ASM. We conclude that changes in the localization of active GBA and ASM correlate with i) altered stratum corneum ceramide composition in NTS patients, ii) local serine protease activity, and iii) the clinical manifestation of NTS.

Patients with Atopic Dermatitis (AD) suffer from inflamed skin and skin barrier defects. Proper formation of the outermost part of the skin, the stratum corneum (SC), is crucial for the skin barrier function. In this study we analyzed the localization and activity of lipid enzymes beta-glucocerebrosidase (GBA) and acid sphingomyelinase (ASM) in the skin of AD patients and controls. Localization of both the expression and activity of GBA and ASM in the epidermis of AD patients was altered, particularly at lesional skin sites. These changes aligned with the altered SC lipid composition. More specifically, abnormal localization of GBA and ASM related to an increase in specific ceramide subclasses [AS] and [NS]. Moreover we related the localization of the enzymes to the amounts of SC ceramide subclasses and free fatty acids (FFAs).We report a correlation between altered localization of active GBA and ASM and a disturbed SC lipid composition. Localization of antimicrobial peptide beta-defensin-3 (HBD-3) and AD biomarker Thymus and Activation Regulated Chemokine (TARC) also appeared to be diverging in AD skin compared to control. This research highlights the relation between correct localization of expressed and active lipid enzymes and a normal SC lipid composition for a proper skin barrier.

Tissue engineering using soft materials has become a very promising approach for repairing damaged cartilage and bone tissue. In this study, novel in situ forming composite hydrogels were synthesized based on chitosan and gelatin biopolymers associated with bioactive glass nanoparticles. The zeta potential at 37 °C ranged from +3.1 ± 1.4 mV to +6.9 ± 3.2 mV and showed the cationic nature of these hydrogels, which are capable of interacting with negatively charged molecules from native extracellular matrix (ECM). Moreover, the zeta potential analysis and FTIR spectra confirmed that network formation of the hydrogels occurs predominantly by molecular interactions. Rheological results revealed that the elastic modulus (G') increased from 5.4 Pa for pure chitosan hydrogels to 12.4 Pa for hydrogel composites with higher contents of gelatin and bioactive glass. Syringeability and injectability tests showed that all formulations can be smoothly expelled from a syringe and needle with the use of moderate force with a maximum value of 4.0 N. These hydrogel nanocomposites were cytocompatible based on the live cell viability responses of human osteosarcoma cell line (SAOS). These results suggest that these nanocomposites are a promising temporary injectable matrix for bone tissue engineering.

Proteasome inhibitors bortezomib, carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM), but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue–specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites, β5i, β1i, and β2i, which are different from their constitutive β5c, β1c, and β2c counterparts. Bortezomib and carfilzomib block β5c and β5i sites. We report here that pharmacologically relevant concentrations of β5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition, increasing immunoproteasome expression by interferon-γ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102, an inhibitor of β2 sites, dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus, immunoproteasome inhibitors, currently in clinical trials for the treatment of autoimmune diseases, should also be considered for the treatment of MM.

Malignant melanoma is the most lethal form of skin cancer in humans which is difficult to treat by conventional surgery and chemotherapeutics. Despite unquestionable progresses in recent years demonstrated by anticancer drug carriers to target tumor local microenvironment, it is growing at a rate of one million new cases being reported annually because overcoming the skin physiological barriers and the side effects associated with chemotherapy still remain threatening challenges. Herein, we designed and developed a novel polysaccharide-based prodrug composed of carboxymethylcellulose (CMC) polymer with anticancer drug doxorubicin (DOX) forming electrostatic nanocomplexes in aqueous solution. The results evidenced the effect of degree of substitution (DS = 0.77 and 1.22) of CMC on the physicochemical properties of the CMC-DOX complexes associated with the formation of supramolecular colloidal nanostructures. They were stabilized by electrostatic interactions between anionic carboxylate groups from CMC and cationic amino groups of DOX while the polysaccharide polymer chain encapsulated the hydrophobic drug in the aqueous medium. Moreover, these polymer-drug nanoparticulate systems were crosslinked with citric acid for producing advanced tuned drug delivery hydrogels. The results demonstrated the effect of DS of CMC and the addition of DOX on the physicochemical properties of the hydrogel network structures produced including the swelling behavior and gel fraction. Moreover, the distinct DS of CMC tailored the DOX release kinetics in vitro showing activity for killing melanoma cancer cells while less cytotoxicity towards normal cells. To this end, an innovative platform was developed based on colloidal polysaccharide-drug nanocomplexes producing anticancer hydrogels offering promising perspectives for skin cancer applications using transdermal drug delivery chemotherapy.

Aldehyde dehydrogenases (ALDHs) convert aldehydes into carboxylic acids and are often upregulated in cancer. They have been linked to therapy resistance and are therefore potential therapeutic targets. However, only a few selective and potent inhibitors are currently available for this group of enzymes. Competitive activity-based protein profiling (ABPP) would aid the development and validation of new selective inhibitors. Herein, a broad-spectrum activity-based probe that reports on several ALDHs is presented. This probe was used in a competitive ABPP protocol against three ALDH inhibitors in lung cancer cells to determine their selectivity profiles and establish their target engagement.

In this work novel three-dimensional (3D) scaffolds were developed with chitosan thiomer derivatives for potential soft tissue repair applications. Amino acid-grafted chitosan (cysteine, CHICys) and N-acylated chitosan (11-mercaptoundecanoic acid, CHIMerc) derivatives were synthesized by covalent coupling reaction and hydrogel scaffolds were produced by freeze-drying process. They were comprehensively characterized by swelling and degradation behaviors, NMR, FTIR and Raman spectroscopy, SEM and X-ray microcomputed tomography. The results demonstrated the synthesis of chitosan thiomers with distinct degree of thiol substitution (CHICys = 5% and CHIMerc = 26%), producing highly porous scaffolds (porosity >80%) with hierarchical interconnected 3D pore structures. Additionally, their physicochemical properties and architectural features were significantly tuned by the thiol-modifier, evidenced by the swelling degrees ranging from approximately 2300% (CHICys) to 1800% (CHIMerc) and chemical stability against degradation. Moreover, they exhibited cytocompatibility based on in vitro bioassays, which hold promise as suitable platform in soft tissue engineering applications.

Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell–cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies.

This study focused on the synthesis and comprehensive characterization of environmentally friendly hydrogel membranes based on carboxymethyl cellulose (CMC) for wound dressing and skin repair substitutes. These new CMC hydrogels were prepared with two degrees of functionalization (DS=0.77 and 1.22) and chemically crosslinked with citric acid (CA) for tuning their properties. Additionally, CMC-based hybrids were prepared by blending with polyethylene glycol (PEG, 10wt.%). The results demonstrated that superabsorbent hydrogels (SAP) were produced with swelling degree typically ranging from 100% to 5000%, which was significantly dependent on the concentration of CA crosslinker and the addition of PEG as network modifier. The spectroscopical characterizations indicated that the mechanism of CA crosslinking was mostly associated with the chemical reaction with CMC hydroxyl groups and that PEG played an important role on the formation of a hybrid polymeric network. These hydrogels presented very distinct morphological features depended on the degree of crosslinking and the surface nanomechanical properties (e.g., elastic moduli) were drastically affected (from approximately 0.08GPa to 2.0GPa) due to the formation of CMC-PEG hybrid nanostructures. These CMC-based hydrogels were cytocompatible considering the in vitro cell viability responses of over 95% towards human embryonic kidney cells (HEK293T) used as model cell line.

This post hoc analysis of a multi-institution cohort study assesses the association between androgen receptor perturbation and poorer outcome in patients with TP53 wild-type metastatic castration-resistant prostate cancer who are receiving abiraterone or enzalutamide.