Graphene Oxide Nanosheets Interact and Interfere with SARS-CoV-2 Surface Proteins and Cell Receptors to Inhibit Infectivity.

Nanotechnology can offer a number of options against coronavirus disease 2019 (COVID-19) acting both extracellularly and intracellularly to the host cells. Here, the aim is to explore graphene oxide (GO), the most studied 2D nanomaterial in biomedical applications, as a nanoscale platform for interaction with SARS-CoV-2. Molecular docking analyses of GO sheets on interaction with three different structures: SARS-CoV-2 viral spike (open state - 6VYB or closed state - 6VXX), ACE2 (1R42), and the ACE2-bound spike complex (6M0J) are performed. GO shows high affinity for the surface of all three structures (6M0J, 6VYB and 6VXX). When binding affinities and involved bonding types are compared, GO interacts more strongly with the spike or ACE2, compared to 6M0J. Infection experiments using infectious viral particles from four different clades as classified by Global Initiative on Sharing all Influenza Data (GISAID), are performed for validation purposes. Thin, biological-grade GO nanoscale (few hundred nanometers in lateral dimension) sheets are able to significantly reduce copies for three different viral clades. This data has demonstrated that GO sheets have the capacity to interact with SARS-CoV-2 surface components and disrupt infectivity even in the presence of any mutations on the viral spike. GO nanosheets are proposed to be further explored as a nanoscale platform for development of antiviral strategies against COVID-19.

Role of oxidative stress mediated by glutathione-s-transferase in thiopurines' toxic effects.

Azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are antimetabolite drugs, widely used as immunosuppressants and anticancer agents. Despite their proven efficacy, a high incidence of toxic effects in patients during standard-dose therapy is recorded. The aim of this study is to explain, from a mechanistic point of view, the clinical evidence showing a significant role of glutathione-S-transferase (GST)-M1 genotype on AZA toxicity in inflammatory bowel disease patients. To this aim, the human nontumor IHH and HCEC cell lines were chosen as predictive models of the hepatic and intestinal tissues, respectively. AZA, but not 6-MP and 6-TG, induced a concentration-dependent superoxide anion production that seemed dependent on GSH depletion. N-Acetylcysteine reduced the AZA antiproliferative effect in both cell lines, and GST-M1 overexpression increased both superoxide anion production and cytotoxicity, especially in transfected HCEC cells. In this study, an in vitro model to study thiopurines' metabolism has been set up and helped us to demonstrate, for the first time, a clear role of GST-M1 in modulating AZA cytotoxicity, with a close dependency on superoxide anion production. These results provide the molecular basis to shed light on the clinical evidence suggesting a role of GST-M1 genotype in influencing the toxic effects of AZA treatment.

V4 C3 MXene Immune Profiling and Modulation of T Cell-Dendritic Cell Function and Interaction.

Although vanadium-based metallodrugs are recently explored for their effective anti-inflammatory activity, they frequently cause undesired side effects. Among 2D nanomaterials, transition metal carbides (MXenes) have received substantial attention for their promise as biomedical platforms. It is hypothesized that vanadium immune properties can be extended to MXene compounds. Therefore, vanadium carbide MXene (V4 C3 ) is synthetized, evaluating its biocompatibility and intrinsic immunomodulatory effects. By combining multiple experimental approaches in vitro and ex vivo on human primary immune cells, MXene effects on hemolysis, apoptosis, necrosis, activation, and cytokine production are investigated. Furthermore, V4 C3 ability is demonstrated to inhibit T cell-dendritic cell interactions, evaluating the modulation of CD40-CD40 ligand interaction, two key costimulatory molecules for immune activation. The material biocompatibility at the single-cell level on 17 human immune cell subpopulations by single-cell mass cytometry is confirmed. Finally, the molecular mechanism underlying V4 C3 immune modulation is explored, demonstrating a MXene-mediated downregulation of antigen presentation-associated genes in primary human immune cells. The findings set the basis for further V4 C3 investigation and application as a negative modulator of the immune response in inflammatory and autoimmune diseases.

Graphene oxide activates B cells with upregulation of granzyme B expression: evidence at the single-cell level for its immune-modulatory properties and anticancer activity.

We recently found by single-cell mass cytometry that ex vivo human B cells internalize graphene oxide (GO). The functional impact of such uptake on B cells remains unexplored. Here, we disclosed the effects of GO and amino-functionalized GO (GONH2) interacting with human B cells in vitro and ex vivo at the protein and gene expression levels. Moreover, our study considered three different subpopulations of B cells and their functionality in terms of: (i) cytokine production, (ii) activation markers, (iii) killing activity towards cancer cells. Single-cell mass cytometry screening revealed the higher impact of GO on cell viability towards naïve, memory, and plasma B cell subsets. Different cytokines such as granzyme B (GrB) and activation markers, like CD69, CD80, CD138, and CD38, were differently regulated by GONH2 compared to GO, supporting possible diverse B cell activation paths. Moreover, co-culture experiments also suggest the functional ability of both GOs to activate B cells and therefore enhance the toxicity towards HeLa cancer cell line. Complete transcriptomic analysis on a B cell line highlighted the distinctive GO and GONH2 elicited responses, inducing pathways such as B cell receptor and CD40 signaling pathways, key players for GrB secretion. B cells were regularly left behind the scenes in graphene biological studies; our results may open new horizons in the development of GO-based immune-modulatory strategies having B cell as main actors.

Immune Profiling and Multiplexed Label-Free Detection of 2D MXenes by Mass Cytometry and High-Dimensional Imaging.

There is a critical unmet need to detect and image 2D materials within single cells and tissues while surveying a high degree of information from single cells. Here, a versatile multiplexed label-free single-cell detection strategy is proposed based on single-cell mass cytometry by time-of-flight (CyTOF) and ion-beam imaging by time-of-flight (MIBI-TOF). This strategy, "Label-free sINgle-cell tracKing of 2D matErials by mass cytometry and MIBI-TOF Design" (LINKED), enables nanomaterial detection and simultaneous measurement of multiple cell and tissue features. As a proof of concept, a set of 2D materials, transition metal carbides, nitrides, and carbonitrides (MXenes), is selected to ensure mass detection within the cytometry range while avoiding overlap with more than 70 currently available tags, each able to survey multiple biological parameters. First, their detection and quantification in 15 primary human immune cell subpopulations are demonstrated. Together with the detection, mass cytometry is used to capture several biological aspects of MXenes, such as their biocompatibility and cytokine production after their uptake. Through enzymatic labeling, MXenes' mediation of cell-cell interactions is simultaneously evaluated. In vivo biodistribution experiments using a mixture of MXenes in mice confirm the versatility of the detection strategy and reveal MXene accumulation in the liver, blood, spleen, lungs, and relative immune cell subtypes. Finally, MIBI-TOF is applied to detect MXenes in different organs revealing their spatial distribution. The label-free detection of 2D materials by mass cytometry at the single-cell level, on multiple cell subpopulations and in multiple organs simultaneously, will enable exciting new opportunities in biomedicine.

Lateral dimension and amino-functionalization on the balance to assess the single-cell toxicity of graphene on fifteen immune cell types.

Given the wide variety of potential applications of graphene oxide (GO), its consequent release into the environment poses serious concerns on its safety. The future production and exploitation of graphene in the years to come should be guided by its specific chemical-physical characteristics. The unparalleled potential of single-cell mass cytometry (CyTOF) to dissect by high-dimensionality the specific immunological effects of nanomaterials, represents a turning point in nanotoxicology. It helps us to identify the safe graphene in terms of physical-chemical properties and therefore to direct its future safe production. Here we present a high-dimensional study to evaluate two historically indicated as key parameters for the safe exploitation: functionalization and dimension. The role of lateral dimension and the amino-functionalization of GO on their immune impact were here evaluated as synergistic players. To this end, we dissected the effects of GO, characterized by a large or small lateral size (GO 1.32 µm and GO 0.13 µm, respectively), and its amino-functionalized counterpart (GONH2 1.32 µm and GONH2 0.13 µm, respectively) on fifteen cell types of human primary peripheral blood mononuclear cells (PBMCs). We describe how the smallest later size not only evokes pronounced toxicity on the pool of PBMCs compared to larger GOs but also towards the distinct immune cell subpopulations, in particular on non-classical monocytes, plasmacytoid dendritic cells (pDCs), natural killer cells (NKs) and B cells. The amino-functionalization was able to improve the biocompatibility of classical and non-classical monocytes, pDCs, NKs, and B cells. Detailed single-cell analysis further revealed a complex interaction of all GOs with the immune cells, and in particular monocyte subpopulations, with different potency depending on their physicochemical properties. Overall, by high-dimensional profiling, our study demonstrates that the lateral dimension is an important factor modulating immune cells and specifically monocyte activation, but a proper surface functionalization is the dominant characteristic in its immune effects. In particular, the amino-functionalization can critically modify graphene impact dampening the immune cell activation. Our study can serve as a guide for the future broad production and use of graphene in our everyday life.

2D MXenes with antiviral and immunomodulatory properties: A pilot study against SARS-CoV-2.

Two-dimensional transition metal carbides/carbonitrides known as MXenes are rapidly growing as multimodal nanoplatforms in biomedicine. Here, taking SARS-CoV-2 as a model, we explored the antiviral properties and immune-profile of a large panel of four highly stable and well-characterized MXenes - Ti3C2Tx, Ta4C3T x , Mo2Ti2C3T x and Nb4C3T x . To start with antiviral assessment, we first selected and deeply analyzed four different SARS-CoV-2 genotypes, common in most countries and carrying the wild type or mutated spike protein. When inhibition of the viral infection was tested in vitro with four viral clades, Ti3C2T x in particular, was able to significantly reduce infection only in SARS-CoV-2/clade GR infected Vero E6 cells. This difference in the antiviral activity, among the four viral particles tested, highlights the importance of considering the viral genotypes and mutations while testing antiviral activity of potential drugs and nanomaterials. Among the other MXenes tested, Mo2Ti2C3T x also showed antiviral properties. Proteomic, functional annotation analysis and comparison to the already published SARS-CoV-2 protein interaction map revealed that MXene-treatment exerts specific inhibitory mechanisms. Envisaging future antiviral MXene-based drug nano-formulations and considering the central importance of the immune response to viral infections, the immune impact of MXenes was evaluated on human primary immune cells by flow cytometry and single-cell mass cytometry on 17 distinct immune subpopulations. Moreover, 40 secreted cytokines were analyzed by Luminex technology. MXene immune profiling revealed i) the excellent bio and immune compatibility of the material, as well as the ability of MXene ii) to inhibit monocytes and iii) to reduce the release of pro-inflammatory cytokines, suggesting an anti-inflammatory effect elicited by MXene. We here report a selection of MXenes and viral SARS-CoV-2 genotypes/mutations, a series of the computational, structural and molecular data depicting deeply the SARS-CoV-2 mechanism of inhibition, as well as high dimensional single-cell immune-MXene profiling. Taken together, our results provide a compendium of knowledge for new developments of MXene-based multi-functioning nanosystems as antivirals and immune-modulators.

Bioinspired Scaffold Action Under the Extreme Physiological Conditions of Simulated Space Flights: Osteogenesis Enhancing Under Microgravity.

Prolonged exposure to microgravity (MG) during long-duration space flights is known to induce severe dysregulation of osteoblast functions connected to a significant bone loss, similar to the condition induced by osteoporosis. Hence, we here present MG as a promising model to challenge the effectiveness of new scaffolds designed for bone regeneration in counteracting bone loss. To this end, we carried out an integrative study aimed to evaluate, in the extreme condition of Random Positioning Machine-simulated MG, the osteoinductive potential of nanocrystalline magnesium-doped hydroxyapatite/type I collagen composite scaffold (MHA/Coll), that we previously demonstrated to be an excellent tool for bone tissue engineering. Initially, to test the osteoinductive properties of our bioinspired-scaffold, MHA/Coll structure was fully characterized under MG condition and compared to its static counterpart. Human bone marrow-derived mesenchymal stem cells were used to investigate the scaffold biocompatibility and ability to promote osteogenic differentiation after long-duration exposure to MG (up to 21 days). The results demonstrate that the nanostructure of MHA/Coll scaffold can alleviate MG-induced osteoblast dysfunction, promoting cell differentiation along the osteogenic lineage, with a consequent reduction in the expression of the surface markers CD29, CD44, and CD90. Moreover, these findings were corroborated by the ability of MHA/Coll to induce the expression of genes linked to osteogenesis, including alkaline phosphatase and osteocalcin. This study confirmed MHA/Coll capabilities in promoting osteogenesis even in extreme long-term condition of MG, suggesting MG as an effective challenging model to apply in future studies to validate the ability of advanced scaffolds to counteract bone loss, facilitating their application in translational Regenerative Medicine and Tissue Engineering.

Graphene and other 2D materials: a multidisciplinary analysis to uncover the hidden potential as cancer theranostics.

Cancer represents one of the main causes of death in the world; hence the development of more specific approaches for its diagnosis and treatment is urgently needed in clinical practice. Here we aim at providing a comprehensive review on the use of 2-dimensional materials (2DMs) in cancer theranostics. In particular, we focus on graphene-related materials (GRMs), graphene hybrids, and graphdiyne (GDY), as well as other emerging 2DMs, such as MXene, tungsten disulfide (WS2), molybdenum disulfide (MoS2), hexagonal boron nitride (h-BN), black phosphorus (BP), silicene, antimonene (AM), germanene, biotite (black mica), metal organic frameworks (MOFs), and others. The results reported in the scientific literature in the last ten years (>200 papers) are dissected here with respect to the wide variety of combinations of imaging methodologies and therapeutic approaches, including drug/gene delivery, photothermal/photodynamic therapy, sonodynamic therapy, and immunotherapy. We provide a unique multidisciplinary approach in discussing the literature, which also includes a detailed section on the characterization methods used to analyze the material properties, highlighting the merits and limitations of the different approaches. The aim of this review is to show the strong potential of 2DMs for use as cancer theranostics, as well as to highlight issues that prevent the clinical translation of these materials. Overall, we hope to shed light on the hidden potential of the vast panorama of new and emerging 2DMs as clinical cancer theranostics.

Skin irritation potential of graphene-based materials using a non-animal test.

Besides inhalation, skin contact may be considered one of the most relevant exposure routes to graphene-based materials (GBMs). However, very few data on the cutaneous toxicity of these materials are available, so far. This study is focused on skin irritation potential of a panel of GBMs: few-layer graphene (FLG), exfoliated by ball milling of graphite, FLG exfoliated by ultrasonication using sodium dodecyl sulfate (FLG-SDS) or sodium dodecylbenzenesulfonate (FLG-SDBS), CVD-graphene, obtained by chemical vapor deposition, graphene oxide (GO) and reduced GO (rGO). Skin irritation was assessed using the SkinEthic™ Reconstructed human Epidermis (RhE), following the Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 439. Even though not validated for nanomaterials, the OCED TG 439 turned out to be applicable also for GBM testing, since no interference with the methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction, used as a final readout, was found. Furthermore, direct epidermal exposure to powdered GBMs mimics the actual human exposure, avoiding interference by the cell culture medium (protein corona formation). Only GBMs prepared with irritant surfactants (FLG-SDS and FLG-SDBS), but not the others, reduced RhE viability at levels lower than those predicting skin irritation (≤50%), suggesting irritant properties. This result was further confirmed by measuring cytokine (IL-1α, IL-6 and IL-8) release by GBM-treated RhE and by histological analysis as additional readouts to implement the guideline. On the whole, these results demonstrate that GBMs prepared with non-irritant exfoliation agents do not induce skin irritation after a single acute exposure.

Toward Nanotechnology-Enabled Approaches against the COVID-19 Pandemic.

The COVID-19 outbreak has fueled a global demand for effective diagnosis and treatment as well as mitigation of the spread of infection, all through large-scale approaches such as specific alternative antiviral methods and classical disinfection protocols. Based on an abundance of engineered materials identifiable by their useful physicochemical properties through versatile chemical functionalization, nanotechnology offers a number of approaches to cope with this emergency. Here, through a multidisciplinary Perspective encompassing diverse fields such as virology, biology, medicine, engineering, chemistry, materials science, and computational science, we outline how nanotechnology-based strategies can support the fight against COVID-19, as well as infectious diseases in general, including future pandemics. Considering what we know so far about the life cycle of the virus, we envision key steps where nanotechnology could counter the disease. First, nanoparticles (NPs) can offer alternative methods to classical disinfection protocols used in healthcare settings, thanks to their intrinsic antipathogenic properties and/or their ability to inactivate viruses, bacteria, fungi, or yeasts either photothermally or via photocatalysis-induced reactive oxygen species (ROS) generation. Nanotechnology tools to inactivate SARS-CoV-2 in patients could also be explored. In this case, nanomaterials could be used to deliver drugs to the pulmonary system to inhibit interaction between angiotensin-converting enzyme 2 (ACE2) receptors and viral S protein. Moreover, the concept of "nanoimmunity by design" can help us to design materials for immune modulation, either stimulating or suppressing the immune response, which would find applications in the context of vaccine development for SARS-CoV-2 or in counteracting the cytokine storm, respectively. In addition to disease prevention and therapeutic potential, nanotechnology has important roles in diagnostics, with potential to support the development of simple, fast, and cost-effective nanotechnology-based assays to monitor the presence of SARS-CoV-2 and related biomarkers. In summary, nanotechnology is critical in counteracting COVID-19 and will be vital when preparing for future pandemics.

Nano-bio interactions: a neutrophil-centric view.

Neutrophils are key components of the innate arm of the immune system and represent the frontline of host defense against intruding pathogens. However, neutrophils can also cause damage to the host. Nanomaterials are being developed for a multitude of different purposes and these minute materials may find their way into the body through deliberate or inadvertent exposure; understanding nanomaterial interactions with the immune system is therefore of critical importance. However, whereas numerous studies have focused on macrophages, less attention is devoted to nanomaterial interactions with neutrophils, the most abundant leukocytes in the blood. We discuss the impact of engineered nanomaterials on neutrophils and how neutrophils, in turn, may digest certain carbon-based materials such as carbon nanotubes and graphene oxide. We also discuss the role of the corona of proteins adsorbed onto the surface of nanomaterials and whether nanomaterials are sensed as pathogens by cells of the immune system.

Photodynamic Therapy Based on Graphene and MXene in Cancer Theranostics.

Cancer is one of the leading causes of death in the world. Therefore, the development of new advanced and targeted strategies in cancer research for early diagnosis and treatment has become essential to improve diagnosis outcomes and reduce therapy side effects. Graphene and more recently, MXene, are the main representatives of the family of two-dimensional (2D) materials and are widely studied as multimodal nanoplatforms for cancer diagnostics and treatment, in particular leveraging their potentialities as photodynamic therapeutic agents. Indeed, due to their irreplaceable physicochemical properties, they are virtuous allies for photodynamic therapy (PDT) in combination with bioimaging, photothermal therapy, as well as drug and gene delivery. In this review, the rapidly progressing literature related to the use of these promising 2D materials for cancer theranostics is described in detail, highlighting all their possible future advances in PDT.

A Novel Sensitive Cell-Based Immunoenzymatic Assay for Palytoxin Quantitation in Mussels.

The marine algal toxin palytoxin (PLTX) and its analogues are some of the most toxic marine compounds. Their accumulation in edible marine organisms and entrance into the food chain represent their main concerns for human health. Indeed, several fatal human poisonings attributed to these compounds have been recorded in tropical and subtropical areas. Due to the increasing occurrence of PLTX in temperate areas such as the Mediterranean Sea, the European Food Safety Authority (EFSA) has suggested a maximum limit of 30 µg PLTX/kg in shellfish meat, and has recommended the development of rapid, specific, and sensitive methods for detection and quantitation of PLTX in seafood. Thus, a novel, sensitive cell-based ELISA was developed and characterized for PLTX quantitation in mussels. The estimated limits of detection (LOD) and quantitation (LOQ) were 1.2 × 10-11 M (32.2 pg/mL) and 2.8 × 10-11 M (75.0 pg/mL), respectively, with good accuracy (bias = 2.5%) and repeatability (15% and 9% interday and intraday relative standard deviation of repeatability (RSDr), respectively). Minimal interference of 80% aqueous methanol extract allows PLTX quantitation in mussels at concentrations lower than the maximum limit suggested by EFSA, with an LOQ of 9.1 µg PLTX equivalent/kg mussel meat. Given its high sensitivity and specificity, the cell-based ELISA should be considered a suitable method for PLTX quantitation.

Graphene and graphene oxide induce ROS production in human HaCaT skin keratinocytes: the role of xanthine oxidase and NADH dehydrogenase.

The extraordinary physicochemical properties of graphene-based nanomaterials (GBNs) make them promising tools in nanotechnology and biomedicine. Considering the skin contact as one of the most feasible exposure routes to GBNs, the mechanism of toxicity of two GBNs (few-layer-graphene, FLG, and graphene oxide, GO) towards human HaCaT skin keratinocytes was investigated. Both materials induced a significant mitochondrial membrane depolarization: 72 h cell exposure to 100 µg mL-1 FLG or GO increased mitochondrial depolarization by 44% and 56%, respectively, while the positive control valinomycin (0.1 µg mL-1) increased mitochondrial depolarization by 48%. Since the effect was not prevented by cyclosporine-A, it appears to be unrelated to mitochondrial transition pore opening. By contrast, it seems to be mediated by reactive oxygen species (ROS) production: FLG and GO induced time- and concentration-dependent cellular ROS production, significant already at the concentration of 0.4 µg mL-1 after 24 h exposure. Among a panel of specific inhibitors of the major ROS-producing enzymes, diphenyliodonium, rotenone and allopurinol significantly reverted or even abolished FLG- or GO-induced ROS production. Intriguingly, the same inhibitors also significantly reduced FLG- or GO-induced mitochondrial depolarization and cytotoxicity. This study shows that FLG and GO induce a cytotoxic effect due to a sustained mitochondrial depolarization. This seems to be mediated by a significant cellular ROS production, caused by the activation of flavoprotein-based oxidative enzymes, such as NADH dehydrogenase and xanthine oxidase.

Differential cytotoxic effects of graphene and graphene oxide on skin keratinocytes.

Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24 h, after 72 h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 62.8 µg/mL (WST-8 assay) and 45.5 µg/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 5.4 and 2.9 µg/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.

Pharmacokinetics and pharmacodynamics of thiopurines in an in vitro model of human hepatocytes: Insights from an innovative mass spectrometry assay.

AIM: To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity. METHODS: Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10-4 M azathioprine and 1.1 × 10-3 M mercaptopurine, concentrations corresponding to the IC50 values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA). RESULTS: Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037). CONCLUSION: Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.

Behavioral Health Providers for Persons Who Are Deaf, Deafblind, or Hard-of-Hearing: A National Survey of the Structural and Process Domains of Care.

Research suggests higher prevalence of mental health problems for those with hearing problems than in the general population. Despite barriers, mental health services for persons who are deaf and hard-of-hearing (HOH) have developed to meet the cultural and communication needs of this population. The authors conducted a national survey of mental health service providers to persons who are deaf, deafblind, or HOH, to learn about their structural and process domains of care. Results indicate that services for persons who are deaf, deafblind, or HOH are inadequate for consumers with serious mental illness. Results also uncovered unique pathways to care and practitioners.

Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly.

The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of ß-d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-ß-d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17 °C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.