Design, synthesis, and biological evaluation of novel halogenated chlorido[N,N'-bis(salicylidene)-1,2-bis(3-methoxyphenyl)ethylenediamine]iron(III) complexes as anticancer agents.

Iron(III) complexes based on N,N´-bis(salicylidene)ethylenediamine (salene) scaffolds have demonstrated promising anticancer features like induction of ferroptosis, an iron dependent cell death. Since poor cellular uptake limits their therapeutical potential, this study aimed to enhance the lipophilic character of chlorido[N,N'-bis(salicylidene)-1,2-bis(3-methoxyphenyl)ethylenediamine]iron(III) complexes by introducing lipophilicity improving ligands such as fluorine (X1), chlorine (X2) and bromine (X3) in 5-position in the salicylidene moieties. After detailed characterization the binding to nucleophiles, logP values and cellular uptake were determined. The complexes were further evaluated regarding their biological activity on MDA-MB 231 mammary carcinoma, the non-tumorous SV-80 fibroblast, HS-5 stroma and MCF-10A mammary gland cell lines. Stability of the complexes in aqueous and biological environments was proven by the lack of interactions with amino acids and glutathione. Cellular uptake was positively correlated with the logP values, indicating that higher lipophilicity enhanced cellular uptake. The complexes induced strong antiproliferative and antimetabolic effects on MDA-MB 231 cells, but were inactive on all non-malignant cells tested. Generation of mitochondrial reactive oxygen species, increase of lipid peroxidation and induction of both ferroptosis and necroptosis were identified as mechanisms of action. In conclusion, halogenation of chlorido[N,N'-bis(salicylidene)-1,2-bis(3-methoxyphenyl)ethylenediamine]iron(III) complexes raises their lipophilic character resulting in improved cellular uptake.

Transferrin Receptor-Mediated Cellular Uptake of Fluorinated Chlorido[N,N'-bis(salicylidene)-1,2-phenylenediamine]iron(III) Complexes.

Fluorinated chlorido[salophene]iron(III) complexes (salophene = N,N'-bis(salicylidene)-1,2-phenylenediamine) are promising anticancer agents. Apoptosis and necrosis induction have already been described as part of their mode of action. However, the involvement of ferroptosis in cell death induction, as confirmed for other chlorido[salophene]iron(III) complexes, has not yet been investigated. Furthermore, the mechanism of cellular uptake of these compounds is unknown. Therefore, the biological activity of the fluorescent chlorido[salophene]iron(III) complexes with a fluorine substituent at positions 3, 4, 5, or 6 at the salicylidene moieties (C1-C4) was evaluated in malignant and nonmalignant cell lines with focus on the involvement of the transferrin receptor-1 (TfR-1) in cellular uptake, the influence of the complexes on mitochondrial function, and the analysis of the molecular mechanism of cell death. All complexes significantly decreased the metabolic activity in the tested ovarian cancer (A2780, A2780cis), breast cancer (MDA-MB 231), and leukemia (HL-60) cell lines, while the nonmalignant human stroma cell line HS-5 at a concentration of 0.5 µM, which represents the IC50 of the complexes in most of the used tumorigenic cell lines, was not affected. The mitochondrial function was impaired, as evidenced by a reduced mitochondrial membrane potential ΔΨm and decreased mitochondrial activity. Besides apoptosis and necroptosis, ferroptosis was identified as part of the mode of action. It was further demonstrated for the first time that fluorinated chlorido[salophene]iron(III) complexes downregulate TfR-1 expression, comparable to ferristatin II, an iron transport inhibitor that acts via TfR-1 degradation. FerroOrange staining further indicated that the complexes strongly increased the intracellular iron(II) level as a driving force to induce ferroptosis. In conclusion, these fluorinated chlorido[salophene]iron(III) complexes are potent, tumor cell-specific chemotherapeutic agents, with the potential to treat various types of cancers.

Analysis of fibrin networks using topological data analysis - a feasibility study.

Blood clot formation, a crucial process in hemostasis and thrombosis, has garnered substantial attention for its implications in various medical conditions. Microscopic examination of blood clots provides vital insights into their composition and structure, aiding in the understanding of clot pathophysiology and the development of targeted therapeutic strategies. This study explores the use of topological data analysis (TDA) to assess plasma clot characteristics microscopically, focusing on the identification of the elements components, holes and Wasserstein distances. This approach should enable researchers to objectively classify fibrin networks based on their topologic architecture. We tested this mathematical characterization approach on plasma clots formed in static conditions from porcine and human citrated plasma samples, where the effect of dilution and direct thrombin inhibition was explored. Confocal microscopy images showing fluorescence labeled fibrin networks were analyzed. Both treatments resulted in visual differences in plasma clot architecture, which could be quantified using TDA. Significant differences between baseline and diluted samples, as well as blood anticoagulated with argatroban, were detected mathematically. Therefore, TDA could be indicative of clots with compromised stability, providing a valuable tool for thrombosis risk assessment. In conclusion, microscopic examination of plasma clots, coupled with Topological Data Analysis, offers a promising avenue for comprehensive characterization of clot microstructure. This method could contribute to a deeper understanding of clot pathophysiology and thereby refine our ability to assess clot characteristics.

Assessment of Mitochondrial Respiration During Hypothermic Storage of Liver Biopsies Following Normothermic Machine Perfusion.

Organ quality can be assessed prior to transplantation, during normothermic machine perfusion (NMP) of the liver. Evaluation of mitochondrial function by high-resolution respirometry (HRR) may serve as a viability assessment concept in this setting. Freshly collected tissue is considered as optimal sample for HRR, but due to technical and personnel requirements, more flexible and schedulable measurements are needed. However, the impact of cold storage following NMP before processing biopsy samples for mitochondrial analysis remains unknown. We aimed at establishing an appropriate storage protocol of liver biopsies for HRR. Wedge biopsies of 5 human livers during NMP were obtained and assessed by HRR. Analysis was performed after 0, 4, 8, and 12 h of hypothermic storage (HTS) in HTK organ preservation solution at 4°C. With HTS up to 4 h, mitochondrial performance did not decrease in HTS samples compared with 0 h (OXPHOS, 44.62 [34.75-60.15] pmol·s-1·mg wet mass-1 vs. 43.73 [40.69-57.71], median [IQR], p > 0.999). However, at HTS beyond 4 h, mitochondrial respiration decreased. We conclude that HTS can be safely applied for extending the biopsy measurement window for up to 4 h to determine organ quality, but also that human liver respiration degrades beyond 4 h HTS following NMP.

Elimination of damaged mitochondria during UVB-induced senescence is orchestrated by NIX-dependent mitophagy.

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress-induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX-dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB-irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB-irradiated NIX-depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB-induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF.

The Predictive Value of Graft Viability and Bioenergetics Testing Towards the Outcome in Liver Transplantation.

Donor organ biomarkers with sufficient predictive value in liver transplantation (LT) are lacking. We herein evaluate liver viability and mitochondrial bioenergetics for their predictive capacity towards the outcome in LT. We enrolled 43 consecutive patients undergoing LT. Liver biopsy samples taken upon arrival after static cold storage were assessed by histology, real-time confocal imaging analysis (RTCA), and high-resolution respirometry (HRR) for mitochondrial respiration of tissue homogenates. Early allograft dysfunction (EAD) served as primary endpoint. HRR data were analysed with a focus on the efficacy of ATP production or P-L control efficiency, calculated as 1-L/P from the capacity of oxidative phosphorylation P and non-phosphorylating respiration L. Twenty-two recipients experienced EAD. Pre-transplant histology was not predictive of EAD. The mean RTCA score was significantly lower in the EAD cohort (-0.75 ± 2.27) compared to the IF cohort (0.70 ± 2.08; p = 0.01), indicating decreased cell viability. P-L control efficiency was predictive of EAD (0.76 ± 0.06 in IF vs. 0.70 ± 0.08 in EAD-livers; p = 0.02) and correlated with the RTCA score. Both RTCA and P-L control efficiency in biopsy samples taken during cold storage have predictive capacity towards the outcome in LT. Therefore, RTCA and HRR should be considered for risk stratification, viability assessment, and bioenergetic testing in liver transplantation.

Development of Cytotoxic GW7604-Zeise's Salt Conjugates as Multitarget Compounds with Selectivity for Estrogen Receptor- Positive Tumor Cells.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a carrier was esterified with alkenols of various lengths and coordinated through the ethylene moiety to PtCl3, similar to Zeise's salt (K[PtCl3(C2H4)]). The resulting GW7604-Alk-PtCl3 complexes (Alk = Prop, But, Pent, Hex) degraded in aqueous solution only by exchange of the chlorido ligands. For example, GW7604-Pent-PtCl3 coordinated the amino acid alanine in the cell culture medium, bound the isolated nucleotide 5'-GMP, and interacted with the DNA (empty plasmid pSport1). It accumulated in estrogen receptor (ER)-positive MCF-7 cells primarily via cytosolic vesicles, while it was only marginally taken up in ER-negative SKBr3 cells. Accordingly, GW7604-Pent-PtCl3 and related complexes were inactive in SKBr3 cells. GW7604-Pent-PtCl3 showed high affinity to ERα and ERß without mediating agonistic or ER downregulating properties. GW7604-Alk ligands also increased the cyclooxygenase (COX)-2 inhibitory potency of the complexes. In contrast to Zeise's salt, the GW7604-Alk-PtCl3 complexes inhibited COX-1 and COX-2 to the same extent.

Prediction of Biliary Complications After Human Liver Transplantation Using Hyperspectral Imaging and Convolutional Neural Networks: A Proof-of-concept Study.

BACKGROUND: Biliary complications (BCs) negatively impact the outcome after liver transplantation. We herein tested whether hyperspectral imaging (HSI) generated data from bile ducts (BD) on reperfusion and machine learning techniques for data readout may serve as a novel approach for predicting BC. METHODS: Tissue-specific data from 136 HSI liver images were integrated into a convolutional neural network (CNN). Fourteen patients undergoing liver transplantation after normothermic machine preservation served as a validation cohort. Assessment of oxygen saturation, organ hemoglobin, and tissue water levels through HSI was performed after completing the biliary anastomosis. Resected BD segments were analyzed by immunohistochemistry and real-time confocal microscopy. RESULTS: Immunohistochemistry and real-time confocal microscopy revealed mild (grade I: 1%-40%) BD damage in 8 patients and moderate (grade II: 40%-80%) injury in 1 patient. Donor and recipient data alone had no predictive capacity toward BC. Deep learning-based analysis of HSI data resulted in >90% accuracy of automated detection of BD. The CNN-based analysis yielded a correct classification in 72% and 69% for BC/no BC. The combination of HSI with donor and recipient factors showed 94% accuracy in predicting BC. CONCLUSIONS: Deep learning-based modeling using CNN of HSI-based tissue property data represents a noninvasive technique for predicting postoperative BC.

Design, Synthesis, Electrochemical, and Biological Evaluation of Fluorescent Chlorido[N,N'-bis(methoxy/hydroxy)salicylidene-1,2-bis(4-methoxyphenyl)ethylenediamine]iron(III) Complexes as Anticancer Agents.

The impact of methoxy and hydroxyl groups at the salicylidene moiety of chlorido[N,N'-bis(methoxy/hydroxy)salicylidene-1,2-bis(4-methoxyphenyl)ethylenediamine]iron(III) complexes was evaluated on human MDA-MB 231 breast cancer and HL-60 leukemia cells. Methoxylated complexes (C1-C3) inhibited proliferation, migration, and metabolic activity in a concentration-dependent manner following the rank order: C2 > C3 > C1. In particular, C2 was highly cytotoxic with an IC50 of 4.2 µM which was 6.6-fold lower than that of cisplatin (IC50 of 27.9 µM). In contrast, hydroxylated complexes C4-C6 were almost inactive up to the highest concentration tested due to lack of cellular uptake. C2 caused a dual mode of cell death, ferroptosis, and necroptosis, whereby at higher concentrations, ferroptosis was the preferred form. Ferroptotic morphology and the presence of ferrous iron and lipid reactive oxygen species proved the involvement of ferroptosis. C2 was identified as a promising lead compound for the design of drug candidates inducing ferroptosis.

Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization.

BACKGROUND: Chronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment. METHODS: We examined CD45-/CD31-/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR. RESULTS: CD45-/CD31-/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs. CONCLUSION: Preadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

Increasing the collection flow rate to 2 mL/min is effective and reduces the procedure time in off-line photopheresis.

BACKGROUND: Extracorporeal photopheresis (ECP) treatment, mostly based on apheresis technology, is used for immunomodulation in various diseases such as cutaneous T-cell lymphoma, graft versus host disease and other (auto)immune disorders. The aim of this study was to collect high cell counts and purity in shorter procedure times using an ECP off-line system with an increased collection flow rate of 2 mL/min to a target volume of 200 mL buffy coat. STUDY DESIGN AND METHODS: In this prospective study, data of routinely performed off-line photopheresis treatments were collected and analyzed at the Central Institute for Blood Transfusion & Department of Immunology (ZIB) of the Tirol Kliniken, to assess absolute cell counts and procedure times and to calculate collection efficiencies (CE2). RESULTS: A total of 22 patients participated in this study. The processed blood volume was 4312 mL, the collection time 120 min, overall procedure time 157 min and the absolute cell counts of treated white blood cells (WBC) and mononuclear cells (MNC) were 5.0 and 4.3 × 109 respectively (median values). The calculated CE2 for WBC and MNC was 21.1% and 58.5%, the proportion of treated MNCs of the total number of MNCs present was 55.0%. CONCLUSION: The data presented in this study show high therapeutically effective cell counts collected with a high MNC purity within a shorter overall collection/procedure time due to an increased collection flow rate.

Synthesis and biological evaluation of salophen nickel(II) and cobalt(III) complexes as potential anticancer compounds.

Recent in vitro investigations of N,N'-bis(salicylidene)-1,2-phenylenediamine (SAP) iron(III) complexes substituted with alkyl (ethyl, propyl, butyl) carboxylates at position 4 in tumor and leukemia cells revealed strong cytotoxic activity. In continuation of this study, analogous nickel(II) and cobalt(III) complexes were synthesized and tested in HL-60 leukemia, and cisplatin-sensitive and -resistant A2780 ovarian cancer cell lines. The biological activity depended on the extent of cellular uptake and the formation of reactive oxygen species (ROS). Inactive [(Ni(II)SAP] complexes (1-3) only marginally accumulated in tumor cells and did not induce ROS. The cellular uptake of [Co(III)SAP]Cl complexes (4-6) into the cells depended on the length of the ester alkyl chain (ethyl, 4 < propyl, 5 < butyl, 6). The cytotoxicity correlated with the presence of ROS. The low cytotoxic complex 4 induced only few ROS, while 5 and 6 caused a good to outstanding antiproliferative activity, exerted high ROS generation, and induced cell death after 48 h. Necrostatin-1 prevented the biological effects, proving necroptosis as part of the mode of action. Interestingly, the effects of 5 and 6 were not reversed by Ferrostatin-1, but even enhanced upon simultaneous application to the tumor cells.

Investigations on the Influence of the Axial Ligand in [Salophene]iron(III) Complexes on Biological Activity and Redox Behavior.

The [N,N'-disalicylidene-1,2-phenylenediamine]iron(III) ([salophene]iron(III)) derivatives 1-4 with anionic axial ligands (A = Cl-, NO3-, SCN-, CH3COO-) and complexes 5 and 6 with neutral ligands (A = imidazole, 1-methylimidazole) as well as the µ-oxo dimer 7 inhibited proliferation, reduced metabolic activity, and increased mitochondrial reactive oxygen species. Ferroptosis as part of the mode of action was identified by inhibitor experiments, together with induction of lipid peroxidation and diminished mitochondrial membrane potential. No differences in activity were observed for all compounds except 4, which was slightly less active. Electrochemical analyses revealed for all compounds a fast attachment of the solvent dimethyl sulfoxide and a release of the axial ligand A. In contrast, in dichloromethane and acetonitrile, ligand exchange did not take place, as analyzed by measurements of the standard potential for the iron(III/II) redox reaction.

Mitochondrial respiration during normothermic liver machine perfusion predicts clinical outcome.

BACKGROUND: Reliable biomarkers for organ quality assessment during normothermic machine perfusion (NMP) are desired. ATP (adenosine triphosphate) production by oxidative phosphorylation plays a crucial role in the bioenergetic homeostasis of the liver. Thus, detailed analysis of the aerobic mitochondrial performance may serve as predictive tool towards the outcome after liver transplantation. METHODS: In a prospective clinical trial, 50 livers were subjected to NMP (OrganOx Metra) for up to 24.ßh. Biopsy and perfusate samples were collected at the end of cold storage, at 1.ßh, 6.ßh, end of NMP, and 1.ßh after reperfusion. Mitochondrial function and integrity were characterized by high-resolution respirometry (HRR), AMP, ADP, ATP and glutamate dehydrogenase analysis and correlated with the clinical outcome (L-GrAFT score). Real-time confocal microscopy was performed to assess tissue viability. Structural damage was investigated by histology, immunohistochemistry and transmission electron microscopy. FINDINGS: A considerable variability in tissue viability and mitochondrial respiration between individual livers at the end of cold storage was observed. During NMP, mitochondrial respiration with succinate and tissue viability remained stable. In the multivariate analysis of the 35 transplanted livers (15 were discarded), area under the curve (AUC) of LEAK respiration, cytochrome c control efficiency (mitochondrial outer membrane damage), and efficacy of the mitochondrial ATP production during the first 6.ßh of NMP correlated with L-GrAFT. INTERPRETATIONS: Bioenergetic competence during NMP plays a pivotal role in addition to tissue injury markers. The AUC for markers of outer mitochondrial membrane damage, ATP synthesis efficiency and dissipative respiration (LEAK) predict the clinical outcome upon liver transplantation. FUNDING: This study was funded by a Grant from the In Memoriam Dr. Gabriel Salzner Stiftung awarded to SS and the Tiroler Wissenschaftsfond granted to TH.

Indications that the Antimycotic Drug Amphotericin B Enhances the Impact of Platelets on Aspergillus.

Platelets are currently thought to harbor antimicrobial functions and might therefore play a crucial role in infections, e.g., those caused by Aspergillus or mucormycetes. The incidence of invasive fungal infections is increasing, particularly during the coronavirus disease 2019 (COVID-19) pandemic, and such infections continue to be life-threatening in immunocompromised patients. For this reason, the interaction of antimycotics with platelets is a key issue to evaluate modern therapeutic regimens. Amphotericin B (AmB) is widely used for the therapy of invasive fungal infections either as deoxycholate (AmB-D) or as a liposomal formulation (L-AmB). We showed that AmB strongly activates platelets within a few minutes. AmB concentrations commonly measured in the blood of patients were sufficient to stimulate platelets, indicating that this effect is highly relevant in vivo. The stimulating effect was corroborated by a broad spectrum of platelet activation parameters, including degranulation, aggregation, budding of microparticles, morphological changes, and enhanced adherence to fungal hyphae. Comparison between the deoxycholate and the liposomal formulation excluded the possibility that the liposomal part of L-Amb is responsible for these effects, as no difference was visible. The induction of platelet activation and alteration by L-AmB resulted in the activation of other parts of innate immunity, such as stimulation of the complement cascade and interaction with granulocytes. These mechanisms might substantially fuel the antifungal immune reaction in invasive mycoses. On the other hand, thrombosis and excessive inflammatory processes might occur via these mechanisms. Furthermore, the viability of L-AmB-activated platelets was consequently decreased, a process that might contribute to thrombocytopenia in patients.

Thin foil body-shield resuscitation barrier device to protect from blood: an experimental study.

In times of collective concern about pandemics, body-shield resuscitation barrier devices are more and more considered to protect against transmission of different pathogens between rescuers and patients. The objective of this experimental study was to investigate the characteristics of blood drops dispersed on the surface of four different foils suitable for blanketing patients during resuscitation. We analyzed run-off characteristics of blood stains depending on surface properties of polyvinyl chloride, polyethylene, polyethylene terephthalate and aluminum-coated polyethylene terephthalate. Confocal fluorescence microscopy revealed less cellular density and lack of fibrin networks in blood stains on the four foil surfaces than on paper towel. Delayed clotting went along with larger areas of contamination indicating a greater likelihood of coming into contact with potential germs but a smaller chance of contracting an infection. Space blankets as obligatory components of first aid kits are readily available for rescuers and serve as a mechanical barrier between rescuers and patients during resuscitation.

Mitochondrial Activity Is Upregulated in Nonlesional Atopic Dermatitis and Amenable to Therapeutic Intervention.

Previous work has shown increased expression of genes related to oxidative stress in nonlesional atopic dermatitis (ADNL) skin. Although mitochondria are key regulators of ROS production, their function in AD has never been investigated. Energy metabolism and the oxidative stress response were studied in keratinocytes (KCs) from patients with ADNL or healthy controls. Moreover, ADNL human epidermal equivalents were treated with tigecycline or MitoQ. We found that pyruvate and glucose were used as energy substrates by ADNL KCs. Increased mitochondrial oxidation of (very) long-chain fatty acids, associated with enhanced complexes I and II activities, was observed in ADNL KCs. Metabolomic analysis revealed increased tricarboxylic acid cycle turnover. Increased aerobic metabolism generated oxidative stress in ADNL KCs. ADNL human epidermal equivalents displayed increased mitochondrial function and an enhanced oxidative stress response compared with controls. Treatment of ADNL human epidermal equivalents with tigecycline or MitoQ largely corrected the AD profile, including high p-65 NF-κB, abnormal lamellar bodies, and cellular damage. Furthermore, we found that glycolysis supports but does not supersede mitochondrial metabolism in ADNL KCs. Thus, aerobic metabolism predominates in ADNL but leads to oxidative stress. Therefore, mitochondria could be a reservoir of potential therapeutic targets in atopic dermatitis.

3D bioprinted, vascularized neuroblastoma tumor environment in fluidic chip devices for precision medicine drug testing.

Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel-tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma-tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Investigations of the reactivity, stability and biological activity of halido (NHC)gold(I) complexes.

The significance of the halido ligand (Cl-, Br-, I-) in halido[3-ethyl-4-phenyl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(I) complexes (2-4) in terms of ligand exchange reactions, including the ligand scrambling to the bis[3-ethyl-4-phenyl-5-(2-methoxypyridin-5-yl)-1-propyl-1,3-dihydro-2H-imidazol-2-ylidene]gold(I) complex (5), was evaluated by HPLC in acetonitrile/water = 50:50 (v/v) mixtures. In the presence of 0.9% NaCl, the bromido (NHC)gold(I) complex 3 was immediately transformed into the chlorido (NHC)gold(I) complex 2. The iodido (NHC)gold(I) complex 4 converted under the same conditions during 0.5 h of incubation by 52.83% to 2 and by 8.77% to 5. This proportion remained nearly constant for 72 h. The halido (NHC)gold(I) complexes also reacted very rapidly with 1 eq. of model nucleophiles, e.g., iodide or selenocysteine (Sec). For instance, Sec transformed 3 in the proportion 73.03% to the (NHC)Au(I)Sec complex during 5 min of incubation. This high reactivity against this amino acid, present in the active site of the thioredoxin reductase (TrxR), correlates with the complete inhibition of the isolated TrxR enzyme at 1 µM. Interestingly, in cellular systems (A2780cis cells), even at a 5-fold higher concentration, no increased ROS levels were detected. The concentration required for ROS generation was about 20 µM. Superficially considered, the antiproliferative and antimetabolic activities of the halido (NHC)Au(I) complexes correlate with the reactivity of the Au(I)-X bond (2 < 3 < 4). However, it is very likely that degradation products formed during the incubation in cell culture medium participated in the biological activity. In particular, the high-cytotoxic [(NHC)2Au(I)]+ complex (5) distorts the results.