Using a multiomics approach to unravel a septic shock specific signature in skeletal muscle.

Sepsis is defined as a dysregulated host response to infection leading to organs failure. Among them, sepsis induces skeletal muscle (SM) alterations that contribute to acquired-weakness in critically ill patients. Proteomics and metabolomics could unravel biological mechanisms in sepsis-related organ dysfunction. Our objective was to characterize a distinctive signature of septic shock in human SM by using an integrative multi-omics approach. Muscle biopsies were obtained as part of a multicenter non-interventional prospective study. Study population included patients in septic shock (S group, with intra-abdominal source of sepsis) and two critically ill control populations: cardiogenic shock (C group) and brain dead (BD group). The proteins and metabolites were extracted and analyzed by High-Performance Liquid Chromatography-coupled to tandem Mass Spectrometry, respectively. Fifty patients were included, 19 for the S group (53% male, 64 ± 17 years, SAPS II 45 ± 14), 12 for the C group (75% male, 63 ± 4 years, SAPS II 43 ± 15), 19 for the BD group (63% male, 58 ± 10 years, SAPS II 58 ± 9). Biopsies were performed in median 3 days [interquartile range 1-4]) after intensive care unit admission. Respectively 31 patients and 40 patients were included in the proteomics and metabolomics analyses of 2264 proteins and 259 annotated metabolites. Enrichment analysis revealed that mitochondrial pathways were significantly decreased in the S group at protein level: oxidative phosphorylation (adjusted p = 0.008); branched chained amino acids degradation (adjusted p = 0.005); citrate cycle (adjusted p = 0.005); ketone body metabolism (adjusted p = 0.003) or fatty acid degradation (adjusted p = 0.008). Metabolic reprogramming was also suggested (i) by the differential abundance of the peroxisome proliferator-activated receptors signaling pathway (adjusted p = 0.007), and (ii) by the accumulation of fatty acids like octanedioic acid dimethyl or hydroxydecanoic. Increased polyamines and depletion of mitochondrial thioredoxin or mitochondrial peroxiredoxin indicated a high level of oxidative stress in the S group. Coordinated alterations in the proteomic and metabolomic profiles reveal a septic shock signature in SM, highlighting a global impairment of mitochondria-related metabolic pathways, the depletion of antioxidant capacities, and a metabolic shift towards lipid accumulation.ClinicalTrial registration: NCT02789995. Date of first registration 03/06/2016.

Inhibition of the replication of SARS-CoV-2 in human cells by the FDA-approved drug chlorpromazine.

INTRODUCTION: Urgent action is needed to fight the ongoing coronavirus disease 2019 (COVID-19) pandemic by reducing the number of infected cases, contagiousness and severity. Chlorpromazine (CPZ), an antipsychotic from the phenothiazine group, is known to inhibit clathrin-mediated endocytosis and has antiviral activity against severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus. The aim of this in-vitro study was to test CPZ against SARS-CoV-2 in monkey and human cells. MATERIALS AND METHODS: Monkey VeroE6 cells and human alveolar basal epithelial A549-ACE2 cells were infected with SARS-CoV-2 in the presence of various concentrations of CPZ. Supernatants were harvested at day 2 and analysed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for the presence of SARS-CoV-2 RNA. Cell viability was assessed in non-infected cells. RESULTS: CPZ was found to have antiviral activity against SARS-CoV-2 in monkey VeroE6 cells, with a half maximal inhibitory concentration (IC50) of 8.2 µM, half maximal cytotoxic concentration (CC50) of 13.5 µM, and selectivity index (SI) of 1.65. In human A549-ACE2 cells, CPZ was also found to have anti-SARS-CoV-2 activity, with IC50 of 11.3 µM, CC50 of 23.1 µM and SI of 2.04. DISCUSSION: Although the measured SI values are low, the IC50 values measured in vitro may translate to CPZ dosages used in routine clinical practice because of the high biodistribution of CPZ in lungs and saliva. Also, the distribution of CPZ in brain could be of interest for treating or preventing neurological and psychiatric forms of COVID-19. CONCLUSIONS: These preclinical findings support clinical investigation of the repurposing of CPZ, a drug with mild side effects, in the treatment of patients with COVID-19.

Macrophages: Checking Toxicity of Fungal Metabolites in the Colon.

It is well known that the intestine absorbs nutrients, electrolytes, and water. Chikina et al. recently demonstrated that it is also able to sense, recognize, and block the absorption of toxins through a very sophisticated interactive cellular cooperation between novel subpopulations of macrophages and epithelial cells.

CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1.

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.

Revisiting Old Questions and New Approaches to Investigate the Fungal Cell Wall Construction.

The beginning of our understanding of the cell wall construction came from the work of talented biochemists in the 70-80's. Then came the era of sequencing. Paradoxically, the accumulation of fungal genomes complicated rather than solved the mystery of cell wall construction, by revealing the involvement of a much higher number of proteins than originally thought. The situation has become even more complicated since it is now recognized that the cell wall is an organelle whose composition continuously evolves with the changes in the environment or with the age of the fungal cell. The use of new and sophisticated technologies to observe cell wall construction at an almost atomic scale should improve our knowledge of the cell wall construction. This essay will present some of the major and still unresolved questions to understand the fungal cell wall biosynthesis and some of these exciting futurist approaches.

GPI Anchored Proteins in Aspergillus fumigatus and Cell Wall Morphogenesis.

Glycosylphosphatidylinositol (GPI) anchored proteins are a class of proteins attached to the extracellular leaflet of the plasma membrane via a post-translational modification, the glycolipid anchor. GPI anchored proteins are expressed in all eukaryotes, from fungi to plants and animals. They display very diverse functions ranging from enzymatic activity, signaling, cell adhesion, cell wall metabolism, and immune response. In this review, we investigated for the first time an exhaustive list of all the GPI anchored proteins present in the Aspergillus fumigatus genome. An A. fumigatus mutant library of all the genes that encode in silico identified GPI anchored proteins has been constructed and the phenotypic analysis of all these mutants has been characterized including their growth, conidial viability or morphology, adhesion and the ability to form biofilms. We showed the presence of different fungal categories of GPI anchored proteins in the A. fumigatus genome associated to their role in cell wall remodeling, adhesion, and biofilm formation.

Microglial production of quinolinic acid as a target and a biomarker of the antidepressant effect of ketamine.

Major depressive disorder is a complex multifactorial condition with a so far poorly characterized underlying pathophysiology. Consequently, the available treatments are far from satisfactory as it is estimated that up to 30% of patients are resistant to conventional treatment. Recent comprehensive evidence has been accumulated which suggests that inflammation may be implied in the etiology of this disease. Here we investigated ketamine as an innovative treatment strategy due to its immune-modulating capacities. In a murine model of LPS-induced depressive-like behavior we demonstrated that a single dose of ketamine restores the LPS-induced depressive-like alterations. These behavioral effects are associated with i/ a reversal of anxiety and reduced self-care, ii/ a decrease in parenchymal cytokine production, iii/ a modulation of the microglial reactivity and iv/ a decrease in microglial quinolinic acid production that is correlated with plasmatic peripheral production. In a translational approach, we show that kynurenic acid to quinolinic acid ratio is a predictor of ketamine response in treatment-resistant depressed patients and that the reduction in quinolinic acid after a ketamine infusion is a predictor of the reduction in MADRS score. Our results suggest that microglia is a key therapeutic target and that quinolinic acid is a biomarker of ketamine response in major depressive disorder.

Plasmid-normalized quantification of relative mitochondrial DNA copy number.

Alterations of mitochondrial DNA (mtDNA) copy number have been associated with a wide variety of phenotypes and diseases. Unfortunately, the literature provides scarce methodical information about duplex targeting of nuclear and mtDNA that meets the quality criteria for qPCR. Therefore, we established a method for mtDNA copy number quantification using a quantitative PCR assay that allows for simultaneous targeting of a single copy nuclear gene (beta-2-microglobulin) and the t-RNALeu gene on the mtDNA. We include a plasmid containing both targets in order to normalize against differences in emission intensities of the fluorescent dyes Yakima Yellow and FAM. Applying the plasmid calibrator on an internal control reduced the intra-assay variability from 21% (uncorrected) to 7% (plasmid-corrected). Moreover, we noted that DNA samples isolated with different methods revealed different numbers of mtDNA copies, thus highlighting an important influence of the pre-analytical procedures. In summary, we developed a precise assay for mitochondrial copy number detection relative to nuclear DNA. Our method is applicable to comparative mitochondrial DNA copy number studies since the use of the dual insert plasmid allows correcting for the unequal emission intensities of the different fluorescent labels of the two targets.

Aspergillus terreus: Novel lessons learned on amphotericin B resistance.

The polyene antifungal amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and in general displays a remarkably low rate of antimicrobial resistance. Aspergillus terreus holds an exceptional position among the Aspergilli due to its intrinsic AmB resistance, in vivo and in vitro. Until now, the underlying mechanisms of polyene resistance were not well understood. This review will highlight the molecular basis of A. terreus and AmB resistance recently gained and will display novel data on the mode of action of AmB. A main focus is set on fundamental stress response pathways covering the heat shock proteins (Hsp) 90/Hsp70 axis, as well as reactive oxygen species detoxifying enzymes in response to AmB. The effect on main cellular functions such as fungal respiration will be addressed in detail and resistance mechanisms will be highlighted. Based on these novel findings we will discuss new molecular targets for alternative options in the treatment of invasive infections due to A. terreus.

Metal-homeostasis in the pathobiology of the opportunistic human fungal pathogen Aspergillus fumigatus.

In contrast to obligate pathogens opportunistic pathogens such as Aspergillus fumigatus do not need a specific host to propagate or survive. However several characteristics of the saprophytic life-style and the selective pressure encountered in the primary ecological niche contribute to the virulence of A. fumigatus. All fungi depend on metals for growth and proliferation, like iron, copper, zinc, manganese or calcium. In the recent past several studies explored the manifold impact of metals modulating virulence of pathogens. Components which might be scarce in the natural environment but also in the host due to nutritional immunity. This review recapitulates molecular constituents of metal ion uptake systems in A. fumigatus, their regulation and their significance at the host-pathogen battlefield.

Impact of Morphological Sectors on Antifungal Susceptibility Testing and Virulence Studies.

Morphological heterogeneity of Aspergillus terreus cultures was observed during continued cultivation of amphotericin B (AMB)-resistant isolates on drug-free medium. Outgrowth leads to the emergence of multiple sectors that might result from increased growth rates at drug-free conditions. We evaluated the differences in AMB susceptibility and virulence between sector subcultures (ATSec), AMB-resistant (ATR) strains, and AMB-susceptible (ATS) strains. By comparing A. terreus AMB-resistant (ATR) strains and A. terreus sector (ATSec) cultures we observed a highly significant reduction of AMB MICs in ATSec (ATR MIC, 2 to 32 µg/ml; ATSec MIC, 0.12 to 5 µg/ml). Furthermore, Galleria mellonella survival studies revealed an enhanced virulence of ATSec, which was comparable with that of AMB-sensitive Aspergillus terreus strains (median survival rates for ATS isolates, 72 h; for ATSec isolate ATSecG1, 84 h; for ATR isolates, 144 h). Our findings clearly demonstrate that spontaneous culture degeneration occurs in A. terreus and, most importantly, crucially impacts drug efficacy and virulence.

Oxidative Stress Response Tips the Balance in Aspergillus terreus Amphotericin B Resistance.

In this study, we characterize the impact of antioxidative enzymes in amphotericin B (AmB)-resistant (ATR) and rare AmB-susceptible (ATS) clinical Aspergillus terreus isolates. We elucidate expression profiles of superoxide dismutase (SOD)- and catalase (CAT)-encoding genes, enzymatic activities of SODs, and superoxide anion production and signaling pathways involved in the oxidative stress response (OSR) in ATS and ATR strains under AmB treatment conditions. We show that ATR strains possess almost doubled basal SOD activity compared to that of ATS strains and that ATR strains exhibit an enhanced OSR, with significantly higher sod2 mRNA levels and significantly increased cat transcripts in ATR strains upon AmB treatment. In particular, inhibition of SOD and CAT proteins renders resistant isolates considerably susceptible to the drug in vitro In conclusion, this study shows that SODs and CATs are crucial for AmB resistance in A. terreus and that targeting the OSR might offer new treatment perspectives for resistant species.

Aspergillus fumigatus responds to natural killer (NK) cells with upregulation of stress related genes and inhibits the immunoregulatory function of NK cells.

Natural Killer (NK) cells are active against Aspergillus fumigatus, which in turn is able to impair the host defense. Unfortunately, little is known on the mutual interaction of NK cells and A. fumigatus. We coincubated human NK cells with A. fumigatus hyphae and assessed the gene expression and protein concentration of selected molecules. We found that A. fumigatus up-regulates the gene expression of pro-inflammatory molecules in NK cells, but inhibited the release of these molecules resulting in intracellular accumulation and limited extracellular availability. A. fumigatus down-regulatedmRNA levels of perforin in NK cells, but increased its intra- and extracellular protein concentration. The gene expression of stress related molecules of A. fumigatus such as heat shock protein hsp90 was up-regulated by human NK cells. Our data characterize for the first time the immunosuppressive effect of A. fumigatus on NK cells and may help to develop new therapeutic antifungal strategies.

Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells.

DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection.

Amphotericin B Resistance in Aspergillus terreus Is Overpowered by Coapplication of Pro-oxidants.

AIMS: Invasive fungal infections have significantly increased over the past decades in immunocompromised individuals and high-risk patients. Amphotericin B (AmB) exerts a powerful and broad activity against a vast array of fungi and has a remarkably low rate of microbial resistance. However, most isolates of Aspergillus terreus developed an intrinsic resistance against AmB, and during this study, we characterized the mode of action of this polyene antifungal drug in more detail in resistant (ATR) and rare susceptible (ATS) clinical isolates of A. terreus. RESULTS: We illustrate that AmB treatment changes cellular redox status and promotes the generation of high levels of reactive oxygen species (ROS) in ATS. In contrast, ATR isolates were able to cope better with AmB-induced oxidative stress. INNOVATION: Most importantly, we demonstrate in this study that coapplication of anti- and pro-oxidants significantly affects AmB efficacy in an antithetic manner--antioxidants and ROS-scavenging agents increase AmB tolerance in susceptible strains, while pro-oxidants render formerly resistant isolates considerably susceptible to the antifungal drug also in vivo in a Galleria animal model. CONCLUSION: Thereby, our study provides novel therapeutic options to treat formerly resistant fungal strains by a combination of AmB and pro-oxidant compounds.

Blocking Hsp70 enhances the efficiency of amphotericin B treatment against resistant Aspergillus terreus strains.

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-µ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.

Differential gene expression in Aspergillus fumigatus induced by human platelets in vitro.

Invasive aspergillosis is characterized by vascular invasion and thrombosis. In order to determine the antifungal activity of human platelets, hyphal elongation and metabolic activity of a clinical A. fumigatus isolate were measured. Genome-wide identification of differentially expressed genes in A. fumigatus was performed after exposure to platelets for 15, 30, 60 and 180 min. Data were analyzed by gene ontology annotation as well as functional categories (FunCat) and KEGG enrichment analyses. Platelets attenuated hyphal elongation and viability of A. fumigatus and in total 584 differentially expressed genes were identified, many of which were associated with regulation of biological processes, stress response, transport and metabolism. FunCat and KEGG enrichment analyses showed stress response and metabolic adaptation to be increased in response to platelets. Our findings demonstrate that A. fumigatus displayed a specific transcriptional response when exposed to platelets, thus reflecting their antifungal activities.

[(68)Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvß3 integrin expression based on a novel siderophore derived chelating scaffold-synthesis and evaluation.

Over the last years Gallium-68 ((68)Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). (68)Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for (68)Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based (68)Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and (68)Ga-radiolabeling of a trimeric FSC-RGD conjugate, [(68)Ga]FSC-(RGD)3, targeting αvß3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with (68)Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvß3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvß3 positive human melanoma M21 and αvß3 negative M21-L cells were used. [(68)Ga]FSC-(RGD)3 was prepared with high radiochemical yield (>98%). Distribution coefficient was -3.6 revealing a hydrophilic character, and an IC50 value of 1.8±0.6 nM was determined indicating a high binding affinity for αvß3 integrin. [(68)Ga]FSC-(RGD)3 was stable in PBS (pH7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37°C for 2hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvß3 positive tumor was 4.3% ID/g 60min p.i. which was 3-fold higher than the monomeric [(68)Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [(68)Ga]FSC-(RGD)3 serves as an example for the feasibility of a novel class of bifunctional chelators based on cyclic peptide siderophores and shows excellent targeting properties for αvß3 integrin in vivo for imaging tumor-induced neovascularization.