Interactions between mTORC2 core subunits Rictor and mSin1 dictate selective and context-dependent phosphorylation of substrate kinases SGK1 and Akt.

Mechanistic target of rapamycin complex 2 (mTORC2) is a multi-subunit kinase complex, central to multiple essential signaling pathways. Two core subunits, Rictor and mSin1, distinguish it from the related mTORC1 and support context-dependent phosphorylation of its substrates. mTORC2 structures have been determined previously; however, important questions remain, particularly regarding the structural determinants mediating substrate specificity and context-dependent activity. Here, we used cryo-EM to obtain high-resolution structures of the human mTORC2 apo-complex in the presence of substrates Akt and SGK1. Using functional assays, we then tested predictions suggested by substrate-induced structural changes in mTORC2. For the first time, we visualized in the apo-state the side chain interactions between Rictor and mTOR that sterically occlude recruitment of mTORC1 substrates and confer resistance to the mTORC1 inhibitor rapamycin. Also in the apo-state, we observed that mSin1 formed extensive contacts with Rictor via a pair of short α-helices nestled between two Rictor helical repeat clusters, as well as by an extended strand that makes multiple weak contacts with Rictor helical cluster 1. In co-complex structures, we found that SGK1, but not Akt, markedly altered the conformation of the mSin1 N-terminal extended strand, disrupting multiple weak interactions while inducing a large rotation of mSin1 residue Arg-83, which then interacts with a patch of negatively charged residues within Rictor. Finally, we demonstrate mutation of Arg-83 to Ala selectively disrupts mTORC2-dependent phosphorylation of SGK1, but not of Akt, supporting context-dependent substrate selection. These findings provide new structural and functional insights into mTORC2 specificity and context-dependent activity.

Designed Antimicrobial Peptides for Recurrent Vulvovaginal Candidiasis Treatment.

Recurrent vulvovaginal candidiasis (RVVC) is a widespread chronic infection that has a substantial negative impact on work and quality of life. The development of antimicrobial resistance and biofilm formation are speculated to contribute to Candida pathogenicity and treatment ineffectiveness. Designed antimicrobial peptides (dAMPs) are chemically modified from endogenous antimicrobial peptides that provide the first line of defense against pathogens. The goal here is to identify a dAMP for the topical treatment of RVVC. The dAMP MICs were determined for 46 fluconazole-susceptible and fluconazole-resistant Candida spp. clinical isolates. The possibility of inducing dAMP drug resistance and comparison of dAMP and fluconazole activity against preformed Candida biofilm and biofilm formation were evaluated. Assessment of mammalian cell viability was determined using bioluminescent human keratinocytes. The dAMP effect on fungus was probed via scanning electron microscopy, and topically applied dAMP activity was evaluated in a rodent vulvovaginal candidiasis (VVC) infection model. dAMPs demonstrated broad-spectrum antimicrobial activity against common causative clinical Candida isolates, reduced preformed biofilm, and inhibited biofilm formation. An evaluated dAMP did not induce resistance after repeated exposure of Candida tropicalis The dAMPs were selective for Candida cells with limited mammalian cytotoxicity with substantial activity in a rodent VVC model. dAMPs are described as having potent antifungal and antibiofilm activity, likely direct membrane action with selectivity for Candida cells, with limited resistance development. Combined with activity in a rodent VVC model, the data support clinical evaluation of dAMPs for topical treatment of VCC and recurrent VVC infections.

The hippocampal extracellular matrix regulates pain and memory after injury.

Chronic pain poses a heavy burden for the individual and society, comprising personal suffering, comorbid psychiatric symptoms, cognitive decline, and disability. Treatment options are poor due in large part to pain centralization, where an initial injury can result in lasting CNS maladaptations. Hippocampal cellular plasticity in chronic pain has become a focus of study due to its roles in cognition, memory, and the experience of pain itself. However, the extracellular alterations that parallel and facilitate changes in hippocampal function have not been addressed to date. Here we show structural and biochemical plasticity in the hippocampal extracellular matrix (ECM) that is linked to behavioral, cellular, and synaptic changes in a mouse model of chronic pain. Specifically, we report deficits in working location memory that are associated with decreased hippocampal dendritic complexity, altered ECM microarchitecture, decreased ECM rigidity, and changes in the levels of key ECM components and enzymes, including increased levels of MMP8. We also report aberrations in long-term potentiation (LTP) and a loss of inhibitory interneuron perineuronal ECM nets, potentially accounting for the aberrations in LTP. Finally, we demonstrate that MMP8 is upregulated after injury and that its genetic downregulation normalizes the behavioral, electrophysiological, and extracellular alterations. By linking specific extracellular changes to the chronic pain phenotype, we provide a novel mechanistic understanding of pain centralization that provides new targets for the treatment of chronic pain.

Integration of electron microscopy and solid-state NMR analysis for new views and compositional parameters of Aspergillus fumigatus biofilms.

The general ability and tendency of bacteria and fungi to assemble into bacterial communities, termed biofilms, poses unique challenges to the treatment of human infections. Fungal biofilms, in particular, are associated with enhanced virulence in vivo and decreased sensitivity to antifungals. Much attention has been given to the complex cell wall structures in fungal organisms, yet beyond the cell surface, Aspergillus fumigatus and other fungi assemble a self-secreted extracellular matrix that is the hallmark of the biofilm lifestyle, protecting and changing the environment of resident members. Elucidation of the chemical and molecular detail of the extracellular matrix is crucial to understanding how its structure contributes to persistence and antifungal resistance in the host. We present a summary of integrated analyses of A. fumigatus biofilm architecture, including hyphae and the extracellular matrix, by scanning electron microscopy and A. fumigatus matrix composition by new top-down solid-state NMR approaches coupled with biochemical analysis. This combined methodology will be invaluable in formulating quantitative and chemical comparisons of A. fumigatus isolates that differ in virulence and are more or less resistant to antifungals. Ultimately, knowledge of the chemical and molecular requirements for matrix formation and function will drive the identification and development of new strategies to interfere with biofilm formation and virulence.

Pseudomonas phage inhibition of Candida albicans.

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

Formation of Polymeric Nanocubes by Self-Assembly and Crystallization of Dithiolane-Containing Triblock Copolymers.

Template-free fabrication of non-spherical polymeric nanoparticles is desirable for various applications, but has had limited success owing to thermodynamic favorability of sphere formation. Herein we present a simple way to prepare cubic nanoparticles of block copolymers by self-assembly from aqueous solutions at room temperature. Nanocubes with edges of 40-200 nm are formed spontaneously on different surfaces upon water evaporation from micellar solutions of triblock copolymers containing a central poly(ethylene oxide) block and terminal trimethylene carbonate/dithiolane blocks. These polymers self-assemble into 28±5 nm micelles in water. Upon drying, micelle aggregation and a kinetically controlled crystallization of central blocks evidently induce solid cubic particle formation. An approach for preserving the structures of these cubes in water by thiol- or photo-induced crosslinking was developed. The ability to solubilize a model hydrophobic drug, curcumin, was also explored.

Pf4 bacteriophage produced by Pseudomonas aeruginosa inhibits Aspergillus fumigatus metabolism via iron sequestration.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

Isolation and trans-differentiation of mesenchymal stromal cells into smooth muscle cells: Utility and applicability for cell-sheet engineering.

BACKGROUND: Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) have shown potential to differentiate into various cell types, including smooth muscle cells (SMCs). The extracellular matrix (ECM) represents an appealing and readily available source of SMCs for use in tissue engineering. In this study, we hypothesized that the ECM could be used to induce MSC differentiation to SMCs for engineered cell-sheet construction. METHODS: Primary MSCs were isolated from the BM of Wistar rats, transferred and cultured on dishes coated with 3 different types of ECM: collagen type IV (Col IV), fibronectin (FN), and laminin (LM). Primary MSCs were also included as a control. The proportions of SMC (a smooth muscle actin [aSMA] and SM22a) and MSC markers were examined with flow cytometry and Western blotting, and cell proliferation rates were also quantified. RESULTS: Both FN and LM groups were able to induce differentiation of MSCs toward smooth muscle-like cell types, as evidenced by an increase in the proportion of SMC markers (aSMA; Col IV 42.3 ± 6.9%, FN 65.1 ± 6.5%, LM 59.3 ± 7.0%, Control 39.9 ± 3.1%; P = 0.02, SM22; Col IV 56.0 ± 7.7%, FN 74.2 ± 6.7%, LM 60.4 ± 8.7%, Control 44.9 ± 3.6%) and a decrease in that of MSC markers (CD105: Col IV 64.0 ± 5.2%, FN 57.6 ± 4.0%, LM 60.3 ± 7.0%, Control 85.3 ± 4.2%; P = 0.03). The LM group showed a decrease in overall cell proliferation, whereas FN and Col IV groups remained similar to control MSCs (Col IV, 9.0 ± 2.3%; FN, 9.8 ± 2.5%; LM, 4.3 ± 1.3%; Control, 9.8 ± 2.8%). CONCLUSIONS: Our findings indicate that ECM selection can guide differentiation of MSCs into the SMC lineage. Fibronectin preserved cellular proliferative capacity while yielding the highest proportion of differentiated SMCs, suggesting that FN-coated materials may be facilitate smooth muscle tissue engineering.

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy.

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The (13)C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional (15)N and (31)P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.

Dramatic Changes in Mitochondrial Subcellular Location and Morphology Accompany Activation of the CO2 Concentrating Mechanism.

Dynamic changes in intracellular ultrastructure can be critical for the ability of organisms to acclimate to environmental conditions. Microalgae, which are responsible for ~50% of global photosynthesis, compartmentalize their Rubisco into a specialized structure known as the pyrenoid when the cells experience limiting CO2 conditions; this compartmentalization appears to be a component of the CO2 Concentrating Mechanism (CCM), which facilitates photosynthetic CO2 fixation as environmental levels of inorganic carbon (Ci) decline. Changes in the spatial distribution of mitochondria in green algae have also been observed under CO2 limiting conditions, although a role for this reorganization in CCM function remains unclear. We used the green microalgae Chlamydomonas reinhardtii to monitor changes in the position and ultrastructure of mitochondrial membranes as cells transition between high CO2 (HC) and Low/Very Low CO2 (LC/VLC). Upon transferring cells to VLC, the mitochondria move from a central to a peripheral location, become wedged between the plasma membrane and chloroplast envelope, and mitochondrial membranes orient in parallel tubular arrays that extend from the cell's apex to its base. We show that these ultrastructural changes require protein and RNA synthesis, occur within 90 min of shifting cells to VLC conditions, correlate with CCM induction and are regulated by the CCM master regulator CIA5. The apico-basal orientation of the mitochondrial membrane, but not the movement of the mitochondrion to the cell periphery, is dependent on microtubules and the MIRO1 protein, which is involved in membrane-microtubule interactions. Furthermore, blocking mitochondrial electron transport in VLC acclimated cells reduces the cell's affinity for inorganic carbon. Overall, our results suggest that CIA5-dependent mitochondrial repositioning/reorientation functions in integrating cellular architecture and energetics with CCM activities and invite further exploration of how intracellular architecture can impact fitness under dynamic environmental conditions.

Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts.

IMPORTANCE: Environmental factors like climate change and captive breeding can impact the gut microbiota and host health. Therefore, conservation efforts for threatened species may benefit from understanding how these factors influence animal microbiomes. Parabasalid protists are members of the mammalian microbiota that can modulate the immune system and impact susceptibility to infections. However, little is known about parabasalids in reptiles. Here, we profile reptile-associated parabasalids in wild and captive reptiles and find that captivity has minimal impact on parabasalid prevalence or diversity. However, because reptiles are cold-blooded (ectothermic), their microbiotas experience wider temperature fluctuation than microbes in warm-blooded animals. To investigate whether extreme weather patterns affect parabasalid-host interactions, we analyzed the gene expression in reptile-associated parabasalids and found that temperature differences significantly alter genes associated with host health. These results expand our understanding of parabasalids in this vulnerable vertebrate group and highlight important factors to be taken into consideration for conservation efforts.

Commensal protists in reptiles display flexible host range and adaptation to ectothermic hosts.

Parabasalid protists recently emerged as keystone members of the mammalian microbiota with important effects on their host's health. However, the prevalence and diversity of parabasalids in wild reptiles and the consequences of captivity and other environmental factors on these symbiotic protists are unknown. Reptiles are ectothermic, and their microbiomes are subject to temperature fluctuations, such as those driven by climate change. Thus, conservation efforts for threatened reptile species may benefit from understanding how shifts in temperature and captive breeding influence the microbiota, including parabasalids, to impact host fitness and disease susceptibility. Here, we surveyed intestinal parabasalids in a cohort of wild reptiles across three continents and compared these to captive animals. Reptiles harbor surprisingly few species of parabasalids compared to mammals, but these protists exhibited a flexible host-range, suggesting specific adaptations to reptilian social structures and microbiota transmission. Furthermore, reptile-associated parabasalids are adapted to wide temperature ranges, although colder temperatures significantly altered the protist transcriptomes, with increased expression of genes associated with detrimental interactions with the host. Our findings establish that parabasalids are widely distributed in the microbiota of wild and captive reptiles and highlight how these protists respond to temperature swings encountered in their ectothermic hosts.

CryoET reveals organelle phenotypes in huntington disease patient iPSC-derived and mouse primary neurons.

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin gene, yielding a Huntingtin protein with an expanded polyglutamine tract. While experiments with patient-derived induced pluripotent stem cells (iPSCs) can help understand disease, defining pathological biomarkers remains challenging. Here, we used cryogenic electron tomography to visualize neurites in HD patient iPSC-derived neurons with varying CAG repeats, and primary cortical neurons from BACHD, deltaN17-BACHD, and wild-type mice. In HD models, we discovered sheet aggregates in double membrane-bound organelles, and mitochondria with distorted cristae and enlarged granules, likely mitochondrial RNA granules. We used artificial intelligence to quantify mitochondrial granules, and proteomics experiments reveal differential protein content in isolated HD mitochondria. Knockdown of Protein Inhibitor of Activated STAT1 ameliorated aberrant phenotypes in iPSC- and BACHD neurons. We show that integrated ultrastructural and proteomic approaches may uncover early HD phenotypes to accelerate diagnostics and the development of targeted therapeutics for HD.

Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis.

Intracellular bacterial pathogens Francisella novicida and the Live Vaccine Strain (LVS) are recognized in the macrophage cytosol by the AIM2 inflammasome, which leads to the activation of caspase-1 and the processing and secretion of active IL-1ß, IL-18 and pyroptosis. Previous studies have reported that F. novicida and LVS mutants in specific genes (e.g. FTT0584, mviN and ripA) induce elevated inflammasome activation and hypercytotoxicity in host cells, leading to the proposal that F. novicida and LVS may have proteins that actively modulate inflammasome activation. However, there has been no direct evidence of such inflammasome evasion mechanisms. Here, we demonstrate for the first time that the above mutants, along with a wide range of F. novicida hypercytotoxic mutants that are deficient for membrane-associated proteins (ΔFTT0584, ΔmviN, ΔripA, ΔfopA and ΔFTN1217) or deficient for genes involved in O-antigen or LPS biosynthesis (ΔwbtA and ΔlpxH) lyse more intracellularly, thus activating increased levels of AIM2-dependent pyroptosis and other innate immune signalling pathways. This suggests that an inflammasome-specific evasion mechanism may not be present in F. novicida and LVS. Furthermore, future studies may need to consider increased bacterial lysis as a possible cause of elevated stimulation of multiple innate immune pathways when the protein composition or surface carbohydrates of the bacterial membrane is altered.

Visualization of the distribution of covalently cross-linked hydrogels in CLARITY brain-polymer hybrids for different monomer concentrations.

CLARITY is a tissue preservation and optical clearing technique whereby a hydrogel is formed directly within the architectural confines of ex vivo brain tissue. In this work, the extent of polymer gel formation and crosslinking within tissue was assessed using Raman spectroscopy and rheology on CLARITY samples prepared with a range of acrylamide monomer (AAm) concentrations (1%, 4%, 8%, 12% w/v). Raman spectroscopy of individual neurons within hybrids revealed the chemical presence and distribution of polyacrylamide within the mouse hippocampus. Consistent with rheological measurements, lower %AAm concentration decreased shear elastic modulus G', providing a practical correlation with sample permeability and protein retention. Permeability of F(ab)'2 secondary fluorescent antibody changes from 9.3 to 1.4 µm2 s-1 going from 1 to 12%. Notably, protein retention increased linearly relative to standard PFA-fixed tissue from 96.6% when AAm concentration exceeded 1%, with 12% AAm samples retaining up to ~ 99.3% native protein. This suggests that though 1% AAm offers high permeability, additional %AAm may be required to enhance protein. Our quantitative results on polymer distribution, stability, protein retention, and macromolecule permeability can be used to guide the design of future CLARITY-based tissue-clearing solutions, and establish protocols for characterization of novel tissue-polymer hybrid biomaterials using chemical spectroscopy and rheology.

SEM and TEM for identification of capsular fibrosis and cellular behavior around breast implants - a descriptive analysis.

BACKGROUND: Capsular fibrosis (CF) is the most common long-term complication in implant-based breast augmentation. It is well accepted that the foreign body response (FBR) instigates the development of fibrotic disease. Our study aims to compare murine and human samples of CF and describe the cellular and extracellular matrix (ECM) composition using scanning and transmission electron microscopy (SEM and TEM). RESULTS: Miniature microtextured silicone breast implants were implanted in mice and subsequently harvested at days 15, 30, and 90 post-operation. Isolated human capsules with the most aggravated form of CF (Baker IV) were harvested post-operation. Both were analyzed with SEM and TEM to assess cellular infiltration and ECM structure. An architectural shift of collagen fiber arrangement from unidirectional to multidirectional was observed at day 90 when compared to days 15 and 30. Fibrosis was observed with an increase of histiocytic infiltration. Moreover, bacterial accumulation was seen around silicone fragments. These findings were common in both murine and human capsules. CONCLUSIONS: This murine model accurately recapitulates CF found in humans and can be utilized for future research on cellular invasion in capsular fibrosis. This descriptive study helps to gain a better understanding of cellular mechanisms involved in the FBR. Increases of ECM and cellularity were observed over time with SEM and TEM analysis.

Contributions of Francisella tularensis subsp. novicida chitinases and Sec secretion system to biofilm formation on chitin.

Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studying F. tularensis pathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology of Francisella species. F. tularensis has been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show that F. tularensis subsp. novicida forms biofilms during the colonization of chitin surfaces. The ability of F. tularensis to persist using chitin as a sole carbon source is dependent on chitinases, since mutants lacking chiA or chiB are attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1 and secB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-type F. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation by F. tularensis promotes persistence on chitin surfaces. Further study of the interaction of F. tularensis with the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.

Genetically targeted chemical assembly of functional materials in living cells, tissues, and animals.

The structural and functional complexity of multicellular biological systems, such as the brain, are beyond the reach of human design or assembly capabilities. Cells in living organisms may be recruited to construct synthetic materials or structures if treated as anatomically defined compartments for specific chemistry, harnessing biology for the assembly of complex functional structures. By integrating engineered-enzyme targeting and polymer chemistry, we genetically instructed specific living neurons to guide chemical synthesis of electrically functional (conductive or insulating) polymers at the plasma membrane. Electrophysiological and behavioral analyses confirmed that rationally designed, genetically targeted assembly of functional polymers not only preserved neuronal viability but also achieved remodeling of membrane properties and modulated cell type-specific behaviors in freely moving animals. This approach may enable the creation of diverse, complex, and functional structures and materials within living systems.

The antifungal and Cryptococcus neoformans virulence attenuating activity of Pelargonium sidoides extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Limitations of clinical antifungal treatments and drug-resistance are drivers of the search for novel antifungal strategies. Extracts prepared from the tubers of the medicinal plant, Pelargonium sidoides, are known for their antiviral and antibacterial activities and are used in ethnomedicine for the treatment of acute respiratory infections. Their impact on fungi has not been well characterised. Here, we provide a first report on the antifungal activity of a P. sidoides aerial tissue extract against Cryptococcus neoformans as well as the effects of both tuber and aerial tissue extracts on selected virulence factors. AIM OF THE STUDY: Novel antimicrobial strategies that target multiple cellular pathways or make use of anti-pathogenic compounds that inhibit virulence factors have been proposed. This work aimed to evaluate P. sidoides plant parts for their anticryptococcal activity and antipathogenic properties on selected virulence factors. MATERIALS AND METHODS: The antifungal activity of crude P. sidoides tuber and aerial tissue extracts (15% m/m ethanol) were compared using a modified colourimetric antifungal susceptibility test. Fungicidal activity of the extracts was confirmed by plate counts. To test yeast resistance to the extracts, it was conditioned by multiple passages in sub-lethal doses followed by antifungal susceptibility testing. Cytotoxicity of the extracts was tested with a blood agar haemolysis assay. Extracts were evaluated for the presence of multiple bioactive compounds by solid-phase fractionation and visualisation by thin-layer chromatography in combination with bioassays. The influence of extracts on the production of the polysaccharide capsule, ergosterol content as well as laccase and urease activities were also evaluated. Cell surface variations after extract exposure were visualised by scanning electron microscopy (SEM). RESULTS: Both tuber and aerial tissue extracts were fungicidal and contained multiple bioactive compounds which constrained the development of antifungal resistance. No haemolytic activity was observed, and the extracts did not appear to target ergosterol biosynthesis. However, the extracts displayed anti-pathogenic potential by significantly inhibiting laccase and urease activity while also significantly reducing capsule size. SEM revealed notable cell surface variations and provided support for the observed reduction in capsule size. CONCLUSIONS: Our results provide support to the exploration of medicinal plants as sources of alternative antifungal therapies and the potential use of multicomponent inhibition and or virulence attenuation for next-generation treatment strategies. Our data also provide relevant information that may support the further use of P. sidoides in traditional medicines as well as in commercialised phytopharmaceuticals.

A Combination of Itraconazole and Amiodarone Is Highly Effective against Trypanosoma cruzi Infection of Human Stem Cell-Derived Cardiomyocytes.

Trypanosoma cruzi is the etiologic agent of Chagas disease (CD), which can result in severe cardiomyopathy. Trypanosoma cruzi is endemic to the Americas, and of particular importance in Latin America. In the United States and other non-endemic countries, rising case numbers have also been observed. The currently used drugs are benznidazole (BNZ) and nifurtimox, which have limited efficacy during chronic infection. We repurposed itraconazole (ICZ), originally an antifungal, in combination with amiodarone (AMD), an antiarrhythmic, with the goal of interfering with T. cruzi infection. Human pluripotent stem cells (hiPSCs) were differentiated into cardiomyocytes (hiPSC-CMs). Vero cells or hiPSC-CMs were infected with T. cruzi trypomastigotes of the II or I strain in the presence of ICZ and/or AMD. After 48 hours, cells were Giemsa stained, and infection and multiplication were evaluated microscopically. Trypanosoma cruzi infection and multiplication were evalutated also by electron microscopy. BNZ was used as a reference compound. Cell metabolism in the presence of test substances was assessed. Itraconazole and AMD showed strain- and dose-dependent interference with T. cruzi infection and multiplication in Vero cells or hiPSC-CMs. Combinations of ICZ and AMD were more effective against T. cruzi than the single substances, or BNZ, without affecting host cell metabolism, and better preserving host cell integrity during infection. Our in vitro data in hiPSC-CMs suggest that a combination of ICZ and AMD might serve as a treatment option for CD in patients, but that different responses due to T. cruzi strain differences have to be taken into account.