Tryptophol Acetate and Tyrosol Acetate, Small-Molecule Metabolites Identified in a Probiotic Mixture, Inhibit Hyperinflammation.

Probiotic fermented foods are perceived as contributing to human health; however, solid evidence for their presumptive therapeutic systemic benefits is generally lacking. Here we report that tryptophol acetate and tyrosol acetate, small-molecule metabolites secreted by the probiotic milk-fermented yeast Kluyveromyces marxianus, inhibit hyperinflammation (e.g., "cytokine storm"). Comprehensive in vivo and in vitro analyses, employing LPS-induced hyperinflammation models, reveal dramatic effects of the molecules, added in tandem, on mice morbidity, laboratory parameters, and mortality. Specifically, we observed attenuated levels of the proinflammatory cytokines IL-6, IL-1α, IL-1ß, and TNF-α and reduced reactive oxygen species. Importantly, tryptophol acetate and tyrosol acetate did not completely suppress proinflammatory cytokine generation, rather brought their concentrations back to baseline levels, thus maintaining core immune functions, including phagocytosis. The anti-inflammatory effects of tryptophol acetate and tyrosol acetate were mediated through downregulation of TLR4, IL-1R, and TNFR signaling pathways and increased A20 expression, leading to NF-kB inhibition. Overall, this work illuminates phenomenological and molecular details underscoring anti-inflammatory properties of small molecules identified in a probiotic mixture, pointing to potential therapeutic avenues against severe inflammation.

Bcl-2-Homology-Only Proapoptotic Peptides Modulate ß-Amyloid Aggregation and Toxicity.

Aggregation of the ß-Amyloid (Aß) peptide in brain tissues is the hallmark of Alzheimer's disease (AD). While Aß is presumed to be insidiously involved in the disease's pathophysiology, concrete mechanisms accounting for the role of Aß in AD are yet to be deciphered. While Aß has been primarily identified in the extracellular space, the peptide also accumulates in cellular compartments such as mitochondria and lysosomes and impairs cellular functions. Here, we show that prominent proapoptotic peptides associated with the mitochondrial outer membrane, the Bcl-2-homology-only peptides BID, PUMA, and NOXA, exert significant and divergent effects upon aggregation, cytotoxicity, and membrane interactions of Aß42, the main Aß homolog. Interestingly, we show that BID and PUMA accelerated aggregation of Aß42, reduced Aß42-induced toxicity and mitochondrial disfunction, and inhibited Aß42-membrane interactions. In contrast, NOXA exhibited opposite effects, reducing Aß42 fibril formation, affecting more pronounced apoptotic effects and mitochondrial disfunction, and enhancing membrane interactions of Aß42. The effects of BID, PUMA, and NOXA upon the Aß42 structure and toxicity may be linked to its biological properties and affect pathophysiological features of AD.

Sniffing Bacteria with a Carbon-Dot Artificial Nose.

HIGHLIGHTS: Novel artificial nose based upon electrode-deposited carbon dots (C-dots). Significant selectivity and sensitivity determined by "polarity matching" between the C-dots and gas molecules. The C-dot artificial nose facilitates, for the first time, real-time, continuous monitoring of bacterial proliferation and discrimination among bacterial species, both between Gram-positive and Gram-negative bacteria and between specific strains. Machine learning algorithm furnishes excellent predictability both in the case of individual gases and for complex gas mixtures. Continuous, real-time monitoring and identification of bacteria through detection of microbially emitted volatile molecules are highly sought albeit elusive goals. We introduce an artificial nose for sensing and distinguishing vapor molecules, based upon recording the capacitance of interdigitated electrodes (IDEs) coated with carbon dots (C-dots) exhibiting different polarities. Exposure of the C-dot-IDEs to volatile molecules induced rapid capacitance changes that were intimately dependent upon the polarities of both gas molecules and the electrode-deposited C-dots. We deciphered the mechanism of capacitance transformations, specifically substitution of electrode-adsorbed water by gas molecules, with concomitant changes in capacitance related to both the polarity and dielectric constants of the vapor molecules tested. The C-dot-IDE gas sensor exhibited excellent selectivity, aided by application of machine learning algorithms. The capacitive C-dot-IDE sensor was employed to continuously monitor microbial proliferation, discriminating among bacteria through detection of distinctive "volatile compound fingerprint" for each bacterial species. The C-dot-IDE platform is robust, reusable, readily assembled from inexpensive building blocks and constitutes a versatile and powerful vehicle for gas sensing in general, bacterial monitoring in particular.

Inhibition of Staphylococcus aureus biofilm-forming functional amyloid by molecular tweezers.

Biofilms are rigid and largely impenetrable three-dimensional matrices constituting virulence determinants of various pathogenic bacteria. Here, we demonstrate that molecular tweezers, unique supramolecular artificial receptors, modulate biofilm formation of Staphylococcus aureus. In particular, the tweezers affect the structural and assembly properties of phenol-soluble modulin α1 (PSMα1), a biofilm-scaffolding functional amyloid peptide secreted by S. aureus. The data reveal that CLR01, a diphosphate tweezer, exhibits significant S. aureus biofilm inhibition and disrupts PSMα1 self-assembly and fibrillation, likely through inclusion of lysine side chains of the peptide. In comparison, different peptide binding occurs in the case of CLR05, a tweezer containing methylenecarboxylate units, which exhibits lower affinity for the lysine residues yet disrupts S. aureus biofilm more strongly than CLR01. Our study points to a possible role for molecular tweezers as potent biofilm inhibitors and antibacterial agents, particularly against untreatable biofilm-forming and PSM-producing bacteria, such as methicillin-resistant S. aureus.

The pro-apoptotic domain of BIM protein forms toxic amyloid fibrils.

BIM is a key apoptotic protein, participating in diverse cellular processes. Interestingly, recent studies have hypothesized that BIM is associated with the extensive neuronal cell death encountered in protein misfolding diseases, such as Alzheimer's disease. Here, we report that the core pro-apoptotic domain of BIM, the BIM-BH3 motif, forms ubiquitous amyloid fibrils. The BIM-BH3 fibrils exhibit cytotoxicity, disrupt mitochondrial functions, and modulate the structures and dynamics of mitochondrial membrane mimics. Interestingly, a slightly longer peptide in which BIM-BH3 was flanked by four additional residues, widely employed as a model of the pro-apoptotic core domain of BIM, did not form fibrils, nor exhibited cell disruptive properties. The experimental data suggest a new mechanistic role for the BIM-BH3 domain, and demonstrate, for the first time, that an apoptotic peptide forms toxic amyloid fibrils.

Interactions between BIM Protein and Beta-Amyloid May Reveal a Crucial Missing Link between Alzheimer's Disease and Neuronal Cell Death.

Extensive neuronal cell death is among the pathological hallmarks of Alzheimer's disease. While neuron death is coincident with formation of plaques comprising the beta-amyloid (Aß) peptide, a direct causative link between Aß (or other Alzheimer's-associated proteins) and cell toxicity is yet to be found. Here we show that BIM-BH3, the primary proapoptotic domain of BIM, a key protein in varied apoptotic cascades of which elevated levels have been found in brain cells of patients afflicted with Alzheimer's disease, interacts with the 42-residue amyloid isoform Aß42. Remarkably, BIM-BH3 modulated the structure, fibrillation pathway, aggregate morphology, and membrane interactions of Aß42. In particular, BIM-BH3 inhibited Aß42 fibril-formation, while it simultaneously enhanced protofibril assembly. Furthermore, we discovered that BIM-BH3/Aß42 interactions induced cell death in a human neuroblastoma cell model. Overall, our data provide a crucial mechanistic link accounting for neuronal cell death in Alzheimer's disease patients and the participation of both BIM and Aß42 in the neurotoxicity process.

Vesicle-Based Assays to Study Membrane Interactions of Amyloid Peptides.

The growing interest in membrane interactions of amyloidogenic peptides and proteins emanates from the realization that lipid bilayers and membranes play central roles in the toxicity and pathological pathways of amyloid diseases. This chapter presents experimental schemes designed to study membrane interactions and membrane-induced fibrillation of amyloid peptides.

Chiral modulation of amyloid beta fibrillation and cytotoxicity by enantiomeric carbon dots.

Enantiomeric carbon dots (C-dots) synthesized from l-lysine or d-lysine, modulate aggregation and cytotoxicity of amyloid beta-42 (Aß42), the primary constituent of the amyloid plaques associated with Alzheimer's disease. In particular, l-Lys-C-dots dramatically remodeled Aß42 secondary structure and fibril morphologies, as well as inhibited Aß42 cytotoxicity and membrane interactions.

Carbon and Nitrogen Based Nanosheets as Fluorescent Probes with Tunable Emission.

2D carbon and nitrogen based semiconductors (CN) have attracted widespread attention for their possible use as low-cost and environmentally friendly materials for various applications. However, their limited solution-dispersibility and the difficulty in preparing exfoliated sheets with tunable photophysical properties restrain their exploitation in imaging-related applications. Here, the synthesis of carbon and nitrogen organic scaffolds with highly tunable optical properties, excellent dispersion in water and DMSO, and good bioimaging properties is reported. Tailored photophysical and chemical properties are acquired by the synthesis of new starting monomers containing different substituent chemical groups with varying electronic properties. Upon monomer condensation at moderate temperature, 350 °C, the starting chemical groups are fully preserved in the final CN. The low condensation temperature and the effective molecular-level modification of the CN scaffold lead to well-dispersed photoluminescent CN thin sheets with a wide range of emission wavelengths. The good bioimaging properties and the tunable fluorescence properties are exemplified by in situ visualization of giant unilamellar vesicles in a buffered aqueous solution as a model system. This approach opens the possibility for the design of tailor-made CN materials with tunable photophysical and chemical properties toward their exploitation in various fields, such as photocatalysis, bioimaging, and sensing.

Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide (hIAPP).

The dramatic expansion of nanotechnology applications, particularly the advent of nanomaterials and nanoparticles (NPs) into the consumer economy, have led to heightened awareness of their potential health risks. This study examines the impact of several NPs upon membrane-induced aggregation and bilayer interactions of the human Islet amyloid polypeptide (hIAPP). We report that several NPs - polymeric NPs, TiO2 NPs, and Au NPs displaying coating layers exhibiting different electrostatic charges - did not significantly interfere with the fibrillation process and fibril morphology of hIAPP, both in buffer or in biomimetic DMPC:DMPG vesicle solutions. Spectroscopic and microscopic analyses suggest, in fact, that the NPs promoted membrane-induced fibrillation. Importantly, we find that all the NPs examined, regardless of composition or surface properties, gave rise to more pronounced, synergistic bilayer interactions when co-incubated with hIAPP. NP-enhanced bilayer interactions of hIAPP might point to possible toxicity and pathogenicity risks of amyloidogenic peptides in the presence of NPs.

Reciprocal Interactions between Membrane Bilayers and S. aureus PSMα3 Cross-α Amyloid Fibrils Account for Species-Specific Cytotoxicity.

Phenol-soluble modulin α3 (PSMα3) is a functional amyloid secreted by the pathogenic bacterium Staphylococcus aureus. This 22-residue peptide serves as a key virulence determinant, toxic to human cells via the formation of unique cross-α amyloid-like fibrils. We demonstrate that bilayer vesicles accelerated PSMα3 fibril formation, and the fibrils, in turn, inserted deeply into bilayers mimicking mammalian cell membranes, accounting for PSMα3 cellular toxicity. Importantly, a mere amphipathic helical conformation was not a sufficient determinant for membrane-activity of PSMα3, pointing to the functional role of cross-α fibrils. In contrast to deep insertion of PSMα3 into mammalian membrane bilayers, the peptide only interacted with the surface of bilayers mimicking bacterial membranes, which might be related to its lack of antibacterial activity. Together, our data provide mechanistic insight into species-specific toxicity of a key bacterial amyloid virulence factor via reciprocal interactions with membranes, and open new perspectives into amyloid-related cytotoxicity mediated by helical fibril structures.

Bacterial Model Membranes Reshape Fibrillation of a Functional Amyloid Protein.

Biofilms are aggregates of cells that form surface-associated communities. The cells in biofilms are interconnected with an extracellular matrix, a network that is made mostly of polysaccharides, proteins, and sometimes nucleic acids. Some extracellular matrix proteins form fibers, termed functional amyloid or amyloid-like, to differentiate their constructive function from disease-related amyloid fibers. Recent functional amyloid assembly studies have neglected their interaction with membranes, despite their native formation in a cellular environment. Here, we use TasA, a major matrix protein in biofilms of the soil bacterium Bacillus subtilis, as a model functional amyloid protein and ask whether the bacterial functional amyloid interacts with membranes. Using biochemical, spectroscopic, and microscopic tools, we show that TasA interacts distinctively with bacterial model membranes and that this interaction mutually influences the morphology and structure of the protein and the membranes. At the protein level, fibers of similar structure and morphology are formed in the absence of membranes and in the presence of eukaryotic model membranes. However, in the presence of bacterial model membranes, TasA forms disordered aggregates with a different ß sheet signature. At the membrane level, fluorescence microscopy and anisotropy measurements indicate that bacterial membranes deform more considerably than eukaryotic membranes upon interaction with TasA. Our findings suggest that TasA penetrates bacterial more than eukaryotic model membranes and that this leads to membrane disruption and to reshaping the TasA fiber formation pathway. Considering the important role of TasA in providing integrity to biofilms, our study may direct the design of antibiofilm drugs to the protein-membrane interface.

Membrane Determinants Affect Fibrillation Processes of ß-Sheet Charged Peptides.

Assembly of fibrillar peptide structures is dependent both upon their intrinsic propensities toward ß-structure formation, as well as on structural modulation by external molecular factors. ß-sheet structures may either be designed to form useful assemblies or be the undesired consequence of protein denaturation to toxic amyloid structures in several neurodegenerative diseases. Membrane bilayers have been implicated as primary initiators and modulators of amyloid fibrillation and the reasons for this effect are yet to be elucidated. Here, we employed a set of three charged peptides having the tendency to form ß-sheet fibrils, to investigate the effect of zwitterionic and negatively charged bilayer vesicles on their assembly structures. Microscopic and spectroscopic experiments revealed intimate relationship between peptide/membrane charges and fibrillation properties. Electrostatic attraction was apparent between oppositely charged peptides and vesicles; however, such interactions did not appear to significantly modulate fibril morphologies of either the net anionic peptide or the cationic one. Yet, a dramatic structural effect was observed when the nominal zwitterionic peptide underwent fibrillation in the presence of negatively charged vesicles. Assemblies of this peptide display a net positive charge, which facilitated the counterionic interactions with the vesicles. Furthermore, these interactions templated a unique twisted fiber morphology demonstrating the dramatic effect membrane-mediated interactions exert on fibril morphologies.

Bacoside-A, an Indian Traditional-Medicine Substance, Inhibits ß-Amyloid Cytotoxicity, Fibrillation, and Membrane Interactions.

Bacoside-A, a family of compounds extracted from the Bacopa monniera plant, is a folk-medicinal substance believed to exhibit therapeutic properties, particularly enhancing cognitive functions and improving memory. We show that bacoside-A exerted significant inhibitory effects upon cytotoxicity, fibrillation, and particularly membrane interactions of amyloid-beta (1-42) (Aß42), the peptide playing a prominent role in Alzeheimer's disease progression and toxicity. Specifically, preincubation of bacoside-A with Aß42 significantly reduced cell toxicity and inhibited fibril formation both in buffer solution and, more significantly, in the presence of membrane vesicles. In parallel, spectroscopic and microscopic analyses reveal that bacoside-A blocked membrane interactions of Aß42, while formation of Aß42 oligomers was not disrupted. These interesting phenomena suggest that inhibition of Aß42 oligomer assembly into mature fibrils, and blocking membrane interactions of the oligomers are likely the underlying factors for ameliorating amyloid toxicity by bacoside-A and its putative physiological benefits.

Bacoside-A, an anti-amyloid natural substance, inhibits membrane disruption by the amyloidogenic determinant of prion protein through accelerating fibril formation.

Bacosides, class of compounds extracted from the Bacopa monniera plant, exhibit interesting therapeutic properties, particularly enhancing cognitive functions and putative anti-amyloid activity. We show that bacoside-A exerted significant effects upon fibrillation and membrane interactions of the amyloidogenic fragment of the prion protein [PrP(106-126)]. Specifically, when co-incubated with PrP(106-126), bacoside-A accelerated fibril formation in the presence of lipid bilayers and in parallel inhibited bilayer interactions of the peptide aggregates formed in solution. These interesting phenomena were studied by spectroscopic and microscopic techniques, which suggest that bacoside A-promoted fibrillation reduced the concentration of membrane-active pre-fibrillar species of the prion fragment. This study suggests that induction of fibril formation and corresponding inhibition of membrane interactions are likely the underlying factors for ameliorating amyloid protein toxicity by bacoside-A.

Lipid-Bilayer Dynamics Probed by a Carbon Dot-Phospholipid Conjugate.

Elucidating the dynamic properties of membranes is important for understanding fundamental cellular processes and for shedding light on the interactions of proteins, drugs, and viruses with the cell surface. Dynamic studies of lipid bilayers have been constrained, however, by the relatively small number of pertinent molecular probes and the limited physicochemical properties of the probes. We show that a lipid conjugate comprised of a fluorescent carbon dot (C-dot) covalently attached to a phospholipid constitutes a versatile and effective vehicle for studying bilayer dynamics. The C-dot-modified phospholipids readily incorporated within biomimetic membranes, including solid-supported bilayers and small and giant vesicles, and inserted into actual cellular membranes. We employed the C-dot-phospholipid probe to elucidate the effects of polymyxin-B (a cytolytic peptide), valproic acid (a lipophilic drug), and amyloid-ß (a peptide associated with Alzheimer's disease) upon bilayer fluidity and lipid dynamics through the application of various biophysical techniques.

Toxicity inhibitors protect lipid membranes from disruption by Aß42.

Although the precise molecular factors linking amyloid ß-protein (Aß) to Alzheimer's disease (AD) have not been deciphered, interaction of Aß with cellular membranes has an important role in the disease. However, most therapeutic strategies targeting Aß have focused on interfering with Aß self-assembly rather than with its membrane interactions. Here, we studied the impact of three toxicity inhibitors on membrane interactions of Aß42, the longer form of Aß, which is associated most strongly with AD. The inhibitors included the four-residue C-terminal fragment Aß(39-42), the polyphenol (-)-epigallocatechin-3-gallate (EGCG), and the lysine-specific molecular tweezer, CLR01, all of which previously were shown to disrupt different steps in Aß42 self-assembly. Biophysical experiments revealed that incubation of Aß42 with each of the three modulators affected membrane interactions in a distinct manner. Interestingly, EGCG and CLR01 were found to have significant interaction with membranes themselves. However, membrane bilayer disruption was reduced when the compounds were preincubated with Aß42, suggesting that binding of the assembly modulators to the peptide attenuated their membrane interactions. Importantly, our study reveals that even though the three tested compounds affect Aß42 assembly differently, membrane interactions were significantly inhibited upon incubation of each compound with Aß42, suggesting that preventing the interaction of Aß42 with the membrane contributes substantially to inhibition of its toxicity by each compound. The data suggest that interference with membrane interactions is an important factor for Aß42 toxicity inhibitors and should be taken into account in potential therapeutic strategies, in addition to disruption or remodeling of amyloid assembly.

Unilamellar vesicles from amphiphilic graphene quantum dots.

Graphene quantum dots (GQDs) have attracted considerable interest due to their unique physicochemical properties and various applications. For the first time it is shown that GQDs surface-functionalized with hydrocarbon chains (i.e., amphiphilic GQDs) self-assemble into unilamellar spherical vesicles in aqueous solution. The amphiphilic GQD vesicles exhibit multicolor luminescence that can be readily exploited for membrane studies by fluorescence spectroscopy and microscopy. The GQD vesicles were used for microscopic analysis of membrane interactions and disruption by the peptide beta-amyloid.

Membrane analysis with amphiphilic carbon dots.

Newly-synthesized amphiphilic carbon dots were used for spectroscopic analysis and multicolour microscopic imaging of membranes and live cells. We show that Förster resonance energy transfer (FRET) occurred from the amphiphilic carbon dots to different membrane-associated fluorescence acceptors. The amphiphilic carbon dots enabled imaging of membrane disruption by the beta-amyloid peptide.