Caenorhabditis elegans endorse bacterial nanocellulose fibers as functional dietary Fiber reducing lipid markers.

Bacterial nanocellulose (BNC) is a promising dietary fiber with potential as a functional food additive. We evaluated BNC fibers (BNCf) in the Caenorhabditis elegans model to obtain insight into the BNCf's biointeraction with its gastrointestinal tract while reducing the variables of higher complex animals. BNCf were uptaken and excreted by worms without crossing the intestinal barrier, confirming its biosafety regarding survival rate, reproduction, and aging for concentrations up to 34 µg/ml BNCf. However, a slight decrease in the worms' length was detected. A possible nutrient shortage or stress produced by BNCf was discarded by measuring stress and chemotactic response pathways. Besides, we detected a lipid-lowering effect of BNCf in N2 C. elegans in normal and high-caloric diets. Oxidative damage was computed in N2 worms and Rac1/ced-10 mutants. The GTPase Rac1 is involved in neurological diseases, where its dysregulation enhances ROS production and neuronal damage. BNCf reduced the lipid oxidative markers produced by ROS species in this worm strain. Finally, we detected that BNCf activated the genetic expression of the immunological response and lipid catabolic process. These results strengthen the use of BNCf as a functional dietary fiber and encourage the potential treatment of neurological disease by modulating diet.

Arrhythmic Effects Evaluated on Caenorhabditis elegans: The Case of Polypyrrole Nanoparticles.

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes' pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs' biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs.

Roadmap for Postnatal Brain Maturation: Changes in Gray and White Matter Composition during Development Measured by Fourier Transformed Infrared Microspectroscopy.

Key events in postnatal brain development, such as neuronal migration, synaptogenesis, and myelination, shape the adult brain. These events are reflected in changes in gray and white matter (GM and WM) occurring during this period. Therefore, precise knowledge of GM and WM composition in perinatal brain development is crucial to characterizing brain formation as well as the neurodevelopmental disruption observed in diseases such as autism and schizophrenia. In this study, we combined histochemical and immunohistochemical staining with biochemical and biophysical analyses using Fourier transform infrared (IR) microspectroscopy (µFTIR) to better understand the chemical changes during postnatal developmental myelination. For this purpose, we analyzed the GM and WM in the mouse brain and cerebellum (strain C57BL/6) from postnatal day 0 (P0) to day P28 and established presumed correlations between staining and IR data. IR spectra allowed the (i) quantification of lipid and protein content through the CH2/amide I ratio, (ii) determination of chemical characteristics of lipids, such as the presence of unsaturated bonds in the carbonate chain or carbonyls from ester groups in the polar head, and (iii) determination of the protein secondary structure (α-helix and intramolecular ß-sheets). The results indicate that the increase in the CH2/amide I ratio calculated from the µFTIR data correlates well with lipid histochemical staining. IR data indicated a change in the lipid composition in WM since carbonyl and unsaturated olefinic groups do not increase when lipids accumulate during myelination. Our correlation analysis between IR data and immunohistochemical staining of myelin-associated proteins revealed that myelin oligodendrocyte protein correlated well with lipid accumulation, while myelin basic protein appeared before lipid modifications, which indicated that myelin-associated proteins and lipid deposition were not synchronic. These events were related to a decrease in the intramolecular ß/α protein ratio. Our results indicate that lipids and proteins in WM substantially change their composition due to primary myelination, and according to results obtained from staining, these modifications are better described by lipid histochemical staining than by immunohistochemistry against myelin-related proteins. In conclusion, µFTIR can be a useful technique to study WM during perinatal development and provide detailed information about alterations in the chemical composition related to neurodevelopmental diseases.

Properties and cellular uptake of photo-triggered mixed metallosurfactant vesicles intended for controlled CO delivery in gas therapy.

The scientific relevance of carbon monoxide has increased since it was discovered that it is a gasotransmitter involved in several biological processes. This fact stimulated research to find a secure and targeted delivery and lead to the synthesis of CO-releasing molecules. In this paper we present a vesicular CO delivery system triggered by light composed of a synthetized metallosurfactant (TCOL10) with two long carbon chains and a molybdenum-carbonyl complex. We studied the characteristics of mixed TCOL10/phosphatidylcholine metallosomes of different sizes. Vesicles from 80 to 800 nm in diameter are mainly unilamellar, do not disaggregate upon dilution, in the dark are physically and chemically stable at 4 °C for at least one month, and exhibit a lag phase of about 4 days before they show a spontaneous CO release at 37 °C. Internalization of metallosomes by cells was studied as function of the incubation time, and vesicle concentration and size. Results show that large vesicles are more efficiently internalized than the smaller ones in terms of the percentage of cells that show TCOL10 and the amount of drug that they take up. On balance, TCOL10 metallosomes constitute a promising and viable approach for efficient delivery of CO to biological systems.

Aß40 Aggregation under Changeable Conditions.

Homeostasis is crucial for cell function, and disturbances in homeostasis can lead to health disorders. Under normal conditions, intracellular pH is maintained between 7.35 and 7.45. Altered endosomal and lysosomal pH together with a general drop in brain pH are associated with the aggregation of amyloid-ß-peptide (Aß) and the development of Alzheimer's disease. Under acidic conditions, close to the Aß isoelectric point, the absence of charges favors the formation of intermolecular contacts and promotes aggregation. Here, we analyzed how pH levels affect the aggregation of Aß40 considering the variations in brain pH and the coexistence of different aggregated conformations. Our results suggest that different macromolecular conformations can interact with each other and influence the aggregation process. In addition, we showed that neutral pH and physiological salt concentrations favor a slow aggregation, resulting in ordered, stable fibrils, with low cytotoxic effects. Overall, we highlight the complexity of the aggregation processes occurring in different physiological and pathological environments.

Monitoring reversion of hepatitis C virus-induced cellular alterations by direct-acting antivirals using cryo soft X-ray tomography and infrared microscopy.

Hepatitis C virus (HCV) is an enveloped RNA virus. One of the hallmarks of HCV infection is a rearrangement of the host cell membranes, known as the `membranous web'. Full-field cryo soft X-ray tomography (cryo-SXT) in the water-window energy range (284-543 eV) was performed on the MISTRAL beamline to investigate, in whole unstained cells, the morphology of the membranous rearrangements induced in HCV replicon-harbouring cells in conditions close to the living physiological state. All morphological alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved antivirals (direct-acting antivirals; DAAs) for HCV infection. Correlatively combining cryo-SXT and 2D synchrotron-based infrared microscopy provides critical information on the chemical nature of specific infection-related structures, which allows specific patterns of the infection process or the DAA-mediated healing process to be distinguished.

In situ identification and G4-PPI-His-Mal-dendrimer-induced reduction of early-stage amyloid aggregates in Alzheimer's disease transgenic mice using synchrotron-based infrared imaging.

Amyloid plaques composed of Aß amyloid peptides and neurofibrillary tangles are a pathological hallmark of Alzheimer Disease. In situ identification of early-stage amyloid aggregates in Alzheimer's disease is relevant for their importance as potential targets for effective drugs. Synchrotron-based infrared imaging is here used to identify early-stage oligomeric/granular aggregated amyloid species in situ in the brain of APP/PS1 transgenic mice for the first time. Also, APP/PS1 mice show fibrillary aggregates at 6 and 12 months. A significant decreased burden of early-stage aggregates and fibrillary aggregates is obtained following treatment with poly(propylene imine) dendrimers with histidine-maltose shell (a neurodegenerative protector) in 6-month-old APP/PS1 mice, thus demonstrating their putative therapeutic properties of in AD models. Identification, localization, and characterization using infrared imaging of these non-fibrillary species in the cerebral cortex at early stages of AD progression in transgenic mice point to their relevance as putative pharmacological targets. No less important, early detection of these structures may be useful in the search for markers for non-invasive diagnostic techniques.

Specific microglial phagocytic phenotype and decrease of lipid oxidation in white matter areas during aging: Implications of different microenvironments.

Physiological aging is characterized by an imbalance of pro-inflammatory and anti-inflammatory mediators leading to neuroinflammation. Microglial cells, which are highly regulated by the local microenvironment, undergo specific changes depending upon the brain area during aging. The aim of this study was to evaluate the influence of age over microglial cells along different brain areas and microenvironments. For this purpose, transgenic mice with overproduction of either the anti-inflammatory IL-10 cytokine or the pro-inflammatory IL-6 cytokine were used. Our results show that, during aging, microglial cells located in white matter (WM) areas maintain their phagocytic capacity but present a specific phagocytic phenotype with receptors involved in myelin recognition, arguing for aging-derived myelin damage. Whereas IL-10 overproduction anticipates the age-related microglial phagocytic phenotype, maintaining it over time, IL-6 overproduction exacerbates this phenotype in aging. These modifications were linked with a higher efficiency of myelin engulfment by microglia in aged transgenic animals. Moreover, we show, in a novel way, lower lipid oxidation during aging in WM areas, regardless of the genotype. The novelty of the insights presented in this study open a window to deeply investigate myelin lipid oxidation and the role of microglial cells in its regulation during physiological aging.

Synchrotron X-ray Fluorescence and FTIR Signatures for Amyloid Fibrillary and Nonfibrillary Plaques.

Amyloid plaques are one of the principal hallmarks of Alzheimer's disease and are mainly composed of Aß amyloid peptides together with other components such as lipids, cations, or glycosaminoglycans. The structure of amyloid peptide's aggregates is related to the peptide toxicity and highly depends on the aggregation conditions and the presence of cofactors. While fibrillary aggregates are nowadays considered nontoxic, oligomeric/granular (nonfibrillary) aggregates have been found to be toxic. In this work we have characterized in situ two different types of amyloid deposits analyzing sections of the cortex of patients in advanced stages of Alzheimer disease. By combining SR-µFTIR for the study of the secondary structure of the peptide and ThS fluorescence as an indicator of fibrillary structures, we found two types of plaques: ThS positive plaques with a clear infrared band at 1630 cm-1 that would correspond to fibrillary plaques and ThS negative plaques showing a mixture of nonfibrillar ß-sheet and unordered aggregated structures that would correspond to the nonfibrillary plaques (plaques with increased unordered structure). The analysis of the FTIR spectra has allowed correlation of lipid oxidation with the presence of nonfibrillary plaques. The metal composition of the two types of plaques has been analyzed using SR-nano-XRF and XANES. The results have shown higher accumulation of iron (mainly Fe2+) in fibrillary plaques than in nonfibrillary ones. However, in nonfibrillary plaques Fe3+ has been found to predominate over Fe2+. The identification of different types of aggregated forms and the different composition of metals found in the different types of plaques could be of paramount importance for the understanding of the development of Alzheimer disease.

Retraction Note: In situ structural characterization of early amyloid aggregates in Alzheimer's disease transgenic mice and Octodon degus.

Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-76208-w.

From Mouse to Human: Comparative Analysis between Grey and White Matter by Synchrotron-Fourier Transformed Infrared Microspectroscopy.

Fourier Transform Infrared microspectroscopy (µFTIR) is a very useful method to analyze the biochemical properties of biological samples in situ. Many diseases affecting the central nervous system (CNS) have been studied using this method, to elucidate alterations in lipid oxidation or protein aggregation, among others. In this work, we describe in detail the characteristics between grey matter (GM) and white matter (WM) areas of the human brain by µFTIR, and we compare them with the mouse brain (strain C57BL/6), the most used animal model in neurological disorders. Our results show a clear different infrared profile between brain areas in the lipid region of both species. After applying a second derivative in the data, we established a 1.5 threshold value for the lipid/protein ratio to discriminate between GM and WM areas in non-pathological conditions. Furthermore, we demonstrated intrinsic differences of lipids and proteins by cerebral area. Lipids from GM present higher C=CH, C=O and CH3 functional groups compared to WM in humans and mice. Regarding proteins, GM present lower Amide II amounts and higher intramolecular ß-sheet structure amounts with respect to WM in both species. However, the presence of intermolecular ß-sheet structures, which is related to ß-aggregation, was only observed in the GM of some human individuals. The present study defines the relevant biochemical properties of non-pathological human and mouse brains by µFTIR as a benchmark for future studies involving CNS pathological samples.

In situ structural characterization of early amyloid aggregates in Alzheimer's disease transgenic mice and Octodon degus.

Amyloid plaques composed of Aß amyloid peptides and neurofibrillary tangles are a pathological hallmark of Alzheimer's disease. In situ identification of early-stage amyloid aggregates in Alzheimer's disease is relevant for their importance as potential targets for effective drugs. Synchrotron-based infrared imaging is here used to identify early-stage oligomeric/granular aggregated amyloid species in situ in the brain of APP/PS1 transgenic mice and Octodon degus for the first time. Also, APP/PS1 mice show fibrillary aggregates at 6 and 12 months whereas very little formation of fibrils is found in aged Octodon degus. Finally, significant decreased burden of early-stage aggregates and fibrillary aggregates is obtained following treatment with G4-His-Mal dendrimers (a neurodegenerative protector) in 6-month-old APP/PS1 mice, thus demonstrating putative therapeutic properties of G4-His-Mal dendrimers in AD models. Identification, localization, and characterization using infrared imaging of these non-fibrillary species in the cerebral cortex at early stages of AD progression in transgenic mice point to their relevance as putative pharmacological targets. No less important, early detection of these structures may be useful in the search for markers for non-invasive diagnostic techniques.

Extracellular matrix components modulate different stages in ß2-microglobulin amyloid formation.

Amyloid deposition of WT human ß2-microglobulin (WT-hß2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. In vitro, WT-hß2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-hß2m aggregation and dialysis-related amyloidosis onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-hß2m in physiologically relevant conditions. Using thioflavin T fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-hß2m assembly. Our results revealed that LMW-heparin strongly promotes WT-hß2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hß2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-hß2m amyloid fibrils attenuates surface-mediated growth of WT-hß2m fibrils, demonstrating a key role of secondary nucleation in WT-hß2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hß2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hß2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hß2m into amyloid, which lead to dramatic effects on the time course of assembly.

Myoglobinopathy is an adult-onset autosomal dominant myopathy with characteristic sarcoplasmic inclusions.

Myoglobin, encoded by MB, is a small cytoplasmic globular hemoprotein highly expressed in cardiac myocytes and oxidative skeletal myofibers. Myoglobin binds O2, facilitates its intracellular transport and serves as a controller of nitric oxide and reactive oxygen species. Here, we identify a recurrent c.292C>T (p.His98Tyr) substitution in MB in fourteen members of six European families suffering from an autosomal dominant progressive myopathy with highly characteristic sarcoplasmic inclusions in skeletal and cardiac muscle. Myoglobinopathy manifests in adulthood with proximal and axial weakness that progresses to involve distal muscles and causes respiratory and cardiac failure. Biochemical characterization reveals that the mutant myoglobin has altered O2 binding, exhibits a faster heme dissociation rate and has a lower reduction potential compared to wild-type myoglobin. Preliminary studies show that mutant myoglobin may result in elevated superoxide levels at the cellular level. These data define a recognizable muscle disease associated with MB mutation.

Poly(propylene imine) dendrimers with histidine-maltose shell as novel type of nanoparticles for synapse and memory protection.

Poly(propylene imine) dendrimers have been shown to be promising 3-dimensional polymers for the use in the pharmaceutical and biomedical applications. Our aims of this study were first, to synthesize a novel type of dendrimer with poly(propylene imine) core and maltose-histidine shell (G4HisMal) assessing if maltose-histidine shell can improve the biocompatibility and the ability to cross the blood-brain barrier, and second, to investigate the potential of G4HisMal to protect Alzheimer disease transgenic mice from memory impairment. Our data demonstrate that G4HisMal has significantly improved biocompatibility and ability to cross the blood-brain barrier in vivo. Therefore, we suggest that a maltose-histidine shell can be used to improve biocompatibility and ability to cross the blood-brain barrier of dendrimers. Moreover, G4HisMal demonstrated properties for synapse and memory protection when administered to Alzheimer disease transgenic mice. Therefore, G4HisMal can be considered as a promising drug candidate to prevent Alzheimer disease via synapse protection.

Synchrotron-Based Fourier Transform Infrared Microspectroscopy (µFTIR) Study on the Effect of Alzheimer's Aß Amorphous and Fibrillar Aggregates on PC12 Cells.

Amyloid plaques made of aggregated Aß amyloid peptide are a pathological hallmark in brains affected by Alzheimer's disease (AD). Moreover, the amyloid peptide may play a major role in the onset and development of the disease in association to other factors such as oxidative stress. Although the molecular nature of the amyloid toxic species is still unknown, there is experimental evidence pointing to their nonfibrillar nature. In the present paper, we report the use of synchrotron Fourier transform infrared microspectroscopy (µFTIR) for the study of the effect of two different types of Alzheimer's Aß(1-40) aggregates (amyloid fibrils and granular nonfibrillar aggregates) on PC12 cultured cells. The principal component analysis (PCA) of the infrared spectra has been complemented with a correlation analysis, which permits one to study different spectroscopic parameters as a function of peptide aggregation. The results show that the treatment of PC12 cells with amorphous aggregates generates a higher degree of oxidation in the vicinity of the amyloid aggregates than the treatment with preformed amyloid fibrils. These results, which permit, for the first time, the in situ colocalization of amyloid aggregates and oxidized macromolecules in cell culture, are in agreement with previous data from our group, showing that oxidation was higher in regions surrounding amyloid plaques in human brain samples affected by AD.

Toxicogenomics of iron oxide nanoparticles in the nematode C. elegans.

We present a mechanistic study of the effect of iron oxide nanoparticles (SPIONs) in Caenorhabditis elegans combining a genome-wide analysis with the investigation of specific molecular markers frequently linked to nanotoxicity. The effects of two different coatings were explored: citrate, an anionic stabilizer, and bovine serum albumin, as a pre-formed protein corona. The transcriptomic study identified differentially expressed genes following an exposure to SPIONs. The expression of genes involved in oxidative stress, metal detoxification response, endocytosis, intestinal integrity and iron homeostasis was quantitatively evaluated. The role of oxidative stress was confirmed by gene expression analysis and by synchrotron Fourier Transform infrared microscopy based on the higher tissue oxidation of NP-treated animals. The observed transcriptional modulation of key signaling pathways such as MAPK and Wnt suggests that SPIONs might be endocytosed by clathrin-mediated processes, a putative mechanism of nanotoxicity which deserves further mechanistic investigations.

Fourier transform infrared spectroscopy (FTIR) characterization of the interaction of anti-cancer photosensitizers with dendrimers.

The systemic or local administration of a photosensitizer for photodynamic therapy is highly limited by poor selectivity, rapid deactivation and long-lasting skin toxicity due to unfavorable biodistribution. Drug delivery systems based on nanocarriers may help specific and effective delivery of photosensitizers. In the present paper, the interaction of two photosensitizers, methylene blue and rose bengal, with phosphorous cationic and anionic dendrimers as potential nanocarriers, has been characterized. A novel method is presented based on the analysis of the infrared spectra of mixtures of photosensitizer and dendrimer. The capacity of dendrimers to bind the photosensitizers has been evaluated by obtaining the corresponding binding curves. It is shown that methylene blue interacts with both cationic and anionic dendrimers, whereas rose bengal only binds to the cationic ones. Dendrimers are shown to be potential nanocarriers for a specific delivery of both photosensitizers.

Microspectroscopy (µFTIR) reveals co-localization of lipid oxidation and amyloid plaques in human Alzheimer disease brains.

Amyloid peptides are the main component of one of the characteristic pathological hallmarks of Alzheimer's disease (AD): senile plaques. According to the amyloid cascade hypothesis, amyloid peptides may play a central role in the sequence of events that leads to neurodegeneration. However, there are other factors, such as oxidative stress, that may be crucial for the development of the disease. In the present paper, we show that it is possible, by using Fourier tranform infrared (FTIR) microscopy, to co-localize amyloid deposits and lipid peroxidation in tissue slides from patients affected by Alzheimer's disease. Plaques and lipids can be analyzed in the same sample, making use of the characteristic infrared bands for peptide aggregation and lipid oxidation. The results show that, in samples from patients diagnosed with AD, the plaques and their immediate surroundings are always characterized by the presence of oxidized lipids. As for samples from non-AD individuals, those without amyloid plaques show a lower level of lipid oxidation than AD individuals. However, it is known that plaques can be detected in the brains of some non-AD individuals. Our results show that, in such cases, the lipid in the plaques and their surroundings display oxidation levels that are similar to those of tissues with no plaques. These results point to lipid oxidation as a possible key factor in the path that goes from showing the typical neurophatological hallmarks to suffering from dementia. In this process, the oxidative power of the amyloid peptide, possibly in the form of nonfibrillar aggregates, could play a central role.

Spectroscopic investigation of local mechanical impedance of living cells.

We studied nanoscale mechanical properties of PC12 living cells with a Force Feedback Microscope using two experimental approaches. The first one consists in measuring the local mechanical impedance of the cell membrane while simultaneously mapping the cell morphology at constant force. As the interaction force is increased, we observe the appearance of the sub-membrane cytoskeleton. We compare our findings with the outcome of other techniques. The second experimental approach consists in a spectroscopic investigation of the cell while varying the tip indentation into the membrane and consequently the applied force. At variance with conventional dynamic Atomic Force Microscopy techniques, here it is not mandatory to work at the first oscillation eigenmode of the cantilever: the excitation frequency of the tip can be chosen arbitrary leading then to new spectroscopic AFM techniques. We found in this way that the mechanical response of the PC12 cell membrane is found to be frequency dependent in the 1 kHz - 10 kHz range. In particular, we observe that the damping coefficient consistently decreases when the excitation frequency is increased.