Donepezil-Loaded Nanocarriers for the Treatment of Alzheimer's Disease: Superior Efficacy of Extracellular Vesicles Over Polymeric Nanoparticles.

Introduction: Drug delivery across the blood-brain barrier (BBB) is challenging and therefore severely restricts neurodegenerative diseases therapy such as Alzheimer's disease (AD). Donepezil (DNZ) is an acetylcholinesterase (AChE) inhibitor largely prescribed to AD patients, but its use is limited due to peripheral adverse events. Nanodelivery strategies with the polymer Poly (lactic acid)-poly(ethylene glycol)-based nanoparticles (NPs-PLA-PEG) and the extracellular vesicles (EVs) were developed with the aim to improve the ability of DNZ to cross the BBB, its brain targeting and efficacy. Methods: EVs were isolated from human plasma and PLA-PEG NPs were synthesized by nanoprecipitation. The toxicity, brain targeting capacity and cholinergic activities of the formulations were evaluated both in vitro and in vivo. Results: EVs and NPs-PLA-PEG were designed to be similar in size and charge, efficiently encapsulated DNZ and allowed sustained drug release. In vitro study showed that both formulations EVs-DNZ and NPs-PLA-PEG-DNZ were highly internalized by the endothelial cells bEnd.3. These cells cultured on the Transwell® model were used to analyze the transcytosis of both formulations after validation of the presence of tight junctions, the transendothelial electrical resistance (TEER) values and the permeability of the Dextran-FITC. In vivo study showed that both formulations were not toxic to zebrafish larvae (Danio rerio). However, hyperactivity was evidenced in the NPs-PLA-PEG-DNZ and free DNZ groups but not the EVs-DNZ formulations. Biodistribution analysis in zebrafish larvae showed that EVs were present in the brain parenchyma, while NPs-PLA-PEG remained mainly in the bloodstream. Conclusion: The EVs-DNZ formulation was more efficient to inhibit the AChE enzyme activity in the zebrafish larvae head. Thus, the bioinspired delivery system (EVs) is a promising alternative strategy for brain-targeted delivery by substantially improving the activity of DNZ for the treatment of AD.

Implication of Circulating Extracellular Vesicles-Bound Amyloid-ß42 Oligomers in the Progression of Alzheimer's Disease.

BACKGROUND: The perplex interrelation between circulating extracellular vesicles (cEVs) and amyloid-ß (Aß) deposits in the context of Alzheimer's disease (AD) is poorly understood. OBJECTIVE: This study aims to 1) analyze the possible cross-linkage of the neurotoxic amyloid-ß oligomers (oAß) to the human cEVs, 2) identify cEVs corona proteins associated with oAß binding, and 3) analyze the distribution and expression of targeted cEVs proteins in preclinical participants converted to AD 5 years later (Pre-AD). METHODS: cEVs were isolated from 15 Pre-AD participants and 15 healthy controls selected from the Canadian Study of Health and Aging. Biochemical, clinical, lipid, and inflammatory profiles were measured. oAß and cEVs interaction was determined by nanoparticle tracking analysis and proteinase K digestion. cEVs bound proteins were determined by ELISA. RESULTS: oAß were trapped by cEVs and were topologically bound to their external surface. We identified surface-exposed proteins functionally able to conjugate oAß including apolipoprotein J (apoJ), apoE and RAGE, with apoJ being 30- to 130-fold higher than RAGE and apoE, respectively. The expression of cEVs apoJ was significantly lower in Pre-AD up to 5 years before AD onset. CONCLUSION: Our findings suggest that cEVs might participate in oAß clearance and that early dysregulation of cEVs could increase the risk of conversion to AD.

Effect of APOE ε4 allele on levels of apolipoproteins E, J, and D, and redox signature in circulating extracellular vesicles from cognitively impaired with no dementia participants converted to Alzheimer's disease.

INTRODUCTION: The substantial link between apolipoprotein E (APOE) ε4 allele and oxidative stress may underlie enhanced Alzheimer's disease (AD) risk. Here, we studied the impact of APOE ε4 on the level of apolipoproteins with antioxidant activities along with oxidative markers in circulating extracellular vesicles (cEVs) and plasma from cognitively impaired-not demented (CIND) individuals converted to AD (CIND-AD). METHODS: Apolipoproteins E, J, and D and antioxidant response markers were determined in cEVs and plasma using immunoblotting, electrochemical examination, and spectrofluorimetry. RESULTS: Total antioxidant capacity and apolipoprotein D levels in cEVs, as judged by regression analysis and cognitive performance correlations, allowed us to differentiate CIND APOE ε4 carriers from controls and to predict their progression to AD 5 years later. DISCUSSION: Our findings support the pathological redox linkage between APOE ε4 and AD onset and suggest the use of cEVs oxidative signature in early AD diagnosis.

Levels of Receptor for Advanced Glycation End Products and Glyoxalase-1 in the Total Circulating Extracellular Vesicles from Mild Cognitive Impairment and Different Stages of Alzheimer's Disease Patients.

BACKGROUND: Growing evidence supports that receptor for advanced glycation end products (RAGE) and glyoxalase-1 (GLO-1) are implicated in the pathophysiology of Alzheimer's disease (AD). Extracellular vesicles (EVs) are nanovesicles secreted by almost all cell types, contribute to cellular communication, and are implicated in AD pathology. Recently, EVs are considered as promising tools to identify reliable biomarkers in AD. OBJECTIVE: The aim of our study was to determine the levels of RAGE and GLO-1 in circulating EVs from mild cognitive impairment (MCI) and AD patients and to analyze their correlation with the clinical Mini-Mental State Examination and Montreal Cognitive Assessment scores. We have studied the possibility that neuronal cells could release and transfer GLO-1 through EVs. METHODS: RAGE and GLO-1 levels were measured in circulating EVs, respectively, by Luminex assay and western blot. Released-EVs from SK-N-SH neuronal cells were isolated and GLO-1 levels were determined by western blot. RESULTS: Our data showed higher levels of RAGE in EVs from late AD patients while GLO-1 levels in EVs from early AD were lower as compared to control and MCI patients. Interestingly, levels of RAGE and GLO-1 in EVs were correlated with the cognitive scores regardless of age. For the first time, we demonstrated that GLO-1 was released from neuronal cells through EVs. CONCLUSION: Although more samples will be needed, our preliminary results support the use of peripheral EVs cargo as new tools for the discovery of peripheral AD biomarkers.

Apolipoprotein E4-driven effects on inflammatory and neurotrophic factors in peripheral extracellular vesicles from cognitively impaired, no dementia participants who converted to Alzheimer's disease.

INTRODUCTION: In brain, extracellular vesicles (EVs) play an essential role in the neuron-glia interface and ensure the crosstalk between the brain and the periphery. Some studies now link the pathway dysfunction of the EVs to apolipoprotein E gene variant (APOE ε4) and the risk of progression to Alzheimer's disease (AD). To better understand the role of APOE ε4 in pre-clinical AD, we have determined levels of pathogenic, neurotrophic and inflammatory proteins in peripheral EVs (pEVs) and in plasma from cognitively impaired, no dementia (CIND) participants stratified upon the absence (APOE ε4-) or the presence (APOE ε4+ ) of the ε4 allele of APOE. METHODS: Levels of 15 neurodegenerative, neurotrophic and neuroinflammatory proteins were quantified in pEVs and compared to their plasma levels from cognitively normal and CIND participants. RESULTS: Levels of neurotrophic and inflammatory markers were reduced in pEVs from APOE ε4+. The pentraxin-2/α-synuclein ratio measured in pEVs was able to predict AD 5 years before the onset among APOE ε4+-CIND individuals. DISCUSSION: Our findings suggest an alteration of the endosomal pathway in APOE ε4+ and that pEVs pentraxin-2/α-synuclein ratio could serve as a useful early biomarker for AD susceptibility.

Glutathione: An Old and Small Molecule with Great Functions and New Applications in the Brain and in Alzheimer's Disease.

Significance: Glutathione (GSH) represents the most abundant and the main antioxidant in the body with important functions in the brain related to Alzheimer's disease (AD). Recent Advances: Oxidative stress is one of the central mechanisms in AD. We and others have demonstrated the alteration of GSH levels in the AD brain, its important role in the detoxification of advanced glycation end-products and of acrolein, a by-product of lipid peroxidation. Recent in vivo studies found a decrease of GSH in several areas of the brain from control, mild cognitive impairment, and AD subjects, which are correlated with cognitive decline. Critical Issues: Several strategies were developed to restore its intracellular level with the l-cysteine prodrugs or the oral administration of γ-glutamylcysteine to prevent alterations observed in AD. To date, no benefit on GSH level or on oxidative biomarkers has been reported in clinical trials. Thus, it remains uncertain if GSH could be considered a potential preventive or therapeutic approach or a biomarker for AD. Future Directions: We address how GSH-coupled nanocarriers represent a promising approach for the functionalization of nanocarriers to overcome the blood/brain barrier (BBB) for the brain delivery of GSH while avoiding cellular toxicity. It is also important to address the presence of GSH in exosomes for its potential intercellular transfer or its shuttle across the BBB under certain conditions. Antioxid. Redox Signal. 35, 270-292.

Inhibition of Protein Glycation by Combined Antioxidant and Antiglycation Constituents from a Phenolic Fraction of Sage (Salvia officinalis L.).

Disturbed advanced glycation end products (AGEs)-oxidative stress axis is strongly linked to vascular complications observed in diabetes and other metabolic conditions. Salvia officinalis L. (sage) is a medicinal plant used as an ingredient in foods and beverages and displays a wide range of biological and pharmacological activities including anti-diabetic effects. However, no study has assessed its anti-glycative potential. The aim of this study is to determine the phenolic compounds associated with the anti-glycation and antioxidant potential of sage methanol extract (SME). SME shows similar effects to aminoguanidine on fluorescent AGEs inhibition. It protects albumin damage from glycation (52.9 vs. 50.3%, respectively) by preventing the loss of protein thiol groups (50.0 vs. 44.3%, respectively) and by reducing protein carbonyl accumulation (67.4 vs. 70.5%, respectively). Moreover, linear regression and multivariate analysis support the efficient contribution of SME antioxidant capacity, as judged by DPPH, TBARS and iron chelating tests, in AGEs suppression. Furthermore, HPLC analysis revealed the presence of verbascoside as a novel phenolic constituent identified in sage leaves and suggests that the protective activity is mostly assigned to the presence of rosmarinic acid, resveratrol, quercetin, rutin and luteolin-7-O-glucoside. Likewise, the screening of SME phenolic content supports the contribution of various antioxidant substances to the observed effects. Therefore, a polyphenol enriched sage extract was able to inhibit the formation of AGEs and protein glycation. Our data unveils the promising properties of sage and its bioactive principles in the management of AGEs-mediated vascular complications observed in diabetes and other metabolic disorders.

Methylglyoxal and Glyoxal as Potential Peripheral Markers for MCI Diagnosis and Their Effects on the Expression of Neurotrophic, Inflammatory and Neurodegenerative Factors in Neurons and in Neuronal Derived-Extracellular Vesicles.

Methylglyoxal (MG) and glyoxal (GO) are suggested to be associated with the development of neurodegenerative pathologies. However, their peripheral levels in relation to cognitive decline and their effects on key factors in neuronal cells are poorly investigated. The aim of this study was to determine their serum levels in MCI (mild cognitive impairment) and Alzheimer's disease (AD) patients, to analyze their effects on the neurotrophic and inflammatory factors, on neurodegenerative markers in neuronal cells and in neuronal derived-extracellular vesicles (nEVs). Our results show that MG and GO levels in serum, determined by HPLC, were higher in MCI. ROC (receiver-operating characteristic curves) analysis showed that the levels of MG in serum have higher sensitivity to differentiate MCI from controls but not from AD. Meanwhile, serum GO levels differentiate MCI from control and AD groups. Cells and nEVs levels of BDNF, PRGN, NSE, APP, MMP-9, ANGPTL-4, LCN2, PTX2, S100B, RAGE, Aß peptide, pTau T181 and alpha-synuclein were quantified by luminex assay. Treatment of neuronal cells with MG or GO reduced the cellular levels of NSE, PRGN, APP, MMP-9 and ANGPTL-4 and the nEVs levels of BDNF, PRGN and LCN2. Our findings suggest that targeting MG and GO may be a promising therapeutic strategy to prevent or delay the progression of AD.

Epidemiology and Whole-Genome Analysis of NDM-1-Producing Klebsiella pneumoniae KP3771 from Tunisia.

Objectives: The whole-genome sequence (WGS) of Klebsiella pneumoniae KP3771 isolate was characterized. This strain was recovered from the urine sample of an 80-year-old man hospitalized in an intensive care unit of the University Hospital Tahar Sfar in Tunisia. Materials and Methods: WGS using a MiSeq platform was used. The assembled genome was subjected to several software analyses. Results: K. pneumoniae KP3771 was resistant to all antibiotics but colistin and tigecycline. WGS analysis found 18 transmissible genes encoding resistance markers, including blaNDM-1 and blaCTX-M-15 genes, which were carried by four plasmids belonging to the Inc Ib, IIk, and R groups. Three families of genes encoding virulence factors were detected, including adhesins (fimH, fimA, fimB, fimC, mrkD, Kpn, and ycfM), siderophores (enterobactin, aerobactin, and yersiniabactin siderophores), and protectin/invasin (traT). The strain was assigned to the sequence type 147. Conclusions: This study describes the genome of a carbapenemase-producing K. pneumoniae clinical isolate recovered in Tunisia. Bacteria WGS has become the reference technology to address epidemiological issues; this high level of information is particularly well suited to enrich epidemiological workflows' output.

Preventive effects of Salvia officinalis leaf extract on insulin resistance and inflammation in a model of high fat diet-induced obesity in mice that responds to rosiglitazone.

BACKGROUND: Salvia officinalis (sage) is a native plant to the Mediterranean region and has been used for a long time in traditional medicine for various diseases. We investigated possible anti-diabetic, anti-inflammatory and anti-obesity effects of sage methanol (MetOH) extract in a nutritional mouse model of obesity, inflammation and insulin resistance, as well as its effects on lipolysis and lipogenesis in 3T3-L1 cells. METHODS: Diet-induced obese (DIO) mice were treated for five weeks with sage methanol extract (100 and 400 mg kg-1/day bid), or rosiglitazone (3 mg kg-1/day bid), as a positive control. Energy expenditure, food intake, body weight, fat mass, liver glycogen and lipid content were evaluated. Blood glucose, and plasma levels of insulin, lipids leptin and pro- and anti-inflammatory cytokines were measured throughout the experiment. The effects of sage MetOH extract on lipolysis and lipogenesis were tested in vitro in 3T3-L1 cells. RESULTS: After two weeks of treatment, the lower dose of sage MetOH extract decreased blood glucose and plasma insulin levels during an oral glucose tolerance test (OGTT). An insulin tolerance test (ITT), performed at day 29 confirmed that sage improved insulin sensitivity. Groups treated with low dose sage and rosiglitazone showed very similar effects on OGTT and ITT. Sage also improved HOMA-IR, triglycerides and NEFA. Treatment with the low dose increased the plasma levels of the anti-inflammatory cytokines IL-2, IL-4 and IL-10 and reduced the plasma level of the pro-inflammatory cytokines IL-12, TNF-α, and KC/GRO. The GC analysis revealed the presence of two PPARs agonist in sage MetOH extract. In vitro, the extract reduced in a dose-related manner the accumulation of lipid droplets; however no effect on lipolysis was observed. CONCLUSIONS: Sage MetOH extract at low dose exhibits similar effects to rosiglitazone. It improves insulin sensitivity, inhibits lipogenesis in adipocytes and reduces inflammation as judged by plasma cytokines. Sage presents an alternative to pharmaceuticals for the treatment of diabetes and associated inflammation.

Role of disturbed fatty acids metabolism in the pathophysiology of diabetic erectile dysfunction.

BACKGROUND: Vasculogenic erectile dysfunction (VED) is considered as a common complication among people with type 2 diabetes (T2D). We tested whether changes in fatty acid (FAs) classes measured in erythrocytes are associated with increased risk of diabetic VED along with related risk factors. METHODS: We assessed erythrocyte FAs composition, lipid peroxidation parameters and inflammatory cytokines among 72 T2D men with VED, 78 T2D men without VED and 88 healthy volunteers with similar age. Biochemical, hepatic, lipid and hormonal profiles were measured. RESULTS: T2D people with VED had significant decrease in the indexes of Δ6-desaturase and elongase activities compared to the other studied groups. The same group of participants displayed lower erythrocytes levels of dihomo-γ-linolenic acid (C20:3n-6) (P < .001), precursor of the messenger molecule PGE1 mainly involved in promoting erection. Moreover, absolute SFAs concentration and HOMA IR levels were higher in T2D people with VED when compared to controls and associated with impaired NO concentration (1.43 vs 3.30 ng/L, P < .001). Our results showed that IL-6 and TNF-α were significantly increased and positively correlated with MDA levels only in T2D people with VED (r = 0.884, P = .016 and r = 0.753, P = .035; respectively) suggesting a decrease in the relative availability of vasodilator mediators and an activation of vasoconstrictors release. CONCLUSION: Our findings show that the deranged FAs metabolism represents a potential marker of VED in progress, or at least an indicator of increased risk within men with T2D.

[Virulence factors in Pseudomonas aeruginosa: mechanisms and modes of regulation]. / Les facteurs de virulence de Pseudomonas aeruginosa : mécanismes et modes de régulations.

Pseudomonas aeruginosa is a bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients. The bacterium's virulence depends on a large number of cell-associated and extracellular factors. The virulence factors play an important pathological role in the colonization, the survival of the bacteria and the invasion of tissues. There are two types of virulence factors: (1) factors involved in the acute infection: these factors are either on the surface of P. aeruginosa, either secreted. The pili allow adherence to the epithelium. The exoenzyme S and other adhesins reinforce the adherence to epithelial cells. The exotoxin A is responsible of tissue necrosis. Phospholipase C is a thermolabile haemolysin. The pathogenic role of exoenzyme S is attributable to the disruption of normal cytoskeletal organization, the destruction of immunoglobulin G and A, leads to depolymerization of actin filaments and contributes to the resistance to macrophages. P. aeruginosa produces at least four proteases causing bleeding and tissue necrosis; (2) factors involved in the chronic infection: siderophores (pyoverdin and pyochelin), allow the bacteria to multiply in the absence of ferrous ions. The strains isolated from patients with cystic fibrosis have a pseudocapsule of alginate that protects the bacterium from phagocytosis, dehydration and antibiotics. Moreover, it improves adherence to epithelial cells forming a biofilm. Two different types of regulation systems control the expression of the majority of these virulence factors: the two-component transcriptional regulatory system and the quorum sensing system. These two mechanisms are necessary to the survival and the proliferation of this microorganism in the host.

Evaluation of the maturity of organic waste composts.

Olive-mill wastes represent a significant environmental problem in Mediterranean areas due to their significant production during a short period of time. Their high polyphenol, lipid and organic acid concentrations make them phytotoxic wastes. Composting is one of the technologies used for the valorization of those wastes, producing a fertilizer useful for poor soils. The present study is an attempt to elaborate upon organic matter transformations and define the parameters for product maturity by adapting chemical and spectroscopic methods during composting. The aim of this work was to study the changes involved in the composting process of four piles during 200 days, and follow up the maturity of the final product during composting. Fourier transform infrared (FTIR) spectroscopy has been proven to be an appropriate analytical method for the qualitative assessment of compost stability. FTIR spectroscopy results revealed enrichment in aromatic structures and a degradation of the aliphatic and alcoholic structures indicating stabilization of the final compost. The results showed that stability of the final product was reached after 7 months of composting. The phytotoxic effects of olive mill wastes and animal manures was assessed by germination index. Indeed, the germination indices of piles 1, 2, 3 and 4 reached 131.31%, 72%, 90.56%, and 105.37%, respectively, at the end of the process. This demonstrated the absence of phytotoxicity in the majority of mature composts.