Simultaneous structural and elemental nano-imaging of human brain tissue

Examining chemical and structural characteristics of micro-features in complex tissue matrices is essential for understanding biological systems. Advances in multimodal chemical and structural imaging using synchrotron radiation have overcome many issues in correlative imaging, enabling the characterization of distinct microfeatures at nanoscale resolution in ex vivo tissues. We present a nanoscale imaging method that pairs X-ray ptychography and X-ray fluorescence microscopy (XFM) to simultaneously examine structural features and quantify elemental content of microfeatures in complex ex vivo tissues. We examined the neuropathological microfeatures Lewy bodies, aggregations of superoxide dismutase 1 (SOD1) and neuromelanin in human post-mortem Parkinson''s disease tissue. Although biometals play essential roles in normal neuronal biochemistry, their dyshomeostasis is implicated in Parkinson''s disease aetiology. Here we show that Lewy bodies and SOD1 aggregates have distinct elemental fingerprints yet are similar in structure, whilst neuromelanin exhibits different elemental composition and a distinct, disordered structure. The unique approach we describe is applicable to the structural and chemical characterization of a wide range of complex biological tissues at previously unprecedented levels of detail.

Structural and chemical characterisation of microfeatures in unadulterated Parkinson''s disease brain tissue using synchrotron nanoscale XFM and ptychography.     

Determination of D,L‐serine in midbrain of Parkinson's disease mouse by capillary electrophoresis with in‐column light‐emitting diode induced fluorescence detection

A capillary electrophoresis method with in‐column light‐emitting diode induced fluorescence detection is described for simultaneous determination of D ,L ‐serine in the midbrain of a Parkinson's disease mouse. D ,L ‐Serine was derivatized with fluorescein isothiocyanate, and chiral separation and determination of D ,L ‐serine derivatives were performed on a laboratory‐built capillary electrophoresis system with in‐column light‐emitting diode induced fluorescence detector using γ‐cyclodextrin as chiral selector. Using this method, the levels of D ‐ and L ‐serine in the midbrains of Parkinson's disease mice were determined. When compared to controls, the levels of D ‐ and L ‐serine showed significant differences. The result suggested that the biosynthesis and the transportation of endogenous D ,L ‐serine may participate in Parkinson's disease pathogenesis.     

Targeted resequencing of candidate genes reveals novel variants associated with severe Beh?et's uveitis

Behçet''s disease (BD) is a chronic systemic inflammatory disorder characterized by four major manifestations: recurrent uveitis, oral and genital ulcers and skin lesions. To identify some pathogenic variants associated with severe Behçet''s uveitis, we used targeted and massively parallel sequencing methods to explore the genetic diversity of target regions. A solution-based target enrichment kit was designed to capture whole-exonic regions of 132 candidate genes. Using a multiplexing strategy, 32 samples from patients with a severe type of Behçet''s uveitis were sequenced with a Genome Analyzer IIx. We compared the frequency of each variant with that of 59 normal Korean controls, and selected five rare and eight common single-nucleotide variants as the candidates for a replication study. The selected variants were genotyped in 61 cases and 320 controls and, as a result, two rare and seven common variants showed significant associations with severe Behçet''s uveitis (P<0.05). Some of these, including rs199955684 in KIR3DL3, rs1801133 in MTHFR, rs1051790 in MICA and rs1051456 in KIR2DL4, were predicted to be damaging by either the PolyPhen-2 or SIFT prediction program. Variants on FCGR3A (rs396991) and ICAM1 (rs5498) have been previously reported as susceptibility loci of this disease, and those on IFNAR1, MTFHR and MICA also replicated the previous reports at the gene level. The KIR3DL3 and KIR2DL4 genes are novel susceptibility genes that have not been reported in association with BD. In conclusion, this study showed that target enrichment and next-generation sequencing technologies can provide valuable information on the genetic predisposition for Behçet''s uveitis.     

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student''s t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn''t. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.     

Receptors for organochlorine pesticides based on calixarenes

The water soluble 4-sulfocalix[n]arenes (with n?=?4,6,8) have been investigated as potential synthetic receptors for cyclodiene organochlorine pesticides. Steady state fluorescence experiments in ethanol solution have shown that only the cavitands with n equal to 6 and 8 form complexes, of comparable stability, with heptachlor. Electrochemical data, obtained in water solution, confirmed the ability of 4-sulfocalix[6]arene to bind the heptachlor, unlike the smaller calixarene. Moreover, a significant increase in the stability constant is observed in water solutions. This stability is caused by the sterical hindrance of pesticides with respect to the cavity dimension of the calixarene. This results in a selective interaction of this molecule with other organochlorine pesticides. Binding experiments, carried out with endosulfan have shown that, despite of chemical similarity, 4-sulfocalix[6]arene and 4-sulfocalix[8]arene behave in a very different way: the former is unable to bind this pesticide, while the latter shows a binding constant of 4.7?×?10⁵ with endosulfan. To investigate the molecular features of the interactions, molecular dynamic simulations of 4-sulfocalix[6]arene in presence of heptachlor in water solution have been performed. These simulations show that different configurations of heptachlor inside the calixarene cavity are equally populated and easily interconverting, suggesting that a non specific hydrophobic interaction plays a key role in the complex stability. These studies have permitted to individuate versatile synthetic receptors for organochlorine pesticides.     

Photoinduced elimination of senescent microglia cells in vivo by chiral gold nanoparticles

Parkinson''s disease (PD) is an age-related neurodegenerative disease, and the removal of senescent cells has been proved to be beneficial for improving age-associated pathologies in neurodegeneration disease. In this study, chiral gold nanoparticles (NPs) with different helical directions were synthesized to selectively induce the apoptosis of senescent cells under light illumination. By modifying anti-B2MG and anti-DCR2 antibodies, senescent microglia cells could be cleared by chiral NPs without damaging the activities of normal cells under illumination. Notably, l-P⁺ NPs exhibited about a 2-fold higher elimination efficiency than d-P⁻ NPs for senescent microglia cells. Mechanistic studies revealed that the clearance of senescent cells was mediated by the activation of the Fas signaling pathway. The in vivo injection of chiral NPs successfully confirmed that the elimination of senescent microglia cells in the brain could further alleviate the symptoms of PD mice in which the alpha-synuclein (α-syn) in cerebrospinal fluid (CFS) decreased from 83.83 ± 4.76 ng mL⁻¹ to 8.66 ± 1.79 ng mL⁻¹ after two months of treatment. Our findings suggest a potential strategy to selectively eliminate senescent cells using chiral nanomaterials and offer a promising strategy for alleviating PD.

The apoptosis pathways of senescent microglia cells induced by chiral NPs under the irradiation of 808 nm laser in the brain of PD mice.     

Dysfunction of vesicular storage in young-onset Parkinson's patient-derived dopaminergic neurons and organoids revealed by single cell electrochemical cytometry

Electrochemical cytometry based on nano-tip microelectrodes was used to quantify the vesicular storage at the single-cell level in human neurons and midbrain organoids which acted as disease models of young-onset Parkinson''s disease (YOPD). Human dopaminergic (DA) neurons and midbrain organoids were derived from an induced pluripotent stem cell line from one YOPD patient. We show a significant deficiency in vesicular catecholamine storage and a slower pore forming process on the surface of the microelectrode in the DA neurons derived from the YOPD patient. The upregulation of α-synuclein in both neurons and organoids derived from the YOPD patient is associated with vesicular storage dysfunction, revealing a correlation between the pathogenesis of YOPD and vesicular chemical storage deficiency, a novel chemical insight into the potential pathology of YOPD. Notably, efficacy evaluation and drug testing were performed with our platform to demonstrate that both amantadine, a clinical drug for Parkinson''s disease (PD), and phorbol 12-myristate 13-acetate, an attractive candidate, ameliorate the dysfunction of vesicular storage in DA neurons derived from the YOPD patient. Our platform offers promising avenues for new drug discovery for PD and other neurodegenerative disorders.

Deficient vesicular storage at the single-cell level in human neurons and midbrain organoids derived from an iPSC line from one YOPD patient was revealed via electrochemical cytometry at nanotip microelectrodes.     

In situ tissue regeneration through host stem cell recruitment

The field of tissue engineering has made steady progress in translating various tissue applications. Although the classical tissue engineering strategy, which involves the use of culture-expanded cells and scaffolds to produce a tissue construct for implantation, has been validated, this approach involves extensive cell expansion steps, requiring a lot of time and laborious effort before implantation. To bypass this ex vivo process, a new approach has been introduced. In situ tissue regeneration utilizes the body''s own regenerating capacity by mobilizing host endogenous stem cells or tissue-specific progenitor cells to the site of injury. This approach relies on development of a target-specific biomaterial scaffolding system that can effectively control the host microenvironment and mobilize host stem/progenitor cells to target tissues. An appropriate microenvironment provided by implanted scaffolds would facilitate recruitment of host cells that can be guided to regenerating structural and functional tissues.     

Legionella lipoprotein activates toll-like receptor 2 and induces cytokine production and expression of costimulatory molecules in peritoneal macrophages

Legionella bacterium, an intracellular pathogen of mononuclear phagocytes, causes acute fatal pneumonia, especially in patients with impaired cellular immune responses. Until recently, however, the toll-like receptor (TLR) engagement of bacterial proteins derived from Legionella is uncertain. We previously showed that a 19-kDa highly conserved peptidoglycan-associated lipoprotein (PAL) of Legionella pneumophila induced the PAL-specific B cell and T cell responses in mice. In this study, we observed that the rPAL antigen of L. pneumophila, as an effector molecule, activated murine macrophages via TLR2 and produced proinflammatory cytokines such as IL-6 and TNF-α. In both BALB/c and TLR4-deficient C3H/HeJ mice, pretreatment of macrophages with anti-TLR2 mAb showed severely impaired cytokine production in response to the rPAL. In addition, in vitro the rPAL treatment increased the cell surface expression of CD40, CD80, CD86 and MHC I/II molecules. We further showed that the synthetic CpG-oligodeoxynucleotides (CpG ODN) coadministered with the rPAL enhanced IL-12 and IL-6 production and expression of CD40, CD80 and MHC II compared to the rPAL treatment alone. In conclusions, these results indicate that Legionella PAL might activate macrophages via a TLR2-dependent mechanism which thus induce cytokine production and expression of costimulatory and MHC molecules.     

Molecular mechanisms of amyloid formation in living systems

Fibrillar protein aggregation is a hallmark of a variety of human diseases. Examples include the deposition of amyloid-β and tau in Alzheimer''s disease, and that of α-synuclein in Parkinson''s disease. The molecular mechanisms by which soluble proteins form amyloid fibrils have been extensively studied in the test tube. These investigations have revealed the microscopic steps underlying amyloid formation, and the role of factors such as chaperones that modulate these processes. This perspective explores the question to what extent the mechanisms of amyloid formation elucidated in vitro apply to human disease. The answer is not yet clear, and may differ depending on the protein and the associated disease. Nevertheless, there are striking qualitative similarities between the aggregation behaviour of proteins in vitro and the development of the related diseases. Limited quantitative data obtained in model organisms such as Caenorhabditis elegans support the notion that aggregation mechanisms in vivo can be interpreted using the same biophysical principles established in vitro. These results may however be biased by the high overexpression levels typically used in animal models of protein aggregation diseases. Molecular chaperones have been found to suppress protein aggregation in animal models, but their mechanisms of action have not yet been quantitatively analysed. Several mechanisms are proposed by which the decline of protein quality control with organismal age, but also the intrinsic nature of the aggregation process may contribute to the kinetics of protein aggregation observed in human disease.

The molecular mechanisms of amyloid formation have been studied extensively in test tube reactions. This perspective article addresses the question to what extent these mechanisms apply to the complex situation in living cells and organisms.     

The extent of protein hydration dictates the preference for heterogeneous or homogeneous nucleation generating either parallel or antiparallel β-sheet α-synuclein aggregates

α-Synuclein amyloid self-assembly is the hallmark of a number of neurodegenerative disorders, including Parkinson''s disease, although there is still very limited understanding about the factors and mechanisms that trigger this process. Primary nucleation has been observed to be initiated in vitro at hydrophobic/hydrophilic interfaces by heterogeneous nucleation generating parallel β-sheet aggregates, although no such interfaces have yet been identified in vivo. In this work, we have discovered that α-synuclein can self-assemble into amyloid aggregates by homogeneous nucleation, without the need of an active surface, and with a preference for an antiparallel β-sheet arrangement. This particular structure has been previously proposed to be distinctive of stable toxic oligomers and we here demonstrate that it indeed represents the most stable structure of the preferred amyloid pathway triggered by homogeneous nucleation under limited hydration conditions, including those encountered inside α-synuclein droplets generated by liquid–liquid phase separation. In addition, our results highlight the key role that water plays not only in modulating the transition free energy of amyloid nucleation, and thus governing the initiation of the process, but also in dictating the type of preferred primary nucleation and the type of amyloid polymorph generated depending on the extent of protein hydration. These findings are particularly relevant in the context of in vivo α-synuclein aggregation where the protein can encounter a variety of hydration conditions in different cellular microenvironments, including the vicinity of lipid membranes or the interior of membraneless compartments, which could lead to the formation of remarkably different amyloid polymorphs by either heterogeneous or homogeneous nucleation.     

Extraction and Detection of Pesticides by Cholinesterase Inhibition in a Two‐Phase System: a Strategy to Avoid Heavy Metal Interference

Abstract

A novel method of extraction has been developed to avoid the presence of heavy metals during the measurement of pesticides based on acetylcholinesterase (AchE) inhibition. Heavy metals have been in fact demonstrated in this article to interfere when the assay is performed by using the classic spectrophotometric Ellman's method. We present the results obtained with an assay system using two different phases, one organic and the other aqueous, in which the pesticide and the enzyme are, respectively, solubilized. In a first step, the concentration of the substrate acetylthiocholine (1 mM), of the enzyme (7 mU mL^?1), and the reaction time (20 min) for measurement of enzyme activity were optimized in aqueous solution. Next, the effect of an organic phase on the enzyme activity was studied by the addition of various solvents with the activity being evaluated after 10 min of mixing. It was found that by using hexane, the enzyme retained almost 100% of its activity, and this solvent was chosen for further development of the pesticide assay. Hexane was spiked with different concentrations of pesticides and then added to the enzyme aqueous phase. The pesticides were shown to be able to inhibit the enzyme by interaction at the interface between the two solutions. The degree of inhibition obtained with increasing amounts of pesticide was evaluated. A 50% inhibition was observed for a paraoxon solution of 9×10^?7 M.     

Darwin and Molecular Biology

Darwin's “idea of thehe century”, the principle of selection, is as important in the age of molecular biology as it was a hundred years ago. As a natural law it is open to rigorous physical proof, if certain prerequisites are met, and to quantitative experimental test—in vitro and in vivo—under defined laboratory conditions.     

Neuronal RNA granule contains ApCPEB1, a novel cytoplasmic polyadenylation element binding protein, in Aplysia sensory neuron

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3'' untranslated regions (3''UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.     

Rapid detection of pesticides not amenable to multi-residue methods by flow injection–tandem mass spectrometry

Flow injection combined with tandem mass spectrometry (MS/MS) was investigated for the rapid detection of highly polar pesticides that are not amenable to multi-residue methods because they do not partition into organic solvents and require dedicated chromatographic conditions. The pesticides included in this study were amitrole, chlormequat, cyromazine, daminozide, diquat, ethephon, fosetyl-Al, glufosinate, glyphosate and its metabolite aminomethylphosphonic acid, maleic hydrazide, mepiquat and paraquat. The composition of the flow-injection solvent was optimized to achieve maximum MS/MS sensitivity. Instrumental limits of detection varied between <0.05 and 1 pg. Fruit, vegetable, cereal, milk and kidney samples were extracted with water (1 % formic acid in case of paraquat/diquat) and ten times diluted in either methanol/0.1 % formic acid, methanol/0.1 % ammonia or acetonitrile/0.1 % ammonia, depending on the pesticide. The ion suppression observed depended strongly on both the matrix and the pesticide. This could be largely compensated for by matrix-matched calibration, but more accurate quantification was obtained by using isotopically labelled standards (commercially available for most of the pesticides studied). The method detection limits ranged from 0.02 mg/kg for chlormequat and mepiquat to 2 mg/kg for maleic hydrazide and were 0.05–0.2 mg/kg for most other pesticide/matrix combinations. This was sufficiently low to test compliance with EU maximum residue limits for many relevant pesticide/commodity combinations. The method substantially reduces the liquid chromatography–MS/MS capacity demand which for many laboratories is prohibitive for inclusion of these pesticides in their monitoring and surveillance programmes. Figure

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Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells

Background

Insulin is a hormone that regulates blood glucose homeostasis and is a central protein in a medical condition termed insulin injection amyloidosis. It is intimately associated with glycaemia and is vulnerable to glycation by glucose and other highly reactive carbonyls like methylglyoxal, especially in diabetic conditions. Protein glycation is involved in structure and stability changes that impair protein functionality, and is associated with several human diseases, such as diabetes and neurodegenerative diseases like Alzheimer's disease, Parkinson's disease and Familiar Amyloidotic Polyneuropathy. In the present work, methylglyoxal was investigated for their effects on the structure, stability and fibril formation of insulin.

Results

Methylglyoxal was found to induce the formation of insulin native-like aggregates and reduce protein fibrillation by blocking the formation of the seeding nuclei. Equilibrium-unfolding experiments using chaotropic agents showed that glycated insulin has a small conformational stability and a weaker dependence on denaturant concentration (smaller m-value). Our observations suggest that methylglyoxal modification of insulin leads to a less compact and less stable structure that may be associated to an increased protein dynamics.

Conclusions

We propose that higher dynamics in glycated insulin could prevent the formation of the rigid cross-β core structure found in amyloid fibrils, thereby contributing to the reduction in the ability to form fibrils and to the population of different aggregation pathways like the formation of native-like aggregates.     

α-Synuclein as a Target for Metallo-Anti-Neurodegenerative Agents

The unique thermodynamic and kinetic coordination chemistry of ruthenium allows it to modulate key adverse aggregation and membrane interactions of α-synuclein (α-syn) associated with Parkinson's disease. We show that the low-toxic Ru^III complex trans-[ImH][RuCl₄(Me₂SO)(Im)] (NAMI-A) has dual inhibitory effects on both aggregation and membrane interactions of α-syn with submicromolar affinity, and disassembles pre-formed fibrils. NAMI-A abolishes the cytotoxicity of α-syn towards neuronal cells and mitigates neurodegeneration and motor impairments in a rat model of Parkinson's. Multinuclear NMR and MS analyses show that NAMI-A binds to residues involved in protein aggregation and membrane binding. NMR studies reveal the key steps in pro-drug activation and the effect of activated NAMI-A species on protein folding. Our findings provide a new basis for designing ruthenium complexes which could mitigate α-syn-induced Parkinson's pathology differently from organic agents.     

In Vivo Detection of Copper Ions by Magnetic Resonance Imaging Using a Prion-Based Contrast Agent

Abnormal distributions of transition metals inside the body are potential diagnostic markers for several diseases, including Alzheimer's disease, Parkinson's disease, Wilson??s disease, and cancer. In this article, we demonstrate that P57/Gd, a novel prion-based contrast agent, can selectively image tissues with excessive copper accumulation using magnetic resonance imaging (MRI). P57/Gd selectivity binds copper(II) over other physiologically relevant cations such as zinc, iron, manganese, and calcium. To simulate a metabolic copper disorder, we treated mice with an intraperitoneal injection of a CuSO₄ solution to induce a renal copper overload. The MRI signal intensities from the renal cortex and medulla of copper spiked animals that were administered P57/Gd were found to correlate with the ex vivo copper concentrations determined by inductively coupled plasma mass spectrometry.     

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid''s antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL⁻¹, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL⁻¹). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid''s N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.

Structure–activity relationship studies of the natural product cystobactamid at four different positions led to novel imaging probes and analogs with superior antibacterial activities and in vivo efficacy.