Visualizing alpha-synuclein and iron deposition in M83 mouse model of Parkinson's disease in vivo.

Background: Abnormal alpha-synuclein and iron accumulation in the brain play an important role in Parkinson's disease (PD). Herein, we aim at visualizing alpha-synuclein inclusions and iron deposition in the brains of M83 (A53T) mouse models of PD in vivo. Methods: Fluorescently labelled pyrimidoindole-derivative THK-565 was characterized by using recombinant fibrils and brains from 10-11 months old M83 mice, which subsequently underwent in vivo concurrent wide-field fluorescence and volumetric multispectral optoacoustic tomography (vMSOT) imaging. The in vivo results were verified against structural and susceptibility weighted imaging (SWI) magnetic resonance imaging (MRI) at 9.4 Tesla and scanning transmission X-ray microscopy (STXM) of perfused brains. Brain slice immunofluorescence and Prussian blue staining were further performed to validate the detection of alpha-synuclein inclusions and iron deposition in the brain, respectively. Results: THK-565 showed increased fluorescence upon binding to recombinant alpha-synuclein fibrils and alpha-synuclein inclusions in post-mortem brain slices from patients with Parkinson's disease and M83 mice. i.v. administration of THK-565 in M83 mice showed higher cerebral retention at 20 and 40 minutes post-injection by wide-field fluorescence compared to non-transgenic littermate mice, in congruence with the vMSOT findings. SWI/phase images and Prussian blue indicated the accumulation of iron deposits in the brains of M83 mice, presumably in the Fe3+ form, as evinced by the STXM results. Conclusion: We demonstrated in vivo mapping of alpha-synuclein by means of non-invasive epifluorescence and vMSOT imaging assisted with a targeted THK-565 label and SWI/STXM identification of iron deposits in M83 mouse brains ex vivo.

A NAC domain mutation (E83Q) unlocks the pathogenicity of human alpha-synuclein and recapitulates its pathological diversity.

The alpha-synuclein mutation E83Q, the first in the NAC domain of the protein, was recently identified in a patient with dementia with Lewy bodies. We investigated the effects of this mutation on the aggregation of aSyn monomers and the structure, morphology, dynamic, and seeding activity of the aSyn fibrils in neurons. We found that it markedly accelerates aSyn fibrillization and results in the formation of fibrils with distinct structural and dynamic properties. In cells, this mutation is associated with higher levels of aSyn, accumulation of pS129, and increased toxicity. In a neuronal seeding model of Lewy body (LB) formation, the E83Q mutation significantly enhances the internalization of fibrils into neurons, induces higher seeding activity, and results in the formation of diverse aSyn pathologies, including the formation of LB-like inclusions that recapitulate the immunohistochemical and morphological features of brainstem LBs observed in brains of patients with Parkinson's disease.

Health risks of inhalation exposure to BTEX in a municipal wastewater treatment plant in Middle East city: Shiraz, Iran.

Benzene, toluene, ethylbenzene, and xylene, known as BTEX, adversely affect human health. This study aimed to measure BTEX concentration and assess the health risk through inhalation exposure to these compounds in a municipal wastewater treatment plant. The ambient air samples were collected using the active sampling method in summer and winter. Sampling sites were close to wastewater treatment units, including bar screen & grit chamber (Site-1), aeration tank & secondary clarifiers (Site-2), anaerobic sludge digester (Site-3), and office & control building (Site-4). Results indicate that the mean levels of benzene and toluene in winter were higher than in summer. The levels of benzene and toluene were below the occupational exposure levels of the American Conference of Governmental Industrial Hygienists (ACGIH) and Occupational Safety and Health Administration (OSHA). The carcinogenic risk (CR) and hazard quotient (HQ) were assessed using the United States Environmental Protection Agency (U.S.EPA) method, and Monte Carlo simulation (MCS) was applied to quantify the uncertainty and sensitivity analysis. The CRs for all sampling sites were below the U.S.EPA threshold limit (1.0 × 10-4). The HQ values for benzene nearby Site-2 were above 1.0, indicating inhalation exposure could pose a health hazard. Besides, the HQ results for exposure to toluene in all sampling sites were less than 1.0. The sensitivity analysis had shown that the risk determinant for carcinogenic and non-carcinogenic risk was exposure time, followed by concentration.

Investigating Crosstalk Among PTMs Provides Novel Insight Into the Structural Basis Underlying the Differential Effects of Nt17 PTMs on Mutant Httex1 Aggregation.

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntington's disease. Here, we expand on these studies by investigating the effect of methionine eight oxidation (oxM8) and its crosstalk with lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1 (mHttex1). We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mHttex1aggregates. The presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization. Circular dichroism spectroscopy and molecular dynamics simulation studies show that PTMs that lower the mHttex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and pS13) result in increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation rate (AcK6) of mHttex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mHttex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mHttex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mHttex1 aggregation.

Empowering the Emission of Upconversion Nanoparticles for Precise Subcellular Imaging.

Upconversion nanoparticles (UCNPs) are a class of inorganic fluorophores that follow the anti-Stokes mechanism, to which the wavelength of emission is shorter than absorption. This unique optical behavior generates relatively long-lived intermediate energy levels of lanthanides that stabilize the excitation state in the fluorescence process. Longer-wavelength light sources, e.g., near-infrared (NIR), penetrate deeper into biological materials such as tissue and cells that provide a larger working space for cell biology applications and imaging, whereby UCNPs have recently gained increasing interest in medicine. In this report, the emission intensity of a gadolinium-based UCNP was screened by changing the concentrations of the constituents. The optimized condition was utilized as a luminescent nanoprobe for targeting the mitochondria as a distinguished subcellular organelle within differentiated neuroblastoma cells. The main goal of this study is to illustrate the targeting process within the cells in a native state using modified UCNPs. Confocal microscopy on the cells treated with the functionalized UCNPs indicated a selective accumulation of UCNPs after immunolabeling. To tackle the insolubility of as-synthesized particles in water-based media, the optimized UCNPs were surface-coated with polyamidoamine (PAMAM) dendrimers that due to peripheral amino groups are suitable for functionalizing with peptides and antibodies. Ultimately, we concluded that UCNPs are potentially versatile and ideal tools for NIR bioimaging and capable of making adequate contrast against biomaterials to be detectable in electron microscopy (EM) imaging.

A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation.

Huntington's disease is a neurodegenerative disorder caused by the expansion of a polyglutamine repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Httexon1 (Httex1). Herein, we investigated the aggregation properties of mutant N-terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1 (mHttex1). We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded insights into how post-translational modifications (PTMs) and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slows the aggregation of mHtt171, whereas phosphorylation at T107 and S116 accelerates the aggregation, underscoring the importance of crosstalk between different PTMs. The mHtt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mHttex1. These observations suggest that different N-terminal fragments could have distinct aggregation mechanisms and that a single polyQ-targeting antiaggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the need for further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity and disease progression.

Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson's Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography.

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 µm3. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson's diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

Covalent organic framework membrane for size selective release of small molecules and peptide in vitro.

The ability to control small drug release is crucial in biomedicine, especially for inhibiting the side effects of drugs, but it is still challenging. Herein, to mimic the controlled release of drugs, the release of organic molecules, e.g., small organic dyes and peptides, through Covalent Organic Framework (COF) membranes with ordered nanoscale pores has been investigated, showing constant zero-order release behaviours. Meanwhile, biological assessments show the good biocompatibility of the COF membrane-based release system, and the high stability of the COF membrane was manifested by the long-term release of small molecules in aqueous media.

Enhanced blood-brain-barrier penetrability and tumor-targeting efficiency by peptide-functionalized poly(amidoamine) dendrimer for the therapy of gliomas.

Glioblastoma is one of the most common primary tumor types of central nervous system (CNS) with high malignance and lethality. Although many treatment options are currently available, the therapy of brain cancers remains challenging because of blood-brain-barrier (BBB) which prevents most of the chemotherapeutics into the CNS. In this work, a poly(amidoamine) dendrimer-based carrier was fabricated and modified with angiopep-2 (Ang2) peptide that has been demonstrated to bind to low density lipoprotein receptor-relative protein-1 (LRP1) on the endothelial cells of BBB and could therefore induce BBB penetration of the carrier. To improve tumor-targeting effect towards the glioma sites, the dendrimer was simultaneously functionalized with an epidermal growth factor receptor (EGFR)-targeting peptide (EP-1) which was screened from a "one-bead one-compound" (OBOC) combinatorial library. EP-1 peptide was demonstrated to have high affinity and specificity to EGFR at both the molecular and cellular levels. The dual-targeting dendrimer exhibited outstanding BBB penetrability and glioma targeting efficiency both in vitro and in vivo, which strikingly enhanced the anti-gliomas effect of the drugs and prolonged the survival of gliomas-bearing mice. These results show the potential of the dual-targeting dendrimer-based carrier in the therapy of gliomas through enhancing BBB penetrability and tumor targeting.

Breakthroughs in medicine and bioimaging with up-conversion nanoparticles.

Nanomedicine is a medical application of biochemistry incorporated with materials chemistry at the scale of nanometer for the purpose of diagnosis, prevention, and treatment. New models and approaches are typically associated with nanomedicine for precise multifunctional diagnostic systems at molecular level. Hence, employing nanoparticles (NPs) has unveiled new opportunities for efficient therapies and remedy of difficult-to-cure diseases. Among all types of inorganic NPs, lanthanide-doped up-conversion nanoparticles (UCNPs) have shown excellent potential for biomedical applications, especially for multimodal bioimaging including fluorescence and electron microscopy. Association of these visualization techniques plus the capability for transporting biomaterials and drugs make them superior agents in the field of nanomedicine. Accordingly, in this review, we firstly presented a fundamental understanding of physical and optical properties of UCNPs and secondly, we illustrated some of the prominent associations with bioimaging, theranostics, cancer therapy, and optogenetics.

pH-Triggered Peptide Self-Assembly for Targeting Imaging and Therapy toward Angiogenesis with Enhanced Signals.

Mild acidic environment and angiogenesis are two typical characteristics of tumor. The specific response toward both lower pH and angiogenesis may enhance the targeting ability both for drug and diagnostic probe delivery. Herein, we present a kind of dual responding self-assembled nanotransformation material that is tumor angiogenesis targeting and pH triggered based on amphiphilic conjugation between peptides (STP) and aromatic molecules (tetraphenylethylene (TPE)). The morphology of the self-assembled peptide conjugates is responsibly changed from nanoparticles in neutral condition to nanofibers in acidic condition, which "turn on" the in vivo targeting imaging and accelerate the efficient drug delivery and in vivo therapy. On the basis of the well-controlled nanotransformation both in vitro and in vivo, we envisioned the successful demonstration of the responding materials would open a new avenue in turn on targeting imaging diagnostics and specific cancer therapeutics.

Energy Migration Engineering of Bright Rare-Earth Upconversion Nanoparticles for Excitation by Light-Emitting Diodes.

A novel Nd(3+) -sensitized upconversion nanoparticle (UCNP) that can be excited by near-infrared 740 nm light-emitting diode (LED) lamps with bright upconversion luminescence is designed. Yb(3+) ion distribution is engineered to increase the energy migration efficiency. The benefit of the novel LED-excited UCNPs is demonstrated by imaging of breast cancer cells and enabling an economic handheld semiquantitative visual measurement device.

Hydrophilic alginate based multidentate biopolymers for surface modification of CdS quantum dots.

We report that sodium alginate and its graft-copolymers with (2-dimethylaminoethyl) methacrylate may attach to the surface of colloidal CdS-thioglycerol quantum dots (QDs) via a ligand exchange process and result in water-soluble and highly stable QD supramolecules. Optical properties of modified QDs in water showed that the emission intensity of QDs was still high after surface passivation.