Subcellular protein turnover in human neural progenitor cells revealed by correlative electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Protein turnover is a critical process for accurate cellular function, in which damaged proteins in the cells are gradually replaced with newly synthesized ones. Many previous studies on cellular protein turnover have used stable isotopic labelling by amino acids in cell culture (SILAC), followed by proteomic bulk analysis. However, this approach does not take into account the heterogeneity observed at the single-cell and subcellular levels. To address this, we investigated the protein turnover of neural progenitor cells at the subcellular resolution, using correlative TEM and NanoSIMS imaging, relying on a pulse-chase analysis of isotopically-labelled protein precusors. Cellular protein turnover was found significantly heterogenous across individual organelles, which indicates a possible relation between protein turnover and subcellular activity. In addition, different isotopically-labelled amino acids provided different turnover patterns, in spite of all being protein precursors, suggesting that they undergo distinct protein synthesis and metabolic pathways at the subcellular level.

Lipid organization and turnover in the plasma membrane of human differentiating neural progenitor cells revealed by time-of-flight secondary ion mass spectrometry imaging.

Membrane lipids have been known to influence multiple signalling and cellular processes. Dysregulation of lipids at the neuronal membrane is connected to a significant alteration of the brain function and morphology, leading to brain diseases and neurodegeneration. Understanding the lipid composition and turnover of neuronal membrane will provide a significant insight into the molecular events underlying the regulatory effects of these biomolecules in a neuronal system. In this study, we aimed to characterize the composition and turnover of the plasma membrane lipids in human neural progenitor cells (NPCs) at an early differentiation stage into midbrain neurons using ToF-SIMS imaging. Lipid composition of the native plasma membrane was explored, followed by an examination of the lipid turnover using different isotopically labelled lipid precursors, including 13C-choline, 13C-lauric acid, 15N-linoleic, and 13C-stearic. Our results showed that differentiating NPCs contain a high abundance of ceramides, glycerophosphoserines, neutral glycosphingolipids, diradylglycerols, and glycerophosphocholines at the plasma membrane. In addition, different precursors were found to incorporate into different membrane lipids which are specific for the short- or long-carbon chains, and the unsaturation or saturation stage of the precursors. The lipid structure of neuronal membrane reflects the differentiation status of NPCs, and it can be altered significantly using a particular lipid precursor. Our study illustrates a potential of ToF-SIMS imaging to study native plasma membrane lipids and elucidate complex cellular processes by providing molecular -rich information at a single cell level.

Single Exosome Amperometric Measurements Reveal Encapsulation of Chemical Messengers for Intercellular Communication.

In multicellular organisms, cells typically communicate by sending and receiving chemical signals. Chemical messengers involved in the exocytosis of neuroendocrine cells or neurons are generally assumed to only originate from the fusing of intracellular large dense core vesicles (LDCVs) or synaptic vesicles with the cellular membrane following stimulation. Accumulated evidence suggests that exosomes─one of the main extracellular vesicles (EVs)─carrying cell-dependent DNA, mRNA, proteins, etc., play an essential role in cellular communication. Due to experimental limitations, it has been difficult to monitor the real-time release of individual exosomes; this restricts a comprehensive understanding of the basic molecular mechanisms and the functions of exosomes. In this work, we introduce amperometry with microelectrodes to capture the dynamic release of single exosomes from a single living cell, distinguish them from other EVs, and differentiate the molecules inside exosomes and those secreted from LDCVs. We show that, similar to many LDCVs and synaptic vesicles, exosomes released by neuroendocrine cells also contain catecholamine transmitters. This finding reveals a different mode of chemical communication via exosome-encapsulated chemical messengers and a potential interconnection between the two release pathways, changing the canonical view of exocytosis of neuroendocrine cells and possibly neurons. This defines a new mechanism for chemical communication at the fundamental level and opens new avenues in the research of the molecular biology of exosomes in the neuroendocrine and central nervous systems.

A Systematic Review on the Potential of Aspirin to Reduce Cardiovascular Risk in Schizophrenia.

Cardiovascular disease (CVD), including heart disease and stroke, continues to be the leading cause of death worldwide. Patients with mental health disorders, including schizophrenia (SCZ) are known to have an increased risk for CVD. Given the association with metabolic syndrome, patients with SCZ are often prescribed metformin and statins but its impact remains unsatisfactory. The use of aspirin (ASA) to decrease cardiovascular risk in the general population has been thoroughly investigated and clear guidelines are currently in place. Since adjuvant treatment with ASA could possibly decrease CVD risk and mortality in SCZ, we conducted a systematic review of the literature to determine the state of the current literature on this subject. Our systematic review points to gaps in the literature on CVD prevention in SCZ and illustrates an obvious need for further research. Although several studies have shown increased CVD risk in SCZ, to date, no research has been conducted on the utilization of CVD preventative treatment such as ASA for SCZ.

Characterization of Stress Granule Protein Turnover in Neuronal Progenitor Cells Using Correlative STED and NanoSIMS Imaging.

Stress granules (SGs) are stress-induced biomolecular condensates which originate primarily from inactivated RNA translation machinery and translation initiation factors. SG formation is an important defensive mechanism for cell survival, while its dysfunction has been linked to neurodegenerative diseases. However, the molecular mechanisms of SG assembly and disassembly, as well as their impacts on cellular recovery, are not fully understood. More thorough investigations into the molecular dynamics of SG pathways are required to understand the pathophysiological roles of SGs in cellular systems. Here, we characterize the SG and cytoplasmic protein turnover in neuronal progenitor cells (NPCs) under stressed and non-stressed conditions using correlative STED and NanoSIMS imaging. We incubate NPCs with isotopically labelled (15N) leucine and stress them with the ER stressor thapsigargin (TG). A correlation of STED and NanoSIMS allows the localization of individual SGs (using STED), and their protein turnover can then be extracted based on the 15N/14N ratio (using NanoSIMS). We found that TG-induced SGs, which are highly dynamic domains, recruit their constituents predominantly from the cytoplasm. Moreover, ER stress impairs the total cellular protein turnover regimen, and this impairment is not restored after the commonly proceeded stress recovery period.

Chemical Imaging and Analysis of Single Nerve Cells by Secondary Ion Mass Spectrometry Imaging and Cellular Electrochemistry.

A nerve cell is a unit of neuronal communication in the nervous system and is a heterogeneous molecular structure, which is highly mediated to accommodate cellular functions. Understanding the complex regulatory mechanisms of neural communication at the single cell level requires analytical techniques with high sensitivity, specificity, and spatial resolution. Challenging technologies for chemical imaging and analysis of nerve cells will be described in this review. Secondary ion mass spectrometry (SIMS) allows for non-targeted and targeted molecular imaging of nerve cells and synapses at subcellular resolution. Cellular electrochemistry is well-suited for quantifying the amount of reactive chemicals released from living nerve cells. These techniques will also be discussed regarding multimodal imaging approaches that have recently been shown to be advantageous for the understanding of structural and functional relationships in the nervous system. This review aims to provide an insight into the strengths, limitations, and potentials of these technologies for synaptic and neuronal analyses.

An iodine-containing probe as a tool for molecular detection in secondary ion mass spectrometry.

We developed here an iodine-containing probe that can be used to identify the molecules of interest in secondary ion mass spectrometry (SIMS) by simple immunolabelling procedures. The immunolabelled iodine probe was readily combined with previously-developed SIMS probes carrying fluorine, to generate dual-channel SIMS data. This probe should provide a useful complement to the currently available SIMS probes, thus expanding the scope of this technology.

White matter integrity in mice requires continuous myelin synthesis at the inner tongue.

Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.

Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells.

For decades, "all-or-none" and "kiss-and-run" were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically.

Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy.

In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 µm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis.

Gold-Conjugated Nanobodies for Targeted Imaging Using High-Resolution Secondary Ion Mass Spectrometry.

Nanoscale imaging with the ability to identify cellular organelles and protein complexes has been a highly challenging subject in the secondary ion mass spectrometry (SIMS) of biological samples. This is because only a few isotopic tags can be used successfully to target specific proteins or organelles. To address this, we generated gold nanoprobes, in which gold nanoparticles are conjugated to nanobodies. The nanoprobes were well suited for specific molecular imaging using NanoSIMS at subcellular resolution. They were demonstrated to be highly selective to different proteins of interest and sufficiently sensitive for SIMS detection. The nanoprobes offer the possibility of correlating the investigation of cellular isotopic turnover to the positions of specific proteins and organelles, thereby enabling an understanding of functional and structural relations that are currently obscure.

Correlative fluorescence microscopy, transmission electron microscopy and secondary ion mass spectrometry (CLEM-SIMS) for cellular imaging.

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches.

Secondary Ion Mass Spectrometry Imaging Reveals Changes in the Lipid Structure of the Plasma Membranes of Hippocampal Neurons following Drugs Affecting Neuronal Activity.

The cellular functions of lipids in the neuronal plasma membranes have been increasingly acknowledged, particularly their association to neuronal processes and synaptic plasticity. However, the knowledge of their regulatory mechanisms in neuronal cells remains sparse. To address this, we investigated the lipid organization of the plasma membranes of hippocampal neurons in relation to neuronal activity using secondary ion mass spectrometry imaging. The neurons were treated with drugs, particularly tetrodotoxin (TTX) and bicuculline (BIC), to induce chronic activation and silencing. Distinct lipid organization was found in the plasma membrane of the cell body and the neurites. Moreover, significant alterations of the levels of the membrane lipids, especially ceramides, phosphatidylserines, phosphatidic acids, and triacylglycerols, were observed under the TTX and BIC treatments. We suggest that many types of membrane lipids are affected by, and may be involved in, the regulation of neuronal function.

Electrochemical Measurements Reveal Reactive Oxygen Species in Stress Granules*.

Stress granules (SGs) are membrane-less organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arsenite-induced SGs, and H2 O2 is the main species. The release kinetics of H2 O2 from single SGs and the number of H2 O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple endpoint. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases (NDs), cancers and viral infections.

Localization and Absolute Quantification of Dopamine in Discrete Intravesicular Compartments Using NanoSIMS Imaging.

The absolute concentration and the compartmentalization of analytes in cells and organelles are crucial parameters in the development of drugs and drug delivery systems, as well as in the fundamental understanding of many cellular processes. Nanoscale secondary ion mass spectrometry (NanoSIMS) imaging is a powerful technique which allows subcellular localization of chemical species with high spatial and mass resolution, and high sensitivity. In this study, we combined NanoSIMS imaging with spatial oversampling with transmission electron microscopy (TEM) imaging to discern the compartments (dense core and halo) of large dense core vesicles in a model cell line used to study exocytosis, and to localize 13C dopamine enrichment following 4-6 h of 150 µM 13C L-3,4-dihydroxyphenylalanine (L-DOPA) incubation. In addition, the absolute concentrations of 13C dopamine in distinct vesicle domains as well as in entire single vesicles were quantified and validated by comparison to electrochemical data. We found concentrations of 87.5 mM, 16.0 mM and 39.5 mM for the dense core, halo and the whole vesicle, respectively. This approach adds to the potential of using combined TEM and NanoSIMS imaging to perform absolute quantification and directly measure the individual contents of nanometer-scale organelles.

Adsorption and biodegradation removal of methylene blue in a down-flow hanging filter reactor incorporating natural adsorbent.

This study was carried out to explore the importance of adsorption and biodegradation mechanisms for methylene blue (MB) removal by a novel natural adsorbent (purified coconut fibre; PCF) incorporated to a down-flow hanging fibre (DHF) reactor. An adsorption DHF (Ads-DHF) reactor demonstrated the adsorption removal mechanism, while a combined adsorption-biological DHF (Bio-DHF) reactor simulated the processes of both adsorption and biodegradation were investigated for the MB removal capability. PCF prepared from coconut fibre waste was applied as a media in the DHF reactors. The process performance and the removal mechanisms of the DHF reactors were evaluated for 62 days. The results showed that a total MB removal efficiency of 93 ± 7% was achieved for the Bio-DHF reactor and 36 ± 25% for the Ads-DHF reactor. The combined adsorption and biological degradation in the Bio-DHF reactor enhanced the removal efficiency and the life-time of the reactor compared with the performance of the adsorption process alone in the Ads-DHF reactor. Moreover, microbial community analysis revealed that microorganisms, commonly involved in the biodegradation of dyes, were predominant in the Bio-DHF reactor. The PCF media of the Bio-DHF reactor was essential to keep the dye degrading bacteria in the reactor. Therefore, it can be concluded that the Bio-DHF reactor is an appropriate treatment system for treating dyes wastewater. This research is significant and useful for environmental protection and reuse of biomass wastes.

Sulfur-mediated annulation of 1,2-phenylenediamines towards benzofuro- and benzothieno-quinoxalines.

We report a method for condensation between ortho-phenylenediamines and ortho-hydroxyacetophenones to afford benzofuroquinoxalines. The reactions proceeded in the presence of an elemental sulfur mediator, DABCO base, and DMSO solvent. Functionalities such as nitrile, ester, and halogen groups were compatible. The conditions could be applicable for the synthesis of benzothienoquinoxalines from ortho-chloroacetophenones.

Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles.

Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.

Interplay between Cocaine, Drug Removal, and Methylphenidate Reversal on Phospholipid Alterations in Drosophila Brain Determined by Imaging Mass Spectrometry.

Cocaine dependence displays a broad impairment in cognitive performance including attention, learning, and memory. To obtain a better understanding of the action of cocaine in the nervous system, and the relation between phospholipids and memory, we have investigated whether phospholipids recover in the brain following cocaine removal using the fly model, Drosophila melanogaster. In addition, the effects of methylphenidate, a substitute medication for cocaine dependence, on fly brain lipids after cocaine abuse are also determined to see if it can rescue the lipid changes caused by cocaine. Time of flight secondary ion mass spectrometry with a (CO2)6000+ gas cluster ion beam was used to detect intact phospholipids. We show that cocaine has persistent effects, both increasing and decreasing the levels of specific phosphatidylethanolamines and phosphatidylinositols. These changes remain after cocaine withdrawal and are not rescued by methylphenidate. Cocaine is again shown to generally increase the levels of phosphatidylcholines in the fly brain; however, after drug withdrawal, the abundance of these lipids returns to the original level and methylphenidate treatment of the flies following cocaine exposure enhances the reversal of the lipid level reducing them below the original control. The study provides insight into the molecular effects of cocaine and methylphenidate on brain lipids. We suggest that phosphatidylcholines could be a potential target for the treatment of cocaine abuse as well as be a significant hallmark of cognition and memory loss with cocaine.

Adsorption of colour from dye wastewater effluent of a down-flow hanging sponge reactor on purified coconut fibre.

This study was carried out to evaluate the potential application of agricultural waste coconut (Cocos nucifera) fibre in its purified form for decolorization of dye-containing effluent from a down-flow hanging sponge (DHS) reactor. Batch adsorption experiments were conducted by varying adsorbent dose, solution pH, contact time and temperature. The results showed that the adsorption percentage of colour increased with increasing in adsorbent dosage from 0.1 g to 1.3 g, solution pH from 2 to 10 and contact time from 1 h to 72 h. The adsorption process was sensitive to temperature and the adsorption percentage of colour reached the highest value at 30°C but decreased when temperature increased from 30°C to 60°C. Purified coconut fibre (PCF) had a good adsorption capacity under the optimal conditions of an adsorbent dose of 0.7 g, pH 8, temperature of 30°C and 24 h contact time. The equilibrium data were well fitted to both Langmuir and Freundlich isotherms, and the best fit of equilibrium isotherms was described using the Freundlich isotherm model. The Langmuir maximum adsorption capability of the PCF was found to be 303 (Pt/Co)/g at 30°C. The results of this study indicated the applicability of PCF as post-treatment unit of DHS reactor for removal of colour in the industrial dye effluent.