scholarly journals Quantitative secretome analysis establishes the cell type-resolved mouse brain secretome

2020 ◽  
Author(s):  
Johanna Tüshaus ◽  
Stephan A. Müller ◽  
Evans Sioma Kataka ◽  
Jan Zaucha ◽  
Laura Sebastian Monasor ◽  
...  
Keyword(s):  
Mouse Brain ◽  
High Performance ◽  
Brain Slices ◽  
Large Cell ◽  
Cell Type ◽  
Cellular Origin ◽  
Secretome Analysis ◽  
Csf Proteins

AbstractTo understand how cells communicate in the nervous system, it is essential to define their secretome, which is challenging for primary cells because of large cell numbers being required. Here, we miniaturized secretome analysis by developing the high-performance secretome-protein-enrichment-with-click-sugars method (hiSPECS). To demonstrate its broad utility, hiSPECS was used to identify the secretory response of brain slices upon LPS-induced neuroinflammation and to establish the cell type-resolved mouse brain secretome resource using primary astrocytes, microglia, neurons and oligodendrocytes. This resource allowed mapping the cellular origin of CSF proteins and revealed that an unexpectedly high number of secreted proteins in vitro and in vivo are proteolytically-cleaved membrane protein ectodomains. Two examples are neuronally secreted ADAM22 and CD200, which we identified as substrates of the Alzheimer-linked protease BACE1. hiSPECS and the brain secretome resource can be widely exploited to systematically study protein secretion, brain function and to identify cell type-specific biomarkers for CNS diseases.

scholarly journals Determination of Diffusion Kinetics of Ketamine in Brain Tissue: Implications for in vitro Mechanistic Studies of Drug Actions

2021 ◽  
Vol 15 ◽  
Author(s):  
Zachary Geiger ◽  
Brett VanVeller ◽  
Zarin Lopez ◽  
Abdel K. Harrata ◽  
Kathryn Battani ◽  
...  
Keyword(s):  
Brain Tissue ◽  
High Performance ◽  
Brain Slices ◽  
General Anesthetic ◽  
Adult Mice ◽  
Drug Concentrations ◽  
Drug Actions ◽  
Diffusion Properties

Ketamine has been in use for over 50 years as a general anesthetic, acting primarily through blockade of N-methyl-D-aspartate receptors in the brain. Recent studies have demonstrated that ketamine also acts as a potent and rapid-acting antidepressant when administered at sub-anesthetic doses. However, the precise mechanism behind this effect remains unclear. We examined the diffusion properties of ketamine in brain tissue to determine their effects in in vitro studies related to the antidepressant action of ketamine. Brain slices from adult mice were exposed to artificial cerebrospinal fluid (aCSF) containing ∼17 μM ketamine HCl for varying amounts of time. The amount of ketamine within each slice was then measured by tandem high-performance liquid chromatography – mass spectrometry to characterize the diffusion of ketamine into brain tissue over time. We successfully modeled the diffusion of ketamine into brain tissue using a mono-exponential function with a time constant of τ = 6.59 min. This curve was then compared to a one-dimensional model of diffusion yielding a diffusion coefficient of approximately 0.12 cm2⋅s–1 for ketamine diffusing into brain tissue. The brain:aCSF partition coefficient for ketamine was determined to be approximately 2.76. Our results suggest that the diffusion properties of ketamine have a significant effect on drug concentrations achieved within brain tissue during in vitro experiments. This information is vital to determine the ketamine concentration necessary for in vitro slice preparation to accurately reflect in vivo doses responsible for its antidepressant actions.

scholarly journals A Cre-dependent massively parallel reporter assay allows for cell-type specific assessment of the functional effects of genetic variants in vivo

2021 ◽  
Author(s):  
Tomas Lagunas ◽  
Stephen P Plassmyer ◽  
Ryan Z Friedman ◽  
Michael A Rieger ◽  
Anthony D Fischer ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Genetic Variants ◽  
Massively Parallel ◽  
Psychiatric Disease ◽  
Functional Screening ◽  
Cell Type ◽  
Cell Type Specific ◽  
The Impact

Human genetic studies have identified a large number of disease-associated de novo variants in presumptive regulatory regions of the genome that pose a challenge for interpretation of their effects: the impact of regulatory variants is highly dependent on the cellular context, and thus for psychiatric diseases these would ideally be studied in neurons in a living brain. Furthermore, for both common and rare variants, it is expected that only a subset fraction will affect gene expression. Massively Parallel Reporter Assays (MPRAs) are molecular genetic tools that enable functional screening of hundreds of predefined sequences in a single experiment. These assays have been used for functional screening of several different types of regulatory sequences in vitro. However, they have not yet been adapted to query specific cell types in vivo in a complex tissue like the mouse brain. Here, using a test-case 3′UTR MPRA library with variants from ASD patients, we sought to develop a method to achieve reproducible measurements of variant effects in vivo in a cell type-specific manner. We implemented a Cre-dependent design to control expression of our library and first validated our system in vitro. Next, we measured the effect of >500 3′UTR variants in excitatory neurons in the mouse brain. Finally, we report >40 variants with significant effects on transcript abundance in the context of the brain. This new technique should enable robust, functional annotation of genetic variants in the cellular contexts most relevant to psychiatric disease.

scholarly journals Cell type specific profiling of alternative translation identifies novel protein isoforms in the mouse brain

2018 ◽  
Author(s):  
Darshan Sapkota ◽  
Allison M. Lake ◽  
Wei Yang ◽  
Chengran Yang ◽  
Hendrik Wesseling ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Stop Codon ◽  
Affinity Purification ◽  
Protein Isoforms ◽  
Cell Type ◽  
Alternative Translation ◽  
Cell Type Specific ◽  
Ribosome Footprinting

AbstractTranslation canonically begins at a single AUG and terminates at the stop codon, generating one protein species per transcript. However, some transcripts may use alternative initiation sites or sustain translation past their stop codon, generating multiple protein isoforms. Through other mechanisms such as alternative splicing, both neurons and glia exhibit remarkable transcriptional diversity, and these other forms of post-transcriptional regulation are impacted by neural activity and disease. Here, using ribosome footprinting, we demonstrate that alternative translation is likewise abundant in the central nervous system and modulated by stimulation and disease. First, in neuron/glia mixed cultures we identify hundreds of transcripts with alternative initiation sites and confirm the protein isoforms corresponding to a subset of these sites by mass spectrometry. Many of them modulate their alternative initiation in response to KCl stimulation, indicating activity-dependent regulation of this phenomenon. Next, we detect several transcripts undergoing stop codon readthrough thus generating novel C-terminally-extended protein isoforms in vitro. Further, by coupling Translating Ribosome Affinity Purification to ribosome footprinting to enable cell-type specific analysis in vivo, we find that several of both neuronal and astrocytic transcripts undergo readthrough in the mouse brain. Functional analyses of one of these transcripts, Aqp4, reveals readthrough confers perivascular localization, indicating readthrough can be a conserved mechanism to modulate protein function. Finally, we show that AQP4 readthrough is disrupted in multiple gliotic disease models. Our study demonstrates the extensive and regulated use of alternative translational events in the brain and indicates that some of these events alter key protein properties.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Phytochemical, Analytical and Medicinal Studies of Holoptelea integrifolia Roxb. Planch - A Review

2019 ◽  
Vol 5 (4) ◽  
pp. 270-277 ◽  
Author(s):  
Vijay Kumar ◽  
Simranjeet Singh ◽  
Ragini Bhadouria ◽  
Ravindra Singh ◽  
Om Prakash
Keyword(s):  
Liquid Chromatography ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Biologically Active ◽  
Toxicological Studies ◽  
Wide Range ◽  
Layer Chromatography

Holoptelea integrifolia Roxb. Planch (HI) has been used to treat various ailments including obesity, osteoarthritis, arthritis, inflammation, anemia, diabetes etc. To review the major phytochemicals and medicinal properties of HI, exhaustive bibliographic research was designed by means of various scientific search engines and databases. Only 12 phytochemicals have been reported including biologically active compounds like betulin, betulinic acid, epifriedlin, octacosanol, Friedlin, Holoptelin-A and Holoptelin-B. Analytical methods including the Thin Layer Chromatography (TLC), High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography With Mass Spectral (LC-MS) analysis have been used to analyze the HI. From medicinal potency point of view, these phytochemicals have a wide range of pharmacological activities such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor. In the current review, it has been noticed that the mechanism of action of HI with biomolecules has not been fully explored. Pharmacology and toxicological studies are very few. This seems a huge literature gap to be fulfilled through the detailed in-vivo and in-vitro studies.

scholarly journals A novel PET tracer 18F-deoxy-thiamine: synthesis, metabolic kinetics, and evaluation on cerebral thiamine metabolism status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Changpeng Wang ◽  
Siwei Zhang ◽  
Yuefei Zou ◽  
Hongzhao Ma ◽  
Donglang Jiang ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
High Accumulation ◽  
Metabolic Kinetics ◽  
Thiamine Metabolism ◽  
Pet Tracer ◽  
The Status ◽  
The Brain

Abstract Background Some neuropsychological diseases are associated with abnormal thiamine metabolism, including Korsakoff–Wernicke syndrome and Alzheimer’s disease. However, in vivo detection of the status of brain thiamine metabolism is still unavailable and needs to be developed. Methods A novel PET tracer of 18F-deoxy-thiamine was synthesized using an automated module via a two-step route. The main quality control parameters, such as specific activity and radiochemical purity, were evaluated by high-performance liquid chromatography (HPLC). Radiochemical concentration was determined by radioactivity calibrator. Metabolic kinetics and the level of 18F-deoxy-thiamine in brains of mice and marmosets were studied by micro-positron emission tomography/computed tomography (PET/CT). In vivo stability, renal excretion rate, and biodistribution of 18F-deoxy-thiamine in the mice were assayed using HPLC and γ-counter, respectively. Also, the correlation between the retention of cerebral 18F-deoxy-thiamine in 60 min after injection as represented by the area under the curve (AUC) and blood thiamine levels was investigated. Results The 18F-deoxy-thiamine was stable both in vitro and in vivo. The uptake and clearance of 18F-deoxy-thiamine were quick in the mice. It reached the max standard uptake value (SUVmax) of 4.61 ± 0.53 in the liver within 1 min, 18.67 ± 7.04 in the kidney within half a minute. The SUV dropped to 0.72 ± 0.05 and 0.77 ± 0.35 after 60 min of injection in the liver and kidney, respectively. After injection, kidney, liver, and pancreas exhibited high accumulation level of 18F-deoxy-thiamine, while brain, muscle, fat, and gonad showed low accumulation concentration, consistent with previous reports on thiamine distribution in mice. Within 90 min after injection, the level of 18F-deoxy-thiamine in the brain of C57BL/6 mice with thiamine deficiency (TD) was 1.9 times higher than that in control mice, and was 3.1 times higher in ICR mice with TD than that in control mice. The AUC of the tracer in the brain of marmosets within 60 min was 29.33 ± 5.15 and negatively correlated with blood thiamine diphosphate levels (r = − 0.985, p = 0.015). Conclusion The 18F-deoxy-thiamine meets the requirements for ideal PET tracer for in vivo detecting the status of cerebral thiamine metabolism.

scholarly journals In Vitro Antibacterial Effect of the Methanolic Extract of the Korean Soybean Fermented Product Doenjang against Staphylococcus aureus

Animals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 2319
Author(s):  
Klara Lalouckova ◽  
Lucie Mala ◽  
Petr Marsik ◽  
Eva Skrivanova
Keyword(s):  
Staphylococcus Aureus ◽  
High Performance ◽  
Methanolic Extract ◽  
Bioactive Substances ◽  
Microdilution Method ◽  
Antibacterial Compound ◽  
Fermented Product ◽  
Broth Microdilution Method

Ultra-high performance liquid chromatography/mass spectrometry showed soyasaponin I and the isoflavones daidzein, genistein, and glycitein to be the main components of the methanolic extract of the Korean soybean fermented product doenjang, which is known to be a rich source of naturally occurring bioactive substances, at average contents of 515.40, 236.30, 131.23, and 29.00 ng/mg, respectively. The antimicrobial activity of the methanolic extract of doenjang against nine Staphylococcusaureus strains was determined in vitro by the broth microdilution method to investigate its potential to serve as an alternative antibacterial compound. The results suggest that the extract is an effective antistaphylococcal agent at concentrations of 2048–4096 µg/mL. Moreover, the tested extract also showed the ability to inhibit the growth of both methicillin-sensitive and methicillin-resistant animal and clinical S. aureus isolates. The growth kinetics of the chosen strains of S. aureus at the minimum inhibitory concentration of the methanolic extract of doenjang support the idea that the tested extract acts as an antibacterial compound. To the best of our knowledge, this is the first report on the antistaphylococcal action of the methanolic extract of doenjang thus, additional studies including in vivo testing are necessary to confirm this hypothesis.

scholarly journals High-Throughput Identification of Organic Compounds from Polygoni Multiflori Radix Praeparata (Zhiheshouwu) by UHPLC-Q-Exactive Orbitrap-MS

Molecules ◽  
2021 ◽  
Vol 26 (13) ◽  
pp. 3977
Author(s):  
Shaoyun Wang ◽  
Xiaozhu Sun ◽  
Shuo An ◽  
Fang Sang ◽  
Yunli Zhao ◽  
...  
Keyword(s):  
High Performance ◽  
Chemical Constituents ◽  
Water Extract ◽  
Ethanol Extract ◽  
In Vivo Studies ◽  
Polygonum Multiflorum ◽  
Q Exactive ◽  
Orbitrap Ms

Polygoni Multiflori Radix Praeparata (PMRP), as the processed product of tuberous roots of Polygonum multiflorum Thunb., is one of the most famous traditional Chinese medicines, with a long history. However, in recent years, liver adverse reactions linked to PMRP have been frequently reported. Our work attempted to investigate the chemical constituents of PMRP for clinical research and safe medication. In this study, an effective and rapid method was established to separate and characterize the constituents in PMRP by combining ultra-high performance liquid chromatography with hybrid quadrupole-orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS). Based on the accurate mass measurements for molecular and characteristic fragment ions, a total of 103 compounds, including 24 anthraquinones, 21 stilbenes, 15 phenolic acids, 14 flavones, and 29 other compounds were identified or tentatively characterized. Forty-eight compounds were tentatively characterized from PMRP for the first time, and their fragmentation behaviors were summarized. There were 101 components in PMRP ethanol extract (PMRPE) and 91 components in PMRP water extract (PMRPW). Simultaneously, the peak areas of several potential xenobiotic components were compared in the detection, which showed that PMRPE has a higher content of anthraquinones and stilbenes. The obtained results can be used in pharmacological and toxicological research and provided useful information for further in vitro and in vivo studies.

scholarly journals Phytochemical Study and In Vitro Screening Focusing on the Anti-Aging Features of Various Plants of the Greek Flora

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1206
Author(s):  
Aimilia D. Sklirou ◽  
Maria T. Angelopoulou ◽  
Aikaterini Argyropoulou ◽  
Eliza Chaita ◽  
Vasiliki Ioanna Boka ◽  
...  
Keyword(s):  
Plant Species ◽  
High Performance ◽  
High Resolution Mass Spectrometry ◽  
Ultra Performance Liquid Chromatography ◽  
Skin Aging ◽  
Rosa Damascena ◽  
Phytochemical Study ◽  
Age Related

Skin health is heavily affected by ultraviolet irradiation from the sun. In addition, senile skin is characterized by major changes in the collagen, elastin and in the hyaluronan content. Natural products (NPs) have been shown to delay cellular senescence or in vivo aging by regulating age-related signaling pathways. Moreover, NPs are a preferable source of photoprotective agents and have been proven to be useful against the undesirable skin hyperpigmentation. Greek flora harvests great plant diversity with approximately 6000 plant species, as it has a wealth of NPs. Here, we report an extensive screening among hundreds of plant species. More than 440 plant species and subspecies were selected and evaluated. The extracts were screened for their antioxidant and anti-melanogenic properties, while the most promising were further subjected to various in vitro and cell-based assays related to skin aging. In parallel, their chemical profile was analyzed with High-Performance Thin-Layer Chromatography (HPTLC) and/or Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS). A variety of extracts were identified that can be of great value for the cosmetic industry, since they combine antioxidant, photoprotective, anti-melanogenic and anti-aging properties. In particular, the methanolic extracts of Sideritis scardica and Rosa damascena could be worthy of further attention, since they showed interesting chemical profiles and promising properties against specific targets involved in skin aging.

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