Distinguishing dopamine and calcium responses using XNA-nanotube sensors for improved neurochemical sensing

AbstractTo date, the engineering of single-stranded DNA-SWCNT (DNA-SWCNT) optical biosensors have largely focused on creating sensors for new applications with little focus on optimising existing sensors for in vitro and in vivo conditions. Recent studies have shown that nanotube fluorescence can be severely impacted by changes in local cation concentrations. This is particularly problematic for neurotransmitter sensing applications as spatial and temporal fluctuations in the concentration of cations, such as Na+, K+, or Ca2+, play a central role in neuromodulation. This can lead to inaccuracies in the determination of neurotransmitter concentrations using DNA-SWCNT sensors, which limits their use for detecting and treating neurological diseases.Herein, we present new approaches using locked nucleic acid (LNA) to engineer SWCNT sensors with improved stability towards cation-induced fluorescence changes. By incorporating LNA bases into the (GT)15-DNA sequence, we create sensors that are not only more resistant towards undesirable fluorescence modulation in the presence of Ca2+ but that also retain their capabilities for the label-free detection of dopamine. The synthetic biology approach presented in this work therefore serves as a complementary means for enhancing nanotube optoelectronic behavior, unlocking previously unexplored possibilities for developing nano-bioengineered sensors with augmented capabilities.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Faculty Opinions recommendation of In vivo capture and label-free detection of early metastatic cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725774602.793510350 ◽

2015 ◽

Author(s):

Martin Gruebele ◽

Max Platkov

Keyword(s):

Label Free ◽

Metastatic Cells ◽

Label Free Detection

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The effect of dipeptidyl peptidase IV on disease-associated microglia phenotypic transformation in epilepsy

Journal of Neuroinflammation ◽

10.1186/s12974-021-02133-y ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Zhicheng Zheng ◽

Peiyu Liang ◽

Baohua Hou ◽

Xin Lu ◽

Qianwen Ma ◽

...

Keyword(s):

Signaling Pathway ◽

Neurological Diseases ◽

Dipeptidyl Peptidase ◽

Dipeptidyl Peptidase Iv ◽

Sequencing Data ◽

Migration Ability ◽

Dpp4 Inhibitor ◽

Phenotypic Transformation

Abstract Background Accumulating evidence suggests that disease-associated microglia (DAM), a recently discovered subset of microglia, plays a protective role in neurological diseases. Targeting DAM phenotypic transformation may provide new therapeutic options. However, the relationship between DAM and epilepsy remains unknown. Methods Analysis of public RNA-sequencing data revealed predisposing factors (such as dipeptidyl peptidase IV; DPP4) for epilepsy related to DAM conversion. Anti-epileptic effect was assessed by electroencephalogram recordings and immunohistochemistry in a kainic acid (KA)-induced mouse model of epilepsy. The phenotype, morphology and function of microglia were assessed by qPCR, western blotting and microscopic imaging. Results Our results demonstrated that DPP4 participated in DAM conversion and epilepsy. The treatment of sitagliptin (a DPP4 inhibitor) attenuated KA-induced epilepsy and promoted the expression of DAM markers (Itgax and Axl) in both mouse epilepsy model in vivo and microglial inflammatory model in vitro. With sitagliptin treatment, microglial cells did not display an inflammatory activation state (enlarged cell bodies). Furthermore, these microglia exhibited complicated intersections, longer processes and wider coverage of parenchyma. In addition, sitagliptin reduced the activation of NF-κB signaling pathway and inhibited the expression of iNOS, IL-1β, IL-6 and the proinflammatory DAM subset gene CD44. Conclusion The present results highlight that the DPP4 inhibitor sitagliptin can attenuate epilepsy and promote DAM phenotypic transformation. These DAM exhibit unique morphological features, greater migration ability and better surveillance capability. The possible underlying mechanism is that sitagliptin can reduce the activation of NF-κB signaling pathway and suppress the inflammatory response mediated by microglia. Thus, we propose DPP4 may act as an attractive direction for DAM research and a potential therapeutic target for epilepsy.

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The Potential Neuroprotective Role of Free and Encapsulated Quercetin Mediated by miRNA against Neurological Diseases

Nutrients ◽

10.3390/nu13041318 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1318

Author(s):

Tarek Benameur ◽

Raffaella Soleti ◽

Chiara Porro

Keyword(s):

Inflammatory Responses ◽

Pathological Condition ◽

Neurological Diseases ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

In Vivo Studies ◽

Innovative Drug ◽

Chronic Neuroinflammation

Chronic neuroinflammation is a pathological condition of numerous central nervous system (CNS) diseases such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis and many others. Neuroinflammation is characterized by the microglia activation and concomitant production of pro-inflammatory cytokines leading to an increasing neuronal cell death. The decreased neuroinflammation could be obtained by using natural compounds, including flavonoids known to modulate the inflammatory responses. Among flavonoids, quercetin possess multiple pharmacological applications including anti-inflammatory, antitumoral, antiapoptotic and anti-thrombotic activities, widely demonstrated in both in vitro and in vivo studies. In this review, we describe the recent findings about the neuroprotective action of quercetin by acting with different mechanisms on the microglial cells of CNS. The ability of quercetin to influence microRNA expression represents an interesting skill in the regulation of inflammation, differentiation, proliferation, apoptosis and immune responses. Moreover, in order to enhance quercetin bioavailability and capacity to target the brain, we discuss an innovative drug delivery system. In summary, this review highlighted an important application of quercetin in the modulation of neuroinflammation and prevention of neurological disorders.

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Y-shaped probe for convenient and label-free detection of microRNA-21 in vitro

Analytical Biochemistry ◽

10.1016/j.ab.2016.01.022 ◽

2016 ◽

Vol 499 ◽

pp. 8-14 ◽

Cited By ~ 13

Author(s):

Kui He ◽

Rong Liao ◽

Changqun Cai ◽

Caishuang Liang ◽

Chan Liu ◽

...

Keyword(s):

Label Free ◽

Label Free Detection

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Distinct roles of tumor associated mutations in collective cell migration

Scientific Reports ◽

10.1038/s41598-021-89130-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rachel M. Lee ◽

Michele I. Vitolo ◽

Wolfgang Losert ◽

Stuart S. Martin

Keyword(s):

Collective Behavior ◽

Metastatic Potential ◽

Breast Epithelial Cell ◽

Collective Cell Migration ◽

Label Free ◽

Collective Migration ◽

Contrast Imaging ◽

Pten Deletion

AbstractRecent evidence suggests that groups of cells are more likely to form clinically dangerous metastatic tumors, emphasizing the importance of understanding mechanisms underlying collective behavior. The emergent collective behavior of migrating cell sheets in vitro has been shown to be disrupted in tumorigenic cells but the connection between this behavior and in vivo tumorigenicity remains unclear. We use particle image velocimetry to measure a multidimensional migration phenotype for genetically defined human breast epithelial cell lines that range in their in vivo behavior from non-tumorigenic to aggressively metastatic. By using cells with controlled mutations, we show that PTEN deletion enhances collective migration, while Ras activation suppresses it, even when combined with PTEN deletion. These opposing effects on collective migration of two mutations that are frequently found in patient tumors could be exploited in the development of novel treatments for metastatic disease. Our methods are based on label-free phase contrast imaging, and thus could easily be applied to patient tumor cells. The short time scales of our approach do not require potentially selective growth, and thus in combination with label-free imaging would allow multidimensional collective migration phenotypes to be utilized in clinical assessments of metastatic potential.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v2 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

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Interaction between Gut Microbiota and Curcumin: A New Key of Understanding for the Health Effects of Curcumin

Nutrients ◽

10.3390/nu12092499 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2499 ◽

Cited By ~ 5

Author(s):

Beatrice Scazzocchio ◽

Luisa Minghetti ◽

Massimo D’Archivio

Keyword(s):

Gut Microbiota ◽

Neurological Diseases ◽

Curcuma Longa ◽

Pharmacological Activities ◽

Systemic Bioavailability ◽

Microbiome Profile ◽

Regulatory Effects ◽

High Concentrations

Curcumin, a lipophilic polyphenol contained in the rhizome of Curcuma longa (turmeric), has been used for centuries in traditional Asian medicine, and nowadays it is widely used in food as dietary spice worldwide. It has received considerable attention for its pharmacological activities, which appear to act primarily through anti-inflammatory and antioxidant mechanisms. For this reason, it has been proposed as a tool for the management of many diseases, among which are gastrointestinal and neurological diseases, diabetes, and several types of cancer. However, the pharmacology of curcumin remains to be elucidated; indeed, a discrepancy exists between the well-documented in vitro and in vivo activities of curcumin and its poor bioavailability and chemical instability that should limit any therapeutic effect. Recently, it has been hypothesized that curcumin could exert direct regulative effects primarily in the gastrointestinal tract, where high concentrations of this polyphenol have been detected after oral administration. Consequently, it might be hypothesized that curcumin directly exerts its regulatory effects on the gut microbiota, thus explaining the paradox between its low systemic bioavailability and its wide pharmacological activities. It is well known that the microbiota has several important roles in human physiology, and its composition can be influenced by a multitude of environmental and lifestyle factors. Accordingly, any perturbations in gut microbiome profile or dysbiosis can have a key role in human disease progression. Interestingly, curcumin and its metabolites have been shown to influence the microbiota. It is worth noting that from the interaction between curcumin and microbiota two different phenomena arise: the regulation of intestinal microflora by curcumin and the biotransformation of curcumin by gut microbiota, both of them potentially crucial for curcumin activity. This review summarizes the most recent studies on this topic, highlighting the strong connection between curcumin and gut microbiota, with the final aim of adding new insight into the potential mechanisms by which curcumin exerts its effects.

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Lipid Labelling in Intact Chloroplasts from Exogenous Nucleotide Precursors

Zeitschrift für Naturforschung C ◽

10.1515/znc-1981-1-213 ◽

1981 ◽

Vol 36 (1-2) ◽

pp. 62-70 ◽

Cited By ~ 22

Author(s):

Margrit Bertrams ◽

Käthe Wrage ◽

Ernst Heinz

Keyword(s):

Fatty Acids ◽

Free Fatty Acids ◽

De Novo ◽

Membrane System ◽

Chloroplast Envelope ◽

Label Free ◽

Intact Chloroplasts ◽

Time Required

Abstract De novo-synthesis of glycerolipids in chloroplasts is initiated by a stroma enzyme which catalyzes the formation of lyso-phosphatidic acid from glycerophosphate and acyl-CoA. When these substrates are added to isolated, intact chloroplasts, only glycerophosphate can readily pass through the chloroplast envelope which represents a permeation barrier for acyl-CoA, although higher thioester concentrations destroy this membrane system. At low concentrations of acyl-CoA, which do not impair the envelope, intact chloroplasts metabolize exogenous acyl-CoA in two ways to give free fatty acids and labelled phosphatidyl choline. This indicates that the envelope thioesterase can use exogenous substrates. Isolated, intact chloroplasts fixing radioactive CO2 label free fatty acids and acylglycerols but not galactolipids, since they cannot convert 3-phosphoglycerate into UDP-galactose which in vivo is supplied by the cytoplasm. This cooperation was simulated in vitro by adding all enzymes and cofactors necessary for conversion of 3-phosphoglycerate into UDP-galactose to intact chloroplasts which then formed labelled monogalactosyl diacylglycerol from labelled CO2. The time required to transfer envelope-made galactolipids from the envelope into thylakoids was studied by incubating intact chloroplasts with radioactive UDP-galactose, subsequent osmotic disruption of organelles with concomitant enzymatic degradation of UDP-galactose followed by separation of envelopes and thylakoids. Only after short times (< 1min) appreciable proportions 920-30%) of radioactive galactolipid export from envelopes into thylakoids.

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Label-Free Split Aptamer Sensor for Femtomolar Detection of Dopamine by Means of Flexible Organic Electrochemical Transistors

Materials ◽

10.3390/ma13112577 ◽

2020 ◽

Vol 13 (11) ◽

pp. 2577 ◽

Cited By ~ 5

Author(s):

Yuanying Liang ◽

Ting Guo ◽

Lei Zhou ◽

Andreas Offenhäusser ◽

Dirk Mayer

Keyword(s):

Detection Limit ◽

Electrochemical Sensors ◽

Limit Of Detection ◽

High Sensitivity ◽

Label Free ◽

Dopamine Detection ◽

Electrochemical Transistor ◽

Sensitivity And Selectivity

The detection of chemical messenger molecules, such as neurotransmitters in nervous systems, demands high sensitivity to measure small variations, selectivity to eliminate interferences from analogues, and compliant devices to be minimally invasive to soft tissue. Here, an organic electrochemical transistor (OECT) embedded in a flexible polyimide substrate is utilized as transducer to realize a highly sensitive dopamine aptasensor. A split aptamer is tethered to a gold gate electrode and the analyte binding can be detected optionally either via an amperometric or a potentiometric transducer principle. The amperometric sensor can detect dopamine with a limit of detection of 1 μM, while the novel flexible OECT-based biosensor exhibits an ultralow detection limit down to the concentration of 0.5 fM, which is lower than all previously reported electrochemical sensors for dopamine detection. The low detection limit can be attributed to the intrinsic amplification properties of OECTs. Furthermore, a significant response to dopamine inputs among interfering analogues hallmarks the selective detection capabilities of this sensor. The high sensitivity and selectivity, as well as the flexible properties of the OECT-based aptasensor, are promising features for their integration in neuronal probes for the in vitro or in vivo detection of neurochemical signals.

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