Förster Resonance Energy Transfer (FRET) analysis is a powerful technique for studying protein-protein interactions; however, manual methods often introduce variability and user dependency. We present the SONLab FRET Analysis Tool, an open-source and automated software that integrates Cellpose for cell segmentation with standardized pipelines for bleed-through correction and FRET efficiency calculation. By minimizing human intervention, the tool improves reproducibility and comparability between experiments. The results demonstrate that the tool achieves FRET efficiencies comparable to those of manual methods but with reduced bias, enabling robust and high-throughput analysis of protein interactions.

Excessive signaling by various GPCRs underlies a variety of human disorders. Suppression of GPCRs by "enhanced" arrestin mutants was proposed as therapy. We hypothesized that GPCR binding of endogenous arrestins can be increased by small molecules stabilizing pre-activated conformation. Using molecular dynamics, we identified potentially druggable pockets in pre-activated conformation of arrestin-3 and discovered a compound targeting one of these pockets. Saturation-transfer difference NMR data showed that the compound binds at the back loop of arrestin-3. FRET- and NanoBiT-based assays in living cells showed that the compound increased in-cell arrestin-3, but not arrestin-2, binding to basal β2-adrenergic receptor and its phosphorylation-deficient mutant, but not to muscarinic M2 receptor. These experiments demonstrated the feasibility of enhancing the binding of endogenous wild type arrestin-3 to GPCRs in a receptor-specific and arrestin-subtype selective manner.

SPOCK1, a member of the SPARC family, is highly expressed in breast, lung, prostate, and liver cancer. Various studies have demonstrated that SPARC family proteins play a crucial role in the development and diseases of the central nervous system (CNS). However, the role of SPOCK1, a member of the SPARC family, in CNS diseases—particularly Alzheimer's disease—remains unknown. Previously, we have reported an association between SPOCK1 variants and LOAD in a genome-wide association studies (GWAS) meta-analysis. Based on our observation and the recent literature on SPOCK1, we explored the role of SPOCK1 in different neuronal cells to understand its possible function in Alzheimer's pathogenesis. Human astroglia (SVGp12), microglial (HMC3), and neuroblastoma (SH-SY5Y) cell lines expressing fluorescently tagged SPOCK1 and un-tagged SPOCK1 were used to investigate the expression and localization of SPOCK1 via immunocytochemistry (ICC) and confocal image analysis. It is well known that Alzheimer's disease is associated with death mechanisms in nerve cells. To investigate the molecular basis of Alzheimer's disease based on the possible unpredicted interactions and roles of SPOCK1 protein in mitochondrial cell death, SPOCK1 gene silencing and over-expression effects on apoptosis were detected via TUNEL assay in human astroglia (SVGp12), microglial (HMC3), and neuroblastoma (SH-SY5Y) cell lines. Our results based on immunocytochemistry (ICC) and confocal image analysis with various organelle markers showed for the first time that SPOCK1 is localized at the mitochondria of all the microglial, neuroblastoma, and astroglia cells. Moreover, apoptosis was detected in SPOCK1 over-expressed astrocyte cells. However in SPOCK1 over-expressing neuroblastoma cells, proliferaiton is observed, and when SPOCK1 was silenced, apoptotic cells were decreased. In microglial cells, no significant change was observed when SPOCK1 was over-expressed or silenced. For the first time we have shown localization of SPOCK1 in mitochondria in astroglia, microglial, and neuroblastoma cell lines, and studied SPOCK1's effect on cell death in these cell lines. The SPOCK1 overexpression in astrocytes increased apoptosis, highlighting its potential role in cell death. Overall our findings suggest a potantial role for SPOCK1 in Alzheimer's etiology and introducing it as a novel molecule in neurodegenerative diseases.

Aim: Small defects after any injury to the periperal nerves results in self-regeneration. However, for larger defects, suturing or grafting are necessary, which may have limitations. Thus, research on nerve guidence conduits is needed without drawbacks. The aim of the study was to develop hydrogel-based conduits containing interpenetrating network (IPN). Methods: Methacrylated gelatin (GelMA)-methacrylated hyaluronic acid (HaMA) IPN was filled the poly(2-hydroxyethylmethacrylate) (pHEMA) the outer conduit. Schwann cells (SCs) were used on the pHEMA and the distal end of the tube was injected with netrin-1 to support model SH-SY5Y cells. Results: 1H-nuclear magnetic resonance (1H-NMR) showed that methacrylation degrees were 94% ± 2% for GelMA and 60% ± 7% for HaMA. The fraction of HaMA increased the degradation rate; pure HaMA degraded in 3 weeks, while pure GelMA in more than 5 weeks. An increase in the fraction of 2-hydroxyethylmethacrylate (HEMA) from 20% to 56% decreased the porosity and the pore size, significantly. SH-SY5Y cells migrated along the conduit in the presence of netrin-1. NeuN expression was increased in 2 weeks indicating neuronal activity. Conclusions: SH-SY5Y cells produced neurites in the IPN. pHEMA conduit including GelMA-HaMA IPN is a good candidate for peripheral nerve regeneration applications. As future studies, the conduit will be tested in vivo for nerve regeneration.

GPR56/ADGRG1 is an adhesion G protein-coupled receptor (GPCR) and mutations on this receptor cause cortical malformation due to the over-migration of neural progenitor cells on brain surface. At pial surface, GPR56 interacts with collagen III, induces Rho-dependent activation through Gα12/13 and inhibits the neuronal migration. In human glioma cells, GPR56 inhibits cell migration through Gαq/11 -dependent Rho pathway. GPR56-tetraspanin complex is known to couple Gαq/11 . GPR56 is an aGPCR that couples with various G proteins and signals through different downstream pathways. In this study, bilateral frontoparietal polymicrogyria (BFPP) mutants disrupting GPR56 function but remaining to be expressed on plasma membrane were used to study receptor signalling through Gα12 , Gα13 and Gα11 with BRET biosensors. GPR56 showed coupling with all three G proteins and activated heterotrimeric G protein signalling upon stimulation with Stachel peptide. However, BFPP mutants showed different signalling defects for each G protein indicative of distinct activation and signalling properties of GPR56 for Gα12 , Gα13 or Gα11 . β-arrestin recruitment was also investigated following the activation of GPR56 with Stachel peptide using BRET biosensors. N-terminally truncated GPR56 showed enhanced β-arrestin recruitment; however, neither wild-type receptor nor BFPP mutants gave any measurable recruitment upon Stachel stimulation, pointing different activation mechanisms for β-arrestin involvement.

Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation.

Deviation of the H + concentration from optimum values within the organelles is closely associated with irregular cellular functions that cause the onset of various diseases. Therefore, determining subcellular pH values in live cells and tissues is valuable for diagnostic purposes. In this study, we report a novel ratiometric fluorescence probe 1H-pyrazole-3-carboxylic acid, 4-(benzo[d]thiazol-2-yl)-3-(2,4-dihydroxy-3-methylphenyl)-1H-pyrazole-5-carboxylicacid4-(2-benz othiazolyl)-5-(2,4-dihydroxy-3-methylphenyl), to which we will refer as ThiAKS Green (Thiazole AKyol shifting green), that is pH sensitive. The results presented here show that the probe can penetrate the cell membrane in less than 30 minutes and does not show any detectable toxicity. The measured color shifts up on pH change are linear and most significant around physiological pH (pKa=7.45), thus making this probe suitable for live-cell imaging and intracellular pH measurements. During the long-incubation periods following the application of the probe and the fluorescent microscopy measurements, it shows stable properties and is easy to detect in live cells. In conclusion, the results suggest that ThiAKS Green can be used to obtain precise information on the H + distribution at various compartments of the live cells.

The possible role of somatic copy number variations (CNVs) in Alzheimer's disease (AD) aetiology has been controversial. Although cytogenetic studies suggested increased CNV loads in AD brains, a recent single-cell whole-genome sequencing (scWGS) experiment, studying frontal cortex brain samples, found no such evidence. Here we readdressed this issue using low-coverage scWGS on pyramidal neurons dissected via both laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) across five brain regions: entorhinal cortex, temporal cortex, hippocampal CA1, hippocampal CA3, and the cerebellum. Among reliably detected somatic CNVs identified in 1301 cells obtained from the brains of 13 AD patients and 7 healthy controls, deletions were more frequent compared to duplications. Interestingly, we observed slightly higher frequencies of CNV events in cells from AD compared to similar numbers of cells from controls (4.1% vs. 1.4%, or 0.9% vs. 0.7%, using different filtering approaches), although the differences were not statistically significant. On the technical aspects, we observed that LCM-isolated cells show higher within-cell read depth variation compared to cells isolated with FACS. To reduce within-cell read depth variation, we proposed a principal component analysis-based denoising approach that significantly improves signal-to-noise ratios. Lastly, we showed that LCM-isolated neurons in AD harbour slightly more read depth variability than neurons of controls, which might be related to the reported hyperploid profiles of some AD-affected neurons.

Spinal muscular atrophy (SMA) is a rare neurodegenerative disease caused by the absence of survival motor neuron (SMN) protein. SMN loss results in impairments of the cytoskeleton, including microtubules and regulatory proteins. However, the contribution of microtubule-associated proteins (MAPs) to microtubule dysregulations in SMA is not fully understood. In this study, we investigated neuronal MAPs responsible for the microtubule stability and growth, including MAP1A, MAP2, MAP6, MAP7, EB1, and EB3 using an in vitro model of SMA. Decreased MAP2 and EB3 levels were found in SMN-deficient motor neuron-like cells, and EB3 protein level was also relevant to MAP1B. SMN loss leads to an increase in EB3 comet numbers at proximal neurites, indicating increased microtubule growth. Our findings suggest that SMN deficiency simultaneously causes dysregulations of several MAPs, contributing to the perturbations of microtubule dynamics in SMA.

Oligomerization of G protein-coupled receptors (GPCRs) may play important roles in maturation, internalization, signaling, and pharmacology of these receptors. However, the nature and extent of their oligomerization is still under debate. In our study, Ste2p, a yeast mating pheromone GPCR, was tagged with enhanced green fluorescent protein (EGFP), mCherry, and with split florescent protein fragments at the receptor C-terminus. The Förster resonance energy transfer (FRET) technique was used to detect receptors' oligomerization by calculating the energy transfer from EGFP to mCherry. Stimulation of Ste2p oligomers with the receptor ligand did not result in any significant change on observed FRET values. The bimolecular fluorescence complementation (BiFC) assay was combined with FRET to further investigate the tetrameric complexes of Ste2p. Our results suggest that in its quiescent (nonligand-activated) state, Ste2p is found at least as a tetrameric complex on the plasma membrane. Intriguingly, receptor tetramers in their active form showed a significant increase in FRET. This study provides a direct in vivo visualization of Ste2p tetramers and the pheromone effect on the extent of the receptor oligomerization.

Background The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. Methods Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (− 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. Results Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. Conclusion Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.

The interaction of neural cells with silicon surfaces is important for basic research as well as for various possible applications, such as silicon-based neural implants and neurochips. Laser structuring of silicon provides a quick and versatile method for the generation of complex, hierarchical topographies on precise locations of the substrate. The behaviour ofNeuro-2A cells with laser-structured silicon substrates was studied using a live-imaging setup with fluorescence microscopy. Neuro-2A cells were able to adhere to polished silicon, ripples and microcolumns to different extents, depending on the substrate topography and incubation time. Initially, cells adhere much better to structured areas, resulting in visible cell patterning on the substrates. Time-lapse microscopy revealed cell exploration and motility behaviours on the substrates. Cell motility was significantly decreased on structured substrates, with whole area microcolumns having the slowest cell motility. On polished silicon, cells were found to interact with the substrates using lamellipodia and filopodia. After 24 or 48 hours, cells were better able to adhere to polished as well as structured silicon. Neurite alignment xl.as observed on microcolumn and trench substrates. On the other hand, highly processed substrates were inhibitory to cell growth and resulted in poor cell health.

Objective: To investigate and compare the levels of efficacy of dexamethasone and nimodipine that are known for their neuroprotective properties on cisplatin ototoxicity in a rat model.Methods: The study included 24 male Wistar Albino rats that generated distortion product otoacoustic emission (DPOAE) response.The rats were randomized into three groups.Group 1 (n=8) was taken as the Control Group and administered intraperitoneal cisplatin at a dose of 5 mg/kg on days 1, 3 and 5, adding to a cumulative dose of 15 mg/kg in total.Afterwards, intratympanic saline solution of 0.1-0.3ml was administered on days 5, 10 and 15.In Group 2 (n=8) the same cisplatin protocol was used followed by intratympanic dexamethasone administered on days 5, 10 and 15 at a dose of 0.1-0.3ml.In Group 3 (n=8) the same cisplatin protocol was used followed by intratympanic nimodipine administered on days 5, 10 and 15 at a dose of 0.1-0.3mL.In all groups DPOAE was measured before (Day 1) and after (Day 5) cisplatin administration and after intratympanic treatment (Day 20).After the final measurement all rats were sacrificed and histopathologically examined.The study complied with the guidelines for the care and utilization of animals set forth by the Declaration of Helsinki.Results: Cisplatin have caused hearing loss at all frequencies, with more significance at higher frequencies (p<0.005).In the group that received intratympanic nimodipine, improvement was identified in the mean emission levels at 4000, 6000 and 8000 Hz.The improvement at 8000 Hz was found statistically significant compared to the control group (p<0.005).In the group that received intratympanic dexamethasone, improvement was seen in the mean emission levels at 1500, 3000, 4000, 6000 and 8000 Hz.The improvement levels at 6000 Hz and 8000 Hz were found statistically significant compared to the control group (p<0.005).When the efficacy outcomes of intratympanic nimodipine and dexamethasone were compared, the improvement in the dexamethasone group at 8000 Hz was found significant.Comparison of all groups for degeneration and TUNEL staining showed that the control group which was given cisplatin + saline solution had the highest level of degeneration and apoptotic cells.It was concluded that dexamethasone was the best application in terms of degenerative changes as well as in reducing apoptotic cells.Conclusion: Evaluation of DPOAE and histologic data showed that dexamethasone and nimodipine were autoprotective in rats which autotoxicity was induced by cisplatin.Our study is the first to demonstrate that nimodipine, both histologically and at high frequencies, has a significant healing effect, albeit less than dexamethasone.Accordingly, further experimental and clinical studies are needed on this subject.

Panomycocin is a naturally produced potent antimycotic/antifungal protein secreted by the yeast Wickerhamomyces anomalus NCYC 434 with an exo-β-1,3-glucanase activity. In this study the three dimensional structure of panomycocin was predicted and the computational site-directed mutagenesis was performed to enhance its thermal stability in liquid formulations over the body temperature for topical therapeutic applications. Homology modeling was performed with MODELLER and I-TASSER. Among the generated models, the model with the lowest energy and DOPE score was selected for further loop modeling. The loop model was optimized and the reliability of the model was confirmed with ERRAT, Verify 3D and Ramachandran plot values. Enhancement of the thermal stability of the model was done using contemporary servers and programs such as SPDBViewer, CNA, I-Mutant2.0, Eris, AUTO-MUTE and MUpro. In the region outside the binding site of the model Leu52 Arg, Phe223Arg and Gly254Arg were found to be the best thermostabilizing mutations with 6.26 K, 6.26 K and 8.27 K increases, respectively. In the binding site Glu186Arg was found to be the best thermostabilizer mutation with a 9.58 K temperature increase. The results obtained in this study led us to design a mutant panomycocin that can be used as a novel antimycotic/antifungal drug in a liquid formulation for topical applications over the normal body temperature.

The γ-Aminobutyric acid type A receptors (GABAARs) are heteropentameric chloride channels responsible for primary inhibition in the mammalian brain. Studies have shown the expression of recombinant GABAAR subunits tagged with the green fluorescent protein (GFP), a 26.9 kDa protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. This allows the formation of recombinant proteins essential for the development of relevant in-vitro and in-vivo methodologies. Among the GABAAR subunits, the δ subunit was never tagged in its cytoplasmic domain, an evolutionary conserved domain found in between the third and the fourth transmembrane domains. In this study, first, we have cloned the mouse cDNAs encoding for the δ, α1, β2 subunits of GABAARs, and then developed two fusion proteins of δ subunit each tagged with the GFP variant, EGFP (enhanced GFP) at unique sites in the cytoplasmic domain. The recombinant proteins were expressed alone or in combination with α1 and/or β2 subunits in neuroblastoma 2a cells. Live cell confocal microscopy indicated that the cytoplasmically tagged δ subunits were targeted to the cell membrane when expressed in the presence of α1 and β2 subunits in neuroblastoma 2a cells. However, this was not observed when they were expressed alone or only with α1 or β2 subunits in the same cell line. These results confirm the general oligomerization and targeting pattern of GABAAR subtypes described in the other in-vitro studies in the literature. Thus, our results suggest that the EGFP tagging in the ctoplasmic domain did not interfere with the oligomerization and cell surface expression of recombinant δ subunits. To our knowledge, this is the first study showing the generation, expression and preliminary analysis of the δ-GABAARs tagged in the cytoplasmic domain of the δ subunit which can be further elaborated to probe intracellular protein interactions of GABAARs via the δ subunit.

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (α-factor). Upon the activation of the receptors with α-factor, no significant change in BRET signal was observed. The location of Ste2p-Gpa1p heterodimer was investigated using confocal fluorescence microscopy and bimolecular fluorescence complementation (BiFC) assay, a technique where two non-fluorescent fragments of a fluorescent protein reassemble in vivo to restore fluorescence property thereby directly reporting a protein-protein interaction. BiFC experiments resulted in a dimerization signal intracellularly during biosynthesis on the endoplasmic reticulum (ER) and on the plasma membrane (PM). The constitutive BRET and BiFC signals observed on ER between Ste2p and Gpa1p in their quiescent and activated states are indicative of pre-coupling between these two proteins. This study is the first to show that the extreme N-terminus of yeast G protein alpha subunit is in close proximity to its receptor. The data suggests a pre-coupled heterodimer prior to receptor activation. The images presented in this study are the first direct in vivo evidence showing the localization of receptor - G protein heterodimers during biosynthesis and before reaching the plasma membrane.

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast 9-alpha (Gpalp) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (alpha-factor). Upon the activation of the receptors with alpha-factor, no significant change in BRET signal was observed. The location of Ste2p-Gpalp heterodimer was investigated using confocal fluorescence microscopy and bimolecular fluorescence cornplementation (BiFC) assay, a technique where two non-fluorescent fragments of a fluorescent protein reassemble in vivo to restore fluorescence property thereby directly reporting a protein-protein interaction. BiFC experiments resulted in a dimerization signal intracellularly during biosynthesis on the endoplasmic reticulum (ER) and on the plasma membrane (PM). The constitutive BRET and BiFC signals observed on ER between Ste2p and Gpalp in their quiescent and activated states are indicative of pre-coupling between these two proteins. This study is the first to show that the extreme N-terminus of yeast G protein alpha subunit is in close proximity to its receptor. The data suggests a pre-coupled heterodimer prior to receptor activation. The images presented in this study are the first direct in vivo evidence showing the localization of receptor - G protein heterodimers during biosynthesis and before reaching the plasma membrane.

Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) assay was used to determine where receptor dimerization occurred during protein trafficking by monitoring generation of EGFP fluorescence, which occurred upon GPCR dimerization. Our results suggest that these tagged receptors traffic to the membrane as monomers, undergo dimerization or higher ordered oligomerization predominantly on the plasma membrane, and are internalized as dimers/oligomers. This study is the first to provide direct in vivo visualization of GPCR dimerization/oligomerization, during trafficking to and from the plasma membrane.