Potential Perturbation Research Articles

Cord plasma metabolomic signatures of prenatal per- and polyfluoroalkyl substance (PFAS) exposures in the Boston Birth Cohort

BackgroundPrenatal per- and polyfluoroalkyl substance (PFAS) exposures are associated with adverse offspring health outcomes, yet the underlying pathological mechanisms are unclear. Cord blood metabolomics can identify potentially important pathways associated with prenatal PFAS exposures, providing mechanistic insights that may help explain PFAS' long-term health effects. MethodsThe study included 590 mother-infant dyads from the Boston Birth Cohort. We measured PFAS in maternal plasma samples collected 24–72 h after delivery and metabolites in cord plasma samples. We used metabolome-wide association studies and pathway enrichment analyses to identify metabolites and pathways associated with individual PFAS, and quantile-based g-computation models to examine associations of metabolites with the PFAS mixture. We used False Discovery Rate to account for multiple comparisons. ResultsWe found that 331 metabolites and 18 pathways were associated with ≥ 1 PFAS, and 38 metabolites were associated with the PFAS mixture, predominantly amino acids and lipids. Amino acids such as alanine and lysine and their pathways, crucial to energy generation, biosynthesis, and bone health, were associated with PFAS and may explain PFAS’ effects on fetal growth restriction. Carnitines and carnitine shuttle pathway, associated with 7 PFAS and the PFAS mixture, are involved in mitochondrial fatty acid β-oxidation, which may predispose higher risks of fetal and child growth restriction and cardiovascular diseases. Lipids, such as glycerophospholipids and their related pathway, can contribute to insulin resistance and diabetes by modulating transporters on cell membranes, participating in β-cell signaling pathways, and inducing oxidative damage. Neurotransmission-related metabolites and pathways associated with PFAS, including cofactors, precursors, and neurotransmitters, may explain the PFAS’ effects on child neurodevelopment. We observed stronger associations between prenatal PFAS exposures and metabolites in males. ConclusionsThis prospective birth cohort study contributes to the limited literature on potential metabolomic perturbations for prenatal PFAS exposures. Future studies are needed to replicate our findings and link prenatal PFAS associated metabolomic perturbations to long-term child health outcomes.

Abstract A017: IGF2BP1 is essential for ETO2/GLIS2 leukemogenesis via stabilization of primitive myeloid transcriptional programs

Abstract Certain subtypes of pediatric leukemia continue to have dismal cure rates despite advances in targeted therapy. One such disease is acute megakaryoblastic leukemia (AMKL) with a rearrangement of ETO2/GLIS2 (also known as CBFA2T3/GLIS2.) Based on our preliminary data utilizing CRISPR/Cas9 screens in vivo in patient-derived xenografts, we have nominated IGF2BP1 as a potential vulnerability in ETO2/GLIS2-rearranged AMKL. For our discovery phase, we utilized a patient-derived E2G2r AMKL which successfully engrafts in NSGS mice. We created a stable Cas9-expressing subclone using lentiviral transduction followed by FACS enrichment and limiting dilution transplants. We then generated a custom library targeting the top 100 overexpressed genes between E2G2r leukemias versus other leukemia PDXs which we previously characterized, including eight transcription factors with motifs enriched in differentially expressed genes, as well as eight pan-essential positive controls. Mice were sacrificed at humane endpoints, and spleens and bone marrow underwent PCR amplification for gRNA sequences followed by sequencing and analysis with MAGeCK-VISPR (Wei L, et al. Genome Biol 2015). We observed the dropout of 38 genes with a false discovery rate < 0.25, including all positive control gRNAs. Among the most significantly depleted genes was the RNA binding protein IGF2BP1. We subsequently characterized phenotypic changes using shRNAs in the E2G2r cell line M07e and ETO2/GLIS2-transformed CD34+ umbilical cord blood cells. Notably, we observed impaired cell growth with IGF2BP1 knockdown in both cell lines as well as the parent xenograft. We also generated M07e lines expressing a degron (FKBPF36V, aka “dTAG”, Nabet et al. Nat Chem Biol 2018) knocked in at the endogenous IGF2BP1 locus and observed similar phenomena. Through flow cytometry of M07e following IGF2BP1 depletion, we observed changes in the leukemic immunophenotype as well, suggesting perturbation of megakaryocyte differentiation potential. To determine the mechanism underlying our observations, we performed enhanced UV crosslinking with IP and sequencing (Blue SM et al. Nat Protoc 2022) against both IGF2BP1 and m6A residues based on the putative role of IGF2BP1 as an m6A reader. We observed binding of IGF2BP1 predominantly in 3’UTRs, as well as the presence of m6A-modified bases at IGF2BP1 binding sites. Gene ontology (GO) analysis of IGF2BP1-bound transcripts demonstrated enrichment for GO terms pertaining to embryonic hematopoiesis and megakaryocyte maturation/development. We further evaluated global changes in gene expression following IGF2BP1 loss, and performed RNAseq 72 hours following shRNA-mediated knockdown. GO analysis showed that downregulated genes were highly enriched for E2F-mediated transcriptional programs. We hypothesize that IGF2BP1 regulates myeloid developmental transcripts essential for E2G2r leukemogenesis, likely via stabilization in an m6A-dependent fashion, and thus may represent a selective therapeutic target. Work is ongoing to further elucidate this proposed mechanism. Citation Format: Jason Clark, Mark Wunderlich, Jing Chen, Daniel Starczynowski, Lynn Lee. IGF2BP1 is essential for ETO2/GLIS2 leukemogenesis via stabilization of primitive myeloid transcriptional programs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A017.

Prediction of performance and turbulence in ITER burning plasmas via nonlinear gyrokinetic profile prediction

Abstract Burning plasma performance, transport, and the effect of hydrogen isotope (H, D, D-T fuel mix) on confinement has been predicted for ITER baseline scenario (IBS) conditions using nonlinear gyrokinetic profile predictions. Accelerated by surrogate modeling (Rodriguez-Fernandez et al 2022 Nucl. Fusion 62 076036), high fidelity, nonlinear gyrokinetic simulations performed with the CGYRO code (Candy et al 2016 J. Comput. Phys. 324 73), were used to predict profiles of Ti , Te , and ne while including the effects of alpha heating, auxiliary power (NBI + ECH), collisional energy exchange, and radiation losses inside of r / a = 0.9. Predicted profiles and resulting energy confinement are found to produce fusion power and gain that are approximately consistent with mission goals ( P fusion = 500 MW at Q = 10) for the baseline scenario and exhibit energy confinement that is within 1σ of the H-mode energy confinement scaling. The power of the surrogate modeling technique is demonstrated through the prediction of alternative ITER scenarios with reduced computational cost. These scenarios include conditions with maximized fusion gain and an investigation of potential resonant magnetic perturbation (RMP) effects on performance with a minimal number of gyrokinetic profile iterations required (3–6). These predictions highlight the stiff ITG nature of the core turbulence predicted in the ITER baseline and demonstrate that Q > 17 conditions may be accessible by reducing auxiliary input power while operating in IBS conditions. Prediction of full kinetic profiles allowed for the projection of hydrogen isotope effects around ITER baseline conditions. The gyrokinetic fuel ion species was varied from H, D, and 50/50 D-T and kinetic profiles were predicted. Results indicate that a weak or negligible isotope effect will be observed to arise from core turbulence in IBS conditions. The resulting energy confinement, turbulence, and density peaking, and the implications for ITER operations will be discussed.

Skeletal muscle reactive oxygen species and microvascular endothelial function in age-related hypertension: a study protocol using a microdialysis technique.

Increased reactive oxygen species (ROS) generation and microvascular endothelial disruptions occur with natural ageing, but often transpire before the detection of cardiometabolic conditions including hypertension. Age-related increases in blood pressure are driven by complex systemic changes with poorly understood integrated mechanisms. The deconditioning experienced by ageing skeletal muscle from mid-life is associated with reduced microvascular blood flow and increased peripheral resistance, suggesting that vasodilatory decrements in the muscle may precede the age-related increases in blood pressure. Structural and functional changes within the vascular and skeletal muscle systems with advancing age can influence redox homeostasis, and vice versa, further compounding microvascular endothelial dysfunction. Therefore, comparisons between the microvascular environments of healthy and hypertensive cohorts can provide insights into the changes that occur during significant periods of functional decline. This comprehensive study protocol describes a microdialysis technique to assess the interactions of microvascular health and functional changes in the muscle, which currently cannot be otherwise addressed. Here, we detail an experimental protocol to simultaneously detect skeletal muscle ROS (H2O2 and indirect O2 -), determine nutritive blood flow and assess microvascular endothelial function in response to acetylcholine stimulation. We expect that healthy middle-aged individuals should not have increased ROS generation in the muscle at rest, compared to their hypertensive or older counterparts, but may exhibit perturbed microvascular function. The described technique allows for intricate exploration of microvascular physiology that will provide a critically novel insight into benchmarking potential age-related mechanisms involved in the development of age-related hypertension, and aid in early identification and prevention. KEY POINTS: Increased reactive oxygen species (ROS) production and microvascular endothelial dysfunction precede the onset of age-related cardiometabolic and vascular conditions such as hypertension. The profound structural and functional changes that occur within the vasculature and in skeletal muscle from middle age prompt a need to mechanistically explore the microvascular environment in healthy and hypertensive individuals. Using a novel microdialysis technique, we detail an experimental protocol to simultaneously detect skeletal muscle ROS (H2O2 and indirect O2 -), determine nutritive blood flow and assess microvascular endothelial function in response to acetylcholine stimulation. With this technique and study protocol, we can reveal functional insights into potential perturbations in ROS generation at rest and the microvascular endothelium, which play important roles in the development of age-related hypertension.

Statistics of protein electrostatics.

Molecular dynamics simulations of a small redox-active protein plastocyanin address two questions. (i) Do protein electrostatics equilibrate to the Gibbsian ensemble? (ii) Do the electrostatic potential and electric field inside proteins follow the Gaussian distribution? The statistics of electrostatic potential and electric field are probed by applying small charge and dipole perturbations to different sites within the protein. Nonergodic (non-Gibbsian) sampling is detectable through violations of exact statistical rules constraining the first and second statistical moments (fluctuation-dissipation relations) and the linear relation between free-energy surfaces of the collective coordinate representing the Hamiltonian electrostatic perturbation. We find weakly nonergodic statistics of the electrostatic potential (simulation time of 0.4-1.0 μs) and non-Gibbsian and non-Gaussian statistics of the electric field. A small dipolar perturbation of the protein results in structural instabilities of the protein-water interface and multi-modal distributions of the Hamiltonian energy gap. The variance of the electrostatic potential passes through a crossover at the glass transition temperature Ttr ≃ 170 K. The dipolar susceptibility, reflecting the variance of the electric field inside the protein, strongly increases, with lowering temperature, followed by a sharp drop at Ttr. The linear relation between free-energy surfaces can be directly tested by combining absorption and emission spectra of optical dyes. It was found that the statistics of the electrostatic potential perturbation are nearly Gibbsian/Gaussian, with little deviations from the prescribed statistical rules. On the contrary, the (nonergodic) statistics of dipolar perturbations are strongly non-Gibbsian/non-Gaussian due to structural instabilities of the protein hydration shell.

Accuracy enhancement of metabolic index-based blood glucose estimation with a screening process for low-quality data.

Many researchers have proposed various non-invasive glucose monitoring (NIGM) approaches using wearable or portable devices. However, due to the limited capacity of detectors for such compact devices and the movement of the body during measurement, the precision of the acquired data frequently diminishes, which can cause problems during actual use in daily life. In addition, intensive smoothing is often used in post-processing to mitigate the effects of erroneous values. However, this requires a considerable amount of data and results in a delay in the response to the actual blood glucose level (BGL). Instead of just applying data smoothing in the post-process of the data acquisition, we propose an active low-quality data screening method in the pre-process. In the proposal phase of the screening process, we employ an analytical approach to examine and formulate factors that might affect the BGL estimation accuracy. A signal quality index inspired by the standard deviation concept is introduced to detect visually apparent noise on signals. Furthermore, the total estimation error in the metabolic index (MI) is calculated based on potential perturbations defined by the signal-to-noise ratio (SNR) and the uncertainty due to discrete sampling. Thereafter, the acquired data were screened by these quality indices. By applying the proposed data screening process to the data obtained from a commercially available smartwatch device in the pre-process, the estimation accuracy of the MI-based BGL was improved significantly. Adopting the proposed screen process improves BGL estimation accuracy in the smartwatch-based prototype. Applying the proposed screen process will facilitate the integration of wearable and continuous BGL monitoring into size- and SNR-limited devices such as smartwatches and smart rings.

Countering adversarial perturbations in graphs using error correcting codes.

We consider the problem of a graph subjected to adversarial perturbations, such as those arising from cyber attacks, where edges are covertly added or removed. The adversarial perturbations occur during the transmission of the graph between a sender and a receiver. To counteract potential perturbations, this study explores a repetition coding scheme with sender-assigned noise and majority voting on the receiver's end to rectify the graph's structure. The approach operates without prior knowledge of the attack's characteristics. We analytically derive a bound on the number of repetitions needed to satisfy probabilistic constraints on the quality of the reconstructed graph. The method can accurately and effectively decode Erdős-Rényi graphs that were subjected to nonrandom edge removal, namely, those connected to vertices with the highest eigenvector centrality, in addition to random addition and removal of edges by the attacker. The method is also effective against attacks on large scale-free graphs generated using the Barabási-Albert model but require a larger number of repetitions than needed to correct Erdős-Rényi graphs.

(In)stability of de Sitter quasinormal mode spectra

We consider how the quasinormal spectrum for the conformal wave operator on the static patch of de Sitter changes in response to the addition of a small potential. Since the quasinormal modes and co-modes are explicitly known, we are able to give explicit formulae for the instantaneous rate of change of each frequency in terms of the perturbing potential. We verify these exact computations numerically using a novel technique extending the spectral hyperboloidal approach of Jaramillo et al. (2021). We propose a definition for a family of pseudospectra that we show capture the instability properties of the quasinormal frequencies.

PerturbAtlas: a comprehensive atlas of public genetic perturbation bulk RNA-seq datasets.

Manipulating gene expression is crucial for understanding gene function, with high-throughput sequencing techniques such as RNA-seq elucidating the downstream mechanisms involved. However, the lack of a standardized metadata format for small-scale perturbation expression datasets in public repositories hinders their reuse. To address this issue, we developed PerturbAtlas, an add-value resource that re-analyzes publicly archived RNA-seq libraries to provide quantitative data on gene expression, transcript profiles, and alternative splicing events following genetic perturbation. PerturbAtlas assists users in identifying trends at the gene and isoform levels in perturbation assays by re-analyzing a curated set of 122801 RNA-seq libraries across 13 species. This resource is freely available at https://perturbatlas.kratoss.site as both raw data tables and an interactive browser, allowing searches by species, tissueor genomic features. The results provide detailed information on alterations following perturbations, accessible through both forward and reverse approaches, thereby enabling the exploration of perturbation consequences and the identification of potential causal perturbations.

A tutorial on ordinary differential equations in behavioral science: What does physics teach us?

The present tutorial proposes to use concepts of physics and mathematics to help behavioral scientists to use differential equations in their studies. It focuses on the first-order and the second-order (damped oscillator) differential equation. Simple examples allow to detail the meaning of the coefficients, the conditions of applicability of these differential equations, the underlying hypothesis, and their consequences for the researcher willing to use them. More complex psychological examples demonstrate the importance of parameters' interpretation. Particular attention is paid to how potential external perturbations should be considered. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Determination of electronic resonances by analytic continuation using barycentric formula

Numerical analytical continuation of a function is used to determine its complex roots. The analytical continuation is carried out by means of a barycentric formula. From the knowledge of the complex roots the energy and width of shape resonances as well as of quantum virtual states can be determined. The roots are calculated for several realistic models and the results are compared with other approaches. We also explore and discuss a stability of the predicted resonant roots with respect to changes of the perturbation potential.

Paternal zinc deficiency alters offspring metabolic status in Drosophila melanogaster

BackgroundThis study delves into the understudied yet potentially crucial role of paternal zinc deficiency in programming offspring metabolic outcomes. By examining paternal zinc deficiency, we aim to shed light on a previously unexplored avenue with the potential to significantly impact future generations. We investigated the intergenerational effects of paternal zinc deficiency on metabolic parameters in Drosophila melanogaster. MethodsDietary zinc deficiency was induced by supplementing the diet of Drosophila F0 male flies with TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine) from egg stage. The F0 male flies after eclosion were mated with age-matched virgin female flies from the control group, resulting in the F1 offspring generation. The F1 generation were then cultured on a standard diet for subsequent metabolic analyses, including assessments of body weight, locomotion, and levels of glucose, trehalose, glycogen, and triglycerides as well as the expression of related genes. ResultsWe observed an increase (p<0.05) in body weight in male parent flies and female offspring. Negative geotaxis performance was also impaired in the female offspring. Paternal zinc deficiency exerted distinct effects on carbohydrate and lipid metabolism, as evidenced by a significant (p<0.05) increase in trehalose and triglyceride levels in both parent and offspring. Additionally, zinc deficiency led to alterations in the expression of key metabolic genes, including significant (p<0.05) increase in DILP2 mRNA levels, highlighting potential links to insulin signaling. Also, there were reduced mRNA levels of SOD1 and CAT in both parental and offspring generations. Parental zinc deficiency also increased the expression of Eiger and UPD2 mRNA in the offspring, suggesting potential perturbations in the immune response system. ConclusionThese findings underscore the link between zinc status and various physiological and molecular processes, revealing both immediate and intergenerational impacts on metabolic, antioxidant, and inflammatory pathways and providing valuable insights on the implications of paternal zinc deficiency in Drosophila melanogaster.

Localized Surface Modification during Alternating Current Scanning Electrochemical Microscopy: Origin and Mechanism

In the current study, we report for the first time the observation of unintended localized surface modification on commercially pure aluminum (Al) during an alternating current scanning electrochemical microscopy (AC-SECM) analysis, its origin, and the probable mechanism responsible for it. Application of an AC perturbation potential (∼100 mV amplitude at ∼100 kHz frequency) to the Platinum ultramicroelectrode (Pt UME), during AC-SECM in acidic, neutral chloride, tap water, and alkaline electrolytes was found to cause surface modification on the scanned region of Al. An increase in the local pH of the electrolyte between the UME and the Al substrate, irrespective of the electrolyte pH (3–11) and UME biasing conditions, led to the local surface modification. The reason for the enhancement of local pH is attributed to the occurrence of higher rates of cathodic reduction reactions than that of anodic oxidation reactions. The reduction of dissolved oxygen/protons/water in the electrolytes led to the generation or consumption of OH−/H+ ions, respectively, and thus increased the pH, whereas the oxidation of Pt UME/Al surfaces decreased the pH with the generation of H+ or consumption of OH− ions. These results contribute significantly to accurately analyzing Al and its alloys using the AC-SECM technique.

Wave-Current Interaction Effects on the OC4 DeepCwind Semi-Submersible Floating Offshore Wind Turbine

In order to investigate the hydrodynamic performances of semi-submersible type floating offshore wind turbines (FOWTs), particularly the effect of body-wave-current interaction, the OC4 FOWT is considered in the presence of co-existing regular wave and uniform current fields. The wind loads are not considered at this stage. The problem is treated in the framework of potential-flow theory in the frequency domain, assuming waves of small steepness, and the solution is obtained by using a perturbation expansion method for the diffraction potential with respect to the normalized current speed. Analytical and numerical formulations have been used to treat the inhomogeneous free-surface boundary condition involved in the hydrodynamic problem formulation for the derivation of the associated perturbation potential. The hydrodynamic loads were obtained after evaluating the pressure field around the multi-body configuration using three different computer codes. The results from the three computer codes compare very well with each other and with the numerical predictions of other investigators. Finally, the mean second-order drift forces are calculated by superposing their zero-current values with the corresponding current-dependent first-order corrections, with the latter being evaluated using a ‘heuristic’ approach.

Responses to brief perturbations of stance: EMG, midline cortical, and subcortical changes.

We studied simultaneous EMG and midline EEG responses, including over the cerebellum, in 10 standing subjects (35 ± 15 yr; 5 females, 5 males). Recordings were made following repeated taps to the sternum, stimuli known to evoke short-latency EMG responses in leg muscles, consistent with postural reflexes. EEG power had relatively more high-frequency components (>30 Hz) when recorded from electrodes over the cerebellum (Iz and SIz) compared with other midline electrodes. We confirmed a previous report using a similar stimulus that evoked short-latency potentials over the cerebellum. We showed clear midline-evoked EEG potentials occurring at short latency over the cerebellum (P23, N31, N42, and P54) and frontally (N28 and N57) before the previously described perturbation-evoked potential (P1/N1/P2). The P23 response correlated with the subsequent EMG response in the tibialis anterior muscles (r = 0.72, P = 0.018), confirming and extending previous observations. We did not find a correlation with the N1 amplitude. We conclude that early activity occurs from electrodes over the inion in response to a brief tap to the sternum. This is likely to represent cerebellar activity and it appears to modulate short-latency postural EMG responses.NEW & NOTEWORTHY We studied the effects of a brief tap to the sternum in human subjects, known to evoke short-latency postural responses. Using an extended EEG recording system, we showed early evoked responses over the midline cerebellum, including the P23 potential, which correlated with the EMG responses in tibialis anterior, consistent with a cerebellar role in postural reflexes. The stimulus also evoked later EEG responses, including the perturbation potential.

Quantum Big-Bounce as a phenomenology of RQM in the Mini-superspace

We investigate the emergence of a quantum Big-Bounce in the context of an isotropic Universe, filled by a self-interacting scalar field, which plays the role of a physical clock. The bouncing cosmology is the result of a scattering process, driven by the scalar field potential, which presence breaks down the frequency separation of the Wheeler-DeWitt equation, treated in strict analogy to a relativistic quantum system. Differently from previous analyses, we consider a really perturbative self-interaction potential, affecting the dynamics in a finite range of the time labeled by the scalar clock (and in particular we remove the divergent character previously allowed). The main result of the present analysis is that, when the Relativistic Quantum Mechanics formalism is properly implemented in the Mini-superspace analogy, the probability amplitude for the bounce is, both in the standard and polymerized case, characterized by a maximum in correspondence of the quasi-classical condition of a Universe minimum volume.

Large-Scale Field Trials of an Eimeria Vaccine Induce Positive Effects on the Production Index of Broilers.

Live coccidiosis vaccines have mainly been used to reduce Eimeria species infection, which is considered the most economically important disease in the poultry industry. Evaluation data on vaccine effectiveness through large-scale field experiments are lacking, especially in broilers. Thus, the effectiveness of a commercial coccidiosis vaccine was evaluated in approximately 900,000 chicks reared on three open-broiler farms where coccidiosis is prevalent. The vaccine's effectiveness after vaccination of 1-day-old chicks was monitored using three parameters (lesion score, fecal oocyst shedding, and production index, PI) in nine trials performed three times on each farm. Lesion scores were confirmed in three different areas of the intestine because the vaccine contained four Eimeria species. The average lesion scores were 0.36 to 0.64 in the duodenal region, 0.30 to 0.39 in the jejuno-ileal region, and 0.18 to 0.39 in the cecal region. The average fecal oocyst shedding rate ranged from 19,766 to 100,100 oocysts per gram, showing large variations depending on farms and buildings within the farm. Compared with the PI of the previous 9-10 trials on each farm, the PI increased by 2.45 to 23.55. Because of the potential for perturbation of the fecal microbiota by live coccidiosis vaccines, the fecal microbiota was investigated using 16S rRNA microbial profiling. Although the β-diversity was significantly different in distribution and relative abundance among farms (PERMANOVA, pseudo-F = 4.863, p = 0.009), a Kyoto Encyclopedia of Genes and Genomes pathway analysis found no significant bacterial invasion of the epithelial cell pathway across farms. This large-scale field trial of a live Eimeria vaccine indicates that coccidiosis vaccines can have meaningful effects on the poultry industry and could be used as an alternative to the prophylactic use of anticoccidial drugs under field conditions.

Live Cell Protein Imaging of Tandem Complemented-GFP11-Tagged Coiled-Coil Domain-Containing Protein-124 Identifies this Factor in G3BP1-Induced Stress-Granules.

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

Quercetin alleviates 6-OHDA-caused apoptosis in SH-SY5Y cells.

This study is primarily designed to investigate the potential neuroprotective effects of polyphenol against 6-OHDAcaused neurotoxicity on SH-SY5Y cells. Cytotoxic effect of 6-OHDA and valuable role of quercetin, myricetin and kaempferol on SH-SY5Y cells were analyzed by MTT assay. Generation of 6-OHDA-stimulated reactive oxygen species (ROS) was measured using DCFDA fluorescence dye. Alteration of 6-OHDA-caused mitochondrial membrane potential and nuclear condensation was investigated with the help of rhodamine-123 and hoechest stain. Immunoblotting was performed to detect the expression level of 6-OHDA-caused alpha-synuclein (á-syn), Bcl-2 associated protein X (BAX), caspase 3, cleaved Poly ADP - ribose polymerase (PARP) and Bcell lymphoma 2 proteins (Bcl-2). Through MTT assay, quercetin was selected over myricetin and kaempferol to counter 6-OHDA-caused cell death. The research delves into unraveling the intricate mechanisms underlying 6-OHDA-induced neurotoxicity, encompassing alterations in cellular morphology, escalation of oxidative stress, perturbation in mitochondrial membrane potential, and nuclear condensation. Exposure to 6-OHDA is implicated in the upregulation of á-syn protein, contributing to the aggravation of neurotoxicity. Concurrently, 6-OHDA orchestrates the apoptotic pathway by upregulating the expression of proapoptotic proteins such as BAX, caspase 3, and PARP, while down regulating the expression of the Bcl-2, affirming its role in apoptosis induction. Quercetin demonstrated ability to attenuate the expression of á-syn in the presence of 6-OHDA-caused injury in SH-SY5Y cells. Taken together, these findings collectively underscore the therapeutic potential of quercetin as a promising agent against neurotoxicity caused by 6-OHDA.

Transgenic Targeting of Fcrls Creates a Highly Efficient Constitutively Active Microglia Cre Line with Differentiated Specificity.

Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines can achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specific Fcrls gene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis in Fcrls-2A-Cre mice. Crossing Fcrls-2A-Cre mice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border-associated macrophages are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line, Cx3cr1-CreM Fcrls-2A-Cre mice are immediately available (JAX #036591) and serve as a valuable addition to the community's microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable.