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AbstractEvent-related potentials (ERPs) extracted from electroencephalography (EEG) data in response to stimuli are widely used in psychological and neuroscience experiments. A major goal is to link ERP characteristic components to subject-level covariates. Existing methods typically follow two-step approaches, first identifying ERP components using peak detection methods and then relating them to the covariates. This approach, however, can lead to loss of efficiency due to inaccurate estimates in the initial step, especially considering the low signal-to-noise ratio of EEG data. To address this challenge, we propose a semiparametric latent ANOVA model (SLAM) that unifies inference on ERP components and their association with covariates. SLAM models ERP waveforms via a structured Gaussian process (GPs) prior that encode ERP latency in its derivative and links the subject-level latencies to covariates using a latent ANOVA. This unified Bayesian framework provides estimation at both population- and subject-levels, improving the efficiency of the inference by leveraging information across subjects. We automate posterior inference and hyperparameter tuning using a Monte Carlo expectation–maximization (MCEM) algorithm. We demonstrate the advantages of SLAM over competing methods via simulations. Our method allows us to examine how factors or covariates affect the magnitude and/or latency of ERP components, which in turn reflect cognitive, psychological, or neural processes. We exemplify this via an application to data from an ERP experiment on speech recognition, where we assess the effect of age on two components of interest. Our results verify the scientific findings that older people take a longer reaction time to respond to external stimuli because of the delay in perception and brain processes.

Abstract Background Cholesterol plays a vital role in multiple physiological processes. Cellular uptake of cholesterol is mediated primarily through endocytosis of low-density lipoprotein (LDL) receptor. New modifiers of this process remain to be characterized. Particularly, the role of fasting- and CREB-H-induced (FACI) protein in cholesterol homeostasis merits further investigation. Methods Interactome profiling by proximity labeling and affinity purification − mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed. Results FACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn’t affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice. Conclusions FACI facilitates LDL endocytosis through its interaction with the AP2 complex.

Background & Aims: CREB-H is a key liver-enriched transcription factor governing lipid metabolism. Additional targets of CREB-H remain to be identified and characterized. Here, we identified a novel fasting- and CREB-H–induced (FACI) protein that inhibits intestinal lipid absorption and alleviates diet-induced obesity in mice. Methods: FACI was identified by reanalysis of existing transcriptomic data. Faci-/- mice were generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated genome engineering. RNA sequencing was performed to identify differentially expressed genes in Faci-/- mice. Lipid accumulation in the villi was assessed by triglyceride measurement and Oil red O staining. In vitro fatty acid uptake assay was performed to verify in vivo findings. Results: FACI expression was enriched in liver and intestine. FACI is a phospholipid-binding protein that localizes to plasma membrane and recycling endosomes. Hepatic transcription of Faci was regulated by not only CREB-H, but also nutrient-responsive transcription factors sterol regulatory element-binding protein 1 (SREBP1), hepatocyte nuclear factor 4α (HNF4α), peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α), and CREB, as well as fasting-related cyclic adenosine monophosphate (cAMP) signaling. Genetic knockout of Faci in mice showed an increase in intestinal fat absorption. In accordance with this, Faci deficiency aggravated high-fat diet–induced obesity, hyperlipidemia, steatosis, and other obesity-related metabolic dysfunction in mice. Conclusions: FACI is a novel CREB-H–induced protein. Genetic disruption of Faci in mice showed its inhibitory effect on fat absorption and obesity. Our findings shed light on a new target of CREB-H implicated in lipid homeostasis.

Matrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force.

Background/Purpose: Desmosome gene mutations have been reported in patients with arrhythmogenic right ventricular dysplasia (ARVD). However, there are hardly any genetic studies in Asians. We studied the clinical characteristics, cardiac manifestations and desmosome gene mutations in ARVD patients in Taiwan. Methods: Medical records of five ARVD patients were reviewed and genomic DNA was obtained from peripheral blood samples. Mutation screening in desmoplakin (DSP), plakophilin-2, desmoglein-2 (DSG2) and desmocollin-2 genes was performed using polymerase chain reaction and DNA sequencing techniques. Results: Among the five patients, three presented with palpitations followed by loss of consciousness, and the other two had palpitations or chest tightness without loss of consciousness. Electrocardiogram (ECG), magnetic resonance imaging and signal averaged ECG results were similar to those reported in Western countries. Mutations in the desmosome genes were identified in four of the five patients (three with a DSG2 mutation and one with a DSP mutation). Five gene mutations were noted in four patients and all mutations were novel (one patient had a DSG2 double mutation). The mutation types were missense in four and splicing mutation in one. Conclusion: Patients with ARVD in Taiwan had similar clinical and cardiac manifestations as reported in the Western literature. More than half of the patients had desmosome gene mutations.

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3epsilon on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.

Objective: The effect of oil pulling on oral health has not yet been fully demonstrated. Therefore, we performed a meta-analysis to investigate the effect of oil pulling on oral health. Methods: We searched PubMed, the Cochrane Library, and the EMBASE database, limiting the search to human patients and articles written in English and published before 31 July 2022. We included randomized controlled trials (RCTs) comparing the effect of oil pulling on improving dental health and oral hygiene. The outcomes of this study were salivary bacteria count, plaque index, and gingival index. Results: In total, nine RCTs were included in this study. The study showed that salivary bacterial colony (BC) counts were significantly reduced in the oil pulling group compared to the control group [mean difference (MD): 17.55, 95% CI 2.56, 32.55]. There was no significant difference between the two groups (MD: −0.10, 95% CI −0.33, 0.14; −0.05, 95% CI −0.12, 0.02) in plaque index and gingival index score. Conclusions: Based on the results of this meta-analysis, the oil pulling may have a beneficial effect on reducing salivary BC count compared to the control group. There was no significant difference in the plaque index and gingival index score between the oil pulling and the control group. Therefore, future clinical trials should be more rigorous and better reported. [ABSTRACT FROM AUTHOR]

Invadopodia are integrin-mediated adhesions with abundant PI(3,4) P2. However, the functional role of PI(3,4)P2 in adhesion signaling remains unclear. Here, we find that the PI(3,4)P2 biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P2 is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P2-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P2-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P2 promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium. [ABSTRACT FROM AUTHOR]

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to lasermicroirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B-EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin. [ABSTRACT FROM AUTHOR]

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSIinduced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPAbound DSBs. Accordingly, we found that RNF169 promotes CtIPdependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates singlestrand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways. [ABSTRACT FROM AUTHOR]

Recent studies have revealed pronounced effects of the spatial distribution of EphA2 receptors on cellular response to receptor activation. However, little is known about molecular mechanisms underlying this spatial sensitivity, in part due to lack of experimental systems. Here, we introduce a hybrid live-cell patterned supported lipid bilayer experimental platform in which the sites of EphA2 activation and integrin adhesion are spatially controlled. Using a series of live-cell imaging and single-molecule tracking experiments, we map the transmission of signals from ephrinA1:EphA2 complexes. Results show that ligand-dependent EphA2 activation induces localized myosin-dependent contractions while simultaneously increasing focal adhesion dynamics throughout the cell. Mechanistically, Src kinase is activated at sites of ephrinA1:EphA2 clustering and subsequently diffuses on the membrane to focal adhesions, where it up-regulates FAK and paxillin tyrosine phosphorylation. EphrinA1:EphA2 signaling triggers multiple cellular responses with differing spatial dependencies to enable a directed migratory response to spatially resolved contact with ephrinA1 ligands. [ABSTRACT FROM AUTHOR]

Podosomes represent a class of integrin-mediated cell-matrix adhesions formed by migrating and matrix-degrading cells. We demonstrate that in macrophage-like THP1 cells and fibroblasts stimulated to produce podosomes, down-regulation of the G-protein ARF1 or the ARF1 guanine nucleotide exchange factor, ARNO, by small, interfering RNA or pharmacological inhibitors led to striking podosome elimination. Concomitantly, treatments inducing podosome formation increased the level of guanosine triphosphate (GTP)-bound ARF1. ARNO was found to colocalize with the adhesive rings of podosomes, whereas ARF1 was localized to vesicular structures transiently contacting podosome rings. Inhibition of ARF1 led to an increase in RhoA-GTP levels and triggered assembly of myosin-IIA filaments in THP1 cells, whereas the suppression of myosin-IIA rescued podosome formation regardless of ARF1 inhibition. Finally, expression of constitutively active ARF1 in fibroblasts induced formation of putative podosome precursors: actin-rich puncta coinciding with matrix degradation sites and containing proteins of the podosome core but not of the adhesive ring. Thus, ARNO-ARF1 regulates formation of podosomes by inhibition of RhoA/myosin-II and promotion of actin core assembly. [ABSTRACT FROM AUTHOR]

Dramatic photochromic-change of 2,5-bis-(triphenylamine)-substituted N,N'-diphenylpyrrolo-[3,2-b]pyrrole ( l) with halocarbons provides an effective route for halocarbon analysis with the naked eye. The visual detection range can reach as low as 10-4 ∼ 10-5 M ( l-10 ppm) in CH3CN. This method can also be applied for detection of CHC13 in water. Fabrication of a disposable paper test cartridge along with using a camera flash as the light source allows on-site halocarbon detection in seconds. Quantitative analysis for CHCl3 and CH2C12 have also been demonstrated. [ABSTRACT FROM AUTHOR]

Epithelial (E)-cadherin-mediated cell--cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role. [ABSTRACT FROM AUTHOR]

Actin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm. [ABSTRACT FROM AUTHOR]

Cell membranes exhibit multiple layers of complexity, ranging from their specific molecular content to their emergent mechanical properties and dynamic spatial organization. Both compositional and geometrical organizations of membrane components are known to play important roles in life processes, including signal transduction. Supported membranes, comprised of a bilayer assembly of phospholipids on the solid substrate, have been productively served as model systems to study wide range problems in cell biology. Because lateral mobility of membrane components is readily preserved, supported lipid membranes with signaling molecules can be utilized to effectively trigger various intercellular reactions. The spatial organization and mechanical deformation of supported membranes can also be manipulated by patterning underlying substrates with modern micro- and nano-fabrication techniques. This article focuses on various applications and methods to spatially patterned biomembranes by means of curvature modulations and spatial reorganizations, and utilizing them to interface with live cells. The integration of biological components into synthetic devices provides a unique approach to investigate molecular mechanisms in cell biology. [ABSTRACT FROM AUTHOR]

Podosomes play crucial roles in macrophage adhesion and migration. Wiskott–Aldrich syndrome protein (WASP; also known as WAS)-mediated actin polymerization is one of the key events initiating podosome formation. Nevertheless, membrane signals to trigger WASP activation at macrophage podosomes remain unclear. Here, we show that phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] lipids are enriched at the podosome and stably recruit WASP rather than the WASP-5KE mutant. Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit β (PIK3CB) is spatially located at the podosome core. Inhibition of PIK3CB and overexpression of phosphatase and tensin homolog (PTEN) impede F-actin polymerization of the podosome. PIK3CB activation is regulated by Abl1 and Src family kinases. At the podosome core, Src and Hck promote the phosphorylation of Tyr488 in the consensus Y-x-x-M motif of Abl1, which enables the association of phosphoinositide 3-kinase (PI3K) regulatory subunits. Knockdown of Abl1 rather than Abl2 suppresses the PI3K/Akt pathway, regardless of Src and Hck activities. Reintroduction of wild-type Abl1 rather than the Abl1-Y488F mutant rescues PI3KR1 recruitment and PI3K activation. When PIK3CB, Abl1 or Src/Hck is suppressed, macrophage podosome formation, matrix degradation and chemotactic migration are inhibited. Thus, Src/Hck-mediated phosphorylation of Abl1 Tyr488 triggers PIK3CB-dependent PI(3,4,5)P3 production and orchestrates the assembly and function of macrophage podosomes. [ABSTRACT FROM AUTHOR]

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average. [ABSTRACT FROM AUTHOR]

Oncogenic Ras induces cell transformation and promotes an invasive phenotype. The tumor suppressor p53 has a suppressive role in Ras-driven invasion. However, its mechanism remains poorly understood. Here we show that p53 induces activation of the mitochondrial protease high-temperature requirement A2 (HtrA2; also known as Omi) and prevents Ras-driven invasion by modulating the actin cytoskeleton. Oncogenic Ras increases accumulation of p53 in the cytoplasm, which promotes the translocation of p38 mitogen-activated protein kinase (MAPK) into mitochondria and induces phosphorylation of HtrA2/Omi. Concurrently, oncogenic Ras also induces mitochondrial fragmentation, irrespective of p53 expression, causing the release of HtrA2/Omi from mitochondria into the cytosol. Phosphorylated HtrA2/Omi therefore cleaves β-actin and decreases the amount of filamentous actin (F-actin) in the cytosol. This ultimately down-regulates p130 Crk-associated substrate (p130Cas)-mediated lamellipodia formation, countering the invasive phenotype initiated by oncogenic Ras. Our novel findings provide insights into the mechanism by which p53 prevents the malignant progression of transformed cells. [ABSTRACT FROM AUTHOR]

Because the actin network in active lamellipodia is continuously assembling at the edge, moving inward and disassembling, there is a question as to how actin-binding proteins and other components are transported to the leading edge and how nascent adhesions are stabilized. Active transport could play a significant role in these functions but the components involved are unknown. We show here that Myosin 1E (a long tailed Myosin 1 isoform) rapidly moves to the tips of active lamellipodia and to actin-rich early adhesions, unlike Myosin 1G, 1B or 1C (short tailed isoforms). Myosin 1E co-localizes with CARMIL, FHOD1, Arp3 and β3-integrin in those early adhesions. But these structures precede stable paxillin-rich adhesions. Myosin 1E movement depends upon actin-binding domains and the presence of an SH3 oligomerization domain. Overexpression of a Myosin 1E deletion mutant without the extreme C-terminal interacting (SH3) domain (Myosin 1EΔSH3) increases edge fluctuations and decreases stable adhesion lifetimes. In contrast, overexpression of Myosin 1E full tail domain (TH1+TH2+TH3/SH3) decreases edge fluctuation. In Myosin 1E knockdown cells, and more prominently in cells treated with Myosin 1 inhibitor, cell-matrix adhesions are also short-lived and fail to mature. We suggest that, by moving to actin polymerization sites and early adhesion sites in active lamellipodia, Myosin 1E might play important roles in transporting not only important polymerizing proteins but also proteins involved in adhesion stabilization.