February 2025
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11 Reads
Developmental Cell
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February 2025
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11 Reads
Developmental Cell
February 2025
Biophysical Journal
February 2025
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7 Reads
Biophysical Journal
January 2025
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31 Reads
In a randomized, dose-response trial, we used molecular and phenomic profiling to compare responses to traditional (TRAD) endurance plus resistance training and high-intensity tactical training (HITT). Ninety-four participants (18-27 years) completed 12 weeks of TRAD or HITT followed by 4 weeks of detraining. While in vivo phenotype improvements were not dose-dependent, a few dose-dependent ex vivo muscle adaptations were overshadowed by wide-ranging inter-individual response heterogeneity (IRH). To address this, we established minimum clinically important difference (MCID) scores to classify participants by their attainment of MCIDs for functional muscle quality (fMQ) and cardiorespiratory fitness (CRF). Using differential gene expression (DGE) of muscle and exosomal microRNAs (miRs) and higher-order singular value decomposition (HOSVD), we mapped the molecular and phenomic biocircuitry of IRH. Nine miRs emerged as robust features of training adaptability, providing new insights into the integrated biocircuitry driving exercise adaptations and response heterogeneity. In brief We examined in vivo and ex vivo adaptations to two randomized exercise prescriptions. Individual response variability overshadowed dose dependent effects. Deep phenotyping and miR transcriptomics of serum exosomes and skeletal muscle enabled multidimensional modeling of integrated biocircuits linked to attaining clinically significant outcomes.
December 2024
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22 Reads
Cell Death and Disease
Non-melanoma skin cancer, including basal and squamous cell carcinoma, is the most common form of cancer worldwide, with approximately 5.4 million new cases diagnosed each year in the United States. While the chemotherapeutic drug cisplatin is often used to treat squamous cell carcinoma (SCC) patients, low response rates and disease recurrence are common. In this study, we show that TIP60 and ΔNp63α levels correlate with cisplatin resistance in SCC cell lines, suggesting that TIP60 contributes to the failure of platinum-based drugs in SCC by regulating the stability and transcriptional activity of ΔNp63α. Depletion of endogenous TIP60 or pharmacological inhibition of TIP60 led to a decrease in ΔNp63α protein and acetylation levels in multiple SCC cell lines. We showed that TIP60 upregulates ΔNp63α protein levels in cisplatin-resistant SCC cell lines by protecting it from cisplatin-mediated degradation and increasing its protein stability. Stable expression of TIP60 or ΔNp63α individually promoted resistance to cisplatin and reduced cell death, while loss of either TIP60 or ΔNp63α induced G2/M arrest, increased cell death, and sensitized cells to cisplatin. Moreover, pharmacological inhibition of TIP60 reduced acetylation of ΔNp63α and sensitized resistant cells to cisplatin. Taken together, our study indicates that TIP60-mediated stabilization of ΔNp63α increases cisplatin resistance and provides critical insights into the mechanisms by which ΔNp63α confers cisplatin resistance by promoting cell proliferation and inhibiting apoptosis. Furthermore, our data suggests that inhibition of TIP60 may be therapeutically advantageous in overcoming cisplatin resistance in SCC and other epithelial cancers.
November 2024
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79 Reads
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1 Citation
Nano Letters
Dark-field microscopy (DFM) is a widely used imaging tool, due to its high-contrast capability in imaging label-free specimens. Traditional DFM requires optical alignment to block the oblique illumination, and the resolution is diffraction-limited to the wavelength scale. In this work, we present deep-learning assisted plasmonic dark-field microscopy (DAPD), which is a single-frame super-resolution method using plasmonic dark-field (PDF) microscopy and deep-learning assisted image reconstruction. Specifically, we fabricated a designed PDF substrate with surface plasmon polaritons (SPPs) illuminating specimens on the substrate. Dark field images formed by scattered light from the specimen are further processed by a pretrained convolutional neural network (CNN) using a simulation dataset based on the designed substrate and parameters of the detection optics. We demonstrated a resolution enhancement of 2.8 times on various label-free objects with a large potential for future improvement. We highlight our technique as a compact alternative to traditional DFM with a significantly enhanced spatial resolution.
November 2024
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67 Reads
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1 Citation
Chemical Reviews
Cellular function is controlled through intricate networks of signals, which lead to the myriad pathways governing cell fate. Fluorescent biosensors have enabled the study of these signaling pathways in living systems across temporal and spatial scales. Over the years there has been an explosion in the number of fluorescent biosensors, as they have become available for numerous targets, utilized across spectral space, and suited for various imaging techniques. To guide users through this extensive biosensor landscape, we discuss critical aspects of fluorescent proteins for consideration in biosensor development, smart tagging strategies, and the historical and recent biosensors of various types, grouped by target, and with a focus on the design and recent applications of these sensors in living systems.
October 2024
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66 Reads
The nanoscale organization of enzymes associated with the dynamics of second messengers is critical for ensuring compartmentation and localization of signaling molecules in cells. Specifically, the spatiotemporal orchestration of cAMP and Ca²⁺ oscillations is critical for many cellular functions. Previous experimental studies have shown that the formation of nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates the phase of Ca²⁺-cAMP oscillations from out-of-phase to in-phase. In this work, we develop computational models of the Ca²⁺/cAMP pathway and AKAP/AC nanodomain formation that give rise to the two important predictions: instead of an arbitrary phase difference, the out-of-phase Ca²⁺/cAMP oscillation reaches Ca²⁺ trough and cAMP peak simultaneously, which is defined as inversely out-of-phase; the in-phase and inversely out-of-phase oscillations associated with Ca²⁺-cAMP dynamics on and away from the nanodomains can be explained by an incoherent feedforward loop. Factors such as cellular surface-to-volume ratio, compartment size, and distance between nanodomains do not affect the existence of in-phase or inversely out-of-phase Ca²⁺/cAMP oscillation, but cellular surface-to-volume ratio and compartment size can affect the time delay for the inversely out-of-phase Ca²⁺/cAMP oscillation while the distance between two nanodomains does not. Finally, we predict that both the Turing pattern-generated nanodomains and experimentally measured nanodomains demonstrate the existence of in-phase and inversely out-of-phase Ca²⁺/cAMP oscillation when the AC8 is at a low level, consistent with the behavior of an incoherent feedforward loop. These findings unveil the key circuit motif that governs cAMP and Ca²⁺ oscillations and advance our understanding of how nanodomains can lead to spatial compartmentation of second messengers.
October 2024
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82 Reads
Proceedings of the National Academy of Sciences
Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.
October 2024
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41 Reads
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2 Citations
Nature Chemical Biology
The protein kinase C (PKC) family of serine and threonine kinases, consisting of three distinctly regulated subfamilies, has been established as critical for various cellular functions. However, how PKC enzymes are regulated at different subcellular locations, particularly at emerging signaling hubs, is unclear. Here we present a sensitive excitation ratiometric C kinase activity reporter (ExRai-CKAR2) that enables the detection of minute changes (equivalent to 0.2% of maximum stimulation) in subcellular PKC activity. Using ExRai-CKAR2 with an enhanced diacylglycerol (DAG) biosensor, we uncover that G-protein-coupled receptor stimulation triggers sustained PKC activity at the endoplasmic reticulum and lysosomes, differentially mediated by Ca²⁺-sensitive conventional PKC and DAG-sensitive novel PKC, respectively. The high sensitivity of ExRai-CKAR2, targeted to either the cytosol or partitioning defective complexes, further enabled us to detect previously inaccessible endogenous atypical PKC activity in three-dimensional organoids. Taken together, ExRai-CKAR2 is a powerful tool for interrogating PKC regulation in response to physiological stimuli.
... In general, precisely which metric(s) are chosen for optimisation depends on the application in mind, be it utilising MFE or ODMR, or favouring magnitude or speed of response as was of interest for the lock-in and multiplexing applications we demonstrated. As such -much like fluorescent proteins -we expect MFPs will also be engineered to make general improvements, such as to solubility, photo-stability and quantum-yield [52]. Finally, we hope that the development of MFPs can serve as the starting point for magnetically controlled biological actuators, whereby application of a local magnetic field can have downstream cellular effectssuch a technology would be of significant biomedical and biotechnological interest. ...
November 2024
Chemical Reviews
... This novel approach uses circularly permuted FP as the reporting unit, resulting in phosphorylation-dependent ratiometric changes of emission upon excitation at two different wavelengths. ExRai sensors have demonstrated unprecedented sensitivity compared to FRET-based activity sensors for kinases such as PKA, PKC, AMPK, or Akt [16][17][18][19][20] . ...
October 2024
Nature Chemical Biology
... The idea of PKA sequestration and aggregation in mammalian cells has recently been approached by several investigators. [67][68][69] Our proposal is in line with a recently described non-canonical PKA activation mechanism via aggregation of the R1a subunit in inherited Carney complex mutations, which rendered it incapable of inhibiting the C subunit. 67 Studies from the Zhang laboratory have provided evidence that both the mammalian Ca and R1a subunits are co-recruited into R1a bodies that act as a means of compartmentalizing cAMP. ...
March 2024
Molecular Cell
... This property has been recently exploited for the design of biosensors, based on genetically encoded sensing motifs fused to HaloTag in which an analyte-dependent conformational change alters the equilibrium and hence the fluorescence of the dye. [36][37][38][39] Here, we repurpose this approach to engineer a photoswitchable fluorescent system by integrating a light-responsive protein domain into the HaloTag protein ( Figure 1). Upon illumination, the conformational change of the protein photoswitch alters the dye environment, shifting its equilibrium toward the open, fluorescent state. ...
February 2024
... A diverse array of analytical techniques have been employed to quantify glucose concentrations, encompassing fluorescence [5], electrochemical methods [6], and chemiluminescence [7]. Among these techniques, electrochemical sensors stand out prominently owing to their exceptional sensitivity, remarkable selectivity, and low detection limit. ...
February 2024
Annual Review of Biophysics
... Fluorescent biosensors have been remarkably improved in recent years, making them indispensable tools for studying intracellular signal transduction due to their ability to monitor signaling dynamics in real time within living cells [12][13][14]. Fluorescent biosensor imaging techniques such as fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), and translocation-based biosensors have been developed to analyze cellular signaling and behavior in live cells [15][16][17]. Although obtaining robust FRET and BiFC signals, these techniques require a tedious optimization procedure to determine the relative locations of fluorophores and binding pairs as well as appropriate linker domains. ...
February 2024
... We hypothesized that decreased AMP might be related to reduced generation of adenosine under hypoxic condition. And the decrease of adenosine and AMP may result in pulmonary vascular endothelial dysfunction and increase vascular resistance, prompting pulmonary hypertension (PH) 27 . It has been reported that perturbed purine metabolism is associated with HAPE and hypoxia-induced PH 11,27,28 . ...
February 2024
Circulation
... As a multifunctional enzyme with deacetylase, desuccinylase, defatty-acylase, debutyrylase, deglutarylase, decrotonylase, mono-adenosine 5 ′ -diphosphate (ADP)-ribosyltransferase, and an NAD + -independent RNA deacetylase, SIRT7 is involved in various cellular processes such as transcriptional regulation, cellular metabolism, cell proliferation, and the maintenance of genome stability [6]. In recent years, accumulating evidence has underscored the regulatory role of SIRT7 in angiogenesis in different contexts [7][8][9][10][11][12][13]. However, the substrate-mediated molecular mechanisms remain unclear and fragmented. ...
January 2024
Cardiovascular Research
... Previous studies suggested that Rac1 could be a potential therapeutic target for HNSCC, as evident by its tumor-specific overexpression, its elevated expression in relapsed vs. primary HNSCC [45], and its demonstrated contribution to HNSCC chemo-radioresistance [45], cell invasion (via both ∆Np63α/Rac1 and EGFR/Vav2/Rac1 axes) [22,54], adhesion and migration (via the RAPI-Rac1 axis) [21], etc. Here, our demonstration that RAC1-amplified and RAC1-A159V-mutated HNSCC could display preferential sensitivity to Rac inhibitor targeting in vivo further provides supportive evidence for an expansion of efforts in both preclinical and clinical development of Rac inhibitors for the treatment of HNSCC, as well as other cancers driven by RAC1 aberrations. ...
January 2024
Cell Death Discovery
... As alternatives to a classical second messenger role of nucleosides, physiological activation of the PKA holoenzyme in vivo may be co-activated by nucleoside binding together with a second trigger like a posttranslational modification, redox state, specific protein-protein interaction or kinase regulation by liquid-liquid phase separation (Lopez-Palacios and Andersen 2023 ; Hardy et al. 2023 ). These triggers may allosterically shift the affinity or may be required for the final activating conformational change upon binding (Khamina et al. 2022 ), giving nucleosides a more auxiliary role in the allosteric kinase regulation. ...
December 2023