Cross-correlation Spectroscopy Research Articles

Into the red: An M-band study of the chemistry and rotation of β Pictoris b at high spectral resolution

Abstract High-resolution cross-correlation spectroscopy (HRCCS) combined with adaptive optics has been enormously successful in advancing our knowledge of exoplanet atmospheres, from chemistry to rotation and atmospheric dynamics. This powerful technique now drives major science cases for ELT instrumentation including METIS/ELT, GMTNIRS/GMT and MICHI/TMT, targeting biosignatures on rocky planets at 3–5 μm, but remains untested beyond 3.5 μm where the sky thermal background begins to provide the dominant contribution to the noise. We present 3.51–5.21 μm M-band CRIRES+/VLT observations of the archetypal young directly imaged gas giant β Pictoris b, detecting CO absorption at S/N = 6.6 at 4.73 μm and H2O at S/N = 5.7, and thus extending the use of HRCCS into the thermal background noise dominated infrared. Using this novel spectral range to search for more diverse chemistry we report marginal evidence of SiO at S/N = 4.3, potentially indicative that previously proposed magnesium-silicate clouds in the atmosphere are either patchy, transparent at M-band wavelengths, or possibly absent on the planetary hemisphere observed. The molecular detections are rotationally broadened by the spin of β Pic b, and we infer a planetary rotation velocity of vsin(i) = 22 ± 2 km s−1 from the cross-correlation with the H2O model template, consistent with previous K-band studies. We discuss the observational challenges posed by the thermal background and telluric contamination in the M-band, the custom analysis procedures required to mitigate these issues, and the opportunities to exploit this new infrared window for HRCCS using existing and next-generation instrumentation.

Harnessing extracellular vesicle membrane for gene therapy: EVs-biomimetic nanoparticles

One of the main concerns in oligonucleotide-based therapeutics is achieving a successful cell targeting while avoiding drug degradation and clearance. Nanoparticulated drug delivery systems have emerged as a way of overcoming these issues. Among them, membrane-coated nanoparticles are of increasing relevance mainly due to their enhanced cellular uptake, immune evasion and biocompatibility. In this study, we designed and elaborated a simple and highly tuneable biomimetic drug delivery nanosystem based on a polymeric core surrounded by extracellular vesicles (EVs)-derived membranes. This strategy should allow the nanosystems to benefit from the properties conferred by the membrane proteins present in EVs membrane, key paracrine mediators. The developed systems were able to successfully encapsulate the required oligonucleotides. Also, their characterisation through already well standardised methods (dynamic light scattering, transmission electron microscopy and nanoparticle tracking analysis) and by fluorescence cross-correlation spectroscopy (FCCS) showed the desired core-shell structure. The cellular uptake using different cell types further confirmed the coating though an enhancement in cell internalisation of the developed biomimetic nanoparticles. This study brings up new possibilities for GapmeR delivery as it might be a base for the development of new delivery systems for gene therapy.

Silver Nanoparticles Produced In Vitro by Berberis vulgaris Fruit and Their Antioxidant, Antimicrobial and Ex Ovo Irritation Potential Study

AbstractGreen synthesis was successfully used for silver nanoparticles (Ag NPs) preparation using Berberis vulgaris aqueous extracts. The ultraviolet and visible (UV–Vis) spectroscopy, photon cross-correlation spectroscopy (PCCS), transmission electron microscopy (TEM) and selected area electron diffraction (SAED) techniques were used for characterization of the Ag NPs and confirmed the presence of Ag(0) in nanoparticles. The bimodal morphology was discovered, namely, the larger particles had elongated shape and size around 200 nm, while the smaller ones were spherical with a size of up to ten microns. All the nanoparticles (NPs) showed antioxidant activity against radical DPPH and ferric-reducing antioxidant power. The Ag NPs possess antibacterial effects against Escherichia coli and Staphylococcus aureus comparable to silver nitrate solution. In addition, also irritation potential of the produced Ag NPs has been investigated ex Ovo and no irritation of vessels and their surroundings was found, which shows the harmless character of the products.

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

Inverse design and optimization of an aperiodic multi-notch fiber Bragg grating using neural networks

Recent developments in the application of aperiodic fiber Bragg gratings (AFBGs) in astrophotonics, such as AFBG for astronomical near-infrared OH suppression and gas detection based on cross-correlation spectroscopy, have illuminated the problem that the optimization for AFBG with certain fabrication constraints has not been fully investigated and solved. Previous solutions will either sacrifice part of the spectral features or consume a significant amount of computation resources and time. Inspired by recently successful applications of artificial neural networks (ANNs) in photonics inverse design, we develop an AFBG optimization approach employing ANNs in conjunction with genetic algorithms (GAs) for the first time, to the best of our knowledge. The approach maintains the spectral notch depths and preserves the fourth-order super-Gaussian spectral features with improvements of interline loss by ∼100 times. We also implement, to our knowledge, the first inverse scattering neural network based on a tandem architecture for AFBG, using a first-order Gaussian notch profile. The neural network successfully converges but has a poor predictive capability for the phase part of the design. We discuss possible ways to overcome these limitations.

Abstract LB436: Using FCCS to monitor target occupancy in cells and patient biopsies

Abstract Lack of target engagement is one of the major reasons for failures in clinical studies. We developed an assay platform to detect and quatify target occupancy values in patient and animal biopsies to guide the development and dosing during the drug development process. FCCS (fluorescence cross correlation spectroscopy) is a powerful tool to characterize drug target interaction in physiological environments. Over the last decade this technology was used to understand the binding and kinetics of small molecules and antibodies to challenging target proteins in great details and accuracy. Over the last decade Intana Bioscience expanded the application of FCCS from affinity measurements to comprehensive interaction studies including kinetics, multi component complex formation, assessing stoichiometry and quantification of aggregation, PK studies and target engagement. This presentation will highlight examples of results obtained with FCCS in projects ranging from early stages in the drug discovery up to the support of clinical trials to quantify target occupancies in patient samples. Citation Format: Frank Becker. Using FCCS to monitor target occupancy in cells and patient biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB436.

The β2-adrenergic receptor associates with CXCR4 multimers in human cancer cells

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the β2 adrenergic receptor (β2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-β2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and β2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that β2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-β2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

Searching for NLTE effects in the high-resolution transmission spectrum of WASP-121 b with cloudy for exoplanets

ABSTRACT Ultrahot Jupiters (UHJs) undergo intense irradiation by their host stars and are expected to experience non-local thermodynamic equilibrium (NLTE) effects in their atmospheres. Such effects are computationally intensive to model but, at the low pressures probed by high-resolution cross-correlation spectroscopy (HRCCS), can significantly impact the formation of spectral lines. The UHJ WASP-121 b exhibits a highly inflated atmosphere, making it ideal for investigating the impact of NLTE effects on its transmission spectrum. Here, we formally introduce cloudy for exoplanets, a cloudy-based modelling code, and use it to generate 1D LTE and NLTE atmospheric models and spectra to analyse archival HARPS WASP-121 b transmission spectra. We assessed the models using two HRCCS methods: (i) Pearson cross-correlation, and (ii) a method that aims to match the average observed line depth for given atmospheric species. All models result in strong detections of Fe i (7.5 < S/N < 10.5). However, the highest S/N model (LTE) does not agree with the best-matching model of the average line depth (NLTE). We also find degeneracy, such that increasing the isothermal temperature and metallicity of the LTE models can produce average line depths similar to cooler, less metal rich NLTE models. Thus, we are unable to conclusively remark on the presence of NLTE effects in the atmosphere of WASP-121 b. We instead highlight the need for standardized metrics in HRCCS that enable robust statistical assessment of complex physical models, e.g. NLTE or 3D effects, that are currently too computationally intensive to include in HRCCS atmospheric retrievals.

Influence of Cigarette Aerosol in Alpha-Synuclein Oligomerization and Cell Viability in SH-SY5Y: Implications for Parkinson's Disease.

Although cigarette aerosol exposure is associated with various adverse health issues, its impact on Parkinson's disease (PD) remains elusive. Here, we investigated the effect of cigarette aerosol extract (CAE) on SH-SY5Y cells for the first time, both with and without α-synuclein (α-Syn) overexpression. We found that α-Syn aggravates CAE-induced cell death, oxidative stress, and mitochondrial dysfunction. Fluorescence cross-correlation spectroscopy (FCCS) revealed a dual distribution of α-Syn within the cells, with homogeneous regions indicative of monomeric α-Syn and punctated regions, suggesting the formation of oligomers. Moreover, we observed colocalization of α-Syn oligomers with lysosomes along with a reduction in autophagy activity. These findings suggest that α-Syn overexpression exacerbates CAE-induced intracellular cytotoxicity, mitochondrial dysfunction, and autophagy dysregulation, leading to elevated cell mortality. Our findings provide new insights into the pathogenic mechanisms linking exposure to cigarette aerosols with neurodegenerative diseases.

Optimal parameter selection for the THz-CCS system based on an incoherent light source.

Terahertz cross correlation spectroscopy (THz-CCS) systems using broadband incoherent light as the pumping source have received increasing attention from researchers in recent years. However, a comprehensive and in-depth understanding of THz-CCS is still needed to obtain a detailed optimization scheme. Here we systematically investigate the influences of the detection parameters, light propagation process, and pump source on the CCS signals. The impacts of the filter slopes and time constants in lock-in detection are revealed for optimizing the signal-to-noise ratio and bandwidth of the THz signal. By varying the optical fiber length and dispersion coefficient, the dispersion insensitivity of THz-CCS was experimentally demonstrated. The comparison of different pump sources (SLD and ASE) shows that the over-wide and non-flat pump spectrum may attenuate the CCS signal because of the energy waste brought by the photomixing process under the limited bandwidth of the photomixer. Our research may lead to a deeper understanding and further optimization of the THz-CCS system, which will promote the development and widespread application of what is to the best of our knowledge a new technique.

Small but mighty: High-resolution spectroscopy of ultra-hot Jupiter atmospheres with compact telescopes

When observing transmission spectra produced by the atmospheres of ultra-hot Jupiters (UHJs), large telescopes are typically the instrument of choice given the very weak signal of the planet’s atmopshere. The aim of the present study is to demonstrate that, for favourable targets, smaller telescopes are fully capable of conducting high-resolution cross-correlation spectroscopy. We apply the cross-correlation technique to data from the 2.1 m telescope at the Wendelstein Observatory, using its high-resolution spectrograph FOCES, in order to demonstrate its efficacy in resolving the atmosphere of the UHJ KELT-9 b. Using three nights of observations with the FOCES spectrograph and one with the HARPS-N spectrograph, we conduct a performance comparison between FOCES and HARPS-N. This comparison considers both single-transit and combined observations over the three nights. We then consider the potential of 2 m class telescopes by generalising our results to create a transit emulator capable of evaluating the potential of telescopes of this size. With FOCES, we detected seven species in the atmosphere of KELT-9b: Ti II, Fe I, Fe II, Na I, Mg I, Na II, Cr II, and Sc II. Although HARPS-N surpasses FOCES in performance thanks to the mirror of the TNG, our results reveal that smaller telescope classes are capable of resolving the atmospheres of UHJs given sufficient observing time. This broadens the potential scope of such studies, demonstrating that smaller telescopes can be used to investigate phenomena such as temporal variations in atmospheric signals and the atmospheric loss characteristics of these close-in planets.

JWST Transmission Spectroscopy of HD 209458b: A Supersolar Metallicity, a Very Low C/O, and No Evidence of CH4, HCN, or C2H2

We present the transmission spectrum of the original transiting hot Jupiter HD 209458b from 2.3 to 5.1 μm as observed with the NIRCam instrument on the James Webb Space Telescope (JWST). Previous studies of HD 209458b’s atmosphere have given conflicting results on the abundance of H2O and the presence of carbon- and nitrogen-bearing species, which have significant ramifications on the inferences of the planet’s metallicity (M/H) and carbon-to-oxygen (C/O) ratio. We detect strong features of H2O and CO2 in the JWST transmission spectrum, which when interpreted using a retrieval that assumes thermochemical equilibrium and fractional gray cloud opacity yields 3−1+4 × solar metallicity and C/O = 0.11−0.06+0.12 . The derived metallicity is consistent with the atmospheric metallicity–planet mass trend observed in solar gas giants. The low C/O ratio suggests that this planet has undergone significant contamination by evaporating planetesimals while migrating inward. We are also able to place upper limits on the abundances of CH4, C2H2, and HCN of log( χCH4 ) = −5.6, log( χC2H2 ) = −5.7, and log( χHCN ) = −5.1, which are in tension with the recent claim of a detection of these species using ground-based cross-correlation spectroscopy. We find that HD 209458b has a weaker CO2 feature size than WASP-39b when comparing their scale-height-normalized transmission spectra. On the other hand, the size of HD 209458b’s H2O feature is stronger, thus reinforcing the low C/O inference.

Understanding the molecular mechanism of pathogenic variants of BIR2 domain in XIAP-deficient inflammatory bowel disease

X-linked inhibitor of apoptosis protein (XIAP) deficiency causes refractory inflammatory bowel disease. The XIAP protein plays a pivotal role in the pro-inflammatory response through the nucleotide-binding oligomerization domain-containing signaling pathway that is important in mucosal homeostasis. We analyzed the molecular mechanism of non-synonymous pathogenic variants (PVs) of XIAP BIR2 domain. We generated N-terminally green fluorescent protein-tagged XIAP constructs of representative non-synonymous PVs. Co-immunoprecipitation and fluorescence cross-correlation spectroscopy showed that wild-type XIAP and RIP2 preferentially interacted in live cells, whereas all non-synonymous PV XIAPs failed to interact properly with RIP2. Structural analysis showed that various structural changes by mutations, such as hydrophobic core collapse, Zn-finger loss, and spatial rearrangement, destabilized the two loop structures (174–182 and 205–215) that critically interact with RIP2. Subsequently, it caused a failure of RIP2 ubiquitination and loss of protein deficiency by the auto-ubiquitination of all XIAP mutants. These findings could enhance our understanding of the role of XIAP mutations in XIAP-deficient inflammatory bowel disease and may benefit future therapeutic strategies.

Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) for studying the fusion dynamics of protein phase separation.

Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to β-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation.

CRDBP Protein Phase Separation and Its Recruitment to β-Catenin Protein in a Single Living Cell.

The Coding Region Determinant-Binding Protein (CRDBP) is a carcinoembryonic protein, and it is overexpressed in various cancer cells in the form of granules. We speculated the formation of CRDBP granules possibly through liquid-liquid phase separation (LLPS) processes due to the existence of intrinsically disordered regions (IDRs) in CRDBP. So far, we did not know whether or how phase separation processes of CRDBP occur in single living cells due to the lack of in vivo methods for studying intracellular protein phase separation. Therefore, to develop an in situ method for studying protein phase separation in living cells is a very urgent task. In this work, we proposed an efficient method for studying phase separation behavior of CRDBP in a single living cell by combining in situ fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) with a fluorescence protein fusion technique. We first predicted and confirmed that CRDBP has phase separation in solution by conventional fluorescence imaging and FCS methods. And then, we in situ studied the phase separation behaviors of CRDBP in living cells and observed three states of CRDBP phase separation such as monomer state, cluster state, and granule state. We studied the effects of CRDBP truncated forms and its inhibitor on the CRDBP phase separation. Furthermore, we discovered the recruitment of CRDBP to β-catenin protein in living cells and investigated the effects of CRDBP structures and inhibitor on CRDBP recruitment behavior. This finding may help us to further understand the mechanism of CRDBP protein for regulating Wnt signaling pathway. Additionally, our results documented that FCS/FCCS is an efficient and alternative method for studying protein phase separation in situ in living cells.

Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.

CROCODILE

Context. The investigation of the atmospheres of closely separated, directly imaged gas giant exoplanets is challenging due to the presence of stellar speckles that pollute their spectrum. To remedy this, the analysis of medium- to high-resolution spectroscopic data via cross-correlation with spectral templates (cross-correlation spectroscopy) is emerging as a leading technique. Aims. We aim to define a robust Bayesian framework combining, for the first time, three widespread direct-imaging techniques, namely photometry, low-resolution spectroscopy, and medium-resolution cross-correlation spectroscopy in order to derive the atmospheric properties of close-in directly imaged exoplanets. Current atmospheric characterisation frameworks are indeed either not compatible with all three observing techniques or they lack the commitment to efficient sampling strategies that allow high-dimensional forward models. Methods. Our framework CROCODILE (cross-correlation retrievals of directly imaged self-luminous exoplanets) naturally combines the three techniques by adopting adequate likelihood functions. To validate our routine, we simulated observations of gas giants similar to the well-studied β Pictoris b planet and we explored the parameter space of their atmospheres to search for potential biases. Results. We obtain more accurate measurements of atmospheric properties when combining photometry, low- and medium-resolution spectroscopy into atmospheric retrievals than when using the techniques separately as is usually done in the literature. Indeed, the combined fit is, on average, 20% more accurate than fitting only medium-resolution cross-correlation spectroscopy. We find that medium-resolution (R ≈ 4000) K-band cross-correlation spectroscopy alone is not suitable to constrain the atmospheric properties of our synthetic datasets; however, this problem disappears when simultaneously fitting photometry throughout the Y and M bands and low-resolution (R ≈ 60) spectroscopy between the Y and H bands. Our thorough testing demonstrates that free chemistry is a suitable forward model to retrieve the atmospheric thermal and chemical properties of cloudless gas giants at chemical equilibrium. Conclusions. CROCODILE provides a robust statistical framework to interpret medium-resolution spectroscopic data of close-in directly imaged exoplanets, where speckles originating from stellar stray light render the extraction of the continuum difficult. Our framework allows the atmospheric characterisation of directly imaged exoplanets using the high-quality spectral data that will be provided by the new generation of instruments such as the Enhanced Resolution Imager and Spectrograph (ERIS) at the Very Large Telescope, the Mid-Infrared Instrument (MIRI) aboard the James Webb Space Telescope, and in the future the Mid-infrared ELT Imager and Spectrograph (METIS) at the Extremely Large Telescope.

Deubiquitination Detection of p53 Protein in Living Cells by Fluorescence Cross-Correlation Spectroscopy.

Deubiquitination is a reverse post-translational modification of ubiquitination and plays significant roles in various signal transduction cascades and protein stability. The p53 is a very important tumor-suppressor protein and closely implicates more than 50% of human cancers. Although extracellular studies on the deubiquitination of p53 were reported, the process of p53 deubiquitination in living cells due to the shortage of an efficient in situ method for single living cells is still not clear. In this study, we described an in situ method for studying p53 deubiquitination in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. We first constructed the stable cell line expressing EGFP-Ub-p53-mCherry as the substrate of p53 deubiquitination. Then, we established a method for in situ monitoring of the deubiquitination of p53 in living cells. Based on the amplitudes of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy curves from living cells, we obtained the deubiquitination percentage for evaluating the level of p53 protein deubiquitination. Furthermore, we studied the effects of ubiquitin structures on p53 deubiquitination in living cells and found that the C-terminal Gly75-Gly76 motif of ubiquitin is a key location for p53 deubiquitination and the deubiquitination cannot occur when ubiquitin lacks the C-terminal Gly75-Gly76 motif. Our results documented that the developed strategy is an efficient method for in situ study of deubiquitination of proteins in living cells.

Resonance Light-Scattering Correlation Spectroscopy and Its Application in Analytical Chemistry for Life Science.

Resonance light-scattering correlation spectroscopy (RLSCS) is a new single-particle detection method with its working principle being like fluorescence correlation spectroscopy (FCS). RLSCS is obtained by autocorrelation function analysis on the measured fluctuation of the resonance light scattering (RLS) intensity occurring within a subfemtoliter volume when a single nanoparticle (such as gold nanoparticles (NPs) or silver (SNPs)) freely diffuses through the volume. The RLSCS technique can detect such parameters as concentration, diffusion coefficient (translation and rotation), etc. Compared with the FCS technique, the correlated fluorescence intensity signal in RLSCS is replaced with the RLS signal of the nanoparticles, overcoming some limits of the fluorescent probes such as photobleaching under high-intensity or long-term illumination. In this Account, we showcase RLSCS methods, theoretical models at different optical configurations, and some key applications. First, the RLSCS optical detection system was constructed based on the confocal optics, its theoretical model was proposed, and the diffusion behaviors of the nanoparticles in the solution were studied including the rotational and translational diffusion. And, methods were developed to measure the concentration, size, aspect ratio, and size distribution of the NPs. Second, based on the RLSCS methods, some detection strategies were developed for homogeneous DNA detection, immunoassay, apoptosis assay, self-thermophoresis of the nanomotor, and quantitative assay in single living cells. Meanwhile, a new fluorescence/scattering cross-correlation spectroscopy (FSCCS) method was proposed for monitoring the molecule-particle interaction. This method enriched the conventional fluorescence/fluorescence cross-correlation spectroscopy (FCCS) method. Third, using the EMCCD with high sensitivity and rapid response as an optical detector, two temporospatially resolved scattering correlation spectroscopy methods and their theoretical models were developed: total internal reflection (TIR) configuration-based spatially resolved scattering correlation spectroscopy (SRSCS) and dark-field illumination-based scattering correlation spectroscopy (DFSCS). These methods extended single-spot confocal RLSCS to imaging RLSCS, which makes RLSCS have the ability for multiple channel detection with temporospatial resolution. The method was successfully used for investigating the dynamic behaviors of gold NPs in live cells and obtained its temporospatial concentration distribution and diffusion behaviors. The final section of this Account outlines future directions in the development of RLSCS.

Effects of biophysical membrane properties on recognition of phosphatidylserine, or phosphatidylinositol 4-phosphate by lipid biosensors LactC2, or P4M

Lipid biosensors are molecular tools used both in vivo and in vitro applications, capable of selectively detecting specific types of lipids in biological membranes. However, despite their extensive use, there is a lack of systematic characterization of their binding properties in various membrane conditions. The purpose of this study was to investigate the impact of membrane properties, such as fluidity and membrane charge, on the sensitivity of two lipid biosensors, LactC2 and P4M, to their target lipids, phosphatidylserine (PS) or phosphatidylinositol 4-phosphate (PI4P), respectively. Dual-color fluorescence cross-correlation spectroscopy, employed in this study, provided a useful technique to investigate interactions of these recombinant fluorescent biosensors with liposomes of varying compositions. The results of the study demonstrate that the binding of the LactC2 biosensor to low levels of PS in the membrane is highly supported by the presence of anionic lipids or membrane fluidity. However, at high PS levels, the presence of anionic lipids does not further enhance binding of LactC2. In contrast, neither membrane charge, nor membrane fluidity significantly affect the binding affinity of P4M to PI4P. These findings provide valuable insights into the role of membrane properties on the binding properties of lipid biosensors.