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Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications. Single-cell proteomics is an emerging approach to characterize cell-to-cell differences. Here, the authors develop chips that enable complete proteomic sample processing down to the single-cell level and integrate them with DIA-MS into a streamlined single-cell proteomics workflow.

Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within two hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values. Phosphoproteomics can provide systematic insights into disease-associated cell signaling changes. Here, the authors present a sensitive workflow integrating library-based and direct data-independent acquisition approaches, and a hybrid spectral library resource for in-depth phosphoproteomic profiling.

Cancer stem cells (CSCs) are a promising target for treating cancer, yet how CSC plasticity is maintained in vivo is unclear and is difficult to study in vitro . Here we establish a sustainable primary culture of Oct3/4(+)/Nanog(+) lung CSCs fed with CD90(+) cancer-associated fibroblasts (CAFs) to further advance our knowledge of preserving stem cells in the tumour microenvironment. Using transcriptomics we identify the paracrine network by which CAFs enrich CSCs through de-differentiation and reacquisition of stem cell-like properties. Specifically, we find that IGF1R signalling activation in cancer cells in the presence of CAFs expressing IGF-II can induce Nanog expression and promote stemness. Moreover, this paracrine signalling predicts overall and relapse-free survival in stage I non-small cell lung cancer (NSCLC) patients. IGF-II/IGF1R signalling blockade inhibits Nanog expression and attenuates cancer stem cell features. Our data demonstrate that CAFs constitute a supporting niche for cancer stemness, and targeting this paracrine signalling may present a new therapeutic strategy for NSCLC. Cancer stem cells are a sub-population of tumour cells but how they interact with the tumour microenvironment is unclear. Here, Chen et al. culture lung cancer stem cells with cancer-associated fibroblasts and delineate a signalling pathway between the two cells that helps maintain the cancer stem cell state.

Focused ultrasound (FUS) exposure with the presence of microbubbles has been shown to transiently open the blood-brain barrier (BBB), and thus has potential to enhance the delivery of various kinds of therapeutic agents into brain tumors. The purpose of this study was to assess the preclinical therapeutic efficacy of FUS-BBB opening for enhanced temozolomide (TMZ) delivery in glioma treatment. FUS exposure with microbubbles was delivered to open the BBB of nude mice that were either normal or implanted with U87 human glioma cells. Different TMZ dose regimens were tested, ranging from 2.5 to 25 mg/kg. Plasma and brain samples were obtained at different time-points ranging from 0.5 to 4 hours, and the TMZ concentration within samples was quantitated via a developed LC-MS/MS procedure. Tumor progression was followed with T2-MRI, and animal survival and brain tissue histology were conducted. Results demonstrated that FUS-BBB opening caused the local TMZ accumulation in the brain to increase from 6.98 to 19 ng/mg. TMZ degradation time in the tumor core was found to increase from 1.02 to 1.56 hours. Improved tumor progression and animal survival were found at different TMZ doses (up to 15% and 30%, respectively). In conclusion, this study provides preclinical evidence that FUS-BBB opening increases the local concentration of TMZ to improve the control of tumor progression and animal survival, suggesting the potential for clinical application to improve current brain tumor treatment.

Our objective was to develop a rapid technique for the non-invasive profiling and quantification of major tomato carotenoids using handheld Raman spectroscopy combined with pattern recognition techniques. A total of 106 samples with varying carotenoid profiles were provided by the Ohio State University Tomato Breeding and Genetics program and Lipman Family Farms (Naples, FL, USA). Non-destructive measurement from the surface of tomatoes was performed by a handheld Raman spectrometer equipped with a 1064 nm excitation laser, and data analysis was performed using soft independent modelling of class analogy (SIMCA)), artificial neural network (ANN), and partial least squares regression (PLSR) for classification and quantification purposes. High-performance liquid chromatography (HPLC) and UV/visible spectrophotometry were used for profiling and quantification of major carotenoids. Seven groups were identified based on their carotenoid profile, and supervised classification by SIMCA and ANN clustered samples with 93% and 100% accuracy based on a validation test data, respectively. All-trans-lycopene and β-carotene levels were measured with a UV-visible spectrophotometer, and prediction models were developed using PLSR and ANN. Regression models developed with Raman spectra provided excellent prediction performance by ANN (rpre = 0.9, SEP = 1.1 mg/100 g) and PLSR (rpre = 0.87, SEP = 2.4 mg/100 g) for non-invasive determination of all-trans-lycopene in fruits. Although the number of samples were limited for β-carotene quantification, PLSR modeling showed promising results (rcv = 0.99, SECV = 0.28 mg/100 g). Non-destructive evaluation of tomato carotenoids can be useful for tomato breeders as a simple and rapid tool for developing new varieties with novel profiles and for separating orange varieties with distinct carotenoids (high in β-carotene and high in cis-lycopene).

The demand of accurate, near-real-time radar-based quantitative precipitation estimation (QPE), which is key to feed hydrological models and enable reliable flash flood predictions, was highlighted again by the disastrous floods following after an intense stratiform precipitation field passing western Germany on 14 July 2021. Three state-of-the-art rainfall algorithms based on reflectivity 𝛧, specific differential phase 𝐾DP, and specific attenuation 𝐴 were applied to observations of four polarimetric C-band radars operated by the German Meteorological Service [DWD (Deutscher Wetterdienst)]. Due to the large vertical gradients of precipitation below the melting layer suggesting warm-rain processes, all QPE products significantly underestimate surface precipitation. We propose two mitigation approaches: (i) vertical profile (VP) corrections for 𝛧 and 𝐾DP and (ii) gap filling using observations of a local X-band radar, JuXPol. We also derive rainfall retrievals from vertically pointing Micro Rain Radar (MRR) profiles, which indirectly take precipitation gradients in the lower few hundreds of meters into account. When evaluated with DWD rain gauge measurements, those retrievals result in pronounced improvements, especially for the 𝐴-based retrieval partly due to its lower sensitivity to the variability of raindrop size distributions. The VP correction further improves QPE by reducing the normalized root-mean-square error by 23% and the normalized mean bias by 20%. With the use of gap-filling JuXPol data, the 𝐴-based retrieval gives the lowest errors followed by the 𝛧-based retrievals in combination with VP corrections. The presented algorithms demonstrate the increased value of radar-based QPE application for warm-rain events and related potential flash flooding warnings.

Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture. Standard mass spectrometry analyses often miss key targets required for phospho-signalling reconstruction. Here, authors present an intelligent data acquisition strategy that combines discovery and targeted analysis in one run and apply it to maximize the information from single spheroids drug screenings.

MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHOSPHATE2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in PHOSPHATE TRANSPORTER1 (PHT1) family and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

Recent advances demonstrate the benefits of radar-derived specific attenuation at horizontal polarization (A H) for quantitative precipitation estimation (QPE) at S and X band. To date the methodology has, however, not been adapted for the widespread European C-band radars such as those installed in the network of the German Meteorological Service (DWD, Deutscher Wetterdienst). Simulations based on a large dataset of drop size distributions (DSDs) measured over Germany are performed to investigate the DSD dependencies of the attenuation parameter αH for the A H estimates. The normalized raindrop concentration (Nw) and the change of differential reflectivity (Z DR) with reflectivity at horizontal polarization (Z H) are used to categorize radar observations into regimes for which scan-wise optimized αH values are derived. For heavier continental rain with Z H > 40 dBZ, the A H-based rainfall retrieval R(A H) is combined with a rainfall estimator using a substitute of specific differential phase ( K DP * ) . We also assess the performance of retrievals based on specific attenuation at vertical polarization (A V). Finally, the regime-adapted hybrid QPE algorithms are applied to four convective cases and one stratiform case from 2017 to 2019, and compared to DWD’s operational Radar-Online-Aneichung (RADOLAN) RW rainfall product, which is based on Z h only but adjusted to rain gauge measurements. For the convective cases, our hybrid retrievals outperform the traditional R(Z h) and pure R(A H/V) retrievals with fixed αH/V values when evaluated with gauge measurements and outperform RW when evaluated by disdrometer measurements. Potential improvements using ray-wise αH/V and segment-wise applications of the ZPHI method along the radials are discussed.

Intratumoural hypoxia induces HIF-1α and promotes tumour progression, metastasis and treatment resistance. HIF-1α stability is regulated by VHL-E3 ligase-mediated ubiquitin-dependent degradation; however, the hypoxia-regulated deubiquitinase that stabilizes HIF-1α has not been identified. Here we report that HAUSP (USP7) deubiquitinase deubiquitinates HIF-1α to increase its stability, induce epithelial-mesenchymal transition and promote metastasis. Hypoxia induces K63-linked polyubiquitinated HAUSP at lysine 443 to enhance its functions. Knockdown of HAUSP decreases acetylation of histone 3 lysine 56 (H3K56Ac). K63-polyubiquitinated HAUSP interacts with a ubiquitin receptor CBP to specifically mediate H3K56 acetylation. ChIP-seq analysis of HAUSP and HIF-1α binding reveals two motifs responsive to hypoxia. HectH9 is the E3 ligase for HAUSP and a prognostic marker together with HIF-1α. This report demonstrates that hypoxia-induced K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and causes CBP-mediated H3K56 acetylation on HIF-1α target gene promoters to promote EMT/metastasis, further defining HAUSP as a therapeutic target in hypoxia-induced tumour progression. Hypoxia-induced transcriptional responses mediated by HIF-1a are regulated through the ubiquitin-dependent pathway to control HIF-1a stability. Here the authors show that the deubiquitinase HAUSP modulates the stability of HIF-1a and K63-polyubiquitinated HAUSP serves as an anchor for HIF-1a-induced gene transcription.