Oncogenic Mutations Rewire Signaling Pathways by Switching Protein Recruitment to Phosphotyrosine Sites.

Tyrosine phosphorylation regulates multi-layered signaling networks with broad implications in (patho)physiology, but high-throughput methods for functional annotation of phosphotyrosine sites are lacking. To decipher phosphotyrosine signaling directly in tissue samples, we developed a mass-spectrometry-based interaction proteomics approach. We measured the in vivo EGF-dependent signaling network in lung tissue quantifying >1,000 phosphotyrosine sites. To assign function to all EGF-regulated sites, we determined their recruited protein signaling complexes in lung tissue by interaction proteomics. We demonstrated how mutations near tyrosine residues introduce molecular switches that rewire cancer signaling networks, and we revealed oncogenic properties of such a lung cancer EGFR mutant. To demonstrate the scalability of the approach, we performed >1,000 phosphopeptide pulldowns and analyzed them by rapid mass spectrometric analysis, revealing tissue-specific differences in interactors. Our approach is a general strategy for functional annotation of phosphorylation sites in tissues, enabling in-depth mechanistic insights into oncogenic rewiring of signaling networks.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

Prethermal Fragmentation in a Periodically Driven Fermionic Chain.

We study a fermionic chain with nearest-neighbor hopping and density-density interactions, where the nearest-neighbor interaction term is driven periodically. We show that such a driven chain exhibits prethermal strong Hilbert space fragmentation (HSF) in the high drive amplitude regime at specific drive frequencies ω_{m}^{*}. This constitutes the first realization of HSF for out-of-equilibrium systems. We obtain analytic expressions of ω_{m}^{*} using a Floquet perturbation theory and provide exact numerical computation of entanglement entropy, equal-time correlation functions, and the density autocorrelation of fermions for finite chains. All of these quantities indicate clear signatures of strong HSF. We study the fate of the HSF as one tunes away from ω_{m}^{*} and discuss the extent of the prethermal regime as a function of the drive amplitude.

Nematic-Fluctuation-Mediated Superconductivity Revealed by Anisotropic Strain in Ba(Fe_{1-x}Co_{x})_{2}As_{2}.

Anisotropic strain is an external field capable of selectively addressing the role of nematic fluctuations in promoting superconductivity. We demonstrate this using polarization-resolved elasto-Raman scattering by probing the evolution of nematic fluctuations under strain in the normal and superconducting state of the paradigmatic iron-based superconductor Ba(Fe_{1-x}Co_{x})_{2}As_{2}. In the parent compound BaFe_{2}As_{2} we observe a strain-induced suppression of the nematic susceptibility which follows the expected behavior of an Ising order parameter under a symmetry breaking field. For the superconducting compound, the suppression of the nematic susceptibility correlates with the decrease of the critical temperature T_{c}, indicating a significant contribution of nematic fluctuations to electron pairing. Our results validate theoretical scenarios of enhanced T_{c} near a nematic quantum critical point.

Charge-induced nematicity in FeSe.

The spontaneous appearance of nematicity, a state of matter that breaks rotation but not translation symmetry, is one of the most intriguing properties of the iron-based superconductors (Fe SC), and has relevance for the cuprates as well. Establishing the critical electronic modes behind nematicity remains a challenge, however, because their associated susceptibilities are not easily accessible by conventional probes. Here, using FeSe as a model system, and symmetry-resolved electronic Raman scattering as a probe, we unravel the presence of critical charge nematic fluctuations near the structural/nematic transition temperature, [Formula: see text] 90 K. The diverging behavior of the associated nematic susceptibility foretells the presence of a Pomeranchuk instability of the Fermi surface with d-wave symmetry. The excellent scaling between the observed nematic susceptibility and elastic modulus data demonstrates that the structural distortion is driven by this d-wave Pomeranchuk transition. Our results make a strong case for charge-induced nematicity in FeSe.

Nematic Resonance in the Raman Response of Iron-Based Superconductors.

In a fully gapped superconductor the electronic Raman response has a pair-breaking peak at twice the superconducting gap Δ, if the Bogoliubov excitations are uncorrelated. Motivated by the iron based superconductors, we study how this peak is modified if the superconducting phase hosts a nematic-structural quantum critical point. We show that, upon approaching this point by tuning, e.g., doping, the growth of nematic correlations between the quasiparticles transforms the pair-breaking peak into a nematic resonance. The mode energy is below 2Δ, and stays finite at the quantum critical point, where its spectral weight is sharply enhanced. The latter is consistent with recent experiments on electron-doped iron based superconductors and provides direct evidence of nematic correlations in their superconducting phases.

Publisher's Note: Effects of Lifshitz Transitions on Charge Transport in Magnetic Phases of Fe-Based Superconductors [Phys. Rev. Lett. 114, 097003 (2015)].

This corrects the article DOI: 10.1103/PhysRevLett.114.097003.

OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

BACKGROUND: Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored. METHODS: RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth. RESULTS: Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo. CONCLUSIONS: OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

Quantum Critical Elasticity.

We discuss elastic instabilities of the atomic crystal lattice at zero temperature. Because of long-range shear forces of the solid, at such transitions the phonon velocities vanish, if at all, only along certain crystallographic directions, and, consequently, the critical phonon fluctuations are suppressed to a lower dimensional manifold and governed by a Gaussian fixed point. In the case of symmetry-breaking elastic transitions, a characteristic critical phonon thermodynamics arises that is found, e.g., to violate Debye's T(3) law for the specific heat. We point out that quantum critical elasticity is triggered whenever a critical soft mode couples linearly to the strain tensor. In particular, this is relevant for the electronic Ising-nematic quantum phase transition in a tetragonal crystal as discussed in the context of certain cuprates, ruthenates, and iron-based superconductors.

Effects of Lifshitz transition on charge transport in magnetic phases of Fe-based superconductors.

The unusual temperature dependence of the resistivity and its in-plane anisotropy observed in the Fe-based superconducting materials, particularly Ba(Fe_{1-x}Co_{x})_{2}As_{2}, has been a long-standing puzzle. Here, we consider the effect of impurity scattering on the temperature dependence of the average resistivity within a simple two-band model of a dirty spin density wave metal. The sharp drop in resistivity below the Néel temperature T_{N} in the parent compound can only be understood in terms of a Lifshitz transition following Fermi surface reconstruction upon magnetic ordering. We show that the observed resistivity anisotropy in this phase, arising from nematic defect structures, is affected by the Lifshitz transition as well.

DESP demixes cell-state profiles from dynamic bulk molecular measurements.

Understanding the dynamic expression of proteins and other key molecules driving phenotypic remodeling in development and pathobiology has garnered widespread interest, yet the exploration of these systems at the foundational resolution of the underlying cell states has been significantly limited by technical constraints. Here, we present DESP, an algorithm designed to leverage independent estimates of cell-state proportions, such as from single-cell RNA sequencing, to resolve the relative contributions of cell states to bulk molecular measurements, most notably quantitative proteomics, recorded in parallel. We applied DESP to an in vitro model of the epithelial-to-mesenchymal transition and demonstrated its ability to accurately reconstruct cell-state signatures from bulk-level measurements of both the proteome and transcriptome, providing insights into transient regulatory mechanisms. DESP provides a generalizable computational framework for modeling the relationship between bulk and single-cell molecular measurements, enabling the study of proteomes and other molecular profiles at the cell-state level using established bulk-level workflows.

The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation.

The tumor suppressor, PTEN is key to the regulation of diverse cellular processes, making it a prime candidate to be tightly regulated. The PTEN level is controlled in a major way by E3 ligase-mediated degradation through the Ubiquitin-Proteasome System (UPS). Nedd 4-1, XIAP, and WWP2 have been shown to maintain PTEN turnover. Here, we report that CHIP, the chaperone-associated E3 ligase, induces ubiquitination and regulates the proteasomal turnover of PTEN. It was apparent from our findings that PTEN transiently associates with the molecular chaperones and thereby gets diverted to the degradation pathway through its interaction with CHIP. The TPR domain of CHIP and parts of the N-terminal domain of PTEN are required for their interaction. Overexpression of CHIP leads to elevated ubiquitination and a shortened half-life of endogenous PTEN. On the other hand, depletion of endogenous CHIP stabilizes PTEN. CHIP is also shown to regulate PTEN-dependent transcription presumably through its down-regulation. PTEN shared an inverse correlation with CHIP in human prostate cancer patient samples, thereby triggering the prospects of a more complex mode of PTEN regulation in cancer.

The Outcome of Treatment in Second Branchial Cleft Anomalies: A Case Series.

Background Branchial-cleft anomalies are second only to thyroglossal duct anomalies among congenital malformations of the neck, and second branchial-cleft anomalies are the most common. These include branchial cysts, branchial sinuses, and branchial fistulas. Clinical symptoms include neck swelling and a discharging sinus or fistula opening. In a small number of cases, they can lead to major complications like abscesses or malignant changes. Surgical resection is the treatment of choice. Various approaches to resection and sclerotherapy have been tried. In this study, we present our treatment outcome with branchial cleft anomalies at a rural tertiary medical care hospital. Objectives To document the various presentations, clinical features, and outcomes of treatment with second branchial cleft anomalies. Methods This retrospective observational study included 16 patients operated on for second branchial-cleft anomalies. A detailed medical history was elicited, and an accurate clinical examination was done. A contrast-enhanced computed tomography (CECT) scan was done in all cases. A few cases required a fistulogram. The cysts, sinuses, or fistulas were resected en bloc by a single neck crease incision. Primary closure was done in all cases. A recurrence or pharyngocutaneous fistula required axial flap reconstruction. The complications and recurrences were documented. Result There were six children and 10 adults in our study. Seven cysts, five sinuses, and four fistulas were present, of which four were iatrogenic. In seven patients, imaging could not show the entire tract. There were four fistulas from the oropharynx to a cutaneous opening in the neck. A complete resection was done for all. Two pharyngocutaneous fistulas were treated with a pectoralis major myocutaneous (PMMC) flap. Three patients had wound dehiscence postoperatively. None of the patients had neurological or vascular injuries. Conclusion Second branchial cleft anomalies can be completely excised by a single neck crease incision. Meticulous surgery results in a low recurrence or complication rate. Following complete excision, in type IV anomalies, a purse-string suture at the pharyngeal opening ensures good closure and no recurrences.

Comparison Between Clinical Outcome of Intralesional Human Placental Extract Alone and Topical Application of Placental Extract Gel After Fibrotomy in Oral Submucous Fibrosis.

BACKGROUND: Oral submucous fibrosis (OSMF) is a premalignant condition prevalent in our country. Juxtaepithelial inflammation with progressive hyalinization of the lamina propria results in stiffness and fibrosis of the oral mucosa, characterised by trismus, ankyloglossia, and a burning sensation. Various methods of treatment have been tried in these cases, which include placental extract injections and the cutting of fibrous bands. In this study, we aim to compare the outcome of intra-lesional placental extract injection with fibrotomy and placental extract gel application in OSMF. METHODOLOGY: This prospective interventional study included 58 patients clinically diagnosed with OSMF grades II and III at a rural tertiary care hospital between January 2021 and August 2022. The patients were randomised into two groups: group I received 1 ml of intra-lesional human placental extract injection in the submucosal plane of the buccal mucosa and retro-molar trigone (RMT) once a week for five consecutive weeks, and group II was subjected to a transverse division of fibrotic bands in the submucosal plane under general anaesthesia. The surgical wound was left open, and swabs soaked in human-purified placental extract gel were placed in the wound for two hours twice daily until the surgical wound was epithelialized and healed. The patients in both groups I and II were advised to do jaw opening exercises, and weekly follow-up was done. Findings with regard to maximum mouth opening, colour of mucosa, and burning sensation in the oral cavity based on a Likert scale were documented. At the end of five months, the pre-treatment and post-treatment results documented were compared. RESULTS: All patients were between 20 and 60 years of age and were addicted to chewing areca nuts with tobacco. Bilateral involvement was present in all patients, with extension into the RMT and soft palate seen in 31%. Improvement in mouth opening was between 4 mm and 6 mm in group II, and relief of burning sensation and mucosal colour was better in group I. CONCLUSION: Intra-lesional placental extract injections help in the improvement of the mucosa and relief from the burning sensation. Fibrotomy with placental extract gel application is better at relieving trismus in OSMF. Aggressive mouth-opening exercises may improve mouth opening following the above procedures.

GPS2-mediated regulation of the adipocyte secretome modulates adipose tissue remodeling at the onset of diet-induced obesity.

OBJECTIVE: Dysfunctional, unhealthy expansion of white adipose tissue due to excess dietary intake is a process at the root of obesity and Type 2 Diabetes development. The objective of this study is to contribute to a better understanding of the underlying mechanism(s) regulating the early stages of adipose tissue expansion and adaptation to dietary stress due to an acute, high-fat diet (HFD) challenge, with a focus on the communication between adipocytes and other stromal cells. METHODS: We profiled the early response to high-fat diet exposure in wildtype and adipocyte-specific GPS2-KO (GPS2-AKO) mice at the cellular, tissue and organismal level. A multi-pronged approach was employed to disentangle the complex cellular interactions dictating tissue remodeling, via single-cell RNA sequencing and FACS profiling of the stromal fraction, and semi-quantitative proteomics of the adipocyte-derived exosomal cargo after 5 weeks of HFD feeding. RESULTS: Our results indicate that loss of GPS2 in mature adipocytes leads to impaired adaptation to the metabolic stress imposed by HFD feeding. GPS2-AKO mice are significantly more inflamed, insulin resistant, and obese, compared to the WT counterparts. At the cellular level, lack of GPS2 in adipocytes impacts upon other stromal populations, with both the eWAT and scWAT depots exhibiting changes in the immune and non-immune compartments that contribute to an increase in inflammatory and anti-adipogenic cell types. Our studies also revealed that adipocyte to stromal cell communication is facilitated by exosomes, and that transcriptional rewiring of the exosomal cargo is crucial for tissue remodeling. Loss of GPS2 results in increased expression of secreted factors promoting a TGFß-driven fibrotic microenvironment favoring unhealthy tissue remodeling and expansion. CONCLUSIONS: Adipocytes serve as an intercellular signaling hub, communicating with the stromal compartment via paracrine signaling. Our study highlights the importance of proper regulation of the 'secretome' released by energetically stressed adipocytes at the onset of obesity. Altered transcriptional regulation of factors secreted via adipocyte-derived exosomes (AdExos), in the absence of GPS2, contributes to the establishment of an anti-adipogenic, pro-fibrotic adipose tissue environment, and to hastened progression towards a metabolically dysfunctional phenotype.

Parallelized multidimensional analytic framework applied to mammary epithelial cells uncovers regulatory principles in EMT.

A proper understanding of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. Here we combine state-of-the-art data acquisition platforms and bioinformatics tools to devise PAMAF, a workflow that simultaneously examines twelve omics modalities, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We apply PAMAF in an established in vitro model of TGFß-induced epithelial to mesenchymal transition (EMT) to quantify >61,000 molecules from 12 omics and 10 timepoints over 12 days. Bioinformatics analysis of this EMT-ExMap resource allowed us to identify; -topological coupling between omics, -four distinct cell states during EMT, -omics-specific kinetic paths, -stage-specific multi-omics characteristics, -distinct regulatory classes of genes, -ligand-receptor mediated intercellular crosstalk by integrating scRNAseq and subcellular proteomics, and -combinatorial drug targets (e.g., Hedgehog signaling and CAMK-II) to inhibit EMT, which we validate using a 3D mammary duct-on-a-chip platform. Overall, this study provides a resource on TGFß signaling and EMT.

Imaging the future: the emerging era of single-cell spatial proteomics.

The proteome of a human cell is partitioned within organelles, such as the nucleus, and other subcellular compartments, such as the cytoplasm, forming a myriad of membrane-bound and membrane-free ultrastructures. This compartmentalization allows discrete biochemical processes to occur efficiently in isolation, with relevant proteins localized to appropriate niches to fulfill their biological function(s). Proper delivery and dynamic exchange of proteins between compartments underlie the regulation of many cellular processes, such as cell signaling, division, and programmed cell death. To this end, cells deploy dedicated trafficking mechanisms to ensure correct protein localization, as mis-localization can result in pathology. In addition to trafficking, variation in the expression, modification, and physical associations of proteins within and between cells can result in biological heterogeneity, motivating the need for single-cell measurements. In this review, we introduce diverse platform technologies developed for subcellular proteomics and high-throughput systems biology, with the aim of providing mechanistic insights into fundamental cell biological processes underlying healthy and diseased states, and valuable public data resources. In contrast to the rapidly advancing field of single-cell genomics, the single-cell spatial proteomics toolbox remains in its infancy, but is poised to make considerable advances in the coming years.

PDX-derived organoids model in vivo drug response and secrete biomarkers.

Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired patient-derived xenograft (PDX) and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50%-94% of the relative abundance of all N-glycans detected in each of the models. Last, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicle (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis and discovered that 4 EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically actionable serologic biomarkers.