A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

Tandem SERS and MS/MS Profiling of Plasma Extracellular Vesicles for Early Ovarian Cancer Biomarker Discovery.

Extracellular vesicles (EVs) are vectors of biomolecular cargo that play essential roles in intercellular communication across a range of cells. Protein, lipid, and nucleic acid cargo harbored within EVs may serve as biomarkers at all stages of disease; however, the choice of methodology may challenge the specificity and reproducibility of discovery. To address these challenges, the integration of rigorous EV purification methods, cutting-edge spectroscopic technologies, and data analysis are critical to uncover diagnostic signatures of disease. Herein, we demonstrate an EV isolation and analysis pipeline using surface-enhanced Raman spectroscopy (SERS) and mass spectrometry (MS) techniques on plasma samples obtained from umbilical cord blood, healthy donor (HD) plasma, and plasma from women with early stage high-grade serous carcinoma (HGSC). Plasma EVs were purified by size exclusion chromatography and analyzed by surface-enhanced Raman spectroscopy (SERS), mass spectrometry (MS), and atomic force microscopy. After determining the fraction of highest EV purity, SERS and MS were used to characterize EVs from HDs, pooled donors with noncancerous gynecological ailments (n = 6), and donors with early stage [FIGO (I/II)] with HGSC. SERS spectra were subjected to different machine learning algorithms such as PCA, logistic regression, support vector machine, naïve Bayes, random forest, neural network, and k nearest neighbors to differentiate healthy, benign, and HGSC EVs. Collectively, we demonstrate a reproducible workflow with the potential to serve as a diagnostic platform for HGSC.

Specific quinone reductase 2 inhibitors reduce metabolic burden and reverse Alzheimer's disease phenotype in mice.

Biological aging can be described as accumulative, prolonged metabolic stress and is the major risk factor for cognitive decline and Alzheimer's disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes overexpressed with age, and it is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that, in human cells, genetic removal of QR2 produced a shift in the proteome opposing that found in AD brains while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2is) to enable evaluation of chronic QR2 inhibition as a means to reduce biological age-related metabolic stress and cognitive decline. QR2is replicated results obtained by genetic removal of QR2, while local QR2i microinjection improved hippocampal and cortical-dependent learning in rats and mice. Continuous consumption of QR2is in drinking water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity and pathway function in the healthy and neurodegenerative brain and what we believe to be the great therapeutic potential of QR2is as first-in-class drugs.

Noncanonical functions of Ku may underlie essentiality in human cells.

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but is involved in other cellular functions like telomere regulation and maintenance, in which Ku's role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells using CRISPR/Cas9 editing to study the essentiality of Ku70 function. While we observed loss of cell viability upon Ku depletion, we did not detect significant changes in telomere length, nor did we record lethal levels of DNA damage upon loss of Ku. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study suggests that the driving cause of loss of cell viability in Ku70 knockouts is not linked to the functions of Ku in DNA repair or at telomeres. Moreover, our data shows that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

Investigation of N,N,N-Trimethyl-L-alanyl-L-proline Betaine (TMAP) as a Biomarker of Kidney Function.

Glomerular filtration rate (GFR) is the most widely used tool for the measurement of kidney function, but endogenous biomarkers such as cystatin C and creatinine have limitations. A previous metabolomic study revealed N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP) to be reflective of kidney function. In this study, we developed a quantitative LCMS assay for the measurement of TMAP and evaluated TMAP as a biomarker of GFR. An assay to measure TMAP was developed using liquid chromatography-mass spectrometry. After validation of the method, we applied it to plasma samples from three distinct kidney disease patient cohorts: nondialysis chronic kidney disease (CKD) patients, patients receiving peritoneal and hemodialysis, and living kidney donors. We investigated whether TMAP was conserved in other mammalian and nonmammalian species, by analyzing plasma samples from Wistar rats with diet-induced CKD and searching for putative matches to the m/z for TMAP and its known fragments in the raw sample data repository "Metabolomics Workbench". The assay can measure plasma TMAP at a lower limit of quantitation (100 ng/mL) with an interday precision and accuracy of 12.8 and 12.1%, respectively. In all three patient cohorts, TMAP concentrations are significantly higher in patients with CKD than in controls with a normal GFR. Further, TMAP concentrations are also elevated in rats with CKD and TMAP is present in the sap produced from Acer saccharum trees. TMAP concentration is inversely related to GFR suggesting that it is a marker of kidney function. TMAP is present in nonmammalian species suggesting that it is part of a biologically conserved process.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Probing the effects of matrix-derived microcarrier composition on human adipose-derived stromal cells cultured dynamically within spinner flask bioreactors.

Recognizing the cell-instructive capacity of the extracellular matrix (ECM), this study investigated the effects of expanding human adipose-derived stromal cells (hASCs) on ECM-derived microcarriers fabricated from decellularized adipose tissue (DAT) or decellularized cartilage tissue (DCT) within spinner flask bioreactors. Protocols were established for decellularizing porcine auricular cartilage and electrospraying methods were used to generate microcarriers comprised exclusively of DAT or DCT, which were compositionally distinct, but had matching Young's moduli. Both microcarrier types supported hASC attachment and growth over 14 days within a low-shear spinner culture system, with a significantly higher cell density observed on the DCT microcarriers at 7 and 14 days. Irrespective of the ECM source, dynamic culture on the microcarriers altered the expression of genes and proteins associated with cell adhesion and ECM remodeling. Label-free mass spectrometry analysis showed upregulation of proteins associated with cartilage development and ECM in the hASCs expanded on the DCT microcarriers. Based on Luminex analysis, the hASCs expanded on the DCT microcarriers secreted significantly higher levels of IL-8 and PDGFAA, supporting that the ECM source can modulate hASC paracrine factor secretion. Finally, the hASCs expanded on the microcarriers were extracted for analysis of adipogenic and chondrogenic differentiation relative to baseline controls. The microcarrier-cultured hASCs showed enhanced intracellular lipid accumulation at 7 days post-induction of adipogenic differentiation. In the chondrogenic studies, a low level of differentiation was observed in all groups. Future studies are warranted using alternative cell sources with greater chondrogenic potential to further assess the chondro-inductive properties of the DCT microcarriers.

Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation.

Wnt and Hh are known signalling pathways involved in neural differentiation and recent work has shown the cell cycle regulator, Never in Mitosis Kinase 2 (Nek2) is able to regulate both pathways. Despite its known function in pathway regulation, few studies have explored Nek2 within embryonic development. The P19 embryonal carcinoma cell model was used to investigate Nek2 and neural differentiation through CRISPR knockout and overexpression studies. Loss of Nek2 reduced cell proliferation in the undifferentiated state and during directed differentiation, while overexpression increased cell proliferation. Despite these changes in proliferation rates, Nek2 deficient cells maintained pluripotency markers after neural induction while Nek2 overexpressing cells lost these markers in the undifferentiated state. Nek2 deficient cells lost the ability to differentiate into both neurons and astrocytes, although Nek2 overexpressing cells enhanced neuron differentiation at the expense of astrocytes. Hh and Wnt signalling were explored, however there was no clear connection between Nek2 and these pathways causing the observed changes to differentiation phenotypes. Mass spectrometry was also used during wildtype and Nek2 knockout cell differentiation and we identified reduced electron transport chain components in the knockout population. Immunoblotting confirmed the loss of these components and additional studies showed cells lacking Nek2 were exclusively glycolytic. Interestingly, hypoxia inducible factor 1α was stabilized in these Nek2 knockout cells despite culturing them under normoxic conditions. Since neural differentiation requires a metabolic switch from glycolysis to oxidative phosphorylation, we propose a mechanism where Nek2 prevents HIF1α stabilization, thereby allowing cells to use oxidative phosphorylation to facilitate neuron and astrocyte differentiation.

Distinct nuclear and cytoplasmic assemblies and interactomes of the mammalian CTLH E3 ligase complex.

The C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit E3 ubiquitin ligase and its cellular functions are poorly characterized. Although some CTLH subunits have been found to localize in both the nucleus and cytoplasm of mammalian cells, differences between the compartment-specific complexes have not been explored. Here, we show that the CTLH complex forms different molecular mass complexes in nuclear and cytoplasmic fractions. Loss of WDR26 severely decreased nuclear CTLH complex subunit levels and impaired higher-order CTLH complex formation, revealing WDR26 as a critical determinant of the nuclear stability of the CTLH complex. Through affinity purification coupled to mass spectrometry of endogenous RanBPM (also called RANBP9), a CTLH complex member, from nuclear and cytoplasmic fractions, we identified over 170 compartment-specific interactors involved in various conserved biological processes, such as ribonucleoprotein biogenesis and chromatin assembly. We validated the nuclear-specific RanBPM interaction with macroH2A1 and the cytoplasm-specific interaction with tankyrase-1/2 (encoded by TNKS and TNKS2). Overall, this study provides critical insights into CTLH complex function and composition in both the cytoplasm and nucleus.

Structural and Functional Insights into GID/CTLH E3 Ligase Complexes.

Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.

Stabilization of lithium anode with ceramic-rich interlayer for all solid-state batteries.

The deposition of thin layers of polymer/ceramic on a lithium surface to produce a strong barrier against dendrites was demonstrated. Different forms (needle, sphere, rod) and types of ceramic (Al2O3, Mg2B2O5) were tested and polymer/ceramic interlayers of a few micrometers (4 µm minimum) between the lithium and the PEO-based solid polymer electrolyte (SPE) were deposited. Interlayers with high amounts of ceramic up to 85 wt% were successfully coated on the surface of lithium foil. Compact "polymer in ceramic" layers were observed when Al2O3 spheres were used for instance, providing a strong barrier against the progression of dendrites as well as a buffer layer to alleviate the lithium deformation during stripping/plating cycles. The electrochemical performance of the lithium anodes was assessed in symmetrical Li/SPE/Li cells and in full all-solid-state LiFePO4 (LFP)/SPE/Li batteries. It was observed for all the cells that the charge transfer resistance was significantly reduced after the deposition of the polymer/ceramic layers on the lithium surface. In addition, the symmetrical cells were able to cycle at higher C-rates and the durability at C/4 was even improved by a factor of 8. Microscopic observations of Li/SPE/Li stacks after cycling revealed that the polymer/ceramic interlayer reduces the deformation of lithium upon cycling and avoids the formation of dendrites. Finally, LFP/SPE/Li batteries were cycled and better coulombic efficiencies as well as capacity retentions were obtained with the modified lithium electrodes. This work is patent-pending (WO2021/159209A1).

Chemical Genetic Validation of CSNK2 Substrates Using an Inhibitor-Resistant Mutant in Combination with Triple SILAC Quantitative Phosphoproteomics.

Casein Kinase 2 (CSNK2) is an extremely pleiotropic, ubiquitously expressed protein kinase involved in the regulation of numerous key biological processes. Mapping the CSNK2-dependent phosphoproteome is necessary for better characterization of its fundamental role in cellular signalling. While ATP-competitive inhibitors have enabled the identification of many putative kinase substrates, compounds targeting the highly conserved ATP-binding pocket often exhibit off-target effects limiting their utility for definitive kinase-substrate assignment. To overcome this limitation, we devised a strategy combining chemical genetics and quantitative phosphoproteomics to identify and validate CSNK2 substrates. We engineered U2OS cells expressing exogenous wild type CSNK2A1 (WT) or a triple mutant (TM, V66A/H160D/I174A) with substitutions at residues important for inhibitor binding. These cells were treated with CX-4945, a clinical-stage inhibitor of CSNK2, and analyzed using large-scale triple SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) quantitative phosphoproteomics. In contrast to wild-type CSNK2A1, CSNK2A1-TM retained activity in the presence of CX-4945 enabling identification and validation of several CSNK2 substrates on the basis of their increased phosphorylation in cells expressing CSNK2A1-TM. Based on high conservation within the kinase family, we expect that this strategy can be broadly adapted for identification of other kinase-substrate relationships.

Lactate Enhances Mouse ES Cell Differentiation Toward XEN Cells In Vitro.

Metabolism plays a crucial role for cell survival and function; however, recent evidence has implicated it in regulating embryonic development. In the embryo, the inner cell mass undergoes orchestrated cellular divisions resulting in the formation of pluripotent epiblast stem cells and primitive endoderm cells. However, both lineages can be captured in vitro as embryonic stem (ES) cells and extraembryonic endoderm (XEN) cells. Concomitantly, changes in the metabolic profile occurs during development, and are well documented in the embryonic lineages. However, a comprehensive multi-omic analysis of these features in XEN cells remains lacking. We observed that mouse XEN cells exhibited high sensitivity to glycolytic inhibition in addition to maintaining elevated intra- and extracellular lactate levels in vitro. Extraembryonic endoderm cells maintain high lactate levels by increased LDHA activity, and re-routing pyruvate away from the mitochondria resulting in reduced mitochondrial activity due to disruptions in electron transport chain stoichiometry. Importantly, exogenous lactate supplementation or promoting intracellular lactate accumulation enhances XEN differentiation in vitro. These results highlight how lactate contributes to XEN differentiation in vitro and may serve to enhance reprogramming efficiency of cells used for regenerative medicine.

Characterization of ovarian cancer-derived extracellular vesicles by surface-enhanced Raman spectroscopy.

Ovarian cancer is the most lethal gynecological malignancy, owing to the fact that most cases are diagnosed at a late stage. To improve prognosis and reduce mortality, we must develop methods for the early diagnosis of ovarian cancer. A step towards early and non-invasive cancer diagnosis is through the utilization of extracellular vesicles (EVs), which are nanoscale, membrane-bound vesicles that contain proteins and genetic material reflective of their parent cell. Thus, EVs secreted by cancer cells can be thought of as cancer biomarkers. In this paper, we present gold nanohole arrays for the capture of ovarian cancer (OvCa)-derived EVs and their characterization by surface-enhanced Raman spectroscopy (SERS). For the first time, we have characterized EVs isolated from two established OvCa cell lines (OV-90, OVCAR3), two primary OvCa cell lines (EOC6, EOC18), and one human immortalized ovarian surface epithelial cell line (hIOSE) by SERS. We subsequently determined their main compositional differences by principal component analysis and were able to discriminate the groups by a logistic regression-based machine learning method with ∼99% accuracy, sensitivity, and specificity. The results presented here are a great step towards quick, facile, and non-invasive cancer diagnosis.

Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex-dependent regulation of glycolysis.

Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.

Decellularized adipose tissue scaffolds guide hematopoietic differentiation and stimulate vascular regeneration in a hindlimb ischemia model.

Cellular therapies to stimulate therapeutic angiogenesis in individuals with critical limb ischemia (CLI) remain under intense investigation. In this context, the efficacy of cell therapy is dependent on the survival, biodistribution, and pro-angiogenic paracrine signaling of the cells transplanted. Hematopoietic progenitor cells (HPC) purified from human umbilical cord blood using high aldehyde dehydrogenase-activity (ALDHhi cells) and expanded ex vivo, represent a heterogeneous mixture of progenitor cells previously shown to support limb revascularization in mouse models of CLI. The objectives of this study were to investigate the utility of bioscaffolds derived from human decellularized adipose tissue (DAT) to guide the differentiation of seeded HPC in vitro and harness the pro-angiogenic capacity of HPC at the site of ischemia after implantation in vivo. Probing whether the DAT scaffolds altered HPC differentiation, label-free quantitative mass spectrometry and flow cytometric phenotype analyses indicated that culturing the HPC on the DAT scaffolds supported their differentiation towards the pro-angiogenic monocyte/macrophage lineage at the expense of megakaryopoiesis. Moreover, implantation of HPC in DAT scaffolds within a unilateral hindlimb ischemia model in NOD/SCID mice increased cell retention at the site of ischemia relative to intramuscular injection, and accelerated the recovery of limb perfusion, improved functional limb use and augmented CD31+ capillary density when compared to DAT implantation alone or saline-injected controls. Collectively, these data indicate that cell-instructive DAT scaffolds can direct therapeutic HPC differentiation towards the monocyte/macrophage lineage and represent a promising delivery platform for improving the efficacy of cell therapies for CLI.

Embryonic protein NODAL regulates the breast tumor microenvironment by reprogramming cancer-derived secretomes.

The tumor microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumorigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumor growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple-negative breast cancers and that it directly induces Cancer-associated fibroblasts phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g., CXCL1), cytokines (e.g., IL-6) and growth factors (e.g., PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.

Ultrafiltration and Injection of Islet Regenerative Stimuli Secreted by Pancreatic Mesenchymal Stromal Cells.

The secretome of mesenchymal stromal cells (MSCs) is enriched for biotherapeutic effectors contained within and independent of extracellular vesicles (EVs) that may support tissue regeneration as an injectable agent. We have demonstrated that the intrapancreatic injection of concentrated conditioned media (CM) produced by bone marrow MSC supports islet regeneration and restored glycemic control in hyperglycemic mice, ultimately providing a platform to elucidate components of the MSC secretome. Herein, we extend these findings using human pancreas-derived MSC (Panc-MSC) as "biofactories" to enrich for tissue regenerative stimuli housed within distinct compartments of the secretome. Specifically, we utilized 100 kDa ultrafiltration as a simple method to debulk protein mass and to enrich for EVs while concentrating the MSC secretome into an injectable volume for preclinical assessments in murine models of blood vessel and islet regeneration. EV enrichment (EV+) was validated using nanoscale flow cytometry and atomic force microscopy, in addition to the detection of classical EV markers CD9, CD81, and CD63 using label-free mass spectrometry. EV+ CM was predominately enriched with mediators of wound healing and epithelial-to-mesenchymal transition that supported functional regeneration in mesenchymal and nonmesenchymal tissues. For example, EV+ CM supported human microvascular endothelial cell tubule formation in vitro and enhanced the recovery of blood perfusion following intramuscular injection in nonobese diabetic/severe combined immunodeficiency mice with unilateral hind limb ischemia. Furthermore, EV+ CM increased islet number and ß cell mass, elevated circulating insulin, and improved glycemic control following intrapancreatic injection in streptozotocin-treated mice. Collectively, this study provides foundational evidence that Panc-MSC, readily propagated from the subculture of human islets, may be utilized for regenerative medicine applications.

Pillar-beam structures prevent layered cathode materials from destructive phase transitions.

Energy storage with high energy density and low cost has been the subject of a decades-long pursuit. Sodium-ion batteries are well expected because they utilize abundant resources. However, the lack of competent cathodes with both large capacities and long cycle lives prevents the commercialization of sodium-ion batteries. Conventional cathodes with hexagonal-P2-type structures suffer from structural degradations when the sodium content falls below 33%, or when the integral anions participate in gas evolution reactions. Here, we show a "pillar-beam" structure for sodium-ion battery cathodes where a few inert potassium ions uphold the layer-structured framework, while the working sodium ions could diffuse freely. The thus-created unorthodox orthogonal-P2 K0.4[Ni0.2Mn0.8]O2 cathode delivers a capacity of 194 mAh/g at 0.1 C, a rate capacity of 84% at 1 C, and an 86% capacity retention after 500 cycles at 1 C. The addition of the potassium ions boosts simultaneously the energy density and the cycle life.

ADAM protease inhibition overcomes resistance of breast cancer stem-like cells to γδ T cell immunotherapy.

Gamma delta T cells (γδTc) have tremendous anti-tumoral activity, thus γδTc immunotherapy is currently under development for various malignancies. We targeted breast cancer stem-like cells (BCSC), a rare cell population responsible for patient mortality. BCSC were mostly susceptible to γδTc immunotherapy, yet some escaped. The BCSC secretome rendered γδTc hypo-responsive, and resistant BCSC expressed more PD-L1 and anti-apoptotic protein MCL-1 than non-stem-like cells (NSC). BCSC resistance was partially overcome by dMCL1-2, an MCL-1 degrader, or more fully by blocking PD-1 on γδTc. Increased MICA shedding was prevented by the ADAM inhibitor GW280264X, rendering BCSC as sensitive to γδTc cytotoxicity as NSC. Our data show promising potential for γδTc immunotherapy against BCSC while unraveling immune evasion mechanisms exploited by BCSC, which likely also enable their resistance to cytotoxic T and NK cells. Overcoming this resistance, as we have done here, will improve cancer immunotherapy, leading to better cancer patient outcomes.