Integrative Proteomic Characterization of Human Lung Adenocarcinoma.

Genomic studies of lung adenocarcinoma (LUAD) have advanced our understanding of the disease's biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD remain poorly understood. We carried out a comprehensive proteomics analysis of 103 cases of LUAD in Chinese patients. Integrative analysis of proteome, phosphoproteome, transcriptome, and whole-exome sequencing data revealed cancer-associated characteristics, such as tumor-associated protein variants, distinct proteomics features, and clinical outcomes in patients at an early stage or with EGFR and TP53 mutations. Proteome-based stratification of LUAD revealed three subtypes (S-I, S-II, and S-III) related to different clinical and molecular features. Further, we nominated potential drug targets and validated the plasma protein level of HSP 90ß as a potential prognostic biomarker for LUAD in an independent cohort. Our integrative proteomics analysis enables a more comprehensive understanding of the molecular landscape of LUAD and offers an opportunity for more precise diagnosis and treatment.

Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

Interchain-expanded extra-large-pore zeolites.

Stable aluminosilicate zeolites with extra-large pores that are open through rings of more than 12 tetrahedra could be used to process molecules larger than those currently manageable in zeolite materials. However, until very recently1-3, they proved elusive. In analogy to the interlayer expansion of layered zeolite precursors4,5, we report a strategy that yields thermally and hydrothermally stable silicates by expansion of a one-dimensional silicate chain with an intercalated silylating agent that separates and connects the chains. As a result, zeolites with extra-large pores delimited by 20, 16 and 16 Si tetrahedra along the three crystallographic directions are obtained. The as-made interchain-expanded zeolite contains dangling Si-CH3 groups that, by calcination, connect to each other, resulting in a true, fully connected (except possible defects) three-dimensional zeolite framework with a very low density. Additionally, it features triple four-ring units not seen before in any type of zeolite. The silicate expansion-condensation approach we report may be amenable to further extra-large-pore zeolite formation. Ti can be introduced in this zeolite, leading to a catalyst that is active in liquid-phase alkene oxidations involving bulky molecules, which shows promise in the industrially relevant clean production of propylene oxide using cumene hydroperoxide as an oxidant.

NDF is a transcription factor that stimulates elongation by RNA polymerase II.

RNA polymerase II (Pol II) elongation is a critical step in gene expression. Here we found that NDF, which was identified as a bilaterian nucleosome-destabilizing factor, is also a Pol II transcription factor that stimulates elongation with plain DNA templates in the absence of nucleosomes. NDF binds directly to Pol II and enhances elongation by a different mechanism than that used by transcription factor TFIIS. Moreover, yeast Pdp3, which is related to NDF, binds to Pol II and stimulates elongation. Thus, NDF is a Pol II binding transcription elongation factor that is localized over gene bodies and is conserved from yeast to humans.

A phosphate-sensing organelle regulates phosphate and tissue homeostasis.

Inorganic phosphate (Pi) is one of the essential molecules for life. However, little is known about intracellular Pi metabolism and signalling in animal tissues1. Following the observation that chronic Pi starvation causes hyperproliferation in the digestive epithelium of Drosophila melanogaster, we determined that Pi starvation triggers the downregulation of the Pi transporter PXo. In line with Pi starvation, PXo deficiency caused midgut hyperproliferation. Interestingly, immunostaining and ultrastructural analyses showed that PXo specifically marks non-canonical multilamellar organelles (PXo bodies). Further, by Pi imaging with a Förster resonance energy transfer (FRET)-based Pi sensor2, we found that PXo restricts cytosolic Pi levels. PXo bodies require PXo for biogenesis and undergo degradation following Pi starvation. Proteomic and lipidomic characterization of PXo bodies unveiled their distinct feature as an intracellular Pi reserve. Therefore, Pi starvation triggers PXo downregulation and PXo body degradation as a compensatory mechanism to increase cytosolic Pi. Finally, we identified connector of kinase to AP-1 (Cka), a component of the STRIPAK complex and JNK signalling3, as the mediator of PXo knockdown- or Pi starvation-induced hyperproliferation. Altogether, our study uncovers PXo bodies as a critical regulator of cytosolic Pi levels and identifies a Pi-dependent PXo-Cka-JNK signalling cascade controlling tissue homeostasis.

A proteomic and phosphoproteomic landscape of KRAS mutant cancers identifies combination therapies.

KRAS mutant cancer, characterized by the activation of a plethora of phosphorylation signaling pathways, remains a major challenge for cancer therapy. Despite recent advancements, a comprehensive profile of the proteome and phosphoproteome is lacking. This study provides a proteomic and phosphoproteomic landscape of 43 KRAS mutant cancer cell lines across different tissue origins. By integrating transcriptomics, proteomics, and phosphoproteomics, we identify three subsets with distinct biological, clinical, and therapeutic characteristics. The integrative analysis of phosphoproteome and drug sensitivity information facilitates the identification of a set of drug combinations with therapeutic potentials. Among them, we demonstrate that the combination of DOT1L and SHP2 inhibitors is an effective treatment specific for subset 2 of KRAS mutant cancers, corresponding to a set of TCGA clinical tumors with the poorest prognosis. Together, this study provides a resource to better understand KRAS mutant cancer heterogeneity and identify new therapeutic possibilities.

All-perovskite tandem solar cells with improved grain surface passivation.

All-perovskite tandem solar cells hold the promise of surpassing the efficiency limits of single-junction solar cells1-3; however, until now, the best-performing all-perovskite tandem solar cells have exhibited lower certified efficiency than have single-junction perovskite solar cells4,5. A thick mixed Pb-Sn narrow-bandgap subcell is needed to achieve high photocurrent density in tandem solar cells6, yet this is challenging owing to the short carrier diffusion length within Pb-Sn perovskites. Here we develop ammonium-cation-passivated Pb-Sn perovskites with long diffusion lengths, enabling subcells that have an absorber thickness of approximately 1.2 µm. Molecular dynamics simulations indicate that widely used phenethylammonium cations are only partially adsorbed on the surface defective sites at perovskite crystallization temperatures. The passivator adsorption is predicted to be enhanced using 4-trifluoromethyl-phenylammonium (CF3-PA), which exhibits a stronger perovskite surface-passivator interaction than does phenethylammonium. By adding a small amount of CF3-PA into the precursor solution, we increase the carrier diffusion length within Pb-Sn perovskites twofold, to over 5 µm, and increase the efficiency of Pb-Sn perovskite solar cells to over 22%. We report a certified efficiency of 26.4% in all-perovskite tandem solar cells, which exceeds that of the best-performing single-junction perovskite solar cells. Encapsulated tandem devices retain more than 90% of their initial performance after 600 h of operation at the maximum power point under 1 Sun illumination in ambient conditions.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

Unraveling the molecular mechanism for enhanced gas adsorption in mixed-metal MOFs via solid-state NMR spectroscopy.

The incorporation of multiple metal ions in metal-organic frameworks (MOFs) through one-pot synthesis can induce unique properties originating from specific atomic-scale spatial apportionment, but the extraction of this crucial information poses challenges. Herein, nondestructive solid-state NMR spectroscopy was used to discern the atomic-scale metal apportionment in a series of bulk Mg1-xCox-MOF-74 samples via identification and quantification of eight distinct arrangements of Mg/Co ions labeled with a 13C-carboxylate, relative to Co content. Due to the structural characteristics of metal-oxygen chains, the number of metal permutations is infinite for Mg1-xCox-MOF-74, making the resolution of atomic-scale metal apportionment particularly challenging. The results were then employed in density functional theory calculations to unravel the molecular mechanism underlying the macroscopic adsorption properties of several industrially significant gases. It is found that the incorporation of weak adsorption sites (Mg2+ for CO and Co2+ for CO2 adsorption) into the MOF structure counterintuitively boosts the gas adsorption energy on strong sites (Co2+ for CO and Mg2+ for CO2 adsorption). Such effect is significant even for Co2+ remote from Mg2+ in the metal-oxygen chain, resulting in a greater enhancement of CO adsorption across a broad composition range, while the enhancement of CO2 adsorption is restricted to Mg2+ with adjacent Co2+. Dynamic breakthrough measurements unambiguously verified the trend in gas adsorption as a function of metal composition. This research thus illuminates the interplay between atomic-scale structures and macroscopic gas adsorption properties in mixed-metal MOFs and derived materials, paving the way for developing superior functional materials.

Elf1 promotes Rad26's interaction with lesion-arrested Pol II for transcription-coupled repair.

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a common mechanism to recognize a stalled Pol II, with additional interactions when Pol II is arrested at a lesion. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces further interactions between Rad26 and a lesion-arrested Pol II. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation. Together, our results provide important mechanistic insights into how two conserved transcription-coupled repair factors, Rad26/CSB and Elf1/ELOF1, work together at the initial lesion recognition steps of transcription-coupled repair.

Hypothalamic Menin regulates systemic aging and cognitive decline.

Aging is a systemic process, which is a risk factor for impaired physiological functions, and finally death. The molecular mechanisms driving aging process and the associated cognitive decline are not fully understood. The hypothalamus acts as the arbiter that orchestrates systemic aging through neuroinflammatory signaling. Our recent findings revealed that Menin plays important roles in neuroinflammation and brain development. Here, we found that the hypothalamic Menin signaling diminished in aged mice, which correlates with systemic aging and cognitive deficits. Restoring Menin expression in ventromedial nucleus of hypothalamus (VMH) of aged mice extended lifespan, improved learning and memory, and ameliorated aging biomarkers, while inhibiting Menin in VMH of middle-aged mice induced premature aging and accelerated cognitive decline. We further found that Menin epigenetically regulates neuroinflammatory and metabolic pathways, including D-serine metabolism. Aging-associated Menin reduction led to impaired D-serine release by VMH-hippocampus neural circuit, while D-serine supplement rescued cognitive decline in aged mice. Collectively, VMH Menin serves as a key regulator of systemic aging and aging-related cognitive decline.

split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80.

The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of "split-intein Gal4" in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type-specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm ("Two Against Background" or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.

EMT activates exocytotic Rabs to coordinate invasion and immunosuppression in lung cancer.

Epithelial-to-mesenchymal transition (EMT) underlies immunosuppression, drug resistance, and metastasis in epithelial malignancies. However, the way in which EMT orchestrates disparate biological processes remains unclear. Here, we identify an EMT-activated vesicular trafficking network that coordinates promigratory focal adhesion dynamics with an immunosuppressive secretory program in lung adenocarcinoma (LUAD). The EMT-activating transcription factor ZEB1 drives exocytotic vesicular trafficking by relieving Rab6A, Rab8A, and guanine nucleotide exchange factors from miR-148a-dependent silencing, thereby facilitating MMP14-dependent focal adhesion turnover in LUAD cells and autotaxin-mediated CD8+ T cell exhaustion, indicating that cell-intrinsic and extrinsic processes are linked through a microRNA that coordinates vesicular trafficking networks. Blockade of ZEB1-dependent secretion reactivates antitumor immunity and negates resistance to PD-L1 immune checkpoint blockade, an important clinical problem in LUAD. Thus, EMT activates exocytotic Rabs to drive a secretory program that promotes invasion and immunosuppression in LUAD.

Safety and efficacy of an anti-human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models.

Rebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

Application of mendelian randomization in ocular diseases: a review.

Ocular disorders can significantly lower patients' quality of life and impose an economic burden on families and society. However, for the majority of these diseases, their prevalence and mechanisms are yet unknown, making prevention, management, and therapy challenging. Although connections between exposure factors and diseases can be drawn through observational research, it is challenging to rule out the interference of confounding variables and reverse causation. Mendelian Randomization (MR), a method of research that combines genetics and epidemiology, has its advantage to solve this problem and thus has been extensively utilized in the etiological study of ophthalmic diseases. This paper reviews the implementation of MR in the research of ocular diseases and provides approaches for the investigation of related mechanisms as well as the intervention strategies.

Methane Oxidation to Methanol.

The direct transformation of methane to methanol remains a significant challenge for operation at a larger scale. Central to this challenge is the low reactivity of methane at conditions that can facilitate product recovery. This review discusses the issue through examination of several promising routes to methanol and an evaluation of performance targets that are required to develop the process at scale. We explore the methods currently used, the emergence of active heterogeneous catalysts and their design and reaction mechanisms and provide a critical perspective on future operation. Initial experiments are discussed where identification of gas phase radical chemistry limited further development by this approach. Subsequently, a new class of catalytic materials based on natural systems such as iron or copper containing zeolites were explored at milder conditions. The key issues of these technologies are low methane conversion and often significant overoxidation of products. Despite this, interest remains high in this reaction and the wider appeal of an effective route to key products from C-H activation, particularly with the need to transition to net carbon zero with new routes from renewable methane sources is exciting.

ALDH1+ tumor stem cells promote the progression of malignant fibrous tissue sarcoma by inhibiting SYNPO2 through hsa-mir-206.

This research aims to explore the mechanism by which microRNAs may regulate the biological behavior of tumor cells in ALDH1+ fibrosarcoma. We identified differentially expressed miRNAs in ALDH+ NMFH-1 cells, screened genes related to sarcoma metastasis in the TCGA database, and finally obtained key genes regulated by miRNAs that are involved in metastasis. The function and mechanism of these key genes were then validated at the cellular level. Using the ULCAN database, a significant correlation was found between hsa-mir-206 and mortality in sarcoma patients. WGCNA analysis identified 352 genes related to tumor metastasis. Through Venn diagrams, we obtained 15 metastasis-related genes regulated by hsa-mir-206. Survival analysis showed that SYNPO2 expression is significantly correlated with survival rate and is significantly underexpressed in multiple tumors. SYNPO2 showed a negative correlation with macrophages and a positive correlation with CD8+ T cells. After inhibiting the expression of hsa-mir-206 with siRNA plasmids, the mRNA expression of SYNPO2 was significantly upregulated. The results of CCK8 assay, scratch assay, and transwell assay showed that the proliferation and migration ability of NFMH-1 cells were promoted after SYNPO2 was inhibited. ALDH1+ tumor stem cells promote the proliferation and invasion of malignant fibrous histiocytoma cells by inhibiting SYNPO2 through hsa-mir-206.

RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder.

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im-induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im-induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides-dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im-induced transcriptional arrest.