Mitotic Checkpoint Regulators Control Insulin Signaling and Metabolic Homeostasis.

Insulin signaling regulates many facets of animal physiology. Its dysregulation causes diabetes and other metabolic disorders. The spindle checkpoint proteins MAD2 and BUBR1 prevent precocious chromosome segregation and suppress aneuploidy. The MAD2 inhibitory protein p31(comet) promotes checkpoint inactivation and timely chromosome segregation. Here, we show that whole-body p31(comet) knockout mice die soon after birth and have reduced hepatic glycogen. Liver-specific ablation of p31(comet) causes insulin resistance, hyperinsulinemia, glucose intolerance, and hyperglycemia and diminishes the plasma membrane localization of the insulin receptor (IR) in hepatocytes. MAD2 directly binds to IR and facilitates BUBR1-dependent recruitment of the clathrin adaptor AP2 to IR. p31(comet) blocks the MAD2-BUBR1 interaction and prevents spontaneous clathrin-mediated IR endocytosis. BUBR1 deficiency enhances insulin sensitivity in mice. BUBR1 depletion in hepatocytes or the expression of MAD2-binding-deficient IR suppresses the metabolic phenotypes of p31(comet) ablation. Our findings establish a major IR regulatory mechanism and link guardians of chromosome stability to nutrient metabolism.

Noncoding RNA NORAD Regulates Genomic Stability by Sequestering PUMILIO Proteins.

Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.

CTCF and R-loops are boundaries of cohesin-mediated DNA looping.

Cohesin and CCCTC-binding factor (CTCF) are key regulatory proteins of three-dimensional (3D) genome organization. Cohesin extrudes DNA loops that are anchored by CTCF in a polar orientation. Here, we present direct evidence that CTCF binding polarity controls cohesin-mediated DNA looping. Using single-molecule imaging, we demonstrate that a critical N-terminal motif of CTCF blocks cohesin translocation and DNA looping. The cryo-EM structure of the cohesin-CTCF complex reveals that this CTCF motif ahead of zinc fingers can only reach its binding site on the STAG1 cohesin subunit when the N terminus of CTCF faces cohesin. Remarkably, a C-terminally oriented CTCF accelerates DNA compaction by cohesin. DNA-bound Cas9 and Cas12a ribonucleoproteins are also polar cohesin barriers, indicating that stalling may be intrinsic to cohesin itself. Finally, we show that RNA-DNA hybrids (R-loops) block cohesin-mediated DNA compaction in vitro and are enriched with cohesin subunits in vivo, likely forming TAD boundaries.

Author Correction: Structure of human GABAB receptor in an inactive state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Scc2 Is a Potent Activator of Cohesin's ATPase that Promotes Loading by Binding Scc1 without Pds5.

Cohesin organizes DNA into chromatids, regulates enhancer-promoter interactions, and confers sister chromatid cohesion. Its association with chromosomes is regulated by hook-shaped HEAT repeat proteins that bind Scc1, namely Scc3, Pds5, and Scc2. Unlike Pds5, Scc2 is not a stable cohesin constituent but, as shown here, transiently replaces Pds5. Scc1 mutations that compromise its interaction with Scc2 adversely affect cohesin's ATPase activity and loading. Moreover, Scc2 mutations that alter how the ATPase responds to DNA abolish loading despite cohesin's initial association with loading sites. Lastly, Scc2 mutations that permit loading in the absence of Scc4 increase Scc2's association with chromosomal cohesin and reduce that of Pds5. We suggest that cohesin switches between two states: one with Pds5 bound that is unable to hydrolyze ATP efficiently but is capable of release from chromosomes and another in which Scc2 replaces Pds5 and stimulates ATP hydrolysis necessary for loading and translocation from loading sites.

The BUB3-BUB1 Complex Promotes Telomere DNA Replication.

Telomeres and telomere-binding proteins form complex secondary nucleoprotein structures that are critical for genome integrity but can present serious challenges during telomere DNA replication. It remains unclear how telomere replication stress is resolved during S phase. Here, we show that the BUB3-BUB1 complex, a component in spindle assembly checkpoint, binds to telomeres during S phase and promotes telomere DNA replication. Loss of the BUB3-BUB1 complex results in telomere replication defects, including fragile and shortened telomeres. We also demonstrate that the telomere-binding ability of BUB3 and kinase activity of BUB1 are indispensable to BUB3-BUB1 function at telomeres. TRF2 targets BUB1-BUB3 to telomeres, and BUB1 can directly phosphorylate TRF1 and promote TRF1 recruitment of BLM helicase to overcome replication stress. Our findings have uncovered previously unknown roles for the BUB3-BUB1 complex in S phase and shed light on how proteins from diverse pathways function coordinately to ensure proper telomere replication and maintenance.

Electroregulation of graphene-nanofluid interactions to coenhance water permeation and ion rejection in vertical graphene membranes.

Graphene oxide (GO) membranes with nanoconfined interlayer channels theoretically enable anomalous nanofluid transport for ultrahigh filtration performance. However, it is still a significant challenge for current GO laminar membranes to achieve ultrafast water permeation and high ion rejection simultaneously, because of the contradictory effect that exists between the water-membrane hydrogen-bond interaction and the ion-membrane electrostatic interaction. Here, we report a vertically aligned reduced GO (VARGO) membrane and propose an electropolarization strategy for regulating the interfacial hydrogen-bond and electrostatic interactions to concurrently enhance water permeation and ion rejection. The membrane with an electro-assistance of 2.5 V exhibited an ultrahigh water permeance of 684.9 L m-2 h-1 bar-1, which is 1-2 orders of magnitude higher than those of reported GO-based laminar membranes. Meanwhile, the rejection rate of the membrane for NaCl was as high as 88.7%, outperforming most reported graphene-based membranes (typically 10 to 50%). Molecular dynamics simulations and density-function theory calculations revealed that the electropolarized VARGO nanochannels induced the well-ordered arrangement of nanoconfined water molecules, increasing the water transport efficiency, and thereby resulting in improved water permeation. Moreover, the electropolarization effect enhanced the surface electron density of the VARGO nanochannels and reinforced the interfacial attractive interactions between the cations in water and the oxygen groups and π-electrons on the VARGO surface, strengthening the ion-partitioning and Donnan effect for the electrostatic exclusion of ions. This finding offers an electroregulation strategy for membranes to achieve both high water permeability and high ion rejection performance.

Serum Proteomics Identifies Biomarkers Associated With the Pathogenesis of Idiopathic Pulmonary Fibrosis.

The heterogeneity of idiopathic pulmonary fibrosis (IPF) limits its diagnosis and treatment. The association between the pathophysiological features and the serum protein signatures of IPF currently remains unclear. The present study analyzed the specific proteins and patterns associated with the clinical parameters of IPF based on a serum proteomic dataset by data-independent acquisition using MS. Differentiated proteins in sera distinguished patients with IPF into three subgroups in signal pathways and overall survival. Aging-associated signatures by weighted gene correlation network analysis coincidently provided clear and direct evidence that aging is a critical risk factor for IPF rather than a single biomarker. Expression of LDHA and CCT6A, which was associated with glucose metabolic reprogramming, was correlated with high serum lactic acid content in patients with IPF. Cross-model analysis and machine learning showed that a combinatorial biomarker accurately distinguished patients with IPF from healthy individuals with an area under the curve of 0.848 (95% CI = 0.684-0.941) and validated from another cohort and ELISA assay. This serum proteomic profile provides rigorous evidence that enables an understanding of the heterogeneity of IPF and protein alterations that could help in its diagnosis and treatment decisions.

Fluorinated Hafnium and Zirconium Coenable the Tunable Biodegradability of Core-Multishell Heterogeneous Nanocrystals for Bioimaging.

Upconversion (UC)/downconversion (DC)-luminescent lanthanide-doped nanocrystals (LDNCs) with near-infrared (NIR, 650-1700 nm) excitation have been gaining increasing popularity in bioimaging. However, conventional NIR-excited LDNCs cannot be degraded and eliminated eventually in vivo owing to intrinsic "rigid" lattices, thus constraining clinical applications. A biodegradability-tunable heterogeneous core-shell-shell luminescent LDNC of Na3HfF7:Yb,Er@Na3ZrF7:Yb,Er@CaF2:Yb,Zr (abbreviated as HZC) was developed and modified with oxidized sodium alginate (OSA) for multimode bioimaging. The dynamic "soft" lattice-Na3Hf(Zr)F7 host and the varying Zr4+ doping content in the outmoster CaF2 shell endowed HZC with tunable degradability. Through elaborated core-shell-shell coating, Yb3+/Er3+-coupled UC red and green and DC second near-infrared (NIR-II) emissions were, respectively, enhanced by 31.23-, 150.60-, and 19.42-fold when compared with core nanocrystals. HZC generated computed tomography (CT) imaging contrast effects, thus enabling NIR-II/CT/UC trimodal imaging. OSA modification not only ensured the exemplary biocompatibility of HZC but also enabled tumor-specific diagnosis. The findings would benefit the clinical imaging translation of LDNCs.

High-speed AFM imaging reveals DNA capture and loop extrusion dynamics by cohesin-NIPBL.

3D chromatin organization plays a critical role in regulating gene expression, DNA replication, recombination, and repair. While initially discovered for its role in sister chromatid cohesion, emerging evidence suggests that the cohesin complex (SMC1, SMC3, RAD21, and SA1/SA2), facilitated by NIPBL, mediates topologically associating domains and chromatin loops through DNA loop extrusion. However, information on how conformational changes of cohesin-NIPBL drive its loading onto DNA, initiation, and growth of DNA loops is still lacking. In this study, high-speed atomic force microscopy imaging reveals that cohesin-NIPBL captures DNA through arm extension, assisted by feet (shorter protrusions), and followed by transfer of DNA to its lower compartment (SMC heads, RAD21, SA1, and NIPBL). While binding at the lower compartment, arm extension leads to the capture of a second DNA segment and the initiation of a DNA loop that is independent of ATP hydrolysis. The feet are likely contributed by the C-terminal domains of SA1 and NIPBL and can transiently bind to DNA to facilitate the loading of the cohesin complex onto DNA. Furthermore, high-speed atomic force microscopy imaging reveals distinct forward and reverse DNA loop extrusion steps by cohesin-NIPBL. These results advance our understanding of cohesin by establishing direct experimental evidence for a multistep DNA-binding mechanism mediated by dynamic protein conformational changes.

Understanding heterogeneous mechanisms of heart failure with preserved ejection fraction through cardiorenal mathematical modeling.

In contrast to heart failure (HF) with reduced ejection fraction (HFrEF), effective interventions for HF with preserved ejection fraction (HFpEF) have proven elusive, in part because it is a heterogeneous syndrome with incompletely understood pathophysiology. This study utilized mathematical modeling to evaluate mechanisms distinguishing HFpEF and HFrEF. HF was defined as a state of chronically elevated left ventricle end diastolic pressure (LVEDP > 20mmHg). First, using a previously developed cardiorenal model, sensitivities of LVEDP to potential contributing mechanisms of HFpEF, including increased myocardial, arterial, or venous stiffness, slowed ventricular relaxation, reduced LV contractility, hypertension, or reduced venous capacitance, were evaluated. Elevated LV stiffness was identified as the most sensitive factor. Large LV stiffness increases alone, or milder increases combined with either decreased LV contractility, increased arterial stiffness, or hypertension, could increase LVEDP into the HF range without reducing EF. We then evaluated effects of these mechanisms on mechanical signals of cardiac outward remodeling, and tested the ability to maintain stable EF (as opposed to progressive EF decline) under two remodeling assumptions: LV passive stress-driven vs. strain-driven remodeling. While elevated LV stiffness increased LVEDP and LV wall stress, it mitigated wall strain rise for a given LVEDP. This suggests that if LV strain drives outward remodeling, a stiffer myocardium will experience less strain and less outward dilatation when additional factors such as impaired contractility, hypertension, or arterial stiffening exacerbate LVEDP, allowing EF to remain normal even at high filling pressures. Thus, HFpEF heterogeneity may result from a range of different pathologic mechanisms occurring in an already stiffened myocardium. Together, these simulations further support LV stiffening as a critical mechanism contributing to elevated cardiac filling pressures; support LV passive strain as the outward dilatation signal; offer an explanation for HFpEF heterogeneity; and provide a mechanistic explanation distinguishing between HFpEF and HFrEF.

Magic Acts with the Cohesin Ring.

Recent studies, including two in this issue of Molecular Cell (Elbatsh et al., 2016; Beckouët et al., 2016), cast light on how cohesin regulators harness the energy of ATP hydrolysis to open the cohesin ring and enable dynamic, regulated entrapment of chromosomes.

Structural Basis and IP6 Requirement for Pds5-Dependent Cohesin Dynamics.

The ring-shaped cohesin complex regulates transcription, DNA repair, and chromosome segregation by dynamically entrapping chromosomes to promote chromosome compaction and sister-chromatid cohesion. The cohesin ring needs to open and close to allow its loading to and release from chromosomes. Cohesin dynamics are controlled by the releasing factors Pds5 and Wapl and the cohesin stabilizer Sororin. Here, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Our structure establishes the basis for how Wapl and Sororin antagonistically influence cohesin dynamics. The structure further reveals that Pds5 can bind inositol hexakisphosphate (IP6). The IP6-binding segment of Pds5B is shaped like the jaw of a plier lever and inhibits the binding of Scc1 to Smc3. We propose that Pds5 stabilizes a transient, open state of cohesin to promote its release from chromosomes.

Highly Efficient Acidic Electrosynthesis of Hydrogen Peroxide at Industrial-Level Current Densities Promoted by Alkali Metal Cations.

Acidic H2O2 synthesis through electrocatalytic 2e- oxygen reduction presents a sustainable alternative to the energy-intensive anthraquinone oxidation technology. Nevertheless, acidic H2O2 electrosynthesis suffers from low H2O2 Faradaic efficiencies primarily due to the competing reactions of 4e- oxygen reduction to H2O and hydrogen evolution in environments with high H+ concentrations. Here, we demonstrate the significant effect of alkali metal cations, acting as competing ions with H+, in promoting acidic H2O2 electrosynthesis at industrial-level currents, resulting in an effective current densities of 50-421 mA cm-2 with 84-100 % Faradaic efficiency and a production rate of 856-7842 µmol cm-2 h-1 that far exceeds the performance observed in pure acidic electrolytes or low-current electrolysis. Finite-element simulations indicate that high interfacial pH near the electrode surface formed at high currents is crucial for activating the promotional effect of K+. In situ attenuated total reflection Fourier transform infrared spectroscopy and ab initio molecular dynamics simulations reveal the central role of alkali metal cations in stabilizing the key *OOH intermediate to suppress 4e- oxygen reduction through interacting with coordinated H2O.

Yueomyces silvicola sp. nov., a novel ascomycetous yeast species unable to utilize ammonium, glutamate, and glutamine as sole nitrogen sources.

Five yeast strains isolated from tree bark and rotten wood collected in central and southwestern China, together with four Brazilian strains (three from soil and rotting wood collected in an Amazonian rainforest biome and one from Bromeliad collected in Alagoas state) and one Costa Rican strain isolated from a flower beetle, represent a new species closely related with Yueomyces sinensis in Saccharomycetaceae, as revealed by the 26S ribosomal RNA gene D1/D2 domain and the internal transcribed spacer region sequence analysis. The name Yueomyces silvicola sp. nov. is proposed for this new species with the holotype China General Microbiological Culture Collection Center 2.6469 (= Japan Collection of Microorganisms 34885). The new species exhibits a whole-genome average nucleotide identity value of 77.8% with Y. sinensis. The two Yueomyces species shared unique physiological characteristics of being unable to utilize ammonium and the majority of the amino acids, including glutamate and glutamine, as sole nitrogen sources. Among the 20 amino acids tested, only leucine and tyrosine can be utilized by the Yueomyces species. Genome sequence comparison showed that GAT1, which encodes a GATA family protein participating in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae, is absent in the Yueomyces species. However, the failure of the Yueomyces species to utilize ammonium, glutamate, and glutamine, which are generally preferred nitrogen sources for microorganisms, implies that more complicated alterations in the central nitrogen metabolism pathway might occur in the genus Yueomyces.

Improving the Performance of the Lamellar Reduced Graphene Oxide/Molybdenum Sulfide Nanofiltration Membrane through Accelerated Water-Transport Channels and Capacitively Enhanced Charge Density.

Graphene is promising in the construction of next-generation nanofiltration membranes for wastewater treatment and water purification. However, the application of graphene-based membranes has still been prohibited by their deficiencies in permeability and ion rejection. Herein, regulating the 2D channel and enhancing the charge density are co-adopted for simultaneous enhancement of the water flux and salt rejection of reduced graphene oxide (rGO) membranes through the intercalation of molybdenum sulfide (MoS2) nanosheets and external electrical assistance. The fabricated rGO/MoS2 membranes possess expanded nanochannels with less friction and a higher water molecule transport velocity gradient (from 8.57 to 14.07 s-1) than those of rGO membranes. Consequently, their water permeance increases from 0.92 to 34.9 L m-2 h-1 bar-1. Meanwhile, benefiting from the high capacitance and negative potential of -1.1 V versus the saturated calomel electrode given to the membranes, their rejection rates toward NaCl reach 87.2% and those toward Na2SO4 reach 93.7%. The Donnan steric pore model analysis indicates that the capacitively and electrically increased surface charge density make great contributions to the higher ion rejection rate. This work gives new insights into membrane design for high water flux and salt rejection efficiency.

Highly Efficient Hydroxyl Radicals Production Boosted by the Atomically Dispersed Fe and Co Sites for Heterogeneous Electro-Fenton Oxidation.

The heterogeneous electro-Fenton (hetero-e-Fenton)-coupled electrocatalytic oxygen reduction reaction (ORR) is regarded as a promising strategy for ·OH production by simultaneously driving two-electron ORR toward H2O2 and stepped activating the as-generated H2O2 to ·OH. However, the high-efficiency electrogeneration of ·OH remains challengeable, as it is difficult to synchronously obtain efficient catalysis of both reaction steps above on one catalytic site. In this work, we propose a dual-atomic-site catalyst (CoFe DAC) to cooperatively catalyze ·OH electrogeneration, where the atomically dispersed Co sites are assigned to enhance O2 reduction to H2O2 intermediates and Fe sites are responsible for activation of the as-generated H2O2 to ·OH. The CoFe DAC delivers a higher ·OH production rate of 2.4 mmol L-1 min-1 gcat-1 than the single-site catalyst Co-NC (0.8 mmol L-1 min-1 gcat-1) and Fe-NC (1.0 mmol L-1 min-1 gcat-1). Significantly, the CoFe DAC hetero-e-Fenton process is demonstrated to be more energy-efficient for actual coking wastewater treatment with an energy consumption of 19.0 kWh kg-1 COD-1 than other electrochemical technologies that reported values of 29.7∼68.0 kW h kg-1 COD-1. This study shows the attractive advantages of efficiency and sustainability for ·OH electrogeneration, which should have fresh inspiration for the development of new-generation wastewater treatment technology.

Mitotic Transcription Installs Sgo1 at Centromeres to Coordinate Chromosome Segregation.

Human sister chromatids at metaphase are primarily linked by centromeric cohesion, forming the iconic X shape. Premature loss of centromeric cohesion disrupts orderly mitotic progression. Shugoshin (Sgo1) binds to and protects cohesin at inner centromeres. The kinetochore kinase Bub1 phosphorylates histone H2A at T120 (H2A-pT120) and recruits Sgo1 to kinetochores, 0.5 µm from inner centromeres. Here, we show that Sgo1 is a direct reader of the H2A-pT120 mark. Bub1 also recruits RNA polymerase II (Pol II) to unattached kinetochores and promotes active transcription at mitotic kinetochores. Mitosis-specific inactivation of Pol II traps Sgo1 at kinetochores and weakens centromeric cohesion. Sgo1 interacts with Pol II in human cells and with RNA in vitro. We propose that Pol II-dependent transcription enables kinetochore-bound Sgo1 initially recruited by H2A-pT120 to reach cohesin embedded in centromeric chromatin. Our study implicates mitotic transcription in targeting regulatory factors to highly compacted mitotic chromatin.

Responses of transcriptome and metabolome in the roots of Pugionium cornutum (L.) Gaertn to exogenously applied phthalic acid.

BACKGROUND: The yield and quality of Pugionium cornutum (L.) Gaertn., a healthy, green vegetable with low sugar and high protein contents and high medicinal value, is severely affected by autotoxicity, which is a leading factor in the formation of plant disease. To help characterize the autotoxicity mechanism of P. cornutum (L.) Gaertn., we performed transcriptomic and metabolic analysis of the roots of P. cornutum (L.) Gaertn. response to phthalic acid, an autotoxin from P. cornutum (L.) Gaertn. RESULTS: In this study, high-throughput sequencing of nine RNA-seq libraries generated from the roots.of P. cornutum (L.) Gaertn. under different phthalic acid treatments yielded 37,737 unigenes. In total, 1085 (703 upregulated and 382 downregulated) and 5998 (4385 upregulated and 1613 downregulated) DEGs were identified under 0.1 and 10 mmol·L- 1 phthalic acid treatment, respectively, compared with the control treatment. Glutathione metabolism was among the top five important enriched pathways. In total, 457 and 435 differentially accumulated metabolites were detected under 0.1 and 10 mmol·L- 1 phthalic acid treatment compared with the control, respectively, of which 223 and 253, respectively, increased in abundance. With the increase in phthalic acid concentration, the accumulation of ten metabolites increased significantly, while that of four metabolites decreased significantly, and phthalic acid, dambonitol, 4-hydroxy-butyric acid, homocitrulline, and ethyl ß-D-glucopyranoside were 100 times more abundant under the 10 mmol·L- 1 phthalic acid treatment than under the control. Seventeen differentially expressed genes significantly associated with phthalic acid content were identified. In addition, the L-histidinol content was highest under 0.1 mmol·L- 1 phthalic acid, and a total of eleven differentially expressed genes were significantly positively correlated with the L-histidinol content, all of which were annotated to heat shock proteins, aquaporins and cysteine proteases. CONCLUSIONS: Accumulation of autotoxins altered the metabolic balance in P. cornutum (L.) Gaertn. and influenced water absorption and carbon and nitrogen metabolism. These important results provide insights into the formation mechanisms of autotoxicity and for the subsequent development of new control measures to improve the production and quality of replanted plants.