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Most bacterial and all archaeal cells are encapsulated by a paracrystalline, protective, and cell-shape-determining proteinaceous surface layer (S-layer). On Gram-negative bacteria, S-layers are anchored to cells via lipopolysaccharide. Here, we report an electron cryomicroscopy structure of the Caulobacter crescentus S-layer bound to the O-antigen of lipopolysaccharide. Using native mass spectrometry and molecular dynamics simulations, we deduce the length of the O-antigen on cells and show how lipopolysaccharide binding and S-layer assembly is regulated by calcium. Finally, we present a near-atomic resolution in situ structure of the complete S-layer using cellular electron cryotomography, showing S-layer arrangement at the tip of the O-antigen. A complete atomic structure of the S-layer shows the power of cellular tomography for in situ structural biology and sheds light on a very abundant class of self-assembling molecules with important roles in prokaryotic physiology with marked potential for synthetic biology and surface-display applications.
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Proteínas da Membrana Bacteriana Externa/ultraestrutura , Caulobacter crescentus/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/ultraestrutura , Microscopia Crioeletrônica/métodos , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/metabolismo , Tomografia/métodosRESUMO
Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.
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Lisofosfolipídeos , Esfingosina , Humanos , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismoRESUMO
Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1Δ19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.
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Cloretos/metabolismo , Nucleotídeos/metabolismo , Potássio/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Tamanho Celular , Humanos , Fosforilação/fisiologia , Células Sf9 , Transdução de Sinais/fisiologia , Cotransportadores de K e Cl-RESUMO
The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides.
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Proteínas Oncogênicas Virais , Peptídeos , Animais , Humanos , Coelhos , Sequência de Aminoácidos , Peptídeos/genética , Epitopos de Linfócito B , Alinhamento de Sequência , Imunoglobulina G , Mapeamento de Epitopos/métodos , MamíferosRESUMO
Understanding stakeholders' perceptions about human well-being and ecosystem services is essential for designing efficient public policies and sustainable environmental management that help to improve people's quality of life. Despite the fragile ecosystem and poverty concentration in the agro-pastoral ecotone, research in this field remains scarce. We selected a typical agro-pastoral ecotone, Duolun County, Inner Mongolia, China, to explore how socioeconomic and demographic factors affect subjective well-being and perceptions of ecosystem services through structural equation modelling (SEM) and canonical correspondence analysis (CCA). Our results showed that health had the highest correlation with subjective well-being among the five dimensions, but respondents were least satisfied with it. Formal education had the greatest effect on subjective well-being, followed by age, income, and livelihood. Gender had no effect on subjective well-being. Older respondents with lower formal education who had a lower level of subjective well-being considered supporting and provisioning services more important for well-being. In contrast, younger respondents with higher education levels (mostly jobs not associated with working the land) mainly valued cultural services. Finally, we discussed the factors that influence subjective well-being and perceptions of ecosystem services and their implications for local management decision-making.
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Conservação dos Recursos Naturais , Ecossistema , Agricultura/métodos , China , Conservação dos Recursos Naturais/métodos , Humanos , Percepção , Qualidade de VidaRESUMO
BACKGROUND: To delineate the distribution and course layer of the perforator vessels using a modified technique. METHODS: Twelve perforator flaps were obtained from the crura of six fresh adult cadavers. The flaps were randomized into three groups (n = 4 per group): the full-thickness flap group; the deep fascia-free flap group, and the subcutaneous adipose layer-free group. The flaps were smoothened on a silk screen on a batten frame and the isolated flaps were perfused at a perfusion pressure of 140 mmHg for 10 min via the trunk of the posterior tibial artery. Perforator flaps were photographed using a digital camera and radiographed using a mammography device. The imaging data were processed by digital software system. RESULTS: The mean number of the posterior tibial artery perforator was 4.17 ± 0.94. The three relatively constant perforators varied in the projection points as well as the diameter and the length. The vascular branches and courses of the perforators were clearly visible on the mammograms. Elimination of all the deep fascia or the subcutaneous adipose tissues in the distal portion had no significant impact on the blood supply of posterior tibial artery perforator flaps while the vascular areas of the artery perforators were significantly reduced after the subcutaneous adipose tissue was eliminated in the proximal portion. CONCLUSIONS: We developed an effective modified technique for delineating the vascular territory on perforator flaps of different thicknesses. Our results provide significant guidance for clinical surgeons by providing them with more detailed anatomical knowledge of perforator flaps.
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Retalho Perfurante , Artérias da Tíbia/anatomia & histologia , Adulto , HumanosRESUMO
Antibodies targeting a single epitope of the cystic fibrosis transmembrane conductance regulator (CFTR) have been reported to influence the validity of immunological analyses; however, autoimmune mechanisms associated with CFTR epitopes are not well understood. In this study, antiserum raised against a multi-epitope recombinant protein composed of three peptide fragments of CFTR (r-CFTR-3P) was prepared and B cell epitope mapping of the protein was carried out using biosynthetic peptides. The r-CFTR-3P gene was cloned into the pSY621 expression plasmid and the protein was expressed in the BL21 strain of Escherichia coli. The rabbit r-CFTR-3P antiserum recognized the native CFTR antigen extracted from human sperm and the GST188 fusion peptides CFTR(25-36), CFTR(103-117), and CFTR(1387-1480) spanning different regions of CFTR. Four novel r-CFTR-3P B cell epitopes were identified: (29)RQRLEL(34), (104)RIIASY(109), (111)PDN(113), and (1447)VKLF(1450) of CFTR. Other proteins from various species shared sequence homology with the identified epitopes based on NCBI BLAST alignment. This study provides new tools for detecting CFTR protein and insight into the characteristics of minimal B cell epitopes of CFTR and associated immunological mechanisms.
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Regulador de Condutância Transmembrana em Fibrose Cística , Epitopos de Linfócito B , Fragmentos de Peptídeos , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Escherichia coli/genética , Humanos , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Espermatozoides/químicaRESUMO
The mammalian membrane is composed of various eukaryotic lipids interacting with extensively post-translationally modified proteins. Probing interactions between these mammalian membrane proteins and their diverse and heterogeneous lipid cohort remains challenging. Recently, native mass spectrometry (MS) combined with bottom-up 'omics' approaches has provided valuable information to relate structural and functional lipids to membrane protein assemblies in eukaryotic membranes. Here we provide a step-by-step protocol to identify and provide relative quantification for endogenous lipids bound to mammalian membrane proteins and their complexes. Using native MS to guide our lipidomics strategies, we describe the necessary sample preparation steps, followed by native MS data acquisition, tailored lipidomics and data interpretation. We also highlight considerations for the integration of different levels of information from native MS and lipidomics and how to deal with the various challenges that arise during the experiments. This protocol begins with the preparation of membrane proteins from mammalian cells and tissues for native MS. The results enable not only direct assessment of copurified endogenous lipids but also determination of the apparent affinities of specific lipids. Detailed sample preparation for lipidomics analysis is also covered, along with comprehensive settings for liquid chromatography-MS analysis. This protocol is suitable for the identification and quantification of endogenous lipids, including fatty acids, sterols, glycerolipids, phospholipids and glycolipids and can be used to interrogate proteins from recombinant sources to native membranes.
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Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.
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Cromatina/metabolismo , Metilação de DNA , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Cromatina/genética , Epigenômica , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/isolamento & purificação , Células HEK293 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/isolamento & purificação , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/isolamento & purificação , Humanos , Modelos Moleculares , S-Adenosilmetionina/biossíntese , S-Adenosilmetionina/químicaRESUMO
Azacytidine is an inhibitor of DNA methyltransferase and is known to be an anti-leukemic agent to induce cancer cell apoptosis. In the present study, multiple myeloma cells were treated with azacytidine at clinically relevant concentrations to induce necrosis through oxidative stress. Necrotic myeloma cells exhibit unique characteristics, including enrichment of the cell-bound albumin and overexpression of endoplasmic reticulum (ER)- and mitochondrial-specific chaperones, which were not observed in other necrotic cells, including HUH-7, A2780, A549, and Hoc1a. Proteomic analysis shows that HSP60 is the most abundant up-regulated mitochondrial specific chaperone, and azacytidine-induced overexpression of HSP60 is confirmed by western blot analysis. In contrast, expression levels of cytosolic chaperones such as HSP90 and HSP71 were down-regulated in azacytidine-treated myeloma cells, concomitant with an increase of these chaperones in the cell culture medium, suggesting that mitochondrial chaperones and cytosolic chaperones behave differently in necrotic myeloma cells; ER- and mitochondrial-chaperones being retained, and cytosolic chaperones being released into the cell culture medium through the ruptured cell membrane. Our data suggest that HSP60 is potentially a new target for multiple myeloma chemotherapy.
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For the ecologically vulnerable Qinghai-Tibet Plateau (QTP), hypoxia is increasingly becoming an extremely important environmental risk factor that significantly affects the health of both humans and livestock in the plateau region, as well as hindering high-quality development. To focus on the problem of hypoxia, it is especially urgent to study the surface oxygen concentration (i.e., oxygen concentration). However, the existing research is not sufficient, and there is a lack of oxygen concentration data collected on the QTP. In this study, through the Second Tibetan Plateau Scientific Expedition and Research and field measurements, the oxygen concentration data and corresponding geographic environmental data were collected at 807 measurement points on the QTP from 2017 to 2022, and the spatiotemporal oxygen concentration patterns were estimated. This work filled the gaps in the measurement and research of oxygen concentrations on the QTP while providing data support for analyses of the influencing factors and spatiotemporal characteristics of oxygen concentrations, which is of great significance for promoting the construction of ecological civilization in the QTP region.
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In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTBinh-14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters.
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Proteínas de Membrana Transportadoras , Ureia , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Sítios de Ligação , Ureia/farmacologia , Ureia/metabolismo , Transportadores de UreiaRESUMO
The human zona pellucida glycoprotein-3 (hZP3) by virtue of its critical role during fertilization has been proposed as a promising candidate antigen to develop a contraceptive vaccine. In this direction, it is imperative to map minimal motifs of the B cell epitopes (BCEs) so as to avoid ZP-specific oophoritogenic T cell epitopes (TCEs) in the ZP3-based immunogens. In this study, based on known results of mapping marmoset and bonnet monkey ZP3 (mstZP3 and bmZP3), two predictable epitopes(23-30 and 301-320) on hZP3 were first confirmed and five minimal motifs within four epitopes on hZP3 were defined using serum to recombinant hZP3a(22-176) or hZP3b(177-348) as well as a biosynthetic peptide strategy. These defined minimal motifs were QPLWLL(23-28) for hZP3(23-30), MQVTDD(103-108) for hZP3(93-110), EENW(178-181) for hZP3(172-190), as well as SNSWF(306-310) and EGP(313-315) for hZP3(301-320), respectively. Furthermore, the antigenicity of two peptides for hZP3(172-187) and hZP3(301-315) and specificity of the antibody response to these peptides were also evaluated, which produced high-titer antibodies in immunized animals that were capable of reacting to ZP on human oocytes, r-hZP3b(177-348) protein, as well as r-hZP3(172-190), r-hZP3(303-310), and r-hZP3(313-320) epitope peptides fused with truncated GST188 protein.
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Proteínas do Ovo/imunologia , Epitopos de Linfócito B/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Zona Pelúcida/metabolismo , Animais , Proteínas do Ovo/metabolismo , Mapeamento de Epitopos , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Coelhos , Receptores de Superfície Celular/metabolismo , Zona Pelúcida/imunologia , Glicoproteínas da Zona PelúcidaRESUMO
Lipids play important roles in regulating membrane protein function, but the molecular mechanisms used are elusive. Here we investigated how anionic lipids modulate SthK, a bacterial pacemaker channel homolog, and HCN2, whose activity contributes to pacemaking in the heart and brain. Using SthK allowed the reconstitution of purified channels in controlled lipid compositions for functional and structural assays that are not available for the eukaryotic channels. We identified anionic lipids bound tightly to SthK and their exact binding locations and determined that they potentiate channel activity. Cryo-EM structures in the most potentiating lipids revealed an open state and identified a nonannular lipid bound with its headgroup near an intersubunit salt bridge that clamps the intracellular channel gate shut. Breaking this conserved salt bridge abolished lipid modulation in SthK and eukaryotic HCN2 channels, indicating that anionic membrane lipids facilitate channel opening by destabilizing these interactions. Our findings underline the importance of state-dependent protein-lipid interactions.
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Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Lipídeos de Membrana , ÂnionsRESUMO
OBJECTIVES: This study aimed to use a machine-learning method to identify HTR1A/1B methylation and resting-state functional connectivity (rsFC) related to the diagnosis of MDD, then try to build classification models for MDD diagnosis based on the identified features. METHODS: Peripheral blood samples were collected from all recruited participants, and part of the participants underwent the resting-state fMRI scan. Features including HTR1A/1B methylation and rsFC were calculated. Then, the initial feature sets of epigenetics and neuroimaging were separately input into an all-relevant feature selection to generate significant discriminative power for MDD diagnosis. Random forest classifiers were constructed and evaluated based on identified features. In addition, the SHapley Additive exPlanations (SHAP) method was adapted to interpret the diagnostic model. RESULTS: A combination of selected HTR1A/1B methylation and rsFC feature sets achieved better performance than using either one alone - a distinction between MDD and healthy control groups was achieved at 81.78% classification accuracy and 0.8948 AUC. CONCLUSION: A high classification accuracy can be achieved by combining multidimensional information from epigenetics and cerebral radiomic features in MDD. Our approach can be helpful for accurate clinical diagnosis of MDD and further exploring the pathogenesis of MDD.
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Conectoma , Metilação de DNA , Transtorno Depressivo Maior , Receptor 5-HT1A de Serotonina , Receptor 5-HT1B de Serotonina , Humanos , Imageamento por Ressonância Magnética/métodos , Receptor 5-HT1A de Serotonina/genética , Epigênese Genética , Receptor 5-HT1B de Serotonina/genéticaRESUMO
The Ecosystem services (ESs), which play an important role in the balance of the natural ecosystem and social-economic development, are suffering from degradation caused by human activities and climate change. However, the manner in which the ESs respond to the land use/cover changes (LUCCs) and the climatic factors respectively remain elusive, especially in the forest-steppe ecotone, which is highly sensitive to climate change and anthroponotic activities. Based on the remote sensing data and in situ meteorological data, we comprehensively modeled and compared 4 key ESs changes caused by 3 LUCC types, land-use change fraction, and climate changes through two simple comparative experiments. Our results showed that: the Grain for the Green Project improved the mean soil conservation, carbon sequestration, and water yield but reduced the sand fixation. The cropland expansion had a positive influence on the water yield and sand fixation, but it induced a decline in soil conservation and carbon sequestration. The urbanization very likely increased the water yield and decreased soil conservation, carbon sequestration, and sand fixation. The unequal change fractions of the same land-use conversion may affect the ESs differently. The ESs changes have different responses to climate change in different landscapes due to the ecological process. The water yield could be well explained by the temperature, precipitation, radiation, and wind speed. Climate change had a stronger effect on the water yield and carbon sequestration than the land use/cover changes but sand fixation and soil conservation were more likely to be affected by LUCCs. The impact of three types of land-use changes and climate change on the ecosystem services should be considered when formulating land-use policies. This paper might aid the decision-makers in achieving ESs sustainable management and develop land-use strategies in the forest-steppe ecotone.
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BACKGROUND: Osteosarcoma is the most common bone cancer that significantly affects the quality of life of patients. Studies have shown that overexpression of BAIAP2L2 elevates the proliferation and growth of some types of cancer cells. However, the role of BAIAP2L2 in osteosarcoma is unclear. This study aimed to investigate the functions of BAIAP2L2 in the development of osteosarcoma. METHODS: We used immunohistochemical and Western blot analysis to determine the expression levels of endogenic BAIAP2L2 in osteosarcoma cells. Cell counting kit-8 assay and colony formation assay were performed to investigate cell proliferation of tumor cells. Transwell assay was performed to detect cell migration. Flow cytometry assay was used to analyze cell apoptosis. The role of BAIAP2L2 in tumor growth was further explored in vivo. RESULTS: We found that BAIAP2L2 was significantly upregulated in human osteosarcoma, and inhibition of BAIAP2L2 suppressed the proliferation of osteosarcoma cells. In addition, down-regulation of BAIAP2L2 could lead to osteosarcoma cancer cell apoptosis, inhibit cell migration and invasion, and induce the inactivation of the Wnt/ß-catenin pathway. In addition, down-regulation of BAIAP2L2 inhibited tumor growth in vivo. CONCLUSION: In conclusion, down-regulation of BAIAP2L2 inhibited the proliferation, migration, and invasion of osteosarcoma associated with the Wnt/ß-catenin pathway.
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Oxygen (O2) is the most abundant molecule in the atmosphere after nitrogen. Previous studies have documented that oxygen concentration remains nearly constant (20.946%) at all altitudes. Here we show for the first time that oxygen concentration varies significantly from earlier consensus and shows strong spatial and seasonal differences. Field observations on the Qinghai-Tibetan Plateau (QTP) indicate oxygen concentration of 19.94-20.66% (2018, n = 80), 19.98-20.78% (2019, n = 166) and 19.97-20.73% (2020, n = 176), all statistically different from earlier reports (p < 0.001) and are lower than the nearly constant. The mean oxygen concentration in summer (20.47%) is 0.31% higher than that of winter (20.16%) (n = 53) at identical locations in 2019, sampled in the Qilian Mountains, northwest QTP. We used LMG (The Lindeman, Merenda and Gold) method to estimate the relative contributions of altitude, air temperature and vegetation index (Fractional Vegetation Cover, FVC and Leaf Area Index, LAI) to oxygen concentration, which are 47%, 32% and 3% (FVC, R2 = 82%); 45%, 30% and 7% (LAI, R2 = 82%), respectively. These findings provide a new perspective for in-depth understanding on population risk in high altitude regions in the context of global climate change, to ensure the health and safety of residents and tourists in high altitude regions and promoting the stability, prosperity and sustainable development of high-altitude regions worldwide.
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Stakeholder perception and supply-demand relations are the main challenges and future directions for research on ecosystem services (ES). Based on spatial data and statistical data, we mapped eight key ES supply between 2005-2015 in the Huailai mountain-basin area. Using data from 507 survey questionnaires, we identified the ES demand and the public perceptions of the changes in ES. In addition, we also reveal the characteristics of the spatial distribution of ES demand, analyze the spatial supply-demand matching of ES, and explore the relationships between ES supply-demand and human well-being. From 2005 to 2015, a general upward trend was observed in the supply of four types of product provisioning services, which is different from the trend perceived by the stakeholders. An increasing trend was observed for carbon sequestration and forest recreation, which was in line with the perceptions of change. A spatial mismatch existed between the ES demand and supply, whereby the supply of carbon sequestration, soil conservation, habitat quality, and forest recreation services exceeded the demand in townships in the mountainous and hilly regions. On the other hand, townships located in the valley plains experienced a high imbalance between the demand and the supply. For the four types of product provisioning services, most towns and villages showed a balance in demand and supply. Linking ES supply-demand and human well-being showed that a threshold may exist in the supply-demand imbalance of regulating and supporting services before it begins to impact human well-being. Our study would enrich the theory and methodology research on relationships between ecosystem services and human well-being, and support knowledge to land allocation and management.
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Ecossistema , Sequestro de Carbono , China , Cidades , Conservação dos Recursos Naturais , HumanosRESUMO
Recent applications of mass spectrometry (MS) to study membrane protein complexes are yielding valuable insights into the binding of lipids and their structural and functional roles. To date, most native MS experiments with membrane proteins are based on detergent solubilization. Many insights into the structure and function of membrane proteins have been obtained using detergents; however, these can promote local lipid rearrangement and can cause fluctuations in the oligomeric state of protein complexes. To overcome these problems, we developed a method that does not use detergents or other chemicals. Here we report a detailed protocol that enables direct ejection of protein complexes from membranes for analysis by native MS. Briefly, lipid vesicles are prepared directly from membranes of different sources and subjected to sonication pulses. The resulting destabilized vesicles are concentrated, introduced into a mass spectrometer and ionized. The mass of the observed protein complexes is determined and this information, in conjunction with 'omics'-based strategies, is used to determine subunit stoichiometry as well as cofactor and lipid binding. Within this protocol, we expand the applications of the method to include peripheral membrane proteins of the S-layer and amyloid protein export machineries overexpressed in membranes from which the most abundant components have been removed. The described experimental procedure takes approximately 3 d from preparation to MS. The time required for data analysis depends on the complexity of the protein assemblies embedded in the membrane under investigation.