Epitope delimitation: A new method for defining epitopes of human IgG-reactive antigenic peptides based on rabbit-recognized epitope motifs.

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.

Surface oxygen concentration on the Qinghai-Tibet Plateau (2017-2022).

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.

Structural characterization of human urea transporters UT-A and UT-B and their inhibition.

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.

The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate.

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.

Anionic lipids unlock the gates of select ion channels in the pacemaker family.

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.

Combined HTR1A/1B methylation and human functional connectome to recognize patients with MDD.

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.

Exploring subjective well-being and ecosystem services perception in the agro-pastoral ecotone of northern China.

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.

BAIAP2L2 promotes the proliferation, migration and invasion of osteosarcoma associated with the Wnt/ß-catenin pathway.

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.

Factors contributing to spatial-temporal variations of observed oxygen concentration over the Qinghai-Tibetan Plateau.

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.

Phospho-regulation, nucleotide binding and ion access control in potassium-chloride cotransporters.

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.

Climate change versus land-use change-What affects the ecosystem services more in the forest-steppe ecotone?

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.

Exploring Connections among Ecosystem Services Supply, Demand and Human Well-Being in a Mountain-Basin System, China.

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.

The use of sonicated lipid vesicles for mass spectrometry of membrane protein complexes.

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.

In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer.

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.

HSP60 silencing promotes Warburg-like phenotypes and switches the mitochondrial function from ATP production to biosynthesis in ccRCC cells.

HSP60 is a major mitochondrial chaperone for maintaining mitochondrial proteostasis. Our previous studies showed that HSP60 was significantly downregulated in clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer characterized by the classic Warburg effect. Here, we analyzed datasets in The Cancer Genome Atlas and revealed that higher HSP60 expression correlated with better overall survival in ccRCC patients. We also stably knocked down or overexpressed HSP60 in ccRCC cells to investigate the effects of HSP60 expression on the transition between oxidative phosphorylation and glycolysis. We confirmed that HSP60 knockdown increased cell proliferation, whereas its overexpression decreased cell growth. Proteomics and metabolomics revealed that HSP60 knockdown promoted Warburg-like phenotypes with enhanced glycolysis and decreased mitochondrial activity. Consistent with this finding, isotope tracing showed that the metabolic flow from glycolysis to TCA was reduced. However, HSP60 silencing enhanced mitochondrial functions in glutamine-directed biosynthesis with increased flow in two parts of the TCA cycle: Gln→αKG→OAA→Asp and Gln→αKG→ISO→acetyl-CoA, resulting in elevated de novo nucleotide synthesis and lipid synthesis. Proteomic analysis indicated that HSP60 silencing activated NRF2-mediated oxidative stress responses, while glutamate generated from glutamine increased glutathione synthesis for quenching excessive reactive oxygen species (ROS) produced upon elevated cell growth. We further found that HSP60 silencing activated the MEK/ERK/c-Myc axis to promote glutamine addiction, and confirmed that ccRCC cells were susceptible to oxidative stress and glutaminase inhibition. Collectively, our data show that HSP60 knockdown drives metabolic reprogramming in ccRCC to promote tumor progression and enhances mitochondrial-dependent biosynthesis.

Isotope tracing assisted metabolic profiling: Application to understanding HSP60 silencing mediated tumor progression.

Isotope-tracing facilitates the understanding of metabolic regulation in biological systems. Depending on the selection of tracers, some essential metabolites cannot be traced. A comprehensive understanding of the regulated pathways can only be achieved with focus beyond labeled metabolites. The isotope tracing assisted metabolic profiling described here is a platform for high throughput mapping of isotope labeled metabolites with simultaneous metabolomics profiling. This approach incorporates an in-house MS/MS library for metabolite identification and ID-based quantitation. An "Isotopic" software was developed to generate potential labeled isotopomers. Using this platform, a total of 394 metabolites were reliably identified based on MS/MS confirmation in 3 million 293 T cells, among which 54 and 43 metabolites were discovered to carry extensive labels (>2%) from 13C6-glucose and 13C5-glutamine respectively. Citrate flowing into malate shuttle was also observed. More interestingly, the rate-limiting step in NAD and UDP-GlcNAc biosynthesis was clearly observed according to time course labeling. In HSP60 knockdown cell lines, enhanced purine and pyrimidine biosynthesis were confirmed by the abundance and labeling percentages of intermediate metabolites.

Responses of soil respiration to soil management changes in an agropastoral ecotone in Inner Mongolia, China.

Studying the responses of soil respiration (Rs) to soil management changes is critical for enhancing our understanding of the global carbon cycle and has practical implications for grassland management. Therefore, the objectives of this study were (1) quantify daily and seasonal patterns of Rs, (2) evaluate the influence of abiotic factors on Rs, and (3) detect the effects of soil management changes on Rs. We hypothesized that (1) most of daily and seasonal variation in Rs could be explained by soil temperature (Ts) and soil water content (Sw), (2) soil management changes could significantly affect Rs, and (3) soil management changes affected Rs via the significant change in abiotic and biotic factors. In situ Rs values were monitored in an agropastoral ecotone in Inner Mongolia, China, during the growing seasons in 2009 (August to October) and 2010 (May to October). The soil management changes sequences included free grazing grassland (FG), cropland (CL), grazing enclosure grassland (GE), and abandoned cultivated grassland (AC). During the growing season in 2010, cumulative Rs for FG, CL, GE, and AC averaged 265.97, 344.74, 236.70, and 226.42 gC m-2 year-1, respectively. The Ts and Sw significantly influenced Rs and explained 66%-86% of the variability in daily Rs. Monthly mean temperature and precipitation explained 78%-96% of the variability in monthly Rs. The results clearly showed that Rs was increased by 29% with the conversion of FG to CL and decreased by 35% and 11% with the conversion of CL to AC and FG to GE. The factors impacting the change in Rs under different soil management changes sequences varied. Our results confirm the tested hypotheses. The increase in Q10 and litter biomass induced by conversion of FG to GE could lead to increased Rs if the climate warming. We suggest that after proper natural restoration period, grasslands should be utilized properly to decrease Rs.

A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping.

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.

Epitomics: IgG-epitome decoding of E6, E7 and L1 proteins from oncogenic human papillomavirus type 58.

To enable rational multi-epitope vaccine and diagnostic antigen design, it is imperative to delineate complete IgG-epitome of the protein. Here, we describe results of IgG-epitome decoding of three proteins from high-risk (HR-) oncogenic human papillomavirus type 58 (HPV58). To reveal their entire epitomes, employing peptide biosynthetic approach, 30 precise linear B-cell epitopes (BCEs) were mapped on E6, E7 and L1 proteins using rabbits antisera to the respective recombinant proteins. Using sequence alignment based on BCE minimal motif, the specificity and conservativeness of each mapped BCE were delineated mainly among known HR-HPVs, including finding 3 broadly antibody cross-reactive BCEs of L1 that each covers almost all HR-HPVs. Western blots revealed that 13 of the 18 BCEs within L1-epitome were recognized by murine antisera to HPV58 virus-like particles, suggesting that these are antibody accessible BCEs. Also, a highly conserved epitope (YGD/XTL) of E6 was found to exist only in known common HR-HPVs, which could be used as the first peptide reference marker for judging HR-HPVs. Altogether, this study provides systemic and exhaustive information on linear BCEs of HR-HPV58 that will facilitate development of novel multi-epitope diagnostic reagents/chips for testing viral antibodies and 'universal' preventive HPV peptide vaccine based on L1 conserved BCEs.