Long noncoding RNA MEG8 induces an imbalance of Th17/Treg cells through the miR-107/STAT3 axis in Henoch-Schonlein purpura rats.

T-helper (Th) 17/ T-regulatory (Treg) cell dysregulation underlies the pathogenesis of Henoch-Schonlein purpura (HSP). This research focused on the implication/s of the long noncoding RNA (lncRNAs) maternally expressed gene 8 (MEG8) in Th17 and Treg cell differentiation in HSP rats. MEG8, miR-107, signal transducer and activator of transcription-3 (STAT3), receptor-related orphan receptor γt (RORγt), and the transcription factor forkhead box P3 (Foxp3) expression levels were detected using real-time quantitative polymerase chain reaction and Western blot analyses. Flow cytometry was employed for measuring Th17 and Treg cells within the CD4+ T cell population. The interaction between miR-107 and MEG8 or STAT3 was examined. A low proportion of MEG8 and Treg cells together with Th17 cells were denoted within HSP rats. Moreover, MEG8 overexpression altered the Th17/Treg imbalance in peripheral blood CD4+ T-cell population, and the miR-107 mimic and STAT3 silencing reversed this effect. Thus, MEG8 served as a sponge for miR-107, lowering binding activity to STAT3 and thus overexpressing the molecule. Taken together, MEG8 induces an imbalance of Th17/Treg cells through the miR-107/STAT3 axis in HSP rats.

SubCellBarCode: integrated workflow for robust spatial proteomics by mass spectrometry.

The molecular functions of a protein are defined by its inherent properties in relation to its environment and interaction network. Within a cell, this environment and network are defined by the subcellular location of the protein. Consequently, it is crucial to know the localization of a protein to fully understand its functions. Recently, we have developed a mass spectrometry- (MS) and bioinformatics-based pipeline to generate a proteome-wide resource for protein subcellular localization across multiple human cancer cell lines ( www.subcellbarcode.org ). Here, we present a detailed wet-lab protocol spanning from subcellular fractionation to MS-sample preparation and analysis. A key feature of this protocol is that it includes all generated cell fractions without discarding any material during the fractionation process. We also describe the subsequent quantitative MS-data analysis, machine learning-based classification, differential localization analysis and visualization of the output. For broad applicability, we evaluated the pipeline by using MS data generated by two different peptide pre-fractionation approaches, namely high-resolution isoelectric focusing and high-pH reverse-phase fractionation, as well as direct analysis without pre-fractionation by using long-gradient liquid chromatography-MS. Moreover, an R package covering the dry-lab part of the method was developed and made available through Bioconductor. The method is straightforward and robust, and the entire protocol, from cell harvest to classification output, can be performed within 1-2 weeks. The protocol enables accurate classification of proteins to 15 compartments and 4 neighborhoods, visualization of the output data and differential localization analysis including treatment-induced protein relocalization, condition-dependent localization or cell type-specific localization. The SubCellBarCode package is freely available at https://bioconductor.org/packages/devel/bioc/html/SubCellBarCode.html .

Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms.

Despite major advancements in lung cancer treatment, long-term survival is still rare, and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune evasion mechanisms. Here we performed in-depth mass spectrometry (MS)-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition MS-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.

Transfection of STAT3 overexpression plasmid mediated through recombinant lentivirus promotes differentiation of bone marrow mesenchymal stem cells into neural cells in fetal rats with spina bifida aperta.

We investigated the influence of signal transducer and activator of transcription-3 (STAT3) on the spinal cord tissue grafts of rat fetuses with spina bifida aperta. In particular, we hoped to identify whether transfection of the STAT3 overexpression plasmid increases the survival of spinal cord transplantation in order to improve therapeutic efficacy. The fetal rat model of spina bifida aperta was established using retinoic acid and treated with a microsurgical injection of bone marrow mesenchymal stem cells (BMSCs). The animals were divided into either the blank control group, negative control group or the experimental group. The optical density (OD) value of BMSCs viability was determined using the Cell Counting Kit-8 (CCK-8). The expression of STAT3, phosphorylated STAT3 (pSTAT3), neural markers and apoptosis-related factors were evaluated using real-time PCR and Western blot. The OD value in the experimental group was highest at eight hours after transplantation using CCK-8. The expression of pSTAT3, glial fibrillary acidic protein, neuron-specific enolase, neurofilament and nestin in the experimental group was significantly higher compared to the blank control group and negative control group (P<0.05). However, STAT3 expression in the experimental group was statistically significantly decreased (P<0.05). The relative expression of caspase-8 and bcl-2 in the experimental group were significantly lower compared to the blank control group and negative control group (P<0.05). Transfection of the recombinant lentivirus-mediated STAT3 overexpression plasmid with BMSCs can help improve the efficiency of transforming into neural cells and provide new seed cells for the treatment of congenital spina bifida aperta.

Subcellular proteomics.

The eukaryotic cell is compartmentalized into subcellular niches, including membrane-bound and membrane-less organelles. Proteins localize to these niches to fulfil their function, enabling discreet biological processes to occur in synchrony. Dynamic movement of proteins between niches is essential for cellular processes such as signalling, growth, proliferation, motility and programmed cell death, and mutations causing aberrant protein localization are associated with a wide range of diseases. Determining the location of proteins in different cell states and cell types and how proteins relocalize following perturbation is important for understanding their functions, related cellular processes and pathologies associated with their mislocalization. In this Primer, we cover the major spatial proteomics methods for determining the location, distribution and abundance of proteins within subcellular structures. These technologies include fluorescent imaging, protein proximity labelling, organelle purification and cell-wide biochemical fractionation. We describe their workflows, data outputs and applications in exploring different cell biological scenarios, and discuss their main limitations. Finally, we describe emerging technologies and identify areas that require technological innovation to allow better characterization of the spatial proteome.

Fingerprinting the Ammonia Synthesis Pathway Using Spatiotemporal Electrostatic Potential Distribution of Intermediates.

It remains a research challenge in determining the catalytic reaction mechanisms primarily caused by the difficulty to experimentally identify active intermediates with current analytic characterizations. Although computational chemistry has provided an alternative approach to simulate the catalysis process and achieve insights into the reaction pathways, the simulation results would not be conclusive without experimental evidence. Herein, we investigate spatiotemporal electrostatic potential (ESP) distribution surrounding reacting molecules during the catalysis process and suggest its use as a fingerprint to help differentiate and identify active intermediates. Our ESP study of ammonia synthesis on the Ru surface shows a high spatial sensitivity of ESP distribution to molecular configuration and structure of intermediate species and only minor temporal ESP oscillation throughout the lifetime of the intermediates, which provides strong theoretical support to use ESP distribution as a new approach to characterize intermediates. With the ESP measurements at the microscale and in real-time, turning feasible, experimental identification of active intermediates and determination of reaction pathways would become possible by measuring the ESP surrounding the reacting molecules. We suggest developing ESP measurement tools to experimentally explore and unveil reaction mechanisms.

Designing Champion Nanostructures of Tungsten Dichalcogenides for Electrocatalytic Hydrogen Evolution.

Fine-tuning strain and vacancies in 2H-phase transition-metal dichalcogenides, although extremely challenging, is crucial for activating the inert basal plane for boosting the hydrogen evolution reaction (HER). Here, atomically curved 2H-WS2 nanosheets with precisely tunable strain and sulfur vacancies (S-vacancies) along with rich edge sites are synthesized via a one-step approach by harnessing geometric constraints. The approach is based on the confined epitaxy growth of WS2 in ordered mesoporous graphene derived from nanocrystal superlattices. The spherical curvature imposed by the graphitic mesopores enables the generation of uniform strain and S-vacancies in the as-grown WS2 nanosheets, and simultaneous manipulation of these two key parameters can be realized by simply adjusting the pore size. In addition, the formation of unique mesoporous WS2 @graphene van der Waals heterostructures ensures the ready access of active sites. Fine-tuning the WS2 layer number, strain, and S-vacancies enables arguably the best-performing HER 2H-WS2 electrocatalysts ever reported. Density functional theory calculations indicate that compared with strain, S-vacancies play a more critical role in enhancing the HER activity.

A vacuum impregnation method for synthesizing octahedral Pt2CuNi nanoparticles on mesoporous carbon support and the oxygen reduction reaction electrocatalytic properties.

Mesoporous carbon (MPC) nanomaterials, with large specific surface area, excellent conductivity and stability, and effective mass transfer are beneficial for use as catalyst support in electrochemical oxygen reduction reaction (ORR) for fuel cell applications. However, MPC utilization was limited by difficulties in loading catalyst nanoparticles within the MPC pores while simultaneously controlling critical particle parameters such as size and distribution. In this study we report a new vacuum impregnation method combined with solid-state chemistry synthesis for preparing highly active ORR catalyst nanoparticles on MPC supports. We confirm the effectiveness of this method by synthesizing octahedral Pt2CuNi nanoparticles on hydrophilic MPC with an even particle distribution in the MPC pores. We also demonstrate the capability of this method in controlling the particle size and morphology by adjusting the synthesis parameters. The synthesized catalysts exhibited excellent ORR activity and promising durability, which proves the goodness of using MPC support in ORR electrocatalysis. The findings offer a new methodology for synthesizing nanoparticles in MPC pores with parameter control and provide an intriguing strategy to develop new ORR catalysts using MPC support structure.

Properties of amorphous iron phosphate in pseudocapacitive sodium ion removal for water desalination.

Capacitive deionization (CDI) is an energy saving and environmentally friendly technology for water desalination. However, classical CDI is challenged by a low salt removal capacity. To improve the desalination capacity, electrode materials utilizing the battery mechanism for salt ion removal have emerged as a new direction more recently. In this work, we report a study of amorphous iron phosphate (FePO4) as a promising electrode material for pseudocapacitive sodium ion removal. Sodium ions can be effectively, reversibly intercalated and de-intercalated upon its electrochemical reduction and oxidation, with an excellent sodium ion capacity under half-cell testing conditions. By assembling a hybrid CDI (HCDI) system utilizing the FePO4 electrode for pseudocapacitive sodium ion removal and active carbon electrode for capacitive chloride ion removal, the cell exhibited a high salt removal capacity and good reversibility and durability, which was attributed to the advantageous features of amorphous FePO4. The HCDI system achieved a high deionization capacity (82 mg g-1) in 10 mM NaCl, a fast deionization rate (0.046 mg g-1 s-1), and good stability and cyclability.

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET.

Designing Highly Efficient and Long-Term Durable Electrocatalyst for Oxygen Evolution by Coupling B and P into Amorphous Porous NiFe-Based Material.

Oxygen evolution reaction (OER) is of great significance for hydrogen production via water electrolysis, which, however, demands development of highly active, durable, and cost-effective electrocatalysts in order to stride into a renewable energy era. Herein, highly efficient and long-term durable OER by coupling B and P into an amorphous porous NiFe-based electrocatalyst is reported, which possesses an amorphous porous metallic bulk structure and high corrosion resistance, and overcomes the issues associated with currently used catalyst nanomaterials. The PB codoping in the activated NiFePB (a-NiFePB) delocalizes both Fe and Ni at Fermi energy level and enhances p-d hybridization as simulated by density functional theory calculations. The harmonized electronic structure and unique porous framework of the a-NiFePB consequently improve the OER activity. The activated NiFePB thus exhibits an extraordinarily low overpotential of 197 mV for harvesting 10 mA cm-2 OER current density and 233 mV for reaching 100 mA cm-2 under chronopotentiometry condition, with the Tafel slope harmoniously conforming to 34 mV dec-1 . Impressive long-term stability of this new catalyst is evidenced by only limited activity decay after 1400 h operation at 100 mA cm-2 . This work strategically directs a way for heading up a promising energy conversion alternative.

Dual-Site Cascade Oxygen Reduction Mechanism on SnO x/Pt-Cu-Ni for Promoting Reaction Kinetics.

Designing highly active oxygen reduction reaction (ORR) catalysts is crucial to boost the fuel cell economy. Previous research has mainly focused on Pt-based alloy catalysts in which surface Pt is the solely active site and the activity improvement was challenged by the discovered scaling relationship. Herein we report a new concept of utilizing dual active sites for the ORR and demonstrate its effectiveness by synthesizing a SnO x/Pt-Cu-Ni heterojunctioned catalyst. A maximum of 40% enhancement in the apparent specific activity, which corresponds to 10-fold enhancement on interface sites, is measured compared with pure Pt-Cu-Ni. Detailed investigations suggest an altered dual-site cascade mechanism wherein the first two steps occur on SnO x sites and the remaining steps occur on adjacent Pt sites, allowing a significant decrease in the energy barrier. This study with the suggested dual-site cascade mechanism shows the potential to overcome the ORR energy barrier bottleneck to develop highly active catalysts.

SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization.

Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.

Deconvolution of octahedral Pt3Ni nanoparticle growth pathway from in situ characterizations.

Understanding the growth pathway of faceted alloy nanoparticles at the atomic level is crucial to morphology control and property tuning. Yet, it remains a challenge due to complexity of the growth process and technical limits of modern characterization tools. We report a combinational use of multiple cutting-edge in situ techniques to study the growth process of octahedral Pt3Ni nanoparticles, which reveal the particle growth and facet formation mechanisms. Our studies confirm the formation of octahedral Pt3Ni initiates from Pt nuclei generation, which is followed by continuous Pt reduction that simultaneously catalyzes Ni reduction, resulting in mixed alloy formation with moderate elemental segregation. Carbon monoxide molecules serve as a facet formation modulator and induce Ni segregation to the surface, which inhibits the (111) facet growth and causes the particle shape to evolve from a spherical cluster to an octahedron as the (001) facet continues to grow.

More accurate depiction of adsorption energy on transition metals using work function as one additional descriptor.

The reaction mechanism and properties of a catalytic process are primarily determined by the interactions between reacting species and catalysts. However, the interactions are often challenging to be experimentally measured, especially for unstable intermediates. Therefore, it is of significant importance to establish an exact relationship between chemical-catalyst interactions and catalyst parameters, which will allow calculation of these interactions and thus advance their mechanistic understanding. Herein we report the description of adsorption energy on transition metals by considering both ionic bonding and covalent bonding contributions and introduce the work function as one additional responsible parameter. We find that the adsorption energy can be more accurately described using a two-dimensional (2D) polynomial model, which shows a significant improvement compared with the current adsorption energy-d-band center linear correlation. We also demonstrate the utilization of this new 2D polynomial model to calculate oxygen binding energy of different transition metals to help understand their catalytic properties in oxygen reduction reactions.

In vivo protein allylation to capture protein methylation candidates.

An approach combining in vivo protein allylation, chemical tagging and affinity enrichment was devised to capture protein methylation candidates in yeast S. cerevisiae. The study identified 167 hits, covering many proteins with known methylation events on different types of amino acid residues.

Specific Enrichment of Peptides with N-Terminal Serine/Threonine by a Solid-Phase Capture-Release Approach for Efficient Proteomics Analysis.

A problem for "shot-gun" proteomics is that the peptides generated in the proteolysis step overwhelm the analytical capacity of current LC-MS/MS systems. A straightforward approach to overcome this problem is to reduce the sample complexity by isolating the representative peptides of each protein. In this study, we presented a facile solid-phase capture-release approach to selectively enrich the peptides with N-terminal serine/threonine from protein digests. This method exploited the highly efficient reaction between an aldehyde group and a hydrazine group. The excellent performance of this approach was validated using synthetic peptides as well as complex protein digests. It was found that high enrichment specificity could be obtained and the identifications for complex samples with and without enrichment were highly complementary. Besides, the enrichment of peptides with serine/threonine adjacent to different protease cleavage sites demonstrated that our method was able to enrich peptides from protein digests in a sequence specific way. As a result, this new approach provides a simple way to reduce sample complexity and facilitates the identification of low-abundance proteins.

The proteomic analysis improved by cleavage kinetics-based fractionation of tryptic peptides.

Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi-stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi-stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

[Effect of the lysine guanidination on proteomic analysis].

The guanidination of lysine side chain was paid great attention in recent years. It plays an important role in qualitative and quantitative proteomics. In this study, based on the results of separated peptides extracted from HeLa cells before and after the guanidination by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the effect of the guanidination of three different kinds of peptides was systematically analyzed. It was found that the selectivity of the guanidination of the lysine side chain was as high as 96.8%. The ratio of identified peptides with lysine at C-term to all peptides increased from 51.7% to 57.3% and more new peptides were identified, while the ratio of peptides with lysine in the middle or without lysine changed little. Further study on the ratio of b and y ions indicated that there were more y ions of peptides with lysine at C-term after the guanidination. The results proved that the selective conversion of lysine to homoarginine by the guanidination could increase the sensitivity and selectivity of mass spectrum. The increased basicity and ability to sequester proton of lysine produced more y ions fragmentation information, which contributed to more identified peptides. It concluded that the lysine guanidination can improve the coverage of proteomic analysis.