A Non-canonical PDK1-RSK Signal Diminishes Pro-caspase-8-Mediated Necroptosis Blockade.

Necroptosis induction in vitro often requires caspase-8 (Casp8) inhibition by zVAD because pro-Casp8 cleaves RIP1 to disintegrate the necrosome. It has been unclear how the Casp8 blockade of necroptosis is eliminated naturally. Here, we show that pro-Casp8 within the necrosome can be inactivated by phosphorylation at Thr265 (pC8T265). pC8T265 occurs in vitro in various necroptotic cells and in the cecum of TNF-treated mice. p90 RSK is the kinase of pro-Casp8. It is activated by a mechanism that does not need ERK but PDK1, which is recruited to the RIP1-RIP3-MLKL-containing necrosome. Phosphorylation of pro-Casp8 at Thr265 can substitute for zVAD to permit necroptosis in vitro. pC8T265 mimic T265E knockin mice are embryonic lethal due to unconstrained necroptosis, and the pharmaceutical inhibition of RSK-mediated pC8T265 diminishes TNF-induced cecum damage and lethality in mice by halting necroptosis. Thus, phosphorylation of pro-Casp8 at Thr265 by RSK is an intrinsic mechanism for passing the Casp8 checkpoint of necroptosis.

TCR-induced sumoylation of the kinase PKC-θ controls T cell synapse organization and T cell activation.

Sumoylation regulates many cellular processes, but its role in signaling via the T cell antigen receptor (TCR) remains unknown. We found that the kinase PKC-θ was sumoylated upon costimulation with antigen or via the TCR plus the coreceptor CD28, with Lys325 and Lys506 being the main sumoylation sites. We identified the SUMO E3 ligase PIASxß as a ligase for PKC-θ. Analysis of primary mouse and human T cells revealed that sumoylation of PKC-θ was essential for T cell activation. Desumoylation did not affect the catalytic activity of PKC-θ but inhibited the association of CD28 with PKC-θ and filamin A and impaired the assembly of a mature immunological synapse and central co-accumulation of PKC-θ and CD28. Our findings demonstrate that sumoylation controls TCR-proximal signaling and that sumoylation of PKC-θ is essential for the formation of a mature immunological synapse and T cell activation.

Fine mapping of TFL, a major gene regulating fruit length in snake gourd (Trichosanthes anguina L).

Fruit length is a crucial agronomic trait of snake gourd (Trichosanthes anguina L); however, genes associated with fruit length have not been characterised. In this study, F2 snake gourd populations were generated by crossing the inbred lines, S1 and S2 (fruit lengths: 110 and 20 cm, respectively). Subsequently, bulk segregant analysis, sequencing, and fine-mapping were performed on the F2 population to identify target genes. Our findings suggest that the fruit length of snake gourd is regulated by a major-effect regulatory gene. Mining of genes regulating fruit length in snake gourd to provide a basis for subsequent selection and breeding of new varieties. Genotype-phenotype association analysis was performed on the segregating F2 population comprising 6,000 plants; the results indicate that the target gene is located on Chr4 (61,846,126-61,865,087 bp, 18.9-kb interval), which only carries the annotated candidate gene, Tan0010544 (designated TFL). TFL belongs to the MADS-box family, one of the largest transcription factor families. Sequence analysis revealed a non-synonymous mutation of base C to G at position 202 in the coding sequence of TFL, resulting in the substitution of amino acid Gln to Glu at position 68 in the protein sequence. Subsequently, an InDel marker was developed to aid the marker-assisted selection of TFL. The TFL in the expression parents within the same period was analysed using quantitative real-time PCR; the TFL expression was significantly higher in short fruits than long fruits. Therefore, TFL can be a candidate gene for determining the fruit length in snake gourd. Collectively, these findings improve our understanding of the genetic components associated with fruit length in snake gourds, which could aid the development of enhanced breeding strategies for plant species.

Quantitative proteomic analysis of exosomes from umbilical cord mesenchymal stem cells and rat bone marrow stem cells.

Exosomes derived from mesenchymal stem cells (MSCs) have been used for cancer treatment, however, an in-depth analysis of the exosomal proteomes is lacking. In this manuscript, we use the diaPASEF (parallel accumulation serial fragmentation combined with the data-independent acquisition) method to quantify exosomes derived from human umbilical cord mesenchymal stem cells (UCMSCs) and rat bone marrow stem cells (BMSCs), resulting in identification of 4200 human proteins and 5362 rat proteins. Comparison of human exosomal proteins and total cellular proteins reveals that some proteins exist in the exosomes exclusively that can be served as potential markers for exosomes. Quantitative proteomic analysis of exosomes from different passages of BMSCs shows that the proteins involved in TGF-ß signaling pathway are regulated in abundance, which could be markers for the therapeutic ability of BMSC exosomes. Collectively, the data presented by this study can be a resource for further study of exosome research.

Evaluation of DDA Library-Free Strategies for Phosphoproteomics and Ubiquitinomics Data-Independent Acquisition Data.

Phosphoproteomics and ubiquitinomics data-independent acquisition (DIA) mass spectrometry (MS) data is typically analyzed by using a data-dependent acquisition (DDA) spectral library. The performance of various library-free strategies for analyzing phosphoproteomics and ubiquitinomics DIA MS data has not been evaluated. In this study, we systematically compare four commonly used DDA library-free approaches including Spectronaut's directDIA, DIA-Umpire, DIA-MSFragger, and in silico-predicted library for analysis of phosphoproteomics SWATH, DIA, and diaPASEF data as well as ubiquitinomics diaPASEF data. Spectronaut's directDIA shows the highest sensitivity for phosphopeptide detection not only in synthetic phosphopeptide samples but also in phosphoproteomics SWATH-MS and DIA data from real biological samples, when compared to the other three library-free strategies. For phosphoproteomics diaPASEF data, Spectronaut's directDIA and the in silico-predicted library based on DIA-NN identify almost the same number of phosphopeptides as a project-specific DDA spectral library. However, only about 30% of the total phosphopeptides are commonly identified, suggesting that the library-free strategies for phospho-diaPASEF data need further improvement in terms of sensitivity. For ubiquitinomics diaPASEF data, the in silico-predicted library performs the best among the four workflows and detects ∼50% more K-GG peptides than a project-specific DDA spectral library. Our results demonstrate that Spectronaut's directDIA is suitable for the analysis of phosphoproteomics SWATH-MS and DIA MS data, while the in silico-predicted library based on DIA-NN shows substantial advantages for ubiquitinomics diaPASEF MS data.

Programmed necrosis: backup to and competitor with apoptosis in the immune system.

Programmed cell death is essential for the development and maintenance of the immune system and its responses to exogenous and endogenous stimuli. Studies have demonstrated that in addition to caspase-dependent apoptosis, necrosis dependent on the kinases RIP1 and RIP3 (also called necroptosis) is a major programmed cell-death pathway in development and immunity. These two programmed cell-death pathways may suppress each other, and necroptosis also serves as an alternative when caspase-dependent apoptosis is inhibited or absent. Here we summarize recent advancements that have identified the molecular mechanisms that underlie necroptosis and explore the mechanisms that regulate the interplay between apoptosis and necroptosis.

Density functional theory based computational investigations on the stability of highly active trimetallic PtPdCu nanoalloys for electrochemical oxygen reduction.

Activity, cost, and durability are the trinity of catalysis research for the electrochemical oxygen reduction reaction (ORR). While studies towards increasing activity and reducing cost of ORR catalysts have been carried out extensively, much effort is needed in durability investigation of highly active ORR catalysts. In this work, we examined the stability of a trimetallic PtPdCu catalyst that has demonstrated high activity and incredible durability during ORR using density functional theory (DFT) based computations. Specifically, we studied the processes of dissolution/deposition and diffusion between the surface and inner layer of Cu species of Pt20Pd20Cu60 catalysts at electrode potentials up to 1.2 V to understand their role towards stabilizing Pt20Pd20Cu60 catalysts. The results show there is a dynamic Cu surface composition range that is dictated by the interplay of the four processes, dissolution, deposition, diffusion from the surface to inner layer, and diffusion from the inner layer to the surface of Cu species, in the stability and observed oscillation of lattice constants of Cu-rich PtPdCu nanoalloys.

SCASP: A Simple and Robust SDS-Aided Sample Preparation Method for Proteomic Research.

SDS is widely used in sample preparation for proteomic research. However, SDS is incompatible with LC and electrospray ionization. SDS depletion is therefore required ahead of LC-MS analysis. Most of current SDS removal strategies are time consuming, laborious, and have low reproducibility. Here, we describe a method, SDS-cyclodextrin (CD)-assisted sample preparation, by which CD can bind to SDS and form CD-SDS complexes in solutions, allowing for direct tryptic digestion. We demonstrate that SDS-CD-assisted sample preparation is a simple, fast, and robust SDS-based sample preparation method for proteomics application.

Fine Mapping and Identification of SmAPRR2 Regulating Rind Color in Eggplant (Solanum melongena L.).

Rind color is an economically important agronomic trait in eggplant that impacts consumer preferences. In this study, bulked segregant analysis and competitive allele-specific PCR were employed to identify the candidate gene for eggplant rind color through constructing a 2794 F2 population generated from a cross between "BL01" (green pericarp) and "B1" (white pericarp). Genetic analysis of rind color revealed that a single dominant gene controls green color of eggplant peel. Pigment content measurement and cytological observations demonstrated that chlorophyll content and chloroplast number in BL01 were higher than in B1. A candidate gene (EGP19168.1) was fine-mapped to a 20.36 Kb interval on chromosome 8, which was predicted to encode the two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2). Subsequently, allelic sequence analysis revealed that a SNP deletion (ACT→AT) in white-skinned eggplant led to a premature termination codon. Genotypic validation of 113 breeding lines using the Indel marker closely linked to SmAPRR2 could predict the skin color (green/white) trait with an accuracy of 92.9%. This study will be valuable for molecular marker-assisted selection in eggplant breeding and provides theoretical foundation for analyzing the formation mechanism of eggplant peel color.

Investigation of Effects of the Spectral Library on Analysis of diaPASEF Data.

Targeted analysis of data-independent acquisition (DIA) data needs a spectral library, which is generated by data-dependent acquisition (DDA) experiments or directly from DIA data. A comparison of the DDA library and DIA library in analyzing DIA data has been reported. However, the effects of different spectral libraries on the analysis of diaPASEF data have not been investigated. Here, we generate different spectral libraries with varying proteome coverage to analyze parallel accumulation-serial fragmentation (diaPASEF) data. Besides, we also employ the library-free strategy. The library, constructed by extensive fractionation DDA experiments, produces the highest numbers of precursors and proteins but with a high percentage of missing values. The library-free strategy identifies 10-20% fewer proteins than the library-based method but with a high degree of data completeness. A further study shows that the library-free strategy, although it identifies fewer proteins than the library-based method, leads to similar biological conclusions as the library-based method.

Silver-Copper Alloy Nanoinks for Ambient Temperature Sintering.

There is an increasing need to reduce the silver content in silver-based inks or pastes and achieve low-temperature sintering for scalable and low-cost production of printed wearable electronics. This need depends on the ability to control the metal composition and the surface properties of the nanoinks. Alloying silver with copper provides a pathway for meeting the need in terms of cost reduction, but little is known about the composition controllability and the low-temperature sintering capability. We report herein a scalable wet chemical synthesis of bimetallic silver-copper alloy nanoinks with room temperature sintering properties. The bimetallic alloy nanoparticles with a controllable composition can be formulated as stable nanoinks. The nanoinks printed on paper substrates are shown to sinter under room temperature. In addition to composition dependence, the results reveal an intriguing dependence of sintering on humidity above the printed nanoink films. These findings are assessed based on theoretical simulation of the sintering processes via surface-mediated sintering and interparticle necking mechanisms in terms of nanoscale adsorption, adhesion and diffusion, and surface free energies. Implications of the findings for room temperature fabrication of wearable sensors are also discussed.

A Low-Current and Multi-Channel Chemiresistor Array Sensor Device.

This paper describes the design of a low-current, multichannel, handheld electronic device integrated with nanostructured chemiresistor sensor arrays. A key design feature of the electronic circuit board is its low excitation current for achieving optimal performance with the arrays. The electronics can rapidly acquire the resistances for different sensors, not only spanning several orders of magnitude, but also as high as several hundreds of megaohms. The device tested is designed using a chemiresistor array with nanostructured sensing films prepared by molecularly-mediated assemblies of gold nanoparticles for detection. The low-current, wide-range, and auto-locking capabilities, along with the effective coupling with the nanostructured chemiresistor arrays, meet the desired performances of a low excitation current and low power consumption, and also address the potential instability of the sensors in a complex sensing environment. The results are promising for potential applications of the device as a portable sensor for the point-of-need monitoring of air quality and as a biosensor for point-of-care human breath screening for disease biomarkers.

QuantPipe: A User-Friendly Pipeline Software Tool for DIA Data Analysis Based on the OpenSWATH-PyProphet-TRIC Workflow.

Targeted analysis of data-independent acquisition (DIA) mass spectrometry data requires elegant software tools and strict statistical control. OpenSWATH-PyProphet-TRIC is a widely used DIA data analysis workflow. The OpenSWATH-PyProphet-TRIC workflow is typically executed by running command lines. Here, we present QuantPipe, which is a graphic interface software tool based on the OpenSWATH-PyProphet-TRIC workflow. In addition to OpenSWATH-PyProphet-TRIC functions, QuantPipe can convert the spectral library to the assay library and output peptides and protein intensities. We demonstrated that QuantPipe can be used to analyze SWATH-MS data from TripleTOF 5600 and TripleTOF 6600, phospho-SWATH-MS data, DIA data from Orbitrap instrument, and diaPASEF data from TimsTOF Pro instrument. The executable files, user manual, and source code of QuantPipe are freely available at https://github.com/tachengxmu/QuantPipe/releases.

Dynamic Core-Shell and Alloy Structures of Multimetallic Nanomaterials and Their Catalytic Synergies.

ConspectusMultimetallic nanomaterials containing noble metals (NM) and non-noble 3d-transition metals (3d-TMs) exhibit unique catalytic properties as a result of the synergistic combination of NMs and 3d-TMs in the nanostructure. The exploration of such a synergy depends heavily on the understanding of the atomic-scale structural details of NMs and 3d-TMs in the nanomaterials. This has attracted a great deal of recent interest in the field of catalysis science, especially concerning the core-shell and alloy nanostructures. A rarely asked question of fundamental significance is how the core-shell and alloy structural arrangements of atoms in the multimetallic nanomaterials dynamically change under reaction conditions, including reaction temperature, surface adsorbate, chemical environment, applied electrochemical potential, etc. The dynamic evolution of the core-shell/alloy structures under the reaction conditions plays a crucial role in the catalytic performance of the multimetallic nanocatalysts.This Account focuses on the dynamic structure characteristics for several different types of composition-tunable alloy and core-shell nanomaterials, including phase-segregated, elemental-enriched, dynamically evolved, and structurally different core-shell structures. In addition to outlining core-shell/alloy structure formation via processes such as seed-mediated growth, thermochemical calcination, adsorbate-induced evolution, chemical dealloying, underpotential deposition/galvanic displacement, etc., this Account will highlight the progress in understanding the dynamic core-shell/alloy structures under chemical or catalytic reaction conditions, which has become an important focal point of the research fronts in catalysis and electrocatalysis. The employment of advanced techniques, especially in situ/operando synchrotron high-energy X-ray diffraction and pair distribution function analyses, has provided significant insights into the dynamic evolution processes of NM/3d-TM nanocatalysts under electrocatalytic or fuel cell operating conditions. Examples will highlight Pt- or Pd-based nanoparticles and nanowires alloyed with various 3d-TMs with a focus on their structural evolution under reaction conditions. While the dynamic process is complex, the ability to gain an insight into the evolution of core-shell and alloy structures under the catalytic reaction condition is essential for advancing the design of multimetallic nanocatalysts. This Account serves as a springboard from fundamental understanding of the core-shell and alloy structural dynamics to the various applications of nanostructured catalysts/electrocatalysts, especially in the fronts of energy and environmental sustainability.

Development of a 3D-Printed Navigational Template for Establishing Rabbit VX2 Lung Cancer Model.

BACKGROUND: The CT-guided percutaneous puncture-inoculation for establishing the rabbit VX2 lung cancer model (LCM) is time-consuming, requires repeated CT scans, and has a high complication rate. Therefore, this study aimed to develop a navigational template using 3D technology to provide an alternative method for establishing the model with improved success and complication rates. MATERIALS AND METHODS: Ideal pressure was determined using chest CT data from 15 anesthetized rabbits fitted with sphygmomanometer cuff around their chests. Subsequently, a preliminary 3D template with a square window and cross-sign to facilitate precise installation was designed. Using another 20 rabbits fixed with the preliminary template, an ideal common puncture point and parameter were determined, a navigational tunnel was set up on the template surface, and the final puncture navigational template was printed out. Eight-four rabbits (42/group) were assigned to the experimental (template-guided puncture) and control (traditional puncutre) groups and underwent VX2 tumor-fragment inoculation to validate the template. Differences in various parameters between two groups were analyzed. RESULTS: The ideal pressure was 30 mmHg. All rabbits were inoculated successfully and the template adequately fit the rabbit chest. The experimental group displayed significantly better operation time (198.93±36.64 vs 735.14±91.19 seconds); number of CT scans (0 vs 7.19±1.64); pneumothorax (11.9% vs 35.7%), chest seeding (16.7% vs 35.7%), and mid-lung field tumor-bearing (88.1% vs 59.5%) rates than the control group (all, P <0.05). The groups did not differ in rib injury, tumor volume or survival time (all, P > 0.05). CONCLUSIONS: We successfully developed a puncture navigational template, providing an alternative method for establishing the rabbit VX2 LCM.

Quantification of Dynamic Protein Interactions and Phosphorylation in LPS Signaling Pathway by SWATH-MS.

Lipopolysaccharide (LPS)-induced macrophage activation is a prototype of innate immune response. Although key effector proteins in LPS signaling pathway have been revealed, the map of dynamic protein interactions and phosphorylation as well as the stoichiometry of protein complexes are lacking. Here we present a dynamic map of protein interactions and phosphorylation in MyD88, TRAF6 and NEMO complexes obtained by SWATH-MS. The comprehensive MS measurement leads to quantification of up to about 3,000 proteins across about 21-40 IP samples. We detected and quantified almost all known interactors of MyD88, TRAF6 and NEMO. By analyzing these quantitative data, we uncovered differential recruitment of IRAK family proteins to LPS-induced signaling complexes and determined the stoichiometry of the Myddosome complex. In addition, quantitative phosphoproteomics analysis identified a number of unreported high-confidence phosphosites on the key proteins in LPS signaling pathway. Collectively, data of dynamic protein interactions and phosphorylation presented by this study could be a resource for further study of the LPS signaling pathway.

Assessing Plasmonic Nanoprobes in Electromagnetic Field Enhancement for SERS Detection of Biomarkers.

The exploration of the plasmonic field enhancement of nanoprobes consisting of gold and magnetic core@gold shell nanoparticles has found increasing application for the development of surface-enhanced Raman spectroscopy (SERS)-based biosensors. The understanding of factors controlling the electromagnetic field enhancement, as a result of the plasmonic field enhancement of the nanoprobes in SERS biosensing applications, is critical for the design and preparation of the optimal nanoprobes. This report describes findings from theoretical calculations of the electromagnetic field intensity of dimer models of gold and magnetic core@gold shell nanoparticles in immunoassay SERS detection of biomarkers. The electromagnetic field intensities for a series of dimeric nanoprobes with antibody-antigen-antibody binding defined interparticle distances were examined in terms of nanoparticle sizes, core-shell sizes, and interparticle spacing. The results reveal that the electromagnetic field enhancement not only depended on the nanoparticle size and the relative core size and shell thicknesses of the magnetic core@shell nanoparticles but also strongly on the interparticle spacing. Some of the dependencies are also compared with experimental data from SERS detection of selected cancer biomarkers, showing good agreement. The findings have implications for the design and optimization of functional nanoprobes for SERS-based biosensors.

Strain-Modulated Platinum-Palladium Nanowires for Oxygen Reduction Reaction.

Electrocatalytic activity of alloy nanocatalytsts can be manipulated effectively by tuning their physical properties (ensemble, geometric, and ligand effects) to afford optimal surface structure and compositions for proton exchange membrane fuel cell (PEMFC) application. Herein, highly catalytic platinum-palladium nanowires (PtnPd100-n NWs) with a subtle lattice strain and Boerdijk-Coxeter helix type morphology are synthesized through a surfactant-free, thermal single phase solvent method. X-ray diffraction results show that PtnPd100-n NWs are exposed through the (111) facets and their shrinking or expanding lattice parameters can be modulated by the alloy compositions. Electrochemical results reveal that their high catalytic activity correlates with the lattice shrinking, facets, and bimetallic compositions, showing higher activity when the ratio of Pt and Pd is ∼78:22, which is further supported by DFT results. Compared to the nanoparticle type platinum-palladium alloyed catalysts with similar metal compositions (PtnPd100-n NPs), the PtnPd100-n NWs exhibit significantly improved electrocatalytic activity and stability for the oxygen reduction reaction. These findings open new strategies to design the highly active and stable alloy nanocatalysts with controllable compositions.

Weighted Gene Co-Expression Analysis Network-Based Analysis on the Candidate Pathways and Hub Genes in Eggplant Bacterial Wilt-Resistance: A Plant Research Study.

Solanum melongena L. (eggplant) bacterial wilt is a severe soil borne disease. Here, this study aimed to explore the regulation mechanism of eggplant bacterial wilt-resistance by transcriptomics with weighted gene co-expression analysis network (WGCNA). The different expression genes (DEGs) of roots and stems were divided into 21 modules. The module of interest (root: indianred4, stem: coral3) with the highest correlation with the target traits was selected to elucidate resistance genes and pathways. The selected module of roots and stems co-enriched the pathways of MAPK signalling pathway, plant pathogen interaction, and glutathione metabolism. Each top 30 hub genes of the roots and stems co-enriched a large number of receptor kinase genes. A total of 14 interesting resistance-related genes were selected and verified with quantitative polymerase chain reaction (qPCR). The qPCR results were consistent with those of WGCNA. The hub gene of EGP00814 (namely SmRPP13L4) was further functionally verified; SmRPP13L4 positively regulated the resistance of eggplant to bacterial wilt by qPCR and virus-induced gene silencing (VIGS). Our study provides a reference for the interaction between eggplants and bacterial wilt and the breeding of broad-spectrum and specific eggplant varieties that are bacterial wilt-resistant.

[Suprapubic lipectomy with a "Ω" incision for buried penis in obese adult men].

Objective: To investigate the application of suprapubic lipectomy with a "Ω" incision to removal of the prepubic fat pad for the management of buried penis in obese adult patients. METHODS: We retrospectively analyzed the clinical data on 20 obese adult patients with buried penis treated by suprapubic lipectomy via a "Ω" incision between August 2016 and September 2019. RESULTS: The operations were successfully completed in all the cases, with a mean operation time of 3.7 ± 0.6 hours and an average hospital stay of 8.3 ± 3.3 days. There were no such severe surgery-related complications as hematoma, urethral injury, or fat embolism in any of the cases. Fat liquefaction-related superficial wound infection developed in 1 patient postoperatively, which was cured by combined topical and systemic antibiotic therapy. A 3-month follow-up showed a 95% satisfaction of the patients with the postoperative appearance of the penis and suprapubic incision, but no complications such as ED, abnormal penile sensation, or penile retraction. CONCLUSIONS: Suprapubic lipectomy with a "Ω" incision to remove the prepubic fat pad is an effective surgical approach to the management of buried penis in obese adult males, which is an anatomy-based surgical correction and has the advantages of slight injury, rapid recovery and few complications./.