Chimeric Antigen Receptor Designed to Prevent Ubiquitination and Downregulation Showed Durable Antitumor Efficacy.

Clinical evidence suggests that poor persistence of chimeric antigen receptor-T cells (CAR-T) in patients limits therapeutic efficacy. Here, we designed a CAR with recyclable capability to promote in vivo persistence and to sustain antitumor activity. We showed that the engagement of tumor antigens induced rapid ubiquitination of CARs, causing CAR downmodulation followed by lysosomal degradation. Blocking CAR ubiquitination by mutating all lysines in the CAR cytoplasmic domain (CARKR) markedly repressed CAR downmodulation by inhibiting lysosomal degradation while enhancing recycling of internalized CARs back to the cell surface. Upon encountering tumor antigens, CARKR-T cells ameliorated the loss of surface CARs, which promoted their long-term killing capacity. Moreover, CARKR-T cells containing 4-1BB signaling domains displayed elevated endosomal 4-1BB signaling that enhanced oxidative phosphorylation and promoted memory T cell differentiation, leading to superior persistence in vivo. Collectively, our study provides a straightforward strategy to optimize CAR-T antitumor efficacy by redirecting CAR trafficking.

DeKinomics pulse-chases kinase functions in living cells.

Cellular context is crucial for understanding the complex and dynamic kinase functions in health and disease. Systematic dissection of kinase-mediated cellular processes requires rapid and precise stimulation ('pulse') of a kinase of interest, as well as global and in-depth characterization ('chase') of the perturbed proteome under living conditions. Here we developed an optogenetic 'pulse-chase' strategy, termed decaging kinase coupled proteomics (DeKinomics), for proteome-wide profiling of kinase-driven phosphorylation at second-timescale in living cells. We took advantage of the 'gain-of-function' feature of DeKinomics to identify direct kinase substrates and further portrayed the global phosphorylation of understudied receptor tyrosine kinases under native cellular settings. DeKinomics offered a general activation-based strategy to study kinase functions with high specificity and temporal resolution under living conditions.

miPEP31 alleviates Ang II-induced hypertension in mice by occupying Cebpα binding sites in the pri-miR-31 promoter.

BACKGROUND: Previous studies have shown that peptides encoded by noncoding RNAs (ncRNAs) can be used as peptide drugs to alleviate diseases. We found that microRNA-31 (miR-31) is involved in the regulation of hypertension and that the peptide miPEP31, which is encoded by the primary transcript of miR-31 (pri-miR-31), can inhibit miR-31 expression. However, the role and mechanism of miPEP31 in hypertension have not been elucidated. METHODS: miPEP31 expression was determined by western blot analysis. miPEP31-deficient mice (miPEP31-/-) were used, and synthetic miPEP31 was injected into Ang II-induced hypertensive mice. Blood pressure was monitored through the tail-cuff method. Histological staining was used to evaluate renal damage. Regulatory T (Treg) cells were assessed by flow cytometry. Differentially expressed genes were analysed through RNA sequencing. The transcription factors were predicted by JASPAR. Luciferase reporter and electrophoretic mobility shift assays (EMSAs) were used to determine the effect of pri-miR-31 on the promoter activity of miPEP31. Images were taken to track the entry of miPEP31 into the cell. RESULTS: miPEP31 is endogenously expressed in target organs and cells related to hypertension. miPEP31 deficiency exacerbated but exogenous miPEP31 administration mitigated the Ang II-induced systolic blood pressure (SBP) elevation, renal impairment and Treg cell decreases in the kidney. Moreover, miPEP31 deletion increased the expression of genes related to Ang II-induced renal fibrosis. miPEP31 inhibited the transcription of miR-31 and promoted Treg differentiation by occupying the Cebpα binding site. The minimal functional domain of miPEP31 was identified and shown to regulate miR-31. CONCLUSION: miPEP31 was identified as a potential therapeutic peptide for treating hypertension by promoting Treg cell differentiation in vivo. Mechanistically, we found that miPEP31 acted as a transcriptional repressor to specifically inhibit miR-31 transcription by competitively occupying the Cebpα binding site in the pri-miR-31 promoter. Our study highlights the significant therapeutic effect of miPEP31 on hypertension and provides novel insight into the role and mechanism of miPEPs.

Identification of novel disulfidptosis-related lncRNA signatures to predict the prognosis and immune microenvironment of skin cutaneous melanoma patients.

BACKGROUND: Skin cutaneous melanoma (SKCM) is an aggressive form of malignant melanoma with poor prognosis and high mortality rates. Disulfidptosis is a newly discovered cell death regulatory mechanism caused by the abnormal accumulation of disulfides. This unique pathway is guiding significant new research to understand cancer progression for targeted treatment. However, the correlation between disulfidptosis with long non-coding RNAs (lncRNAs) in SKCM remains unknown at present. METHODS: The Cancer Genome Atlas database furnished lncRNA expression data and clinical information for SKCM patients. Pearson correlation and Cox regression analyses identified disulfidptosis-related lncRNAs associated with SKCM prognosis. ROC curves and a nomogram validated the model. TME, immune infiltration, GSEA analysis, immune checkpoint gene expression profiling, and drug sensitivity were assessed in high and low-risk groups. Consistent clustering categorized SKCM patients for personalized clinical treatment guidance. RESULTS: A total of twelve disulfidptosis-related lncRNAs were identified for the development of prognosis prediction models. The area under the curve (AUC) values of the ROC curve and the nomogram provided reliable discrimination to evaluate the prognostic potential for SKCM patients. The TME played a crucial role in tumorigenesis, progression and prognosis, and the risk scores were closely related to immune cell infiltration. Meanwhile, the combination of chemotherapy, targeted therapy, and immunotherapy was recommended for low-risk patients based on drug sensitivity and immune efficacy analyses. CONCLUSION: We identified a risk model of twelve disulfidptosis-related lncRNAs that could be used to predict the prognosis of SKCM patients and help guide immunotherapy and chemotherapy for personalized treatment plans.

Duty ratio drive prediction model of lifetime degradation for organic light emitting diode-on-silicon microdisplay.

A duty ratio drive prediction (DRDP) model of luminance degradation for organic light emitting diodes (OLED) microdisplay is proposed in this paper. The traditional stretched exponential decay (SED) model is not applicable for OLED driven by duty ratio. The DRDP model introduces the duty ratio as the variables affecting the lifetime of OLED. By fitting the undetermined coefficients with the measured luminance data, the quantitative relationships among the initial luminance, duty ratio, and OLED lifetime are obtained. Meanwhile, the model quantifies the phenomenon of spontaneous luminance recovery, which occurs when OLED switches from bright to dark. Finally, the DRDP model is used to compensate the luminance degradation of OLED driven by duty ratio. The experimental results show that the average prediction accuracy of DRDP model for white, red, green, and blue (W/R/G/B) OLED degradation trend is 0.9623. The average prediction accuracy of W/R/G/B OLED lifetime is 0.6119, which is greater than that of SED model. The lifetime is extended by 89.83% after compensation.

MicroRNA-31 regulates TNF-α and IL-17A co-induced-endothelial cell apoptosis by repressing E2F6.

Vascular endothelial cell (VEC) apoptosis is the fundamental cause of pulmonary arterial hypertension. MicroRNA-31 (MiR-31) is a novel target for hypertension treatment. However, the role and mechanism of miR-31 in the apoptosis of VECs remain unclear. The purpose of this study is to determine whether miR-31 plays an important role in VEC apoptosis as well as the detailed mechanisms involved. We found that pro-inflammatory cytokines IL-17A and TNF-α were highly expressed in serum and aorta, and the expression of miR-31 was significantly increased in aortic intimal tissue from Angiotensin II (AngII)- induced hypertensive mice (WT-AngII) compared with control mice (WT-NC). In vitro, co-stimulation of VECs with IL-17A and TNF-α resulted in increased expression of miR-31 and VEC apoptosis. MiR-31 inhibition strikingly decreased TNF-α and IL-17A co-induced VEC apoptosis. Mechanistically, in IL-17A and TNF-α co-stimulated VECs (co-induced VECs), we found that the activation of the NF-κB signal effectively increased the expression of miR-31. Dual-luciferase reporter gene assay revealed that miR-31 directly targeted and inhibited the expression of the E2F transcription factor 6 (E2F6). The expression of E2F6 was decreased in Co-induced VECs. MiR-31 inhibition significantly alleviated the decreased expression of E2F6 in co-induced VECs. Consistent with the co-stimulated effect of IL-17A and TNF-α on VECs, transfection of siRNA E2F6 induced cell apoptosis without the stimulation of the above cytokines. In conclusion, TNF-α and IL-17A generated in the aortic vascular tissue and serum from Ang II-induced hypertensive mice could trigger VECs apoptosis by the miR-31/E2F6 axis. To sum up, our study suggests that the key factor between cytokine co-stimulation effect and VEC apoptosis was miR-31/E2F6 axis, which was mainly regulated by NF-қB signaling pathway. This gives us a new sight to treat hypertension-associated VR.

Comparative Investigation on the Phytochemicals and Biological Activities of Jackfruit (Artocarpus heterophyllus Lam.) pulp from Five Cultivars.

Jackfruit is one of the major tropical fruits, but information on the phytochemicals and biological benefits of its pulp is limited. In this study, the phytochemicals and biological activities including antioxidant, antitumor and anti-inflammatory activities of five jackfruit pulp cultivars (M1, M2, M3, M7 and T5) were comparatively investigated. A total of 11 compounds were identified in all cultivars of jackfruit pulp, among which 4-hydroxybenzoic acid, caffeic acid, ferulic acid and tryptophan N-glucoside were reported for the first time in jackfruit. T5 exhibited the highest total phenolic content (7.69 ± 0.73 mg GAE/g DW), antioxidant capacity (109.8, 96.7 and 207 mg VCE/g DW for DPPH, ABTS and FRAP, respectively), antitumor activity (80.31%) and anti-inflammatory activity (78.44%) among five cultivars. These results can provide a reference for growers to choose jackfruit cultivar and offer an insight into the industrial application of jackfruit pulp derived-products.

Sustainable response strategy for COVID-19: Pandemic zoning with urban multimodal transport data.

In the post-COVID-19 era, the pandemic response is increasingly difficult and entails a high cost to society. Existing pandemic control methods, such as lockdowns, greatly affect residents' normal lives. This paper proposes a pandemic control method, consisting of the scientific delineation of urban areas based on multimodal transportation data. An improved Leiden method based on the gravity model is used to construct a preliminary zoning scheme, which is then modified by spatial constraints. The modularity index demonstrates the suitability of this method for community detection. This method can minimize cut-off traffic flows between pandemic control areas. The results show that only 24.8% of travel links are disrupted using our method, which could reduce both the impact of pandemic control on the daily life of residents and its cost. These findings can help develop sustainable strategies and proposals for effective pandemic response.

Integrated and High-Throughput Approach for Sensitive Analysis of Tyrosine Phosphoproteome.

Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 µg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.

Morphological specificity analysis of an image-based 3D model of airway filling in a difficult airway.

BACKGROUND: The purpose of this study was to analyze position-specific morphological changes of the upper airway and to further assess the impact of these changes in difficult airway during intubation. METHODS: This observational comparative study included two groups (n = 20 patients/group): Group A had normal airway and Group B had difficult airway. Data obtained from two-dimensional magnetic resonance imaging were imported to Mimics V20.0 software for processing. We then reconstructed three-dimensional models of upper airway filling in patients in the supine and maximum extension position based on the imaging data. Those models were projected on coronal, sagittal, and horizontal planes to investigate multiple morphological features. We measured the surface area, radial length, and corner angle of the projected areas. RESULTS: Group A had larger upper airway filling volumes compared to Group B The volumes for the supine position were 6,323.83 ± 156.06 mm3 for Group A and 5,336.22 ± 316.13 mm3 for Group B (p = 0.003). The volumes the maximum extension position were 9,186.58 ± 512.61 mm3 for Group A and 6,735.46 ± 794.63 mm3 for Group B (p = 0.003). Airway volume increased in the upper airway filling model as the body position varied from the supine to maximum extension position (Group A: volume increase 2,953.75 ± 524.6 mm3, rate of change 31%; Group B: volume increase 1,632.89 ± 662.66 mm3, rate of change 25%; p = 0.052). CONCLUSION: The three-dimensional reconstruction model developed in this study was used to digitally quantify morphological features of a difficult airway and could be used as a novel airway management assessment tool.

Fully Integrated and Multiplexed Sample Preparation Technology for Sensitive Interactome Profiling.

Affinity purification coupled to mass spectrometry (AP-MS) is a popular approach for deciphering the architecture of protein interaction networks. Protein lysates (100 µg) are typically required for multistep sample processing in large volumes, which often causes sample loss and reduces the MS analysis sensitivity. Herein, we reported a fully integrated spintip-based AP-MS technology, termed FISAP, for multiplexed and sensitive interactome profiling. The FISAP device can be easily employed for routine use by introducing AP beads into a C18 StageTip. Taking advantage of the switchable functionalization of the C18 matrix by sodium dodecyl sulfate, all the sample preparation steps encompassing peptide or antibody-based AP, reduction, alkylation, tryptic digestion, tandem mass tag (TMT) labeling, and desalting can be performed in a single tip with a benchtop centrifuge in 4 h. Using a biotinylated tyrosine phosphorylated (pTyr) peptide as an affinity ligand, we mapped the pTyr-dependent interactome of the pY191 motif on the immune receptor CD28 cytoplasmic domain. When processing 50 µg of protein lysates, FISAP showed a comparable interactome identification performance but better quantification performance and lower background interference compared to the traditional tube-based method. Furthermore, a cost-effective on-column TMT labeling protocol was established and integrated into the FISAP pipeline with increased sensitivity. Compared to the tube-based method, the usage of a synthetic peptide probe and a TMT reagent was both reduced by 20 times. As low as 1 µg of protein lysates could be applied for interactome profiling. Finally, we expanded the applicability of the FISAP technology to epitope tag-based AP-MS for profiling the ILK/PINCH/Parvin complex using 100 times less protein lysate than a previous report. Collectively, FISAP is an easy-to-use and sensitive technology for quantitatively profiling protein complexes when the starting material and affinity reagent are the limitation, especially for applications in biomedical research and chemical biology.

AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation.

With recent advances in LC-MS systems, current MS-based proteomics has an increasing need for automated, high-throughput sample preparation with neglectable sample loss. In this study, we developed a microfluidic system for fully automated proteomics sample preparation. All of the required proteomics sample preparation steps for both protein digestion and peptide fractionation are fully integrated into a disposable plastic chip device (named AutoProteome Chip). The AutoProteome Chip packed with mixed-mode ion exchange beads and C18 membrane in tandem could be fabricated with very low cost and high stability in organic reagents. Benefiting from its low backpressure, the AutoProteome Chip could be precisely driven by gas pressure, which could be easily multiplexed. As low as 2 ng of standard protein BSA could be trapped into the AutoProteome chip and processed within 2 h. Fully automated processing of 10 µg of protein extracts of HEK 293T cells achieved more than 97% of digestion efficiency with missed cleavage less than 2 and comparable performance with conventional approaches. More than 4700 proteins could be readily identified within 80 min of LC-MS analysis with good label-free quantification performance (Pearson correlation coefficient >0.99). Furthermore, deep proteome profiling by integrated high-pH RP fractionation in the same AutoProteome Chip resulted in more than 7500 proteins being identified from only 20 µg of protein extracts of HEK 293T cells and comparable reprodicibility as single-shot analysis. The AutoProteome Chip system provided a valuable prototype for developing a fully automated proteome analysis workflow and for proteomic applications with high demand for processing throughput, reproducibility, and sensitivity.

High-Throughput and Integrated Chemical Proteomic Approach for Profiling Phosphotyrosine Signaling Complexes.

Phosphotyrosine (pTyr) signaling complexes are important resources of biomarkers and drug targets which often need to be profiled with enough throughput. Current profiling approaches are not feasible to meet this need due to either biased profiling by antibody-based detection or low throughput by traditional affinity purification-mass spectrometry approach (AP-MS), as exemplified by our previously developed photo-pTyr-scaffold approach. To address these limitations, we developed a 96-well microplate-based sample preparation and fast data independent proteomic analysis workflow. By assembling the photo-pTyr-scaffold probe into a 96-well microplate, we achieved steric hindrance-free photoaffinity capture of pTyr signaling complexes, selective enrichment under denaturing conditions, and efficient in-well digestion in a fully integrated manner. EGFR signaling complex proteins could be efficiently captured and identified by using 300 times less cell lysate and 100 times less photo-pTyr-scaffold probe as compared with our previous approach operated in an Eppendorf tube. Furthermore, the lifetime of the photo-pTyr-scaffold probe in a 96-well microplate was significantly extended from 1 week up to 1 month. More importantly, by combining with high-flow nano LC separation and data independent acquisition on the Q Exactive HF-X mass spectrometer, LC-MS time could be significantly reduced to only 35 min per sample without increasing sample loading amount and compromising identification and quantification performance. This new high-throughput proteomic approach allowed us to rapidly and reproducibly profile dynamic pTyr signaling complexes with EGF stimulation at five time points and EGFR inhibitor treatment at five different concentrations. We are therefore optimized for its generic application in biomarkers discovery and drug screening in a high-throughput fashion.

Multiomic analysis of a dried single-drop plasma sample using an integrated mass spectrometry approach.

An easy-to-use and fast approach was developed for integrated proteomic and metabolic profiling in a dried single-drop plasma sample. Plasma collection, room temperature storage, and sample preparation for both proteins and metabolites were seamlessly integrated in one spintip device. MS-based multiomic profiling using the same nano LC-MS system identified more than 150 proteins and 160 metabolites from the 1 µL plasma sample in 6 hours. Further combination with micro-flow LC and targeted MS made it a promising approach for the fast profiling of molecular biomarkers with high sensitivity and accuracy.

Membrane rafts-redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial-mesenchymal transition.

KEY POINTS: Membrane rafts (MRs)-redox signalling pathway is activated in response to transforming growth factor-ß1 (TGF-ß1) stimulation in renal tubular cells. This pathway contributes to TGF-1ß-induced epithelial-mesenchymal transition (EMT) in renal tubular cells. The the MRs-redox signalling pathway is activated in renal tubular cells isolated from angiotensin II (AngII)-induced hypertensive rats. Inhibition of this pathway attenuated renal inflammation and fibrosis in AngII-induced hypertension. ABSTRACT: The membrane rafts (MRs)-redox pathway is characterized by NADPH oxidase subunit clustering and activation through lysosome fusion, V-type proton ATPase subunit E2 (encoded by the Atp6v1e2 gene) translocation and sphingomyelin phosphodiesterase 1 (SMPD1, encoded by the SMPD1 gene) activation. In the present study, we hypothesized that the MRs-redox-derived reactive oxygen species (ROS) are involved in renal inflammation and fibrosis by promoting renal tubular epithelial-mesenchymal transition (EMT). Results show that transforming growth factor-ß1 (TGF-ß1) acutely induced MR formation and ROS production in NRK-52E cells, a rat renal tubular cell line. In addition, transfection of Atp6v1e2 small hairpin RNAs (shRNA) and SMPD1 shRNA attenuated TGF-ß1-induced changes in EMT markers, including E-cadherin, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) in NRK-52E cells. Moreover, Erk1/2 activation may be a downstream regulator of the MRs-redox-derived ROS, because both shRNAs significantly inhibited TGF-ß1-induced Erk1/2 phosphorylation. Further in vivo study shows that the renal tubular the MRs-redox signalling pathway was activated in angiotensin II (AngII)-induced hypertension, as indicated by the increased NADPH oxidase subunit Nox4 fraction in the MR domain, SMPD1 activation and increased ROS content in isolated renal tubular cells. Finally, renal transfection of Atp6v1e2 shRNA and SMPD1 shRNA significantly prevented renal fibrosis and inflammation, as indicated by the decrease of α-SMA, fibronectin, collagen I, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and tumour necrosis factor-α (TNF-α) in kidneys from AngII-infused rats. It was concluded that the the MRs-redox signalling pathway is involved in TGF-ß1-induced renal tubular EMT and renal inflammation/fibrosis in AngII-induced hypertension.

Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.

Transplantation of skin mesenchymal stem cells attenuated AngII-induced hypertension and vascular injury.

Skin mesenchymal stem cells (S-MSCs) revealed an important immunomodulatory activity to markedly suppress the formation of the atherosclerosis (AS) plaque by modulating macrophages, and also inhibit the development of experimental autoimmune encephalomyelitis (EAE) by regulating T helper 17 (Th17) cell differentiation. Macrophages and Th17 cells play important roles in hypertension. However, it remains unclear whether S-MSCs are capable of improving angiotensin (AngII)-induced hypertension by acting on inflammatory cells. Therefore, we studied a direct effect of S-MSC treatment on an AngII-induced hypertensive mouse model. Twenty-seven C57BL/6 (WT) mice were divided into three groups: Control group (WT-NC), AngII-infused group (WT-AngII), and S-MSC treatment group (WT-AngII + S-MSCs). In contrast to WT-AngII group, systolic blood pressure (SBP) and vascular damage were strikingly attenuated after tail-vein injection of S-MSCs. Numbers of Th17 cells in mouse peripheral blood of S-MSC treated group were significantly decreased, and IL-17 mRNA and protein levels were also reduced in the aorta and serum compared with WT-AngII group. Furthermore, macrophages in S-MSC treated group were switched to a regulatory profile characterized by a low ability to produce pro-inflammatory cytokine TNF-α and a high ability to produce anti-inflammatory cytokines Arg1 and IL-10. Mechanistically, we found that S-MSCs inhibited Th17 cell differentiation and induced M2 polarization. Moreover, we found proliferation and migration of S-MSCs were elevated, and expression of CXCR4, the receptor for Stromal derivated factor -1(SDF-1), was markedly increased in lipopolysaccharide (LPS)- stimulated S-MSCs. Given that SDF-1 expression was increased in the serum and aorta in AngII- induced hypertensive mice, the immunomodulatory effects exerted by S-MSCs involved the CXCR4/SDF-1 signaling. Collectively, our data demonstrated that S-MSCs attenuated AngII-induced hypertension by inhibiting Th17 cell differentiation and by modulating macrophage M2 polarization, suggesting that S-MSCs potentially have a role in stem cell based therapy for hypertension.

An integrated strategy for highly sensitive phosphoproteome analysis from low micrograms of protein samples.

Phosphoproteomics has become a popular proteomic technology for exploring cellular signaling networks. However, current approaches often require milligrams of protein samples which hamper their applications for translational studies with limited starting materials. In this study, we aimed to challenge the lowest starting material limit for phosphoproteome profiling. By carefully optimizing the well-established high-pH reversed-phase (RP) fractionation plus Ti4+-IMAC enrichment strategy, we achieved the identification of 15 260 and 8936 unique phosphopeptides from only 500 µg and 250 µg predigested peptides, respectively. To further improve the sensitivity of phosphoproteome analysis for low micrograms of protein samples, we developed an integrated strategy, termed Phospho-SISPROT. This technology integrates three tips in tandem for protein digestion by the simple and integrated spintip-based proteomics technology (SISPROT), phosphopeptide enrichment by the Ti4+-IMAC tip, and desalting by the StageTip, respectively, which could dramatically reduce the phosphoproteome analysis time from a couple of days to only 6 hours and improve the system sensitivity. The flow through of Phospho-SISPROT could be reused for the global protein identification, which is very helpful for accurate phosphoproteome analysis with limited starting materials. More than 5500 and 600 unique phosphopeptides were respectively identified from 20 µg and 1 µg pervanadate treated HEK 293T cell lysates processed by the Phospho-SISPROT. To the best of our knowledge, this performance is the highest reported to date by using the standard LC-MS/MS setup. We expect that the Phospho-SISPROT and the optimized high-pH RP fractionation plus Ti4+-IMAC enrichment strategy will be well suited for highly sensitive phosphoproteome analysis of rare biological samples.

Deep Metaproteomics Approach for the Study of Human Microbiomes.

Host-microbiome interactions have been shown to play important roles in human health and diseases. Most of the current studies of the microbiome have been performed by genomic approaches through next-generation sequencing. Technologies, such as metaproteomics, for functional analysis of the microbiome are needed to better understand the intricate host-microbiome interactions. However, significant efforts to improve the depth and resolution of gut metaproteomics are still required. In this study, we combined an efficient sample preparation technique, high resolution mass spectrometry, and metaproteomic bioinformatics tools to perform ultradeep metaproteomic analysis of human gut microbiome from stool. We reported the deepest analysis of the microbiome to date with an average of 20 558 protein groups identified per sample analysis. Moreover, strain resolution taxonomic and pathway analysis using deep metaproteomics revealed strain level variations, in particular for Faecalibacterium prausnitzii, in the microbiome from the different individuals. We also reported that the human proteins identified in stool samples are functionally enriched in extracellular region pathways and in particular those proteins involved in defense response against microbial organisms. Deep metaproteomics is a promising approach to perform in-depth microbiome analysis and simultaneously reveals both human and microbial changes that are not readily apparent using the standard genomic approaches.