Chemical Affinity-Based Isolation of Extracellular Vesicles from Biofluids for Proteomics and Phosphoproteomics Analysis.

Extracellular vesicles (EVs) from biofluids have recently gained significant attention in the field of liquid biopsy. Released by almost every type of cell, they provide a real-time snapshot of host cells and contain a wealth of molecular information, including proteins, in particular those with post-translational modifications (PTMs) such as phosphorylation, as the main player of cellular functions and disease onset and progression. However, the isolation of EVs from biofluids remains challenging due to low yields and impurities from current EV isolation methods, making the downstream analysis of EV cargo, such as EV phosphoproteins, difficult. Here, we describe a rapid and effective EV isolation method based on functionalized magnetic beads for EV isolation from biofluids such as human urine and downstream proteomics and phosphoproteomics analysis following EV isolation. The protocol enabled a high recovery yield of urinary EVs and sensitive profiles of EV proteome and phosphoproteome. Furthermore, the versatility of this protocol and relevant technical considerations are also addressed here.

ALCAM: A Novel Surface Marker on EpCAMlow Circulating Tumor Cells.

Background: Current strategies in circulating tumor cell (CTC) isolation in pancreatic cancer heavily rely on the EpCAM and cytokeratin cell status. EpCAM is generally not considered a good marker given its transitory change during Epithelial to Mesenchymal Transition (EMT) or reverse EMT. There is a need to identify other surface markers to capture the complete repertoire of PDAC CTCs. The primary objective of the study is to characterize alternate surface biomarkers to EpCAM on CTCs that express low or negligible levels of surface EpCAM in pancreatic cancer patients. Methods: Flow cytometry and surface mass spectrometry were used to identify proteins expressed on the surface of PDAC CTCs in culture. CTCs were grown under conditions of attachment and in co-culture with naïve neutrophils. Putative biomarkers were then validated in GEMMs and patient samples. Results: Surface proteomic profiling of CTCs identified several novel protein biomarkers. ALCAM was identified as a novel robust marker in GEMM models and in patient samples. Conclusions: We identified several novel surface biomarkers on CTCs expressed under differing conditions of culture. ALCAM was validated and identified as a novel alternate surface marker on EpCAMlow CTCs.

Identification of novel early pancreatic cancer biomarkers KIF5B and SFRP2 from "first contact" interactions in the tumor microenvironment.

BACKGROUND: Pancreatic cancer is one of the most difficult cancers to detect early and most patients die from complications arising due to distant organ metastases. The lack of bona fide early biomarkers is one of the primary reasons for late diagnosis of pancreatic cancer. It is a multifactorial disease and warrants a novel approach to identify early biomarkers. METHODS: In order to characterize the proteome, Extracellular vesicles (EVs) isolated from different in vitro conditions mimicking tumor-microenvironment interactions between pancreatic cancer epithelial and stromal cells were analyzed using high throughput mass spectrometry. The biological activity of the secreted EVome was analyzed by investigating changes in distant organ metastases and associated early changes in the microbiome. Candidate biomarkers (KIF5B, SFRP2, LOXL2, and MMP3) were selected and validated on a mouse-human hybrid Tissue Microarray (TMA) that was specifically generated for this study. Additionally, a human TMA was used to analyze the expression of KIF5B and SFRP2 in progressive stages of pancreatic cancer. RESULTS: The EVome of co-cultured epithelial and stromal cells is different from individual cells with distinct protein compositions. EVs secreted from stromal and cancer cells cultures could not induce significant changes in Pre-Metastatic Niche (PMN) modulation, which was assessed by changes in the distant organ metastases. However, they did induce significant changes in the early microbiome, as indicated by differences in α and ß-diversities. KIF5B and SFRP2 show promise for early detection and investigation in progressive pancreatic cancer. These markers are expressed in all stages of pancreatic cancer such as low grade PanINs, advanced cancer, and in liver and soft tissue metastases. CONCLUSIONS: Proteomic characterization of EVs derived from mimicking conditions of epithelial and stromal cells in the tumor-microenvironment resulted in the identification of several proteins, some for the first time in EVs. These secreted EVs cannot induce changes in distant organ metastases in in vivo models of EV education, but modulate changes in the early murine microbiome. Among all the proteins that were analyzed (MMP3, KIF5B, SFRP2, and LOXL2), KIF5B and SFRP2 show promise as bona fide early pancreatic cancer biomarkers expressed in progressive stages of pancreatic cancer.

Microcystin-LR Induces NLRP3 Inflammasome Activation via FOXO1 Phosphorylation, Resulting in Interleukin-1ß Secretion and Pyroptosis in Hepatocytes.

Microcystin-LR (MC-LR), the most common and toxic microcystin (MC) present in freshwater, poses a substantial threat to human health, especially hepatotoxicity. Recent evidence reveals that the NLRP3 inflammasome plays an important role in liver injury by activating caspase-1 to promote interleukin-1ß (IL-1ß) secretion. In this study, we investigated the possible role of NLRP3 inflammasome activation in MC-LR-induced mouse liver inflammatory injury. We found that MC-LR administered to mice by oral gavage mainly accumulated in liver and induced the activation of the NLRP3 inflammasome and production of mature IL-1ß. Additionally, we observed an increase in the levels of NLRP3 inflammasome-related proteins and the proportion of pyroptosis in MC-LR-treated AML-12 cells. We also found that inhibition of NLRP3 in mice attenuated MC-LR-induced IL-1ß production, indicating an essential role for NLRP3 in MC-LR-induced liver inflammatory injury. In addition, we found that inhibition of FOXO1 by AKT-mediated hyperphosphorylation, due to protein phosphatase 2A (PP2A) inhibition, is required for MC-LR-induced expression of NLRP3. Taken together, our in vivo and in vitro findings suggest a model in which the NLRP3 inflammasome activation, a result of AKT-mediated hyperphosphorylation of FOXO1 through inhibition of PP2A, plays a key role in MC-LR-induced liver inflammatory injury via IL-1ß secretion and pyroptotic cell death.

Comparison of the efficacy and impact of GEMOX and GDP in the treatment of patients with non-Hodgkin's lymphoma.

PURPOSE: To compare the efficacy and impact of GEMOX and GDP in the treatment of patients with non-Hodgkin's lymphoma (NHL). METHODS: A total of 68 patients with NHL admitted to the hospitals of the authors from February 2013 to April 2016 were equally distributed into the GEMOX Group (treated with Gemcitabine and Oxaliplatin) and the GDP Group (treated with Gemcitabine, Cisplatin, and Dexamethasone), with cycle repetition every 3 weeks. The efficacy was analyzed every two weeks. The side effects were analyzed once a week. Comparison of survival was performed using Kaplan-Meier method and log-rank test and Cox univariate and multivariate regression analyses. RESULTS: Efficacy in the two groups was not statistically different (p>0.05). The incidence of III-IV grade of nausea and vomiting in the GDP Group was higher than in the GEMOX Group (p<0.05). The overall incidence decreased hemoglobin, nausea and vomiting, and renal dysfunction of the GDP Group was also higher than in the GEMOX Group (p<0.05). Analysis by multivariate Cox model found that the clinical classification and the grade of malignancy were independent prognostic factors (p<0.05). The odds ratio (OR) values of the clinical classification in the GEMOX Group and the GDP Group were 2.874 and 24.074, respectively. The OR values of the grade of malignancy in the GEMOX Group and the GDP Group were 14.034 and 6.873, respectively. CONCLUSION: Both the GEMOX regimen and the GDP regimen had good short-term efficacy on NHL patients, but the GEMOX regimen is to be preferred since as it had fewer side effects than the GDP regimen.

Isolation and analysis of extracellular vesicles in a Morpho butterfly wing-integrated microvortex biochip.

With the function of mediating intercellular communication between cells, extracellular vesicles (EVs) have been intently studied for their physiopathology and clinical application values. However, efficient EV isolation from biological fluids remains a significant challenge. To address this, this work constructs a new microvortex chip that can isolate EVs efficiently by integrating the lipid nanoprobe modified Morpho Menelaus (M. Menelaus) butterfly wing into microfluidic chip. M. Menelaus wing is well known for its orderly arranged periodic nanostructures and can generate microvortex when liquid passes through it, leading to increased interaction between EVs and M. Menelaus wing. In addition, the nanoprobe containing lipid tails can be inserted into EVs through their lipid bilayer membrane structure. Based on the described properties, high-throughput enrichment of EVs with over 70% isolation efficiency was realized. Moreover, it was demonstrated that the nanoprobe system based on M. Menelaus wing enabled downstream biological analysis of nucleic acids and proteins in EVs. Microvortex chips showed potential application value in efficient EV isolation for biomedical research and cancer diagnosis.

An evolutionarily conserved iron-sulfur cluster underlies redox sensory function of the Chloroplast Sensor Kinase.

Photosynthetic efficiency depends on equal light energy conversion by two spectrally distinct, serially-connected photosystems. The redox state of the plastoquinone pool, located between the two photosystems, is a key regulatory signal that initiates acclimatory changes in the relative abundance of photosystems. The Chloroplast Sensor Kinase (CSK) links the plastoquinone redox signal with photosystem gene expression but the mechanism by which it monitors the plastoquinone redox state is unclear. Here we show that the purified Arabidopsis and Phaeodactylum CSK and the cyanobacterial CSK homologue, Histidine kinase 2 (Hik2), are iron-sulfur proteins. The Fe-S cluster of CSK is further revealed to be a high potential redox-responsive [3Fe-4S] center. CSK responds to redox agents with reduced plastoquinone suppressing its autokinase activity. Redox changes within the CSK iron-sulfur cluster translate into conformational changes in the protein fold. These results provide key insights into redox signal perception and propagation by the CSK-based chloroplast two-component system.

Long Noncoding RNA LINC00152 Facilitates the Leukemogenesis of Acute Myeloid Leukemia by Promoting CDK9 Through miR-193a.

The vital role of long noncoding RNAs (lncRNAs) on the acute myeloid leukemia (AML) has been increasingly recognized. This study aims to explore the unknown function of lncRNA LINC00152 in the leukemogenesis of AML. LINC00152 is determined to be upregulated in the AML samples, and the overexpression of LINC00152 is also authenticated in the advanced French-American-British (FAB) AML patients and closely correlated with the poor outcome of AML patients. The functional experiments state that knockdown of LINC00152 suppresses the proliferation, accelerates the apoptosis, and induces the cycle arrest of AML cells. The mechanical experiments state that LINC00152 and CDK9 were both targeted by miR-193a with the complementary binding sites at 3'-UTR. Moreover, in the rescue experiments, the enhanced LINC00152 expression could regain the suppression of tumor behavior induced by LINC00152 knockdown. In conclusion, this research reveals the important role of lncRNA LINC00152 in the AML leukemogenesis through targeting miR-193a/CDK9 axis. This finding could indicate the important pathogenesis of ncRNA and the vital roles of epigenetic regulation.

An Arabidopsis Nucleoporin NUP85 modulates plant responses to ABA and salt stress.

Several nucleoporins in the nuclear pore complex (NPC) have been reported to be involved in abiotic stress responses in plants. However, the molecular mechanism of how NPC regulates abiotic stress responses, especially the expression of stress responsive genes remains poorly understood. From a forward genetics screen using an abiotic stress-responsive luciferase reporter (RD29A-LUC) in the sickle-1 (sic-1) mutant background, we identified a suppressor caused by a mutation in NUCLEOPORIN 85 (NUP85), which exhibited reduced expression of RD29A-LUC in response to ABA and salt stress. Consistently, the ABA and salinity induced expression of several stress responsive genes such as RD29A, COR15A and COR47 was significantly compromised in nup85 mutants and other nucleoporin mutants such as nup160 and hos1. Subsequently, Immunoprecipitation and mass spectrometry analysis revealed that NUP85 is potentially associated with HOS1 and other nucleoporins within the nup107-160 complex, along with several mediator subunits. We further showed that there is a direct physical interaction between MED18 and NUP85. Similar to NUP85 mutations, MED18 mutation was also found to attenuate expression of stress responsive genes. Taken together, we not only revealed the involvement of NUP85 and other nucleoporins in regulating ABA and salt stress responses, but also uncovered a potential relation between NPC and mediator complex in modulating the gene expression in plants.

Ascl2 inhibits myogenesis by antagonizing the transcriptional activity of myogenic regulatory factors.

Myogenic regulatory factors (MRFs), including Myf5, MyoD (Myod1) and Myog, are muscle-specific transcription factors that orchestrate myogenesis. Although MRFs are essential for myogenic commitment and differentiation, timely repression of their activity is necessary for the self-renewal and maintenance of muscle stem cells (satellite cells). Here, we define Ascl2 as a novel inhibitor of MRFs. During mouse development, Ascl2 is transiently detected in a subpopulation of Pax7+ MyoD+ progenitors (myoblasts) that become Pax7+ MyoD- satellite cells prior to birth, but is not detectable in postnatal satellite cells. Ascl2 knockout in embryonic myoblasts decreases both the number of Pax7+ cells and the proportion of Pax7+ MyoD- cells. Conversely, overexpression of Ascl2 inhibits the proliferation and differentiation of cultured myoblasts and impairs the regeneration of injured muscles. Ascl2 competes with MRFs for binding to E-boxes in the promoters of muscle genes, without activating gene transcription. Ascl2 also forms heterodimers with classical E-proteins to sequester their transcriptional activity on MRF genes. Accordingly, MyoD or Myog expression rescues myogenic differentiation despite Ascl2 overexpression. Ascl2 expression is regulated by Notch signaling, a key governor of satellite cell self-renewal. These data demonstrate that Ascl2 inhibits myogenic differentiation by targeting MRFs and facilitates the generation of postnatal satellite cells.

Analytical challenges translating mass spectrometry-based phosphoproteomics from discovery to clinical applications.

Phosphoproteomics is the systematic study of one of the most common protein modifications in high throughput with the aim of providing detailed information of the control, response, and communication of biological systems in health and disease. Advances in analytical technologies and strategies, in particular the contributions of high-resolution mass spectrometers, efficient enrichments of phosphopeptides, and fast data acquisition and annotation, have catalyzed dramatic expansion of signaling landscapes in multiple systems during the past decade. While phosphoproteomics is an essential inquiry to map high-resolution signaling networks and to find relevant events among the apparently ubiquitous and widespread modifications of proteome, it presents tremendous challenges in separation sciences to translate it from discovery to clinical practice. In this mini-review, we summarize the analytical tools currently utilized for phosphoproteomic analysis (with focus on MS), progresses made on deciphering clinically relevant kinase-substrate networks, MS uses for biomarker discovery and validation, and the potential of phosphoproteomics for disease diagnostics and personalized medicine.

ArhGAP15, a Rac-specific GTPase-activating protein, plays a dual role in inhibiting small GTPase signaling.

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase.

Serine/threonine phosphatase Stp1 mediates post-transcriptional regulation of hemolysin, autolysis, and virulence of group B Streptococcus.

Elucidating how serine/threonine phosphatases regulate kinase function and bacterial virulence is critical for our ability to combat these infections. Group B streptococci (GBS) are ß-hemolytic Gram-positive bacteria that cause invasive infections in humans. To adapt to environmental changes, GBS encodes signaling mechanisms comprising two component systems and eukaryotic-like enzymes. We have previously described the importance of the serine/threonine kinase Stk1 to GBS pathogenesis. However, how the presence or absence of the cognate serine/threonine phosphatase Stp1 affects Stk1 function and GBS virulence is not known. Here, we show that GBS deficient only in Stp1 expression are markedly reduced for their ability to cause systemic infections, exhibit decreased ß-hemolysin/cytolysin activity, and show increased sensitivity to autolysis. Although transcription of genes important for ß-hemolysin/cytolysin expression and export is similar to the wild type (WT), 294 genes (excluding stp1) showed altered expression in the stp1 mutant and included autolysin genes. Furthermore, phosphopeptide enrichment analysis identified that 35 serine/threonine phosphopeptides, corresponding to 27 proteins, were unique to the stp1 mutant. This included phosphorylation of ATP synthase, DNA and RNA helicases, and proteins important for cell division and protein synthesis. Collectively, our results indicate that Stp1 is important for appropriate regulation of Stk1 function, hemolysin activity, autolysis, and GBS virulence.

Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

Exotoxins, including the hemolysins known as the alpha (alpha) and beta (beta) toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1) were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1) increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU), serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE) and a hypothetical protein (NWMN_1123) were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

[Induction of dendritic cells derived from acute promyelocytic leukemia cells by all trans retinoic acid].

This study was purposed to investigate the possibility of differentiating the acute promyelocytic leukemia (APL) cells into dendritic cells (DCs) induced by all-trans retinoic acid (ATRA) combined with classic cytokines so as to provide a new approach for development of APL-DC vaccine. The bone marrow mononuclear cells from a new diagnosed patient with APL and HL-60 cells were separately cultured in complete culture medium. The cells were treated by ATRA, GM-CSF, IL-4 and TNFalpha in experimental groups and no ATRA was added in control and blank control groups. The cell morphology was observed by light microscopy, the phenotypes of DCs were detected by flow cytometry, the level of IL-12 was measured by using ELISA, the mixed lymphocyte reaction (MLR) and effect of cytotoxic T-lymphocyte (CTL) were assayed by MTT method. The results indicated that in experiment groups, the cells had dendritic appearance and cytogenetic characteristics of APL; expression of CD1a, CD83, CD80, CD86, HLA-DR and CD1d as well as level of IL-12 obviously increased; the MLR and CTL effects were significant, but increase of CD1a expression in HL60-DCs did not show statistical difference from control and blank control groups. It is concluded that ATRA can successfully induce APL cells to differentiate into functionally mature DSs which obviously mediate MLR and CTL effects. The APL-DCs derived by ATRA can notably express CD1d that may activate CD1d-restricted NKT cells and promote proliferation of NRT cells. The exact mechanism of which should be further studied.

[Study on the relationship between changes of adrenomedulin in plasma and vascular resistance during traumatic shock in rats].

OBJECTIVE: To observe the changes of adrenomedulin in plasma and vascular resistance during traumatic shock in rats. METHODS: The concentration of adrenomedulin in plasma of rats after traumatic shock was detected by radio immunization. Mean arterial pressure, total peripheral vascular resistance (TPVR) and cardiac index (CI) were estimated by electrical conductance method. RESULTS: The concentration of adrenomedulin in plasma of traumatic shock without resuscitation group (68.34+/-3.71)ng/L and traumatic shock with resuscitation group (146.27+/-9.83)ng/L were higher than that of control group(32.63+/-7.55)ng/L (P<0.01 and P<0.05). TPVR of traumatic shock with resuscitation group (10.57+/-0.35) kPa.s.L-1 was lower than that of traumatic shock without resuscitation group (16.75+/-0.23) kPa.s.L-1(P<0.01) and its CI (215.59+/-1.29) ml.min-1.kg-1 was higher than that of traumatic shock without resuscitation group (143.11+/-0.86) ml.min-1.kg-1 (P<0.01). CONCLUSION: Adrenomedulin is closely correlated with changes of vascular resistance and plays an important role during pathophysiological processes in rats after traumatic shock.

Proteomic analysis of an extreme halophilic archaeon, Halobacterium sp. NRC-1.

Halobacterium sp. NRC-1 insoluble membrane and soluble cytoplasmic proteins were isolated by ultracentrifugation of whole cell lysate. Using an ion trap mass spectrometer equipped with a C18 trap electrospray ionization emitter/micro-liquid chromatography column, a number of trypsin-generated peptide tags from 426 unique proteins were identified. This represents approximately one-fifth of the theoretical proteome of Halobacterium. Of these, 232 proteins were found only in the soluble fraction, 165 were only in the insoluble membrane fraction, and 29 were in both fractions. There were 72 and 61% previously annotated proteins identified in the soluble and membrane protein fractions, respectively. Interestingly, 57 of previously unannotated proteins found only in Halobacterium NRC-1 were identified. Such proteins could be interesting targets for understanding unique physiology of Halobacterium NRC-1. A group of proteins involved in various metabolic pathways were identified among the expressed proteins, suggesting these pathways were active at the time the cells were collected. This data containing a list of expressed proteins, their cellular locations, and biological functions could be used in future studies to investigate the interaction of the genes and proteins in relation to genetic or environmental perturbations.