Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions

Background

Eukaryotic ubiquitin and SUMO are frequently used as tags to enhance the fusion protein expression in microbial host. They increase the solubility and stability, and protect the peptides from proteolytic degradation due to their stable and highly conserved structures. Few of prokaryotic ubiquitin-like proteins was used as fusion tags except ThiS, which enhances the fusion expression, however, reduces the solubility and stability of the expressed peptides in E. coli. Hence, we investigated if MoaD, a conserved small sulfur carrier in prokaryotes with the similar structure of ubiquitin, could also be used as fusion tag in heterologous expression in E. coli.

Results

Fusion of MoaD to either end of EGFP enhanced the expression yield of EGFP with a similar efficacy of ThiS. However, the major parts of the fusion proteins were expressed in the aggregated form, which was associated with the retarded folding of EGFP, similar to ThiS fusions. Fusion of MoaD to insulin chain A or B did not boost their expression as efficiently as ThiS tag did, probably due to a less efficient aggregation of products. Interestingly, fusion of MoaD to the murine ribonuclease inhibitor enhanced protein expression by completely protecting the protein from intracellular degradation in contrast to ThiS fusion, which enhanced degradation of this unstable protein when expressed in E. coli.

Conclusions

Prokaryotic ubiquitin-like protein MoaD can act as a fusion tag to promote the fusion expression with varying mechanisms, which enriches the arsenal of fusion tags in the category of insoluble expression.     

Assignment of human satellite 1 DNA as revealed by fluorescent in situ hybridization with oligonucleotides

We have used a fluorescent in situ hybridization procedure to detect human satellite 1 DNA, the simple sequence family that constitutes the non-male-specific fraction of classical satellite 1 DNA. Satellite 1 appears to be located on pericentromeric regions of chromosomes 3, 4 and 13, and on satellites of each acrocentric chromosome. These results suggest a possible relationship between quinacrine fluorescence of heterochromatin and DNA composition. Furthermore, by means of multicolour in situ hybridization, we have spatially resolved satellite 1 sequences and centromeric -satellite within heterochromatic blocks.     

In-depth Characterization of the Secretome of Colorectal Cancer Metastatic Cells Identifies Key Proteins in Cell Adhesion,Migration, and Invasion

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.Despite the efforts for colorectal cancer (CRC)¹ prevention using different strategies (1–6), 30–40% of patients have regionally advanced disease or suffer from metastasis when diagnosed (7). Moreover, half of the CRC patients will develop recurrence and liver metastasis within 5 years (8). Although genetic changes leading to the development of sporadic colorectal cancer primary tumors in intestinal cells have been relatively well characterized (9), further efforts are necessary to better understand the biology of CRC metastasis and to identify associated markers that can be used as diagnostic/prognostic biomarkers or potential drug targets. Metastasis is a complex process involving different steps from extravasation to liver colonization and requires the concerted action of a large number of proteins to modulate different effects on adhesion, migration, invasion, and survival at the target organ (10).Cancer cells secrete proteins or protein fragments to body fluids, such as blood, that can be used as biomarkers (11, 12) and/or potential therapeutic targets (13). In the case of CRC, there are only three proteins currently used as biomarkers: the carcinoembryonic antigen (CEA) for recurrence and metastasis (1), deleted in colorectal carcinoma (DCC), and vascular endothelial growth factor (VEGF). The secretome constitutes a rich source of information not only for the identification of biomarkers but for the characterization of altered molecules like growth factors, cytokines, proteases, etc., which are vital for cancer progression and metastasis.We are using the well known human KM12 cell system (14) to study the biology of CRC metastasis. KM12SM cells, which possess high metastatic capacity to liver, were isolated from liver metastases in nude mice after five cycles of intrasplenic injection of the poorly metastatic cell line KM12C (14, 15). Multiple studies support a good correlation between the findings observed in the KM12 cell model and patient samples, indicating that KM12 isogenic cell lines recapitulate quite effectively some of the critical issues in CRC metastasis (16–21). In a previous study, we carried out a characterization of plasma membrane proteins of metastatic KM12 cells using a SILAC assay but with a low accuracy and resolution linear ion trap (17). About 60 proteins that showed ≥1.5-fold-change between both types of cells were identified. Recent studies applied iTRAQ or label-free quantification to other pairs of isogenic, nonmetastatic-metastatic colorectal cancer cell lines, SW480 and SW620, for the characterization of protein differences in the whole cell proteome (22) and secretome (23), respectively. The SW620 cell line was isolated from a metastatic lymph node of the same patient as SW480 (24). In contrast, KM12SM cells were chosen based on their capacity for liver metastasis, which makes them most appropriate for the study of liver homing and late stages of metastasis.We are analyzing different fractions of KM12 cells, including the secretome, for a deeper analysis of functionally relevant proteins in metastasis. In a previous report, we analyzed the cytokine/chemokine profiles released in the conditioned media by colorectal metastatic cancer KM12SM cells compared with KM12C using antibody microarrays (20). We found an important role for T_H2 cytokine IL-13 and its receptor IL13Rα2 in cell adhesion, migration, invasion, and liver colonization. Here, we continued this in-depth characterization of the secretome compartment using SILAC analysis with a high accuracy and resolution mass spectrometer, the linear ion trap Orbitrap Velos. The proteomic characterization resulted in the identification and quantification of 1337 and 1098 proteins, respectively, in the conditioned medium. In silico studies demonstrated a predominant association of deregulated proteins in metastatic cells to adhesion, migration, and invasion processes. Three candidates (GDF15, S100A8/A9, and SERPINI1) showed promise as CRC diagnostic markers in serum samples from CRC patients using ELISA. Functional studies using siRNA silencing and antibody blocking experiments demonstrated important tumorigenic and invasive properties in some previously uncharacterized proteins in CRC. In addition, three proteins, EFNA3, CD137L/TNFSF9, and SOSTDC1, demonstrated a critical role in liver homing for metastasis. Finally, meta-analysis of mRNA alterations data indicated that CD137L/TNFSF9, CTSS, SOSTDC1, ZG16B, EFNA3, and MDK were associated with poor prognosis in colorectal cancer.     

The tick salivary protein, Salp15, inhibits the development of experimental asthma

Activation of Th2 CD4(+) T cells is necessary and sufficient to elicit allergic airway disease, a mouse model with many features of human allergic asthma. Effectively controlling the activities of these cells could be a panacea for asthma therapy. Blood-feeding parasites have devised remarkable strategies to effectively evade the immune response. For example, ticks such as Ixodes scapularis, which must remain on the host for up to 7 days to feed to repletion, secrete immunosuppressive proteins. Included among these proteins is the 15-kDa salivary protein Salp15, which inhibits T cell activation and IL-2 production. Our objective for these studies was to evaluate the T cell inhibitory properties of Salp15 in a mouse model of allergic asthma. BALB/cJ mice were Ag sensitized by i.p. injection of OVA in aluminum hydroxide, with or without 50 mug of Salp15, on days 0 and 7. All mice were challenged with aerosolized OVA on days 14-16 and were studied on day 18. Compared with control mice sensitized with Ag, mice sensitized with Ag and Salp15 displayed significantly reduced airway hyperresponsiveness, eosinophilia, Ag-specific IgG1 and IgE, mucus cell metaplasia, and Th2 cytokine secretion in vivo and by CD4(+) T cells restimulated with Ag in vitro. Our results demonstrate that Salp15 can effectively prevent the generation of a Th2 immune response and the development of experimental asthma. These studies, and those of others, support the notion that a lack of ectoparasitism may contribute to the increasing prevalence of allergic asthma.     

Austrocedrus chilensis growth decline in relation to drought events in northern Patagonia, Argentina

The significant mortality of the Austrocedrus chilensis (D. Don) Pic. Serm. et Bizarri forests, locally known as “Mal del Ciprés”, has been reported since 1945 for most sites across its distribution in Argentina. However, the cause of this decline is still a topic of discussion. In this study, radial growth patterns from symptomatic and asymptomatic A. chilensis trees were analyzed to determine the influence of drought events on tree growth. Fifty pairs of symptomatic and asymptomatic trees with similar DBH, competition, and microsite conditions were cored at five pure A. chilensis stands near El Bolsón, Río Negro, Argentina. A reference chronology from nonaffected trees was used to cross-date all cores and to determine the relationship between A. chilensis radial growth and climate. The growth of A. chilensis is favored by above average precipitation in late spring–early summer (November and December). A strong relationship was also observed between radial growth patterns and the Palmer drought severity index, a measure of the regional water deficit. Significant differences in growth patterns were recorded between symptomatic and asymptomatic trees. Following extreme drought events, the growth of symptomatic trees is consistently lower than in asymptomatic trees. Based on the larger number of droughts recorded during the past decades and on future climatic predictions suggesting increasing trends in the frequency and intensity of drought events in northern Patagonia, a gradual increase in the number of trees affected by “Mal del Ciprés” along the twenty-first century is likely expected.     

METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis

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Aberrant expression of proteins involved in signal transduction and DNA repair pathways in lung cancer and their association with clinical parameters

BACKGROUND: Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues. METHODOLOGY/ PRINCIPAL FINDINGS: We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA) assay. The results showed that 18 molecules were significantly different (p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery. CONCLUSIONS/ SIGNIFICANCE: Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers.     

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.