Bovine serum promotes the formation and phenotype memory retention of persister cells in Escherichia coli liquid cultures.

Persister cells, or persisters, are a fraction of bacterial cells that have become temporarily tolerant to antibiotics despite their lack of typical antibiotic-resistant genes. In a previous study, we found that colony-biofilm culture (i.e., biofilm formed at an air-solid interface) promoted the formation and phenotype memory retention of persisters of Escherichia coli and other bacteria. To assess whether these same effects are caused by other types of stimuli that bacterial cells encounter in the environment, we examined the effects of bovine serum on the formation and phenotype retention of ampicillin-tolerant persisters in E. coli liquid culture. Bovine serum did indeed exert these effects significantly, and its effects were negated by heat treatment. Similar effects were observed with bovine serum albumin, albeit weaker than those of BS. Given that serum is a component of blood and lymph and is thus a general substance within animal and human bodies, our findings suggest that bacteria encountering these body fluids may enhance their abilities for persister formation and phenotype memory retention to allow their longer survival in antibiotic-containing environments.

Paracoccidioides species present distinct fungal adherence to epithelial lung cells and promote different IL-8 secretion levels.

Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.

Histoplasma capsulatum chemotypes I and II induce IL-8 secretion in lung epithelial cells in distinct manners.

The cell wall is one of the most important structures of pathogenic fungi, enabling initial interaction with the host and consequent modulation of immunological responses. Over the years, some researchers have shown that cell wall components of Histoplasma capsulatum vary among fungal isolates, and one of the major differences is the presence or absence of α-(1,3)-glucan, classifying wild-type fungi as chemotypes II or I, respectively. The present work shows that an isolate of H. capsulatum chemotype I induced lower levels of interleukin (IL)-8 secretion by the lung epithelial cell line A549, when compared to chemotype II yeasts. Thus, we expected that the absence of α-glucan in spontaneous variant yeasts, which were isolated from chemotype II cultures, would modify IL-8 secretion by A549 cells, but surprisingly, these fungi promoted similar levels of IL-8 secretion as their wild-type counterpart. Furthermore, when using a specific inhibitor for Syk activation, we observed that this inhibitor reduced IL-8 levels in A549 cell cultures infected with wild type chemotype I fungi. This inhibitor failed to reduce this cytokine levels in A549 cell cultures infected with chemotype II and their spontaneous variant yeasts, which also do not present α-glucan on their surface. The importance of SFKs and PKC δ in this event was also analyzed. Our results show that different isolates of H. capsulatum modulate distinct cell signaling pathways to promote cytokine secretion in host epithelial cells, emphasizing the existence of various mechanisms for Histoplasma pathogenicity.

Secretion of high amounts of hepatocyte growth factor is a characteristic feature of cancer-associated fibroblasts with EGFR-TKI resistance-promoting phenotype: A study of 18 cases of cancer-associated fibroblasts.

Humoral factors from cancer-associated fibroblasts (CAFs) reportedly affect epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance in cancer cells with EGFR mutations. The aim of this study was to identify the robust humoral factors secreted from CAFs that induce the primary resistance to EGFR-TKI. We evaluated the EGFR-TKI sensitivity of EGFR-mutant lung adenocarcinoma cell line (PC-9) treated with condition media (CM) from 18 cases of CAFs and matched non-cancerous-tissue-associated fibroblasts (NCAFs). We measured the expression levels of hepatocyte growth factor (HGF), interleukin-6, fibroblast growth factor-2, insulin-like growth factor-1, and vascular endothelial growth factor-A in CAFs and NCAFs. We examined whether HGF neutralizing antibody could annul the EGFR-TKI resistance induced by CM from CAFs. Compared to CM from NCAFs, CM from CAFs increased the resistance of PC-9 cells to EGFR-TKI in five out of 18 cases. Relative expression ratio of HGF messenger RNA was significantly higher in these five CAFs compared to others (P = 0.0013), whereas other cytokines were not. In four of these five cases, the addition of HGF neutralizing antibody significantly decreased the survival ratio of PC-9 cells. This study suggests that the secretion of higher amounts of HGF is the robust feature of EGFR-TKI resistance-promoting CAFs.

T Cell-Independent Mechanisms Associated with Neutrophil Extracellular Trap Formation and Selective Autophagy in IL-17A-Mediated Epidermal Hyperplasia.

IL-17A has been strongly associated with epidermal hyperplasia in many cutaneous disorders. However, because IL-17A is mainly produced by αß and γδT cells in response to IL-23, the role of T cells and IL-23 has overshadowed any IL-17A-independent actions. In this article, we report that IL-17A gene transfer induces epidermal hyperplasia in Il23r-/-Rag1-/-- and Tcrδ-deficient mice, which can be prevented by neutrophil depletion. Moreover, adoptive transfer of CD11b+Gr-1hi cells, after IL-17A gene transfer, was sufficient to phenocopy the disease. We further show that the IL-17A-induced pathology was prevented in transgenic mice with impaired neutrophil extracellular trap formation and/or neutrophils with conditional deletion of the master regulator of selective autophagy, Wdfy3. Our data demonstrate a novel T cell-independent mechanism that is associated with neutrophil extracellular trap formation and selective autophagy in IL-17A-mediated epidermal hyperplasia.

Paracoccidioides brasiliensis induces cytokine secretion in epithelial cells in a protease-activated receptor-dependent (PAR) manner.

Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.

IL-17A gene transfer induces bone loss and epidermal hyperplasia associated with psoriatic arthritis.

BACKGROUND: Psoriatic arthritis (PsA) is a chronic inflammatory disease characterised by clinical features that include bone loss and epidermal hyperplasia. Aberrant cytokine expression has been linked to joint and skin pathology; however, it is unclear which cytokines are critical for disease initiation. Interleukin 17A (IL-17A) participates in many pathological immune responses; however, its role in PsA has not been fully elucidated. OBJECTIVE: To determine the role of IL-17A in epidermal hyperplasia and bone destruction associated with psoriatic arthritis. DESIGN: An in vivo gene transfer approach was used to investigate the role of IL-17A in animal models of inflammatory (collagen-induced arthritis) and non-inflammatory (receptor activator of NF-κB ligand (RANKL)-gene transfer) bone loss. RESULTS: IL-17A gene transfer induced the expansion of IL-17RA(+)CD11b(+)Gr1(low) osteoclast precursors and a concomitant elevation of biomarkers indicative of bone resorption. This occurred at a time preceding noticeable joint inflammation, suggesting that IL-17A is critical for the induction of pathological bone resorption through direct activation of osteoclast precursors. Moreover, IL-17A induced a second myeloid population CD11b(+)Gr1(high) neutrophil-like cells, which was associated with cutaneous pathology including epidermal hyperplasia, parakeratosis and Munro's microabscesses formation. CONCLUSIONS: Collectively, these data support that IL-17A can play a key role in the pathogenesis of inflammation-associated arthritis and/or skin disease, as observed in PsA.

Discovery of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase involved in the metabolism of N-glycans.

A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, ß-N-acetylhexosaminidase, exo-α-sialidase, and endo-ß-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where ß-1,4-D-mannosyl-N-acetyl-D-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-D-mannose 1-phosphate and N-acetyl-D-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where ß-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the D-mannose residue of ß-1,4-D-mannosyl-N-acetyl-D-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-ß-D-mannopyranosyl-N-acetyl-D-glucosamine:phosphate α-D-mannosyltransferase as the systematic name and ß-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase as the short name for BT1033.

The impaired viability of prostate cancer cell lines by the recombinant plant kallikrein inhibitor.

BACKGROUND: Kallikreins play a pivotal role in establishing prostate cancer. RESULTS: In contrast to the classical Kunitz plant inhibitor SbTI, the recombinant kallikrein inhibitor (rBbKIm) led to prostate cancer cell death, whereas fibroblast viability was not affected. CONCLUSION: rBbKIm shows selective cytotoxic effect and angiogenesis inhibition against prostate cancer cells. SIGNIFICANCE: New actions of rBbKIm may contribute to understanding the mechanisms of prostate cancer. Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145.

Prevalence, genetic characteristics, and antimicrobial resistance of staphylococcal isolates from oral cavity and skin surface of healthy individuals in northern Japan.

BACKGROUND: Oral cavity is an ecological niche for colonization of staphylococci, which are a major bacterial species causing community-acquired infections in humans. In this study, prevalence, and characteristics of staphylococci in oral cavity and skin of healthy individuals were investigated in northern Japan. METHODS: Saliva from oral cavity and swab from skin surface of hand were collected and cultured on selective media. Species of the isolates were identified genetically, and ST was determined for S. aureus and S. argenteus. Genes associated with antimicrobial resistance were detected by PCR. RESULTS: Among 166 participants, a total of 75 S. aureus isolates were obtained from 61 individuals (37 %), and recovered more frequently in oral cavity (n = 48) than skin (n = 27). Among 23 STs identified in S. aureus isolates, ST8 (CC8), ST15 (CC15), and ST188 (CC1) were the most common (10 isolates each), with STs of CC1 being dominant (17 isolates). Methicillin-resistant S. aureus (MRSA) was isolated in the skin of two individuals and belonged to ST1 and ST6. Resistance to erythromycin and gentamicin associated with erm(A) and aac(6')-Ie-aph(2")-Ia, respectively, was more commonly found in ST5 and ST8 isolates. One S. argenteus isolate (ST2250, mecA-negative) was recovered from oral cavity of a participant (0.6 %). A total of 186 isolates of coagulase-negative staphylococci (CoNS) were recovered from 102 participants and identified into 14 species, with S. warneri being the most common (n = 52), followed by S. capitis (n = 42), S. saprophyticus (n = 20) and S. haemolyticus (n = 19). mecA was detected in S. saprophyticus, S. haemolyticus, and S. caprae, while arginine-catabolic mobile element (ACME) in only S. capitis and S. epidermidis. CONCLUSION: S. aureus was more prevalent in oral cavity than skin surface, belonging to three major STs, with CC1 being a dominant lineage. The prevalence of antimicrobial resistance was distinct depending on CoNS species.

Enterolobium contortisiliquum trypsin inhibitor (EcTI), a plant proteinase inhibitor, decreases in vitro cell adhesion and invasion by inhibition of Src protein-focal adhesion kinase (FAK) signaling pathways.

Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin ß1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.

Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity.

Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mßCD). Removal of ergosterol by mßCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mßCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mßCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.

Paracoccidioides brasiliensis Induces α3 Integrin Lysosomal Degradation in Lung Epithelial Cells.

Studies on the pathogen-host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the expression of α3 integrin in lung epithelial cells within 5 h of infection. Interestingly, α3 integrin levels were reduced by approximately 99% after 24 h of infection with P. brasiliensis compared to non-infected cells. In this work, we show that, during infection with this fungus, α3 integrin is increased in the late endosomes of A549 lung epithelial cells. We also observed that the inhibitor of the lysosomal activity bafilomycin A1 was able to inhibit the decrease in α3 integrin levels. In addition, the silencing of the charged multivesicular body protein 3 (CHMP3) inhibited the reduction in α3 integrin levels induced by P. brasiliensis in A549 cells. Thus, together, these results indicate that this fungus induces the degradation of α3 integrin in A549 lung epithelial cells by hijacking the host cell endolysosomal pathway.

Alternative Methods for Pulmonary-Administered Drugs Metabolism: A Breath of Change.

Prediction of pulmonary metabolites following inhalation of a locally acting pulmonary drug is essential to the successful development of novel inhaled medicines. The lungs present metabolic enzymes, therefore they influence drug disposal and toxicity. The present review provides an overview of alternative methods to evaluate the pulmonary metabolism for the safety and efficacy of pulmonary delivery systems. In vitro approaches for investigating pulmonary drug metabolism were described, including subcellular fractions, cell culture models and lung slices as the main available in vitro methods. In addition, in silico studies are promising alternatives that use specific software to predict pulmonary drug metabolism, determine whether a molecule will react with a metabolic enzyme, the site of metabolism (SoM) and the result of this interaction. They can be used in an integrated approach to delineate the major cytochrome P450 (CYP) isoforms to rationalize the use of in vivo methods. A case study about a combination of experimental and computational approaches was done using fluticasone propionate as an example. The results of three tested software, RSWebPredictor, SMARTCyp and XenoSite, demonstrated greater probability of the fluticasone propionate being metabolized by CYPs 3A4 at the S1 atom of 5-S-fluoromethyl carbothioate group. As the in vitro studies were not able to directly detect pulmonary metabolites, those alternatives in silico methods may reduce animal testing efforts, following the principle of 3Rs (Replacement, Reduction and Refinement), and contribute to the evaluation of pharmacological efficacy and safety profiles of new drugs in development.

Rheumatoid and pyrophosphate arthritis synovial fibroblasts induce osteoclastogenesis independently of RANKL, TNF and IL-6.

Bone destruction is a common feature of inflammatory arthritis and is mediated by osteoclasts, the only specialized cells to carry out bone resorption. Aberrant expression of receptor activator of nuclear factor kappa ß ligand (RANKL), an inducer of osteoclast differentiation has been linked with bone pathology and the synovial fibroblast in rheumatoid arthritis (RA). In this manuscript, we challenge the current concept that an increase in RANKL expression governs osteoclastogenesis and bone destruction in autoimmune arthritis. We isolated human fibroblasts from RA, pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients and analyzed their RANKL/OPG expression profile and the capacity of their secreted factors to induce osteoclastogenesis. We determined a 10-fold increase of RANKL mRNA and protein in fibroblasts isolated from RA relative to PPA and OA patients. Peripheral blood mononuclear cells (PBMC) from healthy volunteers were cultured in the presence of RA, PPA and OA synovial fibroblast conditioned medium. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), F-actin ring formation and bone resorption assays. The formation of TRAP(+), VNR(+) multinucleated cells, capable of F-actin ring formation and lacunar resorption in synovial fibroblast conditioned medium cultures occured in the presence of osteoprotegerin (OPG) a RANKL antagonist. Osteoclasts did not form in these cultures in the absence of macrophage colony stimulating factor (M-CSF). Our data suggest that the conditioned medium of pure synovial fibroblast cultures contain inflammatory mediators that can induce osteoclast formation in human PBMC independently of RANKL. Moreover inhibition of the TNF or IL-6 pathway was not sufficient to abolish osteoclastogenic signals derived from arthritic synovial fibroblasts. Collectively, our data clearly show that alternate osteoclastogenic pathways exist in inflammatory arthritis and place the synovial fibroblast as a key regulatory cell in bone and joint destruction, which is a hallmark of autoimmune arthritis.

Sulfatides inhibit adhesion, migration, and invasion of murine melanoma B16F10 cell line in vitro.

Endogenous sulfatide, such as 3-sulfated galactosylceramide (3-sulfatide) has been reported to be involved in neuronal development and regulation of tumor cell metastasis. Recently, a new 6-sulfated glucosylceramide (6-sulfatide) has been isolated from the ascidian, Ciona intestinalis. To determine the antitumor function of the new sulfatide, we examined the effects of synthetic 6-sulfatide and 3-sulfatide on the metastatic features of a murine melanoma cell line, B16F10. Both sulfatides significantly inhibited the adhesion of melanoma cells onto fibronectin-coated tissue plates and, the motility and invasion of the cells, with 6-sulfatide showing stronger inhibitory activities. In addition, both sulfatides inhibited α(5)-, and ß(1)- but not α(v)- or ß(3)-integrin expression. Furthermore, these sulfatides inhibited the activation of focal adhesion kinase, Akt, and extracellular signal-regulated kinase signaling pathways, which are thought to be important for cell migration and invasion. Therefore, these sulfatides may serve as promising drug candidates for the treatment of cancer metastasis.

Respiratory Epithelial Cells: More Than Just a Physical Barrier to Fungal Infections.

The respiratory epithelium is highly complex, and its composition varies along the conducting airways and alveoli. In addition to their primary function in maintaining the respiratory barrier and lung homeostasis for gas exchange, epithelial cells interact with inhaled pathogens, which can manipulate cell signaling pathways, promoting adhesion to these cells or hosting tissue invasion. Moreover, pathogens (or their products) can induce the secretion of chemokines and cytokines by epithelial cells, and in this way, these host cells communicate with the immune system, modulating host defenses and inflammatory outcomes. This review will focus on the response of respiratory epithelial cells to two human fungal pathogens that cause systemic mycoses: Aspergillus and Paracoccidioides. Some of the host epithelial cell receptors and signaling pathways, in addition to fungal adhesins or other molecules that are responsible for fungal adhesion, invasion, or induction of cytokine secretion will be addressed in this review.

Shared Nearest Neighbors Approach and Interactive Browser for Network Analysis of a Comprehensive Non-Small-Cell Lung Cancer Data Set.

PURPOSE: Advances in biological measurement technologies are enabling large-scale studies of patient cohorts across multiple omics platforms. Holistic analysis of these data can generate actionable insights for translational research and necessitate new approaches for data integration and mining. METHODS: We present a novel approach for integrating data across platforms on the basis of the shared nearest neighbors algorithm and use it to create a network of multiplatform data from the immunogenomic profiling of non-small-cell lung cancer project. RESULTS: Benchmarking demonstrates that the shared nearest neighbors-based network approach outperforms a traditional gene-gene network in capturing established interactions while providing new ones on the basis of the interplay between measurements from different platforms. When used to examine patient characteristics of interest, our approach provided signatures associated with and new leads related to recurrence and TP53 oncogenotype. CONCLUSION: The network developed offers an unprecedented, holistic view into immunogenomic profiling of non-small-cell lung cancer, which can be explored through the accompanying interactive browser that we built.

Bone morphogenetic protein-2 functions as a negative regulator in the differentiation of myoblasts, but not as an inducer for the formations of cartilage and bone in mouse embryonic tongue.

BACKGROUND: In vitro studies using the myogenic cell line C2C12 demonstrate that bone morphogenetic protein-2 (BMP-2) converts the developmental pathway of C2C12 from a myogenic cell lineage to an osteoblastic cell lineage. Further, in vivo studies using null mutation mice demonstrate that BMPs inhibit the specification of the developmental fate of myogenic progenitor cells. However, the roles of BMPs in the phases of differentiation and maturation in skeletal muscles have yet to be determined. The present study attempts to define the function of BMP-2 in the final stage of differentiation of mouse tongue myoblast. RESULTS: Recombinant BMP-2 inhibited the expressions of markers for the differentiation of skeletal muscle cells, such as myogenin, muscle creatine kinase (MCK), and fast myosin heavy chain (fMyHC), whereas BMP-2 siRNA stimulated such markers. Neither the recombinant BMP-2 nor BMP-2 siRNA altered the expressions of markers for the formation of cartilage and bone, such as osteocalcin, alkaline phosphatase (ALP), collagen II, and collagen X. Further, no formation of cartilage and bone was observed in the recombinant BMP-2-treated tongues based on Alizarin red and Alcian blue stainings. Neither recombinant BMP-2 nor BMP-2 siRNA affected the expression of inhibitor of DNA binding/differentiation 1 (Id1). The ratios of chondrogenic and osteogenic markers relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a house keeping gene) were approximately 1000-fold lower than those of myogenic markers in the cultured tongue. CONCLUSIONS: BMP-2 functions as a negative regulator for the final differentiation of tongue myoblasts, but not as an inducer for the formation of cartilage and bone in cultured tongue, probably because the genes related to myogenesis are in an activation mode, while the genes related to chondrogenesis and osteogenesis are in a silencing mode.

OsJAR1 and OsJAR2 are jasmonyl-L-isoleucine synthases involved in wound- and pathogen-induced jasmonic acid signalling.

The synthesis of JA-Ile was catalysed by JA-Ile synthase, which is a member of the group I GH3 family of proteins. Here, we showed evidence that OsGH3.5 (OsJAR1) and OsGH3.3 (OsJAR2) are the functional JA-Ile synthases in rice, using recombinant proteins. The expression levels of OsJAR1 and OsJAR2 were induced in response to wounding with the concomitant accumulation of JA-Ile. In contrast, only the expression of OsJAR1 was associated with the accumulation of JA-Ile after blast infection. Our data suggest that these two JA-Ile synthases are differentially involved in the activation of JA signalling in response to wounding and pathogen challenge in rice.