Bronchoalveolar cytokine profile differentiates Pulmonary Langerhans cell histiocytosis patients from other smoking-related interstitial lung diseases.

BACKGROUND: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease (ILD) associated with smoking, whose definitive diagnosis requires the exclusion of other forms of ILD and a compatible surgical lung biopsy. Bronchoalveolar lavage (BAL) is commonly proposed for the diagnosis of ILD, including PLCH, but the diagnostic value of this technique is limited. Here, we have analyzed the levels of a panel of cytokines and chemokines in BAL from PLCH patients, in order to identify a distinct immune profile to discriminate PLCH from other smoking related-ILD (SR-ILD), and comparing the results with idiopathic pulmonary fibrosis (IPF) as another disease in which smoking is considered a risk factor. METHODS: BAL samples were collected from thirty-six patients with different ILD, including seven patients with PLCH, sixteen with SR-ILD and thirteen with IPF. Inflammatory profiles were analyzed using the Human Cytokine Membrane Antibody Array. Principal component analysis (PCA) was performed to reduce dimensionality and protein-protein interaction (PPI) network analysis using STRING 11.5 database were conducted. Finally, Random forest (RF) method was used to build a prediction model. RESULTS: We have found significant differences (p < 0.05) on thirty-two cytokines/chemokines when comparing BAL from PLCH patients with at least one of the other ILD. Four main groups of similarly regulated cytokines were established, identifying distinct sets of markers for each cluster. Exploratory analysis using PCA (principal component analysis) showed clustering and separation of patients, with the two first components capturing 69.69% of the total variance. Levels of TARC/CCL17, leptin, oncostatin M (OSM) and IP-10/CXCL10 were associated with lung function parameters, showing positive correlation with FVC. Finally, random forest (RF) algorithm demonstrates that PLCH patients can be differentiated from the other ILDs based solely on inflammatory profile (accuracy 96.25%). CONCLUSIONS: Our results show that patients with PLCH exhibit a distinct BAL immune profile to SR-ILD and IPF. PCA analysis and RF model identify a specific immune profile useful for discriminating PLCH.

Visualization of Lipid Droplets in the Alveolar Macrophage Cell Line MH-S with Live-cell Imaging by 3D Holotomographic Microscopy (Nanolive).

Lipid droplets (LD), triglycerides and sterol esters among them, are well known for their capacity as lipid storage organelles. Recently, they have emerged as critical cytoplasmic structures involved in numerous biological functions. LD storage is generated de novo by the cell and provides an energy reserve, lipid precursors, and cell protection. Moreover, LD accumulation can be observed in some pathologies as obesity, atherosclerosis, or lung diseases. Fluorescence imaging techniques are the most widely used techniques to visualize cellular compartments in live cells, including LD. Nevertheless, presence of fluorophores can damage subcellular components and induce cytotoxicity, or even alter the dynamics of the organelles. As an alternative to fluorescence microscopy, label-free techniques such as stimulated Raman scattering and coherent anti-stokes Raman scattering microscopy offer a solution to avoid the undesirable effects caused by dyes and fluorescent proteins, but are expensive and complex. Here, we describe a label-free method using live-cell imaging by 3D holotomographic microscopy (Nanolive) to visualize LD accumulation in the MH-S alveolar macrophage cell line after treatment with oleic acid, a monounsaturated fatty acid that promotes lipid accumulation.

A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and about 30% of the pneumococcal clinical isolates show type I pili-like structures. These long proteinaceous polymers extending from the bacterial surface are encoded by pilus islet 1 and play major roles in adhesion and host colonization. Pili expression is bistable and is controlled by the transcriptional activator RlrA. In this work, we demonstrate that the previously identified small noncoding RNA srn135 also participates in pilus regulation. Our findings show that srn135 is generated upon processing of the 5'-UTR region of rrgA messenger and its deletion prevents the synthesis of RrgA, the main pili adhesin. Moreover, overexpression of srn135 increases the expression of all pili genes and rises the percentage of piliated bacteria within a clonal population. This regulation is mediated by the stabilization of rlrA mRNA since higher levels of srn135 increase its half-life to 165%. Our findings suggest that srn135 has a dual role in pilus expression acting both in cis- (on the RrgA levels) and in trans- (modulating the levels of RlrA) and contributes to the delicate balance between pili expressing and non-expressing bacteria.

Local delivery of bone morphogenetic protein-2 from near infrared-responsive hydrogels for bone tissue regeneration.

Achievement of spatiotemporal control of growth factors production remains a main goal in tissue engineering. In the present work, we combined inducible transgene expression and near infrared (NIR)-responsive hydrogels technologies to develop a therapeutic platform for bone regeneration. A heat-activated and dimerizer-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in hydrogels based on fibrin and plasmonic gold nanoparticles that transduced incident energy of an NIR laser into heat. In the presence of dimerizer, photoinduced mild hyperthermia induced the release of bioactive BMP-2 from NIR-responsive cell constructs. A critical size bone defect, created in calvaria of immunocompetent mice, was filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the heat-activated and dimerizer-dependent gene circuit. In animals that were treated with dimerizer, NIR irradiation of implants induced BMP-2 production in the bone lesion. Induction of NIR-responsive cell constructs conditionally expressing BMP-2 in bone defects resulted in the formation of new mineralized tissue, thus indicating the therapeutic potential of the technological platform.

α-Hispanolol Induces Apoptosis and Suppresses Migration and Invasion of Glioblastoma Cells Likely via Downregulation of MMP-2/9 Expression and p38MAPK Attenuation.

α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G1 population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.

Mutations in TrkA Causing Congenital Insensitivity to Pain with Anhidrosis (CIPA) Induce Misfolding, Aggregation, and Mutation-dependent Neurodegeneration by Dysfunction of the Autophagic Flux.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder characterized by insensitivity to noxious stimuli and variable intellectual disability (ID) due to mutations in the NTRK1 gene encoding the NGF receptor TrkA. To get an insight in the effect of NTRK1 mutations in the cognitive phenotype we biochemically characterized three TrkA mutations identified in children diagnosed of CIPA with variable ID. These mutations are located in different domains of the protein; L213P in the extracellular domain, Δ736 in the kinase domain, and C300stop in the extracellular domain, a new mutation causing CIPA diagnosed in a Spanish teenager. We found that TrkA mutations induce misfolding, retention in the endoplasmic reticulum (ER), and aggregation in a mutation-dependent manner. The distinct mutations are degraded with a different kinetics by different ER quality control mechanisms; although C300stop is rapidly disposed by autophagy, Δ736 degradation is sensitive to the proteasome and to autophagy inhibitors, and L213P is a long-lived protein refractory to degradation. In addition L213P enhances the formation of autophagic vesicles triggering an increase in the autophagic flux with deleterious consequences. Mouse cortical neurons expressing L213P showed the accumulation of LC3-GFP positive puncta and dystrophic neurites. Our data suggest that TrkA misfolding and aggregation induced by some CIPA mutations disrupt the autophagy homeostasis causing neurodegeneration. We propose that distinct disease-causing mutations of TrkA generate different levels of cell toxicity, which may provide an explanation of the variable intellectual disability observed in CIPA patients.

Small regulatory RNAs in Streptococcus pneumoniae: discovery and biological functions.

Streptococcus pneumoniae is a prominent human pathogen responsible for many severe diseases and the leading cause of childhood mortality worldwide. The pneumococcus is remarkably adept at colonizing and infecting different niches in the human body, and its adaptation to dynamic host environment is a central aspect of its pathogenesis. In the last decade, increasing findings have evidenced small RNAs (sRNAs) as vital regulators in a number of important processes in bacteria. In S. pneumoniae, a small antisense RNA was first discovered in the pMV158 plasmid as a copy number regulator. More recently, genome-wide screens revealed that the pneumococcal genome also encodes multiple sRNAs, many of which have important roles in virulence while some are implicated in competence control. The knowledge of the sRNA-mediated regulation in pneumococcus remains very limited, and future research is needed for better understanding of functions and mechanisms. Here, we provide a comprehensive summary of the current knowledge on sRNAs from S. pneumoniae, focusing mainly on the trans-encoded sRNAs.

Isolation of a point mutation associated with altered expression of the CmeABC efflux pump in a multidrug-resistant Campylobacter jejuni population of poultry origin.

The objective of this study was to investigate the antibiotic resistance phenotype of Campylobacter jejuni isolates from a poultry flock of broiler production in Spain. Isolates were characterised by RFLP-PCR of the flaA gene and multilocus sequence typing. Minimum inhibitory concentrations of quinolones, aminoglycosides, ß-lactams, tetracyclines, phenicols, macrolides and lincosamides were determined by Etest. Determinants of resistance and the regulatory region of the cmeABC operon were investigated in all isolates by PCR detection and sequencing. Expression of the CmeABC efflux pump was investigated by quantitative RT-PCR and accumulation assay. Based on their molecular markers, two different populations of C. jejuni were identified: one resistant to quinolones, ß-lactams and tetracyclines, considered multidrug-resistant (MDR); and another resistant only to tetracyclines. Both populations possessed the tetO gene, previously associated with tetracycline resistance. The blaOXA-61 gene was also present in both populations, although only the MDR population showed ß-lactamase activity. In addition, MDR isolates possessed the Thr86Ile mutation in the gyrA gene responsible for quinolone resistance. Moreover, sequencing of the regulatory region of the cmeABC operon revealed the presence of the C-32→T mutation in the MDR isolates, which was accompanied by an increase in cmeA mRNA levels compared with the non-mutant population. In conclusion, this is the first report of the mutation C-32→T in the cmeABC operon in C. jejuni isolates of veterinary origin. This mutation is associated with overexpression of the CmeABC efflux pump in a MDR population and is possibly related to enhanced tolerance to antimicrobials that favours the development of resistance.

Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae.

Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5'- and/or 3'-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen.

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold.

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity-dormancy transition.

Cancer abolishes the tissue type-specific differences in the phenotype of energetic metabolism.

Nowadays, cellular bioenergetics has become a central issue of investigation in cancer biology. Recently, the metabolic activity of the cancer cell has been shown to correlate with a proteomic index that informs of the relative mitochondrial activity of the cell. Within this new field of investigation, we report herein the production and characterization of high-affinity monoclonal antibodies against proteins of the "bioenergetic signature" of the cell. The use of recombinant proteins and antibodies against the mitochondrial beta-F1-ATPase and Hsp60 proteins and the enzymes of the glycolytic pathway glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase M2 in quantitative assays provide, for the first time, the actual amount of these proteins in normal and tumor surgical specimens of breast, lung, and esophagus. The application of this methodology affords a straightforward proteomic signature that quantifies the variable energetic demand of human tissues. Furthermore, the results show an unanticipated finding: tumors from different tissues and/or histological types have the same proteomic signature of energetic metabolism. Therefore, the results indicate that cancer abolishes the tissue-specific differences in the bioenergetic phenotype of mitochondria. Overall, the results support that energetic metabolism represents an additional hallmark of the phenotype of the cancer cell and a promising target for the treatment of diverse neoplasias.

Overall alteration of circadian clock gene expression in the chestnut cold response.

Cold acclimation in woody plants may have special features compared to similar processes in herbaceous plants. Recent studies have shown that circadian clock behavior in the chestnut tree (Castanea sativa) is disrupted by cold temperatures and that the primary oscillator feedback loop is not functional at 4 degrees C or in winter. In these conditions, CsTOC1 and CsLHY genes are constantly expressed. Here, we show that this alteration also affects CsPRR5, CsPRR7 and CsPRR9. These genes are homologous to the corresponding Arabidopsis PSEUDO-RESPONSE REGULATOR genes, which are also components of the circadian oscillator feedback network. The practically constant presence of mRNAs of the 5 chestnut genes at low temperature reveals an unknown aspect of clock regulation and suggests a mechanism regulating the transcription of oscillator genes as a whole.

Breast carcinomas fulfill the Warburg hypothesis and provide metabolic markers of cancer prognosis.

The aim of this study was to investigate selected proteomic markers of the metabolic phenotype of breast carcinomas as prognostic markers of cancer progression. For this purpose, a series of 101 breast carcinomas and 13 uninvolved breast samples were examined for quantitative differences in protein expression of mitochondrial and glycolytic markers. The beta-subunit of the mitochondrial H(+)-ATP synthase (beta-F1-ATPase) and heat shock protein 60 (Hsp60), and the glycolytic glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase were identified by immunological techniques. Correlations of the expression level of the protein markers and of the ratios derived from them were established with the clinicopathological information of the tumors and the follow-up data of the patients. The metabolic proteome of breast cancer specimens revealed a pronounced shift towards an enhanced glycolytic phenotype concurrent with a profound alteration on the mitochondrial beta-F1-ATPase/Hsp60 ratio when compared with normal samples. Discriminant analysis using markers of the metabolic signature as predictor variables revealed a classification sensitivity of approximately 97%. Kaplan-Meier survival analysis showed that several of the proteomic variables significantly correlated with overall and disease-free survival of the patients. The expression level of beta-F1-ATPase per se allowed the identification of a subgroup of breast cancer patients with significantly worse prognosis. Multivariate Cox regression analysis indicated that tumor expression of beta-F1-ATPase is a significant marker independent from clinical variables to assess the prognosis of the patients. We conclude that the alteration of the mitochondrial and glycolytic proteomes is a hallmark feature of breast cancer further providing relevant markers to aid in the prognosis of breast cancer patients.

Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: designing new cytotoxic compounds.

Safracin is an antibiotic with anti-tumour activity produced by Pseudomonas fluorescens A2-2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non-ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross-feeding experiments have confirmed that 3-hydroxy-5-methyl-O-methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N-methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti-cancer compounds.

Quantitative detection of Streptococcus pneumoniae cells harbouring single or multiple copies of the gene encoding the green fluorescent protein.

A modified gfp gene from Aequorea victoria, encoding a variant of the green fluorescent protein (GFP), was subcloned into the mobilizable plasmid pMV158. gfp was placed under the control of the inducible P(M) promoter of the Streptococcus pneumoniae gene malM, cloned in plasmid pLS70. The P(M) promoter is regulated by the product of the pneumococcal malR gene, which is inactivated by growing the cells in maltose-containing media. By homologous recombination, the P(M)-gfp construction was integrated into the host chromosome in a single copy. In both conditions (single and multiple copies), the pneumococcal cells were able to express GFP in an inducible or constitutive form, depending on whether the S. pneumoniae strain harboured a wild-type or a mutant malR gene. Quantification of the levels of GFP expressed by cultures supplemented with sucrose or maltose as carbon sources was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed pneumococcal cells expressing GFP in mixed cultures to be distinguished from those not carrying the gfp gene.