Febrile temperature reprograms by redox-mediated signaling the mitochondrial metabolic phenotype in monocyte-derived dendritic cells

Fever-like hyperthermia is known to stimulate innate and adaptive immune responses. Hyperthermia-induced immune stimulation is also accompanied with, and likely conditioned by, changes in the cell metabolism and, in particular, mitochondrial metabolism is now recognized to play a pivotal role in this context, both as energy supplier and as signaling platform. In this study we asked if challenging human monocyte-derived dendritic cells with a relatively short-time thermal shock in the fever-range, typically observed in humans, caused alterations in the mitochondrial oxidative metabolism. We found that following hyperthermic stress (3 h exposure at 39 °C) TNF-α-releasing dendritic cells undergo rewiring of the oxidative metabolism hallmarked by decrease of the mitochondrial respiratory activity and of the oxidative phosphorylation and increase of lactate production. Moreover, enhanced production of reactive oxygen and nitrogen species and accumulation of mitochondrial Ca²⁺ was consistently observed in hyperthermia-conditioned dendritic cells and exhibited a reciprocal interplay. The hyperthermia-induced impairment of the mitochondrial respiratory activity was (i) irreversible following re-conditioning of cells to normothermia, (ii) mimicked by exposing normothermic cells to the conditioned medium of the hyperthermia-challenged cells, (iii) largely prevented by antioxidant and inhibitors of the nitric oxide synthase and of the mitochondrial calcium porter, which also inhibited release of TNF-α. These observations combined with gene expression analysis support a model based on a thermally induced autocrine signaling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells.     

Chromosome Separation and Exit from Mitosis in Budding Yeast: Dependence on Growth Revealed by cAMP-Mediated Inhibition

Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.     

Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco

A total of 274 bacterial strains were isolated from the root nodules of Prosopis juliflora, growing in two arid soils of the eastern area of Morocco. A physiological plate screening allowed the selection of 15 strains that could tolerate NaCl concentrations between 175 and 500 mM. These were compared with 15 strains chosen from among the ones which did not tolerate high salinity. The diversity of strains was first assessed by rep-PCR amplification fingerprinting using BOXA1R and ERIC primers. An analysis of the PCR-amplified 16S rDNA gene digestion profiles using five endonucleases indicated the presence of different lineages among the taxa associated with P. juliflora nodules in the soils studied. Nucleotide sequencing of the small subunit rRNA gene and BLAST analysis showed that P. juliflora could host at least six bacterial species in this region and that the identity of those associated with high salt tolerance was clearly distinct from that of the salt-sensitive ones. Among the former, the first type displayed 99% similarity with different members of the genus Sinorhizobium, the second 97% similarity with species within the genus Rhizobium, while the third ribosomal type had 100% homology to Achromobacter xylosoxidans. Within the salt-sensitive isolates the prevailing type observed showed 98% similarity with Rhizobium multihospitium and R. tropici, a second type had 98% similarity to R. giardinii, and a further case displayed 97% colinearity with the Ensifer group including E. maghrebium and E. xericitae. All of the thirty strains encompassing these types re-nodulated P. juliflora in microbiologically controlled conditions and all of them were shown to possess a copy of the nodC gene. This is the first report detecting the betaproteobacterial genus Achromobacter as nodule-forming species for legumes. The observed variability in symbiont species and the abundance of nodulation-proficient strains is in line with the observation that the plant always appears to be nodulated and efficiently fixing nitrogen in spite of a wide range of soil and environmental conditions.     

HPLC enantioseparation and absolute configuration of novel anti-inflammatory pyrrole derivatives

The assignment of the absolute configuration of novel anti-inflammatory pyrrole derivatives has been accomplished by a combined strategy based on independent physical methods. The key step of our stereochemical characterization approach is the production at mg-scale of enantiomerically pure forms by HPLC on Chiralpak IA stationary phase.     

Gamma proteobacteria can nodulate legumes of the genus Hedysarum

The bacteria hosted in the root nodules of the three Mediterranean wild legume species Hedysarum carnosum, Hedysarum spinosissimum subsp. capitatum, and Hedysarum pallidum, growing in native stands in different habitats in Algeria were isolated. Bacteria were recovered on yeast-mannitol-agar or on minimal media from a total of 52 nodules. Isolates were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) using the enzyme CfoI, and further sorted by RAPD fingerprinting. A total of ten different types were found and their amplified 16S rDNA was sequenced and compared to databases. The BLAST alignment indicates that all the species whose sequences share 98 to 100% identity to the bacteria found in these nodules belong to the class Gammaproteobacteria and include Pantoea agglomerans, Enterobacter kobei, Enterobacter cloacae, Leclercia adecarboxylata, Escherichia vulneris, and Pseudomonas sp. No evidence of any rhizobial-like sequence was found even upon amplifying from the bulk of microbial cells obtained from the squashed nodules, suggesting that the exclusive occupants of the nodules formed by the three plants tested are members of the orders Enterobacteriales or Pseudomonadales. This is the first report of Gammaproteobacteria associated with legume nodules. Despite the presence of the related crop plant Hedysarum coronarium, specifically nodulated by Rhizobium sullae, these three Hedysarum species demonstrate to have undergone a separate path in terms of endophytic interactions with bacteria. An hypothesis to account for differences between the symbiotic relationships engaged by man-managed legumes, and those found in plants whose ecology is independent from human action, is discussed.     

TSPO interacts with VDAC1 and triggers a ROS-mediated inhibition of mitochondrial quality control

The 18-kDa TSPO (translocator protein) localizes on the outer mitochondrial membrane (OMM) and participates in cholesterol transport. Here, we report that TSPO inhibits mitochondrial autophagy downstream of the PINK1-PARK2 pathway, preventing essential ubiquitination of proteins. TSPO abolishes mitochondrial relocation of SQSTM1/p62 (sequestosome 1), and consequently that of the autophagic marker LC3 (microtubule-associated protein 1 light chain 3), thus leading to an accumulation of dysfunctional mitochondria, altering the appearance of the network. Independent of cholesterol regulation, the modulation of mitophagy by TSPO is instead dependent on VDAC1 (voltage-dependent anion channel 1), to which TSPO binds, reducing mitochondrial coupling and promoting an overproduction of reactive oxygen species (ROS) that counteracts PARK2-mediated ubiquitination of proteins. These data identify TSPO as a novel element in the regulation of mitochondrial quality control by autophagy, and demonstrate the importance for cell homeostasis of its expression ratio with VDAC1.     

Multiple Marker Detection in Peripheral Blood for NSCLC Diagnosis

Background

Non-invasive early detection of lung cancer could reduce the number of patients diagnosed with advanced disease, which is associated with a poor prognosis. We analyzed the diagnostic accuracy of a panel of peripheral blood markers in detecting non small cell lung cancer (NSCLC).

Methods

100 healthy donors and 100 patients with NSCLC were enrolled onto this study. Free circulating DNA, circulating mRNA expression of peptidylarginine deiminase type 4 (PAD4/PADI4), pro-platelet basic protein (PPBP) and haptoglobin were evaluated using a Real-Time PCR-based method.

Results

Free circulating DNA, PADI4, PPBP and haptoglobin levels were significantly higher in NSCLC patients than in healthy donors (p<0.0001, p<0.0001, p = 0.0002 and p = 0.0001, respectively). The fitted logistic regression model demonstrated a significant direct association between marker expression and lung cancer risk. The odds ratios of individual markers were 6.93 (95% CI 4.15–11.58; p<0.0001) for free DNA, 6.99 (95% CI 3.75–13.03; p<0.0001) for PADI4, 2.85 (95% CI 1.71–4.75; p<0.0001) for PPBP and 1.16 (95% CI 1.01–1.33; p = 0.031) for haptoglobin. Free DNA in combination with PPBP and PADI4 gave an area under the ROC curve of 0.93, 95% CI = 0.90–0.97, with sensitivity and specificity over 90%.

Conclusions

Free circulating DNA analysis combined with PPBP and PADI4 expression determination appears to accurately discriminate between healthy donors and NSCLC patients. This non-invasive multimarker approach warrants further research to assess its potential role in the diagnostic or screening workup of subjects with suspected lung cancer.