Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.

Summary Zampanolide and its less active analog dactylolide compete with paclitaxel for binding to microtubules and represent a new class of microtubule-stabilizing agent (MSA). Mass spectrometry demonstrated that the mechanism of action of both compounds involved covalent binding to β-tubulin at residues N228 and H229 in the taxane site of the microtubule. Alkylation of N228 and H229 was also detected in α,β-tubulin dimers. However, unlike cyclostreptin, the other known MSA that alkylates β-tubulin, zampanolide was a strong MSA. Modeling the structure of the adducts, using the NMR-derived dactylolide conformation, indicated that the stabilizing activity of zampanolide is likely due to interactions with the M-loop. Our results strongly support the existence of the luminal taxane site of microtubules in tubulin dimers and that microtubule nucleation induction by MSAs may proceed through an allosteric mechanism.

The Pseudomonas aeruginosa genome contains several different multidrug resistance (MDR) efflux pumps. Overproduction of these pumps reduces susceptibility to a variety of antibiotics. Some recently published works have analyzed the effect of the overproduction of MDR efflux pumps on bacterial virulence. Here we have studied the effect of overproduction of the efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY on type III secretion (T3S) in P. aeruginosa. The type III secretion system (T3SS) is used by P. aeruginosa to deliver toxins directly into the cytoplasm of the host cell. Our data indicate that overexpression of either MexCD-OprJ or MexEF-OprN is associated with the impairment of T3S in P. aeruginosa. No effect on overexpression of either MexAB-OprM or MexXY was detected. The observed defect in T3S was due to a lack of expression of genes belonging to the T3SS regulon. Transcription of this regulon is activated by ExsA in response to environmental signals. Overexpression of this transcriptional regulator complemented the defect in T3S observed in the MexCD-OprJ-and MexEF-OprN-overproducing strains. Taken together, these results suggest that overproduction of either MexCD-OprJ or MexEF-OprN is associated with a reduction in the transcription of the T3SS regulon due to the lack of expression of the exsA gene, encoding the master regulator of the system. The relevance of potential metabolic and quorum-sensing imbalances due to overexpression of MDR pumps associated with this phenotype is also discussed.

Summary Like normal hematopoietic stem cells, leukemic stem cells depend on their bone marrow (BM) microenvironment for survival, but the underlying mechanisms remain largely unknown. We have studied the contribution of nestin + BM mesenchymal stem cells (BMSCs) to MLL-AF9-driven acute myeloid leukemia (AML) development and chemoresistance in vivo . Unlike bulk stroma, nestin + BMSC numbers are not reduced in AML, but their function changes to support AML cells, at the expense of non-mutated hematopoietic stem cells (HSCs). Nestin + cell depletion delays leukemogenesis in primary AML mice and selectively decreases AML, but not normal, cells in chimeric mice. Nestin + BMSCs support survival and chemotherapy relapse of AML through increased oxidative phosphorylation, tricarboxylic acid (TCA) cycle activity, and glutathione (GSH)-mediated antioxidant defense. Therefore, AML cells co-opt energy sources and antioxidant defense mechanisms from BMSCs to survive chemotherapy.

Exosomes are gaining importance because they show great promise in therapeutic applications for several diseases. Particularly in stroke, exosomes derived from mesenchymal stem cell (MSC) therapy work as paracrine effectors responsible for promoting neurovascular remodeling and functional recovery. Adult male rats were subjected to intracerebral hemorrhage (ICH) by intrastriatal injection of collagenase type IV; 24 h after surgery, MSC-derived exosomes were administered through the tail vein. The rats were euthanized at 7 or 28 days after treatment. Functional evaluation, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers, biodistribution of exosomes and secretome proteomics were analyzed. DiI-labeled exosomes were found in the brains of the ICH-treated group and in the liver, lung and spleen. Animals receiving treatment with exosomes showed significantly better results in terms of functional recovery, lesion size, fiber tract integrity, axonal sprouting and white matter repair markers compared with the control group 28 days after stroke. Proteomics analysis of the exosomes identified more than 2000 proteins that could be implicated in brain repair function. In conclusion, white matter integrity was partly restored by exosome administration mediated by molecular repair factors. Exosomes as a treatment could be a heterogeneous process by nature and presents many factors that can influence brain plasticity in an adaptable and versatile manner.

Proteomic analysis of omental fat reveals differential expression of several proteins in PCOS patients and non-hyperandrogenic women presenting with morbid obesity. The application of this novel methodology adds further evidence to support the role of visceral adiposity in the pathogenesis of PCOS.

Obesity is increasing exponentially in developed countries and constitutes a public health problem by enhancing the risk for metabolic disorder and cardiovascular disease. Differences in gene expression profiles and in metabolic and biochemical properties have been well-described between omental and subcutaneous adipose tissue in humans. Because omental adipose tissue has been strongly associated with the development of insulin resistance, type 2 diabetes and cardiovascular disease, we searched for proteins differentially expressed in these two fat depots using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). In this analysis, we found 43 proteins, several of which were validated by immunoblotting and immunostaining analyses. Results demonstrated tissue-specific molecular differences in the protein makeup of the two analyzed fat depots mainly related to metabolic processes such as glucose and lipid metabolism, lipid transport, protein synthesis, protein folding, response to stress and inflammation. This suggests higher metabolic activity as well as increased cell stress in the omental compared to the subcutaneous fat. These findings provide some insights into the role of omental fat in abdominal obesity-associated co-morbidities.