An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148 993 and 121 703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25 342 human and 24 109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3′ end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a ∼80% correlation with hybridizations performed on Affymetrix GeneChip™ suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE

Comparative Proteomic Analysis of Transcriptional and Regulatory Proteins Abundances in S. lividans and S. coelicolor Suggests a Link between Various Stresses and Antibiotic Production

Streptomyces coelicolor and Streptomyces lividans constitute model strains to study the regulation of antibiotics biosynthesis in Streptomyces species since these closely related strains possess the same pathways directing the biosynthesis of various antibiotics but only S. coelicolor produces them. To get a better understanding of the origin of the contrasted abilities of these strains to produce bioactive specialized metabolites, these strains were grown in conditions of phosphate limitation or proficiency and a comparative analysis of their transcriptional/regulatory proteins was carried out. The abundance of the vast majority of the 355 proteins detected greatly differed between these two strains and responded differently to phosphate availability. This study confirmed, consistently with previous studies, that S. coelicolor suffers from nitrogen stress. This stress likely triggers the degradation of the nitrogen-rich peptidoglycan cell wall in order to recycle nitrogen present in its constituents, resulting in cell wall stress. When an altered cell wall is unable to fulfill its osmo-protective function, the bacteria also suffer from osmotic stress. This study thus revealed that these three stresses are intimately linked in S. coelicolor. The aggravation of these stresses leading to an increase of antibiotic biosynthesis, the connection between these stresses, and antibiotic production are discussed

A proteomic analysis indicates that oxidative stress is the common feature triggering antibiotic production in Streptomyces coelicolor and in the pptA mutant of Streptomyces lividans

International audienceIn most Streptomyces species, antibiotic production is triggered in phosphate limitation and repressed in phosphate proficiency. However, the model strain, Streptomyces coelicolor , escapes this general rule and produces actinorhoddin (ACT), a polyketide antibiotic, even more abundantly in phosphate proficiency than in phosphate limitation. ACT was shown to bear “anti-oxidant” properties suggesting that its biosynthesis is triggered by oxidative stress. Interestingly, Streptomyces lividans , a strain closely related to S. coelicolor , does not produce ACT in any phosphate condition whereas its pptA/sco4144 mutant produces ACT but only in phosphate limitation. In order to define the potentially common features of the ACT producing strains, these three strains were grown in condition of low and high phosphate availability, and a comparative quantitative analysis of their proteomes was carried out. The abundance of proteins of numerous pathways differed greatly between S. coelicolor and the S. lividans strains, especially those of central carbon metabolism and respiration. S. coelicolor is characterized by the high abundance of the complex I of the respiratory chain thought to generate reactive oxygen/nitrogen species and by a weak glycolytic activity causing a low carbon flux through the Pentose Phosphate Pathway resulting into the low generation of NADPH, a co-factor of thioredoxin reductases necessary to combat oxidative stress. Oxidative stress is thus predicted to be high in S. coelicolor . In contrast, the S. lividans strains had rather similar proteins abundance for most pathways except for the transhydrogenases SCO7622-23, involved in the conversion of NADPH into NADH. The poor abundance of these enzymes in the pptA mutant suggested a deficit in NADPH. Indeed, PptA is an accessory protein forcing polyphosphate into a conformation allowing their efficient use by various enzymes taking polyphosphate as a donor of phosphate and energy, including the ATP/Polyphosphate-dependent NAD kinase SCO1781. In phosphate limitation, this enzyme would mainly use polyphosphate to phosphorylate NAD into NADP, but this phosphorylation would be inefficient in the pptA mutant resulting in low NADP(H) levels and thus high oxidative stress. Altogether, our results indicated that high oxidative stress is the common feature triggering ACT biosynthesis in S. coelicolor and in the pptA mutant of S. lividans

EFA6B regulates a stop signal for collective invasion in breast cancer

International audienceCancer is initiated by somatic mutations in oncogenes or tumor suppressor genes. However, additional alterations provide selective advantages to the tumor cells to resist treatment and develop metastases. Their identification is of paramount importance. Reduced expression of EFA6B (Exchange Factor for ARF6, B) is associated with breast cancer of poor prognosis. Here, we report that loss of EFA6B triggers a transcriptional reprogramming of the cell-to-ECM interaction machinery and unleashes CDC42-dependent collective invasion in collagen. In xenograft experiments, MCF10 DCIS.com cells, a DCIS-to-IDC transition model, invades faster when knocked-out for EFA6B. In addition, invasive and metastatic tumors isolated from patients have lower expression of EFA6B and display gene ontology signatures identical to those of EFA6B knockout cells. Thus, we reveal an EFA6B-regulated molecular mechanism that controls the invasive potential of mammary cells; this finding opens up avenues for the treatment of invasive breast cancer

Autophagy inactivation in osteosarcoma leads to the appearance of poor prognosis-associated factors

Osteosarcoma (OS) is a bone cancer exhibiting a 20% survival rate for metastatic patients, which motivates the development of new therapeutic options. Among the various new treatment approaches, modulation of autophagy is the subject of rising interest. In addition to its pro-survival role in established tumors, autophagy recently emerged as an active player in the crosstalk between tumor and stromal cells. In OS, although the knockdown of key autophagy genes in human cell lines demonstrates a protumoral role of autophagy, the analysis of patient tumors indicates that lack of LC3-positive punctae at resection following neoadjuvant chemotherapy is a poor prognostic marker, suggesting that loss of autophagy is not detrimental for the tumor. In the present work, we analyzed the consequences of autophagy inactivation in OS cells both on tumor development and on bone microenvironment in an orthotopic syngeneic model. We found that inactivation of the autophagy-essential gene Atg5 in OS cells decreases their tumorigenic properties in vitro. However, these effects were no longer observed in vivo, likely due to microenvironment modifications such as overexpression of the major OS-promoting factor TGF-β or increased infiltration of Foxp3-positive and CD31-positive cells in Atg5 KO tumors. In addition, autophagy-deficient tumor cells stimulate the in vitro formation of osteoclast, the cells in charge of bone resorption which can release bone matrix-embedded growth factors thereby stimulating tumor growth. Taken together, these results suggest that Atg5 inactivation in OS cells is associated with microenvironment modifications known as poor prognosis-associated factors in OS, and could thus balance the negative cell-autonomous effects of autophagy suppression. Abbreviations: ACTB -β-actin; Atg -autophagy-related; Baf-A1 - Bafilomycin-A1; CSC -cancer stem cells; Col1A -type 1a collagen; d -day; HBSS -Hank’s balanced salt solution; LC3 -microtubule-associated protein 1 light chain 3 protein; SQSTM1/p62 -sequestosome; OB -osteoblast; OC -osteoclast; OS -osteosarcoma; TEM -transmission electron microscopy; TGF-β -transforming growth factor β; TRAP -acid phosphatase 5, tartrate-resistant

Neuroprognostication of Consciousness Recovery in a Patient with COVID-19 Related Encephalitis: Preliminary Findings from a Multimodal Approach

International audiencePredicting the functional recovery of patients with severe neurological condition due to coronavirus disease 2019 (COVID-19) is a challenging task. Only limited outcome data are available, the pathophysiology is poorly understood, and the time-course of recovery is still largely unknown. Here, we report the case of a patient with COVID-19 associated encephalitis presenting as a prolonged state of unresponsiveness for two months, who finally fully recovered consciousness, functional communication, and autonomy after immunotherapy. In a multimodal approach, a high-density resting state EEG revealed a rich brain activity in spite of a severe clinical presentation. Using our previously validated algorithms, we could predict a possible improvement of consciousness in this patient. This case report illustrates the value of a multimodal approach capitalizing on advanced brain-imaging and bedside electrophysiology techniques to improve prognosis accuracy in this complex and new aetiology