IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.     

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.     

Functional and molecular insights into KSRP function in mRNA decay

KSRP is a single strand nucleic acid binding protein that controls gene expression at multiple levels. In this review we focus on the recent molecular, cellular, and structural insights into the mRNA decay promoting function of KSRP. We discuss also some aspects of KSRP-dependent microRNA maturation from precursors that are related to its mRNA destabilizing function. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Extracellular Sphingosine-1-Phosphate: A Novel Actor in Human Glioblastoma Stem Cell Survival

Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs), a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P) a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ), and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source of extracellular S1P, which might act as an autocrine/paracrine signal contributing to their malignant properties.     

Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.     

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus)

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (N_e) are much smaller than those expected in a fishes with large population census sizes (N_c). Estimates of N_e indicate low effective population sizes, even before the population collapse. The ratio N_e/N_e ranged between 10⁻⁶ and 10⁻⁸, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.     

Biotransformations of Bile Acids with Bacteria from Cayambe Slaughterhouse (Ecuador): Synthesis of Bendigoles

The biotransformations of cholic acid ( 1a ), deoxycholic acid ( 1b ), and hyodeoxycholic acid ( 1c ) to bendigoles and other metabolites with bacteria isolated from the rural slaughterhouse of Cayambe (Pichincha Province, Ecuador) were reported. The more active strains were characterized, and belong to the genera Pseudomonas and Rhodococcus. Various biotransformation products were obtained depending on bacteria and substrates. Cholic acid ( 1a ) afforded the 3‐oxo and 3‐oxo‐4‐ene derivatives 2a and 3a (45% and 45%, resp.) with P. mendocina ECS10, 3,12‐dioxo‐4‐ene derivative 4a (60%) with Rh. erythropolis ECS25, and 9,10‐secosteroid 6 (15%) with Rh. erythropolis ECS12. Bendigole F ( 5a ) was obtained in 20% with P. fragi ECS22. Deoxycholic acid ( 1b ) gave 3‐oxo derivative 2b with P. prosekii ECS1 and Rh. erythropolis ECS25 (20% and 61%, resp.), while 3‐oxo‐4‐ene derivative 3b was obtained with P. prosekii ECS1 and P. mendocina ECS10 (22% and 95%, resp.). Moreover, P. fragi ECS9 afforded bendigole A ( 8b ; 80%). Finally, P. mendocina ECS10 biotransformed hyodeoxycholic acid ( 1c ) to 3‐oxo derivative 2c (50%) and Rh. erythropolis ECS12 to 6α‐hydroxy‐3‐oxo‐23,24‐dinor‐5β‐cholan‐22‐oic acid ( 9c , 66%). Bendigole G ( 5c ; 13%) with P. prosekii ECS1 and bendigole H ( 8c ) with P. prosekii ECS1 and Rh. erythropolis ECS12 (20% and 16%, resp.) were obtained.     

The key role of “Moscato bianco” and “Malvasia aromatica di Parma” in the parentage of traditional aromatic grape varieties

A great number of flavored grape varieties, of significant oenological potential, are traditionally cultivated in north-western Italy, besides the renowned “Moscato bianco” (syn. “Muscat à petits grains blancs”). Understanding their origin, besides its historical and scientific interest, would help to increase market appeal and consequently facilitate the commercial exploitation of these products. Twenty-four aromatic genotypes were investigated for their identity, kinship relations, and genetic origins through molecular markers (SSR and SNPs) supported by plant morphology and historical information. Flavored grape genotypes from other regions, possible ancestors, and reference cultivars of known pedigree were also included in the analysis. Kinship analysis used a likelihood-based approach (IBS, IBD, relatedness coefficients, and likelihood ratios) to achieve strong statistical support. The analyses revealed two possible leading genitors, in turn closely related by a parent/offspring relationship: “Moscato bianco” and “Malvasia aromatica di Parma,” a female grape cultivar that is today almost extinct. The outlined molecular and statistical approach could be applied for the investigation on the origin of ancient traditional cultivars of other vegetative propagated species.