Rapidly mutating Y-STRs in rapidly expanding populations: Discrimination power of the Yfiler Plus multiplex in northern Africa

The male-specific northern African genetic pool is characterised by a high frequency of the E-M81 haplogroup, which expanded in very recent times (2-3 kiloyears ago). As a consequence of their recent coalescence, E-M81 chromosomes often cannot be completely distinguished on the basis of their Y-STR profiles, unless rapidly-mutating Y-STRs (RM Y-STRs) are analysed. In this study, we used the Yfiler® Plus kit, which includes 7 RM Y-STRs and 20 standard Y-STR, to analyse 477 unrelated males coming from 11 northern African populations sampled from Morocco, Algeria, Libya and Egypt. The Y chromosomes were assigned to monophyletic lineages after the analysis of 72 stable biallelic polymorphisms and, as expected, we found a high proportion of E-M81 subjects (about 46%), with frequencies decreasing from west to east. We found low intra-population diversity indexes, in particular in the populations that experienced long-term isolation. The AMOVA analysis showed significant differences between the countries and between most of the 11 populations, with a rough differentiation between northwestern Africa and northeastern Africa, where the Egyptians Berbers from Siwa represented an outlier population. The comparison between the Yfiler® and the Yfiler® Plus network of the E-M81 Y chromosomes confirmed the high power of discrimination of the latter kit, thanks to higher variability of the RM Y-STRs: indeed, the number of chromosomes sharing the same haplotype was drastically reduced from 201 to 81 and limited, in the latter case, to subjects from the same population

Heparan Sulfate: A Potential Candidate for the Development of Biomimetic Immunomodulatory Membranes

Clinical trials have demonstrated that heparan sulfate (HS) could be used as a therapeutic agent for the treatment of inflammatory diseases. Its anti-inflammatory effect makes it suitable for the development of biomimetic innovative strategies aiming at modulating stem cells behavior toward a pro-regenerative phenotype in case of injury or inflammation. Here, we propose collagen type I meshes fabricated by solvent casting and further crosslinked with HS (HS-Col) to create a biomimetic environment resembling the extracellular matrix of soft tissue. HS-Col meshes were tested for their capability to provide physical support to stem cells’ growth, maintain their phenotypes and immunosuppressive potential following inflammation. HS-Col effect on stem cells was investigated in standard conditions as well as in an inflammatory environment recapitulated in vitro through a mix of pro-inflammatory cytokines (tumor necrosis factor-α and interferon-gamma; 20 ng/ml). A significant increase in the production of molecules associated with immunosuppression was demonstrated in response to the material and when cells were grown in presence of pro-inflammatory stimuli, compared to bare collagen membranes (Col), leading to a greater inhibitory potential when mesenchymal stem cells were exposed to stimulated peripheral blood mononuclear cells. Our data suggest that the presence of HS is able to activate the molecular machinery responsible for the release of anti-inflammatory cytokines, potentially leading to a faster resolution of inflammation

RACK1 Is a Ribosome Scaffold Protein for β-actin mRNA/ZBP1 Complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs

Human chorionic villus, amniotic fluid and amniotic membrane: Three different gestational tissues as source of valuable mesenchymal stem cells for regenerative medicine applications

La medicina “rigenerativa” ha come scopo quello di riparare o ripristinare la funzionalità di organi o tessuti danneggiati da malattie, traumi o dal “semplice” invecchiamento mediante l’utilizzo di cellule staminali. Una linea cellulare staminale molto importante e molto utilizzata in approcci di medicina rigenerativa è costituita dalle staminali adulte o staminali mesenchimali. Il midollo osseo e il tessuto adiposo sono stati considerati, per lungo tempo, la principale fonte di staminali mesenchimali adulte. Tuttavia le staminali adulte prevedono tecniche di prelievo invasive, non prive di complicazioni, e la coltura richiede diverse settimane; inoltre, possiedono limitate capacità di proliferazione e differenziaamento che sono inversamente proporzionali all’età del donatore. Da qui l’interesse, per il clinico, di nuove fonti cellulari. La recente scoperta in campo umano dell’esistenza di staminali mesenchimali nei tessuti fetali ha reso queste cellule molto interessanti agli occhi dell’intera comunità scientifica. I tessuti fetali sono di facile accesso perchè solitamente eliminati alla nascita, non richiedono procedure invasive, il loro utilizzo non suscita controversie etico-morali e inoltre le staminali isolte da tali tessuti sono caratterizzate da proliferazione più rapida, una maggiore espansione in vitro e da un notevole potenziale differenziativo. Alla luce di queste considerazioni, l’obiettivo principale di questo studio è stato quello di recuperare cellule staminali mesenchimali da tessuti fetali umani extraembrionali (il villo coriale, il liquido amniotico e la membrana amniotica) durante 3 diverse epoche gestazionali e mantenerle in coltura per un lungo periodo. Allo scopo di verificare se l’età gestazionale influenzasse le proprietà di staminalità, le staminali isolate da tali tessuti sono state confrontate nel fenotipo, nella espressione del markers specifici di pluripotenza e mesenchimalità, nel potenziale proliferativo, differenziativo e immunosoppressivo e immunomodulatorio.Regenerative medicine involves the use of living cells to repair, replace or restore normal function to injured tissues and organ. With the field of mesenchymal stem cell (MSCs) research taking off in the late 1980’s and the early 1990’s, scientists highlighted the importance of adult MSCs for regenerative medicine applications, and identified their presence in all adult tissues. Among MSCs, MSCs derived from fetal tissues (bone marrow, blood and liver) and extra-embryonic compartments (amniotic fluid, umbilical cord blood, amniotic membrane, chorion and placenta) are promising in cell-based therapies because of their beneficial properties in wound healing. Moreover, these tissues are ideal sources for studying the features of stem cell characteristics due to the possibility of harvesting large amount of tissue, without posing ethical debate, following prenatal diagnosis (as in the case of chorion from chorionic villi sampling and amniotic fluid from amniocentesis) or at birth (as in the case of amniotic membrane). In addition, MSCs isolated from fetal sources non only fulfill general characteristic of MSCs but exhibit features of embryonic stem cells including the expression of specific pluripotent markers. In fact, they possess higher proliferation rates, lower immunogenicity and wider differentiation capacity than their adult counterparts as well as immuno- suppressive and modulatory properties. Scarce information on the behavior of MSCs from different stages of human gestation are so far available. The first aim of this study was the isolation of MSCs from fetal (extra-embryonic) tissues during first- second- and third- gestation period and their long-term culturing; secondly, the detection of the common features between chorionic villi (CV), amniotic fluid (AF) and amniotic membrane (AM)-derived MSCs; thirdly, the observation of differences in phenotype, proliferative capacity, differentiation ability as well as in the immuno-suppressive/immuno-modulatory properties among them

Rapidly mutating Y-STRs in rapidly expanding populations:discrimination power of the Yfiler Plus multiplex in northern Africa.

The male-specific northern African genetic pool is characterised by a high frequency of the E-M81 haplogroup, which expanded in very recent times (2-3 kiloyears ago). As a consequence of their recent coalescence, E-M81 chromosomes often cannot be completely distinguished on the basis of their Y-STR profiles, unless rapidly-mutating YSTRs (RM Y-STRs) are analysed. In this study, we used the Yfiler® Plus kit, which includes 7 RM Y-STRs, to analyse 477 unrelated males coming from 11 northern African populations sampled from Morocco, Algeria, Libya and Egypt. The Y chromosomes were assigned to monophyletic lineages after the analysis of 72 stable biallelic polymorphisms and, as expected, we found a high proportion of E-M81 subjects (about 46%), with frequencies decreasing from west to east. We found low intrapopulation diversity indexes, in particular in the populations that experienced long-term isolation. The AMOVA analysis showed significant differences between the countries and between most of the 11 populations, with a rough differentiation between northwestern Africa and northeastern Africa, where the Egyptians Berbers from Siwa represented an outlier population. The comparison between the Yfiler® and the Yfiler® Plus network of the EM81 Y chromosomes confirmed the high power of discrimination of the latter kit, thanks to higher variability of the RM Y-STRs: indeed, the number of chromosomes sharing the same haplotype was drastically reduced from 201 to 81 and limited, in the latter case, to subjects from the same population

RACK1_Y246F induces accumulation of β-actin mRNA in growth cones of cortical cells and reduces its translation.

<p><b>A</b>, Growth cones of cortical cells co-transfected with Flag-ZBP1_wt and GFP-RACK1_Y246F show increased levels of β-actin mRNA as measured by Q-FISH. The method to quantify the signal is reported in supplemental information and the values are summarized in the histogram in the left. <b>B</b>, The translation of β-actin protein in growth cones of cortical neurons co-transfected with Flag-ZBP1_wt and GFP-RACK1_Y246F was reduced, as indicated by in Q-IF. All data are reported as mean ± s.e.m. Significance was set as p≤0.05. When significance was adjusted, it is referred to as alpha. β-tubulin immunofluorescence is shown as a marker for growth cone morphology. Scale bar 10 µm.</p

Src kinase regulates the RACK1/ZBP1 complex and the Y246 of RACK1 is critical for the binding with ZBP1.

<p><b>A</b>, Endogenous RACK1 interacts with Flag-ZBP1 in an anti-Flag immunoprecipitation assay from total lysate of neuroblastoma Flag-ZBP1-transfected cells. Western blotting for endogenous RACK1 and Flag-ZBP1 on proteins eluted from anti-Flag immunoprecipitation assay. Flag transfected cells were used as negative control. Input represents 5% of total lysate. <b>B</b> and <b>C</b>, Src activity regulates the RACK1-ZBP1 complex formation. <b>B</b>, db-cAMP or PACAP treatments of Flag-ZBP1 transfected cells reduced the binding between RACK1 and Flag-ZBP1, whereas PP2 restored the binding, as in untreated cells. <b>C</b> BDNF treatments increased the binding of Flag-ZBP1 to RACK1 in Flag-ZBP1 transfected cells. Src inhibition by Src inhibitor PP2 reduced the RACK1-ZBP1 complex stimulated by BDNF. The density value of immnoprecipitated RACK1 is normalized to that of immunoprecipitated Flag-ZBP1 and summarized in both graphics. Data are graphed as means plus S.D. <b>D</b>, The Src binding and phosphorylation site (Y246) of RACK1 is critical for complex formation. Flag-ZBP1 protein co-immunoprecipitated with GFP-RACK1_wt and GFP-RACK1_Y246F in immunopreciptation assays using anti-GFP antibody, but in the presence of GFP-RACK1_Y246F the binding was reduced. The figures are representative of three independent experiments. The density value of co-immnoprecipitated Flag-ZBP1 is normalized to that of immnoprecipitated GFP-RACK1_wt or GFP-RACK1_Y246F and summarized in both graphic. Data are graphed as means plus S.D.</p

The dendritic branching out induced by GFP-RACK1wt.

<p><b>A</b>, Immunofluorescence for GFP in cortical neurons transfected with GFP, or GFP-RACK1_wt or GFP-RACK1_Y246F. GFP-RACK1_Y246F reduced the dendritic arbors indeced by GFP-RACK1_wt overxpression Scale bar 20 µm <b>B</b>, Graphic reporting the values of dendritic branching seen in <b>A</b>. The values were measured as means of the number of neurite intersections measured by Sholl analysis. Data are graphed as mean ± S.D.</p