Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA-miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs-in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.

Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a hybridoma cell culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a hybridoma cell culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96. (M.R. Aires-Barros). over 98%, as a result of the high specificity of protein A towards the Fc antibody domain . Nevertheless, the use of protein A has some drawbacks which are essentially related to possible leakage from the matrix, and consequent contamination of the final product, and most importantly, to the high cost of protein A resins . As the large number of mAb candidates in preclinical and clinical trials is reaching the process development stage, the need for effective, economical and rapid downstream processes is becoming crucial .

Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not only solely achieved by enhanced efflux capacity but also by supressed intake of the drug, offering an alternative target to overcome drug resistance or potentiate chemotherapy.

Partitioning and purification studies of human IgG were performed in polyethylene glycol (PEG) and citrate aqueous two-phase systems (ATPSs). Initial studies performed with pure IgG solutions showed that increasing the concentration of a neutral salt, such as NaCl up to 15% (w/w), increased the partition coefficient of IgG from 0.14 to 5.21. This partitioning behaviour was also observed first in the presence of a simulated protein mixture, containing albumin and myoglobin, and then in a hybridoma cell culture supernatant. Thus, by changing the concentration of NaCl, it is possible to target the partition of IgG to the phase with fewer impurities. The partial purification of IgG from a hybridoma cell culture supernatant in a serum-containing media was achieved using 15% NaCl and recovering the antibody in the top phase. Using an ATPS composed of 8% (w/w) PEG 3350, 8% (w/w) citrate and 15% (w/w) NaCl at pH 6, IgG was recovered with a 99% yield, 44% HPLC purity and with a IgG/protein ratio of 0.9. IgG can be re-extracted to a new citrate phase by decreasing the overall concentration of NaCl to 5%. This back-extraction step introduces not only a further purification step but also allows the separation of the antibody from the polymer. IgG was recovered with a global yield of 99%, with a HPLC purity of 76% and with a IgG/protein ratio of 0.96. (M.R. Aires-Barros). over 98%, as a result of the high specificity of protein A towards the Fc antibody domain . Nevertheless, the use of protein A has some drawbacks which are essentially related to possible leakage from the matrix, and consequent contamination of the final product, and most importantly, to the high cost of protein A resins . As the large number of mAb candidates in preclinical and clinical trials is reaching the process development stage, the need for effective, economical and rapid downstream processes is becoming crucial .

Kindler Syndrome (KS), characterized by transient skin blistering followed by abnormal pigmentation, skin atrophy, and skin cancer, is caused by mutations in the FERMT1 gene. Although a few KS patients have been reported to also develop ulcerative colitis (UC), a causal link to the FERMT1 gene mutation is unknown. The FERMT1 gene product belongs to a family of focal adhesion proteins (Kindlin-1, -2, -3) that bind several b integrin cytoplasmic domains. Here, we show that deleting Kindlin-1 in mice gives rise to skin atrophy and an intestinal epithelial dysfunction with similarities to human UC. This intestinal dysfunction results in perinatal lethality and is triggered by defective intestinal epithelial cell integrin activation, leading to detachment of this barrier followed by a destructive inflammatory response.

Angiogenesis requires coordinated changes in cell shape of endothelial cells (ECs), orchestrated by the actin cytoskeleton. The mechanisms that regulate this rearrangement in vivo are poorly understood -largely because of the difficulty to visualize filamentous actin (F-actin) structures with sufficient resolution. Here, we use transgenic mice expressing Lifeact-EGFP to visualize F-actin in ECs. We show that in the retina, Lifeact-EGFP expression is largely restricted to ECs allowing detailed visualization of F-actin in ECs in situ. Lifeact-EGFP labels actin associated with cell-cell junctions, apical and basal membranes and highlights actin-based structures such as filopodia and stress fiber-like cytoplasmic bundles. We also show that in the skin and the skeletal muscle, Lifeact-EGFP is highly expressed in vascular mural cells (vMCs), enabling vMC imaging. In summary, our results indicate that the Lifeact-EGFP transgenic mouse in combination with the postnatal retinal angiogenic model constitutes an excellent system for vascular cell biology research. Our approach is ideally suited to address structural and mechanistic details of angiogenic processes, such as endothelial tip cell migration and fusion, EC polarization or lumen formation.