Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells

Using a substrate measuring deletion or inversion of an I-SceI-excised fragment and both accurate and inaccurate rejoining, we determined the impact of non-homologous end-joining (NHEJ) on mammalian chromosome rearrangements. Deletion is 2- to 8-fold more efficient than inversion, independent of the DNA ends structure. KU80 controls accurate rejoining, whereas in absence of KU mutagenic rejoining, particularly microhomology-mediated repair, occurs efficiently. In cells bearing both the NHEJ and a homologous recombination (HR) substrate containing a third I-SceI site, we show that NHEJ is at least 3.3-fold more efficient than HR, and translocation of the I-SceI fragment from the NHEJ substrate locus into the HR-I-SceI site can occur, but 50- to 100-fold less frequently than deletion. Deletions and translocations show both accurate and inaccurate rejoining, suggesting that they correspond to a mix of KU-dependent and KU-independent processes. Thus these processes should represent prominent pathways for DSB-induced genetic instability in mammalian cells.     

Simulated weightlessness in the design and exploitation of a NMR-compatible bioreactor

Mammalian cells cultured in simulated weightlessness take advantage of a favorable environment, experiencing low shear stress and reduced turbulence. NMR spectroscopy allows the on-line noninvasive monitoring of cell growth and metabolism. With this in mind, we developed a novel bioreactor that fits into a NMR instrument and in which the simulated weightlessness conditions are obtained by a suitable medium and a flow-lift suspension. In detail, the gravitational vector acting on cells is counterbalanced by the hydrodynamic thrusts created by a bottom-up spiral flow of a fluid having increased density. We validate its efficiency (a) by calculating the main physical parameters as relative velocity, shear stress, and oxygen transport, and (b) by comparing the experimental results of growing a cell culture in the proposed bioreactor with those obtained using an established simulated weightlessness system (rotating wall vessel, NASA). As a test study we focused on the proliferation of human umbilical vein endothelial cells (HUVEC) in terms of cell viability and organization of their cytoskeleton.     

Anatomical templates for tissue (re)generation and beyond

Induced pluripotent stem cells (iPSCs) represent a valuable alternative to stem cells in regenerative medicine overcoming their ethical limitations, like embryo disruption. Takahashi and Yamanaka in 2006 reprogrammed, for the first time, mouse fibroblasts into iPSCs through the retroviral delivery of four reprogramming factors: Oct3/4, Sox2, c-Myc, and Klf4. Since then, several studies started reporting the derivation of iPSC lines from animals other than rodents for translational and veterinary medicine. Here, we review the potential of using these cells for further intriguing applications, such as “cellular agriculture.” iPSCs, indeed, can be a source of in vitro, skeletal muscle tissue, namely “cultured meat,” a product that improves animal welfare and encourages the consumption of healthier meat along with environmental preservation. Also, we report the potential of using iPSCs, obtained from endangered species, for therapeutic treatments for captive animals and for assisted reproductive technologies as well. This review offers a unique opportunity to explore the whole spectrum of iPSC applications from regenerative translational and veterinary medicine to the production of artificial meat and the preservation of currently endangered species.     

Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h⁻¹ – Relative Fluorescence Unit h⁻¹), in comparison to batch cultivation (174.0 RFU h⁻¹) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L⁻¹.     

A new <Emphasis Type="Italic">Comanthera</Emphasis> (Eriocaulaceae) from the Espinhaço Range,Minas Gerais,Brazil

We describe and illustrate the new species Comanthera brunnea (Eriocaulaceae: Paepalanthoideae). This species is endemic to the sandy fields of the campos rupestres of the northeastern Espinhaço range in Minas Gerais, Brazil. We compare it with Comanthera suberosa, the morphologically most similar species, and with C. brasiliana, which also has similar features. The morphological variation, habitat, geographic distribution, and conservation status of this new species are discussed.     

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.     

Endocytosis and trafficking of human lactoferrin in macrophage-like human THP-1 cells (1)

Different cell types have been reported to internalize lactoferrin (Lf) by specific or nonspecific receptors. Our studies focused on the endocytic pathway of human Lf in macrophage-like THP-1 cells. Lactoferrin was found to be internalized by THP-1 cells differentiated with phorbol myristate acetate. Incubation of cells with chlorpromazine and dansylcadaverine, inhibitors of clathrin-dependent endocytosis, led to a 50% inhibition of Lf internalization compared with untreated cells. Bafilomycin A1 and NH(4)Cl treatment also resulted in 40%-60% inhibition, respectively, suggesting that the internalization of Lf may partly be mediated by acidic endosome-like organelles. Endocytic uptake of Lf was also cholesterol-dependent, as shown by methyl-β-cyclodextrin or nystatin treatment of the cells prior to internalization. Partial colocalization of Lf and EEA-1, a marker specific for early endosomes, could be observed. Colocalization of Lf with a specific endoplasmic reticulum marker was also detected. Our results suggest that Lf is internalized mainly by the clathrin-dependent pathway in THP-1 cells and targets the ER. The physiological consequences of this intracellular trafficking will be the subject of future investigations.     

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt's lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin-proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the alpha/beta subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6' subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.