Optimizing Culture Medium Composition to Improve Oligodendrocyte Progenitor Cell Yields In Vitro from Subventricular Zone-Derived Neural Progenitor Cell Neurospheres

Neural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.     

The Effect of Tear Supplementation on Ocular Surface Sensations during the Interblink Interval in Patients with Dry Eye

Purpose

To investigate the characteristics of ocular surface sensations and corneal sensitivity during the interblink interval before and after tear supplementation in dry eye patients.

Methods

Twenty subjects (41.88±14.37 years) with dry eye symptoms were included in the dry eye group. Fourteen subjects (39.13±11.27 years) without any clinical signs and/or symptoms of dry eye were included in the control group. Tear film dynamics was assessed by non-invasive tear film breakup time (NI-BUT) in parallel with continuous recordings of ocular sensations during forced blinking. Corneal sensitivity to selective stimulation of corneal mechano-, cold and chemical receptors was assessed using a gas esthesiometer. All the measurements were made before and 5 min after saline and hydroxypropyl-guar (HP-guar) drops.

Results

In dry eye patients the intensity of irritation increased rapidly after the last blink during forced blinking, while in controls there was no alteration in the intensity during the first 10 sec followed by an exponential increase. Irritation scores were significantly higher in dry eye patients throughout the entire interblink interval compared to controls (p<0.004). NI-BUT significantly increased after HP-guar (p = 0.003) but not after saline drops (p = 0.14). In both groups, either after saline or HP-guar the shape of symptom intensity curves remained the same with significantly lower irritation scores (p<0.004), however after HP-guar the decrease was significantly more pronounced (p<0.004). Corneal sensitivity to selective mechanical, cold and chemical stimulation decreased significantly in both groups after HP-guar (p<0.05), but not after saline drops (p>0.05).

Conclusion

Ocular surface irritation responses due to tear film drying are considerably increased in dry eye patients compared to normal subjects. Although tear supplementation improves the protective tear film layer, and thus reduce unpleasant sensory responses, the rapid rise in discomfort is still maintained and might be responsible for the remaining complaints of dry eye patients despite the treatment.     

Interferon, a growing cytokine family: 50 years of interferon research

The establishment of an antiviral state in cells is the defining activity of interferons (IFNs) as well as the property that permitted their discovery in 1957 by Isaacs and Lindenmann. In addition, interferons have other cellular functions that have potential clinical applications. Today, interferons are used for the treatment of a variety of malignancies and viral diseases. The publication of this special issue of Biochimie gives us a great opportunity to review the state of the art in knowledge about interferons and to explore possible future directions. This commentary text will introduce the reviews written by colleagues who are experts in different aspects of interferon research, to mark the 50th anniversary of the discovery of interferon.     

Rho-kinase (ROCK) in sea urchin sperm: its role in regulating the intracellular pH during the acrosome reaction

Sperm must undergo the acrosome reaction (AR) in order to fertilize the egg. In sea urchins, this reaction is triggered by the egg jelly (EJ) which, upon binding to its sperm receptor, induces increases in the ion permeability of the plasma membrane and changes in protein phosphorylation. Here, we demonstrated that the sperm expresses ROCK (∼135 kDa), which is a serine/threonine protein kinase. ROCK localized, as RhoGTPase (Rho), in the acrosomal region, midpiece and flagellum. H-1152, a ROCK antagonist, inhibited the two cellular processes defining the AR: the acrosomal exocytosis and the actin polymerization. The ionophores nigericin and A23187 reversed the AR inhibition induced by H-1152, suggesting that ROCK functions at the level of the EJ-induced ion fluxes. Accordingly, H-1152 blocked 70% the intracellular alkalinization induced by EJ. These results indicate that EJ activates a Na⁺-H⁺ exchanger (NHE) in the sperm through a Rho/ROCK-dependent signaling pathway that culminates in the AR.     

Differential expression of the three multicopper oxidases from Myxococcus xanthus

Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells pre-adapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: a versatile eukaryotic system in virology

The yeast Saccharomyces cerevisiae is a well-established model system for understanding fundamental cellular processes relevant to higher eukaryotic organisms. Less known is its value for virus research, an area in which Saccharomyces cerevisiae has proven to be very fruitful as well. The present review will discuss the main achievements of yeast-based studies in basic and applied virus research. These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production.     

A phylogeographical perspective on the ex situ conservation of Aylacostoma (Thiaridae,Gastropoda) from the High Paraná River (Argentina–Paraguay)

Aylacostoma Spix, 1827, contains species that are the subject of focused conservation efforts under the auspices of the ‘Aylacostoma Project’, the only ex situ conservation programme for freshwater gastropods in South America. Two species from the High Paraná River (Argentina–Paraguay) are included in this programme (Aylacostoma chloroticum Hylton Scott, 1954 and Aylacostoma brunneum Vogler & Peso, 2014), as their habitats have disappeared as a consequence of the filling of the Yacyretá Reservoir in the 1990s. At present, A. chloroticum is restricted to only one known wild population in a small and fragile habitat, and wild populations of A. brunneum are presumed to have gone extinct. We used partial sequences of the cytochrome oxidase subunit I gene to provide the first phylogeographical perspective on these species from a limited dataset containing representatives of several wild populations that are successfully being bred in captivity. We found low genetic diversity and two haplotypes in A. chloroticum, and absence of variation with one haplotype in A. brunneum. The reservoir's entry zone was identified to be of great interest for conservation, and is where we suggest re‐introductions and translocations should be targeted, to preserve the future evolutionary potential of the extant diversity. © 2015 The Linnean Society of London     

The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms

The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.