Genomic mapping of human chromosome paints on the threatened masked Titi monkey (Callicebus personatus)

Callicebus is a complex genus of neotropical primates thought to include 29 or more species. Currently, the genus is divided into 5 species groups: donacophilus, cupreus, moloch, torquatus and personatus. However, the phylogenetic relationships among the species are still poorly understood. This genus is karyotypically diverse and shows extensive variation in diploid number (2n = 16 to 50). To foster a better understanding of the chromosomal diversities and phylogenetic relationships among the species of Callicebus, we performed a chromosome-painting analysis on the Callicebus personatus genome using human probes, and compared the resulting hybridization map to those of previously mapped titi species. We detected 38 hybridization signals per haploid autosomal set of C. personatus. Few ancestral syntenies were conserved without rearrangement, but 4 human associations (HSA20/13, 3c/8b, 1b/1c and 21/3a/15a/14) were demonstrated to be apomorphic traits for C. persona tus. G-banding suggested that these associations are shared with C. nigrifrons and C. coimbrai (personatus group), while C. personatus is linked with C. pallescens (donacophilus group) by 2 synapomorphies: HSA10b/11 (submetacentric) and an inversion of HSA1a.     

From in vitro culture to in vivo models to study testis development and spermatogenesis

The testis is a complex organ playing host to one of the most intricate mass cell divisions occurring in postnatal life. Since the beginning of the 20th century, great efforts have been made to recapitulate spermatogenesis and elucidate spermatogonial stem cell function. These efforts have resulted in the development of a variety of model systems that provide invaluable knowledge regarding testis organogenesis, key cell types and their interactions, and signaling pathways controlling testis function. The goal of this review is to elaborate on the evolution of the techniques available from in vitro culture systems to in vivo bioassays by providing up to date information and weighing their particular strengths and weaknesses. Each technique offers a different approach to the elucidation of male reproduction, the enhancement of germ-lineage genetic manipulation, the preservation of gametes, the restoration of fertility, and the improvement in our understanding of stem cell biology.     

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior

Background

Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity.

Methods

MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses.

Results

After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months.

Conclusions

These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.     

The antibody mining toolbox: An open source tool for the rapid analysis of antibody repertoires

In vitro selection has been an essential tool in the development of recombinant antibodies against various antigen targets. Deep sequencing has recently been gaining ground as an alternative and valuable method to analyze such antibody selections. The analysis provides a novel and extremely detailed view of selected antibody populations, and allows the identification of specific antibodies using only sequencing data, potentially eliminating the need for expensive and laborious low-throughput screening methods such as enzyme-linked immunosorbant assay. The high cost and the need for bioinformatics experts and powerful computer clusters, however, have limited the general use of deep sequencing in antibody selections. Here, we describe the AbMining ToolBox, an open source software package for the straightforward analysis of antibody libraries sequenced by the three main next generation sequencing platforms (454, Ion Torrent, MiSeq). The ToolBox is able to identify heavy chain CDR3s as effectively as more computationally intense software, and can be easily adapted to analyze other portions of antibody variable genes, as well as the selection outputs of libraries based on different scaffolds. The software runs on all common operating systems (Microsoft Windows, Mac OS X, Linux), on standard personal computers, and sequence analysis of 1–2 million reads can be accomplished in 10–15 min, a fraction of the time of competing software. Use of the ToolBox will allow the average researcher to incorporate deep sequence analysis into routine selections from antibody display libraries.     

Control of sulphide during anaerobic treatment of S-containing wastewaters by adding limited amounts of oxygen or nitrate

Sulphide generated during anaerobic treatment of S-containing wastewaters represents an environmental problem. Adding limited amounts of oxygen or nitrate (or nitrite) to biologically (or chemically) oxidise sulphide forms a simple process-level strategy to control this problem. This short review evaluates the feasibility and limitations of this strategy on the basis of the results of bioreactor studies.     

Sturgeon orphanin, a molecular "fossil" that bridges the gap between the opioids and orphanin FQ/nociceptin

The elucidation of the cDNA sequence for sturgeon proorphanin provides a unique window for interpreting the evolutionary history of the opioid/orphanin gene family. The molecular "fossil" status of this precursor can be seen in several ancestral sequence characteristics that point to its origin as a duplication of either a prodynorphin- or proenkephalin-like gene. The sturgeon proorphanin cDNA encodes a precursor protein of 194 residues, and the orphanin heptadecapeptide itself binds not only the opioid receptor-like 1 (ORL1) receptor but also the classical (mu, kappa, and delta) opioid receptors with near equal affinity. Allowing for this broad receptor specificity are several amino acid substitutions at key positions in the heptadecapeptide sequence, relative to its mammalian orthologs, that have been linked by amino acid scans and site-directed mutagenic studies to the exclusion of mammalian orphanin FQ/nociceptin from classic opioid ligands (i.e. F1Y and L14W). The unique receptor binding profile of sturgeon orphanin not only provides insight into the evolutionary history of the opioid and opioid-related peptides but also provides an ideal context in which to investigate the underlying mechanisms by which novel and often divergent physiological functions arise in receptor-ligand systems.     

Elevation of plasma cortisol during the spawning migration of landlocked kokanee salmon (Oncorhynchus nerka kennerlyi)

Kokanee salmon (Oncorhynchus nerka kennerlyi ), a landlocked subspecies of sockeye salmon, exhibited hypothalamic-pituitary interrenal (HPI, adrenal homologue) axis activation and an increase in plasma cortisol concentration up to 639 +/- 55.9 ng/ml in association with upstream migration in the upper Colorado River even though they were not exposed to a change in salinity and lengthy migration. Kokanee salmon were collected at various stages of migration and concomitant sexual maturation. The pattern of cortisol elevation in kokanee is similar to that in ocean-run sockeye salmon (O. nerka nerka). The presence of plasma cortisol elevation in an upstream migrating, landlocked Pacific salmon suggests that stressors previously considered to cause the cortisol increase, such as long-distance migration and changes in salinity, may not be primary causes of the HPI axis activation.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Quantification of the virus-host interaction in human T lymphotropic virus I infection

Background

Cellular infection with human immunodeficiency virus (HIV) both in vitro and in vivo requires a member of the chemokine receptor family to act as a co-receptor for viral entry. However, it is presently unclear to what extent the interaction of HIV proteins with chemokine receptors generates intracellular signals that are important for productive infection.

Results

In this study we have used a recently described family of chemokine inhibitors, termed BSCIs, which specifically block chemokine-induced chemotaxis without affecting chemokine ligands binding to their receptors. The BSCI termed Peptide 3 strongly inhibited CCR5 mediated HIV infection of THP-1 cells (83 ± 7% inhibition assayed by immunofluoresence staining), but had no effect on gp120 binding to CCR5. Peptide 3 did not affect CXCR4-dependent infection of Jurkat T cells.

Conclusion

These observations suggest that, in some cases, intracellular signals generated by the chemokine coreceptor may be required for a productive HIV infection.