Estimated comparative integration hotspots identify different behaviors of retroviral gene transfer vectors

Integration of retroviral vectors in the human genome follows non random patterns that favor insertional deregulation of gene expression and may cause risks of insertional mutagenesis when used in clinical gene therapy. Understanding how viral vectors integrate into the human genome is a key issue in predicting these risks. We provide a new statistical method to compare retroviral integration patterns. We identified the positions where vectors derived from the Human Immunodeficiency Virus (HIV) and the Moloney Murine Leukemia Virus (MLV) show different integration behaviors in human hematopoietic progenitor cells. Non-parametric density estimation was used to identify candidate comparative hotspots, which were then tested and ranked. We found 100 significative comparative hotspots, distributed throughout the chromosomes. HIV hotspots were wider and contained more genes than MLV ones. A Gene Ontology analysis of HIV targets showed enrichment of genes involved in antigen processing and presentation, reflecting the high HIV integration frequency observed at the MHC locus on chromosome 6. Four histone modifications/variants had a different mean density in comparative hotspots (H2AZ, H3K4me1, H3K4me3, H3K9me1), while gene expression within the comparative hotspots did not differ from background. These findings suggest the existence of epigenetic or nuclear three-dimensional topology contexts guiding retroviral integration to specific chromosome areas.     

L-ascorbic acid producing yeasts learn from plants how to recycle it

Microorganisms employed in industrial fermentation processes are often subjected to a variety of stresses that negatively affect growth, production and productivity. Therefore, stress robustness is an important property for their application. Reactive Oxygen Species (ROS) accumulation is a common denominator to a lot of these stress factors. Ascorbic acid (L-AA) acts as ROS scavenger, thus potentially protecting cells from harmful oxidative products. We have previously reported the development of Saccharomyces cerevisiae strains able to produce L-AA. This was obtained by expressing the known plant pathway genes and by complementing the missing step with an animal activity. Here, we show that L-AA accumulation inside yeast cells can be improved by expressing the complete biosynthetic plant pathway and, even further, by recycling its oxidation products. These new strains can be seen in a double perspective of exploitation: as novel organisms for vitamin C production and as novel cell factories for industrial processes.     

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.     

Association of the metastatic phenotype with CCN family members among breast and oral cancer cells

The CCN family of proteins consists of six members with conserved structural features. These proteins play several roles in the physiology and pathology of cells. Among the pathological roles of the CCN family, one of the most important and controversial ones is their role in the expansion and metastasis of cancer. Up to now a number of reports have described the possible role of each CCN family member independently. In this study, we comprehensively analyzed the roles of all six CCN family members in cell growth, migration and invasion of breast cancer cells in vitro and in vivo. As a result, we found the CCN2/CCN3 ratio to be a parameter that is associated with the metastatic phenotype of breast cancer cells that are highly metastatic to the bone. The same analysis with cell lines from oral squamous carcinomas that are not metastatic to the bone further supported our notion. These results suggest the functional significance of the interplay between CCN family members in regulating the phenotype of cancer cells.     

Heterologous production of five Hepatitis C virus-derived antigens in three Saccharomyces cerevisiae host strains

In this study, the production of recombinant Hepatitis C virus (HCV) derived proteins from transformed Saccharomyces cerevisiae yeast cells is reported. Three different yeast strains (GRF18U, BY4743-4A and CENPK 113-5D) have been transformed for the intracellular expression of five antigens of different dimensions (from 32.8 to 85.2 kDa), all derived from the non-structural (NS) region of different HCV viruses' genotypes and posed under the control of a glycolytic promoter. The putative trans-membrane domains contained in four antigens seem responsible of their accumulation as protein aggregates. Good productions of the smaller and of the bigger antigens (50 and 30 mgl(-1), respectively) have been observed in simple flask batch cultures. Productions are strongly dependent from the genetic background of the yeast host and from the cellular localization of the antigen, while they appear independent from the growth rate of the transformed hosts. For every recombinant antigen tested, the highest production levels were achieved with the CENPK 113-5D-host strain, while the GRF18U strain shows symptoms of a heavily stressed phenotype.     

Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.     

Recombinant protein production in yeasts

Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.     

Does Monitoring Matter? A Quantitative Assessment of Management Decisions from Locally-based Monitoring of Protected Areas

Biodiversity monitoring is criticized for being insufficiently relevant to the needs of managers and ineffective in integrating information into decision-making. We examined conservation management interventions resulting from 2½ years of monitoring by 97 rangers and 350 community volunteers over 1 million hectares of Philippine protected areas. Before this monitoring scheme was established, there was little collaboration between local people and park authorities, and park monitoring was restricted to assessments of the quantity of extracted timber. As a result of the scheme, 156 interventions were undertaken in terrestrial, marine and freshwater ecosystems. In total 98% of these interventions were meaningful and justified, 47% targeted the three most serious threats to biodiversity at the site, and 90% were implemented without external support, suggesting that the interventions were relevant and could be sustained over time at the local level. The mean time from sampling to decision-making was only 97 days, probably because 82% of the interventions were initiated by the same people and institutions that had compiled the underlying data, bypassing potential government bureaucracy. Many of the interventions were jointly undertaken by community members and the management authorities or consisted of local bylaws in support of park management. As a result of the monitoring, indigenous resource use regulation schemes were re-established with government recognition in several parks. The monitoring led to more diversified and realistic management responses on the part of the authorities, including a more socially acceptable and effective approach to enforcement. Of the four field monitoring techniques used, the most participatory one generated more interventions aimed at ensuring a continued resource supply for local communities (χ²₃ = 69.1, p <0.01). Although this suggests that the interest of community members is associated with their possibilities to influence the flow of ecosystem goods and services, the 156 interventions targeted, directly or indirectly, all known globally threatened species of mammals, birds and butterflies in the parks.