Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s Disease

There is much interest in the use of mesenchymal stem cells/marrow stromal cells (MSC) to treat neurodegenerative disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease. MSC present a promising tool for cell therapy and are currently being tested in FDA-approved phase I-III clinical trials for many disorders. In preclinical studies of neurodegenerative disorders, MSC have demonstrated efficacy, when used as delivery vehicles for neural growth factors. A number of investigators have examined the potential benefits of innate MSC-secreted trophic support and augmented growth factors to support injured neurons. These include overexpression of brain-derived neurotrophic factor and glial-derived neurotrophic factor, using genetically engineered MSC as a vehicle to deliver the cytokines directly into the microenvironment. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals, and regulate inflammation. These abilities are primarily modulated through paracrine actions. Clinical trials for MSC injection into the central nervous system to treat amyotrophic lateral sclerosis, traumatic brain injury, and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of Huntington's disease is discussed.     

Genome-Wide Association Studies in an Isolated Founder Population from the Pacific Island of Kosrae

It has been argued that the limited genetic diversity and reduced allelic heterogeneity observed in isolated founder populations facilitates discovery of loci contributing to both Mendelian and complex disease. A strong founder effect, severe isolation, and substantial inbreeding have dramatically reduced genetic diversity in natives from the island of Kosrae, Federated States of Micronesia, who exhibit a high prevalence of obesity and other metabolic disorders. We hypothesized that genetic drift and possibly natural selection on Kosrae might have increased the frequency of previously rare genetic variants with relatively large effects, making these alleles readily detectable in genome-wide association analysis. However, mapping in large, inbred cohorts introduces analytic challenges, as extensive relatedness between subjects violates the assumptions of independence upon which traditional association test statistics are based. We performed genome-wide association analysis for 15 quantitative traits in 2,906 members of the Kosrae population, using novel approaches to manage the extreme relatedness in the sample. As positive controls, we observe association to known loci for plasma cholesterol, triglycerides, and C-reactive protein and to a compelling candidate loci for thyroid stimulating hormone and fasting plasma glucose. We show that our study is well powered to detect common alleles explaining ≥5% phenotypic variance. However, no such large effects were observed with genome-wide significance, arguing that even in such a severely inbred population, common alleles typically have modest effects. Finally, we show that a majority of common variants discovered in Caucasians have indistinguishable effect sizes on Kosrae, despite the major differences in population genetics and environment.     

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.     

Oxidative stress attenuates NO-induced modulation of sympathetic neurotransmission in the mesenteric arterial bed of spontaneously hypertensive rats

Current evidence suggests that hyperactivity of the sympathetic nervous system and endothelial dysfunction are important factors in the development and maintenance of hypertension. Under normal conditions the endothelial mediator nitric oxide (NO) negatively modulates the activity of the norepinephrine portion of sympathetic neurotransmission, thereby placing a "brake" on the vasoconstrictor ability of this transmitter. This property of NO is diminished in the isolated, perfused mesenteric arterial bed taken from the spontaneously hypertensive rat (SHR), resulting in greater nerve-stimulated norepinephrine and lower neuropeptide Y (NPY) overflow from this mesenteric preparation compared with that of the normotensive Wistar-Kyoto rat (WKY). We hypothesized that increased oxidative stress in the SHR contributes to the dysfunction in the NO modulation of sympathetic neurotransmission. Here we demonstrate that the antioxidant N-acetylcysteine reduced nerve-stimulated norepinephrine and increased NPY overflow in the mesenteric arterial bed taken from the SHR. Furthermore, this property of N-acetylcysteine was prevented by inhibiting nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester, demonstrating that the effect of N-acetylcysteine was due to the preservation of NO from oxidation. Despite a reduction in norepinephrine overflow, the nerve-stimulated perfusion pressure response in the SHR mesenteric bed was not altered by the inclusion of N-acetylcysteine. Studies including the Y(1) antagonist BIBO 3304 with N-acetylcysteine demonstrated that this preservation of the perfusion pressure response was due to elevated NPY overflow. These results demonstrate that the reduction in the bioavailability of NO as a result of elevated oxidative stress contributes to the increase in norepinephrine overflow from the SHR mesenteric sympathetic neuroeffector junction.     

Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells

Immunodeficient CD8 knockout mice were infected with Sarcocystis neurona merozoites, in order to determine the role of CD8 cells in protective immunity. Using a direct agglutination test, all infected mice seroconverted by selected time points. Infected mice developed splenomegaly and bilateral lymphadenopathy. Histological changes included marked follicular development in the spleen, endothelitis and moderate perivascular inflammation in the liver, and meningoencephalitis in the brain. Infected brains were positive for S. neurona by polymerase chain reaction. Corresponding to histopathological changes, there were decreased numbers of B-cells in the spleen. The mice did not have significant memory (CD44hi/CD4) or effector (CD45RBhi/CD4) populations present at the time of euthanasia. Flow cytometry confirmed the lack of CD8 cells. Taken together, these data support previous studies suggesting a critical role for CD8 cells in the prevention of menigoencephalitis in S. neurona-infected mice.     

Population genetic variation in gene expression is associated with phenotypic variation in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background

The relationship between genetic variation in gene expression and phenotypic variation observable in nature is not well understood. Identifying how many phenotypes are associated with differences in gene expression and how many gene-expression differences are associated with a phenotype is important to understanding the molecular basis and evolution of complex traits.

Results

We compared levels of gene expression among nine natural isolates of Saccharomyces cerevisiae grown either in the presence or absence of copper sulfate. Of the nine strains, two show a reduced growth rate and two others are rust colored in the presence of copper sulfate. We identified 633 genes that show significant differences in expression among strains. Of these genes, 20 were correlated with resistance to copper sulfate and 24 were correlated with rust coloration. The function of these genes in combination with their expression pattern suggests the presence of both correlative and causative expression differences. But the majority of differentially expressed genes were not correlated with either phenotype and showed the same expression pattern both in the presence and absence of copper sulfate. To determine whether these expression differences may contribute to phenotypic variation under other environmental conditions, we examined one phenotype, freeze tolerance, predicted by the differential expression of the aquaporin gene AQY2. We found freeze tolerance is associated with the expression of AQY2.

Conclusions

Gene expression differences provide substantial insight into the molecular basis of naturally occurring traits and can be used to predict environment dependent phenotypic variation.

     

Modeling the precision and robustness of Hunchback border during Drosophila embryonic development

During anterior-posterior axis specification in the Drosophila embryo, the Hunchback (Hb) protein forms a sharp boundary at the mid-point of the embryo with great positional precision. While Bicoid (Bcd) is a known upstream regulator for hb expression, there is evidence to suggest that Hb effectively filters out “noisy” data received from varied Bcd gradients. We use mathematical models to explore simple regulatory networks which filter out such noise to produce a precise Hb boundary. We find that in addition to Bcd and Hb, at least one freely evolving protein is necessary. An automated search yields a number of examples of three-protein networks exhibiting the desired precision. In all such networks, Hb diffuses much slower than the third protein. In addition, the action of Hb on the third protein is the opposite of the action of the third protein on hb (i.e. if Hb activates the third protein, then the third protein inhibits hb expression, and vice versa). Most of the discovered systems satisfy the known biological properties, that Bcd activates hb, and that Hb activates its own expression. We find that all network topologies satisfying these constraints arise among the networks exhibiting the desired precision. Investigating the dynamics of these networks, we find that under a general class of non-uniform initial conditions, Bcd can be eliminated from the system and the spatiotemporal evolution of these two proteins alone is sufficient to recapture the dynamics. We hypothesize that Bcd is needed only to spatially disturb the gradient of the third protein, and then becomes unnecessary in the further evolution of the Hb border. This provides a possible explanation as to why the Hb dynamics are robust under perturbations of the Bcd gradient. Under this hypothesis, other proteins would be able to assume the role of Bcd in our simulations (possibly in the case of evolutionary divergences or a redundancy in the process), with the only constraint that they act to positively regulate hb.     

Integrating economic input–output life cycle assessment with risk assessment for a screening-level analysis

Goal, Scope, and Background  The paper describes the integration of the economic input–output life cycle assessment (EIO-LCA) model and the environmental fate and transport model (CHEMGL) with a risk assessment tool. Utilizing the EIO-LCA, instead of a traditional LCA, enables a rapid, screening-level analysis of an emerging chemical of concern, decabromodiphenyl ether (DecaBDE). The risk assessment in this study is evaluated based on the mass of chemical released, estimated concentrations, exposure, and chemical toxicity. Methods  The relative risk from ten economic sectors identified within the EIO-LCA model, 55 chemicals utilized in those sectors and DecaBDE along with four potential DecaBDE breakdown products, were evaluated for the life cycle stages and exposure pathways. The relative risk (expressed as toluene equivalents) of the different chemicals, sectors, and life cycle stages were compared to assess those representing the greatest overall relative risks to humans (via inhalation and ingestion) and fish. Results  The greatest overall risk to human health resulted from the manufacturing and production stages. For fish, the manufacturing stage represented virtually all of the risk. Of the 56 chemicals evaluated, DecaBDE represented the majority of the total risk to humans. However, DecaBDE posed the least risk compared to its potential breakdown products. Discussion  The risk to humans from ingestion, which represented the greatest risk, from the production, manufacturing, and consumption stages can be controlled and reduced through various safety precautions in the workplace. Additionally, the increasing concentration of DecaBDE in anaerobic compartments represents a threat to humans and fish via the higher risk DecaBDE breakdown products. Conclusions  Overall, the manufacturing and production life cycle stages pose the greatest risk to humans and fish. The sediment compartment received the highest DecaBDE concentration for the production, manufacturing, and consumption stages. This case study demonstrates that the integrated EIO-LCA with risk assessment is suitable for screening-level analysis of emerging chemicals due to rapid life cycle inventory analysis. Recommendations  The production and manufacturing stages allow for greater industry control and government regulation, compared to the consumption stage, because there are fewer point sources. This integrated life cycle methodology may allow chemical designers to evaluate each stage and assess areas where risks can be minimized.