Integrated Redox Sensor and Effector Functions for Tetrahydrobiopterin- and Glutathionylation-dependent Endothelial Nitric-oxide Synthase Uncoupling

Endothelial nitric-oxide synthase (eNOS) is a critical regulator of vascular homeostasis by generation of NO that is dependent on the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, eNOS becomes “uncoupled,” resulting in superoxide production in place of NO. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, although the functional relationships between BH4 and S-glutathionylation remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we expressed either WT or mutant eNOS rendered resistant to S-glutathionylation in cells with Tet-regulated expression of human GTP cyclohydrolase I to regulate intracellular BH4 availability. We reveal that S-glutathionylation of eNOS, by exposure to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase-specific siRNA, results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:7,8-dihydrobiopterin ratio. In eNOS uncoupling induced by BH4 deficiency, BCNU exposure further exacerbates superoxide production, BH4 oxidation, and eNOS activity. Following mutation of C908S, BCNU-induced eNOS uncoupling and BH4 oxidation are abolished, whereas uncoupling induced by BH4 deficiency was preserved. Furthermore, BH4 deficiency alone is alone sufficient to reduce intracellular GSH:GSSG ratio and cause eNOS S-glutathionylation. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling, although mechanistically distinct, are functionally related. We propose that uncoupling of eNOS by S-glutathionylation- or by BH4-dependent mechanisms exemplifies eNOS as an integrated redox “hub” linking upstream redox-sensitive effects of BH4 and glutathione with redox-dependent targets and pathways that lie downstream of eNOS.     

Both cell‐autonomous mechanisms and hormones contribute to sexual development in vertebrates and insects

The differentiation of male and female characteristics in vertebrates and insects has long been thought to proceed via different mechanisms. Traditionally, vertebrate sexual development was thought to occur in two phases: a primary and a secondary phase, the primary phase involving the differentiation of the gonads, and the secondary phase involving the differentiation of other sexual traits via the influence of sex hormones secreted by the gonads. In contrast, insect sexual development was thought to depend exclusively on cell‐autonomous expression of sex‐specific genes. Recently, however, new evidence indicates that both vertebrates and insects rely on sex hormones as well as cell‐autonomous mechanisms to develop sexual traits. Collectively, these new data challenge the traditional vertebrate definitions of primary and secondary sexual development, call for a redefinition of these terms, and indicate the need for research aimed at explaining the relative dependence on cell‐autonomous versus hormonally guided sexual development in animals.     

Landing Surface Color Preferences of Spathius agrili (Hymenoptera: Braconidae), a Parasitoid of Emerald Ash Borer, Agrilus planipennis (Coleoptera: Buprestidae)

The color preferences for landing surfaces were examined for Spathius agrili (Hymenoptera: Braconidae), a parasitic wasp introduced for biocontrol of emerald ash borer, Agrilus planipennis (Coleoptera: Buprestidae). Lures with the 3-component pheromone blend of male S. agrili were used to activate upwind flight by virgin female S. agrili in a laminar flow wind tunnel. Paper discs with halves of two different colors (combination pairs of black, white, red, yellow, green, or purple), with the pheromone lure in the center, were tested to quantify preferences for landing on one color over another. Females landed preferentially on green, yellow, and white surfaces, and landed the least frequently on red, black, and purple surfaces. Changes in color preferences due to adjacent colors were observed and discussed.     

Location and Dynamics of the Immunodominant CD8 T Cell Response to SIVΔnef Immunization and SIVmac251 Vaginal Challenge

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.     

How Changes in Extracellular Matrix Mechanics and Gene Expression Variability Might Combine to Drive Cancer Progression

Changes in extracellular matrix (ECM) structure or mechanics can actively drive cancer progression; however, the underlying mechanism remains unknown. Here we explore whether this process could be mediated by changes in cell shape that lead to increases in genetic noise, given that both factors have been independently shown to alter gene expression and induce cell fate switching. We do this using a computer simulation model that explores the impact of physical changes in the tissue microenvironment under conditions in which physical deformation of cells increases gene expression variability among genetically identical cells. The model reveals that cancerous tissue growth can be driven by physical changes in the microenvironment: when increases in cell shape variability due to growth-dependent increases in cell packing density enhance gene expression variation, heterogeneous autonomous growth and further structural disorganization can result, thereby driving cancer progression via positive feedback. The model parameters that led to this prediction are consistent with experimental measurements of mammary tissues that spontaneously undergo cancer progression in transgenic C3(1)-SV40Tag female mice, which exhibit enhanced stiffness of mammary ducts, as well as progressive increases in variability of cell-cell relations and associated cell shape changes. These results demonstrate the potential for physical changes in the tissue microenvironment (e.g., altered ECM mechanics) to induce a cancerous phenotype or accelerate cancer progression in a clonal population through local changes in cell geometry and increased phenotypic variability, even in the absence of gene mutation.     

Integration of stochastic simulation with multivariate analysis: Short‐term facility fit prediction

This article describes a decision‐support tool to help pinpoint the potential root causes of sub‐optimal short‐term facility fit issues in biopharmaceutical facilities. This was achieved by creating a tool that integrated stochastic simulation with advanced multivariate statistical analysis. Process fluctuations in product titers in cell culture, step yields, and chromatography eluate volumes were mimicked using Monte Carlo simulation data derived using a stochastic discrete‐event simulation model. The resulting stochastic datasets, with the computed consequences on key metrics such as product mass loss and cost of goods, were examined using advanced multivariate statistical techniques. Principal component analysis combined with clustering algorithms was used to analyze the complex datasets from complete industrial batch processes for biopharmaceuticals. The challenge of visualizing the multidimensional nature of the dataset was addressed using hierarchical and k‐means clustering as well as stacked parallel co‐ordinate plots to help identify process fingerprints and characteristics of clusters leading to sub‐optimal facility fit issues. Industrially‐relevant case studies are presented that focus on technology transfer challenges for therapeutic antibodies moving from early phase to late phase clinical trials. The case study details how sub‐optimal facility fit can be alleviated by allocating alternative product pool tanks. The impact of this operational change is then assessed by reviewing an updated process fingerprint. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 368–377, 2013     

Behavior Matters—Cognitive Predictors of Survival in Amyotrophic Lateral Sclerosis

Background

It is difficult to longitudinally characterize cognitive impairment in amyotrophic lateral sclerosis (ALS) due to motor deficits, and existing instruments aren’t comparable with assessments in other dementias.

Methods

The ALS Brief Cognitive Assessment (ALS-BCA) was validated in 70 subjects (37 with ALS) who also underwent detailed neuropsychological analysis. Cognitive predictors for poor survival were then analyzed in a longitudinal cohort of 171 ALS patients.

Results

The ALS-BCA was highly sensitive (90%) and specific (85%) for ALS-dementia (ALS-D). ALS-D patients had shorter overall survival, primarily due to the poor survival among ALS-D patients with disinhibited or apathetic behaviors after adjusting for demographic variables, ALS site of onset, medications, and supportive measures. ALS-D without behavioral changes was not a predictor of poor survival.

Conclusion

ALS-D can present with or without prominent behavioral changes. Cognitive screening in ALS patients should focus on behavioral changes for prognosis, while non-behavioral cognitive impairments may impact quality of life without impacting survival.