Preservation of T cell proliferation restricted by protective HLA alleles is critical for immune control of HIV-1 infection

HIV-1-infected persons with HLA-B27 and -B57 alleles commonly remain healthy for decades without antiretroviral therapy. Properties of CD8+ T cells restricted by these alleles considered to confer disease protection in these individuals are elusive but important to understand and potentially elicit by vaccination. To address this, we compared CD8+ T cell function induced by HIV-1 immunogens and natural infection using polychromatic flow cytometry. HIV-1-specific CD8+ T cells from all four uninfected immunized and 21 infected subjects secreted IFN-gamma and TNF-alpha. However, CD8+ T cells induced by vaccination and primary infection, but not chronic infection, proliferated to their cognate epitopes. Notably, B27- and B57-restricted CD8+ T cells from nonprogressors exhibited greater expansion than those restricted by other alleles. Hence, CD8+ T cells restricted by certain protective alleles can resist replicative defects, which permits expansion and antiviral effector activities. Our findings suggest that the capacity to maintain CD8+ T cell proliferation, regardless of MHC-restriction, may serve as an important correlate of disease protection in the event of infection following vaccination.     

Effects of contemporary agricultural land cover on Colorado potato beetle genetic differentiation in the Columbia Basin and Central Sands

Landscape structure, which can be manipulated in agricultural landscapes through crop rotation and modification of field edge habitats, can have important effects on connectivity among local populations of insects. Though crop rotation is known to influence the abundance of Colorado potato beetle (CPB; Leptinotarsa decemlineata Say) in potato (Solanum tuberosum L.) fields each year, whether crop rotation and intervening edge habitat also affect genetic variation among populations is unknown. We investigated the role of landscape configuration and composition in shaping patterns of genetic variation in CPB populations in the Columbia Basin of Oregon and Washington, and the Central Sands of Wisconsin, USA. We compared landscape structure and its potential suitability for dispersal, tested for effects of specific land cover types on genetic differentiation among CPB populations, and examined the relationship between crop rotation distances and genetic diversity. We found higher genetic differentiation between populations separated by low potato land cover, and lower genetic diversity in populations occupying areas with greater crop rotation distances. Importantly, these relationships were only observed in the Columbia Basin, and no other land cover types influenced CPB genetic variation. The lack of signal in Wisconsin may arise as a consequence of greater effective population size and less pronounced genetic drift. Our results suggest that the degree to which host plant land cover connectivity affects CPB genetic variation depends on population size and that power to detect landscape effects on genetic differentiation might be reduced in agricultural insect pest systems.     

Effect of interferon γ and tumour necrosis factor α on sensitivity to cisplatin in ovarian carcinoma cell lines

This study aimed to investigate whether the biological response modifiers (BRM) interferon (IFN) and tumour necrosis factor (TNF) could enhance the cytotoxic action of cisplatin on ovarian tumour cells in vitro. The sensitivity of four cell lines (OAW42, GG, JAM and PE01) to drugs and drug combinations was tested by a radiolabelled-thymidine incorporation assay. Cell lines demonstrated a range of sensitivity to cisplatin and the innate cytotoxic effect of each of the BRM. When IFN was used in combination with cisplatin, a significant enhancement of cisplatin toxicity occurred in three of four cell lines. TNF demonstrated such an effect in two cell lines but diminished the toxicity of cisplatin in one cell line. A purely additive effect of the agents may explain the enhanced toxicity of cisplatin in some of these cases. However, in one cell line at least (PEO1), both TNF and IFN demonstrated a clearly synergistic effect with cisplatin. These BRM used in conjunction with cisplatin may provide better antitumour regimen than cisplatin alone in some patients with ovarian cancer, but the response is likely to be heterogeneous between patients.     

Temporal and ontogenetic aspects of protein induction in foliage of the tomato, Lycopersicon esculentum

Damage to foliage of the tomato, Lycopersicon esculentum, causes the induction of proteinase inhibitors and of the oxidative enzymes polyphenol oxidase, peroxidase, and lipoxygenase. The time courses of induction of these proteins by feeding of two caterpillar species (Manduca sexta and Helicoverpa zea) were studied in a series of experiments. In another series of experiments, the effects of plant age on the inducibility of these proteins were studied. In the time course experiments, induction of proteinase inhibitors and oxidative enzymes in the damaged leaflet was rapid, with higher protein activities evident in damaged leaflets within 12–24 h of damage, depending on the enzyme and the species of insect used to damage the plant. Systemic induction of proteinase inhibitors was also rapid, but systemic induction of polyphenol oxidase was delayed relative to systemic induction of proteinase inhibitors, possibly because high constitutive polyphenol oxidase activities obscured expression of systemic induction at earlier time points. Lipoxygenase and peroxidase were not induced systemically. Induction of all proteins persisted for at least 21 days. In the phenology experiments, inducibility of all proteins decreased in magnitude and was less consistent as plants aged. The results of these experiments exemplify the numerous constraints on induction in tomato plants. Knowledge of these physiological constraints is important to an understanding of the ecological role and causal basis of induced resistance.     

A thin‐walled polydimethylsiloxane bioreactor for high‐density hepatocyte sandwich culture

In vitro drug testing requires long‐term maintenance of hepatocyte liver specific functions. Hepatocytes cultured at a higher seeding density in a sandwich configuration exhibit an increased level of liver specific functions when compared to low density cultures due to the better cell to cell contacts that promote long term maintenance of polarity and liver specific functions. However, culturing hepatocytes at high seeding densities in a standard 24‐well plate poses problems in terms of the mass transport of nutrients and oxygen to the cells. In view of this drawback, we have developed a polydimethylsiloxane (PDMS) bioreactor that was able to maintain the long‐term liver specific functions of a hepatocyte sandwich culture at a high seeding density. The bioreactor was fabricated with PDMS, an oxygen permeable material, which allowed direct oxygenation and perfusion to take place simultaneously. The mass transport of oxygen and the level of shear stress acting on the cells were analyzed by computational fluid dynamics (CFD). The combination of both direct oxygenation and perfusion has a synergistic effect on the liver specific function of a high density hepatocyte sandwich culture over a period of 9 days. Biotechnol. Bioeng. 2013; 110: 1663–1673. © 2012 Wiley Periodicals, Inc.     

Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation

The sterile alpha motif (SAM) and HD domain-containing protein-1 (SAMHD1) inhibits the infection of resting CD4+ T cells and myeloid cells by human and related simian immunodeficiency viruses (HIV and SIV). Vpx inactivates SAMHD1 by promoting its proteasome-dependent degradation through an interaction with CRL4 (DCAF1) E3 ubiquitin ligase and the C-terminal region of SAMHD1. However, the determinants in SAMHD1 that are required for Vpx-mediated degradation have not been well characterized. SAMHD1 contains a classical nuclear localization signal (NLS), and NLS point mutants are cytoplasmic and resistant to Vpx-mediated degradation. Here, we demonstrate that NLS-mutant SAMHD1 K11A can be rescued by wild-type SAMHD1, restoring its nuclear localization; consequently, SAMHD1 K11A became sensitive to Vpx-mediated degradation in the presence of wild-type SAMHD1. Surprisingly, deletion of N-terminal regions of SAMHD1, including the classical NLS, generated mutant SAMHD1 proteins that were again sensitive to Vpx-mediated degradation. Unlike SAMHD1 K11A, these deletion mutants could be detected in the nucleus. Interestingly, NLS-defective SAMHD1 could still bind to karyopherin-β1 and other nuclear proteins. We also determined that the linker region between the SAM and HD domain and the HD domain itself is important for Vpx-mediated degradation but not Vpx interaction. Thus, SAMHD1 contains an additional nuclear targeting mechanism in addition to the classical NLS. Our data indicate that multiple regions in SAMHD1 are critical for Vpx-mediated nuclear degradation and that association with Vpx is not sufficient for Vpx-mediated degradation of SAMHD1. Since the linker region and HD domain may be involved in SAMHD1 multimerization, our results suggest that SAMHD1 multimerization may be required for Vpx-mediation degradation.     

Trophic Tangles through Time? Opposing Direct and Indirect Effects of an Invasive Omnivore on Stream Ecosystem Processes

Omnivores can impact ecosystems via opposing direct or indirect effects. For example, omnivores that feed on herbivores and plants could either increase plant biomass due to the removal of herbivores or decrease plant biomass due to direct consumption. Thus, empirical quantification of the relative importance of direct and indirect impacts of omnivores is needed, especially the impacts of invasive omnivores. Here we investigated how an invasive omnivore (signal crayfish, Pacifastacus leniusculus) impacts stream ecosystems. First, we performed a large-scale experiment to examine the short-term (three month) direct and indirect impacts of crayfish on a stream food web. Second, we performed a comparative study of un-invaded areas and areas invaded 90 years ago to examine whether patterns from the experiment scaled up to longer time frames. In the experiment, crayfish increased leaf litter breakdown rate, decreased the abundance and biomass of other benthic invertebrates, and increased algal production. Thus, crayfish controlled detritus via direct consumption and likely drove a trophic cascade through predation on grazers. Consistent with the experiment, the comparative study also found that benthic invertebrate biomass decreased with crayfish. However, contrary to the experiment, crayfish presence was not significantly associated with higher leaf litter breakdown in the comparative study. We posit that during invasion, generalist crayfish replace the more specialized native detritivores (caddisflies), thereby leading to little long-term change in net detrital breakdown. A feeding experiment revealed that these native detritivores and the crayfish were both effective consumers of detritus. Thus, the impacts of omnivores represent a temporally-shifting interplay between direct and indirect effects that can control basal resources.     

Damage to living trees contributes to almost half of the biomass losses in tropical forests

Accurate estimates of forest biomass stocks and fluxes are needed to quantify global carbon budgets and assess the response of forests to climate change. However, most forest inventories consider tree mortality as the only aboveground biomass (AGB) loss without accounting for losses via damage to living trees: branchfall, trunk breakage, and wood decay. Here, we use ~151,000 annual records of tree survival and structural completeness to compare AGB loss via damage to living trees to total AGB loss (mortality + damage) in seven tropical forests widely distributed across environmental conditions. We find that 42% (3.62 Mg ha⁻¹ year⁻¹; 95% confidence interval [CI] 2.36–5.25) of total AGB loss (8.72 Mg ha⁻¹ year⁻¹; CI 5.57–12.86) is due to damage to living trees. Total AGB loss was highly variable among forests, but these differences were mainly caused by site variability in damage-related AGB losses rather than by mortality-related AGB losses. We show that conventional forest inventories overestimate stand-level AGB stocks by 4% (1%–17% range across forests) because assume structurally complete trees, underestimate total AGB loss by 29% (6%–57% range across forests) due to overlooked damage-related AGB losses, and overestimate AGB loss via mortality by 22% (7%–80% range across forests) because of the assumption that trees are undamaged before dying. Our results indicate that forest carbon fluxes are higher than previously thought. Damage on living trees is an underappreciated component of the forest carbon cycle that is likely to become even more important as the frequency and severity of forest disturbances increase.     

Attenuation of Notch and Hedgehog signaling is required for fate specification in the spinal cord

During the development of the spinal cord, proliferative neural progenitors differentiate into postmitotic neurons with distinct fates. How cells switch from progenitor states to differentiated fates is poorly understood. To address this question, we studied the differentiation of progenitors in the zebrafish spinal cord, focusing on the differentiation of Kolmer-Agduhr″ (KA″) interneurons from lateral floor plate (LFP) progenitors. In vivo cell tracking demonstrates that KA″ cells are generated from LFP progenitors by both symmetric and asymmetric cell divisions. A photoconvertible reporter of signaling history (PHRESH) reveals distinct temporal profiles of Hh response: LFP progenitors continuously respond to Hh, while KA″ cells lose Hh response upon differentiation. Hh signaling is required in LFP progenitors for KA″ fate specification, but prolonged Hh signaling interferes with KA″ differentiation. Notch signaling acts permissively to maintain LFP progenitor cells: activation of Notch signaling prevents differentiation, whereas inhibition of Notch signaling results in differentiation of ectopic KA″ cells. These results indicate that neural progenitors depend on Notch signaling to maintain Hh responsiveness and rely on Hh signaling to induce fate identity, whereas proper differentiation depends on the attenuation of both Notch and Hh signaling.