Linking Material Flow Analysis with Environmental Impact Potential

Technology transition can have significant implications on the evolution of environmental impact potential of disposed electronics over time. Considering technology transition, we quantify the temporal behavior of ecological and human health impact potential from select heavy metals in electronic waste (e‐waste). The case study analyzes product substitution effects in two electronic cohorts from the U.S. market: (1) computers (laptops substituting for desktops) and (2) televisions (flat‐panel liquid crystal displays [LCDs] and plasma displays substituting for cathode‐ray tubes [CRTs]). Quantities of end‐of‐life (EoL) units to year 2030 are forecasted by the unique combination of dynamic material flow analysis, logistic trend analysis, and product lifespan calibration methods. Metal content from EoL units are assessed via a pathway and effect model using USETox? characterization factors to determine the toxicity potential attributed to heavy metal releases into different media (e.g., air, water, and soil) as an indicator of environmental burden. Results show high impact materials such as lead, nickel, and zinc cause changes in human health toxicity potential and copper causes changes in ecological toxicity potential. Effects of dematerialization, such as reduced metal content in laptops over desktops, provide some positive benefits in toxicity potential per product. However, from a market perspective, emerging e‐waste quantities created by increasing per capita penetration rates of electronics and increasing population will offset gains in environmental performance at the product level. The resulting analysis provides guidance on the timing expected for emerging EoL units and an indication of high impact potential materials requiring pollution prevention as product substitution occurs.     

The molecular mechanism of NELL2 movement and secretion in hippocampal progenitor HiB5 cells

Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 μm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 μm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.     

GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors

Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.     

Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

Background

Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.

Methods and Findings

We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.

Conclusions

The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.     

Effects of Treadmill Exercise on Cell Proliferation and Differentiation in the Subgranular Zone of the Dentate Gyrus in a Rat Model of Type II Diabetes

In the present study, we investigated the effects of a treadmill exercise on serum glucose levels and Ki67 and doublecortin (DCX) immunoreactivity, which is a marker of cell proliferation expressed during cell cycles except G0 and early G1 and a marker of progenitors differentiating into neurons, respectively, in the subgranular zone of the dentate gyrus (SZDG) using a type II diabetic model. At 6 weeks of age, Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats were put on a treadmill with or without running for 1 h/day/5 consecutive days at 22 m/min for 5 weeks. Body weight was significantly increased in the control (without running)-ZDF rats compared to that in the other groups. In the control groups blood glucose levels were increased by 392.7 mg/dl in the control-ZDF rats and by 143.3 mg/dl in the control-ZLC rats. However, in the exercise groups, blood glucose levels were similar between the exercise-ZLC and ZDF rats: The blood glucose levels were 110.0 and 118.2 mg/dl, respectively. Ki67 positive nuclei were detected in the SZDG in control and exercise groups. The number of Ki67 positive nuclei was significantly high in exercise groups compared to that in the control groups. In addition, Ki67 positive cells were abundant in ZLC groups compared to those in ZDF groups. DCX-immunoreactive structures in the control-ZDF rats were lower than that in the control-ZLC rats. In the exercise groups, DCX-immunoreactive structures (somata and processes with tertiary dendrites) and DCX protein levels were markedly increased in both the exercise-ZLC and ZDF rats compared to that in the control groups. These results suggest that a treadmill exercise reduces blood glucose levels in ZDF rats and increases cell proliferation and differentiation in the SZDG in ZLC and ZDF rats compared to those in control groups.     

Topology of Endoplasmic Reticulum‐Associated Cellular and Viral Proteins Determined with Split‐GFP

The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER‐associated proteins. A 215‐amino acid fragment of GFP (S1–10) was expressed in the cytoplasm as a free protein or fused to the N‐terminus of calnexin and in the ER as an intraluminal protein or fused to the C‐terminus of calnexin. A 16‐amino acid fragment of GFP (S11) was fused to the N‐ or C‐terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N‐ and C‐termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N‐ and C‐termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.      

Crystal structure of constitutively monomeric E. coli Hsp33 mutant with chaperone activity

Heat shock protein 33 (Hsp33) from Escherichia coli is a redox-regulated molecular chaperone that protects cells from oxidative stress. To understand the molecular basis for the monomer-dimer switch in the functional regulation of E. coli Hsp33, we generated a constitutively monomeric Hsp33 by introducing the Q151E mutation in the dimeric interface and determined its crystal structure. The overall scaffold of the monomeric Hsp33(1-235) (Q151E) mutant is virtually the same as that of the dimeric form, except that there is no domain swapping. The measurement of chaperone activity to thermally denatured luciferase showed that the constitutively monomeric Hsp33 mutant still retains chaperone activity similar to that of wild-type Hsp33(1-235), suggesting that a Hsp33 monomer is sufficient to interact with slowly unfolded substrate.     

Transducin Beta-Like Gene FTL1 Is Essential for Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley. In a previous study, we identified several mutants with reduced virulence by insertional mutagenesis. A transducin beta-like gene named FTL1 was disrupted in one of these nonpathogenic mutants. FTL1 is homologous to Saccharomyces cerevisiae SIF2, which is a component of the Set3 complex involved in late stages of ascospore formation. The Δftl1 mutant was significantly reduced in conidiation and failed to cause typical disease symptoms. It failed to colonize the vascular tissues of rachis or cause necrosis on the rachis of inoculated wheat heads. The Δftl1 mutant also was defective in spreading from infected anthers to ovaries and more sensitive than the wild type to plant defensins MsDef1 and osmotin. However, the activation of two mitogen-activated protein kinases, Mgv1 and Gpmk1, production of deoxynivalenol, and expression of genes known to be important for plant infection in F. graminearum were not affected, indicating that the defect of the Δftl1 mutant in plant infection is unrelated to known virulence factors in this pathogen and may involve novel mechanisms. The Δftl1 deletion mutant was significantly reduced in histone deacetylation, and many members of the yeast Set3 complex are conserved in F. graminearum. FTL1 appears to be a component of this well-conserved protein complex that plays a critical role in the penetration and colonization of wheat tissues.The filamentous ascomycete Fusarium graminearum (teleomorph Gibberella zeae) is the main causal agent of Fusarium head blight (FHB), or scab, which is an important disease on wheat and barley throughout the world (18). It also causes stalk and ear rots of maize and infects other small grains. In addition to causing yield losses, this pathogen often contaminates infested grains with trichothecene and estrogenic mycotoxins, such as deoxynivalenol (DON) and zearalenone. Unfortunately, complete resistance to F. graminearum is lacking in wheat, and fungicide application is not cost-effective for FHB control in wheat and barley.F. graminearum overwinters in infected plant debris and produces ascospores in the spring. Ascospores are forcibly discharged from mature perithecia (52) and function as the primary inoculum for FHB. The multicellular conidia or macroconidia are important for spreading the disease in the field and colonizing plant vegetative tissues. Wheat spikes are most susceptible to FHB at anthesis (34a). Although F. graminearum can colonize glumes, anthers are the main site of primary infection on flowering wheat heads (3, 38). Earlier studies indicated that wheat anther extracts stimulate F. graminearum virulence on wheat. Choline and glycine betaine were identified as two major components in anthers that stimulate fungal growth and predispose wheat to F. graminearum infection (50, 51). Under conducive conditions, the fungus can spread from the infected floret along the rachis and cause severe damage. The production of DON, the first virulence factor identified in F. graminearum (11, 42), is not necessary for the initial infection but is important for the spread of FHB on infected wheat heads (2).In the past few years, genetic and genomic studies of F. graminearum have advanced significantly. The genome of F. graminearum has been sequenced (10) and a whole-genome microarray of this haploid homothallic fungus is commercially available (21). A number of pathogenicity or virulence factors have been identified by insertional mutagenesis or targeted gene deletion approaches. Two mitogen-activated protein (MAP) kinase genes, MGV1 and GPMK1, are essential for pathogenicity in F. graminearum (23, 24). Genes that are important for full virulence in F. graminearum on wheat include FGL1 (54), GzCPS1 (31), FBP1 (22), FSR1 (48), SID1 (19), NPS6 (37), RAS2 (5), GzGPA2 and GzGPB1 (56), and HMR1 (47). These virulence-associated genes encode proteins with various biochemical activities, such as lipase, nonribosomal peptide synthase, Ras protein, and 3-hydroxy 3-methylglutaryl coenzyme A reductase. Several genes involved in the primary metabolism, such as the CBL1, RSY1, GzHIS7, ADE5, and ARG2 genes (29, 44, 46) that are required for methionine, histidine, and arginine syntheses, also have been implicated in plant infection in F. graminearum. Overall, molecular mechanisms underlying F. graminearum pathogenesis appear to be complex and remain to be fully understood.In a previous study, we identified 11 restriction enzyme-mediated integration (REMI) mutants that are defective in plant infection (46). In one of these mutants, the transforming vector was inserted in a predicted gene named FTL1 (for Fusarium transducin beta-like gene 1). FTL1 is homologous to the mammalian TBL1 or TBLR1 genes (40, 55) and the Saccharomyces cerevisiae SIF2 gene (8). The products of these genes are components of protein complexes involving histone deacetylases (HDACs). In mammalian cells, TBL1 and TBLR1 are parts of the N-CoR/SMRT/HDAC complexes (40). In yeast, SIF2 is a part of the Set3 complex regulating ascospore formation. In F. graminearum, the Δftl1 gene replacement mutant was significantly reduced in conidiation and failed to cause typical head blight symptoms on flowering wheat heads. It failed to colonize vascular tissues or cause necrosis on the rachis of inoculated wheat heads. The Δftl1 mutant also was defective in spreading from infected anthers to ovaries and was more sensitive than the wild type to plant defensins MsDef1 and osmotin. Although it was normal in the production of deoxynivalenol and the expression of known virulence factors, the Δftl1 mutant was significantly reduced in HDAC activities. FTL1 appears to be a component of this well-conserved HDAC complex that plays a critical role in the penetration and colonization of wheat tissues.     

Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.     

Microglial Phagocytosis of Dopamine Neurons at Early Phases of Apoptosis

Transection of the medial forebrain bundle caused apoptosis of dopamine neurons in the rat substantia nigra. Immunohistochemical localization of activated microglia and tyrosine hydroxylase in the axotomized substantia nigra showed that activation of microglia was rapid and OX-6 (MHC-II marker)-positive and ED1 (lysosomal phagocytic marker)-positive microglia were apposed to structurally intact tyrosine hydroxylase-positive dopamine neurons, indicating microglial phagocytosis of degenerating dopamine neurons. The occurrence of microglial phagocytosis at early stages of apoptosis may indicate the evolution of apoptosis into an irreversible state. Alternatively, interventions that suppress early activation of microglia might lead to novel mechanisms for neuron protection.