Interferon and Ribavirin Combination Treatment Synergistically Inhibit HCV Internal Ribosome Entry Site Mediated Translation at the Level of Polyribosome Formation

Purpose

Although chronic hepatitis C virus (HCV) infection has been treated with the combination of interferon alpha (IFN-α) and ribavirin (RBV) for over a decade, the mechanism of antiviral synergy is not well understood. We aimed to determine the synergistic antiviral mechanisms of IFN-α and RBV combination treatment using HCV cell culture.

Methods

The antiviral efficacy of IFN-α, RBV alone and in combination was quantitatively measured using HCV infected and replicon cell culture. Direct antiviral activity of these two drugs at the level of HCV internal ribosome entry site (IRES) mediated translation in Huh-7 cell culture was investigated. The synergistic antiviral effect of IFN-α and RBV combination treatment was verified using both the CalcuSyn Software and MacSynergy Software.

Results

RBV combination with IFN-α efficiently inhibits HCV replication cell culture. Our results demonstrate that IFN-α, interferon lambda (IFN-λ) and RBV each inhibit the expression of HCV IRES-GFP and that they have a minimal effect on the expression of GFP in which the translation is not IRES dependent. The combination treatments of RBV along with IFN-α or IFN-λ were highly synergistic with combination indexes <1. We show that IFN-α treatment induce levels of PKR and eIF2α phosphorylation that prevented ribosome loading of the HCV IRES-GFP mRNA. Silencing of PKR expression in Huh-7 cells prevented the inhibitory effect of IFN-α on HCV IRES-GFP expression. RBV also blocked polyribosome loading of HCV-IRES mRNA through the inhibition of cellular IMPDH activity, and induced PKR and eIF2α phosphorylation. Knockdown of PKR or IMPDH prevented RBV induced HCV IRES-GFP translation.

Conclusions

We demonstrated both IFN-α and RBV inhibit HCV IRES through prevention of polyribosome formation. The combination of IFN-α and RBV treatment synergistically inhibits HCV IRES translation via using two different mechanisms involving PKR activation and depletion of intracellular guanosine pool through inhibition of IMPDH.     

Loss of Heterozygosity Drives Clonal Diversity of Phytophthora capsici in China

Phytophthora capsici causes significant loss to pepper (Capsicum annum) in China and our goal was to develop single nucleotide polymorphism (SNP) markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs) identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH). Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.     

Adolescent Changes in Dopamine D1 Receptor Expression in Orbitofrontal Cortex and Piriform Cortex Accompany an Associative Learning Deficit

The orbitofrontal cortex (OFC) and piriform cortex are involved in encoding the predictive value of olfactory stimuli in rats, and neural responses to olfactory stimuli in these areas change as associations are learned. This experience-dependent plasticity mirrors task-related changes previously observed in mesocortical dopamine neurons, which have been implicated in learning the predictive value of cues. Although forms of associative learning can be found at all ages, cortical dopamine projections do not mature until after postnatal day 35 in the rat. We hypothesized that these changes in dopamine circuitry during the juvenile and adolescent periods would result in age-dependent differences in learning the predictive value of environmental cues. Using an odor-guided associative learning task, we found that adolescent rats learn the association between an odor and a palatable reward significantly more slowly than either juvenile or adult rats. Further, adolescent rats displayed greater distractibility during the task than either juvenile or adult rats. Using real-time quantitative PCR and immunohistochemical methods, we observed that the behavioral deficit in adolescence coincides with a significant increase in D1 dopamine receptor expression compared to juvenile rats in both the OFC and piriform cortex. Further, we found that both the slower learning and increased distractibility exhibited in adolescence could be alleviated by experience with the association task as a juvenile, or by an acute administration of a low dose of either the dopamine D1 receptor agonist SKF-38393 or the D2 receptor antagonist eticlopride. These results suggest that dopaminergic modulation of cortical function may be important for learning the predictive value of environmental stimuli, and that developmental changes in cortical dopaminergic circuitry may underlie age-related differences in associative learning.     

Transition between Acute and Chronic Hepatotoxicity in Mice Is Associated with Impaired Energy Metabolism and Induction of Mitochondrial Heme Oxygenase-1

The formation of protein inclusions is frequently associated with chronic metabolic diseases. In mice, short-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to hepatocellular damage indicated by elevated serum liver enzyme activities, whereas only minor morphological changes are observed. Conversely, chronic administration of DDC for several weeks results in severe morphological damage, characterized by hepatocellular ballooning, disruption of the intermediate filament cytoskeleton, and formation of Mallory-Denk bodies consisting predominantly of misfolded keratins, Sqstm1/p62, and heat shock proteins. To evaluate the mechanistic underpinnings for this dichotomy we dissected the time-course of DDC intoxication for up to 10 weeks. We determined body weight change, serum liver enzyme activities, morphologic alterations, induction of antioxidant response (heme oxygenase-1, HO-1), oxidative damage and ATP content in livers as well as respiration, oxidative damage and the presence and activity of HO-1 in endoplasmic reticulum and mitochondria (mtHO-1). Elevated serum liver enzyme activity and oxidative liver damage were already present at early intoxication stages without further subsequent increase. After 2 weeks of intoxication, mice had transiently lost 9% of their body weight, liver ATP-content was reduced to 58% of controls, succinate-driven respiration was uncoupled from ATP-production and antioxidant response was associated with the appearance of catalytically active mtHO-1. Oxidative damage was associated with both acute and chronic DDC toxicity whereas the onset of chronic intoxication was specifically associated with mitochondrial dysfunction which was maximal after 2 weeks of intoxication. At this transition stage, adaptive responses involving mtHO-1 were induced, indirectly leading to improved respiration and preventing further drop of ATP levels. Our observations clearly demonstrate principally different mechanisms for acute and chronic toxic damage.     

Effects of Wild Pig Disturbance on Forest Vegetation and Soils

In North America, wild pigs (Sus scrofa; feral pigs, feral swine, wild boars) are a widespread exotic species capable of creating large-scale biotic and abiotic landscape perturbations. Quantification of wild pig environmental effects has been particularly problematic in northern climates, where they occur only recently as localized populations at low densities. Between 2016 and 2017, we assessed short-term (within ~2 yrs of disturbance) effects of a low-density wild pig population on forest features in the central Lower Peninsula of Michigan, USA. We identified 16 8-ha sites using global positioning system locations from 7 radio-collared wild pigs for sampling. Within each site, we conducted fine-scale assessments at 81 plots and quantified potential disturbance by wild pigs. We defined disturbance as exposure of overturned soil, often resulting from rooting behavior by wild pigs. We quantified ground cover of plants within paired 1-m² frames at each plot, determined effects to tree regeneration using point-centered quarter sampling, and collected soil cores from each plot. We observed less percent ground cover of native herbaceous plants and lower species diversity, particularly for plants with a coefficient of conservatism ≥5, in plots disturbed by wild pigs. We did not observe an increase in colonization of exotic plants following disturbance, though the observed prevalence of exotic plants was low. Wild pigs did not select for tree species when rooting, and we did not detect any differences in regeneration of light- and heavy-seeded tree species between disturbed or undisturbed plots. Magnesium and ammonium content in soils were lower in disturbed plots, suggesting soil disturbance accelerated leaching of macronutrients, potentially altering nitrogen transformation. Our study suggested that disturbances by wild pigs, even at low densities, alters short-term native herbaceous plant diversity and soil chemistry. Thus, small-scale exclusion of wild pigs from vulnerable and rare plant communities may be warranted. © 2020 The Wildlife Society.     

Opportunistic Predation of Wild Turkey Nests by Wild Pigs

Wild pigs (Sus scrofa; i.e., feral hogs, feral swine) are considered an invasive species in the United States. Where they occur, they damage agricultural crops and wildlife habitat. Wild pigs also depredate native wildlife, particularly ground-nesting bird species during nesting season. In areas inhabited by wild turkeys (Meleagris gallopavo), nest destruction caused by wild pigs may affect recruitment. There is debate whether wild pigs actively seek ground-nesting bird nests or depredate them opportunistically. To address this debate, in 2016 we examined the movements of wild pigs relative to artificial wild turkey nests (i.e., control [no artificial nests], moderate density [12.5–25 nests/km²], and high density [25–50 nests/km²]) throughout the nesting season (i.e., early, peak, and late) in south-central Texas, USA. We found no evidence that wild pigs learned to seek and depredate wild turkey nests relative to nest density or nesting periods. Despite wild pigs being important nest predators, depredation was not a functional response to a pulsed food resource and can only be associated with overlapping densities of wild pigs and nests. Protecting reproductive success of wild turkeys will require reducing wild pig densities in nesting habitat prior to nesting season. © 2019 The Wildlife Society.     

Einfluß der Unterbrechung langjähriger Getreide-Fruchtfolgen durch zweijährigen Luzerne-Anbau auf den Kornertrag sowie auf den Befall des Winterweizens durch Pseudocercosporella herpotrichoides (Fron) Deighton

Die herbizide Wirksamkeit von Bromoxynil und Phenmedipham wird in starkem Maße von der Lösungsmittelkomponente der Herbizidformulierung beeinflußt. Es zeigt sich, daß die Photosynthesehemmung — als Maß für die Herbizidwirkung — mit zunehmender Hydrophilie der verwendeten Lösungsmittel sinkt, was insbesondere anhand der untersuchten Essigsäurealkylesterreihe nachweisbar ist. Zwischen der Wirkstoffapplikation auf Ganzblättern und isolierten Chloroplasten treten beträchtliche Wirkungsunterschiede auf. Demnach ist der wirkungsbegrenzende Schritt nicht innerhalb der Thylakoidmembran selbst zu suchen sondern findet auf dem Wege von der Blattoberfläche zum Chloroplasten statt.     

Der Besatz von Beta-Rüubensaatgut rait Wurzelbranderregern und der Einfluß von Umweltfaktoren auf das Auftreten des samenbüurtigen Wurzelbrandes

The root knot nematode Meloidogyne incognita was controlled more effectively when P. lilacinus and G. mosseae were applied together in a pot experiment than either was applied alone. Inoculation of tomato plant with G. mosseae did not markedly increase the growth of plant infected with M. incognita. Inoculation of plant with G. mosseae and P. lilacinus together or alone resulted in a similar shoot and plant height. The highest root development was achieved when mycorrhizal plant were inoculated with P. lilacinus to combat root knot nematode. Inoculation of tomato plant with P. lilacinus suppressed galls/root system and eggs/egg masses, compared to seedling inoculated with M. incognita alone. The mycorrhizal colonization was not affected by inoculation of P. lilacinus.