Cut down by some cowardly miscreants’: Plant maiming, or the Malicious Cutting of Plants as an Act of Protest in Eighteenth and Nineteenth Century Rural England

Since the publication of Hobsbawm and Rudé's Captain Swing our understanding of the role(s) of covert protests in Hanoverian rural England has advanced considerably. Whilst we now know much about the dramatic practices of incendiarism and animal maiming and the voices of resistance in seemingly straightforward acquisitive acts, one major gap remains. Despite the fact that almost thirty years have passed since E. P. Thompson brought to our attention that under the notorious ‘Black Act’ the malicious cutting of trees was a capital offence, no subsequent research has been published. This paper seeks to address this major lacuna by systematically analysing the practices and patterns of malicious attacks on plants (‘plant maiming’) in the context of late eighteenth- and early nineteenth-century southern England. It is shown that not only did plant maiming take many different forms, attacking every conceivable type of flora, but also that it was universally understood and practised. In some communities plant maiming was the protestors' weapon of choice. As a social practice it therefore embodied wider community beliefs regarding the defence of plebeian livelihoods and identities.

Persistent measles virus infection of mouse neural cells lacking known human entry receptors

Aims: Infection of the mouse central nervous system with wild type (WT) and vaccine strains of measles virus (MV) results in lack of clinical signs and limited antigen detection. It is considered that cell entry receptors for these viruses are not present on murine neural cells and infection is restricted at cell entry.

Methods: To examine this hypothesis, virus antigen and caspase 3 expression (for apoptosis) was compared in primary mixed, neural cell cultures infected in vitro or prepared from mice infected intracerebrally with WT, vaccine or rodent neuroadapted viruses. Viral RNA levels were examined in mouse brain by nested and real-time reverse transcriptase polymerase chain reaction.

Results: WT and vaccine strains were demonstrated for the first time to infect murine oligodendrocytes in addition to neurones despite a lack of the known MV cell receptors. Unexpectedly, the percentage of cells positive for viral antigen was higher for WT MV than neuroadapted virus in both in vitro and ex vivo cultures. In the latter the percentage of positive cells increased with time after mouse infection. Viral RNA (total and mRNA) was detected in brain for up to 20 days, while cultures were negative for caspase 3 in WT and vaccine virus infections.

Conclusions: WT and vaccine MV strains can use an endogenous cell entry receptor(s) or alternative virus uptake mechanism in murine neural cells. However, viral replication occurs at a low level and is associated with limited apoptosis. WT MV mouse infection may provide a model for the initial stages of persistent MV human central nervous system infections.

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus

The lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognized, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entry of MV into the body. However, the lack of detectable CD150 on these cells raises the issue of their importance in the pathogenesis of measles. This study utilized a combination of in vitro, ex vivo and in vivo model systems to characterize the susceptibility of epithelial cells to wt-MV of proven pathogenicity. Low numbers of MV-infected epithelial cells in close proximity to underlying infected lymphocytes or myeloid cells suggested infection via the basolateral side of the epithelium in the macaque model. In primary cultures of human bronchial epithelial cells, foci of MV-infected cells were only observed following infection via the basolateral cell surface. The extent of infection in primary cells was enhanced both in vitro and in ex vivo cornea rim tissue by disrupting the integrity of the cells prior to the application of virus. This demonstrated that, whilst epithelial cells may not be the primary target cells for wt-MV, areas of epithelium in which tight junctions are disrupted can become infected using high m.o.i. The low numbers of MV-infected epithelial cells observed in vivo in conjunction with the absence of infectious virus release from infected primary cell cultures suggest that epithelial cells have a peripheral role in MV transmission.

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV) several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question, and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. Additionally, endo-lysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable over-expression of p62 significantly suppressed DENV replication suggesting a novel role for p62 as viral restriction factor. Overall our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an anti-viral role, which is countered by DENV.

IMPORTANCE: Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we employed high-content, imaging-based flow cytometry to quantify autophagic flux and endo-lysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endo-lysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a pro- to an antiviral cellular process, which is counteracted by the virus.

Do leaves of plants on phosphorus-impoverished soils contain high concentrations of phenolic defence compounds?

Prominent theories of plant defence have predicted that plants growing on nutrient-poor soils produce more phenolic defence compounds than those on richer soils. Only recently has the Protein Competition Model (PCM) of phenolic allocation suggested that N and P limitation could have different effects because the nutrients are involved in different cellular metabolic processes. 2. We extend the prediction of the PCM and hypothesize that N will have a greater influence on the production of phenolic defensive compounds than P availability, because N limitation reduces protein production and thus competition for phenylalanine, a precursor of many phenolic compounds. In contrast, P acts as a recyclable cofactor in these reactions, allowing protein and hence phenolic production to continue under low P conditions. 3. We test this hypothesis by comparing the foliar concentrations of phenolic compounds in (i) phenotypes of 21 species growing on P-rich alluvial terraces and P-depleted marine terraces in southern New Zealand, and (ii) 87 species growing under similar climates on comparatively P-rich soils in New Zealand vs. P-depleted soils in Tasmania. 4. Foliar P concentrations of plants from the marine terraces were about half those of plants from alluvial soils, and much lower in Tasmania than in New Zealand. However, foliar concentrations of N and phenolic compounds were similar across sites in both comparisons, supporting the hypothesis that N availability is a more important determinant of plant investment in phenolic defensive compounds than P availability. We found no indication that reduced soil P levels influenced plant concentrations of phenolic compounds. There was wide variation in the foliar N and P concentrations among species, and those with low foliar nutrient concentrations produced more phenolics (including condensed tannins). 5. Our study is the first trait comparison extending beyond standard leaf economics to include secondary metabolites related to defence in forest plants, and emphasizes that N and P have different influences on the production of phenolic defence compounds. © 2009 British Ecological Society.

Development and comparison of a Primer-Probe Energy Transfer based assay and a 5 ' conjugated Minor Groove Binder assay for sensitive real-time PCR detection of infectious laryngotracheitis virus

In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2 x 10(8) to 2 x 10(2) copies/mu l. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV. (C) 2011 Elsevier B.V. All rights reserved.

Analysis of receptor (CD46, CD150) usage by measles virus

This is a collaborative paper between Juergen Schneider-Schaulies's group and ours. It shows the differential use of the two measles receptors in various strains.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (sORNA-soaked M incognita eggs Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH2)-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s. which recovered within 24 h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48 h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-en-I transcript which encodes an RNAi-inhibiting siRNA exonuclease We observe a marked up-regulation of MI-en-I transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RN/NI-based reverse genetic screens in plant parasitic nematodes. (C) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.