Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24 h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4 h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4 h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24 h soaks. However, a 10-fold increase in dsRNA to 1 mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24 h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

Systematic random sampling of the comet assay

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory ‘tail’ DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the ‘randomness’ of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

CD207+ CD103+ dermal dendritic cells cross-present keratinocyte-derived antigens irrespective of the presence of Langerhans cells.

Recent studies have challenged the view that Langerhans cells (LCs) constitute the exclusive antigen-presenting cells of the skin and suggest that the dermal dendritic cell (DDC) network is exceedingly complex. Using knockin mice to track and ablate DCs expressing langerin (CD207), we discovered that the dermis contains five distinct DC subsets and identified their migratory counterparts in draining lymph nodes. Based on this refined classification, we demonstrated that the quantitatively minor CD207+ CD103+ DDC subset is endowed with the unique capability of cross-presenting antigens expressed by keratinocytes irrespective of the presence of LCs. We further showed that Y-Ae, an antibody that is widely used to monitor the formation of complexes involving I-Ab molecules and a peptide derived from the I-E alpha chain, recognizes mature skin DCs that express I-Ab molecules in the absence of I-E alpha. Knowledge of this extra reactivity is important because it could be, and already has been, mistakenly interpreted to support the view that antigen transfer can occur between LCs and DDCs. Collectively, these data revisit the transfer of antigen that occurs between keratinocytes and the five distinguishable skin DC subsets and stress the high degree of functional specialization that exists among them.

Climate change-linked range expansion of Nathusius' pipistrelle bat, Pipistrellus nathusii (Keyserling & Blasius, 1839)

Aim To examine the effect of climate change on the occurrence and distribution of Pipistrellus nathusii (Nathusius' pipistrelle) in the United Kingdom (UK).Location We modelled habitat and climatic associations of P. nathusii in the UK and applied this model to the species' historical range in continental Europe.Methods A binomial logistic regression model was constructed relating the occurrence of P. nathusii to climate and habitat characteristics using historical species occurrence records (1940-2006) and CORINE land cover data. This model was applied to historical and projected climate data to examine changes in suitable range (1940-2080) of this species. We tested the predictive ability of the model with known records in the UK after 2006 and applied the model to the species' known range in Europe.Results The distribution of P. nathusii was related positively to the area of water bodies, woodland and small areas of urbanization, and negatively related to the area of peat/heathland. Species records were associated with higher minimum temperatures, low seasonal variation in temperature and intermediate rainfall. We found that suitable areas have existed in the UK since the 1940s and that these have expanded. The model had high predictive power when applied to new records after 2006, with a correct classification rate of 70%, estimated by receiver operating characteristic analysis. Based on climate projections, our model suggests a potential twofold increase in the area suitable for P. nathusii in the UK by 2050. The single most influential climate variable contributing to range increase was the projected increase in minimum temperature. When applied to Europe, the model predictions had best predictive capability of known records in western areas of the species' range, where P. nathusii is present during the winter.Main conclusions We show that a mobile, migratory species has adapted its range in response to recent climate change on a continental scale. We believe this may be the first study to demonstrate a case of range change linked to contemporary climate change in a mammal species in Europe.

A unique homologue of the eukaryotic protein-modifier ubiquitin present in the bacterium Bacteroides fragilis, a predominant resident of the human gastro-intestinal tract

In the complete genome sequences of Bacteroides fragilis NCTC9343 and 638R, we have discovered a gene, ubb, the product of which has 63% identity to human ubiquitin and cross-reacts with antibodies raised against bovine ubiquitin. The sequence of ubb is closest in identity (76%) to the ubiquitin gene from a Migratory Grasshopper entomopoxvirus, suggesting acquisition by inter-kingdom horizontal gene transfer. We have screened clinical isolates of B. fragilis from diverse geographical regions and found that ubb is present in some, but not all strains. The gene is transcribed and the mRNA translated in B. fragilis, but deletion of ubb did not have a detrimental effect on growth. BfUbb has a predicted signal sequence; both full length and processed forms were detected in whole cell extracts, while the processed form was found in concentrated culture supernatants. Purified recombinant BfUbb inhibited in vitro ubiquitination and was able to covalently bind the human E1 activating enzyme, suggesting it could act as a suicide substrate in vivo. B. fragilis is one of the predominant members of the normal human resident gastro-intestinal microbiota with estimates up to >1011 cells g-1 of faeces by culture. These data indicate that the gastro-intestinal tract of some individuals could contain a significant amount of aberrant ubiquitin with the potential to inappropriately activate the host immune system and/or interfere with eukaryotic ubiquitin activity. This discovery could have profound implications in relation to our understanding of human diseases such as inflammatory bowel and autoimmune diseases.

Langerin(+) Dermal Dendritic Cells Are Critical for CD8(+) T Cell Activation and IgH gamma-1 Class Switching in Response to Gene Gun Vaccines

The C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin(+) dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin(+) DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin(+) DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin(+) cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin(+) DC abrogated the activation of IFN-gamma producing and cytolytic CD8(+) T cells after gene gun vaccination. Moreover, we identified migratory langerin(+) dermal DC as the subset that directly activated CD8(+) T cells in lymph nodes. Langerin(+) DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4(+) T helper cells by langerin(+) or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin(+) DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation. The Journal of Immunology, 2011, 186: 1377-1383.

Differential effects of magnetic pulses on the orientation of naturally migrating birds

In migratory passerine birds, strong magnetic pulses are thought to be diagnostic of the remagnetization of iron minerals in a putative sensory system contained in the beak. Previous evidence suggests that while such a magnetic pulse affects the orientation of migratory birds in orientation cages, no effect was present when pulse-treated birds were tested in natural migration. Here we show that two migrating passerine birds treated with a strong magnetic pulse, designed to alter the magnetic sense, migrated in a direction that differed significantly from that of controls when tested in natural conditions. The orientation of treated birds was different depending on the alignment of the pulse with respect to the magnetic field. These results can aid in advancing understanding of how the putative iron-mineral-based receptors found in birds' beaks may be used to detect and signal the intensity and/or direction of the Earth's magnetic field.

Evidence for repeated independent evolution of migration in the largest family of bats

Background: How migration evolved represents one of the most poignant questions in evolutionary biology. While studies on the evolution of migration in birds are well represented in the literature, migration in bats has received relatively little attention. Yet, more than 30 species of bats are known to migrate annually from breeding to non-breeding locations. Our study is the first to test hypotheses on the evolutionary history of migration in bats using a phylogenetic framework. Methods and Principal Findings: In addition to providing a review of bat migration in relation to existing hypotheses on the evolution of migration in birds, we use a previously published supertree to formulate and test hypotheses on the evolutionary history of migration in bats. Our results suggest that migration in bats has evolved independently in several lineages potentially as the need arises to track resources (food, roosting site) but not through a series of steps from short- to long-distance migrants, as has been suggested for birds. Moreover, our analyses do not indicate that migration is an ancestral state but has relatively recently evolved in bats. Our results also show that migration is significantly less likely to evolve in cave roosting bats than in tree roosting species. Conclusions and Significance: This is the first study to provide evidence that migration has evolved independently in bat lineages that are not closely related. If migration evolved as a need to track seasonal resources or seek adequate roosting sites, climate change may have a pivotal impact on bat migratory habits. Our study provides a strong framework for future research on the evolution of migration in chiropterans. © 2009 Bisson et al.

Estimation of survival rate and extinction probability for stage-structured populations with overlapping life stages

The development of methods providing reliable estimates of demographic parameters (e. g., survival rates, fecundity) for wild populations is essential to better understand the ecology and conservation requirements of individual species. A number of methods exist for estimating the demographics of stage-structured populations, but inherent mathematical complexity often limits their uptake by conservation practitioners. Estimating survival rates for pond-breeding amphibians is further complicated by their complex migratory and reproductive behaviours, often resulting in nonobservable states and successive cohorts of eggs and tadpoles. Here we used comprehensive data on 11 distinct breeding toad populations (Bufo calamita) to clarify and assess the suitability of a relatively simple method [the Kiritani-Nakasuji-Manly (KNM) method] to estimate the survival rates of stage-structured populations with overlapping life stages. The study shows that the KNM method is robust and provides realistic estimates of amphibian egg and larval survival rates for species in which breeding can occur as a single pulse or over a period of several weeks. The study also provides estimates of fecundity for seven distinct toad populations and indicates that it is essential to use reliable estimates of fecundity to limit the risk of under- or overestimating the survival rates when using the KNM method. Survival and fecundity rates for B. calamita populations were then used to define population matrices and make a limited exploration of their growth and viability. The findings of the study recently led to the implementation of practical conservation measures at the sites where populations were most vulnerable to extinction. © 2010 The Society of Population Ecology and Springer.

Reducing the future threat from (liver) fluke: realistic prospect or quixotic fantasy?

The liver fluke remains an economically significant parasite of livestock and is emerging as an important zoonotic infection of humans. The incidence of the disease has increased in the last few years, as a possible consequence of changes to the World's climate. Future predictions suggest that this trend is likely to continue. Allied to the changing pattern of disease, reports of resistance to triclabendazole (TCBZ) have appeared in the literature, although they do not all represent genuine cases of resistance. Nevertheless, any reports of resistance are a concern, because triclabendazole is the only drug that has high activity against the migratory and damaging juvenile stages of infection. How to deal with the twin problems (of increasing incidence and drug resistance) is the overall theme of the session on “Trematodes: Fasciola hepatica epidemiology and control” and of this review to introduce the session.

Greater knowledge of fluke epidemiology and population genetics will highlight those regions where surveillance is most required and indicate how quickly resistant populations of fluke may arise. Models of disease risk are becoming increasingly sophisticated and precise, with more refined data analysis programmes and Geographic Information Systems (GIS) data. Recent improvements have been made in our understanding of the action of triclabendazole and the ways in which flukes have become resistant to it. While microtubules are the most likely target for drug action, tubulin mutations do not seem to be involved in the resistance mechanism. Rather, upregulation of drug uptake and metabolism processes appear to be more important and the data relating to them will be discussed. The information may help in the design of new treatment strategies or pinpoint potential molecular markers for monitoring fluke populations. Advances in the identification of novel targets for drugs and vaccines will be made by the various “-omics” technologies that are now being applied to Fasciola. A major area of concern in the current control of fasciolosis is the lack of reliable tests for the diagnosis of drug (TCBZ) resistance. This has led to inaccurate reports of resistance, which is hindering successful disease management, as farmers may be encouraged to switch to less effective drugs. Progress with the development of a number of new diagnostic tests will be reviewed.

Nathusius' pipistrelle bats (Pipistrellus nathusii, Keyserling & Blasius 1839) breeding in Ireland

We describe the most westerly known maternity colony of Nathusius' pipistrelle bats (Pipistrellus nathusii). The bats were identified by using morphometric measurements and analysis of time-expanded echolocation and social calls. The roost, containing approximately 150 individuals, was located in a mid 19th century farm stable block and store house situated in parkland in County Antrim, Northern Ireland. The roost was visited on 30 April, I May and 22 June 1997. Over this period, 11 bats were caught: one adult male, five pregnant females, four lactating females and a juvenile male. Direct observation of behaviour patterns suggests that mating groups of P. nathusii may occur in Ireland as late as May. The migratory nature of this species is discussed.

Pharmacogenomic Profiling and Pathway Analyses Identify MAPK-Dependent Migration as an Acute Response to SN38 in p53 Null and p53-Mutant Colorectal Cancer Cells.

The topoisomerase I inhibitor irinotecan is used to treat advanced colorectal cancer and has been shown to have p53-independent anticancer activity. The aim of this study was to identify the p53-independent signaling mechanisms activated by irinotecan. Transcriptional profiling of isogenic HCT116 p53 wild-type and p53 null cells was carried out following treatment with the active metabolite of irinotecan, SN38. Unsupervised analysis methods showed that p53 status had a highly significant impact on gene expression changes in response to SN38. Pathway analysis indicated that pathways involved in cell motility [adherens junction, focal adhesion, mitogen-activated protein kinase (MAPK), and regulation of the actin cytoskeleton] were significantly activated in p53 null cells, but not p53 wild-type cells, following SN38 treatment. In functional assays, SN38 treatment increased the migratory potential of p53 null and p53-mutant colorectal cancer cell lines, but not p53 wild-type lines. Moreover, p53 null SN38-resistant cells were found to migrate at a faster rate than parental drug-sensitive p53 null cells, whereas p53 wild-type SN38-resistant cells failed to migrate. Notably, cotreatment with inhibitors of the MAPK pathway inhibited the increased migration observed following SN38 treatment in p53 null and p53-mutant cells. Thus, in the absence of wild-type p53, SN38 promotes migration of colorectal cancer cells, and inhibiting MAPK blocks this potentially prometastatic adaptive response to this anticancer drug.

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro

BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell’s response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel.
METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches.
RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2k) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8
representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2kcells show enhanced migratory ability. At 72 h after paclitaxel, MAD2kcells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2k MCF7 cell line after paclitaxel reflecting the observed increase in senescence.
CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.