Site recognition and substrate screens for PKN family proteins

The PRKs [protein kinase C-related kinases; also referred to as PKNs (protein kinase Ns)] are a kinase family important in diverse functions including migration and cytokinesis. In the present study, we have re-evaluated and compared the specificity of PKN1 and PKN3 and assessed the predictive value in substrates. We analysed the phosphorylation consensus motif of PKNs using a peptide library approach and demonstrate that both PKN1 and PKN3 phosphorylate serine residues in sequence contexts that have an arginine residue in position -3. In contrast, PKN1 and PKN3 do not tolerate arginine residues in position +1 and -1 respectively. To test the predictive value of this motif, site analysis was performed on the PKN substrate CLIP-170 (cytoplasmic linker protein of 170 kDa); a PKN target site was identified that conformed to the predicted pattern. Using a protein array, we identified 22 further substrates for PKN1, of which 20 were previously undescribed substrates. To evaluate further the recognition signature, the site on one of these hits, EGFR (epidermal growth factor receptor), was identified. This identified Thr??? in EGFR as the PKN1 phosphorylation site and this retains an arginine residue at the -3 position. Finally, the constitutive phosphorylation of EGFR on Thr??? is shown to be modulated by PKN in vivo.     

Asynchronous development of honey bee host and Varroa destructor (Mesostigmata: Varroidae) influences reproductive potential of mites

A high proportion of nonreproductive (NR) Varroa destructor Anderson & Trueman (Mesostigmata: Varroidae), is commonly observed in honey bee colonies displaying the varroa sensitive hygienic trait (VSH). This study was conducted to determine the influence of brood removal and subsequent host reinvasion of varroa mites on mite reproduction. We collected foundress mites from stages of brood (newly sealed larvae, prepupae, white-eyed pupae, and pink-eyed pupae) and phoretic mites from adult bees. We then inoculated these mites into cells containing newly sealed larvae. Successful reproduction (foundress laid both a mature male and female) was low (13%) but most common in mites coming from sealed larvae. Unsuccessful reproductive attempts (foundress failed to produce both a mature male and female) were most common in mites from sealed larvae (22%) and prepupae (61%). Lack of any progeny was most common for mites from white-eyed (83%) and pink-eyed pupae (92%). We also collected foundress mites from sealed larvae and transferred them to cells containing newly sealed larvae, prepupae, white-eyed pupae, or pink-eyed pupae. Successful reproduction only occurred in the transfers to sealed larvae (26%). Unsuccessful reproductive attempts were most common in transfers to newly sealed larvae (40%) and to prepupae (25%). Unsuccessful attempts involved the production of immature progeny (60%), the production of only mature daughters (26%) or the production of only a mature male (14%). Generally, lack of progeny was not associated with mites having a lack of stored sperm. Our results suggest that mites exposed to the removal of prepupae or older brood due to hygiene are unlikely to produce viable mites if they invade new hosts soon after brood removal. Asynchrony between the reproductive status of reinvading mites and the developmental stage of their reinvasion hosts may be a primary cause of NR mites in hygienic colonies. Even if reinvading mites use hosts having the proper age for infestation, only a minority of them will reproduce.     

Migration patterns and navigation cues of Atlantic salmon post-smolts migrating from 12 rivers through the coastal zones around the Irish Sea

The freshwater phase of the first seaward migration of juvenile Atlantic salmon (Salmo salar) is relatively well understood when compared with our understanding of the marine phase of their migration. In 2021, 1008 wild and 60 ranched Atlantic salmon smolts were tagged with acoustic transmitters in 12 rivers in England, Scotland, Northern Ireland and Ireland. Large marine receiver arrays were deployed in the Irish Sea at two locations: at the transition of the Irish Sea into the North Atlantic between Ireland and Scotland, and between southern Scotland and Northern Ireland, to examine the early phase of the marine migration of Atlantic salmon smolts. After leaving their natal rivers' post-smolt migration through the Irish Sea was rapid with minimum speeds ranging from 14.03 to 38.56 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal river, to 9.69–39.94 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal estuary. Population minimum migration success through the study area was strongly correlated with the distance of travel, populations further away from the point of entry to the open North Atlantic exhibited lower migration success. Post-smolts from different populations experienced different water temperatures on entering the North Atlantic. This was largely driven by the timing of their migration and may have significant consequences for feeding and ultimately survivorship. The influence of water currents on post-smolt movement was investigated using data from previously constructed numerical hydrodynamic models. Modeled water current data in the northern Irish Sea showed that post-smolts had a strong preference for migrating when the current direction was at around 283° (west-north-west) but did not migrate when exposed to strong currents in other directions. This is the most favorable direction for onward passage from the Irish Sea to the continental shelf edge current, a known accumulation point for migrating post-smolts. These results strongly indicate that post-smolts migrating through the coastal marine environment are: (1) not simply migrating by current following (2) engage in active directional swimming (3) have an intrinsic sense of their migration direction and (4) can use cues other than water current direction to orientate during this part of their migration.     

The plant mitochondrial protein import apparatus — The differences make it interesting

Background

Mitochondria play essential roles in the life and death of almost all eukaryotic cells, ranging from single-celled to multi-cellular organisms that display tissue and developmental differentiation. As mitochondria only arose once in evolution, much can be learned from studying single celled model systems such as yeast and applying this knowledge to other organisms. However, two billion years of evolution have also resulted in substantial divergence in mitochondrial function between eukaryotic organisms.

Scope of Review

Here we review our current understanding of the mechanisms of mitochondrial protein import between plants and yeast (Saccharomyces cerevisiae) and identify a high level of conservation for the essential subunits of plant mitochondrial import apparatus. Furthermore, we investigate examples whereby divergence and acquisition of functions have arisen and highlight the emerging examples of interactions between the import apparatus and components of the respiratory chain.

Major conclusions

After more than three decades of research into the components and mechanisms of mitochondrial protein import of plants and yeast, the differences between these systems are examined. Specifically, expansions of the small gene families that encode the mitochondrial protein import apparatus in plants are detailed, and their essential role in seed viability is revealed.

General significance

These findings point to the essential role of the inner mitochondrial protein translocases in Arabidopsis, establishing their necessity for seed viability and the crucial role of mitochondrial biogenesis during germination. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Optimising and Evaluating the Characteristics of a Multiple Antigen ELISA for Detection of Mycobacterium bovis Infection in a Badger Vaccine Field Trial

A long-term research programme has been underway in Ireland to evaluate the usefulness of badger vaccination as part of the national bTB (bovine tuberculosis) control strategy. This culminated in a field trial which commenced in county Kilkenny in 2009 to determine the effects of badger vaccination on Mycobacterium bovis transmission in badgers under field conditions. In the present study, we sought to optimise the characteristics of a multiplex chemiluminescent assay for detection of M. bovis infection in live badgers. Our goal was to maximise specificity, and therefore statistical power, during evaluation of the badger vaccine trial data. In addition, we also aimed to explore the effects of vaccination on test characteristics. For the test optimisation, we ran a stepwise logistic regression with analytical weights on the converted Relative Light Units (RLU) obtained from testing blood samples from 215 badgers captured as part of culling operations by the national Department of Agriculture, Food and the Marine (DAFM). The optimised test was applied to two other datasets obtained from two captive badger studies (Study 1 and Study 2), and the sensitivity and specificity of the test was attained separately for vaccinated and non-vaccinated badgers. During optimisation, test sensitivity was maximised (30.77%), while retaining specificity at 99.99%. When the optimised test was then applied to the captive badger studies data, we observed that test characteristics did not vary greatly between vaccinated and non-vaccinated badgers. However, a different time lag between infection and a positive test result was observed in vaccinated and non-vaccinated badgers. We propose that the optimized multiplex immunoassay be used to analyse the vaccine trial data. In relation to the difference in the time lag observed for vaccinated and non-vaccinated badgers, we also present a strategy to enable the test to be used during trial evaluation.     

Oral Tolerance to Cancer Can Be Abrogated by T Regulatory Cell Inhibition

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue – JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups - this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.     

Development of a construct-based risk assessment framework for genetic engineered crops

Experience gained in the risk assessment (RA) of genetically engineered (GE) crops since their first experimental introductions in the early nineties, has increased the level of familiarity with these breeding methodologies and has motivated several agencies and expert groups worldwide to revisit the scientific criteria underlying the RA process. Along these lines, the need to engage in a scientific discussion for the case of GE crops transformed with similar constructs was recently identified in Argentina. In response to this need, the Argentine branch of the International Life Sciences Institute (ILSI Argentina) convened a tripartite working group to discuss a science-based evaluation approach for transformation events developed with genetic constructs which are identical or similar to those used in previously evaluated or approved GE crops. This discussion considered new transformation events within the same or different species and covered both environmental and food safety aspects. A construct similarity concept was defined, considering the biological function of the introduced genes. Factors like environmental and dietary exposure, familiarity with both the crop and the trait as well as the crop biology, were identified as key to inform a construct-based RA process.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Vector competence of Australian Culex gelidus Theobald (Diptera: Culicidae) for endemic and exotic arboviruses

The recent recognition of established populations of the mosquito, Culex gelidus Theobald, in Australia has raised concerns about local transmission of arboviruses. The vector competence of a mainland population of Cx. gelidus was investigated for two local alphaviruses, Ross River (RRV) and Barmah Forest (BFV) viruses, and three flaviviruses, Japanese encephalitis (JEV), Kunjin (KUNV) and Murray Valley encephalitis (MVEV) viruses. Colonised mosquitoes were exposed to virus via blood-soaked pledgets and transmission was tested using a capillary-tube method. The important Australian vectors, Aedes vigilax (Skuse) and Culex annulirostris Skuse, were used as internal controls for the alphaviruses and flaviviruses, respectively. Overall, Cx. gelidus was a more efficient vector of flaviviruses than alphaviruses. Culex gelidus was refractory to infection with BFV, and nearly 25% transmitted RRV, which was comparable to Ae. vigilax . Culex gelidus was susceptible to all three flaviviruses, with transmission rates of 96%, 95% and 41% for JEV, KUNV and MVEV, respectively. JEV transmission rates in Cx. annulirostris were unexpectedly low and this was possibly due to differences in susceptibility to JEV genotypes I and II. Considering the high susceptibility to the flaviviruses demonstrated here, and the natural infections with RRV and JEV that have been detected from northern Australian populations, the establishment of the exotic mosquito, Cx. gelidus , in Australia is potentially a significant public health concern.