Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy

ATG4B belongs to the autophagin family of cysteine proteases required for autophagy, an emerging target of cancer therapy. Developing pharmacological ATG4B inhibitors is a very active area of research. However, detailed studies on the role of ATG4B during anticancer therapy are lacking. By analyzing PC-3 and C4-2 prostate cancer cells overexpressing dominant negative ATG4B^C74Ain vitro and in vivo, we show that the effects of ATG4B^C74A are cell type, treatment, and context-dependent. ATG4B^C74A expression can either amplify the effects of cytotoxic therapies or contribute to treatment resistance. Thus, the successful clinical application of ATG4B inhibitors will depend on finding predictive markers of response.     

Ciliate–adenovirus interactions in experimental co-cultures of Euplotes octocarinatus and in wastewater environment

The aim of the present work was to study the dynamics of the interactions between human adenovirus and ciliates under both experimental and field conditions. Experimental co-cultures of the ciliated protozoan Euplotes octocarinatus and human adenovirus (HAdV) type 2 were established and virus internalization was investigated using nested PCR and direct immunofluorescence (IF). In addition, to study protozoa-virus interactions in the field, wild ciliates were isolated from active sludges of a wastewater treatment plant and analyzed for the presence of adenovirus using direct IF. In vitro experiments revealed HAdV type 2 inside Euplotes cells after 15 min of contact and its persistence until at least 35 days post infection. In addition, our results showed the adsorption of adenovirus on the surface of wild ciliates. We conclude that HAdV is taken up by ciliates, however more studies are necessary in order to better investigate the mechanisms, the infectivity of internalized virus and the protective effects of internalization against disinfection.     

Circadian Locomotor Activity Under Artificial Light in the Freshwater Crab Pseudothelphusa americana

Long-term recordings of locomotor activity were obtained from intact freshwater crabs, Pseudothelphusa americana in constant darkness (DD), constant light (LL) and different light-dark (LD) protocols. Bimodal rhythms were typically observed in this crab when subjected to DD or LD, with bouts of activity anticipating lights-on and lights-off, respectively. Freerunning circadian rhythms were expressed in both DD and LL for longer than 30 days. In DD, we observed that some animals presented different period lengths for each activity component. During LL, activity was primarily unimodal, however spontaneous splitting of the rhythms were observed in some animals. When activity was recorded under artificial long days, the morning bouts maintained their phase relationship but the evening bouts changed their phase relationship with the Zeitgeber. Our results indicate that, bimodal locomotor activity rhythm in the crab Pseudothelphusa americana is variable among organisms. The characteristics of phase relationship with LD and responses to LL for morning and evening bouts, suggest that, locomotor activity could be driven by multiple oscillators, and that coupling between these oscillators may be regulated by light.     

The use of statistical tools in field testing of putative effects of genetically modified plants on nontarget organisms

To fulfill existing guidelines, applicants that aim to place their genetically modified (GM) insect‐resistant crop plants on the market are required to provide data from field experiments that address the potential impacts of the GM plants on nontarget organisms (NTO's). Such data may be based on varied experimental designs. The recent EFSA guidance document for environmental risk assessment (2010) does not provide clear and structured suggestions that address the statistics of field trials on effects on NTO's. This review examines existing practices in GM plant field testing such as the way of randomization, replication, and pseudoreplication. Emphasis is placed on the importance of design features used for the field trials in which effects on NTO's are assessed. The importance of statistical power and the positive and negative aspects of various statistical models are discussed. Equivalence and difference testing are compared, and the importance of checking the distribution of experimental data is stressed to decide on the selection of the proper statistical model. While for continuous data (e.g., pH and temperature) classical statistical approaches – for example, analysis of variance (ANOVA) – are appropriate, for discontinuous data (counts) only generalized linear models (GLM) are shown to be efficient. There is no golden rule as to which statistical test is the most appropriate for any experimental situation. In particular, in experiments in which block designs are used and covariates play a role GLMs should be used. Generic advice is offered that will help in both the setting up of field testing and the interpretation and data analysis of the data obtained in this testing. The combination of decision trees and a checklist for field trials, which are provided, will help in the interpretation of the statistical analyses of field trials and to assess whether such analyses were correctly applied.     

Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI‐69A11

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras^(Q61K)::Ink4a^?/?). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras^(Q61K)::Ink4a^?/? tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.     

Ocean Acidification Disrupts Prey Responses to Predator Cues but Not Net Prey Shell Growth in Concholepas concholepas (loco)

Background

Most research on Ocean Acidification (OA) has largely focused on the process of calcification and the physiological trade-offs employed by calcifying organisms to support the building of calcium carbonate structures. However, there is growing evidence that OA can also impact upon other key biological processes such as survival, growth and behaviour. On wave-swept rocky shores the ability of gastropods to self-right after dislodgement, and rapidly return to normal orientation, reduces the risk of predation.

Methodology/Principal Findings

The impacts of OA on this self-righting behaviour and other important parameters such as growth, survival, shell dissolution and shell deposition in Concholepas concholepas (loco) were investigated under contrasting pCO₂ levels. Although no impacts of OA on either growth or net shell calcification were found, the results did show that OA can significantly affect self-righting behaviour during the early ontogeny of this species with significantly faster righting times recorded for individuals of C. concholepas reared under increased average pCO₂ concentrations (± SE) (716±12 and 1036±14 µatm CO₂) compared to those reared at concentrations equivalent to those presently found in the surface ocean (388±8 µatm CO₂). When loco were also exposed to the predatory crab Acanthocyclus hassleri, righting times were again increased by exposure to elevated CO₂, although self-righting times were generally twice as fast as those observed in the absence of the crab.

Conclusions and Significance

These results suggest that self-righting in the early ontogeny of C. concholepas will be positively affected by pCO₂ levels expected by the end of the 21st century and beginning of the next one. However, as the rate of self-righting is an adaptive trait evolved to reduce lethal predatory attacks, our result also suggest that OA may disrupt prey responses to predators in nature.     

The TNF-Family Cytokine TL1A Inhibits Proliferation of Human Activated B Cells

Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.     

IFN-α Regulates Blimp-1 Expression via miR-23a and miR-125b in Both Monocytes-Derived DC and pDC

Type I interferon (IFN-I) have emerged as crucial mediators of cellular signals controlling DC differentiation and function. Human DC differentiated from monocytes in the presence of IFN-α (IFN-α DC) show a partially mature phenotype and a special capability of stimulating CD4+ T cell and cross-priming CD8+ T cells. Likewise, plasmacytoid DC (pDC) are blood DC highly specialized in the production of IFN-α in response to viruses and other danger signals, whose functional features may be shaped by IFN-I. Here, we investigated the molecular mechanisms stimulated by IFN-α in driving human monocyte-derived DC differentiation and performed parallel studies on peripheral unstimulated and IFN-α-treated pDC. A specific miRNA signature was induced in IFN-α DC and selected miRNAs, among which miR-23a and miR-125b, proved to be negatively associated with up-modulation of Blimp-1 occurring during IFN-α-driven DC differentiation. Of note, monocyte-derived IFN-α DC and in vitro IFN-α-treated pDC shared a restricted pattern of miRNAs regulating Blimp-1 expression as well as some similar phenotypic, molecular and functional hallmarks, supporting the existence of a potential relationship between these DC populations. On the whole, these data uncover a new role of Blimp-1 in human DC differentiation driven by IFN-α and identify Blimp-1 as an IFN-α-mediated key regulator potentially accounting for shared functional features between IFN-α DC and pDC.     

Podocyte Expression of Membrane Transporters Involved in Puromycin Aminonucleoside-Mediated Injury

Several complex mechanisms contribute to the maintenance of the intricate ramified morphology of glomerular podocytes and to interactions with neighboring cells and the underlying basement membrane. Recently, components of small molecule transporter families have been found in the podocyte membrane, but expression and function of membrane transporters in podocytes is largely unexplored. To investigate this complex field of investigation, we used two molecules which are known substrates of membrane transporters, namely Penicillin G and Puromycin Aminonucleoside (PA).We observed that Penicillin G pre-administration prevented both in vitro and in vivo podocyte damage caused by PA, suggesting the engagement of the same membrane transporters by the two molecules. Indeed, we found that podocytes express a series of transporters which are known to be used by Penicillin G, such as members of the Organic Anion Transporter Polypeptides (OATP/Oatp) family of influx transporters, and P-glycoprotein, a member of the MultiDrug Resistance (MDR) efflux transporter family.Expression of OATP/Oatp transporters was modified by PA treatment. Similarly, in vitro PA treatment increased mRNA and protein expression of P-glycoprotein, as well as its activity, confirming the engagement of the molecule upon PA administration.In summary, we have characterized some of the small molecule transporters present at the podocyte membrane, focusing on those used by PA to enter and exit the cell. Further investigation will be needed to understand precisely the role of these transporter families in maintaining podocyte homeostasis and in the pathogenesis of podocyte injury.