The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML–CysPc–C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc–C2L domains of land plant calpains form a separate sub‐clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1‐like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1‐3 mutant using CysPc–C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc–C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1‐3 mutant phenotype. In contrast, neither the CysPc–C2L domains from M. viride nor chimeric animal–plant calpains complement this mutant. Co‐evolution analysis identified differences in the interactions between the CysPc–C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1‐3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺‐binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.     

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV‐1 co‐infections. We have delineated the mechanism of cell death induced by the HIV‐1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir–Leishmania interactions, we selected Nelfinavir‐resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir‐resistant and ‐sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir‐resistant and ‐sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time‐dependent manner. Furthermore, high‐resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir‐resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug‐induced intracellular vesicles.     

Plant quality and local adaptation undermine relocation in a bog specialist butterfly

The butterfly Boloria aquilonaris is a specialist of oligotrophic ecosystems. Population viability analysis predicted the species to be stable in Belgium and to collapse in the Netherlands with reduced host plant quality expected to drive species decline in the latter. We tested this hypothesis by rearing B. aquilonaris caterpillars from Belgian and Dutch sites on host plants (the cranberry, Vaccinium oxycoccos). Dutch plant quality was lower than Belgian one conferring lower caterpillar growth rate and survival. Reintroduction and/or supplementation may be necessary to ensure the viability of the species in the Netherlands, but some traits may have been selected solely in Dutch caterpillars to cope with gradual changes in host plant quality. To test this hypothesis, the performance of Belgian and Dutch caterpillars fed with plants from both countries were compared. Dutch caterpillars performed well on both plant qualities, whereas Belgian caterpillars could not switch to lower quality plants. This can be considered as an environmentally induced plastic response of caterpillars and/or a local adaptation to plant quality, which precludes the use of Belgian individuals as a unique solution for strengthening Dutch populations. More generally, these results stress that the relevance of local adaptation in selecting source populations for relocation may be as important as restoring habitat quality.     

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.     

Emerging molecular targets in melanoma invasion and metastasis

Metastatic cutaneous melanoma accounts for the majority of skin cancer deaths due to its aggressiveness and high resistance to current therapies. To efficiently metastasize, invasive melanoma cells need to change their cytoskeletal organization and alter contacts with the extracellular matrix and the surrounding stromal cells. Melanoma cells can use different migratory strategies depending on varying environments to exit the primary tumour mass and invade surrounding and later distant tissues. In this review, we have focused on tumour cell plasticity or the interconvertibility that melanoma cells have as one of the factors that contribute to melanoma metastasis. This has been an area of very intense research in the last 5 yr yielding a vast number of findings. We have therefore reviewed all the possible clinical opportunities that this new knowledge offers to both stratify and treat cutaneous malignant melanoma patients.