The oncolytic peptide LTX-315 triggers necrotic cell death

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.     

The Cytolytic Amphipathic β(2,2)-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma

In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.     

Gene flow, effective population size and selection at major histocompatibility complex genes: brown trout in the Hardanger Fjord, Norway

Brown trout populations in the Hardanger Fjord, Norway, have declined drastically due to increased exposure to salmon lice from salmonid aquaculture. We studied contemporary samples from seven populations and historical samples (1972 and 1983) from the two largest populations, one of which has declined drastically whereas the other remains stable. We analysed 11 microsatellite loci, including one tightly linked to the UBA gene of the major histocompatibility class I complex (MHC) and another locus linked to the TAP2A gene, also associated with MHC. The results revealed asymmetric gene flow from the two largest populations to the other, smaller populations. This has important conservation implications, and we predict that possible future population recoveries will be mediated primarily by the remaining large population. Tests for selection suggested diversifying selection at UBA, whereas evidence was inconclusive for TAP2A. There was no evidence for temporally fluctuating selection. We assessed the distribution of adaptive divergence among populations. The results showed the most pronounced footprints of selection between the two largest populations subject to the least immigration. We suggest that asymmetric gene flow has an important influence on adaptive divergence and constrains local adaptive responses in the smaller populations. Even though UBA alleles may not affect salmon louse resistance, the results bear evidence of adaptive divergence among populations at immune system genes. This suggests that similar genetic differences could exist at salmon louse resistance loci, thus rendering it a realistic scenario that differential population declines could reflect differences in adaptive variation.     

Systematic evaluation of three microRNA profiling platforms: microarray, beads array, and quantitative real-time PCR array

Background

A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA).

Methodology/Principal Findings

The microRNA profiles of 40 human osteosarcoma xenograft samples were generated by LNA array, beads array, and TLDA. Results show that each of the three platforms perform similarly regarding intra-platform reproducibility or reproducibility of data within one platform while LNA array and TLDA had the best inter-platform reproducibility or reproducibility of data across platforms. The endogenous controls/probes contained in each platform have been observed for their stability under different treatments/environments; those included in TLDA have the best performance with minimal coefficients of variation. Importantly, we identify that the proper selection of normalization methods is critical for improving the inter-platform reproducibility, which is evidenced by the application of two non-linear normalization methods (loess and quantile) that substantially elevated the sensitivity and specificity of the statistical data assessment.

Conclusions

Each platform is relatively stable in terms of its own microRNA profiling intra-reproducibility; however, the inter-platform reproducibility among different platforms is low. More microRNA specific normalization methods are in demand for cross-platform microRNA microarray data integration and comparison, which will improve the reproducibility and consistency between platforms.     

Freezing tolerance in two Norway spruce (Picea abies [L.] Karst.) progenies is physiologically correlated with drought tolerance

The goal of the present study was to investigate whether seedlings of Norway spruce (Picea abies [L.] Karst.) from a frost tolerant progeny (P2), were more drought tolerant than seedlings from a less frost tolerant progeny (P1). Progenies differing in freezing tolerance were identified by exposing seedlings in autumn in a large-scale trial to temperatures from -11 to -15 degrees C and scoring the degree of needle injury. Seedlings from P1 and P2 were grown from seeds for about 1 year under controlled conditions in a climatized growth room and were exposed to drought stress by withholding water for about 3 weeks. Drought caused reductions in biomass in both progenies but to a stronger extent in P1 than in P2. Seedlings of P2 were able to fully maintain root biomass. They also showed less water loss in different tissues. Decreases in quantum yield efficiency of photosystem II of dark-adapted plants occurred several days later in P2 than in P1. New proteins of molecular masses of 24.3 and 25.5 kDa appeared during drought stress. Since they occurred in both progenies a role of these proteins in progeny-related differences in drought performance is unlikely. Progeny 2 contained inherently higher superoxide dismutase and lower peroxidase activities than progeny 1. In conclusion, freezing and drought-tolerance respective -sensitivity were co-occurring traits in the spruce progenies studied here. Pre-existing high activities of enzymes protecting against oxidative stress in seedlings may have contributed to increase stress tolerance in P2 compared with P1.     

Secretory protein enrichment and analysis: an optimized approach applied on cancer cell lines using 2D LC-MS/MS

Reliable methods for profiling secretory proteins are highly desirable for the identification of biomarkers of disease progression. Secreted proteins are often masked by high amounts of protein supplements in the culture medium. We have developed an efficient method for the enrichment and analysis of the secretome of different cancer cell lines, free of essential contaminants. The method is based on the optimization of cell incubation conditions in protein-free medium. Secreted proteins are concentrated and fractionated using a reversed-phase tC2 Sorbent, followed by peptide mass fingerprinting for protein identification. An average of 88 proteins were identified in each cancer cell line, of which more than 76% are known to be secreted, possess a signal peptide or a transmembrane domain. Given the importance of secreted proteins as a source for early detection and diagnosis of disease, this approach may help to discover novel candidate biomarkers with potential clinical significance.     

Twisted story of eye migration in flatfish

Early molecular markers for flatfish metamorphosis and eye migration must be linked to the ethmoid region, the earliest part of the flatfish cranium to change, as well as chondral and dermal ossification processes. Serial sections, morphological landmarks, and stereology were used to determine where and when the remodeling of tissues and asymmetry occurs in the head region of metamorphosing Atlantic halibut, Hippoglossus hippoglossus. Not all parts of the head remodel or migrate, and those that do may be asynchronous. Normal metamorphosis limits the torsion of the Atlantic halibut head to the anterior part of the neurocranium and excludes the tip of the snout and the general jaw area. The first cranial structure displaying eye migration-related asymmetric development is the paraethmoid part of the ethmoid cartilage. In early eye migration the medial frontal process moves apace with the eyes, whereas near completion the migrating eye moves significantly closer to the frontal process. Structures of the jaw remain mostly symmetrical, with the exception of the adductor mandibulae muscle and the bone maxillare, which are larger on the abocular than on the ocular side, the muscle occupying the space vacated by the migration of the eye. Thus, normal eye migration involves a series of temperospatially linked events. In juveniles lacking eye migration (arrested metamorphosis), the dermal bone, the prefrontal, does not develop. The two abnormal paraethmoids develop symmetrically as two plate-like structures curving anteriorly, whereas normal elongate fused paraethmoids curve at their posterior. The abocular side retrorbital vesicles are largest in volume only after the completion of normal eye migration. Factors involved in completion of normal metamorphosis and eye migration in flatfish affect chondral and dermal ossification signals in the ethmoid group, as well as remodeling of the mineralized frontal, a series of linked events not involving the entire neurocranium.     

Are responses of herbivores to environmental variability spatially consistent in alpine ecosystems?

Animal responses to global climate variation might be spatially inconsistent. This may arise from spatial variation in factors limiting populations' growth or from differences in the links between global climate patterns and ecologically relevant local climate variation. For example, the North Atlantic Oscillation (NAO) has a spatially consistent relation to temperature, but inconsistent spatial relation to snow depth in Scandinavia. Furthermore, there are multiple mechanistic ways by which climate may limit animal populations, involving both direct effects through thermoregulation and indirect pathways through trophic interactions. It is conceptually appealing to directly model the predicted mechanistic links. This includes the use of climate variables mimicking such interactions, for example, to use growing degree days (GDD) as a proxy for plant growth rather than average monthly temperature. Using a unique database of autumn body mass of 83331 domestic lambs from the period 1992–2007 in four alpine ranges in Norway, we demonstrate the utility of hierarchical, mechanistic path models fitted using a Bayesian approach to analyse explicitly predicted relationships among environmental variables and between lamb body mass and the environmental variables. We found large spatial variation in strength of responses of autumn lamb body mass to the NAO, to a proxy for plant growth in spring (the Normalized Difference Vegetation Index, NDVI) and effects even differed in direction to local summer climate. Average local temperature outperformed GDD as a predictor of the NDVI, whereas the NAO index in two areas outperformed local weather variables as a predictor of lamb body mass, despite the weaker mechanistic link. Our study highlights that spatial variation in strength of herbivore responses may arise from several processes. Furthermore, mechanistically more appealing measures do not always increase predictive power due to scale of measurement and since global measures may provide more relevant “weather packages” for larger scales.