Proteasome inhibition induces developmentally deregulated programs of apoptotic and autophagic cell death during Drosophila melanogaster oogenesis

Ubiquitin/proteasome‐mediated degradation of eukaryotic proteins is critically implicated in a number of signalling pathways and cellular processes. To specifically impair proteasome activities, in vitro developing Drosophila melanogaster egg chambers were exposed to the MG132 or epoxomicin proteasome inhibitors, while a GAL4/UAS binary genetic system was employed to generate double transgenic flies overexpressing β2 and β6 conditional mutant proteasome subunits in a cell type‐specific manner. MG132 and epoxomicin administration resulted in severe deregulation of in vitro developing egg chambers, which was tightly associated with precocious induction of nurse cell‐specific apoptotic and autophagic death programmes, featured by actin cytoskeleton disorganization, nuclear chromatin condensation, DRICE caspase activation and autophagosome accumulation. In vivo targeted overexpression of β2 and β6 conditional mutants, specifically in the nurse cell compartment, led to a notable up‐regulation of sporadic apoptosis potency during early and mid‐oogenesis ‘checkpoints’, thus reasonably justifying the observed reduction in eclosion efficiency. Furthermore, in response to the intracellular abundance of β2 and β6 conditional mutant forms, specifically in numerous tissues of third instar larval stage, the developmental course was arrested, and lethal phenotypes were obtained at this particular embryonic period, with the double transgenic heterozygote embryos being unable to further proceed to complete maturation to adult flies. Our data demonstrate that physiological proteasome function is required to ensure normal oogenesis and embryogenesis in D. melanogaster, since targeted and cell type‐dependent proteasome inactivation initiates developmentally deregulated apoptotic and autophagic mechanisms.     

From research to implementation: Nature conservation in the Eastern Rhodopes mountains (Greece and Bulgaria), European Green Belt

Nature conservation should ideally build on the scientific recommendations that are concluded from applied conservation research, as well as on monitoring schemes that evaluate the effectiveness of recommendations. We considered as a case study a system of six protected areas located in the Eastern Rhodopes mountains in the southern part of the European Green Belt (EGB). To investigate nature conservation effectiveness, we reviewed 196 articles from scientific journals and books, eight doctoral and master theses, and 39 scientific reports regarding the Greek (one protected area, 428 km²) and the Bulgarian (five protected areas, 904 km²) part of the study area. We extracted 743 conservation recommendations, and through questionnaires completed by 10 local experts, we found that 74% of the recommendations were familiar for the experts. In the Greek (GR) and the Bulgarian part (BG) only 52% and 16%, respectively, of the recommendations were implemented, and only 15% (GR) and 3.1% (BG) were implemented and evaluated regarding their effectiveness. According to the experts, the main reasons for non-implementation and non-evaluation were absence or incompetence of the responsible authorities. Some recommendations obtained a remarkable low rate of implementation, such as those regarding agriculture and livestock rearing practices (GR: 29%, BG: 16%) or mammal conservation (GR: 0%, BG: 16%). Some other recommendations obtained rather high rates at least for Greece, such as tourism and environmental education (GR: 57%, BG: 42%) and bird conservation (GR: 57%, BG: 11%). We found that researchers and conservation managers at both sides of the Greek-Bulgarian border face similar implementation problems, related often to the lack of political will for nature conservation and establishment of competent authorities. The role of the EGB is crucial in enhancing the established cross-border collaborations between stakeholders involved in nature conservation.     

STAT-Related Profiles Are Associated with Patient Response to Targeted Treatments in Locally Advanced SCCHN

The anti-epidermal growth factor receptor antibody cetuximab (Erbitux, CTX) is currently used for the treatment of locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN), as yet with modest effectiveness, prompting for the identification of response predictors to this treatment and for the targeting of additional pathways implicated in this disease. Within this scope, we investigated the effect of SRC/STAT pathway components on LA-SCCHN patient outcome. SRC, STAT1, STAT3, STAT5A, STAT5B, ANXA1, CAV1, IGFBP2, EPHA2, EPHB2, and MSN relative gene expression, as well as Stat protein activation, were assessed on LA-SCCHN tumor tissues from 35 patients treated with combined radiotherapy (RT) and CTX-based regimens. Stat1, Stat3, and Stat5 proteins were usually found activated in neoplastic nuclei (70.4%, 85.7%, and 70.8%, respectively). Activated Stat3 and Stat5 were associated with each other (P = .017) and with a CAV1(high)/MSN(high)/IGFBP2(low) profile. All patients with tumors expressing high STAT5A/EPHA2 experienced a complete response on RT-CTX-based treatments (12/15 complete responders, P < .0001) and a longer progression-free survival (P = .024). Few tumors expressed high ANXA1/CAV1/EPHA2 and low IGFBP2, a putative dasatinib response-related profile, whereas high ANXA1 was associated with shorter overall survival (P = .003). In conclusion, Stat activation is common in LA-SCCHN, where overexpression of STAT5A and EPHA2 may predict for response to RT-CTX treatments. The STAT5A/EPHA2 profile seems of particular interest for validation in larger cohorts and in multiple tumor types because markers for the positive selection of patients to benefit from CTX-containing treatments are currently lacking.     

Detection of the TNFSF members BAFF, APRIL, TWEAK and their receptors in normal kidney and renal cell carcinomas

In advanced renal cell carcinoma (RCC), surgery combined with systemic chemotherapy and immunotherapy have had limited effectiveness. Therapeutic modalities targeting VEGF, PDGF, and c-kit using tyrosine kinase inhibitors and m-TOR using specific biologic factors are in development. Therapeutic approaches targeting TNF-alpha have shown limited efficacy, while anti-TRAIL (TNFSF10) antibodies have shown enhanced activity. The presence and potential significance of other members of the TNFSF has not been investigated. Here, we assayed the TNFSF members APRIL, BAFF, TWEAK and their receptors (BCMA, TACI, BAFFR, Fn14) in 86 conventional type clear cell RCC, using immunohistochemistry and correlated our findings with histological data and, in a limited series, follow-up of patients. We observed a differential expression of these TNFSF ligands and receptors in cancerous and non-cancerous structures. BAFF was found in all RCC; APRIL expression is associated with an aggressive phenotype, correlating negatively with patients' disease-free survival, while TWEAK and its receptor Fn14 are heterogeneously expressed, correlating negatively with the grade and survival of RCC patients. This is the first study, presenting together the TNFSF members APRIL, BAFF, TWEAK and their receptors in different areas of normal renal tissue and RCC, suggesting a potential role of these TNFSF members in renal tumor biology.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

Population genetics of the wild yeast Saccharomyces paradoxus

Saccharomyces paradoxus is the closest known relative of the well-known S. cerevisiae and an attractive model organism for population genetic and genomic studies. Here we characterize a set of 28 wild isolates from a 10-km(2) sampling area in southern England. All 28 isolates are homothallic (capable of mating-type switching) and wild type with respect to nutrient requirements. Nine wild isolates and two lab strains of S. paradoxus were surveyed for sequence variation at six loci totaling 7 kb, and all 28 wild isolates were then genotyped at seven polymorphic loci. These data were used to calculate nucleotide diversity and number of segregating sites in S. paradoxus and to investigate geographic differentiation, population structure, and linkage disequilibrium. Synonymous site diversity is approximately 0.3%. Extensive incompatibilities between gene genealogies indicate frequent recombination between unlinked loci, but there is no evidence of recombination within genes. Some localized clonal growth is apparent. The frequency of outcrossing relative to inbreeding is estimated at 1.1% on the basis of heterozygosity. Thus, all three modes of reproduction known in the lab (clonal replication, inbreeding, and outcrossing) have been important in molding genetic variation in this species.     

The sperm-specific proteins of the edible oyster (European flat oyster (Ostrea edulis)) are products of proteolytic processing

Extraction of sperm proteins from the bivalve mollusc Ostrea edulis shows them to contain a normal complement of core histones, together with three sperm-specific proteins, OE1 and OE2, plus the shorter OE3, which shows substantial microheterogeneity. OE1 and OE2 have a very similar amino acid composition, cyanogen bromide (CNBr) cleavage yields products of identical size and possesses a trypsin-resistant core peptide, together indicating that they are closely homologous histone H1-like proteins. Western blotting shows that OE1 and OE2 are closely related to the histone H1-like protein PL-II* of Mytilus trossulus. The amino acid composition of OE3 shows it to be a protamine-like PL-IV type protein. Edman degradation of a CNBr peptide from OE2 gave the sequence (M)KAAFAKGLKSGALVRPKGS-which has 85% identity to a sequence located towards the C-terminal end of the globular domain of the PL-II* protein of M. trossulus. An O. edulis sperm cDNA library yielded a clone of 428 bp. A genomic clone including an open reading frame (ORF) of 750 bp was isolated by PCR amplification from genomic DNA. Hypothetical translation showed the ORF to encode OE1 (or OE2) immediately followed by OE3, separated by a proteolytic processing site. This arrangement (a two-protein ORF) is also found in M. trossulus and Ensis minor.     

Keeping up with Dobzhansky: G. Ledyard Stebbins, Jr., plant evolution, and the evolutionary synthesis

This paper explores the complex relationship between the plant evolutionist G. Ledyard Stebbins and the animal evolutionist Theodosius Dobzhansky. The manner in which the plant evolution was brought into line, synthesized, or rendered consistent with the understanding of animal evolution (and especially insect evolution) is explored, especially as it culminated with the publication of Stebbins's 1950 book Variation and Evolution in Plants. The paper explores the multi-directional traffic of influence between Stebbins and Dobzhansky, but also their social and professional networks that linked plant evolutionists like Stebbins with Edgar Anderson, Carl Epling, and the 'Carnegie team' of Jens Clausen, David Keck, and William Hiesey with collaborators on the animal side like I. Michael Lerner, Sewall Wright and L.C. Dunn and other 'architects' of the synthesis like Ernst Mayr, Julian Huxley and George Gaylord Simpson. The compatibility in training, work styles, methodologies, goals, field sites, levels of analysis, and even choice of organismic systems is explored between Stebbins and Dobzhansky. Finally, the extent to which coevolution between plants and insects is reflected in the relationship is explored, as is the power dynamic in the relationship between two of the most visible figures associated with the evolutionary synthesis.     

Unusual death due to a bleeding from a varicose vein: a case report

ABSTRACT: BACKGROUND: Varicose veins are a common entity presenting a worldwide distribution. Although they are usually benign, sometimes are proved to be a threatening condition. Massive hemorrhage is an unusual complication of this common venous pathology that demands immediate medical intervention. CASE PRESENTATION: We present a case of a 66-year-old woman found dead in her house surrounded by a large quantity of blood. Autopsy revealed a 7 mm ulcer on the internal surface of the left lower leg communicating with a varicose vein, signs of exsanguinations and liver cirrhosis. Toxicological analysis was negative. CONCLUSION: Massive hemorrhage from a ruptured varicosity is a severe medical emergency. Awareness of the risk of massive hemorrhage may provoke preventive treatment to be undertaken so as terminal loss of consciousness and a subsequent unattended death to be averted.     

Interplay between oncogenic K-Ras and wild-type H-Ras in Caco2 cell transformation

Mutation of RAS genes is one of the most common oncogenic alterations in cancer and acquisition of activating RAS mutations has been demonstrated to cause progression of colorectal adenoma to cancer. The aim of this study was to identify changes in the proteome of the intermediate-stage colorectal cancer cell line Caco2, induced by ectopic expression of two distinct RAS proteins, KRAS(V12) and HRAS(V12), in their mutated, constitutively active form. Using 2D-gel electrophoresis, followed by LC-MS/MS we identified almost 200 differentially expressed proteins in pair-wise comparisons of Caco2 vs Caco2-KRAS(V12) and Caco2 vs Caco2-HRAS(V12). Although many of the affected proteins were unique for each pair, there were also substantial similarities. Interestingly, transformation by the mutant KRAS(V12) gene resulted in elevated expression levels and activity of endogenous H-ras protein. Silencing the latter with a specific RNAi reversed several proteomic changes observed in KRAS(V12)-transformed cells, suggesting that oncogenic K-ras partly exerts its effects through endogenous H-ras activation. Alterations in the expression of cytoskeletal and cell adhesion proteins, caused by HRAS siRNA treatment, correlated with a reduction in the invasive properties of Caco2-KRAS(V12) cells. Our data suggest a novel interplay between K-ras and H-ras, with possible implications for colorectal carcinogenesis.