Assessing structural and functional ecosystem condition using leaf breakdown: studies on a polluted river

1. Leaf breakdown rates of Alnus glutinosa were determined and the structure of decomposer assemblages associated with leaves were analysed to assess the effect of pollution on the ecological condition of the Ave River (North‐west Portugal). 2. Increase in organic and inorganic nutrients was associated with an increase in density and a decrease in richness of macroinvertebrates, a dramatic decline in the conidial production of aquatic hyphomycetes, but no major change in the richness of aquatic hyphomycetes. 3. Downstream nutrient enrichment was correlated with accelerated leaf breakdown rates. 4. The degree of functional impairment assessed by the ratio of leaf breakdown rates in coarse‐mesh and fine‐mesh bags was in accordance with the gradient of pollution defined by two biotic indices. 5. This study supports the contention that leaf breakdown experiments are a valuable tool to assess the effect of pollution on the ecological condition of rivers.     

Olfactory Individual Recognition of Mothers by Young Guinea-Pigs (Cavia porcellus)

In mammals, olfactory cues play a major role in individual recognition and urine is one source of potentially individual‐specific olfactory cues. We studied how soon young of the extremely precocial domestic guinea‐pig (Cavia porcellus) establish specific preferences for maternal urine smell by offering 5–30‐d‐old young a simultaneous choice between a urine sample of the mother and urine from an unfamiliar unrelated lactating female. Young showed increasing preference for the smell of maternal urine from day 5 of life onwards. On day 10 of life, they discriminated between maternal urine and that of other lactating females when these were unfamiliar and related, unfamiliar and unrelated or familiar unrelated, but not when the urine of the other female came from a familiar and related lactating animal. The last result is based on fewer litters and, therefore has to be considered as preliminary. As our results are based on spontaneous preferences for just one source of olfactory cues, discrimination of live animals is likely to be even better than demonstrated here. Learning or phenotype matching of individual specific cues enable these precocial young to form a specific bond with their mother soon after birth.     

Structural characterization by NMR of the natively unfolded extracellular domain of beta-dystroglycan: toward the identification of the binding epitope for alpha-dystroglycan

Dystroglycan (DG) is an adhesion molecule playing a crucial role for tissue stability during both early embriogenesis and adulthood and is composed by two tightly interacting subunits: alpha-DG, membrane-associated and highly glycosylated, and the transmembrane beta-DG. Recently, by solid-phase binding assays and NMR experiments, we have shown that the C-terminal domain of alpha-DG interacts with a recombinant extracellular fragment of beta-DG (positions 654-750) independently from glycosylation and that the linear binding epitope is located between residues 550 and 565 of alpha-DG. In order to elucidate which moieties of beta-DG are specifically involved in the complex with alpha-DG, the ectodomain has been recombinantly expressed and purified in a labeled ((13)C,(15)N) form and studied by multidimensional NMR. Although it represents a natively unfolded protein domain, we obtained an almost complete backbone assignment. Chemical shift index, (1)H-(15)N heteronuclear single-quantum coherence and nuclear Overhauser effect (HSQC-NOESY) spectra and (3)J(HN,H)(alpha) coupling constant values confirm that this protein is highly disordered, but (1)H-(15)N steady-state NOE experiments indicate that the protein presents two regions of different mobility. The first one, between residues 659 and 722, is characterized by a limited degree of mobility, whereas the C-terminal portion, containing about 30 amino acids, is highly flexible. The binding of beta-DG(654-750) to the C-terminal region of the alpha subunit, alpha-DG(485-620), has been investigated, showing that the region of beta-DG(654-750) between residues 691 and 719 is involved in the interaction.     

Selection-driven evolution of emergent dengue virus

In the last four decades the incidence of dengue fever has increased 30-fold worldwide, and over half the world's population is now threatened with infection from one or more of four co-circulating viral serotypes (DEN-1 through DEN-4). To determine the role of viral molecular evolution in emergent disease dynamics, we sequenced 40% of the genome of 82 DEN-4 isolates collected from Puerto Rico over the 20 years since the onset of endemic dengue on the island. Isolates were derived from years with varying levels of DEN-4 prevalence. Over our sampling period there were marked evolutionary shifts in DEN-4 viral populations circulating in Puerto Rico; viral lineages were temporally clustered and the most common genotype at a particular sampling time often arose from a previously rare lineage. Expressed changes in structural genes did not appear to drive this lineage turnover, even though these regions include primary determinants of viral antigenic properties. Instead, recent dengue evolution can be attributed in part to positive selection on the nonstructural gene 2A (NS2A), whose functions may include replication efficiency and antigenicity. During the latest and most severe DEN-4 epidemic in Puerto Rico, in 1998, viruses were distinguished by three amino acid changes in NS2A that were fixed far faster than expected by drift alone. Our study therefore demonstrates viral genetic turnover within a focal population and the potential importance of adaptive evolution in viral epidemic expansion.     

Novel adenosine A2A receptor ligands: A synthetic,functional and computational investigation of selected literature adenosine A2A receptor antagonists for extending into extracellular space

Growing evidence has suggested a role in targeting the adenosine A_2A receptor for the treatment of Parkinson’s disease. The literature compounds KW 6002 (2) and ZM 241385 (5) were used as a starting point from which a series of novel ligands targeting the adenosine A_2A receptor were synthesized and tested in a recombinant human adenosine A_2A receptor functional assay. In order to further explore these molecules, we investigated the biological effects of assorted linkers attached to different positions on selected adenosine A_2A receptor antagonists, and assessed their potential binding modes using molecular docking studies. The results suggest that linking from the phenolic oxygen of selected adenosine A_2A receptor antagonists is relatively well tolerated due to the extension towards extracellular space, and leads to the potential of attaching further functionality from this position.     

Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-β5, Survivin, TGF-β, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools.Subject terms: Tumour biomarkers, Protein-protein interaction networks     

Resistance of p53 knockout cells to doxorubicin is related to reduced formation of DNA strand breaks rather than impaired apoptotic signaling

The anthracycline doxorubicin (adriamycin) is an important chemotherapeutic agent used in the treatment of solid epithelial and mesenchymal tumors as well as leukemias. A variety of mechanisms has been proposed to be involved in doxorubicin-induced cytotoxicity such as DNA intercalation, oxidative stress, DNA strand breakage by inhibition of topoisomerase II, activation of death receptors, and altered p53 expression. Concerning doxorubicin resistance and p53 status data reported are contradictory. Here, we show that mouse fibroblasts deficient in p53 (p53(-/-)) are more resistant to doxorubicin than p53 wild-type (p53 wt) cells. This is in contrast to other genotoxic agents (UV-light, alkylating drugs) for which p53(-/-) fibroblasts proved to be more sensitive. Resistance of p53(-/-) cells to doxorubicin is related to reduced induction of apoptosis. This is not likely to be due to altered apoptotic signaling since the expression of Bax and Bcl-2 was unchanged and the induction of Fas/CD95/APO-1 receptor and caspase-8 was the same in p53(-/-) and p53 wt cells on treatment with doxorubicin. However, we observed a clearly lower level of doxorubicin-induced DNA strand breaks in p53(-/-) cells compared to the wt. P170 glycoprotein was equally expressed and the accumulation and elimination of the drug occurred with identical kinetics in both cell types. p53 deficient cells were cross-resistant to another topoisomerase II inhibitor etoposide, which also provoked increased DNA strand breakage in p53 wt cells. Based on the data we conclude that the p53 status significantly impacts the generation of DNA strand breaks because of drug-induced topoisomerase inhibition rather than death receptor signaling. Since human tumors are frequently mutated in p53 the findings bear clinical implications.     

Variable acceleration influences cyclic AMP levels in Paramecium biaurelia

Paramecium is used as a model system to analyse the gravity signal transduction pathway, that leads to gravitaxis and gravikinesis. In order to prove whether gravistimulation is coupled with second messenger production (cyclic AMP: hyperpolarization, cyclic GMP: depolarization) Paramecium was fixated under variable accelerations (1 x g, 9 x g and 10(-4) x g) on a centrifuge and during a sounding rocket flight (TEXUS 39). The analysis of cAMP and cGMP levels revealed an acceleration-dependent change in cAMP, while cGMP-levels showed gravity-independent variations. Hypergravity did not only induce an amplification of gravitaxis and gravikinesis, but also an increase in cAMP compared to the 1 x g-data. We conclude that the increased pressure of the cytoplasm on the lower membrane of upward swimming cells enhance the number of open K+(-)channels, thus causing hyperpolarization and change in cAMP concentration. Consequently, transition to microgravity declines gravitaxis and gravikinesis, and decreases cAMP concentration due to the loss of pressure on the cell membrane.     

Monitoring endoplasmic reticulum-to-Golgi traffic of a plant calreticulin by protein glycosylation analysis

Calreticulin is a ubiquitous and highly conserved Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). Plant calreticulin, in contrast to its animal counterpart, is often glycosylated: its N-glycans have been shown so far to be of the high-mannose type, typical of ER-resident glycoproteins. During the characterization of calreticulin from vegetative and reproductive tissues of Liriodendron tulipifera L., we gained some biochemical evidence that prompted us to investigate the monosaccharide composition and primary structure of the calreticulin N-glycans isolated from the ovary of this dicotyledon tree. The structures of the components of the N-glycan pool were elucidated by HPLC analysis and exoglycosidase sequencing, and further confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The 16 identified oligosaccharide structures, which consisted of both the high-mannose and complex type, are indicative of calreticulin glycan processing through the ER-to-Golgi pathway up to the medial and trans Golgi stacks. Approximately 45% of calreticulin glycan chains are of the complex type, always containing beta(1,2)-xylose, and approximately a third of these also contain alpha(1,3)-fucose in the core. The most complex glycoform harbors the Lewis-a epitope Gal(beta)1-3[Fuc(alpha)1-4]GlcNAc. Immunolocalization of calreticulin with anti-calreticulin antibodies was consistent with protein transit through the Golgi. Thus, although it contains the tetrapeptide HDEL ER retention signal, the reticuloplasmin calreticulin possesses the competence to transit from the ER compartment to the distal Golgi stacks. The final fate of the protein after its complete maturation is still obscure.     

Comparison of the fluorescence kinetics of detergent-solubilized and membrane-reconstituted LH2 complexes from Rps. acidophila and Rb. sphaeroides

Picosecond time-resolved fluorescence spectroscopy has been used in order to compare the fluorescence kinetics of detergent-solubilized and membrane-reconstituted light-harvesting 2 (LH2) complexes from the purple bacteria Rhodopseudomonas (Rps.) acidophila and Rhodobacter (Rb.) sphaeroides. LH2 complexes were reconstituted in phospholipid model membranes at different lipid:protein-ratios and all samples were studied exciting with a wide range of excitation densities. While the detergent-solubilized LH2 complexes from Rps. acidophila showed monoexponential decay kinetics (τ_f = 980 ps) for excitation densities of up to 3·10¹³ photons/(pulse·cm²), the membrane-reconstituted LH2 complexes showed multiexponential kinetics even at low excitation densities and high lipid:protein-ratios. The latter finding indicates an efficient clustering of LH2 complexes in the phospholipid membranes. Similar results were obtained for the LH2 complexes from Rb. sphaeroides. Guest editor: Dr. Conrad Mullineaux.