Sensitivity analysis of coexistence in ecological communities: theory and application

Sensitivity analysis, the study of how ecological variables of interest respond to changes in external conditions, is a theoretically well‐developed and widely applied approach in population ecology. Though the application of sensitivity analysis to predicting the response of species‐rich communities to disturbances also has a long history, derivation of a mathematical framework for understanding the factors leading to robust coexistence has only been a recent undertaking. Here we suggest that this development opens up a new perspective, providing advances ranging from the applied to the theoretical. First, it yields a framework to be applied in specific cases for assessing the extinction risk of community modules in the face of environmental change. Second, it can be used to determine trait combinations allowing for coexistence that is robust to environmental variation, and limits to diversity in the presence of environmental variation, for specific community types. Third, it offers general insights into the nature of communities that are robust to environmental variation. We apply recent community‐level extensions of mathematical sensitivity analysis to example models for illustration. We discuss the advantages and limitations of the method, and some of the empirical questions the theoretical framework could help answer.     

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy.

Methods

Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR.

Results

In vitro binding experiments of ¹²⁵I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney.

Conclusions

These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.     

Microplastic ingestion ubiquitous in marine turtles

Despite concerns regarding the environmental impacts of microplastics, knowledge of the incidence and levels of synthetic particles in large marine vertebrates is lacking. Here, we utilize an optimized enzymatic digestion methodology, previously developed for zooplankton, to explore whether synthetic particles could be isolated from marine turtle ingesta. We report the presence of synthetic particles in every turtle subjected to investigation (n = 102) which included individuals from all seven species of marine turtle, sampled from three ocean basins (Atlantic [ATL]: n = 30, four species; Mediterranean (MED): n = 56, two species; Pacific (PAC): n = 16, five species). Most particles (n = 811) were fibres (ATL: 77.1% MED: 85.3% PAC: 64.8%) with blue and black being the dominant colours. In lesser quantities were fragments (ATL: 22.9%: MED: 14.7% PAC: 20.2%) and microbeads (4.8%; PAC only; to our knowledge the first isolation of microbeads from marine megavertebrates). Fourier transform infrared spectroscopy (FT‐IR) of a subsample of particles (n = 169) showed a range of synthetic materials such as elastomers (MED: 61.2%; PAC: 3.4%), thermoplastics (ATL: 36.8%: MED: 20.7% PAC: 27.7%) and synthetic regenerated cellulosic fibres (SRCF; ATL: 63.2%: MED: 5.8% PAC: 68.9%). Synthetic particles being isolated from species occupying different trophic levels suggest the possibility of multiple ingestion pathways. These include exposure from polluted seawater and sediments and/or additional trophic transfer from contaminated prey/forage items. We assess the likelihood that microplastic ingestion presents a significant conservation problem at current levels compared to other anthropogenic threats.     

Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.     

Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P_BAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 10⁹ unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.     

Determination of turnover of androstenedione to estrone via aromatase in estrogen receptor positive and negative human breast cancer cell lines [Poster 48]

In one estrogen receptor (ER) negative (MDA-MB-231) and two ER positive human breast cancer cell lines (T-47-D,SK-BR-3) we measured aromatase activity by [³H]water assay and estrone (E1) production by thin-layer chromatography. Compared with ether extraction and charcoal method, lyophilization proved to be the most sensitive technique to measure the quantity of [³H]water. The extremely low contamination of the water soluble phase by [1ß-³H]androstenedione (0.02%), as well as the lack of errors due to conjugated steroids, offers the possibility to measure changes of cellular aromatase activity even at very low levels. In contrast to SK-BR-3 and MDA-MB-231 cells, we found no aromatase activity in T-47-D cells. There was no coincidence between ER status and aromatase activity. Proliferation of tumor cells was parallel with a continuous increase of aromatase activity and E1 production during mitogenic growth phase reaching highest levels at the transition from log to plateau-phase.     

Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels.     

Acceptability of Drosophila suzukii as prey for common predators occurring in cherries and berries

The invasive cherry vinegar fly, Drosophila suzukii, has been identified in Europe as a destructive fruit pest since its arrival in 2008. In the present laboratory study, three predatory insects (Orius majusculus, Chrysoperla carnea, and Forficula auricularia) naturally occurring on fruit crops in Europe were investigated for their ability to attack and feed on D. suzukii within and outside fruits. The predators were provided with various D. suzukii life stages (eggs, larvae, pupae or adults) exposed or within infested cherries. The anthocorid bug O. majusculus fed on eggs and larvae, but was not able to attack pupae. Larvae of the lacewing C. carnea preyed upon D. suzukii eggs, larvae and pupae and also captured adult flies. The European earwig F. auricularia was the most voracious predator of these three tested species. Although the earwigs were not able to catch adult flies, they readily preyed upon every other developmental stage. Adult O. majusculus or third instar larvae of C. carnea significantly reduced the offspring of D. suzukii from infested cherries, when these contained the egg stage of the pest. None of the predators were able to attack early larval stages inside the cherries. But pupae that protruded from the fruit epicarp or that had pupated outside the fruit were accessible to lacewing larvae and earwigs and significantly reduced by them. Orius bugs, lacewing larvae and earwigs were able, under laboratory conditions, to capture and prey upon various life stages of the invasive pest, if not completely concealed inside the fruit. Our findings suggest that these generalist predators may have some control capacity on infested fruit in cultivated fruit crops and also in non‐crop habitats.     

Identifying and characterizing the most significant β-glucosidase of the novel species Aspergillus saccharolyticus

The newly discovered fungal species Aspergillus saccharolyticus was found to produce a culture broth rich in β-glucosidase activity. In this present work, the main β-glucosidase of A.?saccharolyticus responsible for the efficient hydrolytic activity was identified, isolated, and characterized. Ion exchange chromatography was used to fractionate the culture broth, yielding fractions with high β-glucosidase activity and only 1 visible band on an SDS-PAGE gel. Mass spectrometry analysis of this band gave peptide matches to β-glucosidases from aspergilli. Through a polymerase chain reaction approach using degenerate primers and genome walking, a 2919 bp sequence encoding the 860 amino acid BGL1 polypeptide was determined. BGL1 of A.?saccharolyticus has 91% and 82% identity with BGL1 from Aspergillus aculeatus and BGL1 from Aspergillus niger , respectively, both belonging to Glycoside Hydrolase family 3. Homology modeling studies suggested β-glucosidase activity with preserved retaining mechanism and a wider catalytic pocket compared with other β-glucosidases. The bgl1 gene was heterologously expressed in Trichoderma reesei QM6a, purified, and characterized by enzyme kinetics studies. The enzyme can hydrolyze cellobiose, p-nitrophenyl-β-d-glucoside, and cellodextrins. The enzyme showed good thermostability, was stable at 50?°C, and at 60?°C it had a half-life of approximately 6?h.     

Faster cytotoxicity with age: Increased perforin and granzyme levels in cytotoxic CD8 + T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8⁺ T cells responses in the elderly has come back into focus since the COVID‐19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8⁺ T cells in aging. We focused on the different subpopulations and time‐resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8⁺ T cells from elderly mice are much faster than those of CD8⁺ T cells from adult mice. This is true not only in the total CD8⁺ T cell population but also for their effector (T_EM) and central memory (T_CM) T cell subpopulations. TIRF experiments reveal that CD8⁺ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8⁺ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell‐intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked.