Development of subfossil <Emphasis Type="Italic">Daphnia</Emphasis> and <Emphasis Type="Italic">Chaoborus</Emphasis> assemblages in relation to progressive acidification and fish community alterations in SW Sweden

Dated sediment cores from acidified and fishless Lake Gaffeln and Lake Härsvatten, SW Sweden, were analyzed for Daphnia ephippia and Chaoborus mandibles to test whether acidification history and fish extirpations could be reconstructed in a paleo-study using these easily identifiable animal remains. According to monitoring data fish were lost in both lakes from the 1950s to the 1970s. Progressive acidification prior to monitoring was confirmed by a gradual decrease and eventual loss of Daphnia ephippia in both study lakes during the first half of the twentieth century. In Lake Gaffeln mandibles of C. obscuripes appeared immediately after fish loss in 1973, and the regular presence of this species confirmed the succeeding fishless state of this lake. In Lake Härsvatten sediments C. obscuripes appeared only recently, i.e. three decades after fish extirpation, showing that the absence of C. obscuripes mandibles is not a trustworthy indicator of fish presence. Hence, the appearance of C. obscuripes was not temporally related to fish loss but confirmed the present fishless condition. Known historical presence of cyprinid fish in Lake Gaffeln was confirmed by a significantly higher proportion of fragmented mandibles of C. flavicans compared to the historically cyprinid-free Lake Härsvatten. In addition, both lake profiles displayed zero-proportions of fragmented mandibles during fishless periods. We conclude that acidification history and fish extirpations can be inferred by integrated studies on subfossil Daphnia ephippia and Chaoborus mandibles. However, during extreme ultra-oligotrophic conditions in acidified clear-water lakes subfossil Chaoborus mandibles may be too scarce to infer fish absence/presence.     

The potential to use QSAR to populate ecotoxicity characterisation factors for simplified LCIA and chemical prioritisation

Purpose

Today’s chemical society use and emit an enormous number of different, potentially ecotoxic, chemicals to the environment. The vast majority of substances do not have characterisation factors describing their ecotoxicity potential. A first stage, high throughput, screening tool is needed for prioritisation of which substances need further measures.

Methods

USEtox characterisation factors were calculated in this work based on data generated by quantitative structure-activity relationship (QSAR) models to expand substance coverage where characterisation factors were missing. Existing QSAR models for physico-chemical data and ecotoxicity were used, and to further fill data gaps, an algae QSAR model was developed. The existing USEtox characterisation factors were used as reference to evaluate the impact from the use of QSARs to generate input data to USEtox, with focus on ecotoxicity data. An inventory of chemicals that make up the Swedish societal stock of plastic additives, and their associated predicted emissions, was used as a case study to rank chemicals according to their ecotoxicity potential.

Results and discussion

For the 210 chemicals in the inventory, only 41 had characterisation factors in the USEtox database. With the use of QSAR generated substance data, an additional 89 characterisation factors could be calculated, substantially improving substance coverage in the ranking. The choice of QSAR model was shown to be important for the reliability of the results, but also with the best correlated model results, the discrepancies between characterisation factors based on estimated data and experimental data were very large.

Conclusions

The use of QSAR estimated data as basis for calculation of characterisation factors, and the further use of those factors for ranking based on ecotoxicity potential, was assessed as a feasible way to gather substance data for large datasets. However, further research and development of the guidance on how to make use of estimated data is needed to achieve improvement of the accuracy of the results.

     

A double role for a strictly conserved serine: further insights into the dUTPase catalytic mechanism

Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine, and the properties of the mutant have been investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases (including the bifunctional dCTP deaminase:dUTPases), and it has been proposed to promote catalysis by balancing negative charge at the oxygen that bridges the alpha- and beta-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue alpha,beta-imido-dUTP.Mg, the serine beta-OH is indeed hydrogen bonded to the alpha,beta-bridging nitrogen of the analogue. However, in the complex of the Asp90 --> Asn mutant dUTPase with the true substrate dUTP.Mg, the serine beta-OH points in the opposite direction and may form a hydrogen bond to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the beta-OH by hydrogen reduces k cat from 5.8 to 0.008 s (-1) but also k -1 , the rate of substrate dissociation, from 6.2 to 0.1 s (-1) ( K M = 6 x 10 (-9) M). We conclude that the serine beta-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. With experimental support, we argue that the beta-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation of the TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the beta-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the beta-OH to promote both deamination and hydrolysis in the bifunctional deaminases. We find that the E. coli dUTPase does not catalyze the hydrolysis of the alpha,beta-imido-dUTP.Mg, suggesting that the analogue provides the hydrogen in the bond to the serine beta-OH.     

Case-control cohort study of patients' perceptions of disability in mastocytosis

Background

Indolent forms of mastocytosis account for more than 90% of all cases, but the types and type and severity of symptoms and their impact on the quality of life have not been well studied. We therefore performed a case-control cohort study to examine self-reported disability and impact of symptoms on the quality of life in patients with mastocytosis.

Methodology/Principal Findings

In 2004, 363 mastocytosis patients and 90 controls in France were asked to rate to their overall disability (OPA score) and the severity of 38 individual symptoms. The latter was used to calculate a composite score (AFIRMM score). Of the 363 respondents, 262 were part of an ongoing pathophysiological study so that the following data were available: World Health Organization classification, standard measures of physical and psychological disability, existence of the D816V KIT mutation, and serum tryptase level. The mean OPA and AFIRMM scores and the standard measures of disability indicated that most mastocytosis patients suffer from disabilities due to the disease. Surprisingly, the patient''s measurable and perceived disabilities did not differ according to disease classification or presence or absence of the D816V KIT mutation or an elevated (≥20 ng/mL) serum tryptase level. Also, 32 of the 38 AFIRMM symptoms were more common in patients than controls, but there were not substantial differences according to disease classification, presence of the D816V mutation, or the serum tryptase level.

Conclusions

On the basis of these results and for the purposes of treatment, we propose that mastocytosis be first classified as aggressive or indolent and that indolent mastocytosis then be categorized according to the severity of patients'' perceived symptoms and their impact on the quality of life. In addition, it appears that mastocytosis patients suffer from more symptoms and greater disability than previously thought, that mastocytosis may therefore be under-diagnosed, and that the symptoms of the indolent forms of mastocytosis might be due more to systemic release of mediators than mast cell burden.     

Three-dimensional cancer cell culture in high-yield multiscale scaffolds by shear spinning

Polymeric scaffolds comprising two size scales of microfibers and submicron fibers can better support three-dimensional (3D) cell growth in tissue engineering, making them an important class of healthcare material. However, a major manufacturing barrier hampers their translation into wider practical use: scalability. Traditional production of two-scale scaffolds by electrospinning is slow and costly. For day-to-day cell cultures, the scaffolds need to be affordable, made in high yield to drive down cost. Combining expertise from academia and industry from the United Kingdom and United States, this study uses a new series of high-yield, low-cost scaffolds made by shear spinning for tissue engineering. The scaffolds comprise interwoven submicron fibers and microfibers throughout as observed under scanning electron microscopy and demonstrate good capability to support cell culturing for tumor modeling. Three model human cancer cell lines (HEK293, A549 and MCF-7) with stable expression of GFP were cultured in the scaffolds and found to exhibit efficient cell attachment and sustained 3D growth and proliferation for 30 days. Cryosection and multiphoton fluorescence microscopy confirmed the formation of compact 3D cell clusters throughout the scaffolds. In addition, comparative growth curves of 2D and 3D cultures show significant cell-type-dependent differences. This work applies high-yield shear-spun scaffolds in mammalian tissue engineering and brings practical, affordable applications of multiscale scaffolds closer to reality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2750, 2019.     

Disease-causing mutations in cardiac troponin T: identification of a critical tropomyosin-binding region

Fifteen percent of the mutations causing familial hypertrophic cardiomyopathy are in the troponin T gene. Most mutations are clustered between residues 79 and 179, a region known to bind to tropomyosin at the C-terminus near the complex between the N- and C-termini. Nine mutations were introduced into a troponin T fragment, Gly-hcTnT(70-170), that is soluble, alpha-helical, binds to tropomyosin, promotes the binding of tropomyosin to actin, and stabilizes an overlap complex of N-terminal and C-terminal tropomyosin peptides. Mutations between residues 92 and 110 (Arg92Leu, Arg92Gln, Arg92Trp, Arg94Leu, Ala104Val, and Phe110Ile) impair tropomyosin-dependent functions of troponin T. Except for Ala104Val, these mutants bound less strongly to a tropomyosin affinity column and were less able to stabilize the TM overlap complex, effects that were correlated with increased stability of the troponin T, measured using circular dichroism. All were less effective in promoting the binding of tropomyosin to actin. Mutations within residues 92-110 may cause disease because of altered interaction with tropomyosin at the overlap region, critical for cooperative actin binding and regulatory function. A model for a five-chained coiled-coil for troponin T in the tropomyosin overlap complex is presented. Mutations outside the region (Ile79Asn, Delta 160Glu, and Glu163Lys) functioned normally and must cause disease by another mechanism.     

Striking a new path in reducing cartilage breakdown: combination of antioxidative therapy and chondroanabolic stimulation after blunt cartilage trauma

Cartilage injury can trigger crucial pathomechanisms, including excessive cell death and expression of matrix‐destructive enzymes, which contribute to the progression of a post‐traumatic osteoarthritis (PTOA). With the intent to create a novel treatment strategy for alleviating trauma‐induced cartilage damage, we complemented a promising antioxidative approach based on cell and chondroprotective N‐acetyl cysteine (NAC) by chondroanabolic stimulation. Overall, three potential pro‐anabolic growth factors – IGF‐1, BMP7 and FGF18 – were tested comparatively with and without NAC in an ex vivo human cartilage trauma‐model. For that purpose, full‐thickness cartilage explants were subjected to a defined impact (0.59 J) and subsequently treated with the substances. Efficacy of the therapeutic approaches was evaluated by cell viability, as well as various catabolic and anabolic biomarkers, representing the present matrix turnover. Although monotherapy with NAC, FGF18 or BMP7 significantly prevented trauma‐induced cell dead and breakdown of type II collagen, combination of NAC and one of the growth factors did not yield significant benefit as compared to NAC alone. IGF‐1, which possessed only moderate cell protective and no chondroprotective qualities after cartilage trauma, even reduced NAC‐mediated cell and chondroprotection. Despite significant promotion of type II collagen expression by IGF‐1 and BMP7, addition of NAC completely suppressed this chondroanabolic effect. All in all, NAC and BMP7 emerged as best combination. As our findings indicate limited benefits of the simultaneous multidirectional therapy, a sequential application might circumvent adverse interferences, such as suppression of type II collagen biosynthesis, which was found to be reversed 7 days after NAC withdrawal.     

Comparative studies on partial microsequence-analysis of Bence-Jones proteins

1. Sequence analyses of Bence-Jones proteins up to 15 amino acids from the N-terminus provide decision of subgroups. 2. Investigation of primary structure of 3 Bence-Jones proteins, monomer and dimer of a kappa type, is limited at position 9-10 using DABITC reagent, whereas DABITC/PITC double coupling method allows sequencing up to 20-21 amino acids. 3. Sequencing of tryptic peptides allows only determination of 6-9 amino acids. 4. Microsequencing of tryptic peptide T13a shows the allotypic variant, inv b+, of Bence-Jones proteins TRA and GAN.     

Biochemical and molecular characterization of the biosynthesis of glutamine and glutamate, two major compatible solutes in the moderately halophilic bacterium Halobacillus halophilus

The moderately halophilic, chloride-dependent bacterium Halobacillus halophilus produces glutamate and glutamine as main compatible solutes at external salinities of 1.0 to 1.5 M NaCl. The routes for the biosynthesis of these solutes and their regulation were examined. The genome contains two genes potentially encoding glutamate dehydrogenases and two genes for the small subunit of a glutamate synthase, but only one gene for the large subunit. However, the expression of these genes was not salt dependent, nor were the corresponding enzymatic activities detectable in cell extracts of cells grown at different salinities. In contrast, glutamine synthetase activity was readily detectable in H. halophilus. Induction of glutamine synthetase activity was strictly salt dependent and reached a maximum at 3.0 M NaCl; chloride stimulated the production of active enzyme by about 300%. Two potential genes encoding a glutamine synthetase, glnA1 and glnA2, were identified. The expression of glnA2 but not of glnA1 was increased up to fourfold in cells adapted to high salt, indicating that GlnA2 is the glutamine synthetase involved in the synthesis of the solutes glutamate and glutamine. Furthermore, expression of glnA2 was stimulated twofold by the presence of chloride ions. Chloride exerted an even more pronounced effect on the enzymatic activity of preformed enzyme: in the absence of chloride in the assay buffer, glutamine synthetase activity was decreased by as much as 90%. These data demonstrate for the first time a regulatory role of a component of common salt, chloride, in the biosynthesis of compatible solutes.     

Defense suppression benefits herbivores that have a monopoly on their feeding site but can backfire within natural communities

Background

Plants have inducible defenses to combat attacking organisms. Hence, some herbivores have adapted to suppress these defenses. Suppression of plant defenses has been shown to benefit herbivores by boosting their growth and reproductive performance.

Results

We observed in field-grown tomatoes that spider mites (Tetranychus urticae) establish larger colonies on plants already infested with the tomato russet mite (Aculops lycopersici). Using laboratory assays, we observed that spider mites have a much higher reproductive performance on russet mite-infested plants, similar to their performance on the jasmonic acid (JA)-biosynthesis mutant def-1. Hence, we tested if russet mites suppress JA-responses thereby facilitating spider mites. We found that russet mites manipulate defenses: they induce those mediated by salicylic acid (SA) but suppress those mediated by JA which would otherwise hinder growth. This suppression of JA-defenses occurs downstream of JA-accumulation and is independent from its natural antagonist SA. In contrast, spider mites induced both JA- and SA-responses while plants infested with the two mite species together display strongly reduced JA-responses, yet a doubled SA-response. The spider mite-induced JA-response in the presence of russet mites was restored on transgenic tomatoes unable to accumulate SA (nahG), but russet mites alone still did not induce JA-responses on nahG plants. Thus, indirect facilitation of spider mites by russet mites depends on the antagonistic action of SA on JA while suppression of JA-defenses by russet mites does not. Furthermore, russet mite-induced SA-responses inhibited secondary infection by Pseudomonas syringae (Pst) while not affecting the mite itself. Finally, while facilitating spider mites, russet mites experience reduced population growth.

Conclusions

Our results show that the benefits of suppressing plant defenses may diminish within communities with natural competitors. We show that suppression of defenses via the JA-SA antagonism can be a consequence, rather than the cause, of a primary suppression event and that its overall effect is determined by the presence of competing herbivores and the distinct palette of defenses these induce. Thus, whether or not host-defense manipulation improves an herbivore’s fitness depends on interactions with other herbivores via induced-host defenses, implicating bidirectional causation of community structure of herbivores sharing a plant.