How non-target chironomid communities respond to mosquito control: Integrating DNA metabarcoding and joint species distribution modelling.

The conservation and management of riparian ecosystems rely on understanding the ecological consequences of anthropogenic stressors that impact natural communities. In this context, studies investigating the effects of anthropogenic stressors require reliable methods capable of mapping the relationships between taxa occurrence or abundance and environmental predictors within a spatio-temporal framework. Here, we present an integrative approach using DNA metabarcoding and Hierarchical Modelling of Species Communities (HMSC) to unravel the intricate dynamics and resilience of chironomid communities exposed to Bacillus thuringiensis var. israelensis (Bti). Chironomid emergence was sampled from a total of 12 floodplain pond mesocosms, half of which received Bti treatment, during a 16-week period spanning spring and summer of 2020. Subsequently, we determined the community compositions of chironomids and examined their genus-specific responses to the Bti treatment, considering their phylogenetic affiliations and ecological traits of the larvae. Additionally, we investigated the impact of the Bti treatment on the body size distribution of emerging chironomids. Our study revealed consistent responses to Bti among different chironomid genera, indicating that neither phylogenetic affiliations nor larval feeding strategies significantly contributed to the observed patterns. Both taxonomic and genetic diversity were positively correlated with the number of emerged individuals. Furthermore, our findings demonstrated Bti-related effects on chironomid body size distribution, which could have relevant implications for size-selective terrestrial predators. Hence, our study highlights the value of employing a combination of DNA metabarcoding and HMSC to unravel the complex dynamics of Bti-related non-target effects on chironomid communities. The insights gained from this integrated framework contribute to our understanding of the ecological consequences of anthropogenic stressors and provide a foundation for informed decision-making regarding the conservation and management of riparian ecosystems.

The high tibial osteotomy, open versus closed wedge, a comparison of methods in 108 patients.

INTRODUCTION: One hundred and eight patients with varus gonarthrosis were treated with high tibial osteotomy (HTO) in 2001. Fifty one patients received an open wedge osteotomy by using the 'Puddu' plate and 57 patients received a Coventry-type closing wedge osteotomy. For both groups the follow-up examination period was 22.5 months (253-1009 days). MATERIAL AND METHODS: To evaluate the study, radiological and subjective criteria as well as the Lysholm and the Tegner Activity Score were used. Altogether 84 % of the patients were included in the follow-up examination study. RESULTS: In both groups a significant improvement of both scores were achieved. Both methods obtained safe and reproducible results for the correction considering the different operation techniques. There were no differences in outcome between the two methods. Satisfactory results were also achieved for early arthrosis of the femoropatellar and the lateral compartment. CONCLUSION: Open and closed wedge HTOs obtain significant improvement in patients with medial osteoarthritis of the knee. Using the right technique is very important for good results. For stabilization of the medial ligament we recommend the open wedge osteotomy. The patient should be informed about the routine removal of the metal plate.

Dominant negative inhibitors of signalling through the phosphoinositol 3-kinase pathway for gene therapy of pancreatic cancer.

BACKGROUND: Ras signalling is frequently aberrant in pancreatic cancer so that there is constitutive activation of the phosphatidylinositol 3-kinase (PI3K) and AKT/protein kinase B pathway, as well as the RAF/MEK/ERK pathway. AIMS: In the present study we investigated the role of the PI3K/AKT pathway in malignant transformation of pancreatic cancer cells. METHODS: A genetic approach was used to interfere with signal transduction in vitro and in vivo. RASN17, a dominant negative mutant of RAS, was applied to inhibit the PI3K/AKT pathway upstream of PI3K. The regulatory p85beta subunit of PI3K and the negative regulator PTEN were utilised to inhibit the pathway at the level of PI3K, and AAA-AKT, a dominant negative mutant of AKT was employed to interfere with PI3K/AKT signalling at the level of AKT. RESULTS: Antiproliferative, proapoptotic, and anticancer effects were documented, showing that inhibition of the PI3K pathway in these cell lines suppresses tumour cell growth in vitro and reduces growth in nude mice. CONCLUSIONS: The PI3K/AKT pathway represents a potential therapeutic target for pancreatic cancer, and gene therapy may be one approach to produce selective inhibition.

[Application of the "Taylor Spatial Frame" with unilateral implantation of a medial sledge prosthesis after posttraumatic dislocation of the femur. A complicated progression]. / Die Anwendung des "Taylor Spatial Frame" mit einzeitiger Implantation einer medialen Schlittenprothese nach posttraumatischer Fehlstellung des Femurs. Ein komplizierter Verlauf.

For years the Ilizarov fixator has been an established method for the treatment of orthopedic and trauma patients. The Taylor Spatial Frame is a software-navigated hexapod construction on the basis of the Ilizarov fixator. Unicompartmental arthroplasty of the knee is an option especially in medial osteoarthritis. The therapeutic procedure with a complicated development is illustrated by a case report on a patient with post-traumatic deformity of the femur and medial osteoarthritis of the knee.

Novel alpha- and beta-amino acid inhibitors of influenza virus neuraminidase.

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 alpha- and beta-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K(i) values in the 50 microM range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the alpha-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the beta-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the alpha- or beta-amino groups were replaced with hydroxyl groups were 365- and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.

A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization.

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of approximately 3.5 microM, 19.0 microM, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD+, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 A resolution at a synchrotron radiation source.

Differences in binding modes of enantiomers of 1-acetamido boronic acid based protease inhibitors: crystal structures of gamma-chymotrypsin and subtilisin Carlsberg complexes.

In order to probe the structural basis of stereoselectivity in the serine protease family, a series of enantiomeric boronic acids RCH2CH(NHCOCH3)B(OH)2 has been synthesized and kinetically characterized as transition-state analog inhibitors using alpha-chymotrypsin and subtilisin Carlsberg as model systems. When the R-substituent in this series was changed from a p-chlorophenyl to a 1-naphthyl group, alpha-chymotrypsin, but not subtilisin, reversed its usual preference for l-enantiomers and bound more tightly to the D-enantiomer [Martichonok, V., & Jones, J. B. (1996) J. Am. Chem. Soc. 118, 950-958]. The structural factors responsible for the differences in stereoselectivity between the two enzymes have been explored by X-ray crystallographic examination of subtilisin Carlsberg and gamma-chymotrypsin complexes of the L- and D-enantiomers of p-chlorophenyl and 1-naphthyl boronic acid derivatives. In both enzymes, the L-isomers of the inhibitors, which are more closely related to the natural L-amino acid substrates, form tetrahedral adducts, covalently linking the central boron atom and Ogamma of the catalytic serine. The d-isomers, however, differ in the way they interact with subtilisin or gamma-chymotrypsin. With subtilisin, both the D-p-chlorophenyl and D-1-naphthyl inhibitor complexes form covalent Ser Ogamma-to-boron bonds, but with gamma-chymotrypsin, the same inhibitors lead to novel tetrahedral adducts covalently linking both Ser195 Ogamma and His57 Nepsilon2 covalently via the boron atom.

Glutathione reductase turned into trypanothione reductase: structural analysis of an engineered change in substrate specificity.

Trypanosoma and Leishmania, pathogens responsible for diseases such as African sleeping sickness, Chagas' heart disease, or Oriental sore, are two of the very few genera that do not use the ubiquitous glutathione/glutathione reductase system to keep a stable cellular redox balance. Instead, they rely on trypanothione and trypanothione reductase to protect them from oxidative stress. Trypanothione reductase (TR) and the corresponding host enzyme, human red blood cell glutathione reductase (GR), belong to the same flavoprotein family. Despite their closely related three-dimensional structures and although their natural substrates share the common structural glutathione core, the two enzymes are mutually exclusive with respect to their disulfide substrates. This makes the parasite enzyme a potential target for antitrypanosomal drug design. While a large body of structural data on GR complexes is available, information on TR-ligand interactions is very limited. When the two amino acid changes Ala34Glu and Arg37Trp are introduced into human GR, the resulting mutant enzyme (GRTR) prefers trypanothione 700-fold over its original substrate, effectively converting a GR into a TR [Bradley, M., Bücheler, U. S., & Walsh, C. T. (1991) Biochemistry 30, 6124-6127]. The crystal structure of GRTR has been determined at 2.3 A resolution and refined to a crystallographic R factor of 20.9%. We have taken advantage of the ease with which ligand complexes can be produced in GR crystals, a property that extends to the isomorphous GRTR crystals, and have produced and analyzed crystals of GRTR complexes with glutathione, trypanothione, glutathionylspermidine and of a true catalytic intermediate, the mixed disulfide between trypanothione and the enzyme. The corresponding molecular structures have been characterized at resolutions between 2.3 and 2.8 A with R factors ranging from 17.1 to 19.7%. The results indicate that the Ala34Glu mutation causes steric hindrance leading to a large displacement of the side chain of Arg347. This movement combined with the change in charge introduced by the mutations modifies the binding cavity, forcing glutathione to adopt a nonproductive binding mode and permitting trypanothione and to a certain degree also the weak substrate glutathionylspermidine to assume a productive mode.

Insights into substrate binding by D-2-ketoacid dehydrogenases from the structure of Lactobacillus pentosus D-lactate dehydrogenase.

BACKGROUND: D-Lactate dehydrogenases (D-LDHs) and L-lactate dehydrogenases (L-LDHs) catalyze a reaction differing only in the chirality of the product. Both enzymes utilize the same kind of amino acid side chains in substrate binding and catalysis. Models based on D-LDH-related enzymes propose that these side chains assume identical roles in both enzymes with their active sites related by a simple geometrical relationship such as a mirror plane. RESULTS: The crystal structure of the homodimeric D-LDH from Lactobacillus pentosus has been determined to 2.6 A resolution by multiple isomorphous replacement methods and the resulting molecular model refined to an R-factor of 19.1%. Topologically, the enzyme is closely related to other D-2-ketoacid dehydrogenase enzymes. Each subunit comprises two domains enclosing a deep cleft containing the active site. Substrate binding and domain closure have been modelled. CONCLUSIONS: Comparison of the D-LDH structure with other members of the protein family and with the L-specific enzyme has confirmed that no overall structural relationship exists between the L-LDH and D-LDH enzymes - they belong to distinct protein classes. The small size of the ketoacid substrate and the very restricted number of functionally appropriate side chains will constrain the choice of amino acids and their placement in the active site. Our models imply that although the same kinds of amino acids are involved in substrate binding their exact chemical role might differ in the two dehydrogenases.

Chemical mechanism and rate-limiting steps in the reaction catalyzed by Streptococcus faecalis NADH peroxidase.

The pH dependence of the kinetic parameters V, V/KNADH, and V/KH2O2 has been determined for the flavoenzyme NADH peroxidase. Both V/KNADH and V/KH2O2 decrease as groups exhibiting pK's of 9.2 and 9.9, respectively, are deprotonated. The V profile decreases by a factor of 5 as a group exhibiting a pK of 7.2 is deprotonated. Primary deuterium kinetic isotope effects on NADH oxidation are observed on V only, and the magnitude of DV is independent of H2O2 concentration at pH 7.5. DV/KNADH is pH independent and equal to 1.0 between pH 6 and pH 9.5, but DV is pH dependent, decreasing from a value of 7.2 at pH 5.5 to 1.9 at pH 9.5. The shape of the DV versus pH profile parallels that observed in the V profile and yields a similar pK of 6.6 for the group whose deprotonation decreases DV. Solvent kinetic isotope effects obtained with NADH or reduced nicotinamide hypoxanthine dinucleotide as the variable substrate are observed on V only, while equivalent solvent kinetic isotope effects on V and V/K are observed when H2O2 is used as the variable substrate. In all cases linear proton inventories are observed. Primary deuterium kinetic isotope effects on V for NADH oxidation decrease as the solvent isotopic composition is changed from H2O to D2O. These data are consistent with a change in the rate-limiting step from a step in the reductive half-reaction at low pH to a step in the oxidative half-reaction at high pH. Analysis of the multiple kinetic isotope effect data suggests that at high D2O concentrations the rate of a single proton transfer step in the oxidative half-reaction is slowed. These data are used to propose a chemical mechanism involving the pH-dependent protonation of a flavin hydroxide anion, following flavin peroxide bond cleavage.

Crystallization and preliminary x-ray diffraction study of the flavoprotein NADH peroxidase from Streptococcus faecalis 10C1.

NADH peroxidase from Streptococcus faecalis 10C1 has been crystallized from ammonium sulfate solutions using the hanging drop vapor diffusion method. Depending on pH, the crystals grew in the orthorhombic space group I222 or one of its subgroups P222 or P2(1)2(1)2 (or one of its two permutations). In both cases the unit cell axes are a = 76.6 A, b = 132.9 A, and c = 145.7 A. There are two monomers/asymmetric unit in the body-centered crystal form and four in the primitive one. The enzyme is catalytically active in the crystalline state. The crystals diffract to at least 2.5 A resolution; they are stable in the x-ray beam and hence suitable for detailed three-dimensional structure determination.

Hypothalamus and puberty.

The effect of a transitory increase in plasma FSH and LH levels during the prepubertal period on puberty has been investigated. Twenty-day-old rats had been bilaterally ovariectomized and 24 hr later they received two ovaries of infantile rats beneath the kidney capsule. These rats exhibited precocious puberty. Animals into which two additional ovaries had been transplanted first and the next day their own ovaries removed showed puberty at the same time as controls. Additional investigations provided evidence that the ovariectomized and ovary implanted rats plasma FSH and LH levels were in the control range four days after implantation. The findings support the assumption that the hypothalamic lesion-induced precocious puberty is due rather to a transitory enhanced release of gonadotropin releasing hormone by the lesion than to the destruction of sex-steroid sensitive structures inhibiting the release of gonadotropic hormones in prepubertal rats.

Kinetic mechanism and nucleotide specificity of NADH peroxidase.

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.

[Transthoracic and transesophageal 2-D echocardiography in the diagnosis of peri- and paracardiac tumors]. / Transthorakale und transösophageale 2-D-Echokardiographie in der Diagnostik peri- und parakardialer Tumoren.

Twelve consecutive patients (five males and seven females, aged 24-61 years) with peri- or paracardial space-occupying lesions were studied by transthoracic and transesophageal 2-D echocardiography (2-DE). The tumor was localized by transthoracic 2-DE in six patients. In four patients the lesion was suspected after transthoracic 2-DE, but only transesophageal 2-DE defined the tumor and separated it from surrounding tissues. In two cases of pericardial tumor transthoracic 2-DE at first misdiagnosed it as pericarditis with pericardial effusion. In eight patients transthoracic and in four transesophageal 2-DE made it possible to assess cardiac function. The findings demonstrate that transthoracic 2-DE may not demonstrate peri- or paracardial tumors; transesophageal 2-DE adds an at times decisive additional diagnostic method.