Plasma concentrations of matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1 and osteopontin reflect severity of heart failure in DOCA-salt hypertensive rat

The matrix metalloproteinases (MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) play a key role in extracellular matrix maintenance and are altered in the failing heart, both in experimental models and in chronic end-stage heart failure in humans. As the common diagnostic markers of heart failure, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) primarily reflect increased pressure loading, determination of soluble, heart-derived MMPs and TIMPs in plasma, as well as the determination of the emerging fibrosis marker osteopontin (OPN) might be valuable tools for detecting heart fibrosis. In this study the effect of spironolactone treatment on plasma MMP-2, TIMP-1 and OPN levels was assessed in a heart failure animal model. Unilaterally nephrectomized Sprague Dawley rats received subcutaneous injection of 100 mg deoxycorticosterone acetate (DOCA) once a week and 1% (w/v) NaCl in drinking water. Blood pressure was monitored weekly and blood samples were collected after 1, 2 and 4 weeks. After 6 weeks, left ventricular contractility (LVC) and heart weight-to-body weight ratio (HW/BW) were assessed. DOCA treatment increased plasma MMP-2, TIMP-1 and OPN concentrations. Alterations of plasma marker levels were correlated with changes of HW/BW and paralleled impaired LVC. Furthermore, beneficial effects of spironolactone treatment were observed. In DOCA-salt hypertensive rats, plasma concentrations of MMP-2, TIMP-1 and OPN reflected heart failure associated with haemodynamic, functional and morphological changes. Based on these findings, it appears reasonable to use plasma markers of fibrosis to monitor the development of heart failure.     

OligoDesign: Optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.     

Identification of the last unknown genes in the fermentation pathway of lysine

Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a l-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.     

Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.     

The Role of Self-Compassion in Buffering Symptoms of Depression in the General Population

Self-compassion, typically operationalized as the total score of the Self-Compassion Scale (SCS; Neff, 2003b), has been shown to be related to increased psychological well-being and lower depression in students of the social sciences, users of psychology websites and psychotherapy patients. The current study builds on the existing literature by examining the link between self-compassion and depressive symptomatology in a sample representative of the German general population (n = 2,404). The SCS subscales of self-judgment, isolation, and over-identification, and the “self-coldness”, composite score, which encompass these three negative subscales, consistently differed between subsamples of individuals without any depressive symptoms, with any depressive syndromes, and with major depressive disorder. The contribution of the positive SCS subscales of self-kindness, common humanity, and mindfulness to the variance in depressive symptomatology was almost negligible. However, when combined to a “self-compassion composite”, the positive SCS subscales significantly moderated the relationship between “self-coldness” and depressive symptoms in the general population. This speaks for self-compassion having the potential to buffer self-coldness related to depression—providing an argument for interventions that foster self-caring, kind, and forgiving attitudes towards oneself.     

Optimized folding and activation of recombinant procathepsin L and S produced in Escherichia coli

Large scale production of the recombinant human cathepsins L and S was optimized. Final purity was nearly 100%, yield 65% and 41%, respectively. Cost-effective expression in Escherichia coli, inclusion body purification and solubilization followed modified standard protocols. Most contribution to the remarkable increase in over-all efficiency came from the subsequent steps: folding by dilution, selective HIC-capturing of the folded proenzymes, and auto-activation. The effort to optimize the process parameters for folding and activation was greatly reduced by central composite fractional factorial experimental design, considering curved responses as well as factor interactions. Theoretical and practical features of this powerful tool for experimental design are given. Yield in procathepsin S folding could be further increased by addition of an excess of its own native propeptide with known intramolecular chaperone function. This corroborates literature data on proenzyme folding and is broadly discussed in the light of the lower conformational stability of the prodomain compared to the catalytic unit. Auto-activation kinetics was largely different between the two related proenzymes; from its time course contribution of uni- and bimolecular events in proregion hydrolysis and rate constants were estimated.