Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173

Background and Aims

The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn²⁺ and Cu²⁺ concentration during hemodialysis.

Methods and Results

Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn²⁺ or Cu²⁺ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn²⁺ and Cu²⁺ concentrations after dialysis.

Conclusions

Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.     

Intra-Rater,Inter-Rater and Test-Retest Reliability of an Instrumented Timed Up and Go (iTUG) Test in Patients with Parkinson’s Disease

Background

The “Timed Up and Go” (TUG) is a widely used measure of physical functioning in older people and in neurological populations, including Parkinson’s Disease. When using an inertial sensor measurement system (instrumented TUG [iTUG]), the individual components of the iTUG and the trunk kinematics can be measured separately, which may provide relevant additional information.

Objective

The aim of this study was to determine intra-rater, inter-rater and test-retest reliability of the iTUG in patients with Parkinson’s Disease.

Methods

Twenty eight PD patients, aged 50 years or older, were included. For the iTUG the DynaPort Hybrid (McRoberts, The Hague, The Netherlands) was worn at the lower back. The device measured acceleration and angular velocity in three directions at a rate of 100 samples/s. Patients performed the iTUG five times on two consecutive days. Repeated measurements by the same rater on the same day were used to calculate intra-rater reliability. Repeated measurements by different raters on the same day were used to calculate intra-rater and inter-rater reliability. Repeated measurements by the same rater on different days were used to calculate test-retest reliability.

Results

Nineteen ICC values (15%) were ≥ 0.9 which is considered as excellent reliability. Sixty four ICC values (49%) were ≥ 0.70 and < 0.90 which is considered as good reliability. Thirty one ICC values (24%) were ≥ 0.50 and < 0.70, indicating moderate reliability. Sixteen ICC values (12%) were ≥ 0.30 and < 0.50 indicating poor reliability. Two ICT values (2%) were < 0.30 indicating very poor reliability.

Conclusions

In conclusion, in patients with Parkinson’s disease the intra-rater, inter-rater, and test-retest reliability of the individual components of the instrumented TUG (iTUG) was excellent to good for total duration and for turning durations, and good to low for the sub durations and for the kinematics of the SiSt and StSi. The results of this fully automated analysis of instrumented TUG movements demonstrate that several reliable TUG parameters can be identified that provide a basis for a more precise, quantitative use of the TUG test, in clinical practice.     

Confirmatory Factor Analysis and Differential Relationships of the Two Subdomains of Negative Symptoms in Chronically Ill Psychotic Patients

Research suggests a two factor structure for negative symptoms in patients with psychotic disorders: social amotivation (SA) and expressive deficits (ED). Applying this two-factor structure in clinical settings may provide valuable information with regard to outcomes and to target treatments. We aimed to investigate 1) whether the factor structure is also supported in chronically ill patients with a psychotic disorder and 2) what the relationship is between these factors and functioning (overall functioning and living situation), depressive symptoms and quality of life. 1157 Patients with a psychotic disorder and a duration of illness of 5 years or more were included in the analysis (data selected from the Pharmacotherapy Monitoring Outcome Survey; PHAMOUS). A confirmatory factor analysis was performed using items of the Positive and Negative Syndrome Scale that were previously identified to reflect negative symptoms (N1-4, N6, G5, G7, G13, G16). Subsequently, regression analysis was performed on outcomes. The results confirmed the distinction between SA (N2, N4, G16) and ED (N1, N3, N6, G5, G7, G13) in chronically ill patients. Both factors were related to worse overall functioning as measured with the Health of the Nation Outcome Scales, ED was uniquely associated with residential living status. Higher scores for SA were associated with more depressive symptoms and worse quality of life. Thus, SA is most strongly related to level of social-emotional functioning, while ED are more related to living situation and thereby are indicative of level of everyday functioning. This subdivision may be useful for research purposes and be a valuable additional tool in clinical practice and treatment development.     

Energy metabolism of bloodstream form Trypanosoma theileri

Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production. This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate availability determines the type of energy metabolism of trypanosomatids.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Contact-based sequence alignment

This paper introduces the novel method of contact-based protein sequence alignment, where structural information in the form of contact mutation probabilities is incorporated into an alignment routine using contact-mutation matrices (CAO: Contact Accepted mutatiOn). The contact-based alignment routine optimizes the score of matched contacts, which involves four (two per contact) instead of two residues per match in pairwise alignments. The first contact refers to a real side-chain contact in a template sequence with known structure, and the second contact is the equivalent putative contact of a homologous query sequence with unknown structure. An algorithm has been devised to perform a pairwise sequence alignment based on contact information. The contact scores were combined with PAM-type (Point Accepted Mutation) substitution scores after parameterization of gap penalties and score weights by means of a genetic algorithm. We show that owing to the structural information contained in the CAO matrices, significantly improved alignments of distantly related sequences can be obtained. This has allowed us to annotate eight putative Drosophila IGF sequences. Contact-based sequence alignment should therefore prove useful in comparative modelling and fold recognition.     

Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants

The phylogeny and distribution of filamentous Alphaproteobacteria, morphologically similar to “Nostocoida limicola” and Eikelboom Type 021N that cause the solids separation problem of bulking in industrial activated sludge plants is described here. A combination of culture-dependent and culture-independent molecular methods has characterized 5 novel species. 16S rRNA targeted oligonucleotide probes were designed for their in situ identification by fluorescence in situ hybridisation (FISH) and used to monitor their presence in 86 WWTPs treating different industrial effluents in four European countries. The involvement of these bacteria in bulking in these plants was confirmed. Filaments hybridising with the ALF-968 probe for the Alphaproteobacteria were present in 65% of the WWTPs examined. They were dominant and therefore probably responsible for bulking in 25.5% of them. The heterogeneous filamentous alphaproteobacterial populations in these communities could be completely identified after application of the oligonucleotide probes used in this study in 91% of the plants containing them. The only filamentous Alphaproteobacteria retrieved in pure culture was isolated from three different industrial WWTPs plants. None of these isolates could grow anaerobically on glucose or denitrify, but all grew aerobically and heterotrophically on a range of carbon sources. Although morphologically similar to the Eikelboom Type 021N morphotype, they were not involved in sulphur metabolism. These bacteria accumulated lipidic storage granules that were associated with their presence under the unbalanced growth conditions existing in these plants.     

Liquid chromatographic assays for DE-310, a novel camptothecin analog, and two major enzymatic products in human matrices

Assays were developed for determination of DE-310, a carboxymethyldextran polyalcohol conjugate of the topoisomerase I inhibitor DX-8951 (exatecan) and two enzymatic products (i.e. glycyl-DX-8951 and unconjugated DX-8951) in human whole blood, erythrocytes and saliva. Sample pretreatment involved a single protein-precipitation step, followed by a thermolysin-mediated deconjugation for the parent molecule. Separation of the compounds was achieved on an Inertsil ODS-80A column (150 mm x 4.6 mm i.d.; 5 microm PS), using isocratic elution. The column effluent was monitored at excitation and emission wavelengths of 375 and 445 nm, respectively. Validation results indicated that the methods are accurate and precise at lower limits of quantitation of 0.5-6.9 ng/ml. The methods were used to study the blood distribution and salivary concentrations in patients receiving DE-310.