Metabolomics, lipidomics and proteomics profiling of myoblasts infected with Trypanosoma cruzi after treatment with different drugs against Chagas disease.

INTRODUCTION: Chagas disease, the most important parasitic infection in Latin America, is caused by the intracellular protozoan Trypanosoma cruzi. To treat this disease, only two nitroheterocyclic compounds with toxic side effects exist and frequent treatment failures are reported. Hence there is an urgent need to develop new drugs. Recently, metabolomics has become an efficient and cost-effective strategy for dissecting drug mode of action, which has been applied to bacteria as well as parasites, such as different Trypanosome species and forms. OBJECTIVES: We assessed if the metabolomics approach can be applied to study drug action of the intracellular amastigote form of T. cruzi in a parasite-host cell system. METHODS: We applied a metabolic fingerprinting approach (DI-MS and NMR) to evaluate metabolic changes induced by six different (candidate) drugs in a parasite-host cell system. In a second part of our study, we analyzed the impact of two drugs on polar metabolites, lipid and proteins to evaluate if affected pathways can be identified. RESULTS: Metabolic signatures, obtained by the fingerprinting approach, resulted in three different clusters. Two can be explained by already known of mode actions, whereas the three experimental drugs formed a separate cluster. Significant changes induced by drug action were observed in all the three metabolic fractions (polar metabolites, lipids and proteins). We identified a general impact on the TCA cycle, but no specific pathways could be attributed to drug action, which might be caused by a high percentage of common metabolome between a eukaryotic host cell and a eukaryotic parasite. Additionally, ion suppression effects due to differences in abundance between host cells and parasites may have occurred. CONCLUSION: We validated the metabolic fingerprinting approach to a complex host-cell parasite system. This technique can potentially be applied in the early stage of drug discovery and could help to prioritize early leads or reconfirmed hits for further development.

Studying the impact of geometrical and cellular cues on myogenesis with a skeletal muscle-on-chip.

In the skeletal muscle tissue, cells are organized following an anisotropic architecture, which is both required during myogenesis when muscle precursor cells fuse to generate myotubes and for its contractile function. To build an in vitro skeletal muscle tissue, it is therefore essential to develop methods to organize cells in an anisotropic fashion, which can be particularly challenging, especially in 3D. In this study, we present a versatile muscle-on-chip system with adjustable collagen hollow tubes that can be seeded with muscle precursor cells. The collagen acts both as a tube-shaped hollow mold and as an extracellular matrix scaffold that can house other cell types for co-culture. We found that the diameter of the channel affects the organization of the muscle cells and that proper myogenesis was obtained at a diameter of 75 µm. In these conditions, muscle precursor cells fused into long myotubes aligned along these collagen channels, resulting in a fascicle-like structure. These myotubes exhibited actin striations and upregulation of multiple myogenic genes, reflecting their maturation. Moreover, we showed that our chip allowed muscle tissue culture and maturation over a month, with the possibility of fibroblast co-culture embedding in the collagen matrix.

Self-management-competency as a new target in Hidradenitis suppurativa care.

Background: Hidradenitis suppurativa affects approximately 1% of the population.Objective: Highlighting the relevance of self-management-competency as a new therapeutic target.Method: 258 patients from the 'Epidemiology and Care in Acne inversa (EpiCAi)' project were included in the study. Disease burden was measured by patient-rated questionnaires in terms of disease activity, pain, quality of life, depression and insomnia and correlated with the domains of the health education impact questionnaire (heiQ) measuring self-management-competency.Results: 66 male (25.6%) and 192 female (74.4%) patients, with a mean age of 40.3 ± 10.24 years were included. Mean scores of pain on the numeric rating scale (NRS), Dermatology Life Quality Index (DLQI) and Hospital Anxiety and Depression Scale (HADS) were 5.11 ± 2.68, 11.35 ± 7.79 and 13.71 ± 7.57, respectively. The Insomnia severity index (ISI) showed a mean of 9.58 ± 5.76. The HADS has the highest increased total risk across all heiQ domains. With respect to the heiQ domains, the highest exposure can be attributed to improving constructive attitudes and approaches as well as decreasing emotional distress.Conclusion: There is a clear association of self-management-competency with overall disease burden, which underlines the need for psychoeducational support. This study provides ideas to develop new possible strategies of care.

Stick-slip dynamics of cell adhesion triggers spontaneous symmetry breaking and directional migration of mesenchymal cells on one-dimensional lines.

Directional cell motility relies on the ability of single cells to establish a front-rear polarity and can occur in the absence of external cues. The initiation of migration has often been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cell-substrate interaction forces has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics the complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters of NIH-3T3 fibroblasts and RPE1 epithelial cells reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to trigger cell motility in 1D in the absence of pre-established polarity. A theoretical model validates the crucial role of adhesion dynamics, proposing that front-rear polarity can emerge independently of a complex self-polarizing system.

Kinetically Selective Inhibitors of Histone Deacetylase 2 (HDAC2) as Cognition Enhancers.

Aiming towards the development of novel nootropic therapeutics to address the cognitive impairment common to a range of brain disorders, we set out to develop highly selective small molecule inhibitors of HDAC2, a chromatin modifying histone deacetylase implicated in memory formation and synaptic plasticity. Novel ortho-aminoanilide inhibitors were designed and evaluated for their ability to selectively inhibit HDAC2 versus the other Class I HDACs. Kinetic and thermodynamic binding properties were essential elements of our design strategy and two novel classes of ortho-aminoanilides, that exhibit kinetic selectivity (biased residence time) for HDAC2 versus the highly homologous isoform HDAC1, were identified. These kinetically selective HDAC2 inhibitors (BRD6688 and BRD4884) increased H4K12 and H3K9 histone acetylation in primary mouse neuronal cell culture assays, in the hippocampus of CK-p25 mice, a model of neurodegenerative disease, and rescued the associated memory deficits of these mice in a cognition behavioural model. These studies demonstrate for the first time that selective pharmacological inhibition of HDAC2 is feasible and that inhibition of the catalytic activity of this enzyme may serve as a therapeutic approach towards enhancing the learning and memory processes that are affected in many neurological and psychiatric disorders.

Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis.

Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [gamma-(32)P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.

Inherited diseases in North American Mennonites: focus on Old Colony (Chortitza) Mennonites.

The patterns of migration and the genetic disorders occurring among North American Mennonites are reviewed, and inherited conditions recently recognized in a religious and genetic isolate, the Old Colony (Chortitza) Mennonites, are described. Old Colony Mennonites are of Dutch/German origin and descend from approximately 400 founding families who settled in the Old Colony, Chortitza (the Ukraine, USSR) in the late 1700s, and then migrated to Canada and Central and South America in the past century. We investigated over 6 generations of a Canadian Old Colony kindred in which there was extensive intermarriage, and in whom 28 individuals developed diabetes mellitus. Insulin-dependent diabetes mellitus (IDDM) occurred in 14 affected individuals in 10 closely related sibships; the 11 living IDDM patients were all concordant for the immunogenetic marker HLA-DR4. Fourteen close relatives had other disorders of carbohydrate metabolism, including gestational diabetes and non-insulin-dependent diabetes mellitus. Other close relatives had autoimmune diseases, including rheumatoid arthritis, hyper- and hypothyroidism, multiple sclerosis, and red cell aplasia. Other inherited diseases, including Alport syndrome, congenital defects, and inborn errors of metabolism were also found in the kindred. In the almost exclusively (99%) Old Colony Mennonite public health district in which the kindred was ascertained, there were multiple cases of Tourette syndrome, of malformations (including congenital heart defects and cleft lip +/- palate), and familial clusters of inborn errors of metabolism. We report this Old Colony (Chortitza) Mennonite isolate because 1) there are large familial aggregations of tissue-specific autoimmune diseases, malformations, inborn errors of metabolism, and of some other conditions whose genetic basis is still unknown; 2) there are multiple cases of rare genetic conditions, 3) we have established a computerized genealogic data base on over 1,000 kindred members as well as a cryopreserved lymphocyte/DNA bank on over 100 closely related individuals with various genetic conditions; and 4) this religious isolate, which extends across North, Central, and South America, offers an excellent opportunity for studying the epidemiology and molecular genetics of both common and rare inherited diseases.

The consolidation/transition model in moral reasoning development.

The consolidation/transition model conceptualizes development as entailing a cyclical pattern of alternating consolidation and transition phases and posits that stage advance is predicted by a specific distribution of reasoning across stages indicative of disequilibrium (more reasoning above than below the mode, with a high degree of mixture). The validity of this model was examined in the context of moral reasoning development with the use of standard statistical techniques as well as Bayesian techniques that can better account for classification error. In this longitudinal study. 64 children and adolescents participated in 5 annual administrations of the Moral Judgment Interview. The distribution of their reasoning across stages was used to predict subsequent development. The results support the hypotheses regarding cyclical patterns of change and predictors of stage transition and demonstrate the utility of Bayesian techniques for evaluating developmental change.

[Sources of error in the interpretation of bone scans with 99mTc-diphosphonate (author's transl)]. / Fehlermöglichkeiten bei der Interpretation der Knochenszintigramme mit 99mTc-Diphosphonat

Errors in the interpretation of bone scans with 99mTc-diphosphonate may be due to summation of radio-activity from superimposition of various bone structures. Lesions with increased bone turn-over as well as reactive hyperaemia result in increased uptake. The possibility of extraosseous uptake of diphosphonate must be borne in mind during the evaluation of bone scans. The kidneys and lower urinary tract must be considered. Absent or slight uptake by the kidneys with good demonstration of bone suggests increased diphosphonate uptake by the bones. Special attention should also be paid to zones of decreased uptake, since these are also due to abnormalities in the bone.

X-ray and fine-needle biopsy in diagnosis of non-palpable breast lesions.

Using mammography and xeroradiography among 78 patients with non-palpable breast lesions, three cases of suspected carcinoma were detected. The diagnosis was confirmed by cytology after x-ray guidance of the fine-needle biopsy.

[Total replacement of humerus including shoulder and elbow joints]. / Totalersatz des Humerus einschliesslich Schulter- und Ellenbogengelenk

A case of total alloplastic replacement of the humerus, including the neighboring joints, is reported. The method may be used for treatment of large defects of the humerus due to tumor, osteomyelitis, or trauma. The alloplastic replacement seems superior to autoplastic fibula transplantation.

[Experience report on the intraoperative use of the ultrasonic Doppler Probe]. / Erfahrungsbericht über den intraoperativen Einsatz der Ultraschall-Dopplersonde.

Experiences in the intraoperative use of an ultrasonic doppler-flowmeter are described. The method can be helpful when performing various operations within the field of generalsurgery and vascular surgery.

[Arterial involvement in elbow-proximal injuries]. / Arterienbeteiligung bei ellenbogennahen Verletzungen.

Nine cases of distal brachial artery injury were discussed. We refer to case No. 7 in which a complete dorsal displacement of the artery occurred through the fracture-line of the epicondyle. Trauma caused dislocation, laceration, or spasm of the artery, with or without intravascular thrombosis. Vascular spasm should not be diagnosed unless an arteriogram is available. If not done preoperatively, it can be done during operation. In this case inspection of the vessel during operation seems essential. Venous interposition is satisfactory for bridging defects of the distal brachial artery even in the elbow region.

A metabolomics approach to identify factors influencing glucosinolate thermal degradation rates in Brassica vegetables.

Thermal processing of Brassica vegetables can lead to substantial loss of potential health-promoting glucosinolates (GLs). The extent of thermal degradation of a specific GL varies in different vegetables, possibly due to differences in the composition of other metabolites within the plant matrices. An untargeted metabolomics approach followed by random forest regression was applied to identify metabolites associated to thermal GL degradation in a segregating Brassica oleracea population. Out of 413 metabolites, 15 were associated with the degradation of glucobrassicin, six with that of glucoraphanin and two with both GLs. Among these 23 metabolites three were identified as flavonols (one kaempferol- and two quercetin-derivatives) and two as other GLs (4-methoxyglucobrassicin, gluconasturtiin). Twenty quantitative trait loci (QTLs) for these metabolites, which were associated with glucoraphanin and glucobrassicin degradation, were identified on linkage groups C01, C07 and C09. Two flavonols mapped on linkage groups C07 and C09 and co-localise with the QTL for GL degradation determined previously.

Pitfalls in the desulphation of glucosinolates in a high-throughput assay.

Glucosinolates are phytochemicals with health promoting properties. Determination as desulpho-glucosinolates is widely used and a desulphation in microtiter plates has been applied to reach high throughput. The use of various sulphatase concentrations and volumes throughout literature necessitates the identification of an appropriate desulphation procedure in microtiter plates. High sulphatase concentrations (≥15 mg/ml) decreased the concentration of the internal standard glucotropaeolin, whereas the other glucosinolates were less affected. Due to the calculation based on the recovery of glucotropaeolin, this leads to an overestimation of GL concentrations after desulphation with high sulphatase concentrations. A glucosidase side-activity, present in the crude sulphatase powder, is likely causing this phenomenon. At lower sulphatase concentrations (1 mg/ml) glucoiberin and glucoraphanin were insufficiently desulphated. Combining these effects results in a small range of applicable sulphatase concentrations. A purified sulphatase preparation resulted in good recoveries for a diversity of samples and is hence recommended for high throughput desulphation in microtiter plates.