Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

[Adverse drug reactions in elderly people : First data from the Leipzig Research Center for Civilization Diseases (LIFE)]. / Unerwünschte Arzneimittelreaktionen beim älteren Menschen : Erste Daten aus dem Leipziger Forschungszentrum für Zivilisationserkrankungen (LIFE).

BACKGROUND: Few data exist on adverse drug reactions (ADR) in elderly people. In this group, pharmacotherapy represents a challenge with regard to comorbidities, drug interactions and compliance. OBJECTIVE: The aim of this article is to highlight the characteristics of ADR in elderly patients. METHODS: In addition to a literature review we present the first data from the Leipzig Research Center for Civilization Diseases (LIFE). Between 2011 and 2015 a total of 9537 subjects aged 40-79 years were randomly included in this population-based, age and sex standardized investigation in the inhabitants of Leipzig, Germany and special emphasis was placed on allergies including questions with regard to ADR. RESULTS: Of the 9537 subjects, data on allergies were available from 8979 subjects. Female gender, comorbidities and the use of multiple drugs were significantly associated with an increased risk of ADR. Women also reported ADR significantly more frequently than men. Of the subjects 22% reported suffering from some form of ADR as a result of medications, while in 2.3% this reaction had occurred within the previous 12 months. Less than 15% of LIFE patients with ADR were in possession of a document giving details of the ADR. DISCUSSION: The occurrence of ADR significantly contributes to morbidity in elderly patients. For prevention of ADR knowledge of patient-related factors, underlying diseases, drug characteristics and drug interactions are necessary.

Genetic risk variants for dyslexia on chromosome 18 in a German cohort.

Dyslexia is characterized by impaired reading and spelling. The disorder has a prevalence of about 5% in Germany, and a strong hereditary component. Several loci are thought to be involved in the development of dyslexia. Scerri et al. identified eight potential dyslexia-associated single nucleotide polymorphisms (SNPs) in seven genes on chromosome 18 in an English-speaking population. Here, we present an association analysis that explores the relevance of these SNPs in a German population comprising 388 dyslexia cases and 364 control cases. In case-control analysis, three nominal SNP associations were replicated. The major alleles of NEDD4L-rs12606138 and NEDD4L-rs8094327 were risk associated [odds ratio (OR) = 1.35, 95% confidence interval (CI) = 1.0-1.7, P-value = 0.017 and OR = 1.39, 95% CI = 1.1-1.7, P-value = 0.007, respectively], and both SNPs were in strong linkage disequilibrium (r(2) = 0.95). For MYO5B-rs555879, the minor allele was risk associated (OR = 1.31, 95% CI = 1.1-1.6, P-value = 0.011). The combined analysis of SNP sets using set enrichment analysis revealed a study-wide significant association for three SNPs with susceptibility for dyslexia. In summary, our results substantiate genetic markers in NEDD4L and MYO5B as risk factors for dyslexia and provide first evidence that the relevance of these markers is not restricted to the English language.

The role of gene DCDC2 in German dyslexics.

Dyslexia is a complex reading and writing disorder with a strong genetic component. In a German case-control cohort, we studied the influence of the suspected dyslexia-associated gene DCDC2. For the first time in a German cohort, we describe association of a 2445 basepair deletion, first identified in an American study. Evidence of association for three DCDC2 single nucleotide polymorphisms (rs807724, rs793862, rs807701), previously identified in German or American cohorts, was replicated. A haplotype of these polymorphisms showed evidence for association as well. Thus, our data further corroborate association of DCDC2 with dyslexia. Analysis of functional subgroups suggests association of investigated DCDC2 variants mainly with nondysphonetic, nonsevere, but probably dyseidetic (surface) dyslexia. Based on the presumed function of DCDC2, our findings point to a role of impaired neuronal migration in the etiology of the disease.

[Relevance of the gene variant PTPN22 620W for rheumatology]. / Zur Bedeutung der Genvariante PTPN22 620W für die Rheumatologie.

PTPN22 620W is regarded as the second most important risk factor for type 1 diabetes and rheumatoid arthritis. Here we describe aspects of the molecular biology of the enzyme and its function, the geographical distribution of the 620W variant, as well as its importance in less frequent rheumatic diseases.

A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein.

We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.

DNA binding in the central channel of bacteriophage T7 helicase-primase is a multistep process. Nucleotide hydrolysis is not required.

Many helicases assemble into ring-shaped hexamers and bind DNA in their central channel. This raises the question as to how the DNA gets into the central channel to form a topologically linked complex. We have used the presteady-state stopped-flow kinetic method and protein fluorescence changes to investigate the mechanism of single-stranded DNA (ssDNA) binding to the bacteriophage T7 helicase-primase, gp4A'. We have found that the kinetics of 30-mer ssDNA binding to a preformed gp4A' hexamer in the presence of both Mg-dTMP-PCP and Mg-dTTP are similar, indicating that Mg-dTTP binding is sufficient and hydrolysis is not necessary for efficient DNA binding. Multiple transient changes in gp4A' fluorescence revealed a four-step mechanism for DNA binding with Mg-dTTP. These transient changes were analyzed by global fitting and kinetic simulation to determine the intrinsic rate constants of this four-step mechanism. The initial steps, including the bimolecular encounter of the DNA with the helicase and a subsequent conformational change, were fast. We propose that these initial steps of DNA binding occur at a readily accessible site, which is likely to be on the outside of the hexamer ring. The binding of the 30-mer ssDNA at this loading site is followed by slower conformational changes that allow the DNA to transit into the central channel of gp4A' via a ring-opening or threading pathway.

Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate.

Bacteriophage T7 DNA helicase requires two noncomplementary single-stranded DNA (ssDNA) tails next to a double-stranded DNA (dsDNA) region to initiate DNA unwinding. The interactions of the helicase with the DNA were investigated using a series of forked DNAs. Our results show that the helicase interacts asymmetrically with the two tails of the forked DNA. When the helicase was preassembled on the forked DNA before the start of unwinding, a DNA with 15-nucleotide (nt) 3'-tail and 35-nt 5'-tail was unwound with optimal rates close to 60 base pairs/s at 18 degrees C. When the helicase was not preassembled on the DNA, a >65-nt long 5'-tail was required for maximal unwinding rates of 12 base pairs/s. We show that the helicase interacts specifically with the ssDNA region and maintains contact with both ssDNA strands during DNA unwinding, since conversion of the two ssDNA tails to dsDNA structures greatly inhibited unwinding, and the helicase was unable to unwind past a nick in the dsDNA region. These studies have provided new insights into the mechanism of DNA unwinding. We propose an exclusion model of DNA unwinding in which T7 helicase hexamer interacts mainly with the ssDNA strands during DNA unwinding, encircling the 5'-strand and excluding the 3'-strand from the hole.

Acute and chronic effects of winter swimming on LH, FSH, prolactin, growth hormone, TSH, cortisol, serum glucose and insulin.

The present investigation is based on a 2.5 months selbstversuch (self-experiment) of the authors, between October 21 1992, and January 6 1993. 11 healthy students, five females and six males, age 24 to 29 years, and their teachers underwent regular winter swimming at least once a week, for 2 to 10 minutes, at the natural water temperature (6.8 degrees C (October 1992) to 2.0 degrees C (January 1993)) in the southern Baltic Sea. Blood samples were drawn before and 30 and 60 minutes after the cold bath, both at the first and the last day of the swimming season. TSH increased from 0.96 mU/l to 1.42 mU/l (p < 0.01) in the untrained, and from 0.93 mU/l to 1.43 mU/l (p < 0.01) in the cold-trained persons, and decreased thereafter (p < 0.01). Similar changes occurred in cortisol serum concentrations, though psychological stress seemed to interfere with cold stress. Cortisol increased from 99 ng/ml to 133 ng/ml in the untrained, and from 101 ng/ml to 137 ng/ml (p < 0.05) in the cold-trained persons within 30 minutes after cold water immersion, and decreased thereafter (p < 0.01). There were mild decreases in prolactin serum levels after cold stress, whereas FSH, LH and growth hormone remained unaltered. There was a mild initial elevation of serum glucose after cold stress (plus 12 mg/dl, (p < 0.01)) which disappeared after training. There were long term training effects besides the effects on glucose: Basal prolactin levels increased by almost the factor two, and insulin serum levels dropped by almost 50%.(ABSTRACT TRUNCATED AT 250 WORDS)