Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform.

The bacterial CRISPR/Cas system has proven to be an efficient gene-targeting tool in various organisms. Here we employ CRISPR/Cas for accurate and efficient genome editing in rats. The synthetic chimeric guide RNAs (gRNAs) discriminate a single-nucleotide polymorphism (SNP) difference in rat embryonic fibroblasts, allowing allele-specific genome editing of the dominant phenotype in (F344 × DA)F1 hybrid embryos. Interestingly, the targeted allele, initially assessed by the allele-specific gRNA, is repaired by an interallelic gene conversion between homologous chromosomes. Using single-stranded oligodeoxynucleotides, we recover three recessive phenotypes: the albino phenotype by SNP exchange; the non-agouti phenotype by integration of a 19-bp DNA fragment; and the hooded phenotype by eliminating a 7,098-bp insertional DNA fragment, evolutionary-derived from an endogenous retrovirus. Successful in vivo application of the CRISPR/Cas system confirms its importance as a genetic engineering tool for creating animal models of human diseases and its potential use in gene therapy.

Characterization of the response to low pH of Lactobacillus casei ΔRR12, a mutant strain with low D-alanylation activity and sensitivity to low pH.

AIMS: To identify the differences that account for the acid sensitivity of Lactobacillus casei ΔRR12. RR12 controls the expression of the dlt operon, and its inactivation leads to a diminished teichoic acid D-alanylation activity. To this end, a comparison of its response of ΔRR12 to low pH with the parental strain Lact. casei BL23 was carried out. METHODS AND RESULTS: The ability to induce an acid tolerance response (ATR), fatty acid (FA) composition and proteome changes induced in both strains in response to acid were investigated. Results obtained showed that both strains induce a growth-phase-dependent ATR. However, significant differences in the content of FAs and membrane-associated proteins were detected. CONCLUSIONS: The greater abundance of cytoplasmic proteins in the membrane fraction of the mutant strain ΔRR12 suggests an increased permeability of the cell membrane in this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The analysis of the response to low pH of strain ΔRR12 indicated that the inactivation of TCS12 affected the content of FAs and proteins associated to the cell envelope. Increased abundance of cytoplasmic proteins suggested that low alanylation of teichoic acids affected the permeability of the cell membrane and possibly accounts for the acid sensitivity of strain ΔRR12.

Diabetic nephropathy but not HbA1c is predictive for frequent complications of Charcot feet - long-term follow-up of 164 consecutive patients with 195 acute Charcot feet.

AIMS: To investigate the clinical characteristics, treatment and long-term outcome of patients with acute Charcot feet (CF). METHODS: Single-center retrospective analysis. Treatment of CF: stage-dependent immobilization/weight-off therapy, orthopaedic/adjusted shoes, foot surgery. 164 consecutive participants (type 1 vs. type 2 diabetes): 12 vs. 150, non-diabetic peripheral neuropathy: n=2, presented with 195 (17 vs. 176) CF. Mean follow-up: 4.7 ± 2.5 (range 2.2-9.8) vs. 5.4 ± 2.9 (range 0.8-18.8) years, vital at follow-up: 100 vs. 88%. RESULTS: Baseline characteristics: age: 43.7 ± 10.9 vs. 57.9 ± 8.9 years (p<0.001), male gender: 66.7 vs. 77.3%, diabetes duration: 19.2 ± 9.1 vs. 13 ± 8.6 years (p=0.018), GHb: 8.1 ± 2.4 vs. 7.6 ± 1.6%, BMI: 24 ± 5.3 vs. 33.7 ± 6.5 kg/m2 (p<0.001), Levine 1: 18.2 vs. 7.4%, Levine 2: 45.5 vs. 65.9%, Sanders 2: 58.3 vs. 68.5%, Sanders 3: 33.3 vs. 45%. THERAPY: immobilization for 6 ± 4.2 vs. 5.4 ± 4.5 months, orthopaedic/adjusted shoes: 27.3 vs. 20.5%, foot surgery: 11.8 vs. 18.2%. Major complications: 50 vs. 56% (rocker bottom deformities: 23.5 vs. 46.3%, foot ulcerations: 17.6 vs. 24.6%, CF amputations: 0 vs. 6%), not CF amputations: 16.7 vs. 15.3%, second episodes of CF: 41.6 vs. 18.3% after 5-132 months. Diabetic nephropathy was associated with an increase, intensive antihypertensive therapy with a decrease of complications. CONCLUSIONS: Patients with CF are middle-aged, overweight males with type 2 diabetes above 10 years. Patients with type 1 diabetes are younger, have normal BMI and longer diabetes duration. Major complications and second episodes of CF are frequent. Diabetic nephropathy could be a risk factor for CF related complications. The awareness for CF must be improved.The study was conducted due to the Declaration of Helsinki.

The breast care nurse: the care specialist in breast centres.

BACKGROUND: In Europe, breast cancer is the most common form of cancer in women; 26.5 per cent of all new malignant diseases and 17.5 per cent of all cancer deaths are due to this type of cancer. In Germany, approximately every tenth woman is diagnosed with breast cancer during her lifetime. OBJECTIVE: In order to increase the quality of life of breast cancer patients, the European Society of Mastology (EUSOMA) requires the availability of, and an integration of qualified and specialized care into the interdisciplinary team. Thus, in Germany, a new type of job profile for 'breast nurse' as a qualified care specialist has to be established and has to be oriented towards the international standards of a breast care nurse (BCN). METHODS: The Charité's Academy of Health--in cooperation with the Interdisciplinary Breast Centre of the Charité--has offered a career advisory service curriculum for the BCN since 2006 in accordance with the EUSOMA guidelines. RESULTS: In three courses, 45 BCNs have been trained. In the first refresher seminar in May 2009, the BCNs were given an opportunity to exchange with fellow BCNs their experiences. CONCLUSION: We were able to set up a new curriculum to train nurses as BCNs as to improve the quality of care for breast cancer patients and to orientate the training towards the international standards of a BCN qualification.

Interlaboratory study of a liquid chromatography method for erythromycin: determination of uncertainty.

Erythromycin is a mixture of macrolide antibiotics produced by Saccharopolyspora erythreas during fermentation. A new method for the analysis of erythromycin by liquid chromatography has previously been developed. It makes use of an Astec C18 polymeric column. After validation in one laboratory, the method was now validated in an interlaboratory study. Validation studies are commonly used to test the fitness of the analytical method prior to its use for routine quality testing. The data derived in the interlaboratory study can be used to make an uncertainty statement as well. The relationship between validation and uncertainty statement is not clear for many analysts and there is a need to show how the existing data, derived during validation, can be used in practice. Eight laboratories participated in this interlaboratory study. The set-up allowed the determination of the repeatability variance, s(2)r and the between-laboratory variance, s(2)L. Combination of s(2)r and s(2)L results in the reproducibility variance s(2)R. It has been shown how these data can be used in future by a single laboratory that wants to make an uncertainty statement concerning the same analysis.

[Two-photon excitation fluorescence spectrum of the light-harvesting complex LH2 from Chromatium minutissimum within 650-745 nm range is determined by two-photon absorption of bacteriochlorophyll rather than of carotenoids]. / Spektr dvukhfotonnogo vozbuzhdeniia fluorestsentsii svetosobiraiushchego kompleksa LH2 iz Chromatium minutissimum v oblasti 650-745 nm opredeliaetsia dvukhfotonnym pogloshcheniem bakteriokhlorofilla, a ne karotinoidov.

Two-photon fluorescence excitation spectra of the peripheral light-harvesting complex LH2 from the purple photosynthetic bacterium Chromatium minutissimum were examined within the expected spectral range of the optically forbidden S1 singlet state of carotenoids. LH2 preparations isolated from wild-type and carotenoid-depleted cells were used. 100-fs laser pulses in the range of 1300-1490 nm with an energy of 7-9 mW (corresponding to one-photon absorption between 650 and 745 nm) were used for two-photon fluorescence excitation. It was shown that two-photon fluorescence excitation spectra of LH2 complex from wild and carotenoid-depleted cells are very similar to each other and to the two-photon fluorescence excitation spectrum of bacteriochlorophyll a in acetone. It was concluded that direct two-photon excitation of bacteriochlorophyll a determines the fluorescence of both samples within the 650-745 nm spectral range.

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically 'dark') S(1) state of carotenoids.

Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were investigated by simultaneous two-photon excited (between 1300-1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2A(g)(-) state of rhodopin [corrected] is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid-depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.

Action of brassinosteroids in the cotton leafworm Spodoptera littoralis.

The two native plant hormones 24-epibrassinolide and 24-epicastasterone showed 50% competition for binding at IC(50) of 1-3.6 microM with [(3)H]ponasterone A using cultured imaginal wing discs from last-instar larvae of the cotton leafworm, Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). However, culture of imaginal wing discs in different concentrations of brassinosteroids, even up to 100 microM, demonstrated no induction of evagination. In contrast, 20E and the non-steroidal agonist RH-5992 competed respectively about 23- and 42-fold more effectively with labeled ponasterone A, and their ability (EC(50)) to induce disc evagination in vitro was 158 and 87 nM, respectively. Injection of 10 microg of brassinosteroids in newly-moulted last-instar larvae did not cause mortality above controls; higher mortalities were scored when brassinosteroids were injected late in the last instar.

The ecdysteroid agonist/antagonist and brassinosteroid-like activities of synthetic brassinosteroid/ecdysteroid hybrid molecules.

A series of synthetic hybrid brassinosteroid/ecdysteroid structures has been assessed for their ecdysteroid agonist/antagonist activities in the Drosophila melanogaster B(II) cell bioassay and for brassinosteroid-like activity in the rice lamina inclination test. Most of the compounds proved inactive for ecdysteroid agonist activity, demonstrating the specificity of the ecdysteroid receptor for compounds closely structurally related to 20-hydroxyecdysone. However, compound 18, with 14alpha-hydroxy-7-en-6-one and 22S-hydroxy functionalities (as in most active ecdysteroids), possessed distinct agonist activity (median effective concentration = 1.4 x 10(-5) M), although this is still almost 2000-fold less active than 20-hydroxyecdysone (25). Compounds 13 and 15 possessed weak agonist activity. Compounds 5, 11 and 14 weakly antagonised the action of 20-hydroxyecdysone (at 5 x 10(-8) M) on B(II) cells. In the brassinosteroid bioassay, most of the tested compounds showed activity. This may reflect the metabolic capability of plant tissue to convert test compounds to more active analogues. However, it is clear that biological activity declines as the structure of the test compound deviates further from that of castasterone (16). Three ecdysteroids (25, 26 and 27) are completely inactive in the rice lamina inclination test. These studies demonstrate the high specificities of the insect ecdysteroid receptor and the plant brassinosteroid receptor and indicate that phytoecdysteroids, even in high concentrations, would not interfere with brassinosteroid signalling pathways in plants where the two classes of compounds co-occur. Equally, brassinosteroids would not interfere with ecdysteroid signalling in insects, especially if one takes into account the low concentrations of brassinosteroids in the diet of phytophagous insects.

A proteomic view on genome-based signal peptide predictions.

The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.

Excitation trap approach to analyze size and pigment-pigment coupling: reconstitution of LH1 antenna of Rhodobacter sphaeroides with Ni-substituted bacteriochlorophyll.

Replacement of the central Mg in chlorophylls by Ni opens an ultrafast (tens of femtoseconds time range) radiationless de-excitation path, while the principal ground-state absorption and coordination properties of the pigment are retained. A method has been developed for substituting the native bacteriochlorophyll a by Ni-bacteriochlorophyll a ([Ni]-BChl) in the light harvesting antenna of the core complex (LH1) from the purple bacterium, Rhodobacter (Rb.) sphaeroides, to investigate its unit size and excited state properties. The components of the complex have been extracted with an organic solvent from freeze-dried membranes of an LH1-only strain of Rb. sphaeroides and transferred into the micelles of n-octyl-beta-glucopyranoside (OG). Reconstitution was achieved by solubilization in 3.4% OG, followed by dilution, yielding a complex nearly identical to the native one, in terms of absorption, fluorescence, and circular dichroism spectra as well as energy transfer efficiency from carotenoid to bacteriochlorophyll. By adding increasing amounts of [Ni]-BChl to the reconstitution mixture, a series of LH1 complexes was obtained that contain increasing levels of this efficient excitation trap. In contrast to the nearly unchanged absorption, the presence of [Ni]-BChl in LH1 markedly affects the emission properties. Incorporation of only 3.2 and 20% [Ni]-BChl reduces the emission by 50% and nearly 100%, respectively. The subnanosecond fluorescence kinetics of the complexes were monoexponential, with the lifetime identical to that of the native complex, and its amplitude decreasing in parallel with the steady-state fluorescence yield. Quantitative analysis of the data, based on a Poisson distribution of the modified pigment in the reconstituted complex, suggests that the presence of a single excitation trap per LH1 unit suffices for efficient emission quenching and that this unit contains 20 +/- 1 BChl molecules.

[Current aspects of defibrillator therapy in congestive heart failure]. / Aktuelle Aspekte der Defibrillatortherapie bei Herzinsuffizienz.

The beneficial effects of implantable cardioverter defibrillator (ICD) therapy in patients (pts) with life-threatening ventricular tachyarrhythmias and impaired left ventricular (LV) function is still unclear. We studied the follow-up of 410 pts (368 males, 42 females, mean age 57 +/- 11 years) after ICD implant. The LV function was assessed by the New York Heart Association functional class of heart failure (NYHA). Fifty pts (12%) were in NYHA I-II, 151 pts (37%) in NYHA II, 117 pts (29%) in NYHA II-III and 92 pts (22%) in NYHA III. Epicardial ICD implantation was performed in 209 pts (51%) and 201 pts (49%) received nonthoracotomy ICDs. Perioperatively (within 30 days after implant), 12 pts (3%) died, significantly more frequent after epicardial (11 of 209 pts, 5%) than after transvenous ICD implant (1 of 201 pts, < 1%) (p < 0.05). During a mean follow-up of 28 +/- 24 months (range < 1 to 114 months), 90 pts (23%) died: 9 pts (2%) died from sudden arrhythmic death and 5 pts (1%) suddenly, but probably not from arrhythmic causes; 55 pts (14%) died from cardiac causes (congestive heart failure, myocardial reinfarction) and 21 pts (5%) from noncardiac causes. The 3-year, 5-year and 7-year survival was 92% to 96% for arrhythmic mortality in NYHA class I, II and III compared to the 3-year survival of 94%, and a 5-year and 7-year survival of 84% in patients with NYHA class II-III. 338 pts (82%) received ICD shocks (mean incidence 21 +/- 43 shocks per pt); pts in NYHA class II (83%), class II-III (84%), class III (90%) received ICD discharges significantly more frequently than in class I-II (64%) (p < 0.05). Our data show that pts with LV dysfunction benefit from ICD therapy and that these pts survive for a considerable time after the first shock. However, survival is clearly influenced by the degree of left ventricular dysfunction and, in addition to ICD therapy, aggressive treatment of heart failure is necessary.

Pigment-protein architecture in the light-harvesting antenna complexes of purple bacteria: does the crystal structure reflect the native pigment-protein arrangement?

Structural analysis of crystallized peripheral (LH2) and core antenna complexes (LH1) of purple bacteria has revealed circular aggregates of high rotational symmetry (C8, C9 and C16, respectively). Quantum-chemical calculations indicate that in particular the waterwheel-like arrangements of pigments should show characteristic structure-sensitive spectroscopic behavior in the near infrared absorption region. Laser-spectroscopic data obtained with non-crystallized, isolated LH2 of Rhodospirillum molischianum are in line with a highly symmetric (C8) circular aggregate, but deviations have been found for LH2 of Rhodobacter sphaeroides and Rhodopseudomonas acidophila. For both the latter, C-shaped incomplete circular aggregates (as seen only recently in electron micrographs of crystallized LH1-reaction center complexes) may be a suitable preliminary model.

Chromosomal mapping of genes for epilepsy in NER: a rat strain with tonic-clonic seizures.

PURPOSE: NER is a mutant rat strain that exhibits spontaneous tonic-clonic convulsions accompanied by epileptic discharges on ictal EEG and serves as a model for generalized tonic-clonic seizures in humans. Our previous experiments have suggested that a major autosomal recessive gene and several minor genes regulate the inheritance of tonic-clonic seizures in NER. The purpose of this study was to confirm the mode of inheritance and to locate the causative genes for epilepsy in NER on the rat genetic map. METHODS: We developed F1 hybrid (F1) and reciprocal back-cross progenies of NER with a seizure-resistant strain, F344, and evaluated their seizure susceptibility under tossing-stimulated and nonstimulated conditions. Backcross animals were genotyped using simple sequence length polymorphism markers for polymerase chain reactions. Linkage between seizure susceptibility and marker loci was analyzed by chi2 statistic tests and by the computer programs MAPMAKER/EXP and MAPMAKER/QTL. RESULTS: Under tossing-stimulating conditions, tonic-clonic seizures were provoked in 90% of NER and 66% of (F1 x NER) backcross animals, but no seizures occurred in the F344, F1, or (F1 x F344) backcross animals. Routine monitoring of nonstimulated animals revealed spontaneous tonic-clonic convulsions in 100% of NER and 64.2% of (F1 x NER) backcross animals, but no seizures in F344 or F1. Gender effect on seizure susceptibility was negligible in (F1 x NER) backcross in both conditions. Preliminary genome-wide scanning and subsequent precise location of the causative genes revealed seizure susceptibility loci, designated Ner1 and Ner2, on rat chromosomes 1 and 3, respectively. CONCLUSIONS: Ner1 is a locus that controls the inheritance of spontaneous tonic-clonic seizures in an autosomal recessive mode, whereas Ner2 affects the occurrence of tossing-induced seizures. Orthologous genes in the vicinity of these loci may be related to epileptogenesis in other species, including humans.

An integrated rat genome map based on genetic and cytogenetic data.

In this study we combined three major rat genome maps, by adding 66 markers to the Kyoto Laboratory Animal Science map (KLAS map), and constructed an integrated map. The resultant integrated map consists of 5,682 redundant markers, spanning a genetic length of 2,028 cM. Eighty genetic markers were anchored to the cytogenetic map, fixing all the genetic maps in the physically correct orientation. This map encapsulates the progress in rat mapping studies in past years and offers useful information for QTL analysis. The map figures are available at http:/(/)www.anim.med.kyoto-u.ac.jp/.

Metabolic transformation of the brassinosteroid 24-epi-castasterone by the cockroach Periplaneta americana.

After feeding of 24-epi-castasterone to the cockroach Periplaneta americana an organ-specific epimerization of the brassinosteroid to 2,24-diepi-castasterone could be detected in female insects. The metabolite being observed only in the ovaries and not in the testes of the insect was identified by GC/MS in comparison with a synthesized authentic sample. Contrary, 24-epi-brassinolide is not metabolized in the sexual organs of Periplaneta americana. This is the first evidence of a metabolic transformation of a brassinosteroid in insects.

[Cerebral perfusion in newborn infants treated with high-frequency oscillation ventilation]. / Zerebrale Perfusion bei Neugeborenen unter Hochfrequenz-Oszillations-Beatmung.

UNLABELLED: The effects of high-frequency oscillatory ventilation (HFOV) on hemodynamic parameters have been shown in animal as well as in clinical studies. In a further study we could demonstrate, that after change of a conventional positive pressure ventilation (CMV) to HFOV flow velocities in cerebral arteries decreased. In the following we added to the dopplerflow method the continuous examination of cerebral oxygenation with near infrared spectroscopy (NIRS). PATIENTS: 19 measurements were prospectively conducted in 18 neonates. The infants were mechanically ventilated with HFOV and were in a stable condition. METHODS: Before change from HFOV to CMV doppler signals of the anterior cerebral artery were measured. We repeated this at the end of the study in each patient. NIRS-optodes were placed on the front and the os parietale of each infant. After stabilization of the system we changed from HFOV to CMV without disconnection of the patient from the machine. PCO2 was registered continuously via a transcutaneous probe, as well as oxygen saturation via pulse oxymetry. Statistical analysis was performed with Wilcoxon test. RESULTS: There were no significant changes of doppler-signals during the study (median vs. 25 cm/s (+/- 6) during HFOV, 28 cm/s (+/-/) during CMV). The parameter of NIRS, oxygenated hemoglobin HbO [-1.5 U (+/- 22.78)] at 15 minutes after change), reduced hemoglobin HbR [-1.17 U (+/- 5.26)] and total hemoglobin HbT [-2.68 U (+/- 18.7)] remained stable during the change from HFOV to CMV, too. In five infants there was a decrease and in two an increase of HbO 15 minutes after change, which correlated with decrease or increase of pCO2. CONCLUSION: In a combined measurement of dopplerflow and NIRS we found no special effect of HFOV on cerebral hemodynamics comparing with CMV. Changes of cerebral oxygenation in NIRS correlated with pCO2.

Inactivation of brassinosteroid biological activity by a salicylate-inducible steroid sulfotransferase from Brassica napus.

Recent discoveries from brassinosteroid-deficient mutants led to the recognition that plants, like animals, use steroids to regulate their growth and development. We describe the characterization of one member of a Brassica napus sulfotransferase gene family coding for an enzyme that catalyzes the O-sulfonation of brassinosteroids and of mammalian estrogenic steroids. The enzyme is specific for the hydroxyl group at position 22 of brassinosteroids with a preference for 24-epicathasterone, an intermediate in the biosynthesis of 24-epibrassinolide. Enzymatic sulfonation of 24-epibrassinolide abolishes its biological activity in the bean second internode bioassay. This mechanism of hormone inactivation by sulfonation is similar to the modulation of estrogen biological activity observed in mammals. Furthermore, the expression of the B. napus steroid sulfotransferase genes was found to be induced by salicylic acid, a signal molecule in the plant defense response. This pattern of expression suggests that, in addition to an increased synthesis of proteins having antimicrobial properties, plants respond to pathogen infection by modulating steroid-dependent growth and developmental processes.

[What is the value of determining walking distance in peripheral arterial occlusive disease on the treadmill and in daily life? Prospective correlation study]. / Was bedeuten Gehstreckenbestimmungen bei peripherer arterieller Verschlusskrankheit auf dem Laufband und im Alltag? Prospektive Korrelationsuntersuchung.

AIM: To investigate the question whether and how walking distances measured under standardized conditions on the treadmill and on the level correlate with the patients walking ability under everyday conditions. PATIENTS AND METHOD: In 49 patients (33 men, 16 women, age 34 to 84 years) with Fontaine Stage II peripheral arterial occlusive disease the pain-free and absolute walking distance on the treadmill were measured under standardized conditions, as also the walking distance on the level at freely selected speed. RESULT: It was found that the pain-free walking distance under everyday conditions was about 2 to 3 times longer than that measured under standardized conditions on the treadmill.