Implementation of a uniform nationwide medical licensing examination in general practice. A feasibility study.

Objective: A competency-based training of medical students that is adapted to the realities of care is required internationally and is being intended in Germany with the Master Plan for Medical Studies 2020. In order to test these competencies, the German National Institute for state examinations in Medicine, Pharmacy and Psychotherapy (IMPP) has developed a concept for the redesign of the final part of the medical licensing examination in Germany. It focuses on general and interprofessional healthcare in the examination with outpatients. The aim of this work is to assess the feasibility of the new final examination on the basis of pilot examinations in family practices and to derive further steps for the national implementation. Methods: Fourteen medical students in their internship year completed a full examination with patients aged 42 to 84 years. Examiners evaluated the examination performance using standardised evaluation forms. Feasibility was qualitatively assessed in terms of compliance with content and time limits, examination results, patient reflections, and implementation in the practice. Results: Students were able to complete all tasks within the given time frame. Based on the evaluation forms, the examiners assessed the performance of the students. Patients appreciated the structured course of the examination in the familiar location of their family practice. For the nationwide implementation of the examination, 2,500 examination practices are required for about 10,000 examinees per year. Four students can then be examined on two days per year in each practice. Conclusions: Oral-practical examinations with outpatients in general medical practices can be carried out successfully throughout the nation. An implementation of the examinations throughout Germany requires that medical studies are restructured and that this new curriculum is implemented as intended by the Master Plan for Medical Studies 2020. Furthermore, training and remuneration of examiners together with a legal framework for the new examination must be established.

Dual redundant sequencing strategy: Full-length gene characterisation of 1056 novel and confirmatory HLA alleles.

The high-throughput department of DKMS Life Science Lab encounters novel human leukocyte antigen (HLA) alleles on a daily basis. To characterise these alleles, we have developed a system to sequence the whole gene from 5'- to 3'-UTR for the HLA loci A, B, C, DQB1 and DPB1 for submission to the European Molecular Biology Laboratory - European Nucleotide Archive (EMBL-ENA) and the IPD-IMGT/HLA Database. Our workflow is based on a dual redundant sequencing strategy. Using shotgun sequencing on an Illumina MiSeq instrument and single molecule real-time (SMRT) sequencing on a PacBio RS II instrument, we are able to achieve highly accurate HLA full-length consensus sequences. Remaining conflicts are resolved using the R package DR2S (Dual Redundant Reference Sequencing). Given the relatively high throughput of this strategy, we have developed the semi-automated web service TypeLoader, to aid in the submission of sequences to the EMBL-ENA and the IPD-IMGT/HLA Database. In the IPD-IMGT/HLA Database release 3.24.0 (April 2016; prior to the submission of the sequences described here), only 5.2% of all known HLA alleles have been fully characterised together with intronic and UTR sequences. So far, we have applied our strategy to characterise and submit 1056 HLA alleles, thereby more than doubling the number of fully characterised alleles. Given the increasing application of next generation sequencing (NGS) for full gene characterisation in clinical practice, extending the HLA database concomitantly is highly desirable. Therefore, we propose this dual redundant sequencing strategy as a workflow for submission of novel full-length alleles and characterisation of sequences that are as yet incomplete. This would help to mitigate the predominance of partially known alleles in the database.

A multi-site study using high-resolution HLA genotyping by next generation sequencing.

The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

The inhibitory neural circuitry as target of antiepileptic drugs.

Impairments and defects in the inhibitory neurotransmission in the CNS can contribute to various seizure disorders, i.e., gamma-aminobutyric acid (GABA) and glycine as the main inhibitory neurotransmitters in the brain play a crucial role in some forms of epilepsy. Recent advances in deciphering the molecular basis of the GABAergic and glycinergic systems has been achieved by means of cloning techniques and gene targeting strategies in animals, contributing to the understanding of drug action. As well, several anticonvulsive substances emerged which target key molecules of the inhibitory systems. Employment of recombinant expression systems, including, but not restricted to the inhibitory circuitry, will further facilitate drug screening and rational approaches to design novel specific antiepileptic drugs, which act highly efficiently to prevent or reduce generation and spread of seizures.

Binding of human lipoproteins (low, very low, high density lipoproteins) to recombinant envelope proteins of hepatitis C virus.

Heterogeneities in the density of hepatitis C virus (HCV)-RNA-carrying material from human sera (1.03-1.20 g/ml) are partially due to the binding of lipoproteins [low density (LDL), very low density (VLDL), high density (HDL) lipoproteins] and immunoglobulins. In this study we demonstrate the binding of recombinant HCV envelope protein (El/E2) to human LDL, VLDL and HDL on a molecular basis. The binding of lipoproteins was restricted to the middle part of the El gene product (amino acids 222-336) and the C-terminal part of the E2 protein (amino acids 523-809). Lipoproteins did not bind to recombinant HCV core protein.

Molecular epidemiology of an outbreak of HCV in a hemodialysis unit: direct sequencing of HCV-HVR1 as an appropriate tool for phylogenetic analysis.

Infection with hepatitis C virus (HCV) is still a serious problem in hemodialysis patients, despite screening of blood products for anti-HCV antibodies. The prevalence of HCV in HD patients is between 15% and 30% in Germany. We report the molecular epidemiology of an HCV outbreak in a hemodialysis unit in 1997 is determined. HCV hypervariable region 1 (HVR1) was amplified from serum samples of 19 patients by polymerase chain reaction (PCR) and sequenced directly. In addition, HCV isolates from 3 of these 19 patients were cloned and sequenced. 14 newly infected patients and two patients, who had been infected for several years had very closely related HCV isolates. Unrelated HCV isolates as well as sequences obtained from an HCV outbreak in a plasmapheresis center were found in different, distantly related branches. These findings provide strong evidence for nosocomial transmission of the virus, despite following strict general hygiene precautions. The production of anti-HCV antibody was delayed significantly or seroconversion did not occur at all during the period of observation in 8 out of 14 newly infected HCV RNA positive patients. Close-meshed reverse transcription-polymerase chain reaction (RT-PCR) analyses on apparently non infected patients within hemodialysis units and upon admission of new patients is strongly recommended for the early detection and prevention of outbreaks of HCV.

Low density lipoprotein receptor as a candidate receptor for hepatitis C virus.

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 microg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS-7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL-receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target.

Characterization of unusual escape variants of hepatitis B virus isolated from a hepatitis B surface antigen-negative subject.

Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee's sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.

[Endogenous digitalis-like factor in liver cirrhosis and cholestasis]. / Endogenous digitalis-like factor bei Leberzirrhose und Cholestase.

Endogenous digitalis-like factor (EDLF), an inhibitor of membrane Na+/K(+)-ATPase, is discussed to be involved in the pathogenesis of cirrhogenic portal hypertension, ascites formation and development of functional hepatorenal failure. Therefore, we investigated the serum content of this mediator in patients with liver cirrhosis Child-Pugh stage A, B, and C (n = 27) by means of enzyme immunoassay with a specific digoxin antibody. Furthermore, a correlation analysis was performed in order to find out correlations between signs of cell injury, cholestasis, synthetic cell function, ascites formation, and hepatorenal failure. Our results demonstrate that EDLF is significantly elevated in Child C cirrhosis (0.61 +/- 0.15 ng/ml) in comparison to Child A cirrhosis (0.013 +/- 0.2 ng/ml) and is also higher than in Child B cirrhosis (0.23 +/- 0.25 ng/ml). In patients without ascites EDLF (0.056 +/- 0.19 ng/ml) differs significantly from that of patients with non-complicated ascites (0.156 +/- 0.176 ng/ml) and from that of patients with therapy refractory ascites (0.66 +/- 0.17 ng/ml) or hepatorenal failure (1.56 ng/ml). There are no correlations between EDLF and renal function. Significant correlations were demonstrated for cholestasis (serum bilirubin), synthesis function (serum protein, Quick's value, cholinesterase, fibrinogen, albumin), and the degree of portasystemic encephalopathy (number connection test). We conclude that EDLF may act as a mediator in the process of progressive portal hypertension and its complications due to cirrhosis. This process of progression is caused by the inhibition of Na+/K(+)-ATPase, vasoconstriction, and endothelin secretion.

Hepatic aldehyde dehydrogenase (E.C. 1.2.1.3.) isoenzymes and malondialdehyde levels in alcoholic liver disease.

To clarify the controversially discussed behaviour of hepatic ALDH isozymes in different liver diseases, 98 liver biopsy specimens were investigated as to their total ALDH and their isozyme activity. Simultaneously the malondialdehyde (MDA) content was measured in both biopsy samples and serum. The results show a significant decrease of the "low Km" isozyme E-2 activity depending on the severity of the liver disease. The tendency for the E-2 activity to decrease was greater in alcoholic than in nonalcoholic liver diseases. Although in all our diagnosis groups elevated MDA concentrations could be measured in both serum and liver tissue, a direct specific inhibitory effect on the E-2 activity could not be confirmed. The change from the low Km pathway to the high Km pathway of aldehyde detoxification, however, may contribute to the progress in alcoholic liver disease.

[Aldehyde-dehydrogenase from human liver: Purification, partial characterization and influence of malondialdehyde]. / Aldehyddehydrogenase aus Humanleber: Isolierung, partielle Charakterisierung und Einfluss von Malondialdehyd.

A procedure to separate the isozymes E1 and E2 of aldehyde dehydrogenase (ALDH, aldehyde: NAD oxidoreductase, EC 1.2.1.3) from human liver, avoiding 5'-AMP-Sepharose, was worked out. It results in fairly purified isozymes. The isoelectric points could be re-estimated for E1 at pH 5.21 +/- 0.04 and for E2 at pH 4.90 +/- 0.05. The pH-optimum of both the isozymes is dependent on the buffer used, the best buffer being sodium pyrophosphate/HCI. In this buffer the enzyme activity is also dependent on ionic strength. Malondialdehyde (MDA), at concentrations which are found in patient serum, is converted by the ALDH. The Km-values of this reaction are 1.71 mmol/l for MDA and 0.19 mmol/l for NAD (E1) and 0.21 mmol/l for MDA and 0.24 mmol/l for NAD (E2) resp. The highest rates of conversion are reached at concentrations of 0.08 mmol/l MDA (E1) and 0.16 mmol/l MDA (E2). At higher concentrations of MDA substrate excess inhibition is observed (Kss = 0.17 mmol/l for E1 and 0.20 mmol/l for E2).

[Diagnostic value of molecular weight related urinary protein electrophoresis in normal total protein excretion]. / Diagnostische Wertigkeit der molekulargewichtsbezogenen Harnproteinelektrophorese bei normaler Gesamteiweissausscheidung.

The electrophoresis of urinary proteins related to the molecular weight in SDS-polyacrylamide gels stood the test for the clarification of the cause of proteinurias. Also in normal excretion of the total protein the analysis of protein is reasonable after concentration of the urine. In the electrophoretic separation of 104 urinary specimens with a maximum protein content of 0.15 g/l in 44 cases a normal protein pattern was found. In 60 cases pathological patterns occurred which in their protein composition greatly corresponded to the damage within the nephron to be expected in the different clinical diagnoses. The results show that the SDS-polyacryl amide gel electrophoresis is suitable for the early recognition and differentiation of injuries of the kidneys.

A low molecular weight alanine aminopeptidase in urine. Biochemical equivalent of tubular atrophy?

Using agarose gel electrophoresis, a faster moving alanine aminopeptidase (EC 3.4.11.2) has been demonstrated in the urine from cases of Fanconi syndrome, endemic (Balkan) nephropathy and advanced renal insufficiency. The enzyme was partially purified and its properties (isoelectric point, molecular weight, substrate specificity, influence of metal ions, Michaelis constant, antigenic behavior) were compared with those of normal kidney alanine aminopeptidase. Isoelectric points and antigenic properties are identical, but the molecular weights differ by a factor of about 2. Therefore, the greater electrophoretic mobility is due to the smaller size of the atypical enzyme.

Nature of the multiple forms of alanine aminopeptidase.

Alanine aminopeptidase (particle-bound aminopeptidase, EC 3.4.11.2) isolated in a highly purified state from human liver, kidney, and pancreas were investigated with regard to their different electrophoretic mobilities. While the molecular weights are identical, different isoelectric points were found. The results of carbohydrate analysis point to N-acetylneuraminic acid as the main factor that causes the differences in electrophoretic behaviour of the alanine aminopeptidases investigated.