Validation and educational impact study of the NANEP high-fidelity simulation model for open preperitoneal mesh repair of umbilical hernia.

OBJECTIVE: The aim of the study was to develop, validate and analyze the educational impact of a high-fidelity simulation model for open preperitoneal mesh repair of an umbilical hernia. The number of surgical simulators available for training residents is limited. Primary for ethical reasons and secondary for the emerging pay-per-quality policies, practicing-on simulators rather than patients is considered gold standard. Validated full-procedural surgical models will become more and more important in training residents. Such models may assure that evidence-based standards regarding technical aspects of the procedures become integral part of the curriculum. Furthermore, they can be employed as a quality control of residents' skills (Fonseca et al. in J Surg Educ 70:129-137, 2013). METHODS: In a repeated measures design, medical students, residents in their last year of training and attending surgeons performed an open preperitoneal mesh repair on the NANEP model [NANEP stands for the German acronym Nabelhernien-Netzimplatation-Präperitonal (English: Umbilical hernia mesh implantation preperitoneal)]. Subjects were categorized as "Beginners" (internship students) or "Experts" (residents and surgeons). Content validity was analyzed by criteria of subject-matter-experts. Blinded raters assessed surgical skills by means of the Competency Assessment Tool (CAT) using the online platform "CATLIVE". Differential validity was measured by group differences. Proficiency gain was analyzed by monitoring the learning curve (Gallagher et al. in Ann Surg 241:364-372, 2005). Post-operative examination of the simulators shed light on criterion validity. RESULTS: The NANEP model-proofed content and construct-valid significant Bonferroni-corrected differences were found between beginners and experts (p < 0.05). Beginners showed a significant learning increase from the first to the second surgery (p < 0.05). Post-operative examination data confirmed criterion validity. CONCLUSION: The NANEP model is an inexpensive, simple and efficient simulation model. It has highly realistic features, it has been shown to be of high-fidelity, full-procedural and benchtop-model. The NANEP model meets the main needs of surgical educational courses at the beginning of residency.

Incisional hernia repair in a high-fidelity silicone model for open retro-muscular mesh implantation with preparation of the fatty triangle: validation and educational impact study.

BACKGROUND: Incisional hernia repair requires detailed anatomic knowledge. Regarding median subxiphoidal hernias, the proper preparation of the fatty triangle is challenging. To foster proficiency-based training, a cost-efficient model for open median retromuscular mesh repair resembling the human body was developed, including the main anatomical structures related to the procedure. The aim is to create and validate a high-fidelity model on open retromuscular mesh repair suitable for "training before doing". MATERIALS AND METHODS: Different types of fabrics for imitation of connective tissue and 2-component silicones were used to construct the incisional hernia model. Sample size for validation of the model was determined by a triangular testing approach. Operations from six beginners and six experts were assessed by three blinded-raters. Reliability and construct-validity were evaluated on a behaviorally anchored rating scale (highest score: 4) for the criteria: "instrument use", "tissue handling", "near misses and errors", and "end-product quality". RESULTS: The model authentically mimicked an open median retromuscular mesh repair. Participants considered the procedure realistic. Reliability was excellent, ranging from 0.811 to 0.974 for "end-product quality", and "tissue handling" respectively. Construct-validity was confirmed with experts significantly outperforming beginners in the "use of instruments" (Mbeg. = 2.33, Mexp. = 3.94, p < 0.001), "tissue handling" (Mbeg. = 2.11, Mexp. = 3.72, p < 0.001), "near misses and errors" (Mbeg. = 2.67, Mexp. = 3.67, p < 0.001), and "end-product quality" (Mbeg. = 2.78, Mexp. = 3.72, p < 0.001). Criterion-validity revealed a paradox effect: beginners performed significantly better than experts (p < 0.05) when preparing the fatty triangle. CONCLUSIONS: The model covers all relevant aspects involved in median-open retromuscular incisional hernia mesh repair. Performance differences between beginners and experts confirm construct-validity and thereby realism of the model. It enables to efficiently improve and practice technical skills of the demanding surgery.

A randomized trial of safety, pharmacokinetics and pharmacodynamics of concizumab in people with hemophilia A.

Essentials explorer™3 was a double-blinded, multiple-dose escalation trial of subcutaneous concizumab. A pharmacodynamic relationship for unbound TFPI and thrombin generation was confirmed. No serious adverse events and no anti-drug antibodies were observed. explorer™3 data support further clinical development of concizumab in people with hemophilia. SUMMARY: Background Concizumab is a humanized mAb targeting tissue factor pathway inhibitor (TFPI), leading to enhanced thrombin generation (TG) potential. explorer™3 (NCT02490787) was a phase 1b, double-blind, multiple-dose escalation trial of subcutaneous concizumab in people with severe hemophilia A without inhibitors. Objectives The primary objective was to evaluate safety. Assessments of pharmacokinetics, pharmacodynamics and subcutaneous concizumab immunogenicity were secondary objectives. Patients/Methods Adverse events (AEs), clinical assessments and bleeding episodes were recorded. Plasma concizumab levels and unbound TFPI levels were measured with ELISAs; residual TFPI activity was measured with a chromogenic assay. Standardized assays were used to assess TG, D-dimer and prothrombin fragment 1 + 2 (F1 + 2 ) levels. explorer™3 was completed after investigation of three dose cohorts (0.25, 0.5 and 0.8 mg kg-1 , once every 4 days) had been completed. Twenty-four patients received 12 doses of concizumab or placebo in a 3 : 1 randomization over a 42-day period. Results No serious AEs and no anti-drug antibodies were observed. Fifty-four mild and two moderate AEs were observed in 19 patients. Concizumab exposure increased with dose in a non-linear manner, confirming target-mediated drug disposition. D-dimer and F1 + 2 levels were increased mostly in the highest dose cohort, in line with previous observations. The level of unbound TFPI decreased in a dose-dependent manner, and was accompanied by a residual TFPI activity decrease and an increase in peak TG. Although the trial was not powered to evaluate efficacy, a trend towards lower bleeding rates was observed in patients in the highest dose cohort. Conclusion explorer™3 data support further clinical development of concizumab for use in people with hemophilia, with or without inhibitors.

Concizumab, an anti-tissue factor pathway inhibitor antibody, induces increased thrombin generation in plasma from haemophilia patients and healthy subjects measured by the thrombin generation assay.

AIMS: Concizumab, a humanized monoclonal antibody against tissue factor pathway inhibitor (TFPI), is being developed as a subcutaneously (s.c.) administered treatment for haemophilia. It demonstrated a concentration-dependent procoagulant effect in functional TFPI assays; however, global haemostatic assays, such as the thrombin generation assay (TGA), offer a more complete picture of coagulation. We investigated how concizumab affects thrombin generation following ex vivo spiking in plasma from haemophilia patients using the TGA, and if the assay can detect the effect of multiple s.c. concizumab doses in healthy subjects. METHODS: For the ex vivo spiking study, platelet-poor plasma (PPP) from 18 patients with severe haemophilia was spiked with 0.001-500 nm concizumab. For the multiple-dosing study, four healthy males received concizumab 250 µg kg-1 s.c. every other day for eight doses; blood was collected before and after dosing and processed into PPP. In both studies, thrombin generation was measured using a Calibrated Automated Thrombogram® system with 1 pm tissue factor. RESULTS: In spiked samples from haemophilia patients, peak thrombin and endogenous thrombin potential (ETP) increased concentration dependently, reaching near-normal levels at concizumab concentrations >10 nm. Repeated s.c. doses of concizumab in healthy subjects increased both peak thrombin and ETP; these effects were sustained throughout the dosing interval. CONCLUSIONS: Thrombin generation assay demonstrated increased thrombin generation with concizumab after ex vivo spiking of haemophilia plasma and multiple s.c. doses in healthy subjects, supporting both the utility of the TGA in evaluating concizumab treatment and the potential of s.c. concizumab as a novel haemophilia therapy.

Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles.

X-linked juvenile retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in the RS1 gene encoding a protein termed retinoschisin. The disease is an excellent candidate for gene replacement therapy as the majority of mutations have been shown to lead to a complete deficiency of the secreted protein in the retinal structures. In this work, we have studied the ability of non-viral vectors based on solid lipid nanoparticles (SLN) to induce the expression of retinoschisin in photoreceptors (PR) after intravitreal administration to Rs1h-deficient mice. We designed two vectors prepared with SLN, protamine, and dextran (DX) or hyaluronic acid (HA), bearing a plasmid containing the human RS1 gene under the control of the murin opsin promoter (mOPS). In vitro, the nanocarriers were able to induce the expression of retinoschisin in a PR cell line. After injection into the murine vitreous, the formulation prepared with HA induced a higher transfection level in PR than the formulation prepared with DX. Moreover, the level of retinoschisin in the inner nuclear layer (INL), where bipolar cells are located, was also higher. Two weeks after vitreal administration into Rs1h-deficient mice, both formulations showed significant improvement of the retinal structure by inducing a decrease of cavities and PR loss, and an increase of retinal and outer nuclear layer (ONL) thickness. HA-SLN resulted in a significant higher increase in the thickness of both retina and ONL, which can be explained by the higher transfection level of PR. In conclusion, we have shown the structural improvement of the retina of Rs1h-deficient mice with PR specific expression of the RS1 gene driven by the specific promoter mOPS, after successful delivery via SLN-based non-viral vectors.

Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model.

X-linked juvenile retinoschisis (XLRS), which results from mutations in the gene RS1 that encodes the protein retinoschisin, is a retinal degenerative disease affecting between 1/5000 and 1/25,000 people worldwide. Currently, there is no cure for this disease and the treatment is based on the application of low-vision aids. The aim of the present work was the in vitro and in vivo evaluation of two different non-viral vectors based on solid lipid nanoparticles (SLNs), protamine and two anionic polysaccharides, hyaluronic acid (HA) or dextran (DX), for the treatment of XLRS. First, the vectors containing a plasmid which encodes both the reporter green fluorescent protein (GFP) and the therapeutic protein retinoschisin, under the control of CMV promoters, were characterized in vitro. Then, the vectors were subretinally or intravitreally administrated to C57BL/6 wild type mice. One week later, GFP was detected in all treated mice and in all retinal layers except in the Outer Nuclear Layer (ONL) and the Inner Nuclear Layer (INL), regardless of the administration route and the vector employed. Finally, two weeks after subretinal or intravitreal injection to Rs1h-deficient mice, GFP and retinoschisin expression was detected in all retinal layers, except in the ONL, which was maintained for at least two months after subretinal administration. The structural analysis of the treated Rs1h-deficient eyes showed a partial recovery of the retina related to the production of retinoschisin. This work shows for the first time a successful RS1 gene transfer to Rs1h-deficient animals using non-viral nanocarriers, with promising results that point to non-viral gene therapy as a feasible future therapeutic tool for retinal disorders.

Safety and pharmacokinetics of anti-TFPI antibody (concizumab) in healthy volunteers and patients with hemophilia: a randomized first human dose trial.

BACKGROUND: Prophylaxis with either intravenous (i.v.) factor VIII (FVIII) or FIX is the gold standard of care for patients with severe hemophilia. A monoclonal antibody (concizumab) targeting tissue factor pathway inhibitor (TFPI) that can be administered subcutaneously (s.c.) has the potential to alter current concepts of prophylaxis in hemophilia. OBJECTIVES: To evaluate the safety and describe the pharmacokinetics and pharmacodynamics of single-dose concizumab in healthy volunteers and patients with hemophilia A or B. METHODS: In this first human dose, phase 1, multicenter, randomized, double-blind, placebo-controlled trial escalating single i.v. (0.5-9000 µg kg(-1) ) or s.c. (50-3000 µg kg(-1) ) doses of concizumab were administered to healthy volunteers (n = 28) and hemophilia patients (n = 24). RESULTS: Concizumab had a favorable safety profile after single i.v. or s.c. administration. There were no serious adverse events and no anti-concizumab antibodies. No clinically relevant changes in platelets, prothrombin time, activated partial thromboplastin time, fibrinogen, or antithrombin were found. A dose-dependent procoagulant effect of concizumab was seen as increased levels of D-dimers and prothrombin fragment 1 + 2. Nonlinear pharmacokinetics of concizumab was observed due to target-mediated clearance. A maximum mean AUC0-∞ of 33 960 h µg mL(-1) and a maximum mean concentration of 247 µg mL(-1) was measured at the highest dose. CONCLUSIONS: Concizumab showed a favorable safety profile after i.v. or s.c. administration and nonlinear pharmacokinetics was observed due to target-mediated clearance. A concentration-dependent procoagulant effect of concizumab was observed, supporting further study into the potential use of s.c. concizumab for hemophilia treatment.

Canine scent detection in the diagnosis of lung cancer: revisiting a puzzling phenomenon.

Patient prognosis in lung cancer largely depends on early diagnosis. The exhaled breath of patients may represent the ideal specimen for future lung cancer screening. However, the clinical applicability of current diagnostic sensor technologies based on signal pattern analysis remains incalculable due to their inability to identify a clear target. To test the robustness of the presence of a so far unknown volatile organic compound in the breath of patients with lung cancer, sniffer dogs were applied. Exhalation samples of 220 volunteers (healthy individuals, confirmed lung cancer or chronic obstructive pulmonary disease (COPD)) were presented to sniffer dogs following a rigid scientific protocol. Patient history, drug administration and clinicopathological data were analysed to identify potential bias or confounders. Lung cancer was identified with an overall sensitivity of 71% and a specificity of 93%. Lung cancer detection was independent from COPD and the presence of tobacco smoke and food odours. Logistic regression identified two drugs as potential confounders. It must be assumed that a robust and specific volatile organic compound (or pattern) is present in the breath of patients with lung cancer. Additional research efforts are required to overcome the current technical limitations of electronic sensor technologies to engineer a clinically applicable screening tool.

Safety and pharmacokinetics of subcutaneously administered recombinant activated factor VII (rFVIIa).

BACKGROUND: Recombinant activated factor VIIa (rFVIIa) is used to treat bleeds in hemophilia patients with inhibitors. A subcutaneous formulation could potentially improve its half-life and make it suitable for prophylactic treatment. OBJECTIVES: A study was conducted to determine the safety of subcutaneously administered rFVIIa in patients with hemophilia and the pharmacokinetic profile (including bioavailability). PATIENTS/METHODS: This was a multicenter, open-label, cross-over comparison of single doses of intravenous rFVIIa 90µgkg(-1) and a new formulation of rFVIIa for subcutaneous injection at dose levels of 45, 90, 180, 270 and 360µgkg(-1) . Sixty subjects (12 per dose cohort) with hemophilia A or B were enrolled. RESULTS: Subcutaneously administered rFVIIa showed lower mean peak plasma concentrations and prolonged FVII activity (C(max) , 0.44-5.16IU mL(-1) [across doses]; t(1/2) , 12.4h; t(max) , 5.6h) compared with intravenously administered rFVIIa (C(max) , 51.7IUmL(-1) ; t(1/2) , 2.7h; t(max) , <10min). The absolute bioavailability of subcutaneous rFVIIa ranged from 21.1 to 30.1% across dose levels. Dose proportionality was observed within a 2-fold dose increase but not across the full dose range. No thromboembolic events, drug-related serious adverse events, severe injection-site reactions or neutralizing antibodies were reported (primary endpoint). Mild and moderate injection-site reactions were more frequent with subcutaneous than with intravenous injections. CONCLUSION: This phase I clinical trial did not identify safety concerns of prolonged exposure to rFVIIa administered subcutaneously in single doses to hemophilia patients.

The immune system in healthy adults and patients with atopic dermatitis seems to be affected differently by a probiotic intervention.

BACKGROUND: Probiotic bacteria are proposed to alleviate atopic dermatitis (AD) in infants. There are few indications about the effect of probiotics on AD in adults. OBJECTIVE: The purpose of this study was to elucidate the influence of a probiotic drink containing a combination of the probiotics Lactobacillus paracasei Lpc-37, Lactobacillus acidophilus 74-2 and Bifidobacterium animalis subsp. lactis DGCC 420 (B. lactis 420) in healthy volunteers and in patients with AD on clinical and immunological parameters and their detection in feces. METHODS: A double-blind, placebo-controlled, randomized cross-over study was conducted in 15 healthy adults and 15 patients with AD. The probiotic product or placebo was given over 8 weeks. A 2-week washout period was interconnected before the intervention was crossed. At the end of each period, blood and stool samples were collected. In patients, the severity of AD was evaluated using the Scoring of Atopic Dermatitis (SCORAD). RESULTS: L. paracasei and B. lactis were recovered in high numbers in feces after supplementation, whereas L. acidophilus marginally increased. In patients, the SCORAD tended to decrease by 15.5% (P=0.081). Major lymphocyte subsets were not affected by the probiotic intervention. However, CD57(+) increased significantly (P=0.034) in healthy subjects after probiotic intake and was not changed in patients, whereas CD4(+)CD54(+) decreased significantly (P=0.031) in patients with AD and remained uninfluenced in healthy subjects. The expression of CD4(+)CD25(+) T cells was similar in healthy subjects and AD patients. The phagocytic activity of monocytes and granulocytes was significantly increased in healthy subjects after probiotic intervention (P=0.014). CONCLUSION: L. paracasei Lpc-37 and B. lactis 420 are able to colonize the intestine transiently. This study reveals that the probiotics differently modulate peripheral immune parameters in healthy subjects and patients with AD.

Lactobacillus acidophilus 74-2 and Bifidobacterium animalis subsp lactis DGCC 420 modulate unspecific cellular immune response in healthy adults.

OBJECTIVE: It was determined whether a combination of Lactobacillus acidophilus (L. acidophilus) 74-2 and Bifidobacterium animalis subsp lactis DGCC 420 (B. lactis 420) affect the faecal microbiota as well as immunological parameters and blood lipids in healthy adults. DESIGN: A placebo-controlled, double-blinded, randomized crossover trial was conducted. SUBJECTS: Twenty-six healthy volunteers (mean age 25 years) were recruited by advertising in academical buildings. All of them completed the study. METHODS: After 3-week run-in period, half of the volunteers consumed 300 g/day of yoghurt supplement containing probiotic strains L. acidophilus 74-2 and B. lactis 420, and the other half received the placebo product for a period of 5 weeks. The two groups were crossed during the following 5-week period. Blood and faecal samples were collected at the end of each period. The faecal content of probiotic bacteria, faecal short-chain fatty acids (SCFA), serum lipids and plasma immune system biomarkers were evaluated. RESULTS: Faecal proportions of L. acidophilus and of B. lactis increased significantly from 0.02 to 0.19 and 0.4 to 1.4% (P<0.05), respectively. Percentages of granulocytes and monocytes showing phagocytic activity were significantly elevated from 92 to 95% during probiotic intervention, whereas their oxidative burst activity and specific immune parameters remained unaffected. Fecal SCFA and serum cholesterol levels were not influenced by the probiotics. However, serum concentrations of triacylglyceroles decreased significantly by 11.6% (P<0.05) in the probiotic supplementation period. CONCLUSIONS: L. acidophilus and B. lactis were recovered in faeces in significantly elevated numbers after supplementation. They are able to modulate unspecific cellular immune response indicated by the increased phagocytic activity.

Gravitational biology within the German Space Program: goals, achievements, and perspectives.

Gravity plays an important role for the evolution, orientation and development of organisms. Most of us, however, tend to overlook its importance because--due to its constant presence from the beginning of evolution some 4 billion years ago--this environmental parameter is almost hardwired into our interpretation of reality. This negligence of gravity is the more surprising as we all have our strong fights with this factor, especially during the very early and again during the late phases of our lives. On the other hand, scientists have been fascinated to observe the effects of gravity especially on plants and microorganisms for more than a hundred years, since Darwin and Sachs demonstrated the role of the root cap for downward growing plants. Different experimental approaches are nowadays available in order to change the influence of gravity and to study the corresponding influences on the physiology of biological systems. With the advent of spaceflight, a long-term nearly nullification of gravity is possible. Utilisation of this so-called "microgravity" condition for research in life sciences thus became an important asset in the space programs of various space agencies around the world. The German Space Life Sciences Program is managed--like all other space programs and activities in Germany--by the German Aerospace Center (DLR) in its role as space agency for Germany. Within the current space program, approved by the German government in May 2001, the overall goal for its life sciences part was defined as to gain scientific knowledge and to disclose new application potential by research under space conditions, especially by utilising the microgravity environment of the International Space Station. Three main scientific fields have been identified in collaboration with the scientific community: integrative human physiology, biotechnological applications of the microgravity environment, and fundamental biology of gravity and radiation responses (i.e., gravitational and radiation biology). In the present contribution, specific goals as well as achievements and perspectives of research in gravitational biology are given. In addition, some information is provided on spaceflight opportunities available.

Breakpoints around the HOXD cluster result in various limb malformations.

BACKGROUND: Characterisation of disease associated balanced chromosome rearrangements is a promising starting point in the search for candidate genes and regulatory elements. METHODS: We have identified and investigated three patients with limb abnormalities and breakpoints involving chromosome 2q31. Patient 1 with severe brachydactyly and syndactyly, mental retardation, hypoplasia of the cerebellum, scoliosis, and ectopic anus, carries a balanced t(2;10)(q31.1;q26.3) translocation. Patient 2, with translocation t(2;10)(q31.1;q23.33), has aplasia of the ulna, shortening of the radius, finger anomalies, and scoliosis. Patient 3 carries a pericentric inversion of chromosome 2, inv(2)(p15q31). Her phenotype is characterised by bilateral aplasia of the fibula and the radius, bilateral hypoplasia of the ulna, unossified carpal bones, and hypoplasia and dislocation of both tibiae. RESULTS: By fluorescence in situ hybridisation, we have mapped the breakpoints to intervals of approximately 170 kb or less. None of the three 2q31 breakpoints, which all mapped close to the HOXD cluster, disrupted any known genes. CONCLUSIONS: Hoxd gene expression in the mouse is regulated by cis-acting DNA elements acting over distances of several hundred kilobases. Moreover, Hoxd genes play an established role in bone development. It is therefore very likely that the three rearrangements disturb normal HOXD gene regulation by position effects.

New prenylated benzoic acid derivatives of Piper hispidum.

Three new 4-hydroxy-benzoic acid derivatives, 4-methoxy-3,5-bis-(3-hydroxy-3-methyl-1-butenyl)benzoate, 3-hydroxy-2-(1-hydroxy-1-methylethyl)-2,3-dihydrobenzofuran-5-carboxylic acid, and 3-hydroxy-2-(1-hydroxy-1-methylethyl)-2,3-dihydrobenzofuran-5-carboxylic acid methyl ester together with eight known compounds, have been isolated from the stems of Piper hispidum. Their structures were elucidated by a detailed spectroscopic analysis. In addition, the cytotoxicity of seven isolated compounds has been evaluated, revealing a moderate activity for three derivatives of dillapiole.

Trisomy 3q25.1-qter and monosomy 8p23.1-pter in a patient: cytogenetic and molecular analysis with delineation of the phenotype.

We describe a 4-year-old boy with partial 3q trisomy and distal 8p monosomy. The patient presented with mental retardation, dysmorphic face, congenital heart defect, brain and genital anomalies, and behavioral problems. The conventional cytogenetic analysis showed a 46,XY,add(8p) karyotype. Reverse painting and microsatellite analysis demonstrated a partial monosomy of 8p23.1 --> pter and a partial trisomy of 3q25.1 --> qter. The data suggest that the chromosomal rearrangement originated from a de novo translocation in a paternal germinal cell. The phenotype observed in our patient resulted from the combination of those defects described in the isolated dup(3q) and distal del(8p) syndromes.

Cytotoxicity of plants used in traditional medicine in Yemen.

Twenty-five extracts obtained from 14 plant species used in the traditional medicine in Yemen have been screened for cytotoxic activity against human ECV-304 cells. Extracts of Dracaena cinnabari, Eucalyptus camaldulensis, Euclea divinorum, Euphorbia cactus, Pulicaria crispa, and Withania somnifera displayed a remarkable activity.

Evidence of decreased risk of cancer in individuals with fragile X.

The occurrence of cancer was studied in a cohort of 223 persons with fragile X syndrome, based on information from the Danish Cytogenetic Registry and the Danish Cancer Registry. Four cases of cancer were found, (carcinoma of pancreas, urinary bladder, uterus, and rectum). None of these cancer types have been reported in patients with fragile X syndrome previously. Compared with cancer rates in the general population, the standardized incidence ratio (SIR) was decreased to 0.28 (95% confidence interval: 0.1-0.8). The possibility that the expanded trinucleotide repeats in fragile X syndrome protect against cancer is discussed.

Application of rRNA-targeted oligonucleotide probes in biotechnology.

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.

Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C.

Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC.