Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk

Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 microL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.

Development and in-vitro characterization of an implantable flow sensing transducer for hydrocephalus

An implantable transducer for monitoring the flow of Cerebrospinal fluid (CSF) for the treatment of hydrocephalus has been developed which is based on measuring the heat dissipation of a local thermal source. The transducer uses passive telemetry at 13.56 MHz for power supply and read out of the measured flow rate. The in vitro performance of the transducer has been characterized using artificial Cerebrospinal Fluid (CSF) with increased protein concentration and artificial CSF with 10\% fresh blood. After fresh blood was added to the artificial CSF a reduction of flow rate has been observed in case that the sensitive surface of the flow sensor is close to the sedimented erythrocytes. An increase of flow rate has been observed in case that the sensitive surface is in contact with the remaining plasma/artificial CSF mix above the sediment which can be explained by an asymmetric flow profile caused by the sedimentation of erythrocythes having increased viscosity compared to artificial CSF. After removal of blood from artificial CSF, no drift could be observed in the transducer measurement which could be associated to a deposition of proteins at the sensitive surface walls of the packaged flow transducer. The flow sensor specification requirement of +-10\% for a flow range between 2 ml/h and 40 ml/h. could be confirmed at test conditions of 37 degrees C.

Basic symptoms and ultrahigh risk criteria: symptom development in the initial prodromal state

Symptom development during the prodromal phase of psychosis was explored retrospectively in first-episode psychosis patients with special emphasis on the assumed time-related syndromic sequence of "unspecific symptoms (UN)-predictive basic symptoms (BS)-attenuated psychotic symptoms (APS)-(transient) psychotic symptoms (PS)." Onset of syndromes was defined by first occurrence of any of their respective symptoms. Group means were inspected for time differences between syndromes and influence of sociodemographic and clinical characteristics on the recalled sequence. The sequence of "UN-BS/APS-PS" was clearly supported, and both BS and, though slightly less, APS were highly sensitive. However, onset of BS and APS did not show significant time difference in the whole sample (N = 126; 90% schizophrenia), although when each symptom is considered independently, APS tended to occur later than first predictive BS. On descriptive level, about one-third each recalled an earlier, equal and later onset of BS compared with APS. Level of education showed the greatest impact on the recall of the hypothesized sequence. Thereby, those with a higher school-leaving certificate supported the assumed sequence, whereas those of low educational background retrospectively dated APS before BS. These findings rather point out recognition and recall bias inherent to the retrospective design than true group characteristics. Future long-term prospective studies will have to explore this conclusively. However, as regards the criteria, the results support the notion of BS as at least a complementary approach to the ultrahigh risk criteria, which may also allow for an earlier detection of psychosis.

Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

Alveoli are formed in the lung by the insertion of secondary tissue folds, termed septa, which are subsequently remodeled to form the mature alveolar wall. Secondary septation requires interplay between three cell types: endothelial cells forming capillaries, contractile interstitial myofibroblasts, and epithelial cells. Here, we report that postnatal lung alveolization critically requires ephrinB2, a ligand for Eph receptor tyrosine kinases expressed by the microvasculature. Mice homozygous for the hypomorphic knockin allele ephrinB2DeltaV/DeltaV, encoding mutant ephrinB2 with a disrupted C-terminal PDZ interaction motif, show severe postnatal lung defects including an almost complete absence of lung alveoli and abnormal and disorganized elastic matrix. Lung alveolar formation is not sensitive to loss of ephrinB2 cytoplasmic tyrosine phosphorylation sites. Postnatal day 1 mutant lungs show extracellular matrix alterations without differences in proportions of major distal cell populations. We conclude that lung alveolar formation relies on endothelial ephrinB2 function.

Monitoring thrombin generation by electrochemistry: development of an amperometric biosensor screening test for plasma and whole blood

BACKGROUND: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. METHODS: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. RESULTS: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. CONCLUSIONS: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 microL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

Evaluating Integration. Research contributions for a gender sensitive migration policy: Research contributions for a gender sensitive migration policy

The integration of academic and non-academic knowledge is a key concern for researchers who aim at bridging the gap between research and policy. Researchers involved in the sustainability-oriented NCCR North-South programme have made the experience that linking different types of knowledge requires time and effort, and that methodologies are still lacking. One programme component was created at the inception of this transdisciplinary research programme to support exchange between researchers, development practitioners and policymakers. After 8 years of research, the programme is assessing whether research has indeed enabled a continuous communication across and beyond academic boundaries and has effected changes in the public policies of poor countries. In a first review of the data, we selected two case studies explicitly addressing the lives of women. In both cases – one in Pakistan, the other in Nepal – the dialogue between researchers and development practitioners contributed to important policy changes for female migration. In both countries, outmigration has become an increasingly important livelihood strategy. National migration policies are gendered, limiting the international migration of women. In Nepal, women were not allowed to migrate to specific countries such as the Gulf States or Malaysia. This was done in the name of positive discrimination, to protect women from potential exploitation and harassment in domestic work. However, women continued to migrate in many other and often illegal and more risky ways, increasing their vulnerability. In Pakistan, female labour migration was not allowed at all and male migration increased the vulnerability of the families remaining back home. Researchers and development practitioners in Nepal and Pakistan brought women’s shared experience of and exposure to the mechanisms of male domination into the public debate, and addressed the discriminating laws. Now, for the first time in Pakistan, the new draft policy currently under discussion would enable broadly-based female labour migration. What can we learn from the two case studies with regard to ways of relating experience- and research-based knowledge? The paper offers insights into the sequence of interactions between researchers, local people, development practitioners, and policy-makers, which eventually contributed to the formulation of a rights-based migration policy. The reflection aims at exploring the gendered dimension of ways to co-produce and share knowledge for development across boundaries. Above all, it should help researchers to better tighten the links between the spheres of research and policy in future.

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

Development of nuclear emulsions with 1 μm spatial resolution for the AEgIS experiment

The main goal of the AEgIS experiment at CERN is to test the weak equivalence principle for antimatter. We will measure the Earth ' s gravitational acceleration g with antihydrogen atoms being launched in a horizontal vacuum tube and traversing a moiré de fl ectometer. We intend to use a position sensitive device made of nuclear emulsions (combined with a time-of- fl ight detector such as silicon μ strips) to measure precisely their annihilation points at the end of the tube. The goal is to determine g with a 1% relative accuracy. In 2012 we tested emulsion fi lms in vacuum and at room temperature with low energy antiprotons from the CERN antiproton decelerator. First results on the expected performance for AEgIS are presented

A new sensitive method for the quantification of glyoxal and methylglyoxal in snow and ice by stir bar sorptive extraction and liquid desorption-HPLC-ESI-MS

In this study, the development of a new sensitive method for the analysis of alpha-dicarbonyls glyoxal (G) and methylglyoxal (MG) in environmental ice and snow is presented. Stir bar sorptive extraction with in situ derivatization and liquid desorption (SBSE-LD) was used for sample extraction, enrichment, and derivatization. Measurements were carried out using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As part of the method development, SBSE-LD parameters such as extraction time, derivatization reagent, desorption time and solvent, and the effect of NaCl addition on the SBSE efficiency as well as measurement parameters of HPLC-ESI-MS/MS were evaluated. Calibration was performed in the range of 1–60 ng/mL using spiked ultrapure water samples, thus incorporating the complete SBSE and derivatization process. 4-Fluorobenzaldehyde was applied as internal standard. Inter-batch precision was <12 % RSD. Recoveries were determined by means of spiked snow samples and were 78.9 ± 5.6 % for G and 82.7 ± 7.5 % for MG, respectively. Instrumental detection limits of 0.242 and 0.213 ng/mL for G and MG were achieved using the multiple reaction monitoring mode. Relative detection limits referred to a sample volume of 15 mL were 0.016 ng/mL for G and 0.014 ng/mL for MG. The optimized method was applied for the analysis of snow samples from Mount Hohenpeissenberg (close to the Meteorological Observatory Hohenpeissenberg, Germany) and samples from an ice core from Upper Grenzgletscher (Monte Rosa massif, Switzerland). Resulting concentrations were 0.085–16.3 ng/mL for G and 0.126–3.6 ng/mL for MG. Concentrations of G and MG in snow were 1–2 orders of magnitude higher than in ice core samples. The described method represents a simple, green, and sensitive analytical approach to measure G and MG in aqueous environmental samples.

Development and validation of a quantitative method to assess pedicle screw loosening in posterior spine instrumentation on plain radiographs.

PURPOSE Currently, the diagnosis of pedicle screw (PS) loosening is based on a subjectively assessed halo sign, that is, a radiolucent line around the implant wider than 1 mm in plain radiographs. We aimed at development and validation of a quantitative method to diagnose PS loosening on radiographs. METHODS Between 11/2004 and 1/2010 36 consecutive patients treated with thoraco-lumbar spine fusion with PS instrumentation without PS loosening were compared with 37 other patients who developed a clinically manifesting PS loosening. Three different angles were measured and compared regarding their capability to discriminate the loosened PS over the postoperative course. The inter-observer invariance was tested and a receiver operating characteristics curve analysis was performed. RESULTS The angle measured between the PS axis and the cranial endplate was significantly different between the early and all later postoperative images. The Spearman correlation coefficient for the measurements of two observers at each postoperative time point ranged between 0.89 at 2 weeks to 0.94 at 2 months and 1 year postoperative. The angle change of 1.9° between immediate postoperative and 6-month postoperative was 75% sensitive and 89% specific for the identification of loosened screws (AUC = 0.82). DISCUSSION The angle between the PS axis and the cranial endplate showed good ability to change in PS loosening. A change of this angle of at least 2° had a relatively high sensitivity and specificity to diagnose screw loosening.

Transient exposure to environmental estrogen affects embryonic development of brown trout (Salmo trutta fario).

Transient exposure of brown trout embryos from fertilization until hatch (70 days) to 17β-estradiol (E2) was investigated. Embryos were exposed to 3.8 and 38.0 ng/L E2 for 2h, respectively, under four scenarios: (A) exposure once at the day of fertilization (0 days post-fertilization, dpf), (B) once at eyeing stage (38 dpf), (C) weekly exposure until hatch or (D) bi-weekly exposure until hatch. Endpoints to assess estrogen impact on embryo development were fertilization success, chronological sequence of developmental events, hatching process, larval malformations, heart rate, body length and mortality. Concentration-dependent acceleration of development until median hatch was observed in all exposure scenarios with the strongest effect observed for embryos exposed once at 0 dpf. In addition, the hatching period was significantly prolonged by 4-5 days in groups receiving single estrogen exposures (scenarios A and B). Heart rate on hatching day was significantly depressed with increasing E2 concentrations, with the strongest effect observed for embryos exposed at eyeing stage. Estrogenic exposure at 0 dpf significantly reduced body length at hatch, not depending on whether this was a single exposure or the first of a series (scenarios A and D). The key finding is that even a single, transient E2 exposure during embryogenesis had significant effects on brown trout development. Median hatch, hatching period, heart rate and body length at hatch were found to be highly sensitive biomarkers responsive to estrogenic exposure during embryogenesis. Treatment effects were observable only at the post-hatch stage.

Novel Features of the Prenatal Horn Bud Development in Cattle (Bos taurus).

Whereas the genetic background of horn growth in cattle has been studied extensively, little is known about the morphological changes in the developing fetal horn bud. In this study we histologically analyzed the development of horn buds of bovine fetuses between ~70 and ~268 days of pregnancy and compared them with biopsies taken from the frontal skin of the same fetuses. In addition we compared the samples from the wild type (horned) fetuses with samples taken from the horn bud region of age-matched genetically hornless (polled) fetuses. In summary, the horn bud with multiple layers of vacuolated keratinocytes is histologically visible early in fetal life already at around day 70 of gestation and can be easily differentiated from the much thinner epidermis of the frontal skin. However, at the gestation day (gd) 212 the epidermis above the horn bud shows a similar morphology to the epidermis of the frontal skin and the outstanding layers of vacuolated keratinocytes have disappeared. Immature hair follicles are seen in the frontal skin at gd 115 whereas hair follicles below the horn bud are not present until gd 155. Interestingly, thick nerve bundles appear in the dermis below the horn bud at gd 115. These nerve fibers grow in size over time and are prominent shortly before birth. Prominent nerve bundles are not present in the frontal skin of wild type or in polled fetuses at any time, indicating that the horn bud is a very sensitive area. The samples from the horn bud region from polled fetuses are histologically equivalent to samples taken from the frontal skin in horned species. This is the first study that presents unique histological data on bovine prenatal horn bud differentiation at different developmental stages which creates knowledge for a better understanding of recent molecular findings.