AMINA-chip : a miniaturized measurement system for ambient ammonia

The development of a miniaturized and integrated measurement system for gaseous ammonia is described in this thesis. The measuring principle, ¿AMINA¿, is an indirect method for selectively measuring ammonia that makes use of pH-transitions, electrolyte conductivity detection and phaseseparating membranes

The Use of a “Hybrid” Trainer in an Established Laparoscopic Skills Program

OBJECTIVESTabletop inanimate trainers have proven to be a safe, inexpensive, and convenient platform for developing laparoscopic skills. Historically, programs that utilize these trainers rely on subjective evaluation of errors and time as the only measures of performance. Virtual reality simulators offer more extensive data collection capability, but they are expensive and lack realism. This study reviews a new electronic proctor (EP), and its performance within the Rosser Top Gun Laparoscopic Skills and Suturing Program. This "hybrid" training device seeks to capture the strengths of both platforms by providing an affordable, reliable, realistic training arena with metrics to objectively evaluate performance. METHODSAn electronic proctor was designed for use in conjunction with drills from the Top Gun Program. The tabletop trainers used were outfitted with an automated electromechanically monitored task arena. Subjects performed 10 repetitions of each of 3 drills: "Cup Drop," "Triangle Transfer," and "Intracorporeal Suturing." In real time, this device evaluates for instrument targeting accuracy, economy of motion, and adherence to the rules of the exercises. A buzzer and flashing light serve to alert the student to inaccuracies and breaches of the defined skill transference parameters. RESULTSBetween July 2001 and June 2003, 117 subjects participated in courses. Seventy-three who met data evaluation criteria were assessed and compared with 744 surgeons who had previously taken the course. The total time to complete each task was significantly longer with the EP in place. The Cup Drop drill with the EP had a mean total time of 1661 seconds (average, 166.10) with 54.49 errors (average, 5.45) vs. 1252 seconds (average, 125.2) without the EP (P = 0.000, t = 6.735, df = 814). The Triangle Transfer drill mean total time was 556 seconds (average, 55.63) and 167.57 errors (average. 16.75) (EP) vs. 454 seconds (non-EP) (average. 45.4) (P = 0.000, t = 4.447, df = 814). The mean total times of the suturing task was 1777 seconds (average, 177.73) and 90.46 errors (average. 9.04) (EP) vs. 1682 seconds (non-EP) (average, 168.2) (P = 0.040, t = 1.150, df = 814). When compared with surgeons who had participated in the Top Gun course prior to EP, the participants in the study collectively scored in the 18.3th percentile with the Cup Drop drill, 22.6th percentile with the Triangle Transfer drill, and 36.7th percentile with the Intracorporeal Suturing exercise. When penalizing for errors recorded by the EP, participants scored collectively in the 9.9th, 0.1th, and 17.7th percentile, respectively. No equipment failures occurred, and the agenda of the course did not have to be modified to accommodate the new platform. CONCLUSIONSThe EP utilized during the Top Gun Course was introduced without modification of the core curriculum and experienced no device failures. This hybrid trainer offers a cost-effective inanimate simulator that brings quality performance monitoring to traditional inanimate trainers. It appears that the EP influenced student performance by alerting them to errors made, thus causing an increased awareness of and focus on precision and accuracy. This suggests that the EP could have internal guidance capabilities. However, validation studies must be done in the future

Biomechanical assessment of the effects of decompressive surgery in non-chondrodystrophic and chondrodystrophic canine multisegmented lumbar spines

Purpose Dogs are often used as an animal model in spinal research, but consideration should be given to the breed used as chondrodystrophic (CD) dog breeds always develop IVD degeneration at an early age, whereas nonchondrodystrophic (NCD) dog breeds may develop IVD degeneration, but only later in life. The aim of this study was to provide a mechanical characterization of the NCD [non-degenerated intervertebral discs (IVDs), rich in notochordal cells] and CD (degenerated IVDs, rich in chondrocyte-like cells) canine spine before and after decompressive surgery (nucleotomy). Methods The biomechanical properties of multisegmented lumbar spine specimens (T13-L5 and L5-Cd1) from 2-year-old NCD dogs (healthy) and CD dogs (early degeneration) were investigated in flexion/extension (FE), lateral bending (LB), and axial rotation (AR), in the native state and after nucleotomy of L2-L3 or dorsal laminectomy and nucleotomy of L7-S1. The range of motion (ROM), neutral zone (NZ), and NZ stiffness (NZS) of L1-L2, L2- L3, L6-L7, and L7-S1 were calculated. Results In native spines in both dog groups, the greatest mobility in FE was found at L7-S1, and the greatest mobility in LB at L2-L3. Surgery significantly increased the ROM and NZ, and significantly decreased the NZS in FE, LB, and AR in both breed groups. However, surgery at L2-L3 resulted in a significantly larger increase in NZ and decrease in NZS in the CD spines compared with the NCD spines, whereas surgery at L7-S1 induced a significantly larger increase in ROM and decrease in NZS in the NCD spines compared with the CD spines. Conclusions Spinal biomechanics significantly differ between NCD and CD dogs and researchers should consider this aspect when using the dog as a model for spinal research. © Springer-Verlag 2012

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

INTRODUCTION: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. METHODS: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. RESULTS: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells. CONCLUSIONS: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration

Environmental Levels of the Antiviral Oseltamivir Induce Development of Resistance Mutation H274Y in Influenza A/H1N1 Virus in Mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals

Epigenetic regulation of CD44 in Hodgkin and non-Hodgkin lymphoma

<p>Abstract</p> <p>Background</p> <p>Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a hallmark of cancer. To assay its extent in human lymphoma, methylation of 24 TSG was analyzed in lymphoma-derived cell lines as well as in patient samples.</p> <p>Methods</p> <p>We screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated <it>CD44 </it>was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of <it>CD44 </it>and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry.</p> <p>Results</p> <p>On average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that <it>CD44 </it>was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: <it>CD44 </it>was not methylated in MCL patients (0/11) whereas <it>CD44 </it>was frequently hypermethylated in BL patients (18/29). In cell lines with <it>CD44 </it>hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of <it>CD44</it>. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44⁺lymphoma cells. <it>CD44 </it>hypermethylated, CD44^-lymphoma cell lines were consistently resistant towards anti-CD44 induced apoptosis.</p> <p>Conclusion</p> <p>Our data show that <it>CD44 </it>is epigenetically regulated in lymphoma and undergoes <it>de novo </it>methylation in distinct lymphoma subtypes like BL. Thus <it>CD44 </it>may be a promising new epigenetic marker for diagnosis and a potential therapeutic target for the treatment of specific lymphoma subtypes.</p

Population structure and linkage disequilibrium unravelled in tetraploid potato

Association mapping is considered to be an important alternative strategy for the identification of quantitative trait loci (QTL) as compared to traditional QTL mapping. A necessary prerequisite for association analysis to succeed is detailed information regarding hidden population structure and the extent of linkage disequilibrium. A collection of 430 tetraploid potato cultivars, comprising two association panels, has been analysed with 41 AFLP® and 53 SSR primer combinations yielding 3364 AFLP fragments and 653 microsatellite alleles, respectively. Polymorphism information content values and detected number of alleles for the SSRs studied illustrate that commercial potato germplasm seems to be equally diverse as Latin American landrace material. Genome-wide linkage disequilibrium (LD)—reported for the first time for tetraploid potato—was observed up to approximately 5 cM using r2 higher than 0.1 as a criterion for significant LD. Within-group LD, however, stretched on average twice as far when compared to overall LD. A Bayesian approach, a distance-based hierarchical clustering approach as well as principal coordinate analysis were adopted to enquire into population structure. Groups differing in year of market release and market segment (starch, processing industry and fresh consumption) were repeatedly detected. The observation of LD up to 5 cM is promising because the required marker density is not likely to disable the possibilities for association mapping research in tetraploid potato. Population structure appeared to be weak, but strong enough to demand careful modelling of genetic relationships in subsequent marker-trait association analyses. There seems to be a good chance that linkage-based marker-trait associations can be identified at moderate marker densities

The appropriate management of persisting pain after spine surgery: a European panel study with recommendations based on the RAND/UCLA method

Purpose: Management of patients with persisting pain after spine surgery (PPSS) shows significant variability, and there is limited evidence from clinical studies to support treatment choice in daily practice. This study aimed to develop patient-specific recommendations on the management of PPSS. Methods: Using the RAND/UCLA appropriateness method (RUAM), an international panel of 6 neurosurgeons, 6 pain specialists, and 6 orthopaedic surgeons assessed the appropriateness of 4 treatment options (conservative, minimally invasive, neurostimulation, and re-operation) for 210 clinical scenarios. These scenarios were unique combinations of patient characteristics considered relevant to treatment choice. Appropriateness had to be expressed on a 9-point scale (1 = extremely inappropriate, 9 = extremely appropriate). A treatment was considered appropriate if the median score was ≥ 7 in the absence of disagreement (≥ 1/3 of ratings in each of the opposite sections 1–3 and 7–9). Results: Appropriateness outcomes showed clear and specific patterns. In 48% of the scenarios, exclusively one of the 4 treatments was appropriate. Conservative treatment was usually considered appropriate for patients without clear anatomic abnormalities and for those with new pain differing from the original symptoms. Neurostimulation was considered appropriate in the case of (predominant) neuropathic leg pain in the absence of conditions that may require surgical intervention. Re-operation could be considered for patients with recurrent disc, spinal/foraminal stenosis, or spinal instability. Conclusions: Using the RUAM, an international multidisciplinary panel established criteria for appropriate treatment choice in patients with PPSS. These may be helpful to educate physicians and to improve consistency and quality of care. Graphical abstract: These slides can be retrieved under Electronic Supplementary Material. [Figure not available: see fulltext.

Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms