Targeted mutagenesis of the Sap47 gene of Drosophila: Flies lacking the synapse associated protein of 47 kDa are viable and fertile

BACKGROUND: Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function. We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila. Sequence comparison identifies homologous proteins in numerous species including C. elegans, fish, mouse and human. First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila. RESULTS: Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful. However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs. Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants. Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits. For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS. In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control. When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels. CONCLUSION: The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function. The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination. RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct. The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila

WHERE ARE THE PARTICIPANTS? INCLUDING MOTIVATIONAL ASPECTS INTO THEORIZING AND DESIGN IN IS RESEARCH

The purpose of this paper is to identify new research prospects for the information systems discipline, based on establishing a link to the body of knowledge in the fields of work psychology and organizational psychology. Since information systems are work systems consisting of information, technologies, and participants, who are supported by information and communication technology (IT), productivity is a function of human performance. Job demands and individual resources in ITsupported work environments should therefore be considered in order to facilitate employee motivation, prevent strain, and cope with the challenges related to an ageing workforce and a high prevalence of burnout in the IT sector. As an initial literature review shows, these research vistas are underrepresented in previous information systems research. The present paper proposes directions for including job demands and individual resources into the analysis, design and operation of ITsupported work environments from two angles. First, the benefits of including psychological theories in typical phases of a business process management project are discussed. Secondly, the contributions of extending five types of information systems theories for the discipline in general are outlined

In situ sounding of radiative flux profiles through the Arctic lower troposphere

In situ profiles and fixed-altitude time series of all four components of net radiation were obtained at Ny-Ålesund, Svalbard (78.9° N, 11.9° E), in the period May 04–21, 2015. Measurements were performed using adapted high-quality instrumentation classified as “secondary standard” carried by a tethered balloon system. Balloon-lifted measurements of albedo under clear-sky conditions demonstrate the local dependence on altitude and on the surface inhomogeneity of this parameter over coastal terrain of Ny-Ålesund. Depending on the surface composition within the sensor’s footprint near the coastline, the albedo over predominantly snow-covered surfaces was found to decrease to 0.548 and 0.452 at 494 m and 881 m altitude compared with 0.731 and 0.788 measured with near-surface references, respectively. Albedo profiles show an all-sky maximum at 150 m above surface level due to local surface inhomogeneity, and an averaged vertical change rate of − 0.040/100 up to 750 m aboveground level (clear sky) and − 0.034/100 m (overcast). Profiling of arctic low-level clouds reveals distinct vertical gradients in all radiative fluxes but longwave upward at the cloud top. Observed radiative cooling at the top of a partly dissolving stratus cloud with heating rates of − 40.4 to − 62.1 Kd−1 in subsequent observations is exemplified

Usefulness of component resolved analysis of cat allergy in routine clinical practice

Background: Cat allergy is of great importance, and its prevalence is increasing worldwide. Cat allergens and house dust mite allergens represent the major indoor allergens;however, they are ubiquitous. Cat sensitization and allergy are known risk factors for rhinitis, bronchial hyperreactivity and asthma. Thus, the diagnosis of sensitization to cats is important for any allergist. Methods: 70 patients with positive skin prick tests for cats were retrospectively compared regarding their skin prick test results, as well as their specific immunoglobulin E antibody profiles with regard to their responses to the native cat extract, rFel d 1, nFel d 2 and rFel d 4. 35 patients were allergic to cats, as determined by positive anamnesis and/or nasal provocation with cat allergens, and 35 patients exhibited clinically non-relevant sensitization, as indicated by negative anamnesis and/or a negative nasal allergen challenge. Results: Native cat extract serology testing detected 100% of patients who were allergic to cats but missed eight patients who showed sensitization in the skin prick test and did not have allergic symptoms. The median values of the skin prick test, as well as those of the specific immunoglobulin E antibodies against the native cat extract, were significantly higher for allergic patients than for patients with clinically non-relevant sensitization. Component based diagnostic testing to rFel d 1 was not as reliable. Sensitization to nFel d 2 and rFel d 4 was seen only in individual patients. Conclusion: Extract based diagnostic methods for identifying cat allergy and sensitization, such as the skin prick test and native cat extract serology, remain crucial in routine clinical practice. In our study, component based diagnostic testing could not replace these methods with regard to the detection of sensitization to cats and differentiation between allergy and sensitization without clinical relevance. However, component resolved allergy diagnostic tools have individual implications, and future studies may facilitate a better understanding of its use and subsequently may improve the clinical management of allergic patients

Evolution of the Definition of Rejection in Kidney Transplantation and Its Use as an Endpoint in Clinical Trials

T cell-mediated rejection; Biopsy; Subclinical rejectionRechazo mediado por células T; Biopsia; Rechazo subclínicoRebuig mediat per cèl·lules T; Biòpsia; Rebuig subclínicThis article outlines the evolving definition of rejection following kidney transplantation. The viewpoints and evidence presented were included in documentation prepared for a Broad Scientific Advice request to the European Medicines Agency (EMA), relating to clinical trial endpoints in kidney transplantation. This request was initiated by the European Society for Organ Transplantation (ESOT) in 2016 and finalized following discussions between the EMA and ESOT in 2020. In ESOT’s opinion, the use of “biopsy-proven acute rejection” as an endpoint for clinical trials in kidney transplantation is no longer accurate, although it is still the approved histopathological endpoint. The spectrum of rejection is now divided into the phenotypes of borderline changes, T cell-mediated rejection, and antibody-mediated rejection, with the latter two phenotypes having further subclassifications. Rejection is also described in relation to graft (dys)function, diagnosed because of protocol (surveillance) or indication (for-cause) biopsies. The ongoing use of outdated terminology has become a potential barrier to clinical research in kidney transplantation. This article presents these perspectives and issues, and provides a foundation on which subsequent articles within this Special Issue of Transplant International build.This initiative was supported by the European Society for Organ Transplantation

Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum

Background African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAG s) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast. Results Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires. Conclusion With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the relatively narrow host range of this subspecies and its apparent long-term clonality. However, our data does not show a clear correlation between size of trypanosome host range and either number of BESs or extent of ESAG genetic diversity

Proposed Definitions of Antibody-Mediated Rejection for Use as a Clinical Trial Endpoint in Kidney Transplantation

Antibody-mediated rejection; Biopsy; Kidney transplantationRechazo mediado por anticuerpos; Biopsia; Trasplante renalRebuig mediat per anticossos; Biòpsia; Trasplantament renalAntibody-mediated rejection (AMR) is caused by antibodies that recognize donor human leukocyte antigen (HLA) or other targets. As knowledge of AMR pathophysiology has increased, a combination of factors is necessary to confirm the diagnosis and phenotype. However, frequent modifications to the AMR definition have made it difficult to compare data and evaluate associations between AMR and graft outcome. The present paper was developed following a Broad Scientific Advice request from the European Society for Organ Transplantation (ESOT) to the European Medicines Agency (EMA), which explored whether updating guidelines on clinical trial endpoints would encourage innovations in kidney transplantation research. ESOT considers that an AMR diagnosis must be based on a combination of histopathological factors and presence of donor-specific HLA antibodies in the recipient. Evidence for associations between individual features of AMR and impaired graft outcome is noted for microvascular inflammation scores ≥2 and glomerular basement membrane splitting of >10% of the entire tuft in the most severely affected glomerulus. Together, these should form the basis for AMR-related endpoints in clinical trials of kidney transplantation, although modifications and restrictions to the Banff diagnostic definition of AMR are proposed for this purpose. The EMA provided recommendations based on this Broad Scientific Advice request in December 2020; further discussion, and consensus on the restricted definition of the AMR endpoint, is required.This initiative was supported by the European Society for Organ Transplantation

Proposed Definitions of T Cell-Mediated Rejection and Tubulointerstitial Inflammation as Clinical Trial Endpoints in Kidney Transplantation

T cell-mediated rejection; Kidney transplantation; OutcomesRebuig mediat per cèl·lules T; Trasplantament renal; ResultatsRechazo mediado por células T; Trasplante renal; ResultadosThe diagnosis of acute T cell-mediated rejection (aTCMR) after kidney transplantation has considerable relevance for research purposes. Its definition is primarily based on tubulointerstitial inflammation and has changed little over time; aTCMR is therefore a suitable parameter for longitudinal data comparisons. In addition, because aTCMR is managed with antirejection therapies that carry additional risks, anxieties, and costs, it is a clinically meaningful endpoint for studies. This paper reviews the history and classifications of TCMR and characterizes its potential role in clinical trials: a role that largely depends on the nature of the biopsy taken (indication vs protocol), the level of inflammation observed (e.g., borderline changes vs full TCMR), concomitant chronic lesions (chronic active TCMR), and the therapeutic intervention planned. There is ongoing variability—and ambiguity—in clinical monitoring and management of TCMR. More research, to investigate the clinical relevance of borderline changes (especially in protocol biopsies) and effective therapeutic strategies that improve graft survival rates with minimal patient morbidity, is urgently required. The present paper was developed from documentation produced by the European Society for Organ Transplantation (ESOT) as part of a Broad Scientific Advice request that ESOT submitted to the European Medicines Agency for discussion in 2020. This paper proposes to move toward refined definitions of aTCMR and borderline changes to be included as primary endpoints in clinical trials of kidney transplantation.This initiative was supported by the European Society for Organ Transplantation

Cytokine patterns in nasal secretion of non-atopic patients distinguish between chronic rhinosinusitis with or without nasal polys

Background: Being one of the most common nasal diseases, chronic rhinosinusitis (CRS) is subdivided into CRS with nasal polyps (NP) and CRS without nasal polyps (CRSsNP). CRSsNP presents itself with a T(H)1 milieu and neutrophil infiltration, while NP is characterised by a mixed T(H)1/T(H)2 profile and an influx of predominantly eosinophils, plasma cells and mast cells. For the purpose of discovering disease-specific cytokine profiles, the present study compares levels of mediators and cytokines in nasal secretions between CRSsNP, NP, and healthy controls. Methods: The study included 45 participants suffering from NP, 48 suffering from CRSsNP and 48 healthy controls. Allergic rhinitis constituted an exclusion criterion. Nasal secretions, sampled using the cotton wool method, were analysed for IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, IL-8, GM-CSF, G-CSF, IFN-gamma, MCP-1, MIP-1 alpha, MIP-1 beta, eotaxin, and RANTES, and for ECP and tryptase, using Bio-Plex Cytokine assay or ELISA, respectively. Results: Elevated levels of IL-5, IL-17, G-CSF, MCP-1, MIP-1 alpha, MIP-1 beta, ECP, and tryptase, as well as decreased levels of IL-10, IL-12, IL-13, and IFN-gamma were detected in NP. CRSsNP presented increased levels of RANTES and MIP-1 beta while IL-13 was decreased. No differences between the three groups were found for IL-4, IL-8, GM-CSF, and eotaxin. Conclusions: The present work suggests a disequilibrium of T(H)1 and T(H)2, together with a down-regulation of regulatory T lymphocytes and up-regulated T(H)17 in NP. Moreover, elevated levels of diverse mediators represent the activation of various inflammatory cells in this disease entity. The inflammation in CRSsNP, however, is only weakly depicted in nasal secretions. Therefore, cytokines in nasal secretions may provide helpful information for differential diagnosis

The Public\u27s Perception of an Earthquake Early Warning System: A Study on Factors Influencing Continuance Intention

This paper investigates the perceptions of the New Zealand public towards the Android Earthquake Alert (AEA) system, a public-facing earthquake early warning system. Specifically, it examines the public’s continuance intention towards the AEA system and the influencing factors of satisfaction, confirmation, perceived usefulness, and perceived trust. To gather insights into the public’s perceptions regarding the AEA system, this study distributed online surveys following two separate earthquake alert events on 12 October and 22 October 2021. A total of 524 and 671 participants responded to the two events’ surveys, providing valuable data for analysis and exploration. Structural Equation Modelling of the two datasets revealed that the continuance model fit the data to some extent, especially on the significance of perceived usefulness and perceived trust to continuance intention. However, the results also showed varying results for satisfaction’s relationship with perceived trust and continuance intention. These findings underscore the need for further investigation into the role of satisfaction and perceived trust, considering the evolving nature of EEW technologies and users’ familiarity over time. The descriptive and inferential analysis results raised concerns about potential confusion around the alerts’ source and highlighted the question of responsibility and liability for EEW. Overall, this study contributes to understanding continuance intention in the EEW context and provides insights into the public’s perception of the AEA system in New Zealand. The findings have implications more broadly for EEW systems’ design, implementation, and communication strategies