Crystallographic and Fluorescence Studies of the Interaction of Haloalkane Dehalogenase with Halide Ions. Studies with Halide Compounds Reveal a Halide Binding Site in the Active Site

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 catalyzes the conversion of 1,2-dichloroethane to 2-chloroethanol and chloride without use of oxygen or cofactors. The active site is situated in an internal cavity, which is accesible from the solvent, even in the crystal. Crystal structures of the dehalogenase enzyme complexed with iodoacetamide, chloroacetamide, iodide, and chloride at pH 6.2 and 8.2 revealed a halide binding site between the ring NH's of two tryptophan residues, Trp-125 and Trp-175, located in the active site. The halide ion lies on the intersection of the planes of the rings of the tryptophans. The binding of iodide and chloride to haloalkane dehalogenase caused a strong decrease in protein fluorescence. The decrease could be fitted to a modified form of the Stern-Volmer equation, indicating the presence of fluorophors of different accessibilities. Halide binding was much stronger at pH 6.0 than at pH 8.2. Assuming ligand binding to Trp-125 and Trp-175 as the sole cause of fluorescence quenching, dissociation constants at pH 6.0 with chloride and iodide were calculated to be 0.49 +/- 0.04 and 0.074 +/- 0.007 mM, respectively. Detailed structural investigation showed that the halide binding site probably stabilizes the halide product as well as the negatively charged transition state occurring during the formation of the covalent intermediate

Reporter gene-expressing bone marrow-derived stromal cells are immune-tolerated following implantation in the central nervous system of syngeneic immunocompetent mice

<p>Abstract</p> <p>Background</p> <p>Cell transplantation is likely to become an important therapeutic tool for the treatment of various traumatic and ischemic injuries to the central nervous system (CNS). However, in many pre-clinical cell therapy studies, reporter gene-assisted imaging of cellular implants in the CNS and potential reporter gene and/or cell-based immunogenicity, still remain challenging research topics.</p> <p>Results</p> <p>In this study, we performed cell implantation experiments in the CNS of immunocompetent mice using autologous (syngeneic) luciferase-expressing bone marrow-derived stromal cells (BMSC-Luc) cultured from ROSA26-L-S-L-Luciferase transgenic mice, and BMSC-Luc genetically modified using a lentivirus encoding the enhanced green fluorescence protein (eGFP) and the puromycin resistance gene (Pac) (BMSC-Luc/eGFP/Pac). Both reporter gene-modified BMSC populations displayed high engraftment capacity in the CNS of immunocompetent mice, despite potential immunogenicity of introduced reporter proteins, as demonstrated by real-time bioluminescence imaging (BLI) and histological analysis at different time-points post-implantation. In contrast, both BMSC-Luc and BMSC-Luc/eGFP/Pac did not survive upon intramuscular cell implantation, as demonstrated by real-time BLI at different time-points post-implantation. In addition, ELISPOT analysis demonstrated the induction of IFN-γ-producing CD8+ T-cells upon intramuscular cell implantation, but not upon intracerebral cell implantation, indicating that BMSC-Luc and BMSC-Luc/eGFP/Pac are immune-tolerated in the CNS. However, in our experimental transplantation model, results also indicated that reporter gene-specific immune-reactive T-cell responses were not the main contributors to the immunological rejection of BMSC-Luc or BMSC-Luc/eGFP/Pac upon intramuscular cell implantation.</p> <p>Conclusion</p> <p>We here demonstrate that reporter gene-modified BMSC derived from ROSA26-L-S-L-Luciferase transgenic mice are immune-tolerated upon implantation in the CNS of syngeneic immunocompetent mice, providing a research model for studying survival and localisation of autologous BMSC implants in the CNS by real-time BLI and/or histological analysis in the absence of immunosuppressive therapy.</p

Examining Educators’ Attitudes of the Common Core State Standards for Mathematics

In October 2013, the House and the Senate officially approved the initiative for statewide implementation of the Common Core State Standards (CCSS) in mathematics, and has been met with much hesitation and opposition from educators. To learn more about secondary mathematics teachers’ views of the mathematics standards and their preparedness in putting them into practice, Michigan Council of Teachers of Mathematics in conjunction with Hope College surveyed 100 secondary math teachers after participating in a 3-day Common Core-related conference. Attendees reported on a range of topics, including how prepared they felt to teach different student demographics, their familiarity with different math subjects’ standards, and what they felt would be helpful for improving their comfort with the CCSS in math classrooms. The results of Chisquared comparisons between the pretests and the posttests suggest results that most teachers felt familiar with what was expected of them. These secondary teachers felt their curricular materials were not aligned with the CCSS, and more targeted preparation was necessary. The student populations causing most concern were ESL and special education students. Finally, educators reported that more planning time, more time to collaborate with colleagues, and access to more CCSS curricular materials would be helpful to prepare for classroom implementation. Information from this study can be used to create more effective professional development sessions in the future

Clinical and microscopic predictors of Entamoeba histolytica intestinal infection in travelers and migrants diagnosed with Entamoeba histolytica/dispar infection

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Accuracy of malaria diagnosis by clinical laboratories in Belgium

BACKGROUND: The Belgian Reference Laboratory for Plasmodium offers a free-of-charge reference testing of malaria-positive or doubtful samples to clinical laboratories. METHODS: The final malaria diagnosis from the Reference Laboratory (microscopy, rapid diagnostic tests (RDTs) and Plasmodium species-specific PCR) were compared with the final diagnosis from peripheral Belgian laboratories. The Reference Laboratory reports were analysed for all samples submitted between 2013 and 2017. Criteria assessed included the diagnosis of malaria, Plasmodium species identification including mixed infections, and in case of Plasmodium falciparum, the parasite density and the presence of sexual and asexual stages. RESULTS: A total of 947 non-duplicate samples were included. Reference testing confirmed 96.3% (893/927) and 90.0% (18/20) samples submitted as positive and negative, respectively, the two missed diagnoses were samples with Plasmodium ovale and Plasmodium malariae. Submitting laboratories had correctly identified P. falciparum in 95.1% (508/534) samples with P. falciparum single infection. They had correctly diagnosed the species in 62.9% (95/151) single non-falciparum samples and had reported 'non-falciparum' in another 26 (17.2%) samples; most errors occurred among P. malariae (n = 8/21, 38.1%) and P. ovale (n = 14/51, 27.5%). Only one of the 21 mixed Plasmodium species infections had been diagnosed as such by the submitting laboratories; in three of them, P. falciparum had been overlooked. Taken single and mixed infections together, P. falciparum was diagnosed in 98.6% (546/554) samples. Among 471 single P. falciparum samples available for comparison, laboratories had correctly reported parasite densities above 2% in 87.5% (70/80) samples; they had incorrectly reported parasite densities > 2% in an extra 52 (8.9%) samples. Laboratories had correctly reported P. falciparum schizonts and gametocytes in 25.6% (11/43) and 56.7% (17/30) samples, respectively. CONCLUSION: Diagnostic laboratories in a malaria non-endemic setting provided excellent diagnosis of malaria and P. falciparum, reasonably good diagnosis of non-falciparum infections and acceptable calculation of P. falciparum parasite density.status: publishe

Dynamic bioluminescence imaging for quantitative tumour burden assessment using IV or IP administration of D-luciferin: effect on intensity, time kinetics and repeatability of photon emission

In vivo bioluminescence imaging (BLI) is a promising technique for non-invasive tumour imaging. D-luciferin can be administrated intraperitonealy or intravenously. This will influence its availability and, therefore, the bioluminescent signal. The aim of this study is to compare the repeatability of BLI measurement after IV versus IP administration of D-luciferin and assess the correlation between photon emission and histological cell count both in vitro and in vivo. Fluc-positive R1M cells were subcutaneously inoculated in nu/nu mice. Dynamic BLI was performed after IV or IP administration of D-luciferin. Maximal photon emission (PEmax) was calculated. For repeatability assessment, every acquisition was repeated after 4 h and analysed using Bland-Altman method. A second group of animals was serially imaged, alternating IV and IP administration up to 21 days. When mice were killed, PEmax after IV administration was correlated with histological cell number. The coefficients of repeatability were 80.2% (IV) versus 95.0% (IP). Time-to-peak is shorter, and its variance lower for IV (p < 0.0001). PEmax was 5.6 times higher for IV. A trend was observed towards lower photon emission per cell in larger tumours. IV administration offers better repeatability and better sensitivity when compared to IP. In larger tumours, multiple factors may contribute to underestimation of tumour burden. It might, therefore, be beneficial to test novel therapeutics on small tumours to enable an accurate evaluation of tumour burden

SARC-F Is Inaccurate to Identify Geriatric Rehabilitation Inpatients at Risk for Sarcopenia: RESORT

Introduction: Sarcopenia is highly prevalent in geriatric rehabilitation inpatients; screening using the Strength, Assistance in walking, Rise from a chair, Climb stairs, Falls history questionnaire (SARC-F) has been recommended. This study assessed the diagnostic accuracy of the SARC-F in identifying sarcopenia according to the European Working Group on Sarcopenia in Older People (EWGSOP), EWGSOP2, and Asian Working Group for Sarcopenia (AWGS) definitions in geriatric rehabilitation inpatients. Methods: REStOring health of acutely unwell adulTs (RESORT) is an observational, longitudinal cohort of geriatric rehabilitation inpatients. The SARC-F was completed for 2 time-points, status at preadmission (1 month before admission) and at admission; a score ≥4 was considered at risk for sarcopenia. Muscle mass (bioelectrical impedance analysis), handgrip strength (handheld dynamometry), and gait speed (4-m walk test) were measured at admission. Diagnostic accuracy was determined by sensitivity, specificity, and area under the curve (AUC). Results: The sarcopenia prevalence (n = 290, median age 84.0 years [IQR 79.0-89.0], 56.9% female) was 40.3% (EWGSOP1), 25.4% (EWGSOP2), and 38.8% (AWGS). For preadmission and admission status, respectively, the SARC-F identified 67.9 and 82.1% (EWGSOP), 66.0 and 81.0% (EWGSOP2), and 67.5 and 81.6% (AWGS) inpatients at risk for sarcopenia. The SARC-F showed fair sensitivity (67-74%), poor specificity (32-37%), and poor AUC (0.411-0.474) to identify inpatients at risk for sarcopenia at preadmission status, and fair-good sensitivity (79-84%), poor specificity (17-20%), and poor AUC (0.401-0.432) to identify inpatients at risk for sarcopenia at admission, according to EWGSOP, EWGSOP2, and AWGS definitions. Conclusion: The SARC-F showed poor diagnostic accuracy in identifying sarcopenia in geriatric rehabilitation inpatients. Assessment of sarcopenia is recommended without screening