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361.
362.
Ndejjo Rawlance Wanyenze Rhoda K. Nuwaha Fred Bastiaens Hilde Musinguzi Geofrey 《Implementation science : IS》2020,15(1):1-20
There is a need to unpack the empirical, practical, and personal challenges within participatory approaches advocated to optimize implementation. The unpredictable, chaotic nature of participatory approaches complicates application of implementation theories, methods, and strategies which do not address researchers’ situatedness within participatory processes. As an implementation scientist, addressing one’s own situatedness through critical reflection is important to unearth how conscious and unconscious approaches, including ontological and epistemological underpinnings, influence the participatory context, process, and outcomes. Therefore, the aim of this exploratory work is to investigate the heretofore blind spot toward the lived experience of implementation researchers within the participatory process. We developed an integrated research-practice partnership (IRPP) to inform the implementation of a gestational weight gain (GWG) control program. Within this IRPP, one investigator conducted a 12-month autoethnography. Data collection and triangulation included field notes, cultural artifacts, and systematic timeline tracking. Data analysis included ethnographic-theoretical dialogue and restorying to synthesize key events and epiphanies into a narrative. Analysis revealed the unpredicted evolution of the GWG program into a maternal health fair and three themes within the researchers’ lived experience: (1) permeable work boundaries, (2) individual and collective blind spots toward the ontological and epistemological underpinnings of implementation paradigms, and (3) maladaptive behaviors seemingly reinforced by the research culture. These themes contributed to the chaos of implementation and to researchers’ experience of inadequate recovery from cognitive, emotional, and practical demands. These themes also demonstrated the importance of contextual factors, subjectivity, and value-based judgments within implementation research. Building on extant qualitative research guidelines, we suggest that researchers anchor their approach to implementation in reflexivity, intentionally and iteratively reflecting on their own situatedness. Through this autoethnography, we have elucidated several strategies based on critical reflection including examining philosophical underpinnings of research, adopting restorative practices that align with one’s values, and embracing personal presence as a foundation of scientific productivity. Within the predominant (post-) positivism paradigms, autoethnography may be criticized as unscientifically subjective or self-indulgent. However, this work demonstrates that autoethnography is a vehicle for third-person observation and first-person critical reflection that is transformative in understanding and optimizing implementation contexts, processes, and outcomes. 相似文献
363.
Ulrike Gerdemann Juan F. Vera Cliona M. Rooney Ann M. Leen 《Journal of visualized experiments : JoVE》2011,(51)
Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture – total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by >90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients. 相似文献
364.
Jan Huebinger Hong-Mei Han Oliver Hofnagel Ingrid?R. Vetter Philippe?I.H. Bastiaens Markus Grabenbauer 《Biophysical journal》2016,110(4):840-849
Complex living systems such as mammalian cells can be arrested in a solid phase by ultrarapid cooling. This allows for precise observation of cellular structures as well as cryopreservation of cells. The state of water, the main constituent of biological samples, is crucial for the success of cryogenic applications. Water exhibits many different solid states. If it is cooled extremely rapidly, liquid water turns into amorphous ice, also called vitreous water, a glassy and amorphous solid. For cryo-preservation, the vitrification of cells is believed to be mandatory for cell survival after freezing. Intracellular ice crystallization is assumed to be lethal, but experimental data on the state of water during cryopreservation are lacking. To better understand the water conditions in cells subjected to freezing protocols, we chose to directly analyze their subcellular water states by cryo-electron microscopy and tomography, cryoelectron diffraction, and x-ray diffraction both in the cryofixed state and after warming to different temperatures. By correlating the survival rates of cells with their respective water states during cryopreservation, we found that survival is less dependent on ice-crystal formation than expected. Using high-resolution cryo-imaging, we were able to directly show that cells tolerate crystallization of extra- and intracellular water. However, if warming is too slow, many small ice crystals will recrystallize into fewer but bigger crystals, which is lethal. The applied cryoprotective agents determine which crystal size is tolerable. This suggests that cryoprotectants can act by inhibiting crystallization or recrystallization, but they also increase the tolerance toward ice-crystal growth. 相似文献
365.
C.J. Kuhlemeier A.A.M. Thomas A. van der Ende R.W. van Leen W.E. Borrias C.A.M.J.J. van den Hondel G.A. van Arkel 《Plasmid》1983,10(2):156-163
We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria. 相似文献