631 rankl knock out mesenchymal stromal cells have an unexpected osteogenic differentiation defect which is improved by a rankl expressing lentiviral vector

Osteoclast-poor RANKL-dependent Autosomal Recessive Osteopetrosis (ARO) is a rare bone disease characterized by an increase in bone density due to the failure of bone resorption by impaired osteoclast formation. Hematopoietic stem cell transplantation is not an effective therapy for this ARO form, since in bone RANKL is produced mainly by cells of mesenchymal origin. Therefore Mesenchymal Stromal Cells (MSC) transplantation together with a gene-therapy strategy to correct RANKL defect in MSC could represent a possible effective therapy. Of note, whether also MSC, besides the osteoclasts, are affected by RANKL deficiency is unknown. To verify this, we established and characterized bone marrow derived MSC (BM-MSC) lines from the Rankl−/− (KO) mouse model, which recapitulates the human disease, and from wild type (WT) mice. No differences were found between KO and WT MSC in terms of morphology, immunophenotype and proliferation capacity. However, KO MSC displayed a reduced clonogenic potential with a decrease in stemness genes expression. KO MSC were able to normally differentiate towards the adipogenic and chondrogenic lineages, while showed a significantly impaired osteogenic differentiation capacity compared to WT MSC, as demonstrated by reduced Alizarin Red staining (ARS) and expression of osteogenic genes. To confirm that this alteration was due to the lack of functional RANKL, we developed a third generation lentiviral vector expressing human soluble RANKL (hsRL) for the genetic correction of KO MSC. We first investigated lentiviral transduction in 293T cells to optimize transduction efficiency at different multiplicity of infection (MOI) ranging from 1 to 100. hsRL production increased proportionally to the MOI and was stable over time. However, the higher the MOI the higher the cytotoxicity observed. Based on these data, we performed a lentiviral hsRL transduction in KO MSC at 20 and 50 MOI, to define the optimal transduction conditions. After transduction 99.5% of MSC were GFP+. While in Rankl−/− control cells the cytokine was not detected, in corrected cells hsRL production and secretion was measurable and comparable to sRL levels in WT mouse. KO MSC stably expressing hsRL showed an improved osteogenic differentiation capacity compared to untransduced KO MSC, as demonstrated by increased ARS and expression of osteogenic genes. Moreover, the expression of RANK receptor in both MSC suggested an autocrine role of sRL as possible mechanism. Our data suggest that restoration of RANKL production in lentiviral-transduced KO MSC might not only allow osteoclast differentiation in Rankl−/− mice upon transplantation, but also improve the osteogenic differentiation defect of KO MSC

Contextual Coding in Qualitative Research Involving Participants with Diverse Sociocultural Backgrounds

Understanding participants’ perspectives in qualitative research is contingent on unravelling the essential meaning of their speech. When data are collected in native language and translated into English language, the underlying sociocultural meaning of participants’ speech can be missed. This paper discusses a new contextual coding approach and illustrates its application in research. The technique was used in a phenomenological study in Pakistan and a mixed methods study in Europe. Contextual coding entails a preliminary coding stage involving data reading in native language, choosing socially and culturally relevant words and phrases, and developing preliminary codes. The concluding coding stage focuses on creating a sociocultural query list, seeking answers through discussions among multilingual individuals, and finding a common language for code description. Contextual coding can enable researchers to understand sociocultural meaning of their data at an early stage, rather than waiting at the later stage of theme development to contextualize the findings

Using the Very Short Form of the Children’s Behavior Questionnaire for Spanish-Speaking Populations in Low- and Middle-Income Countries: A Psychometric Analysis of Dichotomized Variables

While the psychometric properties of the Spanish version of the Very Short Form of the Children’s Behavior Questionnaire (CBQ-VSF) have been assessed in the US and Europe in samples composed of middle- and high-income parents with high levels of education, no studies have tested the instrument in low-income Spanish-speaking populations living in low- and middle- income countries. To fill this gap, our cross-sectional study assessed the psychometric properties of the Spanish version of the CBQ-VSF version in a sample of 315 low-income and low-educated parents with preschool children living in the Caribbean Region of Colombia. While our findings revealed problems that were similar to those identified in previous assessments of the CBQ-VSF Spanish version, they also showed unique problems related to the sociodemographic characteristics of our sample, containing many individuals with a low income and low educational level. Most of the participants gave extreme responses, resulting in a notable kurtosis and skewness of the data. This article describes how we addressed these problems by dichotomizing the variables into binary categories. Additionally, it demonstrates that merely translating the CBQ-VSF is insufficient to be able to capture many of the underlying latent constructs associated with low-income and low-educated Latino/Hispanic populations

731. Hematopoietic Stem Cell Gene Transfer and Integration Site Analysis in Tumor-Prone Mice Uncovers Low Genotoxicity of Lentiviral Vector Integration

Insertional mutagenesis represents a major hurdle to successful gene therapy and mandates for sensitive pre-clinical assays of genotoxicity. Cdkn2a|[minus]|/|[minus]| mice are defective for p53 and Rb pathways, and are susceptible to a broad range of cancer-triggering genetic lesions. We exploited the sensitivity of these tumor-prone mice to develop an in-vivo genotoxicity assay, based on transplantation of Cdkn2a|[minus]|/|[minus]| hematopoietic stem cells (HSC), treated or not with prototypical retroviral (RV) and lentiviral (LV) vectors. In our rationale if RV or LV treatment is genotoxic, then transplanted mice will show a significantly earlier tumor onset. The sensitivity of the model was shown by the ability to detect a vector dose-dependent acceleration in tumor onset in mice transplanted with RV-treated cells. Such acceleration, as in previous studies, is consequent to genetic lesions, produced by vector integration, that cooperate with the germ-line mutation, and is contingent on LTR activity

Taking a critical stance towards mixed methods research: A cross-disciplinary qualitative secondary analysis of researchers’ views

Recent growth and institutionalization in the field of mixed methods research has provided fertile ground for a wide range of thoughtful criticism of how this research approach has been developed and conceptualized by some members of the mixed methods community. This criticism reflects the increasing maturity of the field as well as the different theoretical perspectives and methodological practices of researchers in different disciplines. While debates addressing these criticisms are likely to lead to valuable insights, no empirical studies have been carried out to date that have investigated researchers’ critical views on the development and conceptualization of mixed methods research. This study examines the criticisms of the mixed methods field raised by a cross-national sample of researchers in education, nursing, psychology, and sociology. We carried out a secondary analysis of semi-structured interviews with 42 researchers and identified 11 different criticisms, which we classified in four domains: essence of mixed methods, philosophy, procedures, and politics. The criticisms related to the procedures domain were equally distributed among the four disciplines, while those related to the essence, philosophy and politics domains were more common among sociologists. Based on our findings, we argue that the divergence of views on foundational issues in this field reflects researchers’ affiliation to different communities of practice, each having its own principles, values, and interests. We suggest that a greater awareness of this divergence of perspectives could help researchers establish effective collaboration and anticipate potential challenges when working with researchers having different methodological approaches

Numerical simulation of a rapid fatigue test of high Mn-TWIP steel via a high cycle fatigue constitutive law

The generation of reliable data in the high cycle fatigue domain is crucial to support further metallurgic developments of fatigue optimized steel grades. Commonly employed for this aim, traditional standardized characterization methods are expensive and time-consuming. Thus, to circumvent these limitations, different accelerated fatigue testing methodologies have been proposed. In this work, the rapid fatigue test based on stiffness evolution is numerically reproduced using the finite element method for a specific grade of twinning-induced plasticity steel. A high cycle fatigue constitutive law grounded on the continuum damage mechanics framework is employed for this purpose. To adequately capture the material non-linear behavior observed in the experiments, a novel hardening–softening stress–strain curve for damage is proposed. The entire load history in the fatigue domain is modeled. A cycle-jump algorithm is used to improve the computational efficiency of the simulations. It is shown that a reduction of about 55% in the analysis elapsed time is reached by using this algorithm, while the result accuracy is maintained. Finally, the good agreement between numerical and experimental results, revealed by a maximum relative error smaller than 6.0%, evidences the potential of the present constitutive formulation to model the behavior of metals in the high cycle fatigue domain.This work has been done within the framework of the Fatigue4Light (H2020-LC-GV-06-2020) project: “Fatigue modeling and fast testing methodologies to optimize part design and to boost lightweight materials deployment in chassis parts”. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 101006844. The work has been also supported by the Spanish Government program, Spain FPU17/04196. The authors gratefully acknowledge all the received support. Finally, they acknowledge the support received by the Severo Ochoa Centre of Excellence (2019-2023, Spain under the grant CEX2018-000797-S funded by MCIN/AEI/10.13039/501100011033, Spain .Peer ReviewedPostprint (published version

Epidermal growth factor receptor expression identifies functionally and molecularly distinct tumor-initiating cells in human glioblastoma multiforme and is required for gliomagenesis

Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFR(pos) and EGFR(neg) TIC subpopulations within the same tumor might affect clinical response to treatment

Use of mixed methods research in intervention studies to increase young people’s interest in STEM: A systematic methodological review

IntroductionMixed methods research intervention studies integrate quantitative evaluation approaches, such as randomized controlled trials and quasi-experimental designs, with qualitative research to evaluate the effectiveness, efficacy, or other results of an intervention or program. These types of studies, which have attracted growing attention in recent years, enhance the scope and rigor of the evaluation. While various frameworks that summarize the justifications for carrying out these types of studies and provide implementation guidance have been published in the last few years in the health sciences, we do not know whether such frameworks have been properly implemented in the social and educational sciences. This review examined the methodological features and reporting practices of mixed methods intervention studies aimed at increasing young people’s interest in STEM.MethodsA systematic search was carried out in APA PsycNET, ERIC, ProQuest, Scopus, and Web of Science, and a hand search in 20 journals. We included peer-reviewed English-language articles that reported intervention studies with a quantitative component measuring outcomes specific to increasing secondary school students’ interest in STEM fields, a qualitative component conducted before, during, or after the quantitative component, and evidence of integration of both components. Qualitative content analysis and ideal-type analysis were used to synthesize the findings.ResultsWe found 34 studies; the majority published in the last ten years. Several patterns of mixed methods application were described in these studies, illustrating the unique insights that can be gained by employing this methodology. The reporting quality of the included studies was generally adequate, especially regarding the justification for using a mixed methods intervention design and the integration of the quantitative and qualitative components. Nonetheless, a few reporting issues were observed, such as a lack of detail in the presentation of the mixed methods design, an inadequate description of the qualitative sampling and analysis techniques, and the absence of joint displays for representing integration.DiscussionAuthors must pay attention to these issues to ensure that the insights obtained by the use of mixed methods research are effectively communicated

Fasting glucose and body mass index as predictors of activity in breast cancer patients treated with everolimus-exemestane: the EverExt study

Evidence on everolimus in breast cancer has placed hyperglycemia among the most common high grade adverse events. Anthropometrics and biomarkers of glucose metabolism were investigated in a observational study of 102 postmenopausal, HR + HER2- metastatic breast cancer patients treated with everolimus-exemestane in first and subsequent lines. Best overall response (BR) and clinical benefit rate (CBR) were assessed across subgroups defined upon fasting glucose (FG) and body mass index (BMI). Survival was estimated by Kaplan-Meier method and log-rank test. Survival predictors were tested in Cox models. Median follow up was 12.4 months (1.0-41.0). The overall cohort showed increasing levels of FG and decreasing BMI (p < 0.001). Lower FG fasting glucose at BR was more commonly associated with C/PR or SD compared with PD (p < 0.001). We also observed a somewhat higher BMI associated with better response (p = 0.052). More patients in the lowest FG category achieved clinical benefit compared to the highest (p < 0.001), while no relevant differences emerged for BMI. Fasting glucose at re-assessment was also predictive of PFS (p = 0.037), as confirmed in models including BMI and line of therapy (p = 0.049). Treatment discontinuation was significantly associated with changes in FG (p = 0.014). Further research is warranted to corroborate these findings and clarify the underlying mechanisms