How Do Learner-Directed Scientific Investigations Influence Students’ Questioning and Their Nature of Science Conceptions?

Action research was conducted in two sections of a scientific inquiry class in a Midwestern U.S. university to identify the types of questions that students ask for their investigations, identify emerging patterns regarding students’ questioning, and determine students’ ability to transfer their nature of science (NOS) understanding to a new scientific phenomenon. Participants included 28 non-science major freshmen and sophomores. Data sources included the Earthworm Activity Field Guide, the post-reflection sheet, The Views of Nature of Science Version B Questionnaire (VNOS-B), and audio recordings of small group discussions when students were creating and selecting questions for the first and the second investigations when they were in the field. Findings showed that students improved their ability to ask more investigable and more specific questions after conducting an investigation. Also, students asked fewer descriptive and more cause-and-effect or pattern-seeking questions after the first investigation. The results showed that students were able to apply their understandings of NOS aspects to a new concept. However, some aspects were addressed more than the others. Results suggest that giving students opportunities to ask their own questions following their own interests improves their ability to generate good investigable questions. The results also suggest that reflecting on NOS aspects during content-related inquiry activities enhances students’ NOS understandings

Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal segmental glomerulosclerosis and normal glomeruli

Abstract Morphometric estimates of mean or individual glomerular volume (MGV, IGV) have biological implications, over and above qualitative histologic data. However, morphometry is time‐consuming and requires expertise limiting its utility in clinical cases. We evaluated MGV and IGV using plastic‐ and paraffin‐embedded tissue from 10 control and 10 focal segmental glomerulosclerosis (FSGS) mice (aging and 5/6th nephrectomy models) using the gold standard Cavalieri (Cav) method versus the 2‐profile and Weibel–Gomez (WG) methods and a novel 3‐profile method. We compared accuracy, bias and precision, and quantified results obtained when sampling differing numbers of glomeruli. In both FSGS and controls, we identified an acceptable precision for MGV of 10‐glomerular sampling versus 20‐glomerular sampling using the Cav method, while 5‐glomerular sampling was less precise. In plastic tissue, 2‐ or 3‐profile MGVs showed greater concordance with MGV when using Cav, versus MGV with WG. IGV comparisons using the same glomeruli reported a consistent underestimation bias with both 2‐ or 3‐profile methods versus the Cav method. FSGS glomeruli showed wider variations in bias estimation than controls. Our 3‐profile method offered incremental benefit to the 2‐profile method in both IGV and MGV estimation (improved correlation coefficient, Lin's concordance and reduced bias). In our control animals, we quantified a shrinkage artifact of 52% from tissue processed for paraffin‐embedded versus plastic‐embedded tissue. FSGS glomeruli showed overall reduced shrinkage albeit with variable artifact signifying periglomerular/glomerular fibrosis. A novel 3‐profile method offers slightly improved concordance with reduced bias versus 2‐profile. Our findings have implications for future studies using glomerular morphometry

HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy

Abstract Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis

Multiscale genetic architecture of donor-recipient differences reveals intronic LIMS1 mismatches associated with kidney transplant survival

Donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLA) contribute to kidney allograft loss, but mechanisms remain unclear, specifically for intronic mismatches. We quantified non-HLA mismatches at variant-, gene-, and genome-wide scales from SNP data of D- Rs from two well-phenotyped transplant cohorts: Genomics of Chronic Allograft Rejection (GoCAR; n=385) and Clinical Trials in Organ Transplantation-01/17 (CTOT-01/17; n=146). Unbiased gene-level screening in GoCAR uncovered the LIMS1 locus as the top-ranked gene where D-R mismatches associated with death-censored graft loss (DCGL). A previously unreported, intronic, LIMS1 haplotype of 30 SNPs independently associated with DCGL in both cohorts. Haplotype mismatches showed a dosage effect, and minor-allele introduction to major- allele-carrying recipients showed greater hazard of DCGL. The LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) in immune cells for GCC2 (not LIMS1), which encodes a protein involved in mannose-6-phosphase receptor (M6PR) recycling. Peripheral blood and T-cell transcriptome analyses associated GCC2 gene and LIMS1 SNPs with the TGFB1-SMAD pathway, suggesting a regulatory effect. In vitro GCC2 modulation impacted M6PR-dependent regulation of active TGFB1 and downstream signaling in T-cells. Together, our data link LIMS1 locus D-R mismatches to DCGL via GCC2 eQTLs that modulate TGFB1-dependent effects on T-cells

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.status: publishe

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection

BACKGROUND: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. METHODS: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. RESULTS: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. CONCLUSIONS: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients