Intracellular localization and interaction of mRNA binding proteins as detected by FRET

<p>Abstract</p> <p>Background</p> <p>A number of RNA binding proteins (BPs) bind to A+U rich elements (AREs), commonly present within 3'UTRs of highly regulated RNAs. Individual RNA-BPs proteins can modulate RNA stability, RNA localization, and/or translational efficiency. Although biochemical studies have demonstrated selectivity of ARE-BPs for individual RNAs, less certain is the <it>in vivo </it>composition of RNA-BP multiprotein complexes and how their composition is affected by signaling events and intracellular localization. Using FRET, we previously demonstrated that two ARE-BPs, HuR and AUF1, form stable homomeric and heteromeric associations in the nucleus and cytoplasm. In the current study, we use immuno-FRET of endogenous proteins to examine the intracellular localization and interactions of HuR and AUF1 as well as KSRP, TIA-1, and Hedls. These results were compared to those obtained with their exogenously expressed, fluorescently labeled counterparts.</p> <p>Results</p> <p>All ARE-BPs examined were found to colocalize and to form stable associations with selected other RNA-BPs in one or more cellular locations variably including the nucleus, cytoplasm (in general), or in stress granules or P bodies. Interestingly, FRET based interaction of the translational suppressor, TIA-1, and the decapping protein, Hedls, was found to occur at the interface of stress granules and P bodies, dynamic sites of intracellular RNA storage and/or turnover. To explore the physical interactions of RNA-BPs with ARE containing RNAs, <it>in vitro </it>transcribed Cy3-labeled RNA was transfected into cells. Interestingly, Cy3-RNA was found to coalesce in P body like punctate structures and, by FRET, was found to interact with the RNA decapping proteins, Hedls and Dcp1.</p> <p>Conclusions</p> <p>Biochemical methodologies, such as co-immunoprecipitation, and cell biological approaches such as standard confocal microscopy are useful in demonstrating the possibility of proteins and/or proteins and RNAs interacting. However, as demonstrated herein, colocalization of proteins and proteins and RNA is not always indicative of interaction. To this point, using FRET and immuno-FRET, we have demonstrated that RNA-BPs can visually colocalize without producing a FRET signal. In contrast, proteins that appear to be delimited to one or another intracellular compartment can be shown to interact when those compartments are juxtaposed.</p

Optic flow improves step width and length in older adults while performing dual task

Background Dual-task paradigms are used to investigate gait and cognitive declines in older adults (OA). Optic-flow is a virtual reality environment where the scene flows past the subject while walking on a treadmill, mimicking real-life locomotion. Aims To investigate cost of environment (no optic-flow v. optic-flow) while completing single- and dual-task walking and dual-task costs (DTC; single- v. dual-task) in optic-flow and no optic-flow environments. Methods Twenty OA and seven younger adults (YA) walked on a self-paced treadmill in 3-min segments per task and both environments. Five task conditions included: no task, semantic fluency (category), phonemic fluency (letters), word reading, and serial-subtraction. Results OAs had a benefit of optic-flow compared to no optic-flow for step width (p = 0.015) and step length (p = 0.045) during letters compared to the YA. During letters, OA experienced improvement in step width DTC; whereas YA had a decrement in step width DTC from no optic-flow to optic-flow (p = 0.038). During serial-subtraction, OA had less step width DTC when compared to YA in both environments (p = 0.02). Discussion During letters, step width and step length improved in OA while walking in optic-flow. Also, step width DTC differed between the two groups. Sensory information from optic-flow appears to benefit OA. Letters relies more on verbal ability and word knowledge, which are preserved in aging. However, YA use a complex speech style during dual tasking, searching for complex words and an increased speed of speech. Conclusions OA can benefit from optic-flow by improving spatial gait parameters, specifically, step width, during dual-task walking

Fibroblast Growth Factor Receptor Splice Variants are Stable Markers of Oncogenic Transforming Growth Factor β1 Signaling in Metastatic Breast Cancers.

Introduction Epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle. Methods An in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor β1 (TGF-β1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-β1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398. Results Metastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-β1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-β stably upregulate a truncated FGFR1-β splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-β was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth. Conclusion The results of our study strongly suggest that FGFR1-β is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitor

Consideration of Shared Decision Making in Nursing: A Review of Clinicians’ Perceptions and Interventions

As the number of individuals with chronic illness increases so has the need for strategies to enable nurses to engage them effectively in daily management of their conditions. Shared decision making between patients and nurses is one approach frequently discussed in the literature. This paper reviews recent studies of shared decision making and the meaning of findings for the nurse-patient relationship. Patients likely to prefer to engage in shared decision making are younger and have higher levels of education. However, there is a lack of evidence for the effect of shared decision making on patient outcomes. Further, studies are needed to examine shared decision making when the patient is a child. Nurses are professionally suited to engage their patients fully in treatment plans. More evidence for how shared decision making affects outcomes and how nurses can successfully achieve such engagement is needed

Culturable Root Endophyte Communities are Shaped by Both Warming and Plant Host Identity in the Rocky Mountains, USA

Understanding the biogeographic patterns of root-associated fungi and their sensitivity to temperature may improve predictions of future changes in terrestrial biodiversity and associated ecosystem processes, but data are currently limited. Anticipating change will require combining observational data, which predict how climatic factors limit current species distributions, with direct manipulations of climate, which can isolate responses to specific climate variables. Root endophytes are common symbionts of plants, particularly in arctic and alpine environments, yet their responses to climate warming are not resolved. Here, we directly cultured endophytic fungi from roots collected along altitudinal gradients in replicated mountain watersheds and from a 27 y field warming experiment in the Rocky Mountains, USA, to improve understanding of climate impacts on fungal root endophytes. Fungal taxa that were common at high elevations declined most under climate warming, whereas low elevation dominants responded neutrally or increased with experimental warming. Altitudinal gradients in fungal communities were strongly specific to the plant host species. Specifically, Poa species had 25–60% greater fungal isolate abundance and 25–38% greater fungal diversity at high elevations than at low elevation sites. In contrast, Festuca thurberi had 64% lower fungal diversity on roots at high elevation than at low elevation. Our results help to improve understanding of the potential for climate change to alter plant-fungal interactions in mountain ecosystems

Pathways to Belonging and Engagement: Testing a Tailored Social Belonging Intervention for University Students

Background Prominent theories of motivation suggest that belonging plays a critical role in student success (Connell & Wellborn, 1991). Social-belonging interventions have been shown to improve student belonging, well-being, engagement, and more—especially those from traditionally disadvantaged backgrounds (Walton & Brady, 2017). The current study aimed to explore the effects of a tailored social-belonging intervention delivered in introductory classes at VCU on students’ belonging, engagement, persistence, and achievement. Methods A diverse sample of first-year undergraduate students at VCU participated. To create authentic intervention materials, we collaborated with a diverse group of upper-level undergraduate student researchers who wrote narratives to present vivid stories of how they personally experienced and overcame struggles to belong. Prior to and following the intervention, students completed a survey that assessed student belonging, engagement, and social and academic fit. We also collected student demographics, achievement, and additional data from institutional records. Results Following the implementation of the belonging intervention, data was collected on students’ sense of belonging, their social and academic fit at the university, and other related outcomes. While most students felt as though they belonged at VCU and had the potential to succeed, there were still some students who worried whether they belonged in college. Conclusions From students’ responses, faculty and advisors of first-year students were given an overview on students’ current states of belonging at VCU. As an implication for future research, we argue that including diverse upper-level students as fellow researchers in this work strengthens the authenticity and effectiveness of the belonging intervention.https://scholarscompass.vcu.edu/gradposters/1171/thumbnail.jp

Bodyweight Perceptions among Texas Women: The Effects of Religion, Race/Ethnicity, and Citizenship Status

Despite previous work exploring linkages between religious participation and health, little research has looked at the role of religion in affecting bodyweight perceptions. Using the theoretical model developed by Levin et al. (Sociol Q 36(1):157–173, 1995) on the multidimensionality of religious participation, we develop several hypotheses and test them by using data from the 2004 Survey of Texas Adults. We estimate multinomial logistic regression models to determine the relative risk of women perceiving themselves as overweight. Results indicate that religious attendance lowers risk of women perceiving themselves as very overweight. Citizenship status was an important factor for Latinas, with noncitizens being less likely to see themselves as overweight. We also test interaction effects between religion and race. Religious attendance and prayer have a moderating effect among Latina non-citizens so that among these women, attendance and prayer intensify perceptions of feeling less overweight when compared to their white counterparts. Among African American women, the effect of increased church attendance leads to perceptions of being overweight. Prayer is also a correlate of overweight perceptions but only among African American women. We close with a discussion that highlights key implications from our findings, note study limitations, and several promising avenues for future research

FLNC Gene Splice Mutations Cause Dilated\ua0Cardiomyopathy

OBJECTIVE: To identify novel dilated cardiomyopathy (DCM) causing genes, and to elucidate the pathological mechanism leading to DCM by utilizing zebrafish as a model organism. BACKGROUND: DCM, a major cause of heart failure, is frequently familial and caused by a genetic defect. However, only 50% of DCM cases can be attributed to a known DCM gene variant, motivating the ongoing search for novel disease genes. METHODS: We performed whole exome sequencing (WES) in two multigenerational Italian families and one US family with arrhythmogenic DCM without skeletal muscle defects, in whom prior genetic testing had been unrevealing. Pathogenic variants were sought by a combination of bioinformatic filtering and cosegregation testing among affected individuals within the families. We performed function assays and generated a zebrafish morpholino knockdown model. RESULTS: A novel filamin C gene splicing variant (FLNC c.7251+1 G>A) was identified by WES in all affected family members in the two Italian families. A separate novel splicing mutation (FLNC c.5669-1delG) was identified in the US family. Western blot analysis of cardiac heart tissue from an affected individual showed decreased FLNC protein, supporting a haploinsufficiency model of pathogenesis. To further analyze this model, a morpholino knockdown of the ortholog filamin Cb in zebrafish was created which resulted in abnormal cardiac function and ultrastructure. CONCLUSIONS: Using WES, we identified two novel FLNC splicing variants as the likely cause of DCM in three families. We provided protein expression and in vivo zebrafish data supporting haploinsufficiency as the pathogenic mechanism leading to DCM

Surface Sampling Methods for Bacillus anthracis Spore Contamination

During an investigation conducted December 17–20, 2001, we collected environmental samples from a U.S. postal facility in Washington, D.C., known to be extensively contaminated with Bacillus anthracis spores. Because methods for collecting and analyzing B. anthracis spores have not yet been validated, our objective was to compare the relative effectiveness of sampling methods used for collecting spores from contaminated surfaces. Comparison of wipe, wet and dry swab, and HEPA vacuum sock samples on nonporous surfaces indicated good agreement between results with HEPA vacuum and wipe samples. However, results from HEPA vacuum sock and wipe samples agreed poorly with the swab samples. Dry swabs failed to detect spores >75% of the time they were detected by wipe and HEPA vacuum samples. Wipe samples collected after HEPA vacuum samples and HEPA vacuum samples after wipe samples indicated that neither method completely removed spores from the sampled surfaces

ADRB2 Arg16Gly Polymorphism, Lung Function, and Mortality: Results from the Atherosclerosis Risk in Communities Study

BACKGROUND: Growing evidence suggests that the Arg16Arg genotype of the beta-2 adrenergic receptor gene may be associated with adverse effects of beta-agonist therapy. We sought to examine the association of beta-agonist use and the Arg16Gly polymorphism with lung function and mortality among participants in the Atherosclerosis Risk in Communities study. METHODOLOGY AND PRINCIPAL FINDINGS: We genotyped study participants and analyzed the association of the Arg16Gly polymorphism and beta-agonist use with lung function at baseline and clinical examination three years later and with all-cause mortality during 10 years of follow-up. Lung function was characterized by percent-predicted forced expiratory volume in 1 second. Associations were examined separately for blacks and whites. Black beta-agonist users with the Arg/Arg genotype had better lung function at baseline and at the second clinical visit than those with Arg/Gly and Gly/Gly genotypes. Adjusted mean percent-predicted FEV(1) was 21% higher in Arg/Arg subjects compared to Gly/Gly at baseline (p = 0.01) and 20% higher than Gly/Gly at visit 2 (p = 0.01). Arg/Gly subjects had adjusted percent-predicted FEV(1) 17% lower than Arg/Arg at baseline but were similar to Arg/Arg subjects at visit 2. Although black beta-agonist users with the Arg/Arg genotype appeared to have better crude survival rates, the association between genotype and all-cause mortality was inconclusive. We found no difference in lung function or mortality by genotype among blacks who did not use beta-agonists or among whites, regardless of beta-agonist use. CONCLUSIONS: Black beta-agonist users with the ADRB2 Arg16Arg genotype had better lung function, and, possibly, better overall survival compared to black beta-agonist users with the Gly16Gly genotype. Our findings highlight the need for additional studies of sufficient size and statistical power to allow examination of outcomes among beta-agonist users of different races and genotypes