Parental and professional perceptions of informed consent and participation in a time-critical neonatal trial: a mixed-methods study in India, Sri Lanka and Bangladesh

Introduction Time-critical neonatal trials in low-and-middle-income countries (LMICs) raise several ethical issues. Using a qualitative-dominant mixed-methods design, we explored informed consent process in Hypothermia for encephalopathy in low and middle-income countries (HELIX) trial conducted in India, Sri Lanka and Bangladesh.Methods Term infants with neonatal encephalopathy, aged less than 6 hours, were randomly allocated to cooling therapy or usual care, following informed parental consent. The consenting process was audio-video (A-V) recorded in all cases. We analysed A-V records of the consent process using a 5-point Likert scale on three parameters—empathy, information and autonomy. In addition, we used exploratory observation method to capture relevant aspects of consent process and discussions between parents and professionals. Finally, we conducted in-depth interviews with a subgroup of 20 parents and 15 healthcare professionals. A thematic analysis was performed on the observations of A-V records and on the interview transcripts.Results A total of 294 A-V records of the HELIX trial were analysed. Median (IQR) score for empathy, information and autonomy was 5 (0), 5 (1) and 5 (1), respectively. However, thematic analysis suggested that the consenting was a ceremonial process; and parental decision to participate was based on unreserved trust in the treating doctors, therapeutic misconception and access to an expensive treatment free of cost. Most parents did not understand the concept of a clinical trial nor the nature of the intervention. Professionals showed a strong bias towards cooling therapy and reported time constraints and explaining to multiple family members as key challenges.Conclusion Despite rigorous research governance and consent process, parental decisions were heavily influenced by situational incapacity and a trust in doctors to make the right decision on their behalf. Further research is required to identify culturally and context-appropriate strategies for informed trial participation

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

<div><p><i>ATHB17</i> (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.</p></div

ATHB17 is a member of the α subclass within the HD-Zip II protein family.

<p>(A) represents the dendrogram and the domain architecture of the ATHB17 homologs. ATHB17 contains a typical homeodomain (HD; blue shading) and a leucine zipper motif (LZ; green shading) adjacent to the C-terminus of the HD. Red bars indicate conserved cysteines in the C-terminus. (B) shows the protein sequence of ATHB17. ATHB17 contains a unique N-terminal extension (red shading) rich in cysteines and tyrosines. Additional structural feature identified for ATHB17 is a nuclear localization signal (red boxes). Downstream of the LZ motif is a putative redox sensing motif (CPXCE; red letters).</p

Phenology of <i>ATHB17</i> events and control.

<p>Two independent <i>ATHB17</i> events in three hybrids were used in physiological studies conducted in 2011 and 2012 under standard agricultural practices (SAP) for corn production in the Central Corn Belt. The number of days to 50% silking and anthesis were measured and the number of days between anthesis and silking was calculated (ASI) each year for physiological studies conducted under standard agronomic practices conditions. Differences in phenology between <i>ATHB17</i> events and control were determined using an across year combined analysis using a mixed model ANOVA. N denotes the number of data points included per entry in the statistical analysis. Number of event data points were within ±3 of control data points. Results for individual hybrids per year are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094238#pone.0094238.s004" target="_blank">Table S2</a>.</p

List of differentially expressed genes in ear inflorescence in common between two <i>ATHB17</i> events.

<p>Table lists the fold change (Fc), raw and fdr p-values for common genes in each event.</p

List of differentially expressed genes in ear in common between two <i>ATHB17</i> events.

<p>Table lists the fold change (Fc), raw and fdr p-values for common genes in each event.</p

ATHB17Δ113 can bind both Class II and Class I DNA targets and ATHB17Δ113 containing V182A-Q185A-N186A mutation cannot bind Class II DNA target in <i>in vitro</i> assay (measured by Surface Plasmon Resonance (SPR).

<p>Binding affinities and Kinetic constants of ATHB17Δ113 interacting with Class I and Class II type DNA, measured by Biacore 2000, globally fitted. SPR measurements with Biacore 2000 were at 25°C in HBS-EP, 100 ug/ml BSA (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20,100 ug/ml BSA). Equilibrium dissociation constant K_D = k_off/k_on.</p

ATHB17Δ113 is localized to nucleus.

<p>Fluorescence micrographs of maize leaf protoplasts transformed with (A) GFP alone, (B) AtCBF3 fused to GFP as positive control for nuclear localization or (C) ATHB17Δ113 fused with GFP. Protoplasts from each construct were analyzed by laser scanning microscopy for GFP and chlorophyll fluorescence and acquired images were merged on bright-field image using the Image examiner (See Materials and Methods for details).</p

Full- length ATHB17 protein functions as transcriptional repressor and ATHB17Δ113 can relieve repression caused by full-length ATHB17 protein.

<p>Maize mesophyll protoplasts were transformed (A) with 4 µg cells of reporter (Class II::GUS, Class I::GUS or No BS::GUS) and 0–5 µg cells of effector (Full-length ATHB17) or 5 µg of ATHB17Δ113 and <i>Renilla</i> luciferase (B) with 4 µg reporter (Class II::GUS, Class I::GUS or No BS::GUS), 0–5 µg ATHB17Δ113, and 0 (grey bars) or 0.2 µg (blue bars) of ATHB17 full length. DNA amounts are per 320,000 cells. After 18 h, cells were assayed for GUS and luciferase expression. GUS values were divided by luciferase internal control values for each well and normalized to respective GFP samples. Bars are means and error bars represent 1 SD.</p