Structural and Functional Studies of Proteins Involved in Antigen Processing: A Dissertation

This thesis is comprised of studies of proteins involved in class I and class II major histocompatibility complex (MHC) antigen procressing. In class I MHC processing, structural and functional studies were conducted of an aminopeptidase, ERAP1, that mediates the final step in antigen processing to understand how it is particularly suitable for cleavage of antigenic peptides for class I MHC presentation. In the class II MHC antigen presentation pathway, structural studies were conducted to characterize a fluorogenic peptide that can be used to understand peptide loading events in vivo and in real time. Also structural studies of class II MHC and peptide complexes were conducted to understand the nature of an unique C-terminal secondary structure element exhibited by an HIV derived peptide in the peptide binding groove of class II MHC. The studies discussed in this thesis provide insights into the proteins involved in the class I and class II MHC antigen presentation pathway. The endoplasmic reticulum (ER) aminopeptidase, ERAP1, is a 941 amino acid member of the M1 family of zinc metalloaminopeptidases. Unlike other aminopeptidases, ERAP1 has a length and C-terminal preference for its substrates. Interestingly, ERAP1 has been shown to trim antigenic peptides to lengths of 8 or 9 amino acids long. This length matches the length required to bind into the peptide binding groove of class I MHC molecules. In addition, ERAP1 is upregulated in the ER of cells treated with interferon gamma (IFN-γ). Knock-down of ERAP1 by siRNA results in less overall antigenic presentation during IFN-γ treatment, although the knock-down does not affect all class I MHC epitopes equally. Knock-out studies show that ERAP1 effects the antigen repertoire at the cell surface. These and other data implicate ERAP1 as an important player in class I MHC antigen presentation. A chapter of this thesis will describe the crystallographic work describing the structures of ERAP1 with an aminopeptidase inhibitor, bestatin, and ERAP1 without an inhibitor that suggest possible peptide binding site in ERAP1 that will allow it to generate suitable substrates for a subset of class I MHC alleles. Class II MHC plays a key role in the immune response by presenting antigenic peptides on CD4+ cytotoxic cell surfaces for T-cell response. The binding of peptides onto the MHC is an important step in creating an immune response. Structures of peptide bound MHC class II show conserved side chain binding pockets within the overall peptide-binding groove. In HLA-DR1, a common human class II MHC, the P1 pocket shows a preference for large hydrophobic side chains. Development of environmentally sensitive peptide analogs, that can bind into the class II MHC the same way as native peptides, can assist in visualizing the antigen binding process. A chapter in this thesis describes the crystallographic work showing that (4-DAPA)-HA can be used to study antigen-presenting processes in a cell by visualizing the changes in fluorescence of the synthesized peptide upon antigen loading. Crystallographic analysis of MHC class II, HLA-DR1, in complex with HIV gag-derived peptide, GagP16(PEVIPMFSALSEGATP), and superantigen, SEC3- 3B2, reveals the conventional polyproline conformation up to MHC binding pocket residue, P9, while the C-terminus of GagP16 adopts an unusual β- hairpin loop structure. Additionally, interactions between the leucine at P8 (LeuP8) and other residues on the loop such as ThrP16 and AlaP14 of the hairpin loop, was observed. Importantly, GagP16 requires the last 4 amino acids (P13-P16), which is part of the hairpin loop, for T-cell recognition. Understanding what dictates the C-terminal hairpin loop and the interaction motif of HLA-DR1/GagP16 complex with its TCR will provide insights on why it is important for T cell activation. A chapter in this thesis discusses the structural investigation conducted to understand the determinants of the loop at the C-terminus of GagP16 using designed peptides. It will also discuss work involving HLA-DR1 with the T cell receptor, AC25, that was cloned from T cells that are specific to HLA-DR1 in complex with the GagP16 peptide

Last Semester’s Enduring Impressions

The increased recollection of events occurring at the beginning and end of a period, known as the end point effect, is a phenomenon widely reported in the autobiographical memory literature. We used an interview method to explore the end point effect and to find out what impressions students retain for an academic semester. Participants recounted recollections of events from an academic course that was completed in their immediately preceding semester, and then they rated each recollection on a variety of attributes such as emotionality and vividness. The results showed recollection peaks corresponding to the semester midpoint as well as corresponding to the endpoints of the semester; the results also showed that distinct content defined recollections contributed to the mid- and endpoints. The discussion focuses on the qualitative versus quantitative examination of autobiographical memory

Prediction and confirmation of a switch-like region within the N-terminal domain of hSIRT1

Many proteins display conformational changes resulting from allosteric regulation. Often only a few residues are crucial in conveying these structural and functional allosteric changes. These regions that undergo a significant change in structure upon receiving an input signal, such as molecular recognition, are defined as switch- like regions. Identifying these key residues within switch-like regions can help elucidate the mechanism of allosteric regulation and provide guidance for synthetic regulation. In this study, we combine a novel computational workflow with biochemical methods to identify a switch-like region in the N-terminal domain of human SIRT1 (hSIRT1), a lysine deacetylase that plays important roles in regulating cellular pathways. Based on primary sequence, computational methods predicted a region between residues 186–193 in hSIRT1 to exhibit switch-like behavior. Mutations were then introduced in this region and the resulting mutants were tested for allosteric reactions to resveratrol, a known hSIRT1 allosteric regulator. After fine-tuning the mutations based on comparison of known secondary structures, we were able to pinpoint M193 as the residue essential for allosteric regulation, likely by communicating the allosteric signal. Mutation of this residue maintained enzyme activity but abolished allosteric regulation by resveratrol. Our findings suggest a method to predict switch-like regions in allosterically regulated enzymes based on the primary sequence. If further validated, this could be an efficient way to identify key residues in enzymes for therapeutic drug targeting and other applications

Large-Scale Behavior-Change Initiative for Infant and Young Child Feeding Advanced Language and Motor Development in a Cluster-Randomized Program Evaluation in Bangladesh

Background: Promoting adequate nutrition through interventions to improve infant and young child feeding (IYCF) has the potential to contribute to child development. Objective: We examined whether an intensive intervention package that was aimed at improving IYCF at scale through the Alive & Thrive initiative in Bangladesh also advanced language and gross motor development, and whether advancements in language and gross motor development were explained through improved complementary feeding. Methods: A cluster-randomized design compared 2 intervention packages: intensive interpersonal counseling on IYCF, mass media campaign, and community mobilization (intensive) compared with usual nutrition counseling and mass media campaign (nonintensive). Twenty subdistricts were randomly assigned to receive either the intensive or the nonintensive intervention. Household surveys were conducted at baseline (2010) and at endline (2014) in the same communities (n = ∼4000 children aged 0–47.9 mo for each round). Child development was measured by asking mothers if their child had reached each of multiple milestones, with some observed. Linear regression accounting for clustering was used to derive difference-in-differences (DID) impact estimates, and path analysis was used to examine developmental advancement through indicators of improved IYCF and other factors. Results: The DID in language development between intensive and nonintensive groups was 1.05 milestones (P = 0.001) among children aged 6–23.9 mo and 0.76 milestones (P = 0.038) among children aged 24–47.9 mo. For gross motor development, the DID was 0.85 milestones (P = 0.035) among children aged 6–23.9 mo. The differences observed corresponded to age- and sex-adjusted effect sizes of 0.35 for language and 0.23 for gross motor development. Developmental advancement at 6–23.9 mo was partially explained through improved minimum dietary diversity and the consumption of iron-rich food. Conclusions: Intensive IYCF intervention differentially advanced language and gross motor development, which was partially explained through improved complementary feeding. Measuring a diverse set of child outcomes, including functional outcomes such as child development, is important when evaluating integrated nutrition programs. This trial was registered at clinicaltrials.gov as NCT01678716

CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) for Near-Perfect Selective Transformation

The CRISPR (Clustered, Regularly Interspaced, Short Palindromic Repeats)/Cas9 system has revolutionized genome editing by providing unprecedented DNA-targeting specificity. Here we demonstrate that this system can be also applied in vitro to fundamental cloning steps to facilitate efficient plasmid selection for transformation and selective gene insertion into plasmid vectors by cleaving unwanted plasmid byproducts with a single-guide RNA (sgRNA)-Cas9 nuclease complex. Using fluorescent and chromogenic proteins as reporters, we demonstrate that CRISPR/Cas9 cleavage excludes multiple plasmids as well as unwanted ligation byproducts resulting in an unprecedented increase in the transformation success rate from approximately 20% to nearly 100%. Thus, this CRISPR/Cas9-Assisted Transformation-Efficient Reaction (CRATER) protocol is a novel, inexpensive, and convenient application to conventional molecular cloning to achieve near-perfect selective transformation

Combining Intensive Counseling by Frontline Workers With a Nationwide Mass Media Campaign Has Large Differential Impacts on Complementary Feeding Practices but Not on Child Growth: Results of a Cluster-Randomized Program Evaluation in Bangladesh

Background: Complementary feeding (CF) contributes to child growth and development, but few CF programs are delivered at scale. Alive & Thrive addressed this in Bangladesh through intensified interpersonal counseling (IPC), mass media (MM), and community mobilization (CM). Objective: The objective was to evaluate the impact of providing IPC + MM + CM (intensive) compared with standard nutrition counseling + less intensive MM + CM (nonintensive) on CF practices and anthropometric measurements. Methods: We used a cluster-randomized, nonblinded evaluation with cross-sectional surveys [n = ∼600 and 1090 children 6-23.9 mo and 24-47.9 mo/group, respectively, at baseline (2010) and n = ∼500 and 1100 children of the same age, respectively, at endline (2014)]. We derived difference-in-difference impact estimates (DDEs), adjusting for geographic clustering, infant age, sex, differences in baseline characteristics, and differential change in characteristics over time. Results: Groups were similar at baseline. CF improvements were significantly greater in the intensive than in the nonintensive group [DDEs: 16.3, 14.7, 22.0, and 24.6 percentage points (pp) for minimum dietary diversity, minimum meal frequency, minimum acceptable diet, and consumption of iron-rich foods, respectively]. In the intensive group, CF practices were high: 50.4% for minimum acceptable diet, 63.8% for minimum diet diversity, 75.1% for minimum meal frequency, and 78.5% for consumption of iron-rich foods. Timely introduction of foods improved. Significant, nondifferential stunting declines occurred in intensive (6.2 pp) and nonintensive (5.2 pp) groups in children 24-47.9 mo. Conclusions: The intensive program substantially improved CF practices compared with the nonintensive program. Large-scale program delivery was feasible and, with the use of multiple platforms, reached 1.7 million households. Nondifferential impacts on stunting were likely due to rapid positive secular trends in Bangladesh. Accelerating linear growth further could require accompanying interventions. This study establishes proof of concept for large-scale behavior change interventions to improve child feeding. This trial was registered at clinicaltrials.gov as NCT01678716

Large-Scale Social and Behavior Change Communication Interventions Have Sustained Impacts on Infant and Young Child Feeding Knowledge and Practices: Results of a 2-Year Follow-up Study in Bangladesh

Background: Sustained improvements in infant and young child feeding (IYCF) require continued implementation of effective interventions. From 2010-2014, Alive & Thrive (A&T) provided intensive interpersonal counseling (IPC), community mobilization (CM), and mass media (MM) in Bangladesh, demonstrating impact on IYCF practices. Since 2014, implementation has been continued and scaled up by national partners with support from other donors and with modifications such as added focus on maternal nutrition and reduced program intensity. Objective: We assessed changes in intervention exposure and IYCF knowledge and practices in the intensive (IPC + CM + MM) compared with nonintensive areas (standard nutrition counseling + less intensive CM and MM) 2 y after termination of initial external donor support. Methods: We used a cluster-randomized design with repeated cross-sectional surveys at baseline (2010, n = 2188), endline (2014, n = 2001), and follow-up (2016, n = 2400) in the same communities, among households with children 0-23.9 mo of age. Within-group differences over time and differences between groups in changes were tested. Results: In intensive areas, exposure to IPC decreased slightly between endline and follow-up (88.9% to 77.2%); exposure to CM activities decreased significantly (29.3% to 3.6%); and MM exposure was mostly unchanged (28.1-69.1% across 7 TV spots). Exposure to interventions did not expand in nonintensive areas. Most IYCF indicators in intensive areas declined from endline to follow-up, but remained higher than at baseline. Large differential improvements of 12-17 percentage points in intensive, compared with nonintensive areas, between baseline and follow-up remained for early initiation of and exclusive breastfeeding, timely introduction of foods, and consumption of iron-rich foods. Differential impact in breastfeeding knowledge remained between baseline and follow-up; complementary feeding knowledge increased similarly in both groups. Conclusions: Continued IPC exposure and sustained impacts on IYCF knowledge and practices in intensive areas indicated lasting benefits from A&T\u27s interventions as they underwent major scale-up with reduced intensity. This trial was registered at clinicaltrials.gov as NCT02740842

Engagement of Husbands in a Maternal Nutrition Program Substantially Contributed to Greater Intake of Micronutrient Supplements and Dietary Diversity During Pregnancy: Results of a Cluster-Randomized Program Evaluation in Bangladesh

Background: Although husbands may provide support during pregnancy, limited evidence exists on how to promote husbands\u27 engagement and what impact it has. Alive & Thrive integrated nutrition-focused interventions, targeting both wives and husbands, through an existing Maternal, Neonatal, and Child Health (MNCH) platform in Bangladesh. Objectives: We evaluated 1) the impact of a nutrition-focused MNCH program, compared with the standard MNCH program, on husbands\u27 behavioral determinants (i.e., awareness, knowledge, self-efficacy) and support to wives to adopt optimal nutrition practices and 2) how much of the previously documented impact on women\u27s supplement intake and dietary diversity was explained by husbands\u27 behavioral determinants and support. Methods: We used a cluster-randomized design with cross-sectional surveys at baseline (2015) and endline (2016) (n = ∼1000 women and ∼700 husbands/survey). We used mixed linear regression accounting for clustering to estimate difference-in-differences (DIDs) for impact on husbands\u27 behavioral determinants and path analysis to examine how much these determinants explained the impact on women\u27s nutrition behaviors. Results: Of husbands in the nutrition-focused MNCH group, 62% were counseled by health workers, 66% attended a husbands\u27 forum, and 34% saw video shows. The nutrition-focused MNCH, compared with the standard MNCH group, resulted in greater husbands\u27 awareness (DID: 2.74 of 10 points), knowledge (DID: 1.31), self-efficacy and social norms with regard to optimal nutrition practices (difference: 1.08), and support to their wives (DID: 1.86). Husbands\u27 behavioral determinants and support explained nearly half of the program impact for maternal supplement intake and one-quarter for dietary diversity. Conclusions: A nutrition-focused MNCH program that promoted and facilitated husbands\u27 engagement during their wives\u27 pregnancies significantly improved husbands\u27 awareness, knowledge, self-efficacy, and support. These improvements substantially explained the program\u27s impact on women\u27s intake of micronutrient supplements and dietary diversity. Targeting wives and husbands and designing activities to engage men in maternal nutrition programs are important to maximize impact. This trial was registered at www.clinicaltrials.gov as NCT02745249

Integrating Nutrition Interventions Into an Existing Maternal, Neonatal, and Child Health Program Increased Maternal Dietary Diversity, Micronutrient Intake, and Exclusive Breastfeeding Practices in Bangladesh: Results of a Cluster-Randomized Program Evaluation

Maternal undernutrition is a major concern globally, contributing to poor birth outcomes. Limited evidence exists on delivering multiple interventions for maternal nutrition simultaneously. Alive & Thrive addressed this gap by integrating nutrition-focused interpersonal counseling, community mobilization, distribution of free micronutrient supplements, and weight-gain monitoring through an existing Maternal, Neonatal, and Child Health (MNCH) program in Bangladesh. We evaluated the effect of providing nutrition-focused MNCH compared with standard MNCH (antenatal care with standard nutrition counseling) on coverage of nutrition interventions, maternal dietary diversity, micronutrient supplement intake, and early breastfeeding practices. We used a cluster-randomized design with cross-sectional surveys at baseline (2015) and endline (2016) ( ∼ 300 and 1000 pregnant or recently delivered women, respectively, per survey round). We derived difference-in-difference effect estimates, adjusted for geographic clustering and infant age and sex. Coverage of interpersonal counseling was high; \u3e90% of women in the nutrition-focused MNCH group were visited at home by health workers for maternal nutrition and breastfeeding counseling. The coverage of community mobilization activities was ∼50%. Improvements were significantly greater in the nutrition-focused MNCH group than in the standard MNCH group for consumption of iron and folic acid [effect: 9.8 percentage points (pp); 46 tablets] and calcium supplements (effect: 12.8 pp; 50 tablets). Significant impacts were observed for the number of food groups consumed (effect: 1.6 food groups), percentage of women who consumed ≥5 food groups/d (effect: 30.0 pp), and daily intakes of several micronutrients. A significant impact was also observed for exclusive breastfeeding (EBF; effect: 31 pp) but not for early initiation of breastfeeding. Addressing nutrition during pregnancy by delivering interpersonal counseling and community mobilization, providing free supplements, and ensuring weight-gain monitoring through an existing MNCH program improved maternal dietary diversity, micronutrient supplement consumption, and EBF practices. This trial was registered at clinicaltrials.gov as NCT02745249

Impacts on Breastfeeding Practices of At Scale Strategies That Combine Intensive Interpersonal Counseling, Mass Media, and Community Mobilization: Results of Cluster Randomized Program Evaluations in Bangladesh and Viet Nam

Background Despite recommendations supporting optimal breastfeeding, the number of women practicing exclusive breastfeeding (EBF) remains low, and few interventions have demonstrated implementation and impact at scale. Alive & Thrive was implemented over a period of 6 y (2009–2014) and aimed to improve breastfeeding practices through intensified interpersonal counseling (IPC), mass media (MM), and community mobilization (CM) intervention components delivered at scale in the context of policy advocacy (PA) in Bangladesh and Viet Nam. In Bangladesh, IPC was delivered through a large non-governmental health program; in Viet Nam, it was integrated into government health facilities. This study evaluated the population-level impact of intensified IPC, MM, CM, and PA (intensive) compared to standard nutrition counseling and less intensive MM, CM, and PA (non-intensive) on breastfeeding practices in these two countries. Methods and Findings A cluster-randomized evaluation design was employed in each country. For the evaluation sample, 20 sub-districts in Bangladesh and 40 communes in Viet Nam were randomized to either the intensive or the non-intensive group. Cross-sectional surveys (n ~ 500 children 0–5.9 mo old per group per country) were implemented at baseline (June 7–August 29, 2010, in Viet Nam; April 28–June 26, 2010, in Bangladesh) and endline (June 16–August 30, 2014, in Viet Nam; April 20–June 23, 2014, in Bangladesh). Difference-in-differences estimates (DDEs) of impact were calculated, adjusting for clustering. In Bangladesh, improvements were significantly greater in the intensive compared to the non-intensive group for the proportion of women who reported practicing EBF in the previous 24 h (DDE 36.2 percentage points [pp], 95% CI 21.0–51.5, p \u3c 0.001; prevalence in intensive group rose from 48.5% to 87.6%) and engaging in early initiation of breastfeeding (EIBF) (16.7 pp, 95% CI 2.8–30.6, p = 0.021; 63.7% to 94.2%). In Viet Nam, EBF increases were greater in the intensive group (27.9 pp, 95% CI 17.7–38.1, p \u3c 0.001; 18.9% to 57.8%); EIBF declined (60.0% to 53.2%) in the intensive group, but less than in the non-intensive group (57.4% to 40.6%; DDE 10.0 pp, 95% CI −1.3 to 21.4, p = 0.072). Our impact estimates may underestimate the full potential of such a multipronged intervention because the evaluation lacked a “pure control” area with no MM or national/provincial PA. Conclusions At-scale interventions combining intensive IPC with MM, CM, and PA had greater positive impacts on breastfeeding practices in Bangladesh and Viet Nam than standard counseling with less intensive MM, CM, and PA. To our knowledge, this study is the first to document implementation and impacts of breastfeeding promotion at scale using rigorous evaluation designs. Strategies to design and deliver similar programs could improve breastfeeding practices in other contexts