Transcription factors are central to the developmental and functional regulation of cells through co-ordination of gene expression via interaction with the genome. RFX6 is a winged-helix transcription factor whose expression is highly specific to the pancreas and gastrointestinal tract. Recent developments have highlighted an association between RFX6 and Type 2 Diabetes (T2D), which affects over 500 million people throughout the world. RFX6 controls development in both the pancreas and the gastrointestinal tract, where it is required for the differentiation of islet endocrine and enteroendocrine cells. Coding and non-coding RFX6 variants have been associated with syndromic neonatal diabetes (Mitchell-Riley Syndrome), Maturity Onset Diabetes of the Young (MODY), and T2D. Given the central position of RFX6 in pancreatic development and diabetes, understanding in more detail the regulatory role of RFX6 in different cell types and at different stages of development may open avenues towards patient-specific diabetes treatment and prevention. In this article, we review the literature surrounding RFX6 with respect to its role in development and diabetes pathogenesis.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders. TAAR1 has a rigid consensus binding motif that binds to endogenous trace amine stimuli as well as two extended binding pockets that accommodate diverse chemotypes.

This dataset is part of the manuscript Genetic risk converges on regulatory networks mediating early type 2 diabetes (Walker, Saunders, & Rai et al., 2023), a body of work that includes tissue imaging, sorted islet cell transcriptomics, and islet functional analysis of donors with early-stage type 2 diabetes (T2D) and control donors. Raw imaging data was acquired from 4% PFA-fixed, cryopreserved human pancreas tissue using the CODEX system (now PhenoCycler Open; Akoya Biosciences) integrated with a BZ-X810 epifluorescence microscope (Keyence) with a CFI plan Apo I 20x/0.75 objective (Nikon). Image alignment, stitching, background subtraction, and deconvolution were performed using the CODEX Processor v1.7.0.6 (Akoya Biosciences). Tissue and islet areas were annotated by hand to exclude out-of-focus regions and poor tissue quality. Islets (estimated diameter ≥50 μm; mean 42 islets/donor) were annotated based on DAPI and CHGA channels. Cell segmentation and cell type annotations were performed using the HALO HighPlex FL v3.2.1 module (Indica Labs). For cell neighborhood (CN) analysis, two methods were applied in parallel to CODEX data from annotated islets: a community detection method, termed Dynamic CF-IDF, and a k-means approach. Processed imaging data is available via Pancreatlas (RRID:SCR_018567); packages used for cell neighborhood analyses are published in Github.

We previously showed that α cell function and gene expression in islets from donors with type 1 diabetes (T1D) was significantly compromised. The reason (s) for these intrinsic α cell changes are unknown but may include loss of α-to-β cell contact, chronic hyperglycemia, and/or repeated hypoglycemic events. To test these concurrent hypotheses, we modeled human T1D islets using α cell-enriched (“T1D-like") human pseudoislets. The synchronous acquisition of intracellular Ca2+ and hormone response showed impaired intracellular Ca2+ signaling and glucagon secretion in T1D-like pseudoislets compared to controls containing both α and β cells. For modeling of chronic hyperglycemia, we transplanted pseudoislets into Nod-SCID-IL2Rγnull; RIP-Diphtheria Toxin Receptor mice made diabetic by diphtheria toxin (DT) -induced β cell depletion. In DT-treated mice, α cells in T1D-like pseudoislets maintained expression of MAFB and ARX but began to express the β cell-enriched transcription factor NKX6.1 (control vs. T1D-like, 3.43±2.49 vs. 54.8±20.8% NKX6.1+ α cells; P=0.046, N=3-5 mice, 2 donors/group) . To mimic repeated hypoglycemic events, we intermittently exposed T1D-like pseudoislets to low glucose (1.7 mM) (3 events x 3hr each) in vitro, using those remaining in basal glucose (5 mM) as control. Following multiple low glucose (MLG) exposures, T1D-like pseudoislets had reduced glucagon secretion in the presence of both 1.7 mM (control vs. MLG, 1.51±0.18 vs. 0.79±0.13 % glucagon content; P=0.011, N=4 donors) and 16.7 mM glucose (control vs. MLG, 0.79±0.13 vs. 0.39±0.% glucagon content; P=0.023, N=4) . Native human islets exposed to MLG had similar changes in glucagon response (control vs. MLG, 1.7mM: 0.63±0.vs. 0.22±0.% glucagon content, P=0.007; 16.7mM: 0.18±0.vs. 0.05±0.% glucagon content, P=0.029; N=4 donors) . These data suggest that loss of α-to-β cell contact and repeated exposure to low glucose impair α cell function. Further, exposure to chronic hyperglycemia may lead to changes in α cell identity state after β cell loss. Disclosure Y.D.Pettway: None. C.Dai: None. T.M.Richardson: None. J.T.Walker: None. R.Aramandla: None. G.Poffenberger: None. A.Bradley: None. A.C.Powers: None. M.Brissova: None.

Islet-enriched transcription factors (TFs) exert broad control over cellular processes in pancreatic α and β cells, and changes in their expression are associated with developmental state and diabetes. However, the implications of heterogeneity in TF expression across islet cell populations are not well understood. To define this TF heterogeneity and its consequences for cellular function, we profiled more than 40,000 cells from normal human islets by single-cell RNA-Seq and stratified α and β cells based on combinatorial TF expression. Subpopulations of islet cells coexpressing ARX/MAFB (α cells) and MAFA/MAFB (β cells) exhibited greater expression of key genes related to glucose sensing and hormone secretion relative to subpopulations expressing only one or neither TF. Moreover, all subpopulations were identified in native pancreatic tissue from multiple donors. By Patch-Seq, MAFA/MAFB-coexpressing β cells showed enhanced electrophysiological activity. Thus, these results indicate that combinatorial TF expression in islet α and β cells predicts highly functional, mature subpopulations.

Abstract Clinical and pathologic heterogeneity in type 1 diabetes is increasingly being recognized. Findings in the islets and pancreas of a 22-year-old male with 8 years of type 1 diabetes were discordant with expected results and clinical history (islet autoantibodies negative, hemoglobin A1c 11.9%) and led to comprehensive investigation to define the functional, molecular, genetic, and architectural features of the islets and pancreas to understand the cause of the donor’s diabetes. Examination of the donor’s pancreatic tissue found substantial but reduced β-cell mass with some islets devoid of β cells (29.3% of 311 islets) while other islets had many β cells. Surprisingly, isolated islets from the donor pancreas had substantial insulin secretion, which is uncommon for type 1 diabetes of this duration. Targeted and whole-genome sequencing and analysis did not uncover monogenic causes of diabetes but did identify high-risk human leukocyte antigen haplotypes and a genetic risk score suggestive of type 1 diabetes. Further review of pancreatic tissue found islet inflammation and some previously described α-cell molecular features seen in type 1 diabetes. By integrating analysis of isolated islets, histological evaluation of the pancreas, and genetic information, we concluded that the donor’s clinical insulin deficiency was most likely the result autoimmune-mediated β-cell loss but that the constellation of findings was not typical for type 1 diabetes. This report highlights the pathologic and functional heterogeneity that can be present in type 1 diabetes.

This review focuses on the human pancreatic islet-including its structure, cell composition, development, function, and dysfunction. After providing a historical timeline of key discoveries about human islets over the past century, we describe new research approaches and technologies that are being used to study human islets and how these are providing insight into human islet physiology and pathophysiology. We also describe changes or adaptations in human islets in response to physiologic challenges such as pregnancy, aging, and insulin resistance and discuss islet changes in human diabetes of many forms. We outline current and future interventions being developed to protect, restore, or replace human islets. The review also highlights unresolved questions about human islets and proposes areas where additional research on human islets is needed.

SUMMARYA hallmark of type 2 diabetes (T2D), a major cause of world-wide morbidity and mortality, is dysfunction of insulin-producing pancreatic islet β cells1–3. T2D genome-wide association studies (GWAS) have identified hundreds of signals, mostly in the non-coding genome and overlapping β cell regulatory elements, but translating these into biological mechanisms has been challenging4–6. To identify early disease-driving events, we performed single cell spatial proteomics, sorted cell transcriptomics, and assessed islet physiology on pancreatic tissue from short-duration T2D and control donors. Here, through integrative analyses of these diverse modalities, we show that multiple gene regulatory modules are associated with early-stage T2D β cell-intrinsic defects. One notable example is the transcription factor RFX6, which we show is a highly connected β cell hub gene that is reduced in T2D and governs a gene regulatory network associated with insulin secretion defects and T2D GWAS variants. We validated the critical role of RFX6 in β cells through direct perturbation in primary human islets followed by physiological and single nucleus multiome profiling, which showed reduced dynamic insulin secretion and large-scale changes in the β cell transcriptome and chromatin accessibility landscape. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs, and individuals and thus we anticipate this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits with GWAS data.

Pancreatic islets secrete insulin from β cells and glucagon from α cells, and dysregulated secretion of these hormones is a central component of diabetes. Thus, an improved understanding of the pathways governing coordinated β and α cell hormone secretion will provide insight into islet dysfunction in diabetes. However, the 3D multicellular islet architecture, essential for coordinated islet function, presents experimental challenges for mechanistic studies of intracellular signaling pathways in primary islet cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles. We demonstrate the utility of this experimental approach by studying the effects of Gi and Gq GPCR pathways on insulin and glucagon secretion by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion, which occur through changes in intracellular Ca2+. The experimental approach of combining pseudoislets with a microfluidic system allowed the coregistration of intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.

Selective inhibitors of sodium glucose cotransporter-2 (SGLT2) are widely used for the treatment of type 2 diabetes and act primarily to lower blood glucose by preventing glucose reabsorption in the kidney. However, it is controversial whether these agents also act on the pancreatic islet, specifically the α cell, to increase glucagon secretion. To determine the effects of SGLT2 on human islets, we analyzed SGLT2 expression and hormone secretion by human islets treated with the SGLT2 inhibitor dapagliflozin (DAPA) in vitro and in vivo. Compared to the human kidney, SLC5A2 transcript expression was 1600-fold lower in human islets and SGLT2 protein was not detected. In vitro, DAPA treatment had no effect on glucagon or insulin secretion by human islets at either high or low glucose concentrations. In mice bearing transplanted human islets, 1 and 4 weeks of DAPA treatment did not alter fasting blood glucose, human insulin, and total glucagon levels. Upon glucose stimulation, DAPA treatment led to lower blood glucose levels and proportionally lower human insulin levels, irrespective of treatment duration. In contrast, after glucose stimulation, total glucagon was increased after 1 week of DAPA treatment but normalized after 4 weeks of treatment. Furthermore, the human islet grafts showed no effects of DAPA treatment on hormone content, endocrine cell proliferation or apoptosis, or amyloid deposition. These data indicate that DAPA does not directly affect the human pancreatic islet, but rather suggest an indirect effect where lower blood glucose leads to reduced insulin secretion and a transient increase in glucagon secretion.

Posttransplantation diabetes mellitus (PTDM) is a common and significant complication related to immunosuppressive agents required to prevent organ or cell transplant rejection. To elucidate the effects of 2 commonly used agents, the calcineurin inhibitor tacrolimus (TAC) and the mTOR inhibitor sirolimus (SIR), on islet function and test whether these effects could be reversed or prevented, we investigated human islets transplanted into immunodeficient mice treated with TAC or SIR at clinically relevant levels. Both TAC and SIR impaired insulin secretion in fasted and/or stimulated conditions. Treatment with TAC or SIR increased amyloid deposition and islet macrophages, disrupted insulin granule formation, and induced broad transcriptional dysregulation related to peptide processing, ion/calcium flux, and the extracellular matrix; however, it did not affect regulation of β cell mass. Interestingly, these β cell abnormalities reversed after withdrawal of drug treatment. Furthermore, cotreatment with a GLP-1 receptor agonist completely prevented TAC-induced β cell dysfunction and partially prevented SIR-induced β cell dysfunction. These results highlight the importance of both calcineurin and mTOR signaling in normal human β cell function in vivo and suggest that modulation of these pathways may prevent or ameliorate PTDM.

Human but not mouse islets transplanted into immunodeficient NSG mice effectively accumulate lipid droplets (LDs). Because chronic lipid exposure is associated with islet β-cell dysfunction, we investigated LD accumulation in the intact human and mouse pancreas over a range of ages and states of diabetes. Very few LDs were found in normal human juvenile pancreatic acinar and islet cells, with numbers subsequently increasing throughout adulthood. While accumulation appeared evenly distributed in postjuvenile acinar and islet cells in donors without diabetes, LDs were enriched in islet α- and β-cells from donors with type 2 diabetes (T2D). LDs were also found in the islet β-like cells produced from human embryonic cell-derived β-cell clusters. In contrast, LD accumulation was nearly undetectable in the adult rodent pancreas, even in hyperglycemic and hyperlipidemic models or 1.5-year-old mice. Taken together, there appear to be significant differences in pancreas islet cell lipid handling between species, and the human juvenile and adult cell populations. Moreover, our results suggest that LD enrichment could be impactful to T2D islet cell function.

Identification of cell-surface markers specific to human pancreatic β cells would allow in vivo analysis and imaging. Here we introduce a biomarker, ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), that is expressed on the cell surface of essentially all adult human β cells, including those from individuals with type 1 or type 2 diabetes. NTPDase3 is expressed dynamically during postnatal human pancreas development, appearing first in acinar cells at birth, but several months later its expression declines in acinar cells while concurrently emerging in islet β cells. Given its specificity and membrane localization, we utilized an NTPDase3 antibody for purification of live human β cells as confirmed by transcriptional profiling, and, in addition, for in vivo imaging of transplanted human β cells. Thus, NTPDase3 is a cell-surface biomarker of adult human β cells, and the antibody directed to this protein should be a useful new reagent for β cell sorting, in vivo imaging, and targeting.

Efforts to advance our understanding of human islet biology are hindered by a paucity of approaches to effectively manipulate gene expression in human islets. To address this, we developed a pseudoislet system of human islet dispersion and reaggregation via modified hanging drop with 2500 cells/drop to generate 150-200 μm diameter pseudoislets (n=9 normal donors; 26-73 years). Pseudoislet morphology, dithizone uptake, and expression of markers of α and/or β cell identity (PDX1, NKX6.1, ARX, MAFB, NKX2.2, and PAX6) were similar to native islets; in paired comparisons from the same donor, pseudoislets had slightly fewer β cells and more α cells (53% β, 44% α, 3% δ cells) than native islets (62% β, 35% α, 3% δ cells; p<0.05 β and α cells). In perifusion, pseudoislets were similar to native islets in biphasic glucose-stimulated and cAMP-potentiated insulin secretion as well as in epinephrine and low glucose stimulation of and high glucose inhibition of glucagon release. Adenoviral-transduced dispersed human islet cells (>90% β cell transduction) formed functioning pseudoislets similar to untransduced controls. Using this approach, we virally expressed hM3Dq, a Gq-coupled designer receptor exclusively activated by a designer drug (DREADD), which selectively responds to the otherwise inert ligand clozapine-n-oxide (CNO) and signals through PLC/IP3. In low glucose (2mM G + 10μM CNO), hM3Dq pseudoislets secreted more insulin than control mCherry pseudoislets (19.2 vs. 1.0 ng/100IEQ/hr; p<0.0001). In contrast, hM3Dq and control pseudoislets had similar insulin secretion in high glucose (11mM G + 10μM CNO; 24.8 vs. 23.2 ng/100IEQ/hr; p>0.05), indicating that activation of Gq signaling does not potentiate insulin secretion in high glucose. These data demonstrate how manipulation of gene expression in pseudoislets can provide insight into human islet biology and suggest that activation of Gq signaling in β cells elicits robust insulin secretion that appears to be uncoupled from glucose metabolism. Disclosure J.T. Walker: None. R. Haliyur: None. R. Aramandla: None. G. Poffenberger: None. H.A. Nelson: None. J. Luchsinger: None. P. Wang: None. A. Garcia-Ocaña: None. M. Brissova: None. A.C. Powers: None.

G-protein-coupled-receptors (GPCRs) modulate insulin secretion from β cells and glucagon secretion from α cells. Here, we developed an integrated approach to study the function of primary human islet cells using genetically modified pseudoislets that resemble native islets across multiple parameters. We studied the Gi and Gq GPCR pathways by expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM4Di or hM3Dq. Activation of Gi signaling reduced insulin and glucagon secretion, while activation of Gq signaling stimulated glucagon secretion but had both stimulatory and inhibitory effects on insulin secretion. Further, we developed a microperifusion system that allowed synchronous acquisition of GCaMP6f biosensor signal and hormone secretory profiles and showed that the dual effects for Gq signaling occur through changes in intracellular Ca2+. By combining pseudoislets with a microfluidic system, we co-registered intracellular signaling dynamics and hormone secretion and demonstrated differences in GPCR signaling pathways between human β and α cells.

Single cell RNA-sequencing (scRNA-seq) approaches are continuously improving at the point of data collection and analysis to simultaneously profile all cell types within islet microorgans to better understand heterogeneity and phenotype-function relationships and how these change with aging and disease. To better understand the advantages and limitations of scRNA-seq, we used the ChromiumTMplatform to profile human islets with a robust insulin and glucagon secretion by perifusion (5 nondiabetic donors, ages 14-66 years) and compared these results to scRNA-seq analysis of purified α and β cells. In context of the same donor (n=2), purified α and β cells (using NTPDase3 marker) showed high correlation (r=0.99) with the Seurat-identified α and β subsets originating from dispersed whole islets. Based on the analysis of a very large pool of single cells (79,881 cells; 2316 genes/cell), the Seurat-guided analysis identified cell types that segregated into eight different clusters (17% β, 66% α, 4% δ, 4% ductal, 4% mesenchymal, 2% acinar, 2% endothelial, and 1% immune). Interstingly, in this large single cell context, we did not identify the four β cell subsets previously reported on the basis of alternative cell surface markers, but found a β cell cluster with highly expressed ER stress-associated genes and an α cell cluster with higher cell cycle regulation marker expression. We concurrently investigated expression of functionally important MAFA and MAFB genes, which are expressed only in a fraction of β cells by histological analysis, and found that PERK-mediated UPR transcripts were enriched in the MAFB-expressing β cells, while the MAFA-expressing β cells differentially expressed genes associated with β cell function. Overall, our study indicates high fidelity of α and β cell clustering in dispersed and sorted islet cells and points to a different pattern of β cell heterogeneity when β cells are purified using NTPDase3 expression compared to other cell surface markers. Disclosure S. Shrestha: None. D.C. Saunders: None. J.T. Walker: None. R. Haliyur: None. G. Poffenberger: None. R. Aramandla: None. A. Jones: None. N. Prasad: None. S.E. Levy: None. A.C. Powers: None. M. Brissova: None.

T2D is characterized by progressive pancreatic islet dysfunction; however, dissecting causes of this dysfunction in humans is challenging due to disease heterogeneity and the difficulty of obtaining relevant pancreatic samples. To address these challenges, we used an integrated approach to functionally and molecularly study the native pancreas and isolated islets from the same human T2D cadaveric donor. Using associated donor clinical information, we focused on donors with short disease duration (n=10, age 47-66 years, 2-7-year duration, oral medications). Compared to age-matched control islets, T2D islets had dysfunctional insulin (decreased) and glucagon (increased) secretion despite similar insulin and glucagon content. Tissue-section analysis of T2D pancreata showed no difference in relative endocrine (α, β and δ) cell composition. To assess for changes in the islet microenvironment, we analyzed macrophages by staining with Iba1 and endothelial cells with Caveolin-1 and found a 2-fold increase in intraislet macrophages (T2D vs. control: 3.01% vs. 1.45% by area) but no change in intraislet endothelial cell area (T2D vs. control: 8.97% vs. 8.21% by area). Interestingly, costaining with Iba1 and thioflavin S demonstrated that T2D islets with amyloid did not have increased macrophage infiltration compared to T2D islets without amyloid although a close spatial association of macrophages and amyloid deposits was noted. RNA-sequencing analysis of FACS-purified α and β cells from T2D islets showed increased inflammatory signaling pathways in both cell types. Specifically, transcripts for cytokine receptors such as IL6R, IL1R1, and IFNGR1 as well as downstream regulators including NFKB1 and NFKB2 were elevated in T2D α and/or β cells. Together, these data suggest that early in the course of T2D, increased macrophages in the islet microenvironment may lead to elevated α and β cell inflammatory signaling and contribute to human α and β cell dysfunction. Disclosure J.T. Walker: None. S. Shrestha: None. N. Prasad: None. T. Richardson: None. R. Aramandla: None. G. Poffenberger: None. R. Bottino: None. M. Brissova: None. A.C. Powers: None.