ALBERT

Description: Cu 2 ZnSn(S,Se) 4 (CZTSSe) solar cells typically exhibit high short-circuit current density ( J sc ), but have reduced cell efficiencies relative to other thin film technologies due to a deficit in the open-circuit voltage ( V oc ) , which prevent these devices from becoming commercially competitive. Recent research has attributed the low V oc in CZTSSe devices to small scale disorder that creates band tail states within the absorber band gap, but the physical processes responsible for this V oc reduction have not been elucidated. In this paper, we show that carrier recombination through non-mobile band tail states has a strong voltage dependence and is a significant performance-limiting factor, and including these effects in simulation allows us to simultaneously explain the V oc deficit, reduced fill factor, and voltage-dependent quantum efficiency with a self-consistent set of material parameters. Comparisons of numerical simulations to measured data show that reasonable values for the band tail parameters (characteristic energy, capture rate) can account for the observed low V oc , high J sc , and voltage dependent collection efficiency. These results provide additional evidence that the presence of band tail states accounts for the low efficiencies of CZTSSe solar cells and further demonstrates that recombination through non-mobile band tail states is the dominant efficiency limiting mechanism.

Description: Recent studies demonstrated that IGF2BP1 over-expression (IGF2BP1-OE) in adult erythroblasts has robust effects on fetal hemoglobin (HbF; 〉65% of the total globin levels), accompanied by reversal of the beta-like globin expression patterns to a fetal-like phenotype. Here we investigated if another member of the insulin-like growth factor 2 mRNA-binding protein family, IGF2BP3, also has potential for HbF regulation that may be useful for therapeutic application among patients with beta-hemoglobin disorders. The developmental pattern and expression levels for IGF2BP3 were initially determined in cord blood versus adult blood CD34(+) samples cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. RNA samples were collected at culture day 14 and expression levels were measured by qRT-PCR. IGF2BP3 showed a developmentally regulated expression pattern similar to IGF2BP1 (IGF2BP1: cord blood: 1.3.E+03 ± 4.3.E+02 and adult blood: below detection limits; IGF2BP3: cord blood: 5.8.E+02 ± 2.4.E+02 and adult blood: below detection limits). These results were confirmed in vivo by comparing human fetal liver to adult bone marrow samples (IGF2BP1: fetal liver: 3.5.E+02 ± 5.7.E+01, adult bone marrow: below detection limits and IGF2BP3: fetal liver: 2.0.E+01 ± 2.7.E+00, adult bone marrow: below detection limits). To investigate the effects of IGF2BP3 upon erythropoiesis and globin expression, a lentiviral construct was designed for expression of IGF2BP3 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (IGF2BP3-OE), with a matched empty vector control. Transductions were performed in CD34(+) cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Over-expression of IGF2BP3 was confirmedby qRT-PCR and Western blot analyses at culture day 14. IGF2BP3-OE cells maintained their ability to differentiate and enucleate ex vivo compared to donor-matched controls. The expression levels of globin genes were evaluated at culture day 14 by qRT-PCR and showed that IGF2BP3-OE caused significantly increased gamma-globin expression levels compared to control transductions (control: 7.7.E+05 ± 1.7.E+05; IGF2BP3-OE: 8.4.E+06 ± 3.2.E+06; p=0.018). Consistent with increased gamma-globin, HbF rose to moderately high levels upon IGF2BP3-OE (control: 4.0 ± 2.1%; IGF2BP3-OE: 18.6 ± 1.0%; p=0.0021). In addition, the expression pattern of the erythroid transcription factor BCL11A was investigated by qRT-PCR at culture day 14 and no significant changes were observed (control: 5.6.E+02 ± 2.7.E+02; IGF2BP3-OE: 6.7.E+02 ± 3.5.E+02; p=0.694). However, minor decreases in BCL11A protein levels were detected by Western analysis. These results demonstrate that IGF2BP3 is developmentally regulated in human erythroid tissues with silencing during the fetal-to-adult transition. However, the effects of IGF2BP3-OE on HbF levels were less robust when compared to IGF2BP1-OE in cultured adult erythroblasts. Disclosures No relevant conflicts of interest to declare.

Description: Polymerization of sickle cell hemoglobin S (HbS) is recognized as a key event in the pathophysiology of sickle cell disease (SCD). Repeated HbS polymerization promotes an altered red blood cell (RBC) membrane, hemolysis, and microparticle (MP) formation, which have been shown to play significant roles in the interaction of RBCs with vascular endothelium and progression of vaso-occlusive events. Circulating RBC-derived MPs are elevated in SCD patients and they release a significant portion of their contents including oxidized HbS and heme to the cells of the vasculature. We have recently reported that free HbS oxidizes faster, remains locked in a highly oxidizing form (ferryl) longer, and loses heme faster than normal HbA (Kassa et al., J Biol Chem 290: 27939, 2015). The contributions of HbS higher oxidation states (ferric and ferryl heme) to MP formation, membrane alterations, and heme loss are poorly defined in SCD. RBC-derived MPs (ranging in size between 100-300 nm in diameter) generated by sheer stress or isolated by ultracentrifugation from the plasma (circulating) of SCD patients (N=6), ethnically matched control subjects (N=5), humanized transgenic sickle mice (Townes-SS, N=4), and control wild-type mice (Townes-AA, N=4) were identified by flow cytometry using CD235a glycophorin antibody and annexin V for externalized phosphatidylserine (PS). Time courses of Hb oxidation, obtained during 30 hour incubations of mouse or human MPs were biphasic. The initial levels of oxidized (ferric) Hb (30 to 45%) were slightly reduced within the first ~10 hours, likely due to the presence of RBC residual reductive enzymes within MPs. This was followed by a second phase in which Hb oxidation (ferric Hb) increased linearly and uncontrollably to 65 to 75% of total Hb. SCD MP's contained highly reactive ferryl Hb intermediates, carbonylated membrane proteins, and phosphorylated band 3 proteins. Quantitative proteomic analysis indicated a higher level of protein oxidation in MPs derived from SCD mice and patients. Five-fold higher levels of irreversibly oxidized βCys93 oxidation were found in untreated versus hydroxyurea-treated SCD patients. Intriguingly, HbS β subunits from SCD MPs were ubiquitinated and MPs isolated from untreated SCD patients had 25-fold higher ubiquitination levels than hydroxyurea-treated SCD patients that were comparable to normal controls. MP ubiquitination levels were correlated with HbS and an overall increase in MP oxidative stress, and inversely correlated with HbF. Compared to respective control MPs, incubation of either mice or human SCD MPs with human endothelial cells (HUVEC) activated apoptotic pathways and impacted cellular bioenergetic parameters by lowering mitochondrial oxygen consumption rates to a greater degree in a manner that was correlated with the redox state of Hb iron within MPs. Human endothelial cells incubated with SCD MPs showed greater intracellular reactive oxygen species production and heme oxygenase-1 induction. In summary, Hb transformation to higher oxidation forms is markedly increased in MPs generated from SCD mice and patients, which when incubated with endothelial cells, lead to mitochondrial dysfunction and apoptotic cell death. These mechanistic analyses of RBC-derived SCD microparticles suggest potential anti-oxidative reducing modalities that may interrupt MP heme-mediated pathophysiology in patients with SCD. Disclosures Belcher: Cydan/Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.

Description: Background: The 1000 Genomes Project provides a database of over 80 million genomic variants found across 2504 individuals from 26 populations. A current priority of the genomics field is to design information systems to translate this knowledge into clinical significance and patient care. The applications and advantages of red blood cell (RBC) antigen prediction through genotyping are widely accepted in transfusion medicine. Current technologies address a limited number of single nucleotide polymorphisms (SNPs) in 12 blood group genes, and our background knowledge of RBC phenotype distribution is often limited to a few populations. We analyzed the 1000 Genomes database with 4 objectives: 1) determine allele distributions of 46 blood group-related genes across the 5 genotyped superpopulations: Africa, East Asia, Europe, South Asia and the Americas; 2) identify possible new blood group alleles and their geographic association; 3) determine the feasibility of blood group genotyping by NGS; and 4) establish a scaffold of chromosomal coordinates to interpret NGS output files into a predicted RBC phenotype. Results: From the initial list of 46 blood group-related genes, we eliminated the five genes with known rearrangements and focused only on regions that met the strict criteria for accessibility through short, paired-end NGS reads (77% of 80.4kb). We mapped over 800 known alleles in coding and non-coding regions, and documented the 80 variants that were both present in the 1000 Genomes database and met the strict accessibility criteria. Sixty-four of these 80 variants are not addressed by current RBC genotyping technology. All 80 variants, including the ACKR1 promoter silencing mutation, are located within exon pull-down boundaries. The average low-coverage sequencing depth was 18,424x, with exome-sequencing confirmation at 65.7x depth. Twenty-three alleles had at least one novel population distribution, such as documentation of the Kpaallele for the first time in Africa and South Asia. From a total of 30 novel blood group continental frequencies, 14 correspond to a newfound presence in South Asia. 1000 Genomes identified a total of 926 missense mutations in blood group genes that met strict NGS mapping criteria, as well as multiple deletions. Two novel missense mutations in ERMAP and SLC14A1 are classified as likely antigenic, since they target the same amino acids responsible for the SCER- and Cr(a-) alleles. Six novel deletions involving the Lewis, H, Cromer, Indian and OK systems are also classified as likely-deleterious after careful analysis. For example, a novel in-frame 24bp deletion in SLC14A1 eliminates part of the intracytoplasmic tail, which is required for membrane localization and includes the 28G residue that defines JK*01W.03. Thus, this novel deletion is predicted to alter Kidd protein expression. The 8 novel alleles are distributed throughout the five superpopulations but are most frequently found in Africa. Four standard bioinformatics programs named SIFT, PolyPhen-2, Mutation Taster, and Mutation Assessor failed to detect half of the control known blood group alleles and thus are not adequate for the analysis of novel blood group variants in the transfusion medicine context. Conclusions: NGS can allow comprehensive, fast, and high-throughput RBC antigen prediction. All queried blood group alleles are amenable to targeted exome sequencing, and 77% of blood group coding sequences can be addressed with a short, paired-end NGS strategy. Based on 1000 Genomes, we created a database of the worldwide distribution of 80 known and 8 novel blood group variants, along with their chromosomal coordinates in the hg19 and GRCh38 assemblies. This database is the scaffold for the creation of a new transfusion medicine bioinformatics pipeline that will translate NGS .vcf output files into a predicted RBC phenotype. New algorithms that focus on exposed peptides and antigenicity are required for the analysis of novel variants identified by NGS in the immunohematology context. Disclosures No relevant conflicts of interest to declare.

Description: Pharmacologic and genetic regulation of fetal hemoglobin (HbF) remains a major goal for treatment of the beta-thalassemias and sickle cell disease. Recently, the let-7 microRNA heterochronic pathway of RNA-binding factors was shown to be developmentally correlated with HbF expression in humans. Experimental manipulation of the pathway components (LIN28, let-7, and the IGF2 binding proteins) demonstrated moderate increases in fetal hemoglobin (around 15-35% HbF) with the exception of IGF2BP1, which caused a nearly complete reversal in gamma- and beta-globin gene expression resulting in HbF levels of 60-70% among cultured adult human erythrocytes. To further explore IGF2BP1 effects on HbF and erythropoiesis, lentiviral transduction of CD34(+) cells was performed with let-7 resistant expression of IGF2BP1 driven by the human SPTA1 gene (IGF2BP1-OE). Donor-matched control transductions were studied for comparison. For protein localization studies, confocal imaging of sorted cells was utilized. IGF2BP1-OE caused IGF2BP1 protein expression throughout the cytoplasm and localized to small granules. Granules were unevenly distributed and more abundantly observed in the perinuclear regions. Nuclear detection of IGF2BP1 protein remained at background control levels. We thus hypothesized that IGF2BP1 protein in these cytoplasmic granules may regulate globin gene expression by binding RNAs that encode epigenetic modifiers of the beta-globin locus. To test this hypothesis, RNA-immunoprecipitation (RIP) was performed using a RIP-certified anti-IGF2BP1 antibody. Next generation sequencing and qRT-PCR were used to determine the mRNA species bound by IGF2BP1. RIP-enrichment of BCL11A mRNA was identified (4.6 ± 1.2 fold compared to input sample), as well as mRNA encoding other modifiers of globin gene expression including KLF1 and ZBTB7A. Further analyses of BCL11A showed no significant changes in BCL11A mRNA levels among IGF2BP1-OE cells (control: 6.5.E+03 ± 3.8.E+03, IGF2BP1-OE: 4.1.E+03 ± 1.2.E+02, p=0.403). However, BCL11A protein detection was reduced to background levels by Western analysis compared to transduction control cell lysates. These studies suggest that RNA-binding and cytoplasmic compartmentalization provide IGF2BP1 with a mechanism for increasing fetal hemoglobin via post-transcriptional regulation of globin gene transcription factors. Disclosures No relevant conflicts of interest to declare.