C. elegans as a model for inter-individual variation in metabolism.

Individuals can exhibit differences in metabolism that are caused by the interplay of genetic background, nutritional input, microbiota and other environmental factors1-4. It is difficult to connect differences in metabolism to genomic variation and derive underlying molecular mechanisms in humans, owing to differences in diet and lifestyle, among others. Here we use the nematode Caenorhabditis elegans as a model to study inter-individual variation in metabolism. By comparing three wild strains and the commonly used N2 laboratory strain, we find differences in the abundances of both known metabolites and those that have not to our knowledge been previously described. The latter metabolites include conjugates between 3-hydroxypropionate (3HP) and several amino acids (3HP-AAs), which are much higher in abundance in one of the wild strains. 3HP is an intermediate in the propionate shunt pathway, which is activated when flux through the canonical, vitamin-B12-dependent propionate breakdown pathway is perturbed5. We show that increased accumulation of 3HP-AAs is caused by genetic variation in HPHD-1, for which 3HP is a substrate. Our results suggest that the production of 3HP-AAs represents a 'shunt-within-a-shunt' pathway to accommodate a reduction-of-function allele in hphd-1. This study provides a step towards the development of metabolic network models that capture individual-specific differences of metabolism and more closely represent the diversity that is found in entire species.

O-linked sialic acid residues on platelet membrane glycoprotein IIb mask the human HPA-9b alloepitope.

Sialic acids occupy the terminal position of glycan chains and have the potential to influence the antigenicity of glycoproteins (GP). The polymorphisms of human platelet alloantigens (HPA)-3 and HPA-9, located near the C-terminus of the extracellular domain of platelet membrane GPIIb, are adjacent to sialyl-core 1 O-glycans emanating from serines 845 and 847. Whether the nearby O-glycans affect the antigenicity of HPA-9b or influence the binding of anti-HPA-9b alloantibodies in clinically significant cases of neonatal alloimmune thrombocytopenia is unknown. To address this issue, we generated a series of O-glycan mutant HPA-9 allele-specific induced pluripotent stem cell lines, differentiated them to megakaryocytes (MKs), and examined their ability to bind HPA-9b-specific alloantibodies. We found that both wild-type MKs treated with neuraminidase and those genetically modified to lack the sialidases ST3GAL1 and ST3GAL2 dramatically increased anti-HPA-9b alloantibody binding, indicating that the HPA-9b epitope is partially masked by terminal sialic acids on nearby O-glycans of GPIIb. Interestingly, mutating the serine residues that carry these glycan chains to alanine actually reduced the binding of anti-HPA-9b alloantibodies, indicating that these 2 O-glycan chains contribute to the presentation of the HPA-9b epitope-perhaps by stabilizing the conformation of the GP in this region. Collectively, our data suggest that detection of anti-HPA-9b alloantibodies may be enhanced through the use of HPA-9b-specific MKs that have been genetically altered to lack nearby terminal sialic acid residues but retain the glycan chains to which they are attached.

Parallel pathways for serotonin biosynthesis and metabolism in C. elegans.

The neurotransmitter serotonin plays a central role in animal behavior and physiology, and many of its functions are regulated via evolutionarily conserved biosynthesis and degradation pathways. Here we show that in Caenorhabditis elegans, serotonin is abundantly produced in nonneuronal tissues via phenylalanine hydroxylase, in addition to canonical biosynthesis via tryptophan hydroxylase in neurons. Combining CRISPR-Cas9 genome editing, comparative metabolomics and synthesis, we demonstrate that most serotonin in C. elegans is incorporated into N-acetylserotonin-derived glucosides, which are retained in the worm body and further modified via the carboxylesterase CEST-4. Expression patterns of CEST-4 suggest that serotonin or serotonin derivatives are transported between different tissues. Last, we show that bacterial indole production interacts with serotonin metabolism via CEST-4. Our results reveal a parallel pathway for serotonin biosynthesis in nonneuronal cell types and further indicate that serotonin-derived metabolites may serve distinct signaling functions and contribute to previously described serotonin-dependent phenotypes.

Sleepy without stimulation: subjective and objective sleepiness in actigraphy-verified natural short sleepers.

Natural short sleepers (NSS)-individuals who report minimal sleepiness or daytime dysfunction despite habitually sleeping less than the recommended amount (i.e., <7 h)-are a focus of growing interest in sleep research. Yet, the predominance of research on NSS has relied on subjective reports of functionality. The present study examined subjective and objective sleepiness among actigraphy-verified NSS in comparison with recommended (7-9 h/day) length sleepers (RLS) who reported similarly minimal daytime dysfunction. The study tested the hypothesis that under conditions of low environmental stimulation, NSS have increased risk of drowsiness and sleep onset, regardless of perceived alertness. The NSS and RLS groups were identified via screening and verified with a 14 day assessment with actigraphy, sleep diaries, and morning ratings of sleep restoration. In-laboratory resting electroencephalography (EEG) data were analysed using a computerised EEG-based algorithm (Vigilance Algorithm Leipzig; VIGALL) to classify second-by-second changes in objective sleepiness ranging from cognitively active alertness to sleep onset. Results demonstrated that NSS exhibited significantly higher drowsiness and sleep onset ('microsleeps') across 15 min of resting EEG despite perceptions of lower subjective sleepiness compared to RLS. Findings suggest that irrespective of perceived sleep restoration and alertness, NSS appear to be at high risk of objective sleepiness that is rapidly unmasked under conditions of low environmental stimulation. Such apparent discrepancy between subjective and objective sleepiness has potentially important public health implications. Future research directions, including tests of mechanisms and tailored sleep extension intervention, are discussed.

Posterolateral dislocation and incarceration of the long head of the biceps tendon: a rare cause of irreducible anterior glenohumeral joint dislocation-a case report and brief review of the literature.

Failure of closed reduction of anterior glenohumeral joint dislocation is infrequent. It can be secondary to osseous (e.g., fracture fragments or Hill-Sachs lesion) or soft tissue (e.g., labrum or rotator cuff tendon) impediments. Herein, we present a case of a prolonged irreducible glenohumeral joint secondary to a posterolaterally dislocated and incarcerated long head of the biceps tendon after an episode of anterior instability, highlight the utility of MR imaging for guiding management, and review the literature of this sporadic diagnosis.

Oligonucleotide Catabolism-Derived Gluconucleosides in Caenorhabditis elegans.

Nucleosides are essential cornerstones of life, and nucleoside derivatives and synthetic analogues have important biomedical applications. Correspondingly, production of non-canonical nucleoside derivatives in animal model systems is of particular interest. Here, we report the discovery of diverse glucose-based nucleosides in Caenorhabditis elegans and related nematodes. Using a mass spectrometric screen based on all-ion fragmentation in combination with total synthesis, we show that C. elegans selectively glucosylates a series of modified purines but not the canonical purine and pyrimidine bases. Analogous to ribonucleosides, the resulting gluconucleosides exist as phosphorylated and non-phosphorylated forms. The phosphorylated gluconucleosides can be additionally decorated with diverse acyl moieties from amino acid catabolism. Syntheses of representative variants, facilitated by a novel 2'-O- to 3'-O-dibenzyl phosphoryl transesterification reaction, demonstrated selective incorporation of different nucleobases and acyl moieties. Using stable-isotope labeling, we further show that gluconucleosides incorporate modified nucleobases derived from RNA and possibly DNA breakdown, revealing extensive recycling of oligonucleotide catabolites. Gluconucleosides are conserved in other nematodes, and biosynthesis of specific subsets is increased in germline mutants and during aging. Bioassays indicate that gluconucleosides may function in stress response pathways.

A prospective, blinded study of a PF4-dependent assay for HIT diagnosis.

Heparin-induced thrombocytopenia (HIT) is a life-threatening, prothrombotic, antibody-mediated disorder. To maximize the likelihood of recovery, early and accurate diagnosis is critical. Widely available HIT assays, such as the platelet factor 4 (PF4) heparin enzyme-linked immunosorbent assay (ELISA) lack specificity, and the gold-standard carbon 14-labeled serotonin release assay (SRA) is of limited value for early patient management because it is available only through reference laboratories. Recent studies have demonstrated that pathogenic HIT antibodies selectively activate PF4-treated platelets and that a technically simpler assay, the PF4-dependent P-selectin expression assay (PEA), may provide an option for rapid and conclusive results. Based upon predefined criteria that combined 4Ts scores and HIT ELISA results, 409 consecutive adults suspected of having HIT were classified as disease positive, negative, or indeterminate. Patients deemed HIT indeterminate were considered disease negative in the primary analysis and disease positive in a sensitivity analysis. The ability of PEA and SRA to identify patients judged to have HIT was compared using receiver operating characteristic curve statistics. Using these predefined criteria, the diagnostic accuracy of PEA was high (area under the curve [AUC], 0.94; 95% confidence interval [CI], 0.87-1.0) and similar to that of SRA (AUC, 0.91; 95% CI, 0.82-1.0). In sensitivity analysis, the AUCs of PEA and SRA were also similar at 0.88 (95% CI, 0.78-0.98) and 0.86 (95% CI, 0.77-0.96), respectively. The PEA, a technically simple nonradioactive assay that uses ∼20-fold fewer platelets compared with the SRA, had high accuracy for diagnosing HIT. Widespread use of the PEA may facilitate timely and more effective management of patients with suspected HIT.

HLA antibodies in fetal and neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by antibodies against human platelet antigens (HPA). However, in many cases that meet clinical criteria for the condition, maternal sera do not have HPA antibodies. In studies examining whether human leukocyte antigen (HLA) antibodies cause FNAIT, the results are limited and inconclusive. This study sought to examine whether clinically suspected FNAIT cases with absent maternal HPA antibodies had different HLA antibody strength and specificity compared to controls. STUDY DESIGN AND METHODS: A retrospective case-control study assessed class I HLA antibody strength and specificity in cases submitted for testing to Versiti, Wisconsin. There were 813 cases that met initial screening criteria, but written consent could only be obtained for 50. After review of medical records and expert panel review, 31 cases with clinical criteria of FNAIT and maternal HLA but not HPA antibodies were included. Each case was matched for maternal age, gestational age at delivery, parity, and race/ethnicity to two controls from unaffected pregnancies that had maternal serum HLA antibodies. RESULTS: FNAIT cases were found to have both significantly higher HLA antibody strength, measured by mean fluorescence index (MFI), and broader HLA antibody specificity at antigen epitope level, compared to matched controls (p < .001). p-values remained significant after controlling for parity and gestational age at delivery. DISCUSSION: Additional studies are needed to further examine whether the strong HLA antibodies identified in HPA-antibody-negative cases directly cause neonatal thrombocytopenia and whether prenatal treatment may be warranted in select cases to prevent recurrence.

World human neutrophil antigens investigation survey.

BACKGROUND AND OBJECTIVES: Human neutrophil antigens (HNAs) are categorized into five systems: HNA-1 to HNA-5. Given the importance of neutrophils in immunity, we sought to create awareness of the role of HNA diagnostic services in managing immune neutropenia and transfusion-related acute lung injury. To provide health communities all around the world with access to these services, we conducted a survey to create a directory of these HNA diagnostic services. MATERIALS AND METHODS: An Excel table-based survey was created to capture information on the laboratory's location and was emailed to 55 individuals with known or possible HNA investigation activity. The collected data were then summarized and analysed. RESULTS: Of contacted laboratories, the surveys were returned from 23 (38.2%) laboratories; 17 have already established HNA diagnostic (of them 12 were regular participants of the International Granulocyte Immunobiology Workshop [ISBT-IGIW]), 4 laboratories were in the process of establishing their HNA investigation and the remaining 2 responder laboratories, did not conduct HNA investigations. In established laboratories, investigation for autoimmune neutropenia (infancies and adults) was the most frequently requested, and antibodies against HNA-1a and HNA-1b were the most commonly detected. CONCLUSION: The directory of survey respondents provides a resource for health professionals wanting to access HNA diagnostic services. The present study offers a comprehensive picture of HNA diagnostics (typing and serology), identifying weak points and areas for improvement for the first time. Identifying more laboratories involved in HNA diagnostics with limited access to international societies in the field will globally improve HNA diagnostics.

Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.

Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult-to-procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene-editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1-1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9-puromycin expression vector and transfected into OT1-1. Genotyping was performed to select puromycin-resistant hiPSC KOs. CRISPR/Cas9 gene-editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1-1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34+ CD41+ CD235ab+ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1-1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1-1 as well as the three KO erythroblasts remained positive for RBC markers-CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400-fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene-edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.

Combinatorial Assembly of Modular Glucosides via Carboxylesterases Regulates C. elegans Starvation Survival.

The recently discovered modular glucosides (MOGLs) form a large metabolite library derived from combinatorial assembly of moieties from amino acid, neurotransmitter, and lipid metabolism in the model organism C. elegans. Combining CRISPR-Cas9 genome editing, comparative metabolomics, and synthesis, we show that the carboxylesterase homologue Cel-CEST-1.2 is responsible for specific 2-O-acylation of diverse glucose scaffolds with a wide variety of building blocks, resulting in more than 150 different MOGLs. We further show that this biosynthetic role is conserved for the closest homologue of Cel-CEST-1.2 in the related nematode species C. briggsae, Cbr-CEST-2. Expression of Cel-cest-1.2 and MOGL biosynthesis are strongly induced by starvation conditions in C. elegans, one of the premier model systems for mechanisms connecting nutrition and physiology. Cel-cest-1.2-deletion results in early death of adult animals under starvation conditions, providing first insights into the biological functions of MOGLs.

Bioengineered iPSC-derived megakaryocytes for the detection of platelet-specific patient alloantibodies.

Human platelet membrane glycoprotein polymorphisms can be immunogenic in man and are frequently the cause of clinically important immune reactions responsible for disorders such as neonatal alloimmune thrombocytopenia. Platelets from individuals carrying rare polymorphisms are often difficult to obtain, making diagnostic testing and transfusion of matched platelets challenging. In addition, class I HLA antibodies frequently present in maternal sera interfere with the detection of platelet-reactive alloantibodies. Detection of alloantibodies to human platelet antigen 3 (HPA-3) and HPA-9 is especially challenging, in part because of the presence of cell type-specific glycans situated near the polymorphic amino acid that together form the alloepitope. To overcome these limitations, we generated a series of HLA class I-negative blood group O induced pluripotent stem cell (iPSC) lines that were gene edited to sequentially convert their endogenous HPA-3a alloantigenic epitope to HPA-3b, and HPA-9a to HPA-9b. Subjecting these cell lines, upon differentiation into CD41+/CD42b+ human megakaryocytes (MKs), to flow cytometric detection of suspected anti-HPA-3 and HPA-9 alloantisera revealed that the HPA-3a-positive MKs specifically reacted with HPA-3a patient sera, whereas the HPA-3b MKs lost reactivity with HPA-3a patient sera while acquiring reactivity to HPA-3b patient sera. Importantly, HPA-9b-expressing MKs specifically reacted with anti-HPA-9b-suspected patient samples that had been undetectable using conventional techniques. The provision of specialized iPSC-derived human MKs expressing intact homozygous glycoprotein alloantigens on the cell surface that carry the appropriate endogenous carbohydrate moieties should greatly enhance detection of clinically important and rare HPA-specific alloantibodies that, to date, have resisted detection using current methods.

Syntheses of Amorfrutins and Derivatives via Tandem Diels-Alder and Anionic Cascade Approaches.

We describe two complementary approaches based on a convergent [4+2] logic toward the synthesis of amorfrutins, cannabinoids, and related plant metabolites. An anionic cascade cyclization employing ß-methoxycrotonates and ß-chloro-α,ß-unsaturated esters yielded amorfrutins in four linear steps and demonstrated utility of ß-alkoxycrotonate-derived nucleophiles as functional equivalents of ß-ketoester-derived dianions. Analogously, tandem Diels-Alder/retro-Diels-Alder cycloaddition of dimedone-derived bis(trimethylsiloxy)-dienes and α,ß-alkynyl ester dienophiles provided facile access to resorcinol precursors of amorfrutins and cannabinoids, avoiding late-stage installation of prenyl or geranyl moieties as in previous approaches.

Autonomous conformational regulation of ß3 integrin and the conformation-dependent property of HPA-1a alloantibodies.

Integrin α/ß heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of ß subunit induce the α/ß separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The ß3 integrins, including αIIbß3 and αVß3, are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the ß3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti-HPA-1a alloantibodies can distinguish the αIIbß3 from αVß3 and affect their functions with unknown mechanisms. Here we designed a single-chain ß3 subunit that mimics a separation of α/ß heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain ß3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the ß3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain ß3 integrin, we identified the conformation-dependent property of anti-HPA-1a alloantibodies, which enables them to differently recognize the ß3 in the bent state vs. the extended state and in the complex with αIIb vs. αV This study provides deeper understandings of integrin conformational activation on the cell surface.

Strategies to develop a prophylaxis for the prevention of HPA-1a immunization and fetal and neonatal alloimmune thrombocytopenia.

Anti-HPA-1a-antibodies are the main cause of fetal and neonatal alloimmune thrombocytopenia (FNAIT) which may result in intracranial hemorrhage (ICH) and death among fetuses and newborns. Advances in understanding the pathogenesis of FNAIT and proof of concept for prophylaxis to prevent immunization suggest that development of hyperimmune anti-HPA-1a IgG aimed at preventing immunization against HPA-1a and FNAIT is feasible. Anti-HPA-1a IgG can be obtained either by isolating immunoglobulin from already-immunized women or by development of monoclonal anti-HPA-1a antibodies. Here we discuss recent advances that may lead to the development of a prenatal and postnatal prophylactic treatment for the prevention of HPA-1a-associated FNAIT and life-threatening FNAIT-induced complications.

Pes Anserinus: Anatomy and Pathology of Native and Harvested Tendons.

OBJECTIVE. The purpose of this article is to review the anatomy and pathology of the pes anserinus to increase the accuracy of imaging interpretation of findings affecting these medial knee structures. CONCLUSION. The pes anserinus, consisting of the conjoined tendons of the sartorius, gracilis, and semitendinosus muscles and their insertions at the medial aspect of the knee, is often neglected during imaging assessment. Common pathologic conditions affecting the pes anserinus include overuse, acute trauma, iatrogenic disorders, and tumors and tumorlike lesions.

A case of platelet transfusion refractoriness due to anti-CD36 with a successful treatment outcome.

CONCLUSIONS: Antibodies (Abs) against antigens on platelets (PLTs), including glycoprotein IV (CD36), can cause PLT refractoriness. Transfusing PLTs to patients with anti-CD36 is challenging because of the rarity of CD36-negative (CD36-) donors and the possibility of additional HLA Abs. We report a case of PLT refractoriness due to anti-CD36 and HLA Abs. A 21-year-old man (group O, D+) with assumed drug-induced aplastic anemia received multiple PLT transfusions and developed severe PLT refractoriness. He was found to have anti-CD36 as well as HLA class I Abs, with a CD36- phenotype on both PLTs and monocytes. He was diagnosed with type 1 CD36 deficiency and received intravenous immunoglobulin (IVIG) and rituximab to decrease future Ab production. The PLT corrected count increment (CCI) improved significantly with subsequent transfusions of flow crossmatchcompatible as well as uncrossmatched PLTs. He eventually received a bone marrow transplant and has been doing well since. The mean CCI before and after IVIG/rituximab treatment was 0.2 and 6.2, respectively. Soon after IVIG started, the patient's CCI after receiving CD36-, group AB, D+, and HLA untested PLTs was 0.8, but his CCI after receiving flow crossmatch-compatible PLTs was 12.6. Two months after IVIG was started, the mean CCIs for uncrossmatched apheresis PLTs and crossmatch-compatible PLTs were comparable (6.1 versus 6.0, respectively). Desensitization treatment with IVIG and rituximab lowered anti-CD36 and HLA Ab levels, and the CCI of PLT transfusion improved significantly. This case demonstrates that immune suppression is effective for successful PLT transfusion of patients with anti-CD36.

Individual differences in aesthetic engagement are reflected in resting-state fMRI connectivity: Implications for stress resilience.

OBJECTIVE: Individual differences in aesthetic engagement-the propensity to be moved by art, nature, and beauty-are associated with positive health outcomes, as well as stress resilience. The purpose of the current study was to identify potential neural substrate mechanisms underlying individual differences in aesthetic engagement and reported proneness to aesthetic chill. METHODS: Data from the Human Connectome Project (HCP) 1200 Subjects Release were utilized. Resting-state fMRI connectivity was extracted for 361 regions in the brain including cortical, subcortical and cerebellar regions for each participant, using participant-specific segmentation and parcellation of subcortical gray matter nuclei and a network-based statistics analytical approach. The Aesthetic Interests subcluster of the Openness to Experience scale (NEO-Five Factor Inventory; NEO-FFI) was used to characterize individual differences in aesthetic engagement and chill. RESULTS: Participants reporting higher aesthetic engagement, particularly proneness to aesthetic chill responses, exhibited significantly higher connectivity between the default network and sensory and motor cortices, higher connectivity between the ventral default and salience networks, and decreased connectivity between the cerebellum and somatomotor cortex. CONCLUSIONS: Current findings suggest that greater integration of the default mode network, involving processing of internal narrative, with neural representations of sensory perception and salience detection may be a mechanism underlying individual differences in aesthetic engagement. Thus, these individual differences may reflect general integration of environmental perception with internal emotional experience, which in turn may facilitate comfort with novelty, self-regulation, and positive adaptation to potentially stressful experiences.

CRISPR/Cas9-mediated conversion of human platelet alloantigen allotypes.

Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia and posttransfusion purpura. The HPA-1a/HPA-1b alloantigen system, also known as the Pl(A1)/Pl(A2) polymorphism, is the most frequently implicated HPA among whites, and a single Leu33Pro amino acid polymorphism within the integrin ß3 subunit is responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. HPA-1b/b platelets, like those bearing other low-frequency platelet-specific alloantigens, are relatively rare in the population and difficult to obtain for purposes of transfusion therapy and diagnostic testing. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) gene-editing technology to transform Leu33 (+) megakaryocytelike DAMI cells and induced pluripotent stem cells (iPSCs) to the Pro33 allotype. CD41(+) megakaryocyte progenitors derived from these cells expressed the HPA-1b (Pl(A2)) alloantigenic epitope, as reported by diagnostic NciI restriction enzyme digestion, DNA sequencing, and western blot analysis using HPA-1b-specific human maternal alloantisera. Application of CRISPR/Cas9 technology to genetically edit this and other clinically-important HPAs holds great potential for production of designer platelets for diagnostic, investigative, and, ultimately, therapeutic use.