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Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021.As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented.Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030).The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Genome sequencing holds the promise for great public health benefits. It is currently being used in the context of rare disease diagnosis and novel gene identification, but also has the potential to identify genetic disease risk factors in healthy individuals. Genome sequencing technologies are currently being used to identify genetic factors that may influence variability in symptom severity and immune response among patients infected by SARS-CoV-2. The GENCOV study aims to look at the relationship between genetic, serological, and biochemical factors and variability of SARS-CoV-2 symptom severity, and to evaluate the utility of returning genome screening results to study participants. Study participants select which results they wish to receive with a decision aid. Medically actionable information for diagnosis, disease risk estimation, disease prevention, and patient management are provided in a comprehensive genome report. Using a combination of bioinformatics software and custom tools, this article describes a pipeline for the analysis and reporting of genetic results to individuals with COVID-19, including HLA genotyping, large-scale continental ancestry estimation, and pharmacogenomic analysis to determine metabolizer status and drug response. In addition, this pipeline includes reporting of medically actionable conditions from comprehensive gene panels for Cardiology, Neurology, Metabolism, Hereditary Cancer, and Hereditary Kidney, and carrier screening for reproductive planning. Incorporated into the genome report are polygenic risk scores for six diseases-coronary artery disease; atrial fibrillation; type-2 diabetes; and breast, prostate, and colon cancer-as well as blood group genotyping analysis for ABO and Rh blood types and genotyping for other antigens of clinical relevance. The genome report summarizes the findings of these analyses in a way that extensively communicates clinically relevant results to patients and their physicians. © 2022 Wiley Periodicals LLC. Basic Protocol 1: HLA genotyping and disease association Basic Protocol 2: Large-scale continental ancestry estimation Basic Protocol 3: Dosage recommendations for pharmacogenomic gene variants associated with drug response Support Protocol: System setup.

Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I–low, neuroendocrine carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.

Background The MNS blood group system is defined by three homologous genes: GYPA, GYPB, and GYPE. GYPB encodes for glycophorin B (GPB) carrying S/s and the "universal" antigen U. RBCs of approximately 1% of individuals of African ancestry are U- due to absence of GPB. The U- phenotype has long been attributed to a deletion encompassing GYPB exons 2 to 5 and GYPE exon 1 (GYPB*01N). Study design and methods Samples from two U-individuals underwent Illumina short read whole genome sequencing (WGS) and Nanopore long read WGS. In addition, two existing WGS datasets, MedSeq (n = 110) and 1000 Genomes (1000G, n = 2535), were analyzed for GYPB deletions. Deletions were confirmed by Sanger sequencing. Twenty known U- donor samples were tested by a PCR assay to determine the specific deletion alleles present in African Americans. Results Two large GYPB deletions in U- samples of African ancestry were identified: a 110 kb deletion extending left of GYPB (DEL_B_LEFT) and a 103 kb deletion extending right (DEL_B_RIGHT). DEL_B_LEFT and DEL_B_RIGHT were the most common GYPB deletions in the 1000 Genomes Project 669 African genomes (allele frequencies 0.04 and 0.02). Seven additional deletions involving GYPB were seen in African, Admixed American, and South Asian samples. No samples analyzed had GYPB*01N. Conclusions The U- phenotype in those of African ancestry is primarily associated with two different complete deletions of GYPB (with intact GYPE). Seven additional less common GYPB deletion backgrounds were found. GYPB*01N, long assumed to be the allele commonly encoding U- phenotypes, appears to be rare.

Immune evasion is a hallmark of cancer and a central mechanism underlying acquired resistance to immune therapy. In allogeneic hematopoietic cell transplantation (alloHCT), late relapses can arise after prolonged alloreactive T-cell control, but the molecular mechanisms of immune escape remain unclear. To identify mechanisms of immune evasion, we performed a genetic analysis of serial samples from 25 patients with myeloid malignancies who relapsed ≥1 year after alloHCT. Using targeted sequencing and microarray analysis to determine HLA allele-specific copy number, we identified copy-neutral loss of heterozygosity events and focal deletions spanning class 1 HLA genes in 2 of 12 recipients of matched unrelated-donor HCT and in 1 of 4 recipients of mismatched unrelated-donor HCT. Relapsed clones, although highly related to their antecedent pretransplantation malignancies, frequently acquired additional mutations in transcription factors and mitogenic signaling genes. Previously, the study of relapse after haploidentical HCT established the paradigm of immune evasion via loss of mismatched HLA. Here, in the context of matched unrelated-donor HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention.

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Cohesin is a ring complex composed of four core subunits and seven regulatory subunits. In an effort to comprehensively identify additional cohesin-interacting proteins, we used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in cultured human cells, and we performed MS analyses on dual-affinity purifications. In addition to reciprocally identifying all known components of cohesin, we found that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs). These included diverse components of the U4/U6.U5 tri-small nuclear ribonucleoprotein complex and several splicing factors that are commonly mutated in cancer. The interaction between cohesin and splicing factors/RBPs was RNA- and DNA-independent, occurred in chromatin, was enhanced during mitosis, and required RAD21. Furthermore, cohesin-interacting splicing factors and RBPs followed the cohesin cycle and prophase pathway of cell cycle–regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors and RBPs resulted in aberrant mitotic progression. These results provide a comprehensive view of the endogenous human cohesin interactome and identify splicing factors and RBPs as functionally significant cohesin-interacting proteins.

Background and Objectives: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. Materials and Methods: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. Results: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. Conclusion: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays. (Less)

Emm is a high incidence red cell antigen with eight previously reported Emm− probands. Anti-Emm appears to be naturally occurring yet responsible for a clinically significant acute hemolytic transfusion reaction. Previous work suggests that Emm is located on a GPI-anchored protein, but the antigenic epitope and genetic basis have been elusive. We investigated samples from a South Asian Indian family with two Emm− brothers by whole genome sequencing (WGS). Additionally, samples from four unrelated Emm− individuals were investigated for variants in the candidate gene. Filtering for homozygous variants found in the Emm− brothers and by gnomAD frequency of PIGG [NM_001127178.3:c.2624_2625delTA, p.(Leu875*), rs771819481]. PIGG encodes for a transferase, GPI-ethanolaminephosphate transferase II, which adds ethanolamine phosphate (EtNP) to the second mannose in a GPI-anchor. The four additional unrelated Emm− individuals had various PIGG mutations; deletion of Exons 2–3, deletion of Exons 7–9, insertion/deletion (indel) in Exon 3, and new stop codon in Exon 5. The Emm− phenotype is associated with a rare deficiency of PIGG, potentially defining a new Emm blood group system composed of EtNP bound to mannose, part of the GPI-anchor. The results are consistent with the known PI-linked association of the Emm antigen, and may explain the production of the antibody in the absence of RBC transfusion. Any association with neurologic phenotypes requires further research.

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

BACKGROUND: Although P1 and Xg(a) are known to be associated with the A4GALT and XG genes respectively, the genetic basis of antigen expression has been elusive. Recent reports link both P1 and Xg(a) expression with nucleotide changes in the promotor regions and with antigen negative phenotypes due to disruption of transcription factor binding. STUDY DESIGN AND METHODS: Whole genome sequencing was performed on 113 individuals as part of the MedSeq Project with serologic RBC antigen typing for P1 (n=77) and Xg(a) (n=15). Genomic data were analyzed by two approaches, nucleotide frequency correlation or serologic correlation, to find A4GALT and XG changes associated with P1 and Xg(a) expression. RESULTS: For P1, the frequency approach identified 29 possible associated nucleotide changes, and the serologic approach revealed four among them correlating with the P1+/P1− phenotype: chr22:43,115,523_43,115,520AAAG/delAAAG (rs66781836); chr 22:43,114,551C/T (rs8138197); chr22:43,114,020T/G (rs2143918); and chr22:43,113,793G/T (rs5751348). For Xg(a), the frequency approach identified 82 possible associated nucleotide changes, and among these the serologic approach revealed one correlating with the Xg(a+)/Xg(a–) phenotype: chrX:2,666,384G/C (rs311103). CONCLUSION: A bioinformatics analysis pipeline was created to identify genetic changes responsible for RBC antigen expression. This study, in progress prior to the recently published reports, independently confirms the basis for P1 and Xg(a). Although, this enabled molecular typing of these antigens, the Y chromosome PAR1 region interfered with Xg(a) typing in males. This approach could be used to identify and confirm the genetic basis of antigens, potentially replacing the historical approach using family pedigrees as genomic sequencing becomes common place.

Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on data-sets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation.

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non–self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

OBJECTIVE: To investigate associations among care errors, staffing, and workload in small animal ICUs. DESIGN: Multicenter observational cohort study conducted between January 2017 and September 2018. SETTING: Three small animal teaching hospital ICUs. ANIMALS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Data on patient numbers, illness severity (assesed via the acute patient physiologic and laboratory evaluation [APPLE] score), care burden, staffing levels, technician experience/education level, and care errors were collected at each study site. Care errors were categorized as major (unanticipated arrest or death; patient endangerment through IV line, arterial catheter, chest tube or other invasive device mismanagement, or errors in drug calculation/administration) or minor. Median patient:technician ratio was 4.3 (range: 1–18). Median patient illness severity was 15.1 (4.7–27.1) APPLE score units. A total of 221 major and 3,317 minor errors were observed over the study period. The odds of a major error increased by an average of 11% (odds ratio [OR] = 1.11; 95% confidence interval [CI], 1.02–1.20; P = 0.012) for each 1 patient increase in the patient:technician ratio after averaging by ICU location. The major error incident rate ratio was 2.53 (95% CI, 1.84–3.54; P 4.0 compared with ≤4.0. The odds of a major error increased by 0.5% per total unit APPLE score increase (OR = 1.005; 95% CI, 1.002–1.007; P 73 compared with ≤73. The odds of a major error decreased by 2% (OR = 0.98; 95% CI, 0.97–0.99; P = 0.01) for each year increase in total technician years of ICU work experience. CONCLUSIONS: Substantial reductions in major care errors may be achieved by maintaining ICU patient:technician ratios at ≤4. Technician experience and total unit burden of patient illness severity are also associated with error incidence, and should be taken into consideration when scheduling staff.

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

Objectives To investigate veterinary technician burnout and associations with frequency of self‐reported medical error, resilience, and depression and job‐related risk factors. Design Cross‐sectional observational study using an anonymous survey conducted between November 2017 and June 2018. Setting Four referral teaching hospitals in the United States and Canada. Subjects A total of 344 veterinary technicians were invited to participate. Response rate was 95%. Overall 256 surveys were ultimately analyzed. Interventions Burnout, depression, and resilience were measured using validated instruments. Respondents reported perceptions of workload, working environment, and medical error frequency. Associations between burnout and factors related to physical work environment, workload and schedule, compensation package, interpersonal relationships, intellectual enrichment, and exposure to ethical conflicts were analyzed. Measurements and Main Results Burnout, characterized by high emotional exhaustion, depersonalization, and low sense of personal accomplishment was common, and was positively associated with perceived medical errors, desire to change career, and depression. Burnout levels on all 3 burnout subscales were higher in this population than previously reported for a contemporaneous group of trauma nurses working with human patients (P < 0.05). Burnout was negatively associated with resilience. Respondents’ feelings of fear or anxiety around supervisor communications, perception that patient load was too high to allow for excellent patient care, and perceived lack of available assistance during sudden workload increases were all associated with burnout. Conclusions Burnout in veterinary technicians is common and is associated with numerous undesirable outcomes. Work‐related interventions to reduce burnout should focus on improving supervisor relationships and maintaining an appropriate patient:caregiver ratio.

Background Genotyping has expanded the number red blood cell (RBC) and platelet (PLT) antigens that can readily be typed, but often represents an additional testing cost. The analysis of existing genomic data offers a cost-effective approach. We recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing. Here we extend the algorithm to whole exome sequencing (WES). Study design and methods Whole exome sequencing was performed on samples from 75 individuals. WES-based bloodTyper RBC and PLT typing was compared to conventional polymerase chain reaction (PCR) RHD zygosity testing and serologic and single-nucleotide polymorphism (SNP) typing for 38 RBC antigens in 12 systems (17 serologic and 35 SNPs) and 22 PLT antigens (22 SNPs). Samples from the first 20 individuals were used to modify bloodTyper to interpret WES followed by blinded typing of 55 samples. Results Over the first 20 samples, discordances were noted for C, M, and N antigens, which were due to WES-specific biases. After modification, bloodTyper was 100% accurate on blinded evaluation of the last 55 samples and outperformed both serologic (99.67% accurate) and SNP typing (99.97% accurate) reflected by two Fyb and one N serologic typing errors and one undetected SNP encoding a Jknull phenotype. RHD zygosity testing by bloodTyper was 100% concordant with a combination of hybrid Rhesus box PCR and PCR-restriction fragment length polymorphism for all samples. Conclusion The automated bloodTyper software was modified for WES biases to allow for accurate RBC and PLT antigen typing. Such analysis could become a routing part of future WES efforts.

PURPOSE Determine the incidence of blunt carotid injury (BCAI) in the blunt trauma induced isolated mandible fracture population and determine the utility of computed tomographic angiography (CTA) screening for BCAI in this population. METHODS A retrospective data review was performed on patients presenting to Boston Medical Center from January 2008 to January 2019 with a diagnosis of mandible fracture. Population selected utilizing ICD-9 and ICD-10 diagnosis codes for mandible fracture and BCAI. Excluded populations were pediatric (less than 18 years) and penetrating carotid injuries. RESULTS A total of 1,508 mandible fractures were identified, with 73% (n = 1,103) being isolated injuries. Five BCAIs were identified, and of these, 1 was associated with an isolated mandible fracture (incidence

Recent studies have highlighted the promise of targeting tumor neoantigens to generate potent antitumor immune responses and provide strong motivation for improving our understanding of antigen-T-cell receptor (TCR) interactions. Advances in single-cell sequencing technologies have opened the door for detailed investigation of the TCR repertoire, providing paired information from TCRα and TCRβ, which together determine specificity. However, a need remains for efficient methods to assess the specificity of discovered TCRs. We developed a streamlined approach for matching TCR sequences with cognate antigen through on-demand cloning and expression of TCRs and screening against candidate antigens. Here, we first demonstrate the system’s capacity to identify viral-antigen-specific TCRs and compare the functional avidity of TCRs specific for a given antigen target. We then apply this system to identify neoantigen-specific TCR sequences from patients with melanoma treated with personalized neoantigen vaccines and characterize functional avidity of neoantigen-specific TCRs. Furthermore, we use a neoantigen-prediction pipeline to show that an insertion-deletion mutation in a putative chronic lymphocytic leukemia (CLL) driver gives rise to an immunogenic neoantigen mut-MGA, and use this approach to identify the mut-MGA-specific TCR sequence. This approach provides a means to identify and express TCRs, and then rapidly assess antigen specificity and functional avidity of a reconstructed TCR, which can be applied for monitoring antigen-specific T-cell responses, and potentially for guiding the design of effective T-cell-based immunotherapies.

MutS homolog 2 (MSH2) is an essential DNA mismatch repair (MMR) protein. It interacts with MSH6 or MSH3 to form the MutSα or MutSβ complex, respectively, which recognize base-base mispairs and insertions/deletions and initiate the repair process. Mutation or dysregulation of MSH2 causes genomic instability that can lead to cancer. MSH2 is acetylated at its C terminus, and histone deacetylase (HDAC6) deacetylates MSH2. However, whether other regions of MSH2 can be acetylated and whether other histone deacetylases (HDACs) and histone acetyltransferases (HATs) are involved in MSH2 deacetylation/acetylation is unknown. Here, we report that MSH2 can be acetylated at Lys-73 near the N terminus. Lys-73 is highly conserved across many species. Although several Class I and II HDACs interact with MSH2, HDAC10 is the major enzyme that deacetylates MSH2 at Lys-73. Histone acetyltransferase HBO1 might acetylate this residue. HDAC10 overexpression in HeLa cells stimulates cellular DNA MMR activity, whereas HDAC10 knockdown decreases DNA MMR activity. Thus, our study identifies an HDAC10-mediated regulatory mechanism controlling the DNA mismatch repair function of MSH2.

Detection of somatic mutations in human leukocyte antigen (HLA) genes using whole-exome sequencing (WES) is hampered by the high polymorphism of the HLA loci, which prevents alignment of sequencing reads to the human reference genome. We describe a computational pipeline that enables accurate inference of germline alleles of class I HLA-A, B and C genes and subsequent detection of mutations in these genes using the inferred alleles as a reference. Analysis of WES data from 7,930 pairs of tumor and healthy tissue from the same patient revealed 298 nonsilent HLA mutations in tumors from 266 patients. These 298 mutations are enriched for likely functional mutations, including putative loss-of-function events. Recurrence of mutations suggested that these 'hotspot' sites were positively selected. Cancers with recurrent somatic HLA mutations were associated with upregulation of signatures of cytolytic activity characteristic of tumor infiltration by effector lymphocytes, supporting immune evasion by altered HLA function as a contributory mechanism in cancer.

SummaryPlatelet transfusions are a cornerstone of therapy for patients who develop thrombocytopenia while undergoing Hematopoietic Stem Cell Transplantation (HSCT). Many patients who develop Platelet Transfusion Refractoriness (PTR) require HLA-matched platelets. Identifying these patients early could lead to better utilization of platelets as well as increased platelet counts. We built a SMART on FHIR visualization tool to aid the oncology, blood bank, and blood donor center teams in identifying these patients by showing trends in thrombocytopenia along with a computer generated calculated Panel Reactive Antibody (cPRA) level. To do this, we required a FHIR interface to our HLA database. We describe our methods and outcome for constructing this FHIR interface, as well as the architecture and data flow of HLA data from its proprietary database to the SMART on FHIR environment and application database along with RESTful cPRA web service calculator. Future work will evaluate the clinical impact of this platelet visualization tool and overall success of our FHIR implementation. Citation: Gordon WJ, Baronas J, Lane WJ. A FHIR human leukocyte antigen (HLA) interface for platelet transfusion Support. Appl Clin Inform 2017; 8: 603–611 https://doi.org/10.4338/ACI-2017-01-CR-0010

Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.

Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia mediated by autoantibodies that preferentially react at 4°C. Laboratory testing for cold-reactive autoantibodies is laborious and may not be ordered judiciously, particularly in patients with a negative direct antiglobulin test (DAT). We sought to determine whether a negative DAT using anti-human complement (anti-C3) rules out elevated cold agglutinin (CA) titers and the diagnosis of CAD.We performed a retrospective study of patients with a CA test performed at three major academic medical centers: Barnes-Jewish Hospital (2003-2014), Vanderbilt University Medical Center (2007-2009), and Massachusetts General Hospital (2009-2014).This study included 801 patients, of whom 51% (n = 410) had a DAT within the 7 days before CA testing. A total of 98% of patients with a negative DAT using anti-C3 had a negative CA titer (64). Only five subjects had a negative DAT using anti-C3 and an elevated CA titer.Overutilization of CA testing could be reduced by establishing laboratory acceptance criteria based on a positive DAT using anti-C3. Such acceptance criteria would have reduced CA testing by 68% for those with an available DAT result.

DNA methylation and histone acetylation/deacetylation are distinct biochemical processes that control gene expression. While DNA methylation is a common epigenetic signal that inhibits gene transcription, histone deacetylation similarly represses transcription but can be both an epigenetic and nonepigenetic phenomenon. Here we report that the histone deacetylase SIRT1 regulates the activities of DNMT1, a key enzyme responsible for DNA methylation. In mass spectrometry analysis, 12 new acetylated lysine sites were identified in DNMT1. SIRT1 physically associates with DNMT1 and can deacetylate acetylated DNMT1 in vitro and in vivo. Interestingly, deacetylation of different lysines on DNMT1 has different effects on the functions of DNMT1. For example, deacetylation of Lys1349 and Lys1415 in the catalytic domain of DNMT1 enhances DNMT1's methyltransferase activity, while deacetylation of lysine residues in the GK linker decreases DNMT1's methyltransferase-independent transcriptional repression function. Furthermore, deacetylation of all identified acetylated lysine sites in DNMT1 abrogates its binding to SIRT1 and impairs its capability to regulate cell cycle G(2)/M transition. Finally, inhibition of SIRT1 strengthens the silencing effects of DNMT1 on the expression of tumor suppressor genes ER-α and CDH1 in MDA-MB-231 breast cancer cells. Together, these results suggest that SIRT1-mediated deacetylation of DNMT1 is crucial for DNMT1's multiple effects in gene silencing.

Resource (core) facilities have played an ever-increasing role in furnishing the scientific community with specialized instrumentation and expertise for proteomics experiments in a cost-effective manner. The Proteomics Research Group (PRG) of the Association of Biomolecular Resource Facilities (ABRF) has sponsored a number of research studies designed to enable participants to try new techniques and assess their capabilities relative to other laboratories analyzing the same samples. Presented here are results from three PRG studies representing different samples that are typically analyzed in a core facility, ranging from simple protein identification to targeted analyses, and include intentional challenges to reflect realistic studies. The PRG2008 study compares different strategies for the qualitative characterization of proteins, particularly the utility of complementary methods for characterizing truncated protein forms. The use of different approaches for determining quantitative differences for several target proteins in human plasma was the focus of the PRG2009 study. The PRG2010 study explored different methods for determining specific constituents while identifying unforeseen problems that could account for unanticipated results associated with the different samples, and included (15) N-labeled proteins as an additional challenge. These studies provide a valuable educational resource to research laboratories and core facilities, as well as a mechanism for establishing good laboratory practices.

SUMMARY Circadian rhythms are a hallmark of physiology, but how such daily rhythms organize cellular catabolism is poorly understood. Here, we used proteomics to map daily oscillations in autophagic flux in mouse liver and related these rhythms to proteasome activity. We also explored how systemic inflammation affects the temporal structure of autophagy. Our data identified a globally harmonized rhythm for basal macroautophagy, chaperone-mediated autophagy, and proteasomal activity, which concentrates liver proteolysis during the daytime. Basal autophagy rhythms could be resolved into two antiphase clusters that were distinguished by the subcellular location of targeted proteins. Inflammation induced by lipopolysaccharide reprogrammed autophagic flux away from a temporal pattern that favors cytosolic targets and toward the turnover of mitochondrial targets. Our data detail how daily biological rhythms connect the temporal, spatial, and metabolic aspects of protein catabolism., Graphical Abstract, In Brief How circadian rhythms contribute to cellular quality control is poorly understood. Ryzhikov et al. mapped proteome-wide biological rhythms in liver autophagy and identified synchronous daily oscillations in autophagic and proteasomal activity. Diurnal rhythms play a role in autophagy substrate selection based on subcellular location and inflammation status.

The ataxia telangiectasia group D-complementing (ATDC) gene product, also known as TRIM29, is a member of the tripartite motif (TRIM) protein family. ATDC has been proposed to form homo- or heterodimers and to bind nucleic acids. In cell cultures, ATDC expression leads to rapid growth and resistance to ionizing radiation (IR), whereas silencing of ATDC expression decreases growth rates and increases sensitivity to IR. Although ATDC is overexpressed in many human cancers, the biological significance of ATDC overexpression remains obscure. We report here that ATDC increases cell proliferation via inhibition of p53 nuclear activities. ATDC represses the expression of p53-regulated genes, including p21 and NOXA. Mechanistically, ATDC binds p53, and this interaction is potentially fine-tuned by posttranslational acetylation of lysine 116 on ATDC. The association of p53 and ATDC results in p53 sequestration outside of the nucleus. Together, these results provide novel mechanistic insights into the function of ATDC and offer an explanation for how ATDC promotes cancer cell proliferation.

Background— Performance of prehospital ECGs expedites identification of ST-elevation myocardial infarction and reduces door-to-balloon times for patients receiving reperfusion therapy. To fully realize this benefit, emergency medical service performance must be measured and used in feedback reporting and quality improvement. Methods and Results— This quasi-experimental design trial tested an approach to improving emergency medical service prehospital ECGs using feedback reporting and quality improvement interventions in 2 cities’ emergency medical service agencies and receiving hospitals. All patients age ≥30 years, calling 9-1-1 with possible acute coronary syndrome, were included. In total, 6994 patients were included: 1589 patients in the baseline period without feedback and 5405 in the intervention period when there were feedback reports and quality improvement interventions. Mean age was 66±17 years, and women represented 51%. Feedback and quality improvement increased prehospital ECG performance for patients with acute coronary syndrome from 76% to 93% ( P =P =P =0.001), but the median total emergency medical service run time remained the same at 22 minutes. The proportion of patients with door-to-balloon times of ≤90 minutes increased from 27% to 67% ( P =0.006). Conclusions— Feedback reports and quality improvement improved prehospital ECG performance for patients with acute coronary syndrome and ST-elevation myocardial infarction and increased aspirin administration without prehospital transport delays. Improvements in door-to-balloon times were also seen.

Rapid activation of p53 by ionizing irradiation is a classic DNA damage response mediated by the ATM kinase. However, the major signalling target and mechanism that lead to p53 stabilization are unknown. We show in this report that ATM induces p53 accumulation by phosphorylating the ubiquitin E3 ligase MDM2. Multiple ATM target sites near the MDM2 RING domain function in a redundant manner to provide robust DNA damage signalling. In the absence of DNA damage, the MDM2 RING domain forms oligomers that mediate p53 poly ubiquitination and proteasomal degradation. Phosphorylation by ATM inhibits RING domain oligomerization, specifically suppressing p53 poly ubiquitination. Blocking MDM2 phosphorylation by alanine substitution of all six phosphorylation sites results in constitutive degradation of p53 after DNA damage. These observations show that ATM controls p53 stability by regulating MDM2 RING domain oligomerization and E3 ligase processivity. Promoting or disrupting E3 oligomerization may be a general mechanism by which signalling kinases regulate ubiquitination reactions, and a potential target for therapeutic intervention.

SummaryThe first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis of mitotic dynein revealed a phosphorylation site in the dynein intermediate chains (ICs) that mediates binding to kinetochores. IC phosphorylation directs binding to zw10 rather than dynactin, and this interaction is needed for kinetochore dynein localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown to metaphase, but bioriented microtubule (MT) attachment and chromosome alignment induce IC dephosphorylation. IC dephosphorylation stimulates binding to dynactin and poleward streaming. MT depolymerization, release of kinetochore tension, and a PP1-γ mutant each inhibited IC dephosphorylation, leading to the retention of phosphodynein at kinetochores and reduced poleward streaming. The depletion of kinetochore dynactin by moderate levels of p50(dynamitin) expression disrupted the ability of dynein to remove checkpoint proteins by streaming at metaphase but not other aspects of kinetochore dynein activity. Together, these results suggest a new model for localization of kinetochore dynein and the contribution of kinetochore dynactin.

The antiapoptotic BCL2 family member MCL1 is normally up- and down-modulated in response to environmental signals and conditions, but is constitutively expressed in cancer where it promotes cell survival and drug resistance. A post-translational modification identified here, truncation at the N terminus, was found to act along with previously described ERK- and GSK3-induced phosphorylation events to regulate the turnover of the MCL1 protein and thus its availability for antiapoptotic effects. Although both N-terminally truncated and full-length MCL1 contain sequences enriched in proline, glutamic acid, serine, and threonine and were susceptible to proteasomal degradation, the truncated form decayed less rapidly and was maintained for an extended period in the presence of ERK activation. This was associated with extended cell survival because the truncated form of MCL1 (unlike those of BCL2 and BCLX) retained antiapoptotic activity. N-terminal truncation slightly increased the electrophoretic mobility of MCL1 and differed from the phosphorylation/band shift to decreased mobility, which occurs in the G2/M phase and was not found to affect MCL1 turnover. The N-terminally truncated form of MCL1 was expressed to varying extents in normal lymphoid tissues and was the predominant form present in lymphomas from transgenic mice and human tumor lines of B-lymphoid origin. The degradation versus stabilized expression of antiapoptotic MCL1 is thus controlled by N-terminal truncation as well as by ERK- and GSK3 (but not G2/M)-induced phosphorylation. These modifications may contribute to dysregulated MCL1 expression in cancer and represent targets for promoting its degradation to enhance tumor cell death.

Histone deacetylase 6 (HDAC6) is a tubulin-specific deacetylase that regulates microtubule-dependent cell movement. In this study, we identify the F-actin-binding protein cortactin as a HDAC6 substrate. We demonstrate that HDAC6 binds cortactin and that overexpression of HDAC6 leads to hypoacetylation of cortactin, whereas inhibition of HDAC6 activity leads to cortactin hyperacetylation. HDAC6 alters the ability of cortactin to bind F-actin by modulating a "charge patch" in its repeat region. Introduction of charge-preserving or charge-neutralizing mutations in this cortactin repeat region correlates with the gain or loss of F-actin binding ability, respectively. Cells expressing a charge-neutralizing cortactin mutant were less motile than control cells or cells expressing a charge-preserving mutant. These findings suggest that, in addition to its role in microtubule-dependent cell motility, HDAC6 influences actin-dependent cell motility by altering the acetylation status of cortactin, which, in turn, changes the F-actin binding activity of cortactin.

MRE11-RAD50-NBS1 (MRN) is a conserved nuclease complex that exhibits properties of a DNA damage sensor and is critical in regulating cellular responses to DNA double-strand breaks. NBS1, which is mutated in the human genetic disease Nijmegen breakage syndrome, serves as the regulatory subunit of MRN. Phosphorylation of NBS1 by the ATM kinase is necessary for both activation of the S phase checkpoint and for efficient DNA damage repair response. Here, we report that NBS1 is an acetylated protein and that the acetylation level is tightly regulated by the SIRT1 deacetylase. SIRT1 associates with the MRN complex and, importantly, maintains NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. Our results demonstrate the presence of crosstalk between two different posttranslational modifications in NBS1 and strongly suggest that deacetylation of NBS1 by SIRT1 plays a key role in the dynamic regulation of the DNA damage response and in the maintenance of genomic stability.

International audience; Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the aN -acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD +. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions.

BACKGROUND There are 346 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet (PLT) antigens, most of which have known genetic changes in 45 RBC or six PLT genes that correlate with antigen expression. Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA. This makes antigen prediction from next‐generation sequencing data challenging, since it uses genomic coordinates. STUDY DESIGN AND METHODS The conventional cDNA reference sequences for all known RBC and PLT genes that correlate with antigen expression were aligned to the human reference genome. The alignments allowed conversion of conventional cDNA nucleotide positions to the corresponding genomic coordinates. RBC and PLT antigen prediction was then performed using the human reference genome and whole genome sequencing (WGS) data with serologic confirmation. RESULTS Some major differences and alignment issues were found when attempting to convert the conventional cDNA to human reference genome sequences for the following genes: ABO, A4GALT, RHD, RHCE, FUT3, ACKR1 (previously DARC), ACHE, FUT2, CR1, GCNT2, and RHAG. However, it was possible to create usable alignments, which facilitated the prediction of all RBC and PLT antigens with a known molecular basis from WGS data. Traditional serologic typing for 18 RBC antigens were in agreement with the WGS‐based antigen predictions, providing proof of principle for this approach. CONCLUSION Detailed mapping of conventional cDNA annotated RBC and PLT alleles can enable accurate prediction of RBC and PLT antigens from whole genomic sequencing data.

Recently we identified a novel 250 kDa protein in adipocytes that is a substrate for the insulin-activated protein kinase Akt. We refer to this protein as AS250 for Akt substrate of 250 kDa. AS250 has a predicted GTPase activating protein (GAP) domain at its carboxy terminus. This domain shows some homology to the GAP domains for Rheb at the carboxy terminus of the protein tuberin and for Rap1 in the protein Rap1 GAP. The present study further characterizes AS250. The cDNA sequence for human AS250 is reported, and the sites that undergo phosphorylation upon insulin treatment of adipocytes have been identified by tandem mass spectrometry. We have found that in adipocytes AS250 exists as a complex with a novel protein of 1484 amino acids known as KIAA1219. The complex of AS250 with KIAA1219 is notably similar to the important regulatory complex of the protein tuberin with hamartin (the tuberous sclerosis complex), in the size of its subunits, the location of the GAP domain, and its phosphorylation by Akt. In an effort to detect the cellular role of the AS250/KIAA1219 complex, we generated 3T3-L1 adipocytes that largely lack AS250 by shRNA knockdown and examined several insulin-dependent effects. The knockdown of AS250 had no effect on insulin activation of the kinases, Akt, 70 kDa S6 kinase, or ERK1/2, or on insulin-stimulated actin bundling, and it had only a slight effect on insulin-stimulated GLUT4 translocation.

The third and final diagnostic antigen of the lentil lectin purified glycoproteins (LLGP) extracted from the larval stage of Taenia solium has been characterized, cloned, and expressed. T24 is an integral membrane protein that belongs to the tetraspanin superfamily. It migrates at a position corresponding to 24-kDa and as a homodimer at 42-kDa. Antibodies from cysticercosis patients recognize secondary structure epitopes that are dependent upon correctly formed disulfide bonds. A portion of T24, the large, extracellular loop domain, was expressed in an immunologically reactive form in insect cells. When tested in a Western blot assay with a large battery of serum samples, this protein, T24H, has a sensitivity of 94% (101/107), for detecting cases of cysticercosis with two or more viable cysts, and a specificity of 98% (284/290). The identification and expression of T24H sets the stage for the development of an ELISA suitable for testing single samples and for large-scale serosurveys that is not dependent upon the isolation and purification of antigens from parasite materials.

VP16 is a virion phosphoprotein of herpes simplex virus and a transcriptional activator of the viral immediate-early (IE) genes. We identified four novel VP16 phosphorylation sites (Ser18, Ser353, Ser411, and Ser452) at late times in infection but found no evidence of phosphorylation of Ser375, a residue reportedly phosphorylated when VP16 is expressed from a transfected plasmid. A virus carrying a Ser375Ala mutation of VP16 was viable in cell culture but with a slow growth rate. The association of the mutant VP16 protein with IE gene promoters and subsequent IE gene expression was markedly reduced during infection, consistent with prior transfection and in vitro results. Surprisingly, the association of Oct-1 with IE promoters was also diminished during infection by the mutant strain. We propose that Ser375 is important for the interaction of VP16 with Oct-1, and that the interaction is required to enable both proteins to bind to IE promoters.

Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

Downstream core promoter elements are an expanding class of regulatory sequences that add considerable diversity to the promoter architecture of RNA polymerase II-transcribed genes. We set out to determine the factors necessary for downstream promoter element (DPE)-dependent transcription and find that, against expectations, TFIID and the GTFs are not sufficient. Instead, the protein kinase CK2 and the coactivator PC4 establish DPE-specific transcription in an in vitro transcription system containing TFIID, Mediator, and the GTFs. Chromatin immunoprecipitation analyses using the DPE-dependent IRF-1 and TAF7 promoters demonstrated that CK2, and PC4 are present on these promoters in vivo. In contrast, neither PC4 nor CK2 were detected on the TAF1-dependent cyclin D promoter, which contains a DCE type of downstream element. Our findings also demonstrate that CK2 activity alters TFIID-dependent recognition of DCE sequences. These data establish that CK2 acts as a switch, converting the transcriptional machinery from functioning on one type of downstream element to another.

Background— CD133 (AC133) is a surface antigen that defines a broad population of stem cells, including myogenic and endothelial progenitors. CD133 + cells are rare in adult tissues, and the factors that support their differentiation into mature angiomyogenic cells are not known. These hurdles have hampered the use of CD133 + cells for therapeutic purposes. Because human fetal liver is a rich source of CD133 + cells, we sought to identify the growth factors that promote codifferentiation of these cells into angiogenic and myogenic cells. Methods and Results— Human fetal liver CD133 + and CD133 − cell subpopulations were cultured with 5′-azacytidine or vascular endothelial growth factor (VEGF 165 ) and/or brain-derived nerve growth factor (BDNF). CD133 + but not CD133 − cells from human fetal liver codifferentiated into spindle-shaped cells, as well as flat adherent multinucleated cells capable of spontaneous contractions in culture. The resulting spindle-shaped cells were confirmed to be endothelial cells by immunohistochemistry analysis for von Willebrand factor and by acetylated LDL uptake. Multinucleated cells were characterized as striated muscles by electron microscopy and immunohistochemistry analysis for myosin heavy chain. Presence of VEGF 165 and BDNF significantly enhanced angiomyogenesis in vitro. Inoculation of cells derived from CD133 + cells, but not CD133 − cells, into the ear pinna of NOD/SCID mice resulted in the formation of cardiomyocytes, as identified by immunostaining with cardiac troponin-T antibody. These cells generated electrical action potentials, detectable by ECG tracing. Conclusions— CD133 defines a population of human fetal liver cells capable of differentiating into both angiogenic and myogenic cells. Preconditioning of these CD133 + cells with VEGF 165 and BDNF enhances the angiomyogenesis. CD133 + fetal liver cells ultimately may be used for therapeutic angiomyogenesis.

The c-MYC oncoprotein functions as a sequence-specific transcription factor. The ability of c-MYC to activate transcription relies in part on the recruitment of cofactor complexes containing the histone acetyltransferases mammalian GCN5 (mGCN5)/PCAF and TIP60. In addition to acetylating histones, these enzymes have been shown to acetylate other proteins involved in transcription, including sequence-specific transcription factors. This study was initiated in order to determine whether c-MYC is a direct substrate of mGCN5 and TIP60. We report here that mGCN5/PCAF and TIP60 acetylate c-MYC in vivo. By using nanoelectrospray tandem mass spectrometry to examine c-MYC purified from human cells, the major mGCN5-induced acetylation sites have been mapped. Acetylation of c-MYC by either mGCN5/PCAF or TIP60 results in a dramatic increase in protein stability. The data reported here suggest a conserved mechanism by which acetyltransferases regulate c-MYC function by altering its rate of degradation.

14-3-3 proteins regulate the cell division cycle and play a pivotal role in blocking cell cycle advancement after activation of the DNA replication and DNA damage checkpoints. Here we describe a global proteomics analysis to identify proteins that bind to 14-3-3s during interphase and mitosis. 14-3-3-binding proteins were purified from extracts of interphase and mitotic HeLa cells using specific peptide elution from 14-3-3 zeta affinity columns. Proteins that specifically bound and eluted from the affinity columns were identified by microcapillary high pressure liquid chromatography tandem mass spectrometry analysis. Several known and novel 14-3-3-interacting proteins were identified in this screen. Identified proteins are involved in cell cycle regulation, signaling, metabolism, protein synthesis, nucleic acid binding, chromatin structure, protein folding, proteolysis, nucleolar function, and nuclear transport as well as several other cellular processes. In some cases 14-3-3 binding was cell cycle-dependent, whereas in other cases the binding was shown to be cell cycle-independent. This study adds to the growing list of human 14-3-3-binding proteins and implicates a role for 14-3-3 proteins in a plethora of essential biological processes.

Adult and larval stages of Taenia solium cause 2 diseases in humans, i.e., taeniasis and cysticercosis, respectively. Diagnosis and treatment of taeniasis are the ultimate means to eliminate cysticercosis. A serological taeniasis diagnostic test has been developed for laboratory use. However, recombinant forms of the taeniasis diagnostic proteins are required to overcome the limited supply of native proteins and allow the development of a low-cost and field-applicable test with high sensitivity and specificity. Using 2-dimensional electrophoresis of T. solium excretory and secretory (TSES) products from hamster adult tape-worm in vitro cultures, we have identified 5 T. solium-specific protein spots, with molecular weights of 33 kDa (protein isoelectrofocusing point [pI]: 5.6, 5.3, 5.1) and 38 kDa (pI: 4.6, 4.5). Protein sequencing and molecular cloning of these proteins showed that although endowed with different pls, the proteins with the same molecular weights shared the same protein backbone, named TSES33 and TSES38. Their full-length complementary DNAs encode proteins with 267 and 278 amino acids, respectively. TSES33 and TSES38 were expressed in a baculovirus system. Both recombinant proteins were recognized by a panel of taeniasis, but not cysticercosis patient serum samples, indicating that they can potentially replace the native proteins in the development of a more efficacious taeniasis diagnostic test.

The NuA4 histone acetyltransferase (HAT) multisubunit complex is responsible for acetylation of histone H4 and H2A N-terminal tails in yeast. Its catalytic component, Esa1, is essential for cell cycle progression, gene-specific regulation and has been implicated in DNA repair. Almost all NuA4 subunits have clear homologues in higher eukaryotes, suggesting that the complex is conserved throughout evolution to metazoans. We demonstrate here that NuA4 complexes are indeed present in human cells. Tip60 and its splice variant Tip60b/PLIP were purified as stable HAT complexes associated with identical polypeptides, with 11 of the 12 proteins being homologs of yeast NuA4 subunits. This indicates a highly conserved subunit composition and the identified human proteins underline the role of NuA4 in the control of mammalian cell proliferation. ING3, a member of the ING family of growth regulators, links NuA4 to p53 function which we confirmed in vivo. Proteins specific to the human NuA4 complexes include ruvB-like helicases and a bromodomain-containing subunit linked to ligand-dependent transcription activation by the thyroid hormone receptor. We also demonstrate that subunits MRG15 and DMAP1 are present in distinct protein complexes harboring histone deacetylase and SWI2-related ATPase activities, respectively. Finally, analogous to yeast, a recombinant trimeric complex formed by Tip60, EPC1, and ING3 is sufficient to reconstitute robust nucleosomal HAT activity in vitro. In conclusion, the NuA4 HAT complex is highly conserved in eukaryotes, in which it plays primary roles in transcription, cellular response to DNA damage, and cell cycle control.

The modification of chromatin structure is an important regulatory mechanism for developmental gene expression. Differential expression of the mammalian ISWI genes, SNF2H and SNF2L, has suggested that they possess distinct developmental roles. Here we describe the purification and characterization of the first human SNF2L-containing complex. The subunit composition suggests that it represents the human ortholog of the Drosophila nucleosome-remodeling factor (NURF) complex. Human NURF (hNURF) is enriched in brain, and we demonstrate that it regulates human Engrailed, a homeodomain protein that regulates neuronal development in the mid-hindbrain. Furthermore, we show that hNURF potentiates neurite outgrowth in cell culture. Taken together, our data suggess a role for an ISWI complex in neuronal growth.

The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.

Mitosis requires precise control of microtubule dynamics. The KinI kinesin MCAK, a microtubule depolymerase, is critical for this regulation. In a screen to discover previously uncharacterized microtubule-associated proteins, we identified ICIS, a protein that stimulates MCAK activity in vitro. Consistent with this biochemical property, blocking ICIS function in Xenopus extracts with antibodies caused excessive microtubule growth and inhibited spindle formation. Prior to anaphase, ICIS localized in an MCAK-dependent manner to inner centromeres, the chromosomal region located in between sister kinetochores. From Xenopus extracts, ICIS coimmunoprecipitated MCAK and the inner centromere proteins INCENP and Aurora B, which are thought to promote chromosome biorientation. By immunoelectron microscopy, we found that ICIS is present on the surface of inner centromeres, placing it in an ideal location to depolymerize microtubules associated laterally with inner centromeres. At inner centromeres, MCAK-ICIS may destabilize these microtubules and provide a mechanism that prevents kinetochore-microtubule attachment errors.