Cell Structural Proteins Research Articles

Next generation sequencing of RNA from muscle biopsies shows marked differences between inflammatory myositis, inclusion body myositis, and controls

Inflammatory myositis (IM) is a multi-system inflammatory disorder characterised by a variable triad of muscular weakness, interstitial lung disease, and cutaneous manifestations. A serological profile is now recognised in over 80% of cases and correlates closely with the phenotypic manifestation of the disease. The largest serological subgroup of IM is anti-Jo-1 (an anti-tRNA synthetase antibody). Present standard treatment is with systemic immunosuppression.Inclusion body myositis (IBM) differs from IM by affecting peripheral and proximal muscles and tends to have little in the way of multi-organ involvement. IBM tends to respond poorly to immunosuppression, and currently does not have an as well recognised serological profile as IM.Recent research suggests that non-coding RNA plays a crucial role in orchestrating protein transcription, and aberrant non-coding RNA expression may play a key part in IM and IBM. Advances in sequencing techniques now enable detailed analysis of RNA transcription and may help unravel the pathogenesis of IM and IBM.In this pilot study we investigate the differences in expression of coding and non-coding RNA in IM (anti- Jo-1 antibody positive) and IBM. RNA was extracted from each muscle biopsy and pooled into three samples (IM, IBM, and control) for sequencing. RNA was extracted from five muscle biopsy samples for each of the IM and control group, and two for the IBM group. The muscle transcriptome was determined with next generation sequencing (Illumina Hi-Seq 2000, Poly A+ extraction). Data were mapped onto the human genome and changes in expression were analysed.Preliminary data have shown significant differential changes in transcription of cell structure and inflammatory response proteins, as well as marked variation in skeletal-muscle-specific long non-coding RNAs, between the three groups. FundingNational Institute for Health Research.

Functional interplay between the cell cycle and cell phenotypes.

Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing the cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other's prima facie functions. This study introduces a high-throughput method to simultaneously measure the cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between the cell cycle and cell phenotypes.

Natural Peptides with Potential Applications in Drug Development, Diagnosis, and/or Biotechnology

Natural Peptides with Potential Applications in Drug Development, Diagnosis, and/or Biotechnology

Identificação de genes envolvidos na síntese de proteínas de células musculares lisas com expressão aumentada em placas ateromatosas associados a hiperplasia neointimal após implante de stents não-farmacológicos: estudo GENESIS-R

INTRODUÇÃO: A reestenose coronária é um fenômeno pouco compreendido e que permanece como um desafio mesmo na era dos stents farmacológicos. Este estudo tem como objetivo identificar genes envolvidos na síntese de proteínas estruturais e funcionais de células musculares lisas com expressão aumentada em placas ateromatosas de humanos associados a hiperplasia neointimal após o implante de stents não-farmacológicos. MÉTODOS: Placas ateromatosas foram obtidas mediante aterectomia direcionada, previamente ao implante do stent. A análise da expressão dos genes foi realizada utilizando-se o sistema Affymetrix GeneChip. Os pacientes foram submetidos a ultrassom intracoronário 6 meses após o procedimento para análise volumétrica intrastent. Foi avaliada a correlação entre a expressão gênica de placas ateromatosas e o porcentual de hiperplasia intimal intrastent. RESULTADOS: A maioria dos pacientes era do sexo masculino (85,7%), com 60,2 ± 11,4 anos de idade, 35,7% eram diabéticos e o porcentual de hiperplasia intimal intrastent foi de 29,9 ± 18,7%. Não houve variação do porcentual de hiperplasia intimal intrastent entre os pacientes com ou sem diabetes (29,5% vs. 30,7%; P = 0,89). Não houve correlação entre a extensão do stent e o porcentual de hiperplasia intimal intrastent (r = -0,26; P = 0,26) ou entre o diâmetro do stent e o porcentual de hiperplasia intimal intrastent (r = 0,14; P = 0,56). Oito genes envolvidos na síntese de proteínas estruturais e funcionais de células musculares lisas apresentaram correlação positiva com o porcentual de hiperplasia intimal intrastent. CONCLUSÕES: As lesões coronárias de novo apresentam expressão aumentada de genes relacionados com a síntese de proteínas estruturais e funcionais de células musculares lisas associados a futura hiperplasia neointimal intrastent significativa, surgindo como novos alvos terapêuticos.

MetaMapp: mapping and visualizing metabolomic data by integrating information from biochemical pathways and chemical and mass spectral similarity

BackgroundExposure to environmental tobacco smoke (ETS) leads to higher rates of pulmonary diseases and infections in children. To study the biochemical changes that may precede lung diseases, metabolomic effects on fetal and maternal lungs and plasma from rats exposed to ETS were compared to filtered air control animals. Genome- reconstructed metabolic pathways may be used to map and interpret dysregulation in metabolic networks. However, mass spectrometry-based non-targeted metabolomics datasets often comprise many metabolites for which links to enzymatic reactions have not yet been reported. Hence, network visualizations that rely on current biochemical databases are incomplete and also fail to visualize novel, structurally unidentified metabolites.ResultsWe present a novel approach to integrate biochemical pathway and chemical relationships to map all detected metabolites in network graphs (MetaMapp) using KEGG reactant pair database, Tanimoto chemical and NIST mass spectral similarity scores. In fetal and maternal lungs, and in maternal blood plasma from pregnant rats exposed to environmental tobacco smoke (ETS), 459 unique metabolites comprising 179 structurally identified compounds were detected by gas chromatography time of flight mass spectrometry (GC-TOF MS) and BinBase data processing. MetaMapp graphs in Cytoscape showed much clearer metabolic modularity and complete content visualization compared to conventional biochemical mapping approaches. Cytoscape visualization of differential statistics results using these graphs showed that overall, fetal lung metabolism was more impaired than lungs and blood metabolism in dams. Fetuses from ETS-exposed dams expressed lower lipid and nucleotide levels and higher amounts of energy metabolism intermediates than control animals, indicating lower biosynthetic rates of metabolites for cell division, structural proteins and lipids that are critical for in lung development.ConclusionsMetaMapp graphs efficiently visualizes mass spectrometry based metabolomics datasets as network graphs in Cytoscape, and highlights metabolic alterations that can be associated with higher rate of pulmonary diseases and infections in children prenatally exposed to ETS. The MetaMapp scripts can be accessed at http://metamapp.fiehnlab.ucdavis.edu.

Identification of Genes Involved in Smooth Muscle Cell Protein Synthesis with Increased Expression in Atheromatous Plaques Associated with Neointimal Hyperplasia after Bare-Metal Stenting: A GENESIS-R Study

ABSTRACT Background Coronary restenosis is a poorly understood phenomenon that remains a challenge even in the drug-eluting stent era. This study aimed to identify genes involved in the synthesis of structural and functional proteins in smooth muscle cells with increased expression in human atheromatous plaques associated with neointimal hyperplasia after bare-metal stent implantation. Methods Atheromatous plaques were obtained by directional atherectomy prior to stenting. A gene expression analysis was performed using the Affymetrix GeneChip system. The patients were subjected to intravascular ultrasound six months after the procedure for an in-stent volumetric analysis. The correlation between gene expression in atheromatous plaques and the percentage of in-stent intimal hyperplasia was evaluated. Results Most patients were male (85.7%) with a mean age of 60.2 ± 11.4 years; 35.7% were diabetic, and the percentage of in-stent intimal hyperplasia was 29.9 ± 18.7%. There was no change in the percentage of in-stent intimal hyperplasia in patients with or without diabetes (29.5% vs. 30.7%; P = 0.89). There was no correlation between the stent length and the percentage of in-stent intimal hyperplasia (r = − 0.26; P = 0.26), or between the stent diameter and the percentage of in-stent intimal hyperplasia (r = 0.14; P = 0.56). Eight genes related to smooth uscle cell structural and functional protein synthesis had a positive correlation with the percentage of in-stent intimal hyperplasia. Conclusions De novo coronary lesions exhibited an increased expression of genes related to the synthesis of structural and functional proteins in smooth muscle cells and were associated with future significant in-stent neointimal hyperplasia, thus emerging as novel therapeutic targets.

Construction and analysis of a suppression subtractive hybridization (SSH) library of genic multiple-allele inherited male-sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Utilization of male sterility is a key method for producing crossbred Chinese cabbage (Brassica rapa L. ssp. pekinensis (Lour.) Olsson. In this study, suppression subtractive hybridization (SSH) was used to construct sterility and fertility cDNA libraries, which included differentially, expressed clones between fertile and sterile buds of the A/B line ‘AB01’. The positive clones were randomly selected by polymerase chain reaction amplification (PCR) and 25 high quality sequences (22 from the fertile-tester library and three from the sterile-tester libraries) were generated. The fragment lengths varied from 77 to 469 bp. Differential expression patterns between fertile and sterile buds were selected and verified using five expressed sequence tags (ESTs). Results indicated that, three ESTs were expressed only in fertile buds and two ESTs were down-regulated in sterile buds. According to the Basic Local Alignment Search Tool (BLAST) screening and functional annotation, the 25 ESTs were homologous to known sequences deposited in National Center for Biotechnology Information (NCBI). These genes had homology to known proteins such as flowers/buds development proteins, metabolic-related proteins, cell structure proteins, cell growth/division proteins and secondary metabolic-related proteins. The results suggested that, these proteins played a critical role in nuclear male sterility progression of genic multiple-allele inherited male-sterility in Chinese cabbage.

The Common Modification in αA-Crystallin in the Lens, N101D, Is Associated with Increased Opacity in a Mouse Model

To elucidate the morphological and cellular changes due to introduction of a charge during development and the possible mechanism that underlies cataract development in humans as a consequence of an additional charge, we generated a transgenic mouse model mimicking deamidation of Asn at position 101. The mouse model expresses a human αA-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as αAN101D-transgene and is considered to be "deamidated" in this study. Mice expressing αAN101D-transgene are referred to here CRYAA(N101D) mice. All of the lines showed the expression of αAN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) αA-transgene (referred to as CRYAA(WT) mice), the lenses of CRYAA(N101D) mice showed (a) altered αA-crystallin membrane protein (aquaporin-0 (AQP0), a specific lens membrane protein) interaction, (b) extracellular spaces between outer cortical fiber cells, (c) attenuated denucleation during confocal microscopic examination, (d) disrupted normal fiber cell organization and structure during scanning electron microscopic examination, (e) distorted posterior suture lines by bright field microscopy, and (f) development of a mild anterior lens opacity in the superior cortical region during the optical coherence tomography scan analysis. Relative to lenses with WT αA-crystallin, the lenses containing the deamidated αA-crystallin also showed an aggregation of αA-crystallin and a higher level of water-insoluble proteins, suggesting that the morphological and cellular changes in these lenses are due to the N101D mutation. This study provides evidence for the first time that expression of deamidated αA-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.

Identification of essential genes in C. jejuni genome highlights hyper-variable plasticity regions

A microarray transposon-based tracking approach was used to identify Campylobacter jejuni genes which are required for cell growth at 37°C, under a microaerophilic atmosphere and on a rich Mueller-Hinton medium. A transposon-based mutant library, comprised of 7,201 individual mutants was constructed, representing 4.48× coverage of the genome. An analysis of genes lacking a transposon insertion revealed 195 essential gene candidates. The function of these genes represent many of the expected core functions of the cell, such as energy metabolism, macromolecule and cofactor biosynthesis, cell structural proteins as well as basic cell processes. Forty-nine hypothetical proteins were also identified, further underlining the importance of currently unknown proteins and pathways within C. jejuni. Unlike other bacteria, the essential genes were not uniformly distributed along the chromosome with three main regions lacking essential genes. These particular regions corresponded to known hyper-variable plasticity regions of C. jejuni genome indicating, as expected, that these regions are dispensable in any given C. jejuni strain. Overall, this work identified dispensable and essential genes in C. jejuni that will ultimately lead to a better understanding of Campylobacter physiology.

Isolation of fertility-related genes of multiple-allele-inherited male sterility in Brassica rapa ssp pekinensis by cDNA-AFLP

To better understand the molecular mechanisms of multiple-allele-inherited male sterility in Chinese cabbage (Brassica rapa ssp pekinensis), differentially expressed genes in fertile and sterile plants must be isolated. We used cDNA-AFLP analysis to isolate differentially expressed genes in fertile and sterile buds of the two-type line, AB01. Sixteen high-quality sequences were generated, 11 of which were up-regulated in fertile buds, and five of which were up-regulated in sterile buds. Based on BLAST screening and functional annotation, these genes have homology with genes encoding known flower- or bud-specific proteins, metabolism-related proteins and cell-structure proteins. In addition, the full-length cDNA sequences of the actin gene were cloned from the cabbage plants by RACE and used as an internal standard for semi-quantitative reverse transcription-PCR. Expression of three flower- or bud-specific differentially expressed transcript-derived fragments in fertile and sterile buds was examined using RT-PCR; the expression patterns of these genes were similar to the patterns observed in the cDNA-AFLP analysis.

Identification of proteins overexpressed in papillary thyroid tumors

A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.

A single nucleotide polymorphism in the E-cadherin gene promoter − 160 C/A is associated with risk of nasopharyngeal cancer

Background E-cadherin is a cell structural protein that has a pivotal role in cell–cell adhesion and epithelial development. Up to now, loss of activity of E-cadherin is believed to contribute to progression in several neoplastic diseases of epidermoid origin including nasopharyngeal carcinomas (NPC) by increasing invasion and proliferation. Besides, functional genetic variations in the promoter region of the E-cadherin gene have been associated with susceptibility to several neoplasms. In the current study we investigated the impact of the functional C/A genetic polymorphism at − 160 from transcriptional start site of the E-cadherin gene promoter on susceptibility and prognosis in NPC. Methods A PCR and restriction fragment length polymorphism analysis was used to determine the variation of the − 160C/A promoter region in a Tunisian population consisting of 162 NPC patients and 140 age matched healthy controls. Associations of the genetic markers with the clinicopathological parameters and the rates of the nasopharyngeal carcinoma-specific overall survival and the disease-free survival were also assessed. Results A significantly increased risk of NPC was observed for carriers of E-cadherin − 160A allele (OR = 2.02; P = 0.008). AA and CA genotypes entailed a 4.12 and 1.8 fold high risks, respectively for NPC compared to the CC genotype. Additionally, an association was ascertained between the E-cadherin polymorphism and the young age onset of NPC. Conclusions This is the first report on the studies of functional E-cadherin polymorphisms in NPC and our preliminary results suggest that the − 160 C/A promoter polymorphism is associated with increased risk of nasopharyngeal carcinoma in the Tunisian population, especially in young patients.

Estimation of the efficiency of 2D analysis and bioinformatics search in identification of protein markers for colon tumors

A modification of proteome differential analysis was developed; it comprises initial removal of the cell structural proteins by extraction with buffered saline and protein fractionation by gel filtration with subsequent separation by two-dimensional gel electrophoresis and identification by mass-spectrometry. This approach provided for detection of 12 proteins with significantly elevated expression levels in the majority of the analyzed malignant colorectal tumor specimens as compared with normal tissues. Increased contents of eight proteins were discovered for the first time. The efficiencies of the search for tumor markers by 2D analysis and serial analysis of gene expression (SAGE) were compared using the control panel of 19 potential colorectal tumor markers, identified earlier or independently found by other researchers. The 2D data for the control panel matched the earlier published data, whereas the search of SAGE database succeeded in detection only one-third of the markers.

Screening for therapeutic targets of vorinostat by SILAC‐based proteomic analysis in human breast cancer cells

Histone deacetylases (HDACs) play critical roles in silencing tumor suppressor genes. HDAC inhibitors reactivate tumor suppressor genes and inhibit tumor cell growth in vitro and in vivo, and several HDAC inhibitors are currently being evaluated in clinical trials for cancer therapy. A comprehensive analysis of proteins regulated by HDAC inhibitors would enhance our ability to define and characterize their essential therapeutic targets. Here, we employed stable isotope labeling with amino acids in cell culture-based quantitative proteomics to identify acetylated proteins in human breast cancer cells. Treatment with the clinically relevant HDAC inhibitor, suberoylanilide hydroxamic acid (vorinostat), induces lysine acetylation of 61 proteins in MDA-MB-231 human breast cancer cells. Suberoylanilide hydroxamic acid not only induces lysine acetylation in chromatin-associated proteins, but also acetylates previously unrecognized nonhistone proteins, including transcriptional factors and regulators, chaperones, cell structure proteins, and glycolytic enzymes in a time-dependent manner. Knowledge of the full repertoire of acetylated proteins will provide a foundation for further defining the functions of HDACs in cancer cells.

Identification of Genes Induced in Response to Low-Temperature Treatment in Tea Leaves

A suppression subtractive hybridization analysis approach was undertaken to identify genes involved in cold acclimation of tea (Camellia sinensis (L.) O. Kuntze), a broad-leaved, woody evergreen species. A total of ten complementary DNA (cDNA) clones, induced by low temperature in tea leaves, were identified. These transcripts could be classified into three functional groups. Three transcripts encoded transcriptional factors. Among these, Cs-COR375 contained a putative open reading frame encoding a zinc-finger protein with two EPF-type zinc fingers, while Cs-COR041 and Cs-COR178 were corresponded to CBF-like genes. The predicted products of Cs-COR044, Cs-COR196, and Cs-COR213 cDNAs were cell structure proteins. Cs-COR044 encoded a putative 86 amino acid protein consisting of a hydrophobic N-terminal half and a hydrophilic C-terminal half relatively rich in glycine and proline. The protein was predicted to be exported to the apoplast and likely to be localized in the cell wall. Cs-COR196 and Cs-COR213 encoded for two putative early light-induced proteins, a group of nuclear-encoded thylakoid membrane proteins accumulating in response to various stresses. The predicted proteins encoded by the remaining four transcripts were putatively involved in primary and secondary metabolism. The product encoded by Cs-COR018 was β-amylase, involved in cold acclimation of various plant species. Cs-COR126 coded for chalcone synthase, while Cs-COR054 and Cs-COR113 coded for two different flavonol synthases.

Regulation of Dimethylarginine Dimethylaminohydrolase (DDAH) in Rat Preglomerular Vascular Smooth Muscle Cells (PGVSMCs) by Reactive Oxygen Species (ROS)

DDAH degrades asymmetric dimethylarginine (ADMA) which is an endogenous NOS inhibitor and risk factor for cardiovascular disorders. We tested the hypothesis that ROS regulate the transcription, translation or activity of DDAH. We created a stable rat PGVSMC line overexprissing p22phox (S‐p22phox) using a p22phox‐pcDNA4/HisMax construct (a generous gift from Kathy Griendling). Quantitative real time PCR and Western blotting confirmed a marked increase in p22phox mRNA and protein of 4.7±0.6 and 2.3±0.5 fold, (p<0.01), compared to wild type cells (WT). S‐p22phox cells had elevated superoxide and hydrogen peroxid generation (2.3±0.09 fold and 1.8±0.1 fold p<0.01). The mRNAs for DDAH ‐1 and ‐2 were increased by 2.8 ± 0.2 and 1.8 ± 0.1 fold in S‐p22phox cells p< 0.01). DDAH‐2 promoter activity, determined by transient transfection and luciferase assay, was 4.1 fold increased in S‐p22phox cells (p < 0.01). Interestingly, both DDAH‐1 and ‐2 proteins were significantly lower in S‐p22phox cells (0.9±0.2 and 0.9±0.1 fold; p<0.01), yet β‐actin was not different. DDAH activity measured by a colorimetric assay (Kidney Int. 72:886–9, 2007) was reduced by 62±2% in S‐p22phox(p<0.01). The results reveal for the first time an important effect but complex of p22phox – dependent ROS generation on DDAH. Thus, ROS upregulate the expression of DDAH‐1 and ‐2 genes via activating a promoter, at least for DDAH‐2, yet downregulate both DDAH‐1 and ‐2 proteins and their enzyme activity. This is a specific effect since ROS do not change the expression of cell structure proteins. These findings in PGVSMCs provide a novel regulatory pathway whereby ROS may modulate renal hemodynamics, and thereby BP and salt homeostasis.

Proteomic Analysis of Amniotic Membrane Prepared for Human Transplantation: Characterization of Proteins and Clinical Implications

Amniotic membrane is commonly exploited in several surgical procedures. Despite a freeze preservation period, it is reported to retain wound healing, anti-angiogenic, antiinflammatory and anti-scarring properties; however, little is known about the active protein content. 2-DE analysis of transplant-ready amniotic membrane (TRAM) was performed. The effects of preservation and processing on amnion proteome were investigated, and the major proteins in the TRAM characterized using mass spectrometry and immunoblotting. This identified a spectrum of proteins including thrombospondin, mimecan, BIG-H3, and integrin alpha 6. Preservation compromises cellular viability resulting in selective elution of soluble cellular proteins, leaving behind extracellular matrix-associated and cell structural proteins. A number of key architectural proteins common to the architecture of the ocular surface were demonstrated in AM, which are involved in homeostasis and wound healing. Handling procedures alter the protein composition of amniotic membrane prepared for transplantation. Without standardization, there will be inter-membrane variation, which may compromise the desired therapeutic effect of transplant ready amniotic membrane.

Peptide Aptamer-mediated Inhibition of Target Proteins by Sequestration into Aggresomes

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.

Mini-dystrophin efficiently incorporates into the dystrophin protein complex in living cells

Dystrophin is a critical muscle cell structural protein which when deficient results in Duchenne muscular dystrophy. Recently miniature versions of the dystrophin gene have been constructed that ameliorate the pathology in mouse models. To characterize mini-dystrophin's incorporation into the dystrophin protein complex in living cells, two fusion proteins were constructed where mini-dystrophin is fused to the N- or C-terminus of an enhanced green fluorescent protein reporter gene. Both fusion proteins correctly localize at the plasma membrane in vitro and in vivo. Live cell microscopy establishes that mini-dystrophin translocates directly to the PM of differentiating muscle cells, within 4 h of expression. Latrunculin A treatment, actin and beta-dystroglycan binding domain deletion constructs, and co-immunoprecipitation assays demonstrate that mini-dystrophin is firmly anchored to the sarcolemma primarily through its connections to beta-dystroglycan, mimicking effects seen with wild type dystrophin. Furthermore, point mutations made within the putative beta-dystroglycan anchoring ZZ domain of mini-dystrophin result in an ablation of beta-dystroglycan binding and a nuclear translocation of the protein. These results demonstrate that mini-dystrophin is efficiently bound and incorporated into the dystrophin protein complex, via beta-dystroglycan in living cells, similarly to the full length dystrophin protein.

Post-Transplant Monitoring of Erythroid Lineage Specific Chimerism.

Hematopoietic stem cell transplantation (HSCT) has been increasingly used for treatment of non-malignant hematologic disease, including red blood cell diseases. Critical to the development of novel stem cell therapies is the ability to rapidly evaluate the kinetics of donor stem cell engraftment, lineage specific differentiation and the functional impact of transplantation. Here we report the development of a generalizable method to rapidly assess lineage specific chimerism through the technique of RT-PCR combined with pyrosequencing. To apply this RNA-based method for assessment of RBC lineage engraftment, we used the HapMap and Ensembl databases and direct high-throughput genotyping to identify a panel of expressed single nucleotide polymorphisms (SNPs) of high minor allele frequency (2pq>0.15) in diverse ethnic populations, encoded by genes uniquely expressed on erythroid cells. This panel consisted of 10 SNPs from 7 genes, encoding red cell antigens and structural proteins (Kidd, Duffy, Diego and Lutheran antigens, spectrin, beta-globin, FUT1). Following optimization of PCR amplification and pyrosequencing, mixing studies with samples with defined alleles were used to validate that each SNP could provide a quantitative measure of donor/recipient DNA and RNA. The high minor allele frequency of each of the 10 SNPs was confirmed by direct genotyping of 10 North American normal volunteers and 80 Iranian individuals with thalassemia trait or thalassemia major. This expressed RBC SNP panel was further confirmed to be informative for transplant patient-donor pairs through direct genotyping of 9 North American and 13 Iranian patients who underwent allogeneic HSCT for malignant (CLL, Hodgkin's, NHL) and non-malignant hematologic diseases (sickle cell, polycythemia vera, Blackfan-Diamond Anemia, thalassemia) and their HLA-matched donors (19 related, 3 unrelated). At least 1 informative RBC SNP was identified for each patient-donor pair. A median of 3 and 4 SNPs were informative for matched related and unrelated pairs, respectively. Thirteen of the 22 patients developed mixed hematopoietic chimerism following HSCT. In patient-donor pairs with more than 1 informative SNP, high concordance in serial post-transplant chimerism measurements between individual SNPs was observed. Serial analysis of overall and erythroid lineage specific chimerism among 5 patients with malignant diseases revealed equivalent levels of erythroid and leukocyte donor engraftment. In contrast, differential WBC and RBC engraftment was detected in most of the patients with non-malignant diseases affecting erythropoiesis, in which increased donor representation was observed. RNA pyrosequencing provides rapid measurement of erythroid lineage chimerism, without requiring the laborious process of cell isolation. We report the generation of a RBC-specific SNP panel that is informative across ethnically diverse populations and can provide valuable functional information for diseases in which RBC engraftment is critically important. Similar methods can be applied to generate panels of expressed SNPs informative for other cell lineages to comprehensively assess the impact of multi-lineage engraftment following treatment with novel transplant regimens.