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We have developed a protocol for barcoded cDNA libraries of 48 samples to study gene expression across tissues in the domestic dog, Canis familiaris, by modifying the Single-Cell Tagged Reverse Transcription (STRT) protocol (Islam et al., 2012, 2014). The cDNA reads represent mRNA 5' ends, enabling the study of transcription start sites (TSS). Our modifications include longer UMIs for molecular counting and Globin-Lock® to deplete globin mRNAs that are abundant in blood and blood-rich tissues dominating all reads., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Ethylene oxide (EO), an industrial intermediate and gaseous sterilant for medical devices, is carcinogenic to humans, which warrants minimization of exposure in the workplaces. The principal analytical strategy currently used in biomonitoring of exposure to EO consists in the conversion of N-(2-hydroxyethyl) adduct at the N-terminal valine (HEV) in globin to a specific thiohydantoin derivative accessible to GC-MS analysis (modified Edman degradation, MED). Though highly sensitive, the method is laborious and, at least in our hands, not sufficiently robust. Here we developed an alternative strategy of HEV determination based on acidic hydrolysis (AH) of globin followed directly by HPLC-ESI-MS 2 analysis. Limit of quantitation is ca. 25 pmol HEV/g globin. Comparative analyses of globin samples from EO-exposed workers by both the AH-based and MED-based methods provided results that correlated well with each other (R 2  > 0.95) but those obtained with AH were significantly more accurate (according to external quality control programme G-EQUAS) and repeatible (5% and 6% for intra-day and between-day analyses, respectively). In conclusion, the new AH-based method surpassed MED being similarly sensitive, much less laborious and more reliable, thus applicable as an effective tool for biomonitoring of EO in exposure control and risk assessment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: We evaluated the clinical implications of the albumin to globulin ratio (AGR) in patients with diffuse large B-cell lymphoma (DLBCL) treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP)., Methods: Data of 232 patients with DLBCL treated with first-line R-CHOP from 2004 to 2017 were reviewed retrospectively. Patients with AGR values ≥1.22 and <1.22 were assigned to the high and low AGR groups, respectively. Treatment response, treatment-related toxicity, and survival were compared according to the AGR., Results: The complete response rate was significantly lower in the low AGR group than in the high AGR group (59.1% vs. 81.3%; p < 0.001). Treatment-related mortality was also more frequent in the low AGR group than in the high AGR group (14.0% vs. 4.3%; p =  0.009). The low AGR group (median overall survival [OS] = 26.87 months; 95% confidence interval [CI] = 4.19-49.55) showed a significant decrease in OS compared to the high AGR group (median OS = 148.83 months; 95% CI = 76.26-221.41; p < 0.001). Progression-free survival (PFS) also decreased significantly in the low AGR group (median PFS = 14.29 months; 95% CI = 2.58-26.01) compared to the high AGR group (median PFS = 148.83 months; 95% CI = 76.21-221.45; p < 0.001). In a multivariate analysis, low AGR was an independent poor prognostic factor for OS and PFS., Conclusions: Pretreatment AGR was useful for predicting treatment response, treatment-related toxicity, and prognosis in patients with DLBCL treated with R-CHOP. Further large prospective studies will be necessary to validate our findings., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) develops in some patients who achieve sustained virological response (SVR) against hepatitis C virus (HCV) infection via anti-HCV therapy. To examine the pathogenesis of HCC development after HCV eradication, histopathological changes and clinical markers were evaluated in SVR patients., Methods: Of 654 SVR patients treated with interferon (IFN)-based therapies, 34 patients who had undergone liver biopsy before initiating IFN therapy and after SVR achievement were enrolled: 11 patients with HCC and 23 patients without HCC (male/female, 9/2 and 8/15, respectively: age, 58 ± 5 and 54 ± 11 years, respectively). We compared the clinical and histopathological factors between the two groups. Immunohistochemistry for Cytoglobin (CYGB) and α smooth muscle actin (α-SMA) was also performed., Results: At baseline, prior to initiating the IFN-based therapy, there were significant differences between the SVR-non-HCC and SVR-HCC groups in the male gender, HBc antibody positivity, prothrombin activity, and histological inflammatory grade. Histopathological evaluation, using the new Inuyama classification system, revealed an improvement in the inflammatory grade, from 2.1 ± 0.6 to 1.0 ± 0.6 (p < 0.0001), whereas the fibrosis stage remained unchanged, from 2.3 ± 0.9 to 2.0 ± 1.2 (p = 0.2749), during the 97 ± 72-month observation period in the SVR-HCC group. Both the grade and stage scores were significantly improved in the SVR-non-HCC group. The area of collagen deposition, evaluated using Sirius red staining, showed a marked decrease, from 18.6 ± 7.6% to 7.7 ± 4.6%, in the SVR-non-HCC group, with no change in the SVR-HCC group. CYGB- and α-SMA-positive hepatic stellate cells (HSCs), indicative of the HSC activated phenotype, remained in the fibrotic tissue of livers among patients in the SVR-HCC group., Conclusion: Stagnation of fibrosis regression is associated with a high risk for HCC after SVR. HSC activation may inhibit improvement in fibrosis after SVR and potentially contribute to hepatocarcinogenesis.

Neuroglobin is a monomeric globin containing a six-coordinate heme b, expressed in the nervous system, which exerts an important neuroprotective role. In the human protein (hNgb), Cys46 and Cys55 form an intramolecular disulfide bond under oxidizing conditions, whose cleavage induces a helix-to-strand rearrangement of the CD loop that strengthens the bond between the heme iron and the distal histidine. Hence, it is conceivable that the intramolecular disulfide bridge modulates the functionality of human neuroglobin by controlling exogenous ligand binding. In this work, we investigated the influence of the Cys46/Cys55 disulfide bond on the redox properties and on the pH-dependent conformational equilibria of hNgb, using UV-vis spectroelectrochemistry, cyclic voltammetry, electronic absorption spectroscopy and magnetic circular dichroism (MCD). We found that the SS bridge significantly affects the heme Fe(III) to Fe(II) reduction enthalpy (ΔH°' rc ) and entropy (ΔS°' rc ), mostly as a consequence of changes in the reduction-induced solvent reorganization effects, without affecting the axial ligand-binding interactions and the polarity and electrostatics of the heme environment. Between pH3 and 12, the electronic properties of the heme of ferric hNgb are sensitive to five acid-base equilibria, which are scarcely affected by the Cys46/Cys55 disulfide bridge. The equilibria occurring at extreme pH values induce heme release, while those occurring between pH5 and 10 alter the electronic properties of the heme without modifying its axial coordination and low spin state. They involve the sidechains of non-coordinating aminoacids close to the heme and at least one heme propionate., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The study assessed the effect of conscious halal slaughter and slaughter following minimal anesthesia on bleeding efficiency of goats and keeping quality of goat meat. Ten Boer cross bucks were divided into two groups and subjected to either halal slaughter without stunning (HS) or minimal anesthesia prior to slaughter (AS). The blood lost during exsanguination was measured. Residual blood was further quantified by determination of hemoglobin and myoglobin content in longissimus lumborum muscle. Storage stability of the meat was evaluated by microbiological analysis and lipid oxidation. Blood loss at exsanguination, residual hemoglobin and lipid oxidation were not significantly different (p>0.05) between HS and AS. Lactic acid bacteria was the only microbe that was significantly elevated after 24h of storage at 4°C in the AS group. In conclusion, slaughtering goats under minimal anesthesia or fully conscious did not affect bleeding efficiency and keeping quality of goat meat., (Copyright © 2015. Published by Elsevier Ltd.)

Besides other mechanism(s) 17β-estradiol (E2) facilitates neuronal survival by increasing, via estrogen receptor β (ERβ), the levels of neuroglobin (NGB) an anti-apoptotic protein. In contrast, E2 could exert protective effects in cancer cells by activating apoptosis when the ERβ level prevails on that of ERα as in colon cancer cell lines. These apparently contrasting results raise the possibility that E2-induced NGB up-regulation could regulate the ERβ activities shunning this receptor subtype to trigger an apoptotic cascade in neurons but not in non-neuronal cells. Here, human colorectal adenocarcinoma cell line (DLD-1) that only expresses ERβ and HeLa cells transiently transfected with ERβ encoding vector has been used to verify this hypothesis. In addition, neuroblastoma SK-N-BE cells were used as positive control. Surprisingly, E2 also induced NGB up-regulation, in a dose- and time-dependent manner, in DLD-1 cells. The ERβ-mediated activation of p38/MAPK was necessary for this E2 effect. E2 induced NGB re-allocation in mitochondria where, subsequently to an oxidative stress injury (i.e., 100μM H2O2), NGB interacted with cytochrome c preventing its release into the cytosol and the activation of an apoptotic cascade. As a whole, these results demonstrate that E2-induced NGB up-regulation could act as an oxidative stress sensor, which does not oppose to the pro-apoptotic E2 effect in ERβ-containing colon cancer cells unless a rise of oxidative stress occurs. These results support the concept that oxidative stress plays a critical role in E2-induced carcinogenesis and further open an important scenario to develop novel therapeutic strategies that target NGB against E2-related cancers., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Researchers are regularly interested in interpreting the multipartite structure of data entities according to their functional relationships. Data is often heterogeneous with intricately hidden inner structure. With limited prior knowledge, researchers are likely to confront the problem of transforming this data into knowledge. We develop a new framework, called heat-passing, which exploits intrinsic similarity relationships within noisy and incomplete raw data, and constructs a meaningful map of the data. The proposed framework is able to rank, cluster, and visualize the data all at once. The novelty of this framework is derived from an analogy between the process of data interpretation and that of heat transfer, in which all data points contribute simultaneously and globally to reveal intrinsic similarities between regions of data, meaningful coordinates for embedding the data, and exemplar data points that lie at optimal positions for heat transfer. We demonstrate the effectiveness of the heat-passing framework for robustly partitioning the complex networks, analyzing the globin family of proteins and determining conformational states of macromolecules in the presence of high levels of noise. The results indicate that the methodology is able to reveal functionally consistent relationships in a robust fashion with no reference to prior knowledge. The heat-passing framework is very general and has the potential for applications to a broad range of research fields, for example, biological networks, social networks and semantic analysis of documents.

A laboratory-made INSTCoated fused-silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145,000 theoretical plates per meter for myoglobin to 1,216,000 m(-1) for lysozyme. A total of 49-89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0-86% was obtained in a laboratory polyacrylamide-coated capillary, and only 0-6% was obtained in an uncoated fused-silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1-1.0% (migration time) and 0.3-2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6-1.4% (migration time) and 2.4-5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7-2.1% (migration time) and 2.8-10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and (G)γ chains prepared from newborn blood took 3 min with a peak resolution of 3.6., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neuroglobin and cytoglobin are new members of the heme-globin family. Both globins are primarily expressed in neurons of the brain and retina. Neuroglobin and cytoglobin have been suggested as novel therapeutic targets in various neurodegenerative diseases based on their oxygen binding and cell protecting properties. However, findings in Neuroglobin-deficient mice question the endogenous neuroprotective properties. The expression pattern of neuroglobin and cytoglobin in the rodent brain is also in contradiction to a major role of neuronal protection. In a recent study, neuroglobin was ubiquitously expressed and up-regulated following stroke in the human brain. The present study aimed at confirming our previous observations in rodents using two post-mortem human brains. The anatomical localization of neuroglobin and cytoglobin in the human brain is much like what has been described for the rodent brain. Neuroglobin is highly expressed in the hypothalamus, amygdale and in the pontine tegmental nuclei, but not in the hippocampus. Cytoglobin is highly expressed in the habenula, hypothalamus, thalamus, hippocampus and the pontine tegmental nuclei. We only detected a low expression of neuroglobin and cytoglobin in the cerebral cortex, while no expression in the cerebellar cortex was detectable. We provide a neuroanatomical indication for a different role of neuroglobin and cytoglobin in the human brain.

The aim of this study was to investigate the medial preoptic nucleus (MPN) of the anterior hypothalamus by resonance Raman spectroscopy (514.5 nm) to determine if it is possible to enhance the Raman scattering of hemoproteins in fresh brain tissue slices. The resonance effect was compared with near-infrared Raman spectra. Two groups of male Sprague Dawley rats were studied, one control group on a normal diet and one group on a low-iron diet to evoke iron deficiency. Each group consisted of four rats, 38-41 days old. The diets lasted for 11, 12, and 15 days. The MPN regions of brain tissue slices were analyzed by monitoring raw and pre-processed mean data, by cluster analysis, and by deriving difference spectra from pre-processed mean spectra. Cluster analysis of the resonance Raman spectra could identify different hemoprotein groups, namely, hemoglobin (Hb) and neuroglobin (Ngb). Spectra from randomly distributed spots revealed high Hb content, whereas Ngb was evenly distributed in the MPN. The different spectra showed a decrease of the Ngb and lipid content for the animals on the low-iron diet. The Ngb decrease was approximately 20%. The data show that resonance Raman spectroscopy is well suited to study hemoproteins in fresh brain tissue.

Objective: To study the role of neuroglobin (Ngb) in the pathologic process of contusion and laceration of brain in children., Methods: The proteins in the brain tissue were extracted by two-dimensional gel electrophoresis in 3 children undergoing brain ventricular neoplasms resection (normal brain tissue) and in 8 children with contusion and laceration of brain. The image analysis was done using the PDQuest 7.0 software. The differential protein spots were detected and analyzed with Applied Biosystems Voyager System 4307 MALDI-TOF Mass Spectrometer and bioinformatical skills. Ngb expression in the brain tissue was measured using immunohistochemisty. Ngb expression in plasma was measured using ELISA in 15 children with contusion and laceration of brain and 10 healthy children., Results: Expression maps of the brain tissue were established by two-dimensional gel electrophoresis in children with contusion and laceration of brain and healthy children. Six differential protein spots were found and 5 of them were identified by mass spectrum. Immunohistochemisty assay showed that Ngb expression in the brain tissue in children with contusion and laceration of brain was significantly higher than in normal controls (P<0.05). ELISA results showed that Ngb expression in the plasma increased significantly 6, 12, 18, 24 and 48 hours after trauma in children with contusion and laceration of brain compared with healthy children (P<0.01)., Conclusions: Ngb may play an important role in the pathologic process of contusion and laceration of brain in children.

Colorectal cancer (CRC) is the second most common cause of cancer deaths in Canadian men and women - accounting for almost 12% of all cancer deaths. In Ontario, it is estimated that 8100 persons were diagnosed with CRC in 2011, and 3250 died from the disease. CRC incidence and mortality rates in Ontario are among the highest in the world. Screening offers the best opportunity to reduce this burden of disease. The present report describes the findings and recommendations of Cancer Care Ontario's Fecal Immunochemical Tests (FIT) Guidelines Expert Panel, which was convened in September 2010 by the Program in Evidence-Based Care. The purpose of the present guideline is to evaluate the existing evidence concerning FIT to inform the decision on how to replace the current guaiac fecal occult blood test with FIT in the Ontario ColonCancerCheck Program. Eleven articles were included in the present guideline, comprising two systematic reviews, five articles reporting on three randomized controlled trials, and reports of four other studies. Additionally, one laboratory study was obtained that reported on several parameters of FIT tests that helped to inform the present recommendation. The performance of FIT is superior to the standard guaiac fecal occult blood test in terms of screening participation rates and the detection of CRC and advanced adenoma. Given greater specimen instability with the use of FIT, a pilot study should be undertaken to determine how to implement the FIT in Ontario.

Objective: To investigate mutation spectrums of α- and β-haemoglobin genes in thalassemia patients and carriers in Yunnan province, and to establish procedures on prenatal gene diagnosis., Methods: Totally 10 033 counseling couples and pregnant women, and 22 cases of children with moderate or severe thalassemia were recruited from 5 parts of Yunnan Province, middle, western, eastern, southern and northern areas, during July 2009 to July 2011. Medical records, including results of haemoglobin electrophoresis, blood routine examination, and gene diagnosis of subjects were collected and saved in an database in Excel software by the Key Laboratory for Birth Defects and Genetic Diseases. Using multiple gap-PCR and PCR-reversed dot blotting kits, DNA samples collected from 1077 cases of haematological positive thalassemia patients and carriers were tested to determine common mutations of the α- or β-haemoglobin genes. The codon regions of haemoglobin genes were sequenced by the Sanger sequencing in cases that the mutation tests were negative. Mutation spectrums of α- and β-haemoglobin genes were concluded. Prenatal gene diagnosis was offered to fetuses who had risk of thalassemia major., Results: (1) In 1077 cases of haemological screen positive subjects, deletions and mutations of α-haemoglobin gene were tested in 119 subjects among 347 cases suspected as α-thalassemia patients and carriers. Five kinds of deletions and mutations on α-haemoglobin gene were found. In 104 subjects, four kinds of common deletions and mutations onα-haemoglobin gene were determined: --(SEA), -α(3.7), α(CS)α, -α(4.2). Other 14 subjects were double heterozygotes with haemoglobin H disease and severe α-thalassemia phenotypes. A rare mutation of insertion and deletion in α2 haemoglobin gene intron, α(301-24&#95;301-23 indel), was found in one carrier subject. (2) In 1077 cases of haemological screen positive subjects, deletions and mutations of β-haemoglobin gene were tested in 297 subjects among 730 cases suspected as β-thalassemia patients and carriers. Sixteen kinds of β-haemoglobin gene mutations were found, including 7 cases of rare abnormal haemoglobinopathy patients with β-haemoglobin gene mutations. In one case with β(+) phenotype patient, the Codon 5(-CT) mutation at β-haemoglobin gene was found (firstly reported in China). (3) Three fetuses with high risks of α-thalassemia were accepted for prenatal diagnosis. One case of Hb Bart's hydrops syndrome fetus with the genotype --(SEA)/--(SEA), and one case of mild α-thalassemia fetus with the genotype α(CS)α/αα were found. Another one fetus was found with normal α-haemoglobin. In 6 fetuses accepted for prenatal diagnosis due to high risks of β-thalassemia, one case of β-thalassemia major with the genotype CD(17)(A→T)/-28(A→G) was found, 3 fetuses were heterozygote carriers, and 2 fetuses had normal genotypes without mutations found in their parents. Medical terminations for 2 fetuses with severe thalassemia were made according to the choice of pregnant women. Other 7 pregnancies continued to term. Anemia or growth retardation was not found in the 7 infants when following up after given-birth 6 to 12 months., Conclusions: The mutation spectrums of α- and β-haemoglobin genes of thalassemia patients and carriers in Yunnan province are special, in which β-haemoglobin gene exits more polymorphism in the mutation spectrum. Carrier screening in pregnant women, and offering prenatal gene diagnosis to the high risk pregnancies should be an efficient strategy to prevent thalassemia major.

Hypoxia inducible factor 1(HIF-1α) is the regulator of oxygen homeostasis in tissue correlated with neuroglobin (NGB) a member of the family of globins in vertebrates. The present study investigates, the expression and the location of NGB, HIF-1α in human carotid bodies, sampled at autopsy from children (mean age: 2 year ±), young (mean age: 27.5) and 4 old subjects (mean age: 73.5). The percentage of NGB positive area was higher in the old subjects (4.4 ±2.8%), as compared with the young ones (2.4 ±1.8%) and children (1.0 ±1.8%). Positive HIF-1α nuclei were detected in young and old subjects (1.0 ±0.14% vs 3.0 ±0.28%, respectively), whereas CB tissues from children did not show any HIF-1α reaction. The increase of NGB and HIF-1α expression suggests a possible role of the two oxygen sensors in the aging processes. Even though the physiological role of NGB is not well understood, it could be suggested that is act as a respiratory protein connected with HIF.

Objective: Hereditary spherocytosis (HS), a common inherited hemolytic anemia characterized by decreased deformability, reduced surface to volume ratio, and increased osmotic fragility of the spheroidal erythrocytes, is associated with several mutations of α- and β-spectrin, ankyrin, band 3, band 4.2. HS manifests itself with high degrees of clinical heterogeneity and the molecular events leading to premature hemolysis of the spherocytes are unclear. We have employed proteomic techniques to identify differentially regulated proteins in the membrane and hemoglobin-depleted cytosol of HS erythrocytes., Methods: We have employed 2-D gel electrophoresis and tandem matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry to investigate the differential proteome profiling of membrane and hemoglobin-depleted cytosol of erythrocytes isolated from the peripheral blood samples of HS patients and normal volunteers., Results: Our study showed that redox regulators are up-regulated; while a co-chaperone and a nucleotide kinase are down-regulated in HS erythrocyte cytosol. We observed elevated levels of membrane-associated globin chains and low-molecular weight fragments of several major cytoskeletal proteins., Conclusion: The observed changes in the erythrocyte proteomes indicate altered redox regulation, nucleotide metabolism, protein aggregation and/or degradation, cytoskeletal disorganization, and severe oxidative stress in HS. Taken together, this study could enlighten upon disease progression and pathophysiology of HS., (© 2011 John Wiley & Sons A/S.)

The N-terminal valine adduct (HETE-Val) in globin is believed to behave as a long-lived biomarker after exposure to sulfur mustard (HD). Development of a highly sensitive method for monitoring HETE-Val, particularly at low HD exposure levels or for retrospective detection, would be a significant achievement. In this study, by improving the sample preparation method, a sensitive NCI-GC/MS method was established for the analysis of HETE-Val in globin after HD exposure. To optimize and investigate the sample preparation method, all the relevant HETE-Val chemicals were synthesized, purified, and characterized. By carrying out optimized solid phase extraction (SPE) cleanup followed by modified Edman degradation results in a low detection level and clean baseline. The minimum detectable exposure level of human blood (in vitro) to HD is 20 nmol/L (S/N>3). The interday and intraday precisions of the proposed method were found to be acceptable with less than a 15% relative standard deviation (RSD). A nearly linear dose-effect relationship was observed between HETE-Val and a HD exposure concentration range of 0.1-120 μmol/L. The percentage of HD that reacted with N-terminal valine in globin obtained from human blood (in vitro) was quantified using the proposed method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study describes a rapid and sensitive high-performance liquid chromatography (HPLC) method for the detection of human globin chains in blood. The method involves direct injection of globin chains which prepared by a standard method onto a micro bondapack C18 reversed-phase column (7.8 mm I.D.) with UV detection at 280 nm. The detection limit of hemoglobin (Hb) was 0.1 μg, which is equivalent to about 1 ml of fresh whole blood. We report here the rapid procedure for globin chain analysis. The present method will be useful for the determination of globin chain analysis in clinical laboratories, as well as in thalassemia and sickle cell disease patients.

Relating structure and spectroscopy is fundamental in characterizing the conformational dynamics and elucidating function at an atomistic level in condensed-phase environments. In particular, the combination of infrared spectroscopy and atomistic simulations has provided fundamental insight into structural assignments of spectroscopic bands. Infrared spectroscopy and Molecular Dynamics (MD) simulations on carbonmonoxy myoglobin (MbCO) were able to partially identify three major CO infrared bands (A0, A1 and A3) which are related to different conformational substates in the active site of the protein. Recently, two similar CO bands were identified from experiments in human carbonmonoxy neuroglobin (NgbCO), named N0 and N3. Time-dependent frequency changes found in the N0 band and a large variation of relaxation times for these bands made the characterization of these substates considerably more difficult compared to MbCO. In this work we discuss the structure-spectroscopy relationship for three different His64 protonation states in human and murine NgbCO using MD simulations and density functional theory (DFT) calculations. The present work assigns the N3 band to the His(epsilon)64 tautomer having its side chain in hydrogen bonding contact to CO. Frequencies of the corresponding His64H+ and His(delta)64 tautomers show characteristic contributions to the N0 band.

Introduction: Deep hypothermic circulatory arrest (DHCA) is used in corrective cardiac surgery for complex congenital heart disease. Endogenous protective mechanisms may be responsible for the prevention of brain damage after hypothermic ischemia. Neuroglobin and cytoglobin are expressed in brain cells and appear to modulate hypoxic-ischemic brain injury. However, their neuroprotective potency is still not understood. Thus the aim of this study was to detect the influence exerted by DHCA on their expression., Methods: The effects of DHCA were analyzed in a neonatal piglet model with cardiopulmonary bypass, DHCA of 60 and 120 min and subsequent reperfusion of 6h. Complete histological analysis and changes in the mRNA expression of neuroglobin and cytoglobin were measured in the brain., Results: In comparison to animals without DHCA, neuroglobin expression was stable after 60 min DHCA and neuronal cell necrosis in the cortex was mild (< 10 %). Neuroglobin expression was significantly reduced after 120 min DHCA, which was accompanied by substantial neuronal cell necrosis (> 50 %). Cytoglobin expression did not differ significantly between animals with neuronal necrosis vs. sham., Conclusion: Constitutive expression levels of neuroglobin may explain the mild neuronal injury after 60 min DHCA. Significant neuronal cell death correlates with reduced neuroglobin expression and might reflect a limited capacity to compensate for ischemic injury. Both respiratory cell proteins may constitute attractive targets for therapeutic modulation of gene regulation, but further studies are necessary., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

In this chapter, we describe numerous methods to extract RNA, DNA, and protein from tissue, represented by kidney transplant biopsies, and from peripheral blood cells collected at various clinical sites. Gene expression profiling and SNP-based genome-wide association studies are done using various microarray platforms. In addition, protocols that enable simultaneous protein purification from these clinical samples, enable additional strategies for understanding of the molecular processes involved in organ transplantation, immunosuppressive drug regimens, and the elements determining allograft success and failure. Successfully establishing a multicenter clinical study was essential to meet our objectives for subject enrollment and transplant outcomes. This chapter focuses on our experience setting up and coordinating clinical sample collection from multiple transplant centers for the purpose of microarray analysis.

Phenotype studies still occupy a key position in the diagnosis of hemoglobin (Hb) disorders. An additional dimension to the methods for diagnosis of Hb disorders which are mostly based on difference in charge of the Hb molecules may be brought by studying some properties of the globin chains. Among the methods proposed, reversed-phase liquid-chromatography (RP-LC) reveals differences in hydrophobicity allowing to discriminate between variants displaying identical charges. Thus, abnormal Hbs responsible for hematological disorders, such as chronic hemolytic anemia, erythrocytosis, or thalassemia like presentation, but with a charge similar to HbA or to that of a common variant may be revealed. Also RP-LC, which discriminates between the two types of gamma chains, may be of interest for diagnosis of hereditary persistence of fetal hemoglobin (HPFH) or for suggesting a haplotype in the case of sickle cell anemia.

Blood cells from three different sea turtle species were cultured for approximately 3 weeks in nutrient medium supplemented with recombinant human cytokines known to induce terminal maturation of human hematological stem cells. Cultured turtle erythrocytes were translucent, approximately 10x larger than human erythrocytes, contained a single fluorescent inclusion body, contained nuclear epsilon (embryonic) globin proteins, and, absent of organelles while fresh cells contained few, but well defined mitochondria. Cells with basophilic cytoplasm and in all stages of proliferation were observed in cytokine-supplemented cultures and appeared to possess active protein synthesis. Cultured thrombocytes aggregated in response to agonists for at least 8 days, post-collection, contained P-selectin in the nucleus of 6 day cultured cells which appeared to be released after activation with collagen, and after 6 days had no organelles or open canalicular-like system (OCS) while freshly isolated cells demonstrated few, if any organelles but had a well developed OCS. The response of turtle cells to apparently homologous but unnatural human cytokines and the sustained biological properties of thrombocytes identify this suspension culture system as a powerful tool to explore the evolution of cell types and molecular components of hematopoiesis and hemostasis.

Eosinophilic inclusions in the cytoplasm of protoplasmic astrocytes of the neocortex, usually in the clinical setting of epilepsy and/or psychomotor retardation, were first recognized and illustrated by Alois Alzheimer in 1910. Traditional special stains have failed to elucidate the specific nature of these inclusions. Ultrastructurally, the material was composed predominantly of highly electron-dense, non-membrane-bound, granular material distinct from Rosenthal fibers. Immunohistochemical examination has been informative but also sometimes inconsistent; it has recently been suggested that they may represent a filaminopathy (filamin A). We examined 5 cases with neocortical eosinophilic inclusions (3 autopsies, 2 surgical resections) using a standardized immunohistochemical protocol at a single institution. The specimens were immunostained with 32 antibodies to 30 potentially relevant proteins using several antigen retrieval protocols. We confirmed the presence of filamin A in these inclusions, but several additional proteins, particularly cytoglobin and glutamate transporter 1, were also identified. By electron microscopy in 2 cases, the granular fine structure of the inclusions was confirmed; mitochondria adjacent to, and perhaps within, the inclusions that contained many pleomorphic vesicular and membranous elements were also noted in 1 case. The pathophysiologic relevance of these proteins and the clinical significance of the hyaline inclusions are discussed.

Stroke is predicted by abnormally high cerebral velocities by transcranial doppler (TCD). This study aimed at defining predictive factors for abnormally high velocities (>/= 2 m/sec) based on the Créteil pediatric sickle cell anemia (SCA) cohort composed of 373 stroke-free SCA children. alpha genes and beta-globin haplotypes were determined. Biologic parameters were obtained at baseline. alpha-thalassemia was present in 155 of 325 and G6PD deficiency in 36 of 325 evaluated patients. TCD was abnormal in 62 of 373 patients. Multivariate logistic regression analysis showed that G6PD deficiency (odds ratio [OR] = 3.36, 95% confidence interval [CI] 1.10-10.33; P = .034), absence of alpha-thalassemia (OR = 6.45, 95% CI 2.21-18.87; P = .001), hemoglobin (OR per g/dL = 0.63, 95% CI 0.41-0.97; P = .038), and lactate dehydrogenase (LDH) levels (OR per IU/L = 1.001, 95% CI 1.000-1.002; P = .047) were independent risk factors for abnormally high velocities. This study confirms the protective effect of alpha-thalassemia and shows for the first time that G6PD deficiency and hemolysis independently increase the risk of cerebral vasculopathy.

S-(1,2-dichlorovinyl)- L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by an FMO3-mediated oxidation of the trichloroethylene metabolite S-(1,2-dichlorovinyl)- L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. Because DCVCS incubations with N-acetyl- L-cysteine at pH 7.4, 37 degrees C resulted in the formation of three diastereomeric monoadducts and one diadduct, globin monoadducts and cross-links formed after in vitro incubations of rat erythrocytes with DCVCS (0.9-450 microM) for 2 h and those present at 30 min after in vivo treatment of rats with DCVCS (23 and 230 micromol/kg) were characterized. ESI/MS of intact globin chains revealed adduction of 1 DCVCS moiety on the beta2 chain at the three lowest DCVCS concentrations and on the beta1 chain after the in vivo treatment with 230 micromol/kg DCVCS. Interestingly, intact globin dimers and trimers were detectable by ESI/MS with all DCVCS concentrations in vitro (also by SDS-PAGE) and in vivo. LC/MS and MALDI/FTICR of trypsin digested peptides from globin samples obtained after in vitro (450 microM DCVCS) or in vivo exposure to DCVCS (230 micromol/kg) suggested the formation of DCVCS monoadducts not only with Cys93 and Cys125 of the beta chains but also with Cys13 of the alpha chains, whereas no monoadducted peptides were detected at lower DCVCS concentrations in vitro or in vivo. However, LC/MS and MALDI-TOF/TOF suggested the presence of several DCVCS-derived peptide cross-links both in vivo and in vitro at all DCVCS exposure levels. Collectively, the results indicate at least 4 out of the 5 cysteine moieties of the rat hemoglobin heterodimer may be alkylated by DCVCS, in reactions that could also lead to the formation of multiple cross-links. DCVCS- and N-acetyl-DCVCS (NA-DCVCS)-derived globin cross-links containing GSH and Cys were also detected by mass spectrometry, providing strong evidence for the reactivity and/or cross-linking ability of DCVCS, NA-DCVCS, and their GSH or Cys conjugates in both the in vitro and the in vivo. Thus, hemoglobin adducts and cross-links may be useful biomarkers to investigate the possible presence of DCVCS in circulation after DCVC or trichloroethylene exposure.

RNA interference (RNAi), a process by which target messenger RNA (mRNA) is cleaved by small interfering complementary RNA (siRNA), is widely used for investigations of regulation of gene expression in various cells. In this study, siRNA complementary to 5' region of exon II of alpha-globin mRNA was examined for its role in erythroid colony forming cells (ECFCs) isolated from normal peripheral blood donor. On day 6 of cell culture, 1x10(6) ECFCs were transfected with lipofectamine-containing alpha-globin specific siRNA. After 48h of transfection, alpha-globin specific siRNA produced significantly reduction of alpha-globin mRNA level in a dose-dependent manner, but it did not affect the level of beta-globin mRNA. Significantly, decreased numbers of hemoglobinized erythroid cells relative to the control were observed supporting the inhibitory effect of this alpha-globin mRNA specific siRNA.

The recent proliferation of platforms designed to handle arrays of nano- and picolitre volumes is in response to the need to perform biological assays on discrete entities, such as single cells. However, a critical challenge associated with this trend for in vitro compartmentalization is the need for highly sensitive, yet low-volume detection platforms. In this paper, we coupled confocal fluorescence detection with recirculating microfluidic control to perform single particle DNA assays within five nL chambers. The performance of this low-volume assay was shown to match that of traditional single molecule detection platforms. However, volume requirements per measurement were nearly 3 orders of magnitude less than conventional systems, enabling future integration with lab-on-a-chip systems that require discrete or digitalized sample processing.

Background: Several previous studies reported that the Venezuelan Warao Indians presented unusual genetic characteristics., Aim: The present study checked previous reports of a high frequency of hereditary persistence of fetal hemoglobin (HPFH) and examined other hematological traits., Subjects and Methods: Standard hematology, electrophoresis on cellulose acetate, fetal hemoglobin alkali denaturation, gamma-globin chain, DNA amplification and sequencing, and denaturing gradient gel electrophoresis determinations were performed in 269 individuals living in two localities of the Orinoco River Delta., Results: Two beta(s) genes, in apparently non-related individuals, were found. HPFH, detected in this same population of Warao Indians 25 years ago, was present in heterozygous form in five individuals from a large kindred, with hemoglobin F levels ranging from 3.7% to 8%, and with a pancellular distribution. The HPFH mutation was of the deletional type. beta-globin gene haplotypes were determined by direct counting (through family studies) in 150 chromosomes; 26% of the 150 examined cluster presented haplotype 2, 22% haplotype 6, and 13% a new, Warao haplotype. Haplotype 3, of probable African origin, was also found with a frequency of 5%., Conclusions: The presence of the HPFH mutation was confirmed, and the new beta-globin gene haplotype together with the presence of other rare variants indicates that the Warao are very distinctive in relation to other Native Americans. Evidence was also found of a slight admixture from Africa-derived subjects (Layrisse et al. 1988).

Routine laboratories use a hemoglobin H (HbH) screen to detect alpha-thalassemia carriers of fatal hemoglobin Bart's hydrops fetalis. This test is laborious and has sensitivity concerns. A commercial zeta-globin enzyme-linked immunosorbent assay (ELISA) is effective in detecting Southeast Asian (SEA) alpha-thalassemia. We present results of a study of the effectiveness of carrier detection of ELISA and a shortened HbH screen compared with gap polymerase chain reaction. ELISA was superior to the HbH screen for the SEA alpha0-thalassemia trait. The ELISA and H screen were equal for detection of all carriers encountered and combined were more effective than either test alone. A positive zeta-globin ELISA result is diagnostic of SEA alpha-thalassemia, and routine use of the zeta-globin ELISA in combination with a shortened HbH screen will improve the efficacy of prenatal screening for carriers of hemoglobin Bart's hydrops fetalis through improved detection and referral for follow-up DNA testing.

Implementation of desorption electrospray ionization (DESI) technique on a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is described. Desorption electrospray technique is capable of the direct investigation of natural samples without any need for sample preparation or chromatographic separation. Since the DESI mass spectra of natural samples are very complex owing to the lack of preseparation or cleanup, the ideal mass spectrometric analyzer for these applications is a high-resolution instrument such as FTICR mass spectrometer. DESI was implemented by constructing an electronically controlled source framework comprising six linear moving stages and one rotating stage. A three-dimensional linear stage was used to accommodate samples, while another 3D linear stage equipped with rotating stage was used as a spray mount. A modified electrosonic sprayer was used as a primary electrospray device. DESI-FTICR setup was characterized with regard to geometrical, electrical and flow conditions using deposited peptide samples in range of 1-100 pmol gross deposited amount on glass and polymer surfaces. Optimized conditions enabled the routine acquisition of DESI-MS spectra on the instrument at 130 000 resolution in the broadband mode and with comparable sensitivity to data reported in the literature. Since the main significance of DESI-FTICR MS is the combination of intact tissue analysis, the capabilities of the technique were demonstrated by analyzing murine liver samples. Presence of lysophospholipids in the liver tissue was tentatively associated with the lipid metabolism taking place in liver. DESI-FTICR is also a promising technique in the field of peptide analysis due to capability of top-down sequencing using electron capture dissociation. As a proof-of-principle experiment, a small synthetic polypeptide containing 36 amino acids was ionized using DESI and was sequenced in the FTICR by means of ECD (electron capture dissociation) fragmentation. Spectra gave almost full sequence information in agreement with the known amino acid sequence of the species., ((c) 2008 John Wiley & Sons, Ltd.)

Objectives: Imbalance in alpha/beta-globin chains is an important determinant of thalassemia disease severity. This study examined the relationship between alpha/beta-globin mRNA ratio and disease severity in various thalassemia genotypes., Design and Methods: alpha- and beta-globin mRNA contents of red blood cells of 75 alpha- and 32 beta-thalassemia subjects (5 with beta(0)-thalassemia/Hb E) and 14 normal controls were measured using multiplex quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The alpha/beta-globin mRNA ratio of each sample was calculated based on the 2(-DeltaDeltaC)(T) method., Results: A decrease of alpha/beta-globin mRNA ratios in alpha-thalassemia subjects compared to normal controls correlated with the numbers of defective alpha-globin genes, whereas an increase of the ratios was observed in beta-thalassemia. Subjects with beta(0)-thalassemia/Hb E disease had the highest alpha/beta-globin mRNA ratio, followed by beta(0)-thalassemia trait and then beta(+)-thalassemia trait, which correlated with decrease in severity of anemia. Coinheritance of alpha-thalassemia in beta(0)-thalassemia/Hb E resulted in a more balanced alpha/beta-globin mRNA ratio and an amelioration of the anemia., Conclusions: This study indicates that imbalance in globin gene expression, the major factor affecting clinical severity of thalassemia, could be demonstrated by measuring alpha/beta-globin mRNA ratio, which was conveniently and accurately determined by qRT-PCR. In alpha-thalassemia, alpha/beta-globin mRNA ratio correlated with the number of functional alpha-globin genes present, whereas in beta-thalassemia, the ratio provided a good indicator of disease severity.

The aim of this study was to determine the effect of diet supplemented with Saccharomyces cerevisiae (SC), on the liver morphology, serum activities of aspartateaminotrasferase (AST) and alanineaminotransferase (ALT), and the serum concentration of total proteins, albumin and the different fractions (alpha, beta, gamma) of globulin in chickens. Two groups of Hubbar x Hubbar strain chickens (each = 16), were fed with a diet T1 (commercial food without SC) and T2 (commercial food with SC) for 42 days. Thereafter, animals were sacrificed and samples of blood and liver were obtained. Macroscopic features and relative weight of livers remained in the normal ranges in both groups. High percent of T2 chickens (p < 0.05) showed hepatic alterations with proliferation/dilation of biliary ducts and increased degree of vacuolization. Tricromic stain showed elevated perivascular fibrosis in T2. The ultrastructural study showed fibroblast like cells, increased amount of collagen fibers, cytoplasm vacuolization of hepatocytes and loss of cellular and mitochondrial integrates. No significant differences were observed in the activities of AST and ALT between groups. Increased concentrations of total proteins and alpha2, beta and gamma globulins and decreased albumin were observed in the serum of T2 group. These findings may be related to a hepatotoxic effect of SC and chickens could have the risk of further increased hepatic failure with a prolonged exposition to this diet.

Long double-stranded RNAs (dsRNAs) may undergo extensive modification (hyperediting) by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine (A) residues are changed to inosine (I). Traditionally, consequences of A-to-I editing were thought to be limited to modified RNA itself. We show here, however, that hyperedited dsRNA (I-dsRNA) is able to downregulate gene expression in trans. Furthermore, we show that both endogenous expression and reporter gene expression were substantially reduced in the presence of I-dsRNA. This was due to a reduction in reporter mRNA levels and also translation inhibition. Importantly, we show that I-dsRNA interferes with translation initiation. We also show that I-dsRNA specifically binds a stress-granule-like complex. Stress granules (SGs) are important for translational silencing during stress. Finally, we propose a model whereby editing by ADARs results in downregulation of gene expression via SG formation.

Objective: To investigate the clinical effects and security of YiSuiShengXueGranule (YSSXG) on treating 156 patients with beta-thalassemia major., Methods: YSSXG was given orally to 156 patients with beta-thalassemia in GuangXi Autonomous Region (the high incidence area of beta-thalassemia in China) for 3 months as one therapeutic course, 3 times a day, 10 g each time (for children, the dose should be reduced properly according to their body weight and age), and no blood transfusion used during the course. Clinical symptoms and levels of hemoglobin (Hb), red blood cell (RBC), reticulocyte (Ret) and hemoglobin F (HbF) were observed before and after treatment, and side-effects were observed during the course. A 3-6 months follow up study was performed after withdrawal of YSSXG. And systemic gene analysis was conducted with PCR, SSCP-PCR, RT-PCR and DNA sequences analysis and mRNA differently expression technique, in order to study the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation., Results: Levels of Hb, RBC, Ret and HbF obviously elevated, and clinical symptoms markedly ameliorated in patients after treated with YSSXG from the 1st to 3rd month (all p<0.01). Dynamical observation showed that the improvement of symptoms kept accordance with the elevation of hemorrheological indexes. The treatment was effective in 145 patients and ineffective in 11, and the total effective rate was 92.9%, without any adverse reaction founded. Follow-up studies showed the therapeutic effect could sustain for 3 to 4 months after drug-withdrawal. The molecular mechanism study showed: YSSXG did not change the genetic mutation type, but could obviously increase gamma/(beta+gamma) globin ratio, both gamma-globin mRNA and GM-CSF mRNA expression were significantly enhanced so as to induce HbF synthesis increasing after treated with YSSXG., Conclusion: YSSXG had obvious effects in treating beta-thalassemia by unlocking gamma-gene, increasing the gamma-globin expression and enhancing HbF synthesis so as to compensate for the gene defect. This study has provided a new path for the treatment of beta-thalassemia with Traditional Chinese Medicine.

In medaka, we found a C16orf35-like gene in the region within 1 Kbp 3' downstream of the psibeta end of the 36-Kbp embryonic globin gene cluster ((5')alpha0(3')-(3')beta1(5')-(5')alpha1(3')-(5')beta2(3')-(5')alpha2(3')-(3')alpha3(5')-(5')beta3(3')-(3')beta4(5')-(5')alpha4(3')-(3')psialpha(5')-(5')psibeta(3')). Intron 5 of the gene contained a region having NF-E2 binding sites located between GATA boxes. The region was homologous to human HS-40 in terms of the existence and structure of characteristic transcription-factor binding sites and was named Ol-HS-40. Injection of the fusion gene construct Ol-HS-40-alpha0(up-2)GFP, consisting of Ol-HS-40, a 5' upstream 200-bp minimum promoter for alpha0, and green fluorescent protein (GFP), showed that Ol-HS-40, as in human HS-40, had the ability to strongly enhance GFP expression in erythroid cells of embryos. Further analysis using transgenic technology revealed that Ol-HS-40 had the ability to change the type of the GFP expression from embryo-to-young fish to embryo-to-adult. In addition, the results suggest that Ol-HS-40, although its natural function remains unclear, has strong enhancer activity for the expression of not only the alpha-globin gene but also the beta-globin gene.

Background: Peptide-based analysis of whole blood using electrospray tandem mass spectrometry (MSMS) in multiple reaction monitoring (MRM) mode enables rapid detection and sequence confirmation of clinically significant hemoglobin (Hb) variants. We applied a similar, quantitative approach to the measurement of delta:beta-globin peptide ratios as potential surrogate markers of HbA(2), a biomarker used in population screening for beta-thalassemia trait., Methods: We studied 163 blood samples with normal HbA(2) (%), 105 with increased HbA(2), 43 with delta-chain variants, and 8 with Hb Lepore. All were tested by HPLC. The samples were also incubated with trypsin for 30 min at 37 degrees C for MSMS with flow injection analysis. MRMs for the delta- (T2, T3, and T14) and beta- (T2, T3, and T13) globin tryptic peptides were acquired for 1 min, and delta:beta peptide ratios were calculated. We used HPLC and MSMS to analyze 26 paired whole blood and dried blood spot samples after storage for 1, 8, and 29 days., Results: Within- and between-assay imprecision values (CVs) were <6.1% and <8.4%, respectively, for the delta:beta peptide ratios. Digests were stable at 10 degrees C for 6 days. Significant correlations (P <0.0001) between MSMS delta:beta-globin peptide ratios and HPLC HbA(2) allowed differentiation between increased HbA(2) concentrations and concentrations within the reference interval and identification of Hb Lepore. This differentiation was repeatable by MSMS, but not by HPLC, after blood spot samples had been stored for 1 month., Conclusion: This study validates the quantitative delta:beta-globin peptide ratio as a surrogate marker of HbA(2) and demonstrates the potential of rapid peptide-based MSMS for multiplexed, high-throughput protein biomarker characterization and quantification.

The expression of the cytoglobin/stellate cell activation-associated protein (Cygb/STAP) was recently confirmed in all splanchnic vitamin A-storing cells--including hepatic stellate cells (HSCs)--in normal conditions. In the hepatic fibrous lesion, the expression of Cygb/STAP has been shown to be upregulated in activated HSCs and myofibroblasts (MFs), which have synthesized extracellular matrices. Furthermore, splanchnic vitamin A-storing cells have been reported to be distributed in the kidney. In this study, we clarify the contribution of vitamin A-storing cells to renal fibrosis by focusing on Cygb/ STAP. Adult mice were subjected to unilateral ureteral obstruction (UUO) and kidneys were harvested 1, 3, 7, and 10 days after UUO. Numbers of Cygb/STAP-immunopositive cells as well as Cygb/STAP mRNA 3 days after UUO (UUO day 3 kidney) increased. Vitamin A-autofluorescence was observed in intertubular spaces of controls but gradually declined in a time-dependent manner after UUO. Cygb/STAP+ cells were not completely identical with alpha-smooth muscle actin (alphaSMA)-positive cells in the control or UUO day 7 kidneys. Immunohistochemical analysis for Cygb/STAP and fibulin-2 (Fib), a specific marker for distinguishing MFs from activated HSCs, revealed that the number of Fib+STAP+ cells (MFs) and Fib-STAP+ cells (splanchnic vitamin A-storing cells) significantly increased in UUO day 3 and UUO day 7 kidneys compared with the controls. Our present findings support the concept that Cygb/STAP can be a unique marker for splanchnic fibroblast-like cells, namely the vitamin A-storing cell lineage, and suggest that splanchnic vitamin A-storing cells contribute to renal fibrogenesis in the obstructed kidney.

To clarify the possible pathogenetic role of oxidation products originated from the glycation of proteins, human globins from nephropathic patients have been studied by matrix-assisted laser desorption/ionization mass spectrometry (MALDI), revealing not only unglycated and monoglycated globins, but also a series of different species. For the last ones, structural assignments were tentatively done on the basis of observed masses and expectations for the Maillard reaction pattern. Consequently, they must be considered only propositive, and the discussion which will follow must be considered in this view. In our opinion this approach does not seem to compromise the intended diagnostic use of the data because distinctions are valid even if the assignments are uncertain. We studied nine healthy subjects and 19 nephropathic patients and processed the data obtained from the MALDI spectra using a multivariate analysis. Our results showed that multivariate analytical techniques enable differential aspects of the profile of molecular species to be identified in the blood of end stage nephropathic patients. A correct grouping can be achieved by principal component analysis (PCA) and the results suggest that several products involved in carbonyl stress exist in nephropathic patients. These compounds may have a relevant role as specific markers of the pathological state.

Context: The geographical distributions of hemoglobin S (HbS), hemoglobin C (HbC), and alpha+-thalassemia (-alpha) strongly suggest balancing selection with malaria. However, whereas several studies indicate that the HbS carrier state protects against all major forms of clinical malaria, malaria protection on clinical grounds has been more difficult to confirm for HbC and -alpha, and questions remain as to whether it applies to all forms of the disease., Objective: To assess the association between major clinical forms of severe falciparum malaria and HbS, HbC, and -alpha., Design, Setting, and Participants: Case-control study of 2591 children with severe falciparum malaria enrolled at a tertiary referral center in Ghana, West Africa, and 2048 age-, sex-, and ethnicity-matched control participants recruited by community surveys., Main Outcome Measures: Frequencies of HbS, HbC, and -alpha in patients and controls, including stratifications of patients for signs of disease., Results: Patients presented with partly overlapping signs of disease, including severe anemia (64%), cerebral malaria (22%), respiratory distress (30%), hyperparasitemia (32%), prostration (52%), acidosis (59%), and hyperlactatemia (56%). Carrier states of HbS, HbC, and -alpha were found in 1.4%, 9.4%, and 25.2% of the patients, respectively, and 14.8%, 8.7%, and 27.3% of controls. The HbS carrier state was negatively associated with all forms of the disease studied (overall odds ratio [OR], 0.08; 95% confidence interval [CI], 0.06-0.12). The HbC carrier state showed a negative association selectively with cerebral malaria (OR, 0.64; 95% CI, 0.45-0.91), and the -alpha carrier state showed a negative association selectively with severe anemia (OR, 0.82; 95% CI, 0.69-0.96)., Conclusion: Whereas the HbS carrier state was found to be negatively associated with all major forms of severe falciparum malaria, the negative associations of the carrier states of HbC and -alpha appeared to be limited to cerebral malaria and severe anemia, respectively.

Preterm infants are at risk of developing sepsis, necrotizing enterocolitis (NEC), chronic lung disease (CLD), and retinopathy of prematurity (ROP). We used high-throughput mass spectrometry to investigate whether cord blood proteins can be used to predict development of these morbidities. Cord blood plasma from 44 infants with a birth weight of <1500 g was analyzed by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF). Six infants developed ROP >or=stage II, 10 CLD, three sepsis, and one NEC. We detected 814 protein signals representing 330 distinct protein species. Nineteen biomarkers were associated with development of >or=stage II ROP [false-discovery rate (FDR) <5%] and none with CLD. Several proteins with molecular weight (Mr) 15-16 kD and pI 4-5 were detected with increased abundance in infants with ROP, while similar Mr proteins with pI 7-9 were less abundant in these patients. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and sequence analysis identified these proteins as alpha-, beta-, and gamma-globin chains. Partial deamidation of Asn139 in beta-globin chains was observed only in the pI 4-5 proteins. We conclude that there are several promising biomarkers for the risk of ROP. Deamidation of globin chains is especially promising and may indicate underlying prenatal pathologic mechanisms in ROP. Validation studies will be undertaken to determine their clinical utility.

Primitive erythroblasts (EryPs) are the first hematopoietic cell type to form during mammalian embryogenesis and emerge within the blood islands of the yolk sac. Large, nucleated EryPs begin to circulate around midgestation, when connections between yolk sac and embryonic vasculature mature. Two to 3 days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryPs in the circulation. The development and maturation of EryPs remain poorly defined. Our analysis of embryonic blood at different stages reveals a stepwise developmental progression within the EryP lineage from E9.5 to E12.5. Thereafter, EryDs are also present in the bloodstream, and the 2 lineages are not easily distinguished. We have generated a transgenic mouse line in which the human epsilon-globin gene promoter drives expression of green fluorescent protein exclusively within the EryP lineage. Here, we have used this line to characterize changes in cell morphology and surface-marker expression as EryPs mature and to track EryP numbers and enucleation throughout gestation. This study identifies previously unrecognized synchronous developmental stages leading to the maturation of EryPs in the mouse embryo. Unexpectedly, we find that EryPs are a stable cell population that persists through the end of gestation.

Background: Human placental lactogen (hPL) is synthesised by the placenta and found in maternal serum. We analysed the potential of hPL as a first-trimester maternal serum-screening marker for fetal Down syndrome (DS)., Materials and Methods: hPL was quantified by ELISA in 47 DS pregnancies and 136 controls in gestational weeks 8-13. Distributions of log multiples of the median (MoMs) were established. The quantity of hPL in DS screening was estimated using Monte Carlo simulation methods., Results: The mean log10 MoM hPL was - 0.1995 (SD: 0.1993) in affected and 0.0026 (SD: 0.2129) in control pregnancies. This corresponds to a MoM of 0.63 in DS pregnancies. hPL correlated significantly with log10 MoM values of hCGbeta (r = 0.320) and PAPP-A (r = 0.590) in controls, but not with hCGbeta (r = 0.228) or PAPP-A (r = 0.090) in DS pregnancies. The inclusion of hPL in the double test (PAPP-A + hCGbeta) increased the detection rate from 67 to 75% for a false-positive rate of 5%., Conclusion: hPL is a DS screening marker that is applicable at weeks 9-13 and could be included in multiple marker first-trimester screening for DS., (Copyright 2007 John Wiley & Sons, Ltd.)

Prenatal diagnosis is an option for couples at risk of having a child affected with hemoglobinopathies. Chorionic villus sampling (CVS) and cordocentesis are accurate but a finite risk of fetal loss exists. A non invasive, risk free strategy that has emerged is isolation of fetal erythroblasts from maternal blood. Enrichment of nucleated red blood cells (nRBCs) from 7.0 mL maternal blood was done using a Percoll discontinuous density gradient and isolation by flow sorting using a combination of three monoclonal antibodies: CD45 per CP, glycophorin A-phycoerythrin (PE) and Hb F-fluorescein isothiocyanate (FITC) in 43 cases between 7 and 21 weeks' gestation. The percentage of nRBCs ranged from 0.0001-2.03%. The presence of dual fluorescence (glycophorin A-PE and Hb F-FITC) was confirmed by confocal microscopy. A sufficient number of nRBCs could be isolated in the first and second trimester of pregnancy to provide a simple flow cytometric approach as a potential for non invasive diagnosis of beta-globin defects.

The present study aimed at differentiating rare types of heterozygous beta-thalassemia (thal) with normal Hb A(2) values from alpha-thal in Iranian carriers by globin chain synthesis in addition to other hematological parameters. Our study groups consisted of 51 normal subjects, 24 heterozygous beta- thalassemic subjects with high Hb A(2), 62 alpha-thal-2 subjects, 34 alpha-thal-1 subjects, six Hb H disease thalassemic subjects, 14 silent beta-thal subjects with normal Hb A(2) values, five deltabeta-thal subjects and two subjects with an association of alpha- and deltabeta-thal (total = 198). Analysis of globin chains was performed by high performance liquid chromatography (HPLC). The results showed that the alpha/beta ratio averages were close to the ones in the published literature, but with a greater standard deviation and a wider range. Globin chain synthesis (GCS) could be valuable in differentiating between microcytosis produced by silent beta-thal (heterozygous beta-thal with a normal Hb A(2) level) and that caused by alpha-thal. Since the complex genotype/phenotype relationship can lead to diagnostic difficulties, GCS cannot be used as the only diagnostic tool for thalassemia carrier detection. Therefore, a combination of different tests for each patient is required.