Generation of RNA sequencing libraries for transcriptome analysis of globin-rich tissues of the domestic dog.

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.

Prognostic impact of pretreatment albumin to globulin ratio in patients with diffuse large B-cell lymphoma treated with R-CHOP.

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.

Determination of N-(2-hydroxyethyl)valine in globin of ethylene oxide-exposed workers using total acidic hydrolysis and HPLC-ESI-MS2.

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-MS2 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 (R2 > 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.

Stagnation of histopathological improvement is a predictor of hepatocellular carcinoma development after hepatitis C virus eradication.

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.

The influence of the Cys46/Cys55 disulfide bond on the redox and spectroscopic properties of human neuroglobin.

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.

A comparison of bleeding efficiency, microbiological quality and lipid oxidation in goats subjected to conscious halal slaughter and slaughter following minimal anesthesia.

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.

Heat-passing framework for robust interpretation of data in networks.

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.

ERß-dependent neuroglobin up-regulation impairs 17ß-estradiol-induced apoptosis in DLD-1 colon cancer cells upon oxidative stress injury.

Besides other mechanism(s) 17ß-estradiol (E2) facilitates neuronal survival by increasing, via estrogen receptor ß (ERß), the levels of neuroglobin (NGB) an anti-apoptotic protein. In contrast, E2 could exert protective effects in cancer cells by activating apoptosis when the ERß 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 ERß 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 ERß and HeLa cells transiently transfected with ERß 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 ERß-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 ERß-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.

Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused-silica capillary.

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 ß 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.

Neuroglobin and cytoglobin expression in the human brain.

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.

Resonance micro-Raman investigations of the rat medial preoptic nucleus: effects of a low-iron diet on the neuroglobin content.

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.

Human carotid body HIF and NGB expression during human development and aging.

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.

[Changes of neuroglobin in the pathologic process of contusion and laceration of brain in children].

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.

Fecal immunochemical tests compared with guaiac fecal occult blood tests for population-based colorectal cancer screening.

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.

[A study on gene mutation spectrums of α- and ß-thalassemias in populations of Yunnan Province and the prenatal gene diagnosis].

OBJECTIVE: To investigate mutation spectrums of α- and ß-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 ß-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 ß-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_301-23 indel), was found in one carrier subject. (2) In 1077 cases of haemological screen positive subjects, deletions and mutations of ß-haemoglobin gene were tested in 297 subjects among 730 cases suspected as ß-thalassemia patients and carriers. Sixteen kinds of ß-haemoglobin gene mutations were found, including 7 cases of rare abnormal haemoglobinopathy patients with ß-haemoglobin gene mutations. In one case with ß(+) phenotype patient, the Codon 5(-CT) mutation at ß-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 ß-thalassemia, one case of ß-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 ß-haemoglobin genes of thalassemia patients and carriers in Yunnan province are special, in which ß-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.

Improvements in monitoring the N-terminal valine adduct in human globin after exposure to sulfur mustard and synthesis of reference chemicals.

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.

Elevated levels of redox regulators, membrane-bound globin chains, and cytoskeletal protein fragments in hereditary spherocytosis erythrocyte proteome.

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 ß-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.

Structural characterization of spectroscopic substates in carbonmonoxy neuroglobin.

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.