Hemoglobin Subunit Beta Research Articles

A novel association between hemoglobin subunit beta gene and reproductive performance in Awassi ewes

Abstract This study was performed to assess the polymorphism in the HBB gene and its association with reproductive traits in Awassi ewes. Genomic DNA extracted from 124 and 100 Awassi ewes, respectively, produced single and twins. We amplified three exons in the HBB gene (exon 1, exon 2, and exon 3) to obtain the band (204 bp, 251 bp, and 222 bp) by PCR. Two genotypes were determined in 251 bp amplicon, AA and AG genotypes. Sequencing reactions detected a novel mutation in AG genotype, c.51983514 A > G, with a missense effect of p.K65R. A highly significant (P ≤ 0.01) association of p.K65R SNP with reproductive traits (especially composite reproductive traits, twinning rate, prolificacy and litter size) was reported, in which sheep having p.K65R SNP had a lower composite reproductive traits, twining rate, prolificacy and litter size than those without this SNP. This negative influence of p.K65R SNP was also confirmed by cumulative predictions performed by several in silico tools, which indicated a strongly deleterious impact of p.K65R SNP on hemoglobin. In conclusion, the interesting negative effect of p.K65R SNP has a considerable effect on reproductive performance (especially composite reproductive traits, twinning rate and prolificacy as well as litter size) in Awassi sheep. This manuscript strongly suggests the need to exclude ewes having p.K65R SNP when high values from higher reproductive performance are required.

Salivary Proteome Profile of Women during Fertile Phase of Menstrual Cycle as Characterized by Mass Spectrometry.

Objectives:Ovulation is such a critical physiological process that its noninvasive detection based on salivary constituents has several advantages in humans. Hence, the present study is proposed to identify the ovulatory-specific proteins in saliva in order to detect ovulation phase.Materials and Methods:Samples were collected from women volunteers. The procedure adopted was approved by the Institutional Human Ethical Committee (DM/2014/101/38), Bharathidasan University. The saliva samples were collected from thirty healthy female volunteers, with a prior written consent. One-way analysis of variance was used to calculate protein concentration and band intensity using SPSS 16 software (SPSS Inc., Cary, NC, USA). The salivary protein expression pattern during different phases of menstrual cycle was analyzed using gel-based high resolution-liquid chromatography-mass spectrometry/mass spectrometry and matrix-assisted laser desorption ionization-time of flight/time of flight. Further, bioinformatics tools were adopted to annotate the proteins identified at various phases of menstrual cycle.Results:As many as 530 proteins showed up in the saliva during ovulatory phase, whereas there were only 251 proteins identified during postovulatory phase. The functional annotation of salivary proteins revealed that the proteins got assigned to the class of “extracellular proteins” which are concerned with regulatory functions. The 16 unique and/or differentially expressed protein spots appeared during ovulatory phase, among which Cystatin-S, Prolactin-inducible protein, Cystatin-A, Cystatin-SN, BPI fold-containing family A member 2, Alpha-tubulin N-acetyltransferase 1, Carbonic anhydrase-6, Protein LEG1 homolog, Hemoglobin subunit beta, and Pancreatic alpha-amylase were identified.Conclusion:Total salivary proteome profile has been listed with respect to various phases of menstrual cycle. Among the protein listed, Cystatin-S offers a biomarker protein and/or indicator of ovulatory phase. However, extensive validation is required before arriving to a candidate bio-marker protein.

Investigation of proteins important for microcirculation using in vivo microdialysis after glucose provocation: a proteomic study

Insulin has metabolic and vascular effects in the human body. What mechanisms that orchestrate the effects in the microcirculation, and how the responds differ in different tissues, is however not fully understood. It is therefore of interest to search for markers in microdialysate that may be related to the microcirculation. This study aims to identify proteins related to microvascular changes in different tissue compartments after glucose provocation using in vivo microdialysis. Microdialysis was conducted in three different tissue compartments (intracutaneous, subcutaneous and intravenous) from healthy subjects. Microdialysate was collected during three time periods; recovery after catheter insertion, baseline and glucose provocation, and analyzed using proteomics. Altogether, 126 proteins were detected. Multivariate data analysis showed that the differences in protein expression levels during the three time periods, including comparison before and after glucose provocation, were most pronounced in the intracutaneous and subcutaneous compartments. Four proteins with vascular effects were identified (angiotensinogen, kininogen-1, alpha-2-HS-glycoprotein and hemoglobin subunit beta), all upregulated after glucose provocation compared to baseline in all three compartments. Glucose provocation is known to cause insulin-induced vasodilation through the nitric oxide pathway, and this study indicates that this is facilitated through the interactions of the RAS (angiotensinogen) and kallikrein-kinin (kininogen-1) systems.

Salivary proteome of aphthous stomatitis reveals the participation of vitamin metabolism, nutrients, and bacteria

There are currently no preventative options for recurrent aphthous stomatitis, and the only available treatments are palliative. This is partly due to a poor understanding of its etiopathogenesis. In this case–control study, we characterized the salivary proteome of patients with recurrent aphthous stomatitis in the presence and absence of lesions. Through mass spectrometry-based proteomics and bioinformatics tools, we identified that the presence of oral ulcers is associated with several specific biological processes, including the metabolic pathways of vitamin B9, B12, nitrogen, selenium, and the bacterium Neisseria meningitidis. These changes occurred only in the presence of clinically visible lesions, and there were no relevant differences between patients in anatomical regions unaffected by ulcers. Additionally, using western blot and ELISA assays, we verified that carbonic anhydrase 1 (CA1) and hemoglobin subunit beta (HBB) proteins are highly expressed during the ulcerative and remission phases of recurrent aphthous stomatitis. Our results cumulatively support saliva as an indicator of the pathophysiological changes, which occur during the clinical course of lesions. From a clinical perspective, we suggest that recurrent aphthous stomatitis is a condition triggered by temporary biological changes in people with lesions.

Label-free proteomics of spermatozoa identifies candidate protein markers of idiopathic recurrent pregnancy loss

Recurrent pregnancy loss (RPL) affects a large number of couples worldwide, increasing their mental and financial burdens. While female factors that contribute to RPL have been studied extensively, the role of male factors is largely unknown, and approximately 40 % RPL cases remain unexplained despite thorough clinical examinations. These cases are clinically termed as idiopathic RPL (iRPL). Several studies have recently found that spermatozoa play an important role, beyond fertilization, in iRPL, specifically in early embryonic development. Consequently, scientists explored spermatozoa to understand iRPL and revealed that both oxidative stress and DNA fragmentation contribute to RPL. In this study, we analyzed sperm samples from male partners of iRPL patients and fertile men who recently fathered a child by LC–MS/MS to identify proteomic markers of iRPL. A total of 1,988 proteins were quantified by a label-free method, and stringent statistical analysis was performed for the selection of candidate biomarkers of iRPL. Out of 1,647 proteins quantified, only 7 proteins qualified the selection criteria, which are lactotransferrin, ATP synthase subunit beta mitochondrial, fatty acid synthase, anterior gradient protein 2 homolog, hemoglobin subunit beta, short-chain specific acyl-CoA dehydrogenase mitochondrial, cytoplasmic dynein 1 heavy chain, and 14-3-3 protein sigma. We then performed gene annotations, pathways, and network analyses to gain more biological insights, identifying an association between oxidative stress and iRPL.

Proteomic analysis reveals some common proteins in the kidney stone matrix.

BackgroundProteins are the most abundant component of kidney stone matrices and their presence may reflect the process of the stone’s formation. Many studies have explored the proteomics of urinary stones and crystals. We sought to comprehensively identify the proteins found in kidney stones and to identify new, reliable biomolecules for use in nephrolithiasis research.MethodsWe conducted bioinformatics research in November 2020 on the proteomics of urinary stones and crystals. We used the ClusterProfiler R package to transform proteins into their corresponding genes and Ensembl IDs. In each study we located where proteomic results intersected to determine the 20 most frequently identified stone matrix proteins. We used the Human Protein Atlas to obtain the biological information of the 20 proteins and conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis to explore their biological functions. We also performed immunohistochemistry to detect the expression of the top five stone matrix proteins in renal tissue.ResultsWe included 19 relevant studies for analysis. We then identified 1,409 proteins in the stone matrix after the duplicates were removed. The 20 most-commonly identified stone matrix proteins were: S100A8, S100A9, uromodulin, albumin, osteopontin, lactotransferrin, vitamin K-dependent protein Z, prothrombin, hemoglobin subunit beta, myeloperoxidase, mannan-binding lectin serine protease 2, lysozyme C, complement C3, serum amyloid P-component, cathepsin G, vitronectin, apolipoprotein A-1, eosinophil cationic protein, fibrinogen alpha chain, and apolipoprotein D. GO and KEGG analysis revealed that these proteins were typically engaged in inflammation and immune response.Immunohistochemistry of the top five stone matrix proteins in renal tissue showed that the expression of S100A8, S100A9, and osteopontin increased, while uromodulin decreased in kidney stone patients. Albumin was rarely expressed in the kidney with no significant difference between healthy controls and kidney stone patients.ConclusionProteomic analysis revealed some common inflammation-related proteins in the kidney stone matrix. The role of these proteins in stone formation should be explored for their potential use as diagnostic biomarkers and therapeutic targets for urolithiasis.

Developmental validation of a multiplex proteomic assay for the identification of forensically relevant biological fluids

The aim of this study was to validate a multiplex proteomic assay for the identification of high-specificity protein biomarkers by multiple reaction monitoring mass spectrometry on a triple quadrupole mass spectrometer for the accurate, reliable, and confirmatory identification of bodily fluids commonly encountered in a forensic context. This includes the identification of peripheral blood, semen, saliva, urine, and vaginal/menstrual fluid. The assay is able to efficiently identify pure or mixed stains through the identification of target peptide fragments originating from tissue-specific proteins including: uromodulin from urine; prostatic acid phosphatase, prostate specific antigen and semenogelin-II for semen; statherin, submaxillary gland androgen-regulated protein 3B and amylase for saliva; cornulin, martrigel-induced gene C4 protein, suprabasin and neutrophil gelatinase-associated lipocalin for vaginal/menstrual fluid; and alpha-1 antitrypsin, hemopexin, and hemoglobin subunit beta for peripheral blood. Based on the results of the developmental validation studies which included an assessment of reproducibility and repeatability, sensitivity, species specificity, carryover, mixtures, as well as a series of casework type samples. Only a small selection of case samples was unable to unambiguously identify the target fluid including urine recovered from substrates as well as semen when mixed with personal lubricants. Overall, the mass spectrometry-based workflow offers significant advantages compared to existing serological methods.

Proteomic analysis of lacrimal gland adenoid cystic carcinoma with high-grade transformation

Objective: To analyze the protein expression differences of lacrimal gland adenoid cystic carcinoma (LACC) with high-grade transformation (HGT). Methods: Experimental study. A total of 8 paraffin tissue samples were collected in Tianjin Medical University Eye Hospital from December 2012 to January 2019. According to pathological examination, the samples were divided into the LACC group and the LACC-HGT group, with 4 cases in each group. The LACC group included 2 male samples and 2 female samples, with an average age of 53 years. The LACC-HGT group included 2 male samples and 2 female samples, with an average age of 44 years. Primary cells were cultured from fresh tumor tissue. Isobaric tags for relative and absolute quantification techniques were used to screen the differentially expressed proteins between the two groups, and bioinformatics analysis was conducted for the differentially expressed proteins. Microarray was used to screen differentially expressed mRNAs between LACC and LACC-HGT primary cells. The mass spectrum data were intersected with mRNA microarray data, and quantitative real-time (qRT) PCR was performed to verify the results. Proteomics and microarray data were compared using the independent sample t test. The qRT-PCR data were compared pairwise by one-way analysis of variance. Results: A total of 105 HGT-related differential proteins were detected in this study, including 50 up-regulated proteins and 55 down-regulated proteins. The significantly up-regulated proteins included hemoglobin subunit beta, hemoglobin subunit alpha 1, and collagen type Ⅵ alpha 2 chain; the significantly down-regulated proteins included Cereblon, adenosylhomocysteinase like 2, and ribosomal protein L39 pseudogene 5. Gene ontology analysis results showed that the LACC-HGT differential proteins were mainly located in the cytoplasm, vesicle cavity, and extracellular matrix, had organic acid binding and molecular carrier activity, and participated in the regulation of extracellular matrix composition, immunity, inflammation, apoptosis, and other biological processes. Pathway analysis showed that the LACC-HGT differential proteins were mainly involved in signal pathways such as mitogen-activated protein kinase signal pathway and extracellular matrix proteoglycans and glycan metabolism signal pathway. Protein complex prediction analysis screened out 4 up-regulated protein complexes and 1 down-regulated protein complex. There were 15 LACC-HGT differential proteins that overlapped with mRNA chip differential genes, of which 6 were tumor-related proteins including collagen type XIV alpha 1 chain (COL14A1), EMAP like 4 (EML4), inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), NDRG family member 2 (NDRG2), osteoglycin (OGN) an Ras homolog family member C (RhoC). The main function was the movement and migration of tumor cells. The qRT-PCR results showed that the relative expression levels of COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC in primary LACC-1, LACC-2, LACC-HGT-1, and LACC-HGT-2 cells were significantly different (F=1 675.98, 38.53, 27.37, 16.47, 13.38, 25.22, all P<0.01). For example, the relative expression of COL14A1 in primary LACC-HGT-1 (16.09±0.51) and LACC-HGT-2 (9.96±0.34) cells was significantly higher than that in primary LACC-1 (1.00±0.13) and LACC-2 (0.67±0.08) cells (all P<0.05). Conclusion: There are differentially expressed proteins between LACC-HGT and LACC, among which COL14A1, EML4, ITIH4, NDRG2, OGN, and RhoC may play an important role in LACC-HGT and can be used as potential targets of LACC-HGT in further study. (Chin J Ophthalmol, 2021, 57: 531-539).

Hemoglobin subunit beta interacts with the capsid, RdRp and VPg proteins, and antagonizes the replication of rabbit hemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) causes a highly contagious disease in rabbits that is associated with high mortality. Because of the lack of a suitable cell culture system for RHDV, its pathogenic mechanism and replication remain unclear. This study found that the expression level of host protein rabbit hemoglobin subunit beta (HBB) was significantly downregulated in RHDV-infected cells. To investigate the role of HBB in RHDV replication, small interfering RNAs for HBB and HBB eukaryotic expression plasmids were used to change the expression level of HBB in RK-13 cells and the results showed that the RHDV replication level was negatively correlated with the expression level of HBB. It was also verified that HBB inhibited RHDV replication using constructed HBB stable overexpression cell lines and HBB knockout cell lines. The interaction of HBB with viral capsid protein VP60, replicase RdRp, and VPg protein was confirmed, as was the activation of the expression of interferon γ by HBB. The results of this study indicated that HBB may be an important host protein in host resistance to RHDV infection.

Proteomic Analysis of the Vitreous Body in Proliferative and Non-Proliferative Diabetic Retinopathy

Background: Diabetic Retinopathy (DR), one of the major microvascular complications commonly occurring in diabetic patients, can be classified into Proliferative Diabetic Retinopathy (PDR) and Non-Proliferative Diabetic Retinopathy (NPDR). Currently available therapies are only targeted for later stages of the disease in which some pathologic changes may be irreversible. Thus, there is a need to develop new treatment options for earlier stages of DR through revealing pathological mechanisms of PDR and NPDR. Objective: The purpose of this study was to characterize proteomes of diabetic through quantitative analysis of PDR and NPDR. Methods: Vitreous body was collected from three groups: control (non-diabetes mellitus), NPDR, and PDR. Vitreous proteins were digested to peptide mixtures and analyzed using LC-MS/MS. MaxQuant was used to search against the database and statistical analyses were performed using Perseus. Gene ontology analysis, related-disease identification, and protein-protein interaction were performed using the differential expressed proteins. Results: Twenty proteins were identified as critical in PDR and NPDR. The NPDR group showed different expressions of kininogen-1, serotransferrin, ribonuclease pancreatic, osteopontin, keratin type II cytoskeletal 2 epidermal, and transthyretin. Also, prothrombin, signal transducer and activator of transcription 4, hemoglobin subunit alpha, beta, and delta were particularly up-regulated proteins for PDR group. The up-regulated proteins related to complement and coagulation cascades. Statherin was down-regulated in PDR and NPDR compared with the control group. Transthyretin was the unique protein that increased its abundance in NPDR compared with the PDR and control group. Conclusion: This study confirmed the different expressions of some proteins in PDR and NPDR. Additionally, we revealed uniquely expressed proteins of PDR and NPDR, which would be differential biomarkers: prothrombin, alpha-2-HS-glycoprotein, hemoglobin subunit alpha, beta, and transthyretin.

Haematological abnormalities in children with sickle cell disease and non-severe malaria infection in western Kenya

BackgroundIn Plasmodium falciparum infection, clinical conditions such as anaemia, thrombocytopenia and leukocytosis are common. Mutation in haemoglobin sub-unit beta gene (HBB) may be a genetic factor responsible for these haematological changes during infection. However, the contributions of the carriage of different HBB genotypes on these changes remain largely unknown.MethodologyIn this cross-sectional study, we evaluated haematological abnormalities in P. falciparum-infected children (n = 217, aged 1–192 months) with different haemoglobin sub-unit beta (HBB) genotypes (HbAA, HbAS and HbSS). Children with acute febrile conditions were recruited at Jaramogi Oginga Odinga Teaching and Referral Hospital at the outpatient clinic. Haematological parameters were determined using Beckman Coulter counter ACTdiff2™ while HBB genotyping was done using TaqMan® SNP genotyping assay. Chi-square (χ2) was used to determine differences between proportions. Differences in haematological parameters were compared across groups using Kruskal Wallis test and between groups using Mann Whitney U test. Partial correlation test was used to determine correlation between haematological parameters and sickle cell genotypes while controlling for age and sex.ResultsHaemoglobin (Hb), [median (IQR); 7.3 (1.3), P = 0.001], haematocrit (HCT), [median (IQR); 26.4 (4.4), P = 0.009], red blood cells (RBC), [median (IQR); 3.2 (1.7), P = 0.048] were markedly reduced in HbSS, however, red cell distribution with (RDW) [median (IQR); 14.9 (3.3), P = 0.030] was increased in malaria infected children with HbSS. Severe anaemia was highest in HbSS (23.1%) followed by HbAA (8.6%) and HbAS (7.1%). There were no differences in platelet count (P = 0.399) hence no severe thrombocytopeania across the genotypes. Leukocytosis was highest in HbSS (69.2%), 42% in HbAS and 31% in HbAA. The RBC, HCT and Hb had negative correlation with RDW in HbSS in malarial-infected children (r = − 0.725, P = 0.008), (r = − 0.718, P = 0.009) and (r = − 0.792, P = 0.002), respectively.ConclusionOur study reveals that anaemia is the most common abnormality in malaria-infected children with carriage of HbSS. The RBC, HCT and Hb concentration decrease with increase in RDW levels in infected children with carriage of HbSS compared to other HBB genotypes. Therefore, carriage of HbSS genotype is correlated with severity of haematological abnormalities.

Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1

β-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2β2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat β-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in β-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in β-thalassemia.

HBB gene mutation spectrum in an Indian cohort of 1530 cases using an in-house targeted next-generation sequencing assay

Beta thalassemia major is a common genetic disorder characterized by the reduced production or absence of beta globin, a product of the haemoglobin subunit beta (HBB) gene. Every year, approximately 10,000–12,000 children with thalassemia major are born in India. Molecular methodologies like ARMS (amplification-refractory mutation system)-PCR (polymerase chain reaction) and capillary sequencing are used to detect HBB gene mutations. It is, however, challenging to achieve comprehensive coverage of the HBB gene by these methods. Next-generation sequencing (NGS) can be used to circumvent these problems. Commercial NGS panels are prohibitively expensive and hence are not routinely implemented in most laboratories. We have developed a cost-effective, highly sensitive and specific, indigenous targeted NGS assay for detecting mutations in the HBB gene. Using this custom NGS assay, we processed 1530 samples (3017 alleles), in which we detected a spectrum of 48 pathogenic/likely pathogenic variants (mutations); IVS-I-5 (c.92+5G>C) was the most common mutation detected (allele frequency (AF) 44.55%), followed by the 619-bp deletion (AF 10.74%), c.92+1G>T (AF 6.99%), c.27_28insG (AF 6.23%), c.47G>A (AF 5.77%) and c.126_129delCTTT (AF 4.71%). Additionally, we discovered a novel mutation (c.7delC) that was submitted to the HbVar database (HbVar ID 3193) as a variant of unknown significance (VUS), probably pathogenic. The targeted NGS assay developed during this study was validated using orthogonal methods and showed excellent correlation with currently available molecular methods. Additionally, this targeted NGS assay was used to analyse the mutation spectrum of the largest beta thalassemia cohort from India.

Computational and drug target analysis of functional single nucleotide polymorphisms associated with Haemoglobin Subunit Beta (HBB) gene

There is overwhelming evidence implicating Haemoglobin Subunit Beta (HBB) protein in the onset of beta thalassaemia. In this study for the first time, we used a combined SNP informatics and computer algorithms such as Neural network, Bayesian network, and Support Vector Machine to identify deleterious non-synonymous Single Nucleotide Polymorphisms (nsSNPs) present in the HBB gene. Our findings highlight three major mutation points (R31G, W38S, and Q128P) within the HBB gene sequence that have significant statistical and computational associations with the onset of beta thalassaemia. The dynamic simulation study revealed that R31G, W38S, and Q128P elicited high structural perturbation and instability, however, the wild type protein was considerably stable. Ten compounds with therapeutic potential against HBB were also predicted by structure-based virtual screening. Interestingly, the instability caused by the mutations was reversed upon binding to a ligand. This study has been able to predict potential deleterious mutants that can be further explored in the understanding of the pathological basis of beta thalassaemia and the design of tailored inhibitors.

Assessment of Some Immune Components from The Bioactive Crude Extract Derived from The Epidermal Mucus of Climbing Perch Anabas Testudines

Fish and other members of the aquatic habitats are usually in constant interaction with their habitat and potentially harmful pathogens. This study was undertaken to evaluate some of the immune components in the bioactive crude obtained from epidermal mucus of climbing perch. The enzyme activities were assessed using the spectrophotometer and turbidometric assay for the lysozyme activity, and while antimicrobial peptides were determined via AKTA Purifier 10 FPLC with a Superdex™ peptide 10/300 GL column and Q-TOF LC/MS for characterization. Likewise, the antibacterial activity against P. aeruginosa ATCC 10451 was achieved using chromatography fractions. Results showed that higher activities of lysozyme and protease were present in the crude extract. While values for alkaline phosphatase and esterase activities varied. The data analysis of Q-TOF LC/MS showed apolipoprotein A1, haemoglobin subunit beta (Fragment), elongation factor 1-alpha, cytochrome C oxidase subunit1 (Fragment), Beta-actin (Fragment) and potent antimicrobial peptides including de novo AAGPKGPLGPR peptide in crude. This result emphasizes their role in protecting to climbing perch from foreign invaders as a part of the innate immune system throughout the epidermal mucus. The bioactive crude has a high potential to be used as a natural antimicrobial agent for pharmaceutical applications.

LABEL-FREE QUANTITATIVE PROTEOMICS REVEALS TIME-SERIES PROTEIN SIGNATURE OF FIRST 24 HOURS AFTER CLINICAL HEART TRANSPLANTATION

Purpose: Cardiac allografts are subjected to ischemic preservation before transplantation, and subsequent ischemia-reperfusion injury (IRI) after the surgery. IRI increases the risk for acute rejection development, and more detailed detection methods for early allograft injury and rejection are therefore warranted. Systemic molecular phenotyping by proteomics is a novel method for investigating the underlaying pathophysiology. Plasma protein expressions reflect the ongoing clinical state of the patients, therefore enabling real-time screening of biomarkers for allograft injury or treatment efficacy.Here, we systemically analyze the proteomic signature of recipients within first 24h after transplantation. Methods: This study is a post hoc analysis of data collected from 50 heart transplant recipients that participated in a separate clinical trial. Plasma samples from 1 and 24 hours after transplantation were analyzed with quantitative label-free proteomics in high definition MSE mode (HDMSE). Results: With two or more unique proteins per identification, 463 proteins were quantified. A complete separation between 1h and 24h based on a set of 270 identified proteins (p-value <0.05) was seen in supervised and unsupervised classification techniques, such as orthogonal projections on latent structure-discriminant analysis (OPLS-DA) and self-organizing maps. Identified proteins were studied further by pathway, network, and protein-protein interaction analyses. Using OPLS-DA approach, we obtained a set of 13 S-plot proteins (Hemoglobin subunit beta, Hemoglobin subunit alpha, Keratin type I cytoskeletal 9, Malate dehydrogenase, Electron transfer flavoprotein beta subunit lysine methyltransferase, Zona pellucida-binding protein 1, Carbonic anhydrase 1, 14-3-3 protein epsilon, Rootletin, C-reactive protein, Serum amyloid A-1 protein, Leucine-rich alpha-2 glycoprotein and Alpha-1-antichymotrypsin) that most significantly separated recipients at 1h from 24h. Conclusion: We show the change in proteomic signature of the first 24h after heart transplantation in human blood samples with open-label proteomics. We will further compare the proteomic data to clinical parameters in order to search for prognostic factors for rejection and mortality.

Very preterm birth results in later lower platelet activation markers.

Premature birth entails an adverse cardiovascular risk profile, but the underlying mechanisms are insufficiently understood. Here, we employed an unbiased cardiovascular proteomics approach to profile former very preterm-born preschoolers. This observational study investigated differences in plasma concentrations of 79 proteins, including putative cardiovascular biomarkers between very preterm- and term-born children on average 5.5 years old (53.1% male) using multiple-reaction monitoring mass spectrometry. Very preterm-born (n = 38; median gestational age 29.6 weeks) compared to term-born (n = 26; 40.2 weeks) children featured lower plasma concentrations of platelet factor 4 (PLF4; -61.6%, P < 0.0001), platelet basic protein (CXCL7; -57.8%, P < 0.0001), and hemoglobin subunit beta (-48.3%, P < 0.0001). Results remained virtually unchanged when adjusting for complete blood count parameters, including platelet count. Conversely, whole blood hemoglobin was higher (+7.62%, P < 0.0001) in preterm-born children. Very preterm birth was associated with decreased markers of platelet activation among preschoolers. These findings are consistent with reduced platelet reactivity persisting from very preterm birth to a preschool age. Former very preterm-born preschoolers featured reduced levels of platelet activation markers. While lower platelet reactivity in very preterm-born compared to term-born infants in the first days of life was established, it was unknown when, if at all, reactivity normalizes. The current study suggests that platelet hyporeactivity due to very preterm birth persists at least up to a preschool age. "Immaturity of the hemostatic system" may be a persistent sequel of preterm birth, but larger studies are needed to investigate its potential clinical implications.

Hemoglobin S/Hemoglobin Quebec-Chori Presenting as Sickle Cell Disease: A Case Report

Sickle cell disease is a homozygous hemoglobinopathy with vaso-occlusive complications secondary to abnormal sickling of red blood cells under stressful conditions such as hypoxia. Children with sickle cell trait have a heterozygous genetic state, typically without symptoms. An 8-year-old boy diagnosed with sickle cell trait was found to have multiple complications consistent with sickle cell disease, including pain crises, osteomyelitis, and priapism. Over a 6-year period, he underwent routine laboratory evaluations without a definitive diagnosis. The diagnosis of a compound heterozygous state of hemoglobin S/hemoglobin Quebec-Chori was eventually made on the basis of mass spectrometry and confirmed with hemoglobin subunit beta gene sequencing. Expanding diagnostic evaluation in patients with abnormal clinical presentations is vital to making the correct diagnosis and hence earlier institution of appropriate management of rare hemoglobinopathies.

SAT-035 In Vitro Effect of Different Follicle-Stimulating Hormone Preparations on Sertoli Cells

Follicle-stimulating hormone (FSH) is the main regulator of spermatogenesis and plays a key role in the development and function of the reproductive system. To assess the effects of different FSH preparations in combination with testosterone on porcine pre-pubertal Sertoli cells, we performed Real Time PCR analysis of AMH, inhibin B and FSH-r, Western blotting analysis of AKT-posphoAKT, ERK1/2-posphoERK1/2, ELISA assay for AMH and inhibin B and a high-throughput proteomic analysis.We observed that all three preparations induced a reduction of AMH in terms of mRNA and secreted protein and, an increase of inhibin B in terms of mRNA in all the formulations while, only α-follitropin induced an increase of inhibin B secreted in the culture medium. Proteomic analysis permitted us to identify 46 secreted proteins.Of those, the SPARC protein was down-regulated after the treatment with testosterone associated with α-follitropin, β-follitropin and urofollitropin (vs group stimulated with T alone). 11 proteins were up-regulated by the different FSH preparations. In detail, Hemoglobin subunit beta, TPA and TPI have been observed to be up-regulated by stimulation with testosterone in addition with α-follitropin or with β-follitropin and or with urofollitropin. All preparations induced an increase in the secreted inhibin beta A chain, but in the medium after stimulation with urofollitropin we observed an higher increase in the levels of this protein. β-follitropin, associated with testosterone, specifically induces an up-regulation of 8 specific secreted proteins.Our study, showing that the three FSH preparations were associated with different effects, could offer the opportunity to shed light inside applications to personalized reproductive medicine.

Peptide analysis of mammalian decomposition fluid in relation to the post-mortem interval

We report the results of a semi-quantitative peptide analysis of decomposition fluid under field-based conditions in the absence of a soil matrix. Sixteen domestic pig (Sus scrofa domesticus) cadavers were used to model human decomposition in trials conducted in the summer and winter months in Western Australia. Physical characteristics were recorded and targeted peptide components of decomposition fluid were analysed using high performance liquid chromatography-triple quadrupole mass spectrometry. Principal component analysis identified 29 peptides, originating from haemoglobin subunits alpha and beta, creatine kinase, beta-enolase and lactate dehydrogenase, that contributed to differences in the mean peak areas of samples collected during the early period of decomposition (days 6–12 and day 2 in winter and summer, respectively) and during the later period (days 24–34 and days 8–10 in winter and summer, respectively). Fold changes for 8 peptides between these periods were significantly different. Three peptides derived from haemoglobin subunit beta, one from beta-enolase and two from lactate dehydrogenase displayed consistent trends, in that a notable increase in mean peak area was followed by a marked decrease in both the summer and winter samples. When temperature was accounted for, these trends occurred at different time points in summer and winter, indicating that factors other than temperature had impacted the rate of degradation of the proteins involved. The single peptides derived from haemoglobin subunit alpha and creatine kinase displayed consistent increases in mean peak area for the summer samples, suggesting that temperature played the most significant role in their degradation. Further analyses revealed that 7 peptides (one originating from haemoglobin subunit alpha, three from haemoglobin subunit beta and three from lactate dehydrogenase) displayed consistent trends that could be correlated with total body score and with the early stages of decomposition. The consistent trends (mean peak area versus time) for peptides derived from several proteins during decomposition trials conducted under different temperature regimes further emphasised the potential of peptide analysis in time since death estimation.