Beta-globin Research Articles

Proteomics study the potential targets for Rifampicin-resistant spinal tuberculosis.

Introduction: The escalating global surge in Rifampicin-resistant strains poses a formidable challenge to the worldwide campaign against tuberculosis (TB), particularly in developing countries. The frequent reports of suboptimal treatment outcomes, complications, and the absence of definitive treatment guidelines for Rifampicin-resistant spinal TB (DSTB) contribute significantly to the obstacles in its effective management. Consequently, there is an urgent need for innovative and efficacious drugs to address Rifampicin-resistant spinal tuberculosis, minimizing the duration of therapy sessions. This study aims to investigate potential targets for DSTB through comprehensive proteomic and pharmaco-transcriptomic analyses. Methods: Mass spectrometry-based proteomics analysis was employed to validate potential DSTB-related targets. PPI analysis confirmed by Immunohistochemistry (IHC) and Western blot analysis. Results: The proteomics analysis revealed 373 differentially expressed proteins (DEPs), with 137 upregulated and 236 downregulated proteins. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses delved into the DSTB-related pathways associated with these DEPs. In the context of network pharmacology analysis, five key targets-human leukocyte antigen A chain (HLAA), human leukocyte antigen C chain (HLA-C), HLA Class II Histocompatibility Antigen, DRB1 Beta Chain (HLA-DRB1), metalloproteinase 9 (MMP9), and Phospholipase C-like 1 (PLCL1)-were identified as pivotal players in pathways such as "Antigen processing and presentation" and "Phagosome," which are crucially enriched in DSTB. Moreover, pharmaco-transcriptomic analysis can confirm that 58 drug compounds can regulate the expression of the key targets. Discussion: This research confirms the presence of protein alterations during the Rifampicin-resistant process in DSTB patients, offering novel insights into the molecular mechanisms underpinning DSTB. The findings suggest a promising avenue for the development of targeted drugs to enhance the management of Rifampicin-resistant spinal tuberculosis.

Fibrinogen and Atherosclerosis

Cardiovascular disease has a multifactorial origin. Among these risk markers, fibrinogen is the most prominent. Interest in the role of fibrinogen in the atherogenic process is long-standing. In the 1960s, one of the first studies correlating fibrinogen and coronary artery disease was published. Since then, much evidence has been accumulating establishing elevated plasma levels of fibrinogen as an independent risk factor for coronary artery disease. As in acute myocardial infarction, fibrinogen is also elevated in cases of cerebrovascular accident and peripheral artery disease. It is noteworthy that fibrinogen levels are elevated in patients with transient ischemic attacks, suggesting that fibrinogen levels should be elevated before necrosis occurs. It is known that plasma fibrinogen is a dimer made up of alpha, beta and gamma chains, whose union is made through disulfide bonds. Measures to reduce plasma fibrinogen include diet, alcohol, physical activity, and drugs. Since fibrinogen is a risk factor for coronary artery disease, measures to reduce its levels should be adopted. In the future, with the evolution of genetic studies, the detection of individuals predisposed to hyperfibrinogenemia may be performed. It is up to pharmaceutical research to continue the investigation of effective and easy-to-use drugs to reduce fibrinogen levels.

Study of the types of mutations of Thalassemia in Shanghai area

To analyze the mutations of globin genes among patients suspected for thalassemia from the Shanghai area. A total of 4 644 patients diagnosed at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine between June 2016 and December 2019 were selected as the study subjects. The patients were tested for common mutations associated with thalassemia gene by Gap-PCR and reverse dot blotting (RDB). Patients were suspected to harbor rare mutations based on the inconsistency between hematological phenotypes and results of common mutation detection, and were further analyzed by Gap-PCR and Sanger sequencing. Among the 4 644 patients, 2 194 (47.24%) were found to carry common thalassemia mutations, among which 701 (15.09%) were α-thalassemia, 1 448 (31.18%) were β-thalassemia, and 45 (0.97%) were both α- and β-thalassemia. Forty six samples were found to harbor rare mutations, which included 17 α-globin gene and 29 β-globin gene mutations. CD77(CCC>ACC) (HBA2: c.232C>A) of the α-globin gene, NG_000007.3: g.70567_71015del449, codon 102(-A) (HBB: c.308_308delA) and IVS-Ⅱ-636 (A>G) (HBB: c.316-215A>G) of the β-globin gene were previously unreported new types of globin gene mutations. Among the 4 644 patients, the detection rate for common thalassemia mutations was 47.24%, whilst 46 samples were detected with rare gene mutations. The type of gene mutation types were diverse in the Shanghai area. The study has provided more accurate results for genetic diagnosis and counseling.

Distinct Distribution of HBB Variants in Two Cohorts of Beta Thalassemia Patients, and a Novel Variant from Turkey

Introduction: Beta thalassemia is a serious disease for which mutation-based diagnostic and screening tests are readily available. These tests are based on specific variant profile in the regions of the testing centers. De novo mutations and migration change the distribution of these variants. We aim to update the variant spectrum in the HBB gene in our region. In addition, we present a variant, which not been detected before in Turkey, and also a changed classification of another variant. Methods: This study includes 142 patients (46 of Turkish, 96 of Syrian) who were investigated for defects in their β-globin gene with Sanger sequencing. Clinically, 52 of these patients had thalassemia major, and 90 had thalassemia minor. Results: Twenty three types of pathogenic variants were identified causing beta thalassemia and abnormal hemoglobins. Variant distribution has differed considerably between Turkish and Syrian patients. While the IVSI-110G>A was the most prevalent variant (41.1%) in Turkish patients, the IVSII-1G>A and Codon 39 (C>T) variants were found in 22% and 21.3%, respectively, in Syrian patients. We detected the novel c.31_32insT variant in 3 Syrian patients. Conclusion: The detection of updated regional HBB variant spectrum will contribute to future prenatal and/or postnatal molecular diagnostic tests. Also, our study presents a novel variant that was not previously reported.

Characterization of the plasma proteome from healthy adult dogs.

Bloodwork is a widely used diagnostic tool in veterinary medicine, as diagnosis and therapeutic interventions often rely on blood biomarkers. However, biomarkers available in veterinary medicine often lack sensitivity or specificity. Mass spectrometry-based proteomics technology has been extensively used in the analysis of biological fluids. It offers excellent potential for a more comprehensive characterization of the plasma proteome in veterinary medicine. In this study, we aimed to identify and quantify plasma proteins in a cohort of healthy dogs and compare two techniques for depleting high-abundance plasma proteins to enable the detection of lower-abundance proteins via label-free quantification liquid chromatography-mass spectrometry. We utilized surplus lithium-heparin plasma from 30 healthy dogs, subdivided into five groups of pooled plasma from 6 randomly selected individuals each. Firstly, we used a commercial kit to deplete high-abundance plasma proteins. Secondly, we employed an in-house method to remove albumin using Blue-Sepharose. Among all the samples, some of the most abundant proteins identified were apolipoprotein A and B, albumin, alpha-2-macroglobulin, fibrinogen beta chain, fibronectin, complement C3, serotransferrin, and coagulation factor V. However, neither of the depletion techniques achieved significant depletion of highly abundant proteins. Despite this limitation, we could detect and quantify many clinically relevant proteins. Determining the healthy canine proteome is a crucial first step in establishing a reference proteome for canine plasma. After enrichment, this reference proteome can later be utilized to identify protein markers associated with different diseases, thereby contributing to the diagnosis and prognosis of various pathologies.

An Immunological and Molecular Study to Investigate the Genes (β-globin and HBA1F) in Patients with Thalassemia in Najaf Governorate

Abstract BACKGROUND: Thalassemia, a hereditary blood disorder, poses a significant health challenge globally. This study delves into the prevalence and characteristics of thalassemia, emphasizing the need for enhanced awareness and diagnostic measures, particularly in regions with high prevalence. The primary objective is to comprehensively investigate thalassemia, focusing on genetic mutations and hemoglobin (Hb) dynamics. The specific goals include identifying gene mutations through conventional polymerase chain reaction (PCR) and exploring the correlations between different Hb types. METHODS: Blood samples, totaling three milliliters for ethylenediaminetetraacetic acid tubes and 2 ml for gel tubes, were collected from thalassemia patients and a control group. Genomic DNA was extracted for conventional PCR analysis targeting α and β-globin (HBB) genes. The specific primers were designed, and PCR products were electrophoresed for the gene detection. Demographic characteristics were recorded, and Hb electrophoresis was performed to assess HbA, HbA2, and HbF levels. RESULTS: The study revealed the presence of the HBB gene in 100% of thalassemia patients causing beta-thalassemia, contrasting with the control group. The absence of the HBA1-F gene in alpha-thalassemia patients was observed. Demographic analysis showed no significant age or gender differences between the two groups. Thalassemia patients, analyzed alongside the control group, exhibit elevated HbA2 levels (2.7%–6.6%) and increased HbF levels (2%–12.1%), surpassing normal ranges (HbA2: 2%–3%, HbF: 0.8%–2%). CONCLUSION: The study confirms HBB gene association and proposes HBA1-F absence as an alpha-thalassemia diagnostic marker. Distinctive Hb patterns in thalassemia patients, highlighted by robust negative correlations (HbA-HbA2) and moderate correlations (HbA-HbF), advance diagnostic and management strategies for beta-thalassemia.

Ancestral β-globin gene haplotypes modify β-thalassemia severity in a mouse model

Ancestral β-globin gene haplotypes modify β-thalassemia severity in a mouse model

Molecular Prevalence of Human Papillomavirus Types 16 and 18 in Oral Squamous Cell Carcinoma using Real-time PCR.

Human papillomavirus(HPV) has been established as a causative agent in the development of oral squamous cell carcinoma (OSCC). Specifically, HPV types 16 and 18 are known to be prevalent in oral cancers. This cross-sectional study aimed to determine the prevalence of HPV types 16 and 18 in OSCC cases in Qazvin province, Iran. Thirty-eight paraffin-embedded samples of OSCC were selected, and DNA extraction was performed using the Roche High Pure FFPE DNA isolation kit. The quality of the extracted DNA was assessed through PCR amplification of the human β-Globin gene. The HPV detection was carried out using SYBR green-based real-time PCR with GP5+ and GP6+ primers targeting the L1 region of HPV. The HPV genotyping was conducted on positive samples using specific primers. Statistical analysis was performed between HPV infection in OSCC and age, sex, and anatomical location. This study analyzed 38 biopsy specimens obtained from male and female OSCC patients, with an average age of 64 years. Among these samples, 13 tested positive for HPV, resulting in a prevalence rate of 34.2%. The age group with the highest HPV infection rate was 61-70 (10.5%) years. Notably, HPV type 16 was detected in 21.0% of samples, HPV type 18 in 10.5%, and other viral subtypes in 2.6%. No statistically significant correlation was found between HPV prevalence and gender or age. The findings indicated that 34.2% of OSCC samples in the Qazvin province harbor HPV, with types 16 and 18 being the most common in tumors affecting the tongue. Additionally, no association was observed between HPV infection and age or gender. To address HPV as a risk factor for OSCC, public health initiatives such as vaccination, awareness campaigns, and accessible healthcare services should be implemented. They are, furthermore, incorporating HPV DNA testing into practice.

Frequency Of Beta Thalassemia Major Abo Blood Groups In Different Foundation Of District Peshawar

Background: Thalassemia major (TM) is an inherited hemolytic disorder occur due to abnormal hemoglobin production as a result of HBB (Haemoglobin Subunit Beta) gene mutation because HBB gene provides instructions for making of beta globin. The research study aim is to determine the frequency of beta thalassemia major ABO blood group in different foundation of district Peshawar. Methods: A descriptive cross-sectional study was conducted, involving 100 individuals with Thalassemia major from different foundations in Peshawar, including Hamza Foundation and Fatmid Foundation. The data was analyzed for statistical analysis using SPSS version 22.0 software. Results: The study analyzed the gender-wise distribution of Thalassemia major patients, revealing that out of the 100 individuals, 43 were female (43%) and 57 were male (57%). Moreover, the study examined the ABO blood types among the participants. The most prevalent blood type among the Thalassemia major patients was B+ (35.0%), followed by O+ (34.0%), A+ (26.0%), AB+ (4.0%), and O- (1.0%). Conclusion: Based on the results, it can be said that among those with Thalassemia major in the population under study, the B+ blood type is the most prevalent and well-known ABO blood group.

Genotype-phenotype correlation and mutation spectrum of HBB gene in the Hatay province of Turkey

Purpose: Thalassemia carriage and hemoglobinopathies are quite common disorders in Turkey, especially in the Çukurova region, including Hatay province. Due to the high case population and genetic diversity in our region, this study aimed to investigate the genotype-phenotype correlation in the HBB gene.
 Materials and Methods: The data of patients who applied to Hatay Mustafa Kemal University Medical Genetics or Hematology Clinic between January 2010 and November 2022 were evaluated retrospectively.
 Results: A total of 40 (100%) cases, comprising 25 (62.5%) homozygous and 15 (37.5%) compound heterozygous genotypes, were included in the study based on the mutation profiles in the HBB gene. In the analysis of the cases, it was seen that there were 17 different variants and 22 distinct genotypes. The three most common variants identified in this study were IVS-I-6 (T>C), IVS-I-1 (G>A), and IVS-II-848 (C>A). Of the cases with homozygous genotypes, 13 (52%) had the IVS-I-6 (T>C) variant. The most frequent genotypes observed in cases with compound heterozygous genotype were IVS-I-6 (T>C)/IVS-I-110 (G>A), IVS-I-6 (T>C)/Hb Knossos, and IVS-I-110 (G>A)/-101 C>T, each in 2 (13%) cases.
 Conclusion: This study provides information on the phenotypic characteristics of very rare genotypes. We think that this information will be very beneficial, especially for clinicians interested in prenatal diagnosis, preimplantation genetic diagnosis, and postnatal genetic counseling.

Etiologies and Outcomes of Normocytic Anemia in Children

To characterize the evaluation and outcomes of children referred to pediatric hematology for normocytic anemia. Retrospective cohort study of children aged 0 to 21 years referred to a tertiary pediatric hematology clinic for normocytic anemia from 2019 through 2021. Normocytic anemia was defined as a low hemoglobin and normal mean corpuscular volume, per the referring laboratory reference range. Two-hundred seventy-one patients (48% female, median age 5.4 years) were included. The most common hematologic diagnoses included iron deficiency (n=90, 33%), statistical anemia (n=64, 24%), transient marrow suppression (n=36, 13%), and transient erythroblastopenia of childhood (TEC, n=19, 7%). There were 17 (6%) patients in whom anemia was thought to be secondary to a non-hematologic disorder and therefore were referred to another pediatric specialty. Sixteen patients (6%) had anemia which spontaneously resolved without an underlying etiology being identified. Aside from iron deficient patients, 35 (13%) had diagnoses requiring ongoing hematology care including TEC, hemolytic anemia, Diamond Blackfan Anemia, and abnormal beta globin traits. Two-hundred-fifty-one patients (93%) were discharged from hematology care after a median of 25 days (range 0-2124 days). Pediatric patients with normocytic anemia have diverse underlying etiologies with iron deficiency being most common. These data support initial management within the primary care setting including assessment of a serum ferritin, iron panel, and reticulocyte count, with only a subset of patients requiring ongoing subspecialty care.

Antioxidant activity, metabolic profiling, in-silico molecular docking and ADMET analysis of nano selenium treated sesame seed bioactive compounds as potential novel drug targets against cardiovascular disease related receptors

Sesame (Sesamum indicum) is abundant in a diverse range of lignans, including sesamin, and γ-tocopherol, constituting a cluster of bioactive phenolic compound used for food and medicinal purposes. Cardiovascular diseases remain a leading global health challenge, demanding vigilant prevention and innovative treatments. This study was carried out to evaluate the effect of plant mediated SeNPs on sesame metabolic profile and to screen and check the effect bioactive compounds against CVD via molecular drug docking technique. Three sesame germplasms TS-5, TH-6 and Till-18 were treated with varying concentrations (10, 20, 30, 40 and 50 ppm) of plant-mediated selenium nanoparticles (SeNPs). There were three groups of treatments group-1 got only seed pretreatments of SeNPs, Group-2 with only foliar applications of SeNPs and Group-3 with both seed pretreatments and foliar applications of SeNPs. It was found that plants treated with 40 ppm of SeNPS in group 3 exhibited the highest total phenolic and flavonoid content. Total phenolic content at T4 was highest for TS-5 (134%), TH-6 (132%), and Till-18 (112%). LCMS analysis revealed a total of 276 metabolites, with phenolics, flavonoids, and free fatty acids being most abundant. KEGG analysis indicated enrichment in free fatty acid and phenylalanine tryptophan pathways. ADMET analysis and virtual screening resulted in total of five metabolic compounds as a potential ligand against Hemoglobin beta subunit. Lowest binding energy was achieved by Delta-Tocopherol (−6.98) followed by Lactoflavin (−6.20) and Sesamin (−5.00). Lipinski rule of five revealed that all the compounds completely safe to be used as drug against CVD and specifically for HBB. It was concluded that bioactive compounds from sesame could be an alternative source of drug for CVD related problems and especially for HBB.

Abstract 2062: Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation

Abstract Objectives: To support TCR discovery and enable evaluation of TCR target expression on the surface of cells, we have established a streamlined workflow allowing TCR Discovery, validation and evaluation of MHC-antigen expression on target cells. As proof of concept, MR1 dCODE Dextramer® reagents were used to identify antigen-specific MAIT cells using single-cell sequencing, TCR sequences were extracted from the resulting data and then cloned and expressed as TCR monomers. The specificity of the TCRs was subsequently validated as TCR Dextramer® reagents and used to evaluate target MHC-peptide presentation on cell surface. Methods: Mixed PBMC samples from three healthy donors were stained with antibodies targeting relevant phenotypic markers as well as MR1 dCODE Dextramer® reagents carrying the MAIT cell-specific ligand 5-OP-RU. Following staining, sorting, and partitioning on a BD Rhapsody™ Single-Cell Analysis System, the cells underwent sequencing for a characterization of gene and surface marker expression as well as paired alpha-beta TCR sequences. Single alpha and beta chains were individually expressed, refolded together and purified. Refolded TCR monomer functionality was validated using an artificial cell assay. The functional TCRs can be multimerized on a Dextramer® backbone and used to evaluate expression of target on cell surface. Results: The majority of the identified CD161+ MAIT cells (~70%) expressed typical TCRs consisting of TRAV1-2 and TRAJ33 as well as TRBV6/20, TRBD1/2, and TRBJ2 gene segments. Three alpha-beta TCR sequences were selected: Two TCRs having common TRAV+TRAJ, TRBV+TRBJ combinations and one having a rare TRAV+TRAJ one. TCR alpha and beta chains were successfully expressed for all selected TCRs but only 2 TCRs showed a correctly refolded TCR as demonstrated by proper recognition of MR1/5-OP-RU. Based on these two functional TCRs, TCR Dextramer® reagents were generated and used to evaluate expression of target antigen on cell surface. Conclusion: We demonstrate a workflow allowing: (i) Identification of MAIT cells and their corresponding TCR sequences. (ii) Generation of soluble TCR molecules based on the identified sequences and validation of their specificity. (iii) Generation of TCR Dextramer® reagents allowing identification of target expression on surface of cells. This workflow is also useful for identification of other antigen-specific T cells and creates a new tool for evaluation of antigen-expression by MHC on surface of cells. By combining the high sensitivity of dCODE Dextramer® technology with TCR sequencing and creation of TCR Dextramer® reagents, both low-affinity interactions and rare cell types can be detected and studied in unique detail. Citation Format: Kevin Lenogue, Bjarke Hansen, Kivin Jacobsen, Liselotte Brix, Thomas Holberg Blicher. Identification of functional MAIT-specific TCRs using MR1 dCODE Dextramer® reagents and evaluating antigen presentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2062.

Abstract 2295: Circulating T cell receptor repertoire analysis improves cancer early detection

Abstract Background: Cancer screening by liquid biopsy promises to detect cancer early when it can be cured. However, current liquid biopsy approaches based on circulating tumor DNA (ctDNA) detect only a minority of early-stage cancers, because these tumors shed little ctDNA and plasma sample volume is limited. For liquid biopsy to realize its potential for the early detection of cancer, sensitivity for the earliest stage disease must be improved. Methods: We set out to improve sensitivity of liquid biopsy for cancer early detection by exploiting tumor recognition by T cells through sequencing of the circulating T cell receptor repertoire. Using gDNA extracted from blood buffy coats, we studied a cohort of 451 lung cancer patients (86% with stage I disease) and 525 subjects without cancer, enriched for older individuals with a history of smoking. We performed genomic DNA TCR beta chain sequencing to yield a median of 81,088 TCR clonotypes in cancer patients and 112,588 clonotypes in non-cancer controls. We built a TCR sequence similarity nearest neighbor graph to cluster >68M distinct TCR clonotypes into TCR repertoire functional units (RFUs). The TCR frequencies of RFUs were tested for association with cancer status and significantly associated RFUs were combined using a support vector machine model into a TCR RFU cancer score. This model was evaluated by 10-fold cross-validation (CV) and compared to a ctDNA panel of 237 mutation hotspots in 154 lung cancer driver genes (IDT™) and to a panel of 17 lung cancer related protein biomarkers (Olink®) in 87 subjects. Results: We identified 177 cancer-associated TCR RFUs at FDR≤0.10, including 83 RFUs that were enriched in cancer samples with a TCR count log2-fold change between 0.05 and 1.31, and 94 that were enriched in controls with a log2-fold change between 0.07 and 0.40. We observed 56 of the 83 cancer-enriched RFUs had decreasing TCR counts with increasing age across the cohort, coupled with higher counts in cancer patients relative to age-matched controls. The TCR RFU cancer score achieved a cross-validated ROC AUC of 0.69 for cancer status prediction, with the AUC for stage I cancers similar to that of stage II-IV (0.69 vs. 0.70). 44% of stage I cancers (test samples of each CV fold) were detected by the TCR model at a specificity of 80%. We saw a substantial gain in sensitivity for stage I cancer when TCR RFUs were added to ctDNA and proteins, with a ~15-20%-point increase seen at the 90% target specificity level typical for cancer screening tests. Likewise, sensitivity increased at the 99% specificity level used in multi-cancer early detection, with 35% of stage I lung cancers detected, which could not be achieved by any analyte alone. Conclusion: We demonstrate detection of a significant fraction of early lung cancer cases from blood by analyzing the circulating TCR repertoire and show that this signal is complementary to established analytes such as proteins and ctDNA. Citation Format: Yilong Li, Michelle Nahas, Dennis Stephens, Kate Froburg, Emma Hintz, Devin Champagne, Amaneet Lochab, Markus Brown, Jesse Abhyankar, Jasper Braun, Michelle Dinh, María Antonia Fortuño, María-del-Mar Ocon, Andrea Pasquier, Inés María Luque, Christopher W. Seder, Jeffrey A. Borgia, Luis Miguel Seijo, Luis M. Montuenga, Roman Yelensky. Circulating T cell receptor repertoire analysis improves cancer early detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2295.

Abstract 5216: Peripheral T cell receptor repertoire diversity is associated with outcome in bladder cancer

Abstract Bladder cancer is the sixth most common cancer overall, and treatment often involves removal of the bladder, which comes with a significant reduction in quality of life. Within this cancer type, an easily applicable prognostic biomarker may have clinical utility, potentially stratifying patients into bladder-sparing treatments. Expansion of T cell populations targeting cancer-specific neoantigens is an early response to malignancy. To investigate if the T cell receptor (TCR) repertoire could serve as a biomarker for cancer progression, we analyzed the TCR landscape in a cohort of 119 patients with muscle-invasive bladder cancer, treated with neoadjuvant chemotherapy before undergoing radical cystectomy. Peripheral and tumor TCR repertoires were produced using amplicon-based sequencing of the TCR beta chain. Additionally, T cell fraction was determined from whole exome sequencing data using TcellExTRECT, and latent viral DNA was investigated in plasma whole genome sequencing data using Kraken2. Antigen targets were estimated using GLIPH2. To explore the T cell repertoire subtypes, single-cell sequencing was performed on four fresh blood samples. In pre-treatment peripheral blood, low-diversity TCR repertoires were associated with worse survival (HR=2.3, p=0.024). Particularly poor outcome was observed in a subset of patients with both low TCR diversity and low fraction of circulating T cells (HR=4.7, p=5e-4). Low-diversity repertoires were dominated by expanded T cell clones. TCR target annotation revealed that expanded T cell clones disproportionately targeted latent viral infections (p=2e-13), and cytomegalovirus DNA detection was strongly associated with decreased TCR diversity (p=5e-5). Additionally, tumor TCR sequencing revealed that expanded clones were rarely found in the tumor. Single-cell sequencing of peripheral blood found that most expanded clones consisted of cytotoxic T cells, while non-expanded clones mostly consisted of naive T cells. This indicates that low TCR diversity may represent a contraction of the naive T cell compartment, resulting in reduced immune competence. Finally, analysis of patient-matched longitudinal samples revealed that chemotherapy and cystectomy reduced TCR diversity in patients with high pre-treatment diversity (p=0.001), indicating that treatment negatively affected the immune system in the most immune-competent patients. Our results suggest that both low TCR diversity and low T cell fraction represent decreased immune competency. This could be associated with an elevated risk of progression due to a reduced ability to control cancer growth and limit systemic cancer cell dissemination. These findings underline that immune health is an important factor during malignant disease, even when patients are not treated with immunotherapies. Therefore, improving immune health could be a potential future goal for cancer treatment and prevention. Citation Format: Asbjørn Kjær, Nanna Kristjánsdóttir, Iver Nordentoft, Randi Istrup Juul, Karin Birkenkamp-Demtröder, Johanne Ahrenfeldt, Darren Hodgson, Christopher Abbosh, Hugo JWL Aerts, Mads Agerbæk, Jørgen Bjerggaard Jensen, Nicolai J Birkbak, Lars Dyrskjøt. Peripheral T cell receptor repertoire diversity is associated with outcome in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5216.

Blood Indices of the Patients with β-Thalassemia Minor Compared to the Patients with β-Thalassemia Minor-Alpha-Thalassemia

Objective: Thalassemia, as one of the most common genetic diseases, is a group of hereditary hemoglobin disorders due to a slight disturbance in the production of alpha and beta globin chains in the structure of hemoglobin occurs. There are still no clear criteria for differentiating thalassemia types based on hematological findings. In the current study, we aimed to evaluate the low-grade beta-thalassemia (β-thalassemia) indices in comparison with β-thalassemia minor with alpha-thalassemia (α-thalassemia).
 Methods and materials: In this descriptive-analytic study, 120 patients were enrolled, including 80 patients with minor β-thalassemia and 40 patients with minor β-thalassemia with α-thalassemia. Of all patients, 5cc blood samples were taken. The red blood cell parameters including mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and hemoglobin were determined. The level of MCV> 80 and A2> 3.5 β-thalassemia minor and MCV <80 and A2 <3.5 were considered as elevated thalassemia or iron deficiency anemia.
 Results: The results showed that the mean of hemoglobin, HCT, MCH, MCHC, and MCV in the β-thalassemia group was significantly lower than that of the β-thalassemia with α-thalassemia group (P <0.0001). On the other hand, the level of these indices in the control group was significantly higher than in the two groups of patients (P<0.0001). The results showed that the percentage of hemoglobin A2 in the β-alpha-thalassemia group was 4.5 ± 0.91, significantly higher than the β-thalassemia group. The rate of hemoglobin and MCV was significantly lower in the β-thalassemia group compared to the silent and trait β+αthalassemia group. Also, the rate of hematocrit was significantly lower in the β-thalassemia group compared to the trait, although had no significant difference with the silence group.
 Conclusion: Based on our findings, despite the difference between some hematocrit indices in the patients with β-thalassemia and β-αthalassemia, these indices cannot be used as differential indices.

Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells

Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10-3 ng and 5 × 10-6 ng of target copy per reaction. We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.

Lacticaseibacillus paracasei AD22 Stress Response in Brined White Cheese Matrix: In Vitro Probiotic Profiles and Molecular Characterization.

Functionalizing foods involve discovering and integrating new candidate health-promoting bacteria into the food matrix. This study aimed (i) to reveal the probiotic potential of autochthonous Lacticaseibacillus paracasei AD22 by a series of in vitro tests and molecular characterization and (ii) to evaluate its application to the matrix of brined white cheese, which is the most common cheese in Türkiye, in terms of survival and stress response. To evaluate in vitro probiotic characteristics, L. paracasei AD22 was exposed to functional, technological, and safety tests. Pilot scale production was conducted to integrate L. paracasei AD22 into the brined white cheese matrix. The expression levels of stress-related genes (dnaK, groES, ftsH, argH, and hsp20) were detected by reverse-transcriptase polymerase chain reaction to determine the transcriptional stress response during ripening. The presence of genes encoding stress-related proteins was determined by whole-genome sequence analysis using a subsystem approach; the presence of antibiotic resistance and virulence genes was determined by ResFinder4.1 and VirulenceFinder 2.0 databases. The BAGEL4 database determined the presence of bacteriocin clusters. L. paracasei AD22 was found to survive in pH 2 and medium with 12% NaCl and did not cause hemolysis. Adhesion of the strain to Caco2 cells was 76.26 ± 4.81% and it had coaggregation/autoaggregation properties. It was determined that L. paracasei AD22 exceeded 7 log cfu/g in the cheese matrix at the end of the ripening period. Total mesophilic aerobes decreased in the cheese inoculated with L. paracasei AD22 after the 45th day of ripening. While hsp20 and groES genes were downregulated during ripening, argH was upregulated. Both downregulation and upregulation were observed in dnaK and ftsH. Fold changes indicating the expression levels of dnaK, groES, ftsH, argH, and hsp20 genes were not statistically significant during ripening (p > 0.05). Whole-genome sequence profiles revealed that the strain did not contain antibiotic and virulence genes but bacteriocin clusters encoding Enterolysin A (Class III bacteriocin), Carnosine CP52 (class II bacteriocin), Enterocin X beta chain (Class IIc bacteriocin), and the LanT region. Subsystems approach manifested that the most functional part of the genomic distribution belonged to metabolism, protein processing, and stress response functions. The study findings highlight that L. paracasei AD22 will provide biotechnological innovation as a probiotic adjunct because it contains tolerance factors and probiotic characteristics to produce new functional foods.

Recurrence of macular edema in patients with branch retinal vein occlusion: a proteomic study

BackgroundBranch retinal vein occlusion (BRVO) is a common retinal vascular disease leading to severe vision loss and blindness. This study aimed to investigate and reveal the pathophysiological mechanisms underlying macular edema (ME) recurrence in patients with BRVO through a proteomic approach.MethodsWe detected proteins in the aqueous humor of 14 untreated, four refractory, and four post-operative patients with BRVO-ME and 12 age-matched cataract controls using four-dimensional label-free proteomic and bioinformatics analyses.ResultsIn total, 84 proteins exhibited significant differential expression between the BRVO and control samples (fold change [FC] ≥ 1.2 and adjusted p-value < 0.05). Compared to the control group, 43 and 41 proteins were upregulated and downregulated, respectively, in the BRVO group. These proteins were involved in cell adhesion, visual perception, retina homeostasis, and platelet activation. Several significantly enriched signaling pathways included complement and coagulation cascades and platelet activation. In the protein–protein interaction networks generated using the search tool for retrieval of interacting genes (STRING), the fibrinogen alpha chain and fibrinogen beta chain constituted a tightly connected cluster. Many common protein expression trends, such as the fibrinogen alpha chain and fibrinogen beta chain, were observed in both the recurrent and refractory groups. Differentially expressed proteins in the two groups were involved in complement activation, acute-phase response, platelet activation, and platelet aggregation. Important signaling pathways include the complement and coagulation cascades, and platelet activation. Protein–protein interaction analysis suggested that the fibrinogen alpha chain and fibrinogen beta chain constituted a tightly connected cluster. The expression of some differentially expressed proteins shared by the BRVO and the recurrent and refractory groups was reversed in the post-operative group.ConclusionsOur study is the first to analyze the proteomics of recurrent, refractory, and post-operative groups treated for BRVO-ME, and may potentially provide novel therapeutic interventions for the recurrence of ME.

Molecular characterisation of sickle cell disease and classification of major haplotypes associated with the β-globin cluster (HBB gene) by means of SNP marker sequencing in a group of samples from Bolívar, Colombia

Background Colombia has a mestizo population and the prevalence of haemoglobin variants varies according to each region, but heterozygous carriers can be found in all of them. Aim To characterise sickle cell disease (SCD) haematologically, biochemically, and molecularly, and detect classic haplotypes by DNA sequencing in a group of samples from Bolívar, Colombia. Subjects and methods Blood samples were collected after informed consent from volunteers from eight communities in the Bolívar department, plus samples from the Pacific region, Providencia Island, and Bogotá were included. Data were obtained from: (1) haematological analyses; (2) biochemical tests: dHPLC was used to determine haemoglobin (Hb); and (3) DNA sequencing data through five SNPs. Results 101 samples were identified by rs334 through Sanger’s Sequencing, structural haemoglobinopathies HbAS (34.65%), HbSS (2.97%) and HbAC (1.98%) were found. When contrasting the Hb identification results between SNP rs334 Vs. dHPLC/Isoelectric Focusing (IEF), a coincidence was found in 39/43 samples analysed, therefore, when comparing these techniques, a significant correlation was found (Pearson’s correlation coefficient r = 0.998). 26 samples previously analysed by rs334 were classified into classical haplotypes CAR (50.0%), BEN (30.76%), CAM (7.69%), SEN (3.84%), and ATP-I (7.69%). Conclusions SCD characterisation and SNPs-based classification through Sanger’s DNA sequencing have not been performed before in Colombia. The results of this work will make it possible to expand the data or records of carriers and those affected, which will benefit patients and their families.