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Kritsanawan Sae-khow,1 Awirut Charoensappakit,2 Peerapat Visitchanakun,1 Wilasinee Saisorn,2 Saovaros Svasti,3 Suthat Fucharoen,3 Asada Leelahavanichkul2,4 1Medical Microbiology, Interdisciplinary and International Program, Graduate School, Chulalongkorn University, Bangkok, Thailand; 2Translational Research in Inflammation and Immunology Research Unit (TRIRU), Department of Microbiology, Chulalongkorn University, Bangkok, Thailand; 3Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand; 4Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, ThailandCorrespondence: Asada LeelahavanichkulTranslational Research in Inflammation and Immunology Research Unit (TRIRU), Department of Microbiology, Chulalongkorn University, Bangkok 10330, ThailandTel +66-2-256-4251Fax +66-2-252-6920Email aleelahavanit@gmail.comIntroduction: Systemic inflammation induced by gut translocation of lipopolysaccharide (LPS), a major component of Gram-negative bacteria, in thalassemia with iron-overload worsens sepsis. However, the impact of (1→ 3)-β-D-glucan (BG), a major fungal molecule, in iron-overload thalassemia is still unclear. Hence, the influence of BG was explored in 1) iron-overload mice with sepsis induced by cecal ligation and puncture (CLP) surgery; and 2) in bone marrow-derived macrophages (BMMs).Methods: The heterozygous β-globin-deficient mice, Hbbth3/+ mice, were used as representative thalassemia (TH) mice. Iron overload was generated by 6 months of oral iron administration before CLP surgery- induced sepsis in TH mice and wild-type (WT) mice. Additionally, BMMs from both mouse strains were used to explore the impact of BG.Results: Without sepsis, iron-overload TH mice demonstrated more severe intestinal mucosal injury (gut leakage) with higher LPS and BG in serum, from gut translocation, when compared with WT mice. With CLP in iron-overload mice, sepsis severity in TH mice was more severe than WT as determined by survival analysis, organ injury (kidney and liver), bacteremia, endotoxemia, gut leakage (FITC-dextran) and serum BG. Activation by LPS plus BG (LPS+BG) in BMMs and in peripheral blood-derived neutrophils (both WT and TH cells) demonstrated more prominent cytokine production when compared with LPS activation alone. In parallel, LPS+BG also prominently induced genes expression of M1 macrophage polarization (iNOS, TNF-α and IL-1β) in both WT and TH cells in comparison with LPS activation alone. In addition, LPS+BG activated macrophage cytokine production was enhanced by a high dose of ferric ion (800 mM), more predominantly in TH macrophages compared with WT cells. Moreover, LPS+BG induced higher glycolysis activity with similar respiratory capacity in RAW264.7 (a macrophage cell line) compared with LPS activation alone. These data support an additive pro-inflammatory effect of BG upon LPS.Conclusion: The enhanced-severity of sepsis in iron-overload TH mice was due to 1) increased LPS and BG in serum from iron-induced gut-mucosal injury; and 2) the pro-inflammatory amplification by ferric ion on LPS+BG activation.Keywords: thalassemia, iron overload, leaky-gut, sepsis

Abstract Background Patients with Hb E/β0 thalassemia display remarkable variability in disease severity. To identify genetic modifiers influencing disease severity, we conducted a two-stage genome scan in groups of 207 mild and 305 severe unrelated patients from Thailand with Hb E/β0 thalassemia and normal α-globin genes. Methods First, we estimated and compared the allele frequencies of approximately 110,000 gene-based single nucleotide polymorphisms (SNPs) in pooled DNAs from different severity groups. The 756 SNPs that showed reproducible allelic differences at P < 0.02 by pooling were selected for individual genotyping. Results After adjustment for age, gender and geographic region, logistic regression models showed 50 SNPs significantly associated with disease severity (P < 0.05) after Bonferroni adjustment for multiple testing. Forty-one SNPs in a large LD block within the β-globin gene cluster had major alleles associated with severe disease. The most significant was bthal_bg200 (odds ratio (OR) = 5.56, P = 2.6 × 10-13). Seven SNPs in two distinct LD blocks within a region centromeric to the β-globin gene cluster that contains many olfactory receptor genes were also associated with disease severity; rs3886223 had the strongest association (OR = 3.03, P = 3.7 × 10-11). Several previously unreported SNPs were also significantly associated with disease severity. Conclusions These results suggest that there may be an additional regulatory region centromeric to the β-globin gene cluster that affects disease severity by modulating fetal hemoglobin expression.

Fibroblast growth factor (FGF)-21 is a salient liver-derived endocrine regulator for metabolism of glucose and triglyceride as well as bone remodeling. Previously, certain peptides in the FGF family have been shown to modulate calcium absorption across the intestinal epithelia. Since FGF21 receptor, i.e., FGF receptor-1, is abundantly expressed in the enterocytes, there was a possibility that FGF21 might exert direct actions on the intestine. Herein, a large-scale production of recombinant FGF21 at the multi-gram level was developed in order to minimize variations among various batches. In the oral glucose tolerance test, recombinant FGF21 was found to reduce plasma glucose levels in mice fed high-fat diet. A series of experiments applying radioactive tracer 45 Ca in Ussing chamber showed that FGF21 potentiated the stimulatory effect of low-dose 1,25-dihydroxyvitamin D 3 [10 nM 1,25(OH) 2 D 3 ] on the transepithelial calcium transport across intestinal epithelium-like Caco-2 monolayer. FGF21 + 1,25(OH) 2 D 3 also decreased transepithelial resistance, but had no effect on epithelial potential difference or short-circuit current. Furthermore, 1,25(OH) 2 D 3 alone upregulated the Caco-2 mRNA expression of the major apical calcium channels, i.e., transient receptor potential vanilloid subfamily member 6 (TRPV6), which was further elevated by a combination of FGF21 and 1,25(OH) 2 D 3 , consistent with the upregulated TRPV6 protein expression in enterocytes of FGF21-treated mice. However, FGF21 was without effects on the mRNA expression of voltage-gated calcium channel 1.3, calbindin-D 9k , plasma membrane Ca 2+ -ATPase 1b, claudin-12 or claudin-15. In conclusion, FGF21 did exert a direct action on the intestinal epithelial cells by potentiating the 1,25(OH) 2 D 3 -enhanced calcium transport, presumably through the upregulation of TRPV6 expression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA damage repair (DDR) proteins are well recognized as guardians of the genome that are frequently lost during malignant transformation of normal cells across cancer types. To date, their tumor suppressor functions have been generally regarded as a consequence of their roles in maintaining genomic stability: more genomic instability increases the risk of oncogenic transformation events. However, recent discoveries centering around DNA mismatch repair (MMR) proteins suggest a broader impact of the loss of DDR proteins on cellular processes beyond genomic instability. Here, we explore the clinical implications of nonrepair roles for DDR proteins, using the growing evidence supporting roles for DNA MMR proteins in cell cycle and apoptosis regulation, metabolic function, the cellular secretome, and immunomodulation., Competing Interests: Declaration of interests S.H. serves as a member of the California Breast Cancer Research Program and has two provisional patent applications relating to a role for DNA repair proteins in cell cycle regulation (Serial Nos: 62/670,368, 63/106777). The remaining authors have no interests to declare., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

β-Thalassaemia is one of the most common genetic diseases worldwide. During the past few decades, life expectancy of patients has increased significantly owing to advance in medical treatments. Cognitive impairment, once has been neglected, has gradually become more documented. Cognitive impairment in β-thalassaemia patients is associated with natural history of the disease and socioeconomic factors. Herein, to determined effect of β-thalassaemia intrinsic factors, 22-month-old β-thalassaemia mouse was used as a model to assess cognitive impairment and to investigate any aberrant brain pathology in β-thalassaemia. Open field test showed that β-thalassaemia mice had decreased motor function. However, no difference of neuronal degeneration in primary motor cortex, layer 2/3 area was found. Interestingly, impaired learning and memory function accessed by a Morris water maze test was observed and correlated with a reduced number of living pyramidal neurons in hippocampus at the CA3 region in β-thalassaemia mice. Cognitive impairment in β-thalassaemia mice was significantly correlated with several intrinsic β-thalassaemic factors including iron overload, anaemia, damaged red blood cells (RBCs), phosphatidylserine (PS)-exposed RBC large extracellular vesicles (EVs) and PS-exposed medium EVs. This highlights the importance of blood transfusion and iron chelation in β-thalassaemia patients. In addition, to improve patients' quality of life, assessment of cognitive functions should become part of routine follow-up., (© 2024. The Author(s).)

Splenectomised β-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised β-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Ineffective erythropoiesis is the main cause of anemia in β-thalassemia. The crucial hallmark of ineffective erythropoiesis is the high proliferation of erythroblast. microRNA (miR/miRNA) involves several biological processes, including cell proliferation and erythropoiesis. miR-101 was widely studied and associated with proliferation in several types of cancer. However, the miR-101-3p has not been studied in β-thalassemia/HbE. Therefore, this study aims to investigate the expression of miR-101-3p during erythropoiesis in β-thalassemia/HbE. The results showed that miR-101-3p was upregulated in the erythroblast of β-thalassemia/HbE patients on day 7, indicating that miR-101-3p may be involved with high proliferation in β-thalassemia/HbE. Therefore, the mRNA targets of miR-101-3p including Rac1, SUB1, TET2, and TRIM44 were investigated to determine the mechanisms involved with high proliferation of β-thalassemia/HbE erythroblasts. Rac1 expression was significantly reduced at day 11 in severe β-thalassemia/HbE compared to normal controls and mild β-thalassemia/HbE. SUB1 gene expression was significantly lower in severe β-thalassemia/HbE compared to normal controls at day 9 of culture. For TET2 and TRIM44 expression, a significant difference was not observed among normal and β-thalassemia/HbE. However, the high expression of miR-101-3p at day 7 and these target genes was not correlated, suggesting that this miRNA may regulate ineffective erythropoiesis in β-thalassemia/HbE via other target genes., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cell cycle dysregulation is prerequisite for cancer formation. However, it is unknown whether the mode of dysregulation affects disease characteristics. Here, we conduct comprehensive analyses of cell cycle checkpoint dysregulation using patient data and experimental investigations. We find that ATM mutation predisposes the diagnosis of primary estrogen receptor (ER) + /human epidermal growth factor (HER)2 - cancer in older women. Conversely, CHK2 dysregulation induces formation of metastatic, premenopausal ER + /HER2 - breast cancer ( P = 0.001) that is treatment-resistant (HR = 6.15, P = 0.01). Lastly, while mutations in ATR alone are rare, ATR / TP53 co-mutation is 12-fold enriched over expected in ER + /HER2 - disease ( P = 0.002) and associates with metastatic progression (HR = 2.01, P = 0.006). Concordantly, ATR dysregulation induces metastatic phenotypes in TP53 mutant, not wild-type, cells. Overall, we identify mode of cell cycle dysregulation as a distinct event that determines subtype, metastatic potential, and treatment responsiveness, providing rationale for reconsidering diagnostic classification through the lens of the mode of cell cycle dysregulation..

α-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart's hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (-- SEA ) and the Thai type (-- THAI ) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia., (© 2023. The Author(s).)

Dasatinib, a tyrosine kinase inhibitor, has been shown to produce anti-inflammatory activity and impair vascular integrity in vivo, including during skin wound healing, potentially promoting the repair process. Given that dasatinib is a lipophilic small molecule capable of penetrating skin, topical dasatinib might provide benefits in wound healing. In the present study, we investigated the impact of dasatinib ointments in skin wound healing in mice. A full thickness excisional skin wound (4 mm diameter) was generated on the shaved dorsum of eight-week-old C57BL/6 mice. Dasatinib ointment (0.1 or 0.2% w/w) or ointment base was applied twice daily (every 12 h) for 10 days. Elizabethan collars were used to prevent animal licking. The wound size was monitored daily for 14 days. The results showed that dasatinib ointments, particularly 0.1% dasatinib, promoted a 16-23% reduction in wound size ( p < 0.05) during day 2 to day 6 postinjury compared to controls. Immunohistochemistry analyses demonstrated a reduction in wound neutrophils (38% reduction, p = 0.04), macrophages (47% reduction, p = 0.005), and tumor necrosis factor-α levels (73% reduction, p < 0.01), together with an induction of vascular leakage-mediated fibrin(ogen) accumulation (2.5-fold increase, p < 0.01) in the wound during day 3 postinjury (an early phase of repair) in 0.1% dasatinib-treated mice relative to control mice. The anti-inflammatory and vascular hyperpermeability activities of dasatinib were associated with an enhanced healing process, including increased keratinocyte proliferation (1.8-fold increase in Ki67 + cells, p < 0.05) and augmented angiogenesis (1.7-fold increase in CD31 + area, p < 0.05), compared to the ointment base-treated group. Following treatment with 0.2% dasatinib ointment, minor wound bleeding and scab reformation were observed during the late phase, which contributed to delayed healing. In conclusion, our data suggest that dasatinib ointment, mainly at 0.1%, promotes the repair process by reducing inflammation and producing a local and temporal vascular leakage, leading to an increase in fibrin(ogen) deposition, re-epithelialization, and angiogenesis. Therefore, topical dasatinib might be a potential novel candidate to facilitate skin wound healing., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Despite much vaunted progress in cancer therapeutics and diagnostics, outcomes for many groups of non-White patients with cancer remain worse than those for their White compatriots. One reason for this is the lack of inclusion and representation of non-White patients in clinical trials, preclinical datasets, and among researchers, a shortfall that is gaining wide recognition within the cancer research community and the lay public. Several reviews and editorials have commented on the negative impacts of the status quo on progress in cancer research toward medical breakthroughs that help all communities and not just White patients with cancer. In this perspective, we describe the existence of research silos focused either on the impact of socioeconomic factors proceeding from systemic racism on cancer outcomes, or on genetic ancestry as it affects the molecular biology of cancer developing in specific patient populations. While both these research areas are critical for progress toward precision medicine equity, breaking down these silos will help us gain an integrated understanding of how race and racism impact cancer development, progression, and patient outcomes. Bringing this comprehensive approach to cancer disparities research will undoubtedly improve our overall understanding of how stress and environmental factors affect the molecular biology of cancer, which will lead to the development of new diagnostics and therapeutics that are applicable across cancer patient demographics., (©2023 American Association for Cancer Research.)

Black women in the US have significantly higher breast cancer mortality than White women. Within biomarker-defined tumor subtypes, disparate outcomes seem to be limited to women with hormone receptor positive and HER2 negative (HR+/HER2-) breast cancer, a subtype usually associated with favorable prognosis. In this review, we present data from an array of studies that demonstrate significantly higher mortality in Black compared to White women with HR+/HER2-breast cancer and contrast these data to studies from integrated healthcare systems that failed to find survival differences. Then, we describe factors, both biological and non-biological, that may contribute to disparate survival in Black women.

Hydroxyurea (HU) (hydroxycarbamide) is used as a therapeutic option in β-thalassaemia to increase fetal haemoglobin, which results in a reduced requirement for blood transfusion. However, a potential serious adverse effect of HU is neutropenia. Abnormal neutrophil maturation and function in β-thalassaemia/HbE patients are well documented. This raises questions about the effect of the drug with regards to the immune response these patients. This study investigated the effects of HU treatment on both innate and adaptive immunity in a cross-sectional study of 28 β-thalassaemia/HbE patients who had received HU treatment (BE+HU) as compared with 22 β-thalassaemia/HbE patients who had not received HU (BE-HU) and 26 normal subjects. The expression of PU.1 and C/EBPβ, transcription factors, which are associated with neutrophil maturation, was significantly reduced in BE+HU patients as compared with BE-HU patients and normal subjects. Interestingly, C3bR expression on neutrophils and their oxidative burst activity in BE+HU were restored to close to normal levels when compared with BE-HU. There was no observed effect of HU on monocytes, myeloid derived suppressor cells (both granulocytic and monocytic subsets), CD4 + T cells, CD8 + T cells, complement levels and serum immunoglobulin levels in this study. The full immunophenotyping analysis in this study indicates that HU therapy in β-thalassaemia/HbE patients does not significantly compromise the immune response., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

β-Thalassemia is characterized by ineffective erythropoiesis leading to chronic anemia. Thus, increased iron absorption from the duodenum and via blood transfusions is required to maintain normal blood hemoglobin (Hb) levels and iron chelators in the removal of excessive iron. Certain agents are also needed for the improvement of stress erythropoiesis and iron dysregulation. Green tea extract (GTE), which is rich in epigallocatechin-3-gallate (EGCG), is known to possess radical scavenging and iron-chelating activities. We aimed to assess the effects of green tea extract on erythroid regulators, iron mobilization and anti-lipid peroxidation in the liver, spleen, and kidneys of iron-loaded β-globin gene knockout thalassemic (BKO) mice. Our results indicate that treatments of green tea extract and/or deferiprone (DFP) diminished levels of plasma erythropoietin (EPO) and erythroferrone (ERFE), and consistently suppressed kidney Epo and spleen Erfe mRNA expressions ( p < .05) in iron- loaded BKO mice when compared with untreated mice. Coincidently, the treatments decreased plasma ferritin (Ft) levels, iron content levels in the liver ( p < .05), spleen ( p < .05), and kidney tissues of iron-loaded BKO mice. Furthermore, lipid-peroxidation products in the tissues and plasma were also decreased when compared with untreated mice. This is the first evidence of the orchestral role of green tea extract abundant with epigallocatechin-3-gallate in improving ineffective erythropoiesis, iron dysregulation and oxidative stress in iron-overloaded β-thalassemic mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Settakorn, Kongkarnka, Chompupoung, Svasti, Fucharoen, Porter, Srichairatanakool and Koonyosying.)

β-Thalassaemia results from defects in β-globin chain production, leading to ineffective erythropoiesis and subsequently to severe anaemia and other complications. Apoptosis and autophagy are the main pathways that regulate the balance between cell survival and cell death in response to diverse cellular stresses. Herein, the death of erythroid lineage cells in the bone marrow from both β IVS2-654 -thalassaemic mice and β-thalassaemia/HbE patients was investigated. Phosphatidylserine (PS)-bearing basophilic erythroblasts and polychromatophilic erythroblasts were significantly increased in β-thalassaemia as compared to controls. However, the activation of caspase 8, caspase 9 and caspase 3 was minimal and not different from control in both murine and human thalassaemic erythroblasts. Interestingly, bone marrow erythroblasts from both β-thalassaemic mice and β-thalassaemia/HbE patients had significantly increased autophagy as shown by increased autophagosomes and increased co-localization between LC3 and LAMP-1. Inhibition of autophagy by chloroquine caused significantly increased erythroblast apoptosis. We have demonstrated increased autophagy which led to minimal apoptosis in β-thalassaemic erythroblasts. However, increased PS exposure occurring through other mechanisms in thalassaemic erythroblasts might cause rapid phagocytic removal by macrophages and consequently ineffective erythropoiesis in β-thalassaemia., (© 2022. The Author(s).)

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

β-Thalassemia is one of the most common genetically inherited disorders worldwide, and it is characterized by defective β-globin chain synthesis leading to reduced or absent β-globin chains. The excess α-globin chains are the key factor leading to the death of differentiating erythroblasts in a process termed ineffective erythropoiesis, leading to anemia and associated complications in patients. The mechanism of ineffective erythropoiesis in β-thalassemia is complex and not fully understood. Autophagy is primarily known as a cell recycling mechanism in which old or dysfunctional proteins and organelles are digested to allow recycling of constituent elements. In late stage, erythropoiesis autophagy is involved in the removal of mitochondria as part of terminal differentiation. Several studies have shown that autophagy is increased in earlier erythropoiesis in β-thalassemia erythroblasts, as compared to normal erythroblasts. This review summarizes what is known about the role of autophagy in β-thalassemia erythropoiesis and shows that modulation of autophagy and its interplay with apoptosis may provide a new therapeutic route in the treatment of β-thalassemia. Literature was searched and relevant articles were collected from databases, including PubMed, Scopus, Prospero, Clinicaltrials.gov, Google Scholar, and the Google search engine. Search terms included: β-thalassemia, ineffective erythropoiesis, autophagy, novel treatment, and drugs during the initial search. Relevant titles and abstracts were screened to choose relevant articles. Further, selected full-text articles were retrieved, and then, relevant cross-references were scanned to collect further information for the present review.

Background: The mouse model of human diseases is commonly used for biomedical study, including β-thalassemia (β-thal), an inherited hemoglobin disorder. Maintaining the mice strain by natural mating systems is costly and seems impractical, especially during the COVID-19 pandemic. Sperm-freezing is a cost-effective solution for β-thal mouse colony management., Aim: To determine appropriate cryopreservation media for β-thal mouse spermatozoa to establish a β-thal mouse sperm bank., Methods: The epididymal spermatozoa of C57BL/6 wild-type (WT) and β-globin gene knockout thalassemia (BKO) mice were frozen in four freezing media: I) raffinose-skim milk-monothioglycerol (MTG), II) raffinose-skim milk-glutamine, III) raffinose-egg yolk-glycerol, and IV) egg yolk-TES-Tris. The sperm quality was assessed prior to and following freeze-thawing., Results: Compared with WT counterparts, the viable spermatozoa before freezing exhibiting elevated levels of oxidative stress were significantly greater in BKO ( p = 0.01). After thawing, the membrane integrity of BKO spermatozoa preserved in I was significantly lower ( p = 0.001). The sperm viability and membrane integrity of BKO males were also inferior when media III and IV were used ( p = 0.008-0.027). The amount of oxidative stress in the spermatozoon of BKO mice was significantly greater when preserved in I, III, and IV ( p = 0.002-0.044). Comparing freezing media, the motility and acrosome integrity of WT and BKO spermatozoa preserved in IV were significantly higher than those in other media ( p < 0.001 to p = 0.01). Spermatozoa with the highest mitochondrial membrane potential were observed in I in both genotypes ( p = 0.012 to p > 0.05). The viability, membrane integrity, and oxidative stress of post-thaw BKO spermatozoa did not significantly differ among freezing solutions., Conclusion: Irrespective of freezing media, spermatozoa of BKO males are rather more sensitive to cryopreservation than those of WT. Raffinose-skim milk-MTG/glutamine, raffinose-egg yolk-glycerol, and egg yolk-TES-Tris can all be used to preserve BKO mouse spermatozoa. However, with slightly better sperm characteristics, egg yolk-TES-Tris may be a diluent of choice for BKO mouse sperm cryopreservation. The addition of a reducing agent to thawing media is also strongly recommended to efficiently prevent oxidative stress and therefore improve frozen-thawed sperm survival., Competing Interests: The authors declare that there is no conflict of interest.

Metastatic cancers are chemoresistant, involving complex interplay between disseminated cancer cell aggregates and the distant organ microenvironment (extracellular matrix and stromal cells). Conventional metastasis surrogates (scratch/wound healing, Transwell migration assays) lack 3D architecture and ECM presence. Metastasis studies can therefore significantly benefit from biomimetic 3D in vitro models recapitulating the complex cascade of distant organ invasion and colonization by collective clusters of cells. We aimed to engineer reproducible and quantifiable 3D models of highly therapy-resistant cancer processes: (i) colorectal cancer liver metastasis; and (ii) breast cancer lung metastasis. Metastatic seeds are engineered using 3D tumor spheroids to recapitulate the 3D aggregation of cancer cells both in the tumor and in circulation throughout the metastatic cascade of many cancers. Metastatic soil was engineered by decellularizing porcine livers and lungs to generate biomatrix scaffolds, followed by extensive materials characterization. HCT116 colorectal and MDA-MB-231 breast cancer spheroids were generated on hanging drop arrays to initiate clustered metastatic seeding into liver and lung biomatrix scaffolds, respectively. Between days 3-7, biomatrix cellular colonization was apparent with increased metabolic activity and the presence of cellular nests evaluated via multiphoton microscopy. HCT116 and MDA-MB-231 cells colonized liver and lung biomatrices, and at least 15% of the cells invaded more than 20 μm from the surface. Engineered metastases also expressed increased signatures of genes associated with the metastatic epithelial to mesenchymal transition (EMT). Importantly, inhibition of matrix metalloproteinase-9 inhibited metastatic invasion into the biomatrix. Furthermore, metastatic nests were significantly more chemoresistant (>3 times) to the anti-cancer drug oxaliplatin, compared to 3D spheroids. Together, our data indicated that HCT116 and MDA-MB-231 spheroids invade, colonize, and proliferate in livers and lungs establishing metastatic nests in 3D settings in vitro . The metastatic nature of these cells was confirmed with functional readouts regarding EMT and chemoresistance. Modeling the dynamic metastatic cascade in vitro has potential to identify therapeutic targets to treat or prevent metastatic progression in chemoresistant metastatic cancers.

The types of β-thalassemia mutations, α-thalassemia interactions, and Hb F-associated SNPs have been described in association with variable disease phenotypes. This study aimed to determine the updated spectrum of β-thalassemia mutations and evaluate the contribution of primary and secondary genetic modifiers and SNPs to disease severity, age at onset, and predicted life expectancy in southern Thai β-thalassemia patients. A total of 181 β-thalassemia patients were enrolled and 135 β 0 -thalassemia/Hb E patients without α-thalassemia interactions were divided into three categories according to disease severity, age at onset, and predicted life expectancy. A total of 16 β-thalassemia mutations were identified in this study, and the three most common β-thalassemia mutations accounted for 61.4% of all mutations. It was also found that the Xmn I polymorphism and rs2071348 were associated with age at onset and the predicted life expectancy. More than 82% of β 0 -thalassemia/Hb E patients with CC genotype ( Xmn I) were 3 years old or younger at onset. Additionally, >90% of the higher predicted life expectancy in β 0 -thalassemia/Hb E patients had the T allele of Xmn I. Therefore, genetic prediction for age at onset and life expectancy is beneficial and practical during prenatal diagnosis or newborn screening for better genetic counseling and optimal management., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Nuinoon et al.)

Although patients with either β-thalassemia or chronic kidney disease (CKD) clinically correlate with severe osteoporosis, the mechanism by which CKD exposed to high phosphate affects bone turnover has not been characterized in β-thalassemia. We aimed to determine the effects of renal insufficiency on high phosphate intake induced changes in bone metabolism after 5/6th nephrectomy in hemizygous β-globin knockout (BKO) mice. Male BKO mice manifested severe anemia and osteopenia. Nephrectomy induced renal fibrosis and reduced renal function as assessed by increased serum urea nitrogen levels. Moreover, nephrectomy increased bone turnover leading to bone loss in wild type (WT) but not BKO mice. In nephrectomized BKO, PBS in drinking water induced hyperphosphatemia, and hypercalcemia along with osteopenia in both cancellous and cortical bone. Histomorphometric analysis confirmed reduced cancellous bone volume due to decreased bone formation rate, osteoblast number and osteoclast number. The mRNA levels for Alpl, Sp7, Kl, Tnfsf11, and Tnfsf11/Tnfrsf11b were decreased in nephrectomized BKO mice drinking PBS. Interestingly, Fgf23, a bone-derived hormone produced by osteocytes and osteoblasts in response to hyperphosphatemia, were remarkably increased in nephrectomized BKO mice following PBS intake. Serum FGF23 and erythropoietin levels were markedly elevated in BKO mice. Nephrectomy decreased serum erythropoietin but not FGF23 levels. Hyperphosphatemia in BKO mice increased serum erythropoietin, FGF23, and PTH levels, nominating these factors as candidate mediators of bone loss in thalassemic mice with CKD during phosphate retention., Competing Interests: The authors have declared that no competing interests exist.

Thalassemia causes anemia, ineffective erythropoiesis, bone loss and iron accumulation in several tissues, e.g., liver, bone and heart, the last of which leads to lethal cardiomyopathy and arrhythmia. Although exercise reportedly improves bone density in thalassemic mice, exercise performance is compromised and might pose risk of cardiovascular accident in thalassemic patients. Therefore, we sought to explore whether mild-intensity physical activity (MPA) with 30-50% of maximal oxygen consumption was sufficient to benefit the heart and bone. Herein, male hemizygous β-globin knockout (BKO) mice and wild-type littermates were subjected to voluntary wheel running 1 h/day, 5 days/week for 3 months (MPA group) or kept sedentary (SDN; control). As determined by atomic absorption spectroscopy, BKO-MPA mice had less iron accumulation in heart and bone tissues compared with BKO-SDN mice. Meanwhile, the circulating level of fibroblast growth factor-23-a factor known to reduce serum iron and intestinal calcium absorption-was increased early in young BKO-MPA mice. Nevertheless, MPA did not affect duodenal calcium transport or body calcium retention. Although MPA restored the aberrant bone calcium-phosphorus ratio to normal range, it did not change vertebral calcium content or femoral mechanical properties. Microstructural porosity in tibia of BKO-MPA mice remained unaltered as determined by synchrotron radiation X-ray tomographic microscopy. In conclusion, MPA prevents cardiac and bone iron accumulation, which is beneficial to thalassemic patients with limited physical fitness or deteriorated cardiac performance. However, in contrast to moderate-intensity exercise, MPA does not improve bone mechanical properties or reduce bone porosity., (© 2022. The Author(s).)

Background: Estrogen receptor positive (ER+) breast cancer is one of the most commonly diagnosed malignancies in women irrespective of their race or ethnicity. While Black women with ER+ breast cancer are 42% more likely to die of their disease than White women, molecular mechanisms underlying this disparate outcome are understudied. Recent studies identify DNA damage repair (DDR) genes as a new class of endocrine therapy resistance driver that contributes to poor survival among ER+ breast cancer patients. Here, we systematically analyze DDR regulation in the tumors and normal breast of Black women and its impact on survival outcome., Method: Mutation and up/downregulation of 104 DDR genes in breast tumor and normal samples from Black patients relative to White counterparts was assessed. For DDR genes that were differently regulated in the tumor samples from Black women in multiple datasets associations with survival outcome were tested., Results: Overall, Black patient tumors upregulate or downregulate RNA levels of a wide array of single strand break repair (SSBR) genes relative to their white counterparts and uniformly upregulate double strand break repair (DSBR) genes. This DSBR upregulation was also detectable in samples of normal breast tissue from Black women. Eight candidate DDR genes were reproducibly differently regulated in tumors from Black women and associated with poor survival. A unique DDR signature comprised of simultaneous upregulation of homologous recombination gene expression and downregulation of SSBR genes was enriched in Black patients. This signature associated with cell cycle dysregulation ( p  < 0.001), a hallmark of endocrine therapy resistance, and concordantly, with significantly worse survival outcomes in all datasets analyzed (hazard ratio of 9.5, p  < 0.001)., Conclusion: These results constitute the first systematic analysis of DDR regulation in Black women and provide strong rationale for refining biomarker profiles to ensure precision medicine for underserved populations., Competing Interests: Conflict of interest statement: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dr Haricharan has a provisional patent (US 63/106777) for using DNA repair genes as a biomarker for patient outcome. Dr Freedland is a consultant to Pfizer, Janssen, Astra Zeneca, Merck, and Clovis. All other authors declare no conflicts of interest., (© The Author(s), 2022.)

Neutrophil dysfunction contributes to a high susceptibility to severe bacterial infection which is a leading cause of morbidity and mortality in β-thalassaemia/HbE, especially in splenectomised patients. This study demonstrated another abnormality of neutrophil function, namely neutrophil extracellular trap (NET) formation in splenectomised and non-splenectomised β-thalassaemia/HbE patients who had iron overload. A classification system of morphological NET formation using confocal microscopy was developed, and samples were categorized into early and late phases which were subdivided into web-like and non-web structures. At baseline, neutrophils from non-splenectomised patients (58 ± 4%) and splenectomised patients (65 ± 3%) had higher early phase NETs than those from normal subjects (33 ± 1%). As a mimic of iron overload and infection, haemin/PMA/LPS treatment led to a significant reduction of early NETs and an increase of late NETs in neutrophils from normal and non-splenectomised patients. Interestingly, neutrophils from splenectomised patients had impaired development of late NETs. This suggests that during infection bacteria might not be trapped and may spread from the site of infection resulting in higher susceptibility to severe bacterial infection in splenectomised patients., (© 2022. The Author(s).)

Background: Inflammatory bleeding due to depletion of platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential novel mechanism to promote skin wound healing. Dasatinib inhibits a broad range of tyrosine kinases, including Src and Syk, the signaling molecules downstream of GPVI and CLEC-2., Objectives: To investigate whether dasatinib affects skin wound healing., Methods: A single (4-mm diameter) full-thickness excisional skin wound was generated in mice. Dasatinib (5 or 10 mg/kg) or dimethyl sulfoxide (DMSO) vehicle was intraperitoneally injected daily during the first 4 days. The wound was monitored over 9 days post injury., Results: Dasatinib induced loss of vascular integrity during the inflammatory phase of wound repair (day 1 to day 3 post injury), which was associated with the inhibition of platelet function stimulated by collagen and rhodocytin, the ligands for GPVI and CLEC-2, respectively. Dasatinib-treated mice, particularly at 5 mg/kg, exhibited accelerated wound closure compared to DMSO-treated controls. Transient bleeding into the wound during the inflammatory phase in dasatinib-treated mice allowed for extravasation of fibrinogen. The increased deposition of fibrinogen and fibrin in the wound on day 3 post injury was associated with the augmented progression of re-epithelialization and angiogenesis, attenuated infiltration of neutrophils and macrophages, and decreased levels of tumor necrosis factor-α (TNF-α)., Conclusions: Our data show that dasatinib promotes skin wound healing, and the mechanisms include blocking GPVI- and CLEC-2-mediated platelet activation, leading to self-limited inflammatory bleeding and fibrinogen/fibrin deposition, in association with reduced inflammation, increased re-epithelialization, and enhanced angiogenesis., (© 2021 International Society on Thrombosis and Haemostasis.)

Circulating microRNAs (miRNAs) are useful biomarkers of hemolysis. Since blood cells are the main origins of circulating miRNAs, we evaluated blood cell-related pre-analytical modification of the miRNA signatures during blood drawing and serum processing. The levels of miRNA before and after ex vivo blood drawing were analyzed with the reverse transcriptase-based polymerase chain reaction method. Furthermore, the changes of miRNA signatures caused by different time-lag between blood drawing and serum preparation by 24 h were evaluated. Finally, we compared the miRNA levels between leftover samples and samples of hemolytic diseases. Blood drawing procedure induced increments of red blood cell (RBC)-related miRNAs (miR-451a, miR-486) about 2-fold. One hour standing of blood samples before serum separation induced almost the same increases in RBC-related miRNAs. To test the clinical usefulness of miR-451a as a biomarker of hemolytic diseases, we analyzed miRNAs of samples from 10 normal subjects, 30 leftover samples in the clinical laboratory, and 20 samples from patients with hemolytic diseases. Serum miR-451a significantly increased in patients with hemolytic anemia more than the levels of pre-analytical modification. In conclusion, the pre-analytical modification of serum miRNAs did not disturb the usefulness of RBC-derived miRNAs as biomarkers of hemolytic diseases.

The lethality of estrogen receptor alpha positive (ER+) breast cancer, which is often considered to have better prognosis than other subtypes, is defined by resistance to the standard of care endocrine treatment. Relapse and metastasis are inevitable in almost every patient whose cancer is resistant to endocrine treatment. Therefore, understanding the underlying causes of treatment resistance remains an important biological and clinical focus of research in this area. Growth factor receptor pathway activation, specifically HER2 activation, has been identified as 1 mechanism of endocrine treatment resistance across a range of experimental model systems. However, clinical trials conducted to test whether targeting HER2 benefits patients with endocrine treatment-resistant ER+ breast cancer have consistently and disappointingly shown mixed results. One reason for the failure of these clinical trials could be the complexity of crosstalk between ER, HER2, and other growth factor receptors and the fluidity of HER2 activation in these cells, which makes it challenging to identify stratifiers for this targeted intervention. In the absence of stratifiers that can be assayed at diagnosis to allow prospective tailoring of HER2 inhibition to the right patients, clinical trials will continue to disappoint. To understand stratifiers, it is important that the field invests in key understudied areas of research including characterization of the tumor secretome and receptor activation in response to endocrine treatment, and mapping the ER-HER2 growth factor network in the normal and developing mammary gland. Understanding these mechanisms further is critical to improving outcomes for the hard-to-treat endocrine treatment-resistant ER+ breast cancer cohort., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A primary challenge in lentiviral gene therapy of β-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVβ-shα2, that allows coordinated expression of the therapeutic β A-T87Q -globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and β A-T87Q -globin gene expression identical to the parent vector. Compared with the first β A-T87Q -globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype., Competing Interests: Declaration of interests P.L. is a scientific founder and shareholder of bluebird bio, Inc. He is an inventor of awarded patents that cover the β(A-T87Q)-globin gene and the BB305 LentiGlobin vector., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Redox-active iron generates reactive oxygen species that can cause oxidative organ dysfunction. Thus, the anti-oxidative systems in the body and certain dietary antioxidants, such as anthocyanins, are needed to control oxidative stress. We aimed to investigate the effects of dielectric barrier discharge (DBD) plasma technology in the preparation of Riceberry™ rice flour (PRBF) on iron-induced oxidative stress in mice. PRBF using plasma technology was rich in anthocyanins, mainly cyanidine-3-glucoside and peonidine-3-glucoside. PRBF (5 mg AE/mg) lowered WBC numbers in iron dextran (FeDex)-loaded mice and served as evidence of the reversal of erythrocyte superoxide dismutase activity, plasma total antioxidant capacity, and plasma and liver thiobarbituric acid-reactive substances in the loading mice. Consequently, the PRBF treatment was observed to be more effective than NAC treatment. PRBF would be a powerful supplementary and therapeutic antioxidant product that is understood to be more potent than NAC in ameliorating the effects of iron-induced oxidative stress.

Extracellular vesicles (EVs) are bioactive, submicron-sized membrane vesicles released from all cell types upon activation or apoptosis. EVs including microparticles (MPs) and exosomes have emerged as important mediators of cell-to-cell communication in both normal and pathological states including thalassemia (thal). However, the role of EVs derived from β-thal patients with iron overload (+ IO) and without iron overload (-IO) on cardiac cells is unclear. We hypothesized plasma EVs in thal patients containing ferritin (iron storage protein) and a denaturated hemoglobin-hemichrome that induce cardiac cell proliferation. The origins and numbers of EVs isolated from plasma of normal, thal (+ IO), and (- IO) patients were compared and determined for their iron and iron-containing proteins along with their effects on cardiac and endothelial cells. Data shows that MPs were originated from many cell sources with marked numbers of platelet origin. Only the number of RBC-derived MPs in thal (+ IO) patients was significantly high when compared to normal controls. Although MPs derived from both normal and thal patients promoted cardiac cell proliferation in a dose-dependent manner, only exosomes from thal patients promoted cardiac cell proliferation compared to the untreated. Moreover, the exosomes from thal (+ IO) potentially induce higher cardiac cell proliferation and angiogenesis in terms of tube number than thal (- IO) and normal controls. Interestingly, ferritin content in the exosomes isolated from thal (+ IO) was higher than that found in the MPs isolated from the same patient. The exosomes of thal patients with higher serum ferritin level also contained greater level of ferritin inside the exosomes. Apart from ferritin, there were trends of increasing hemichrome and iron presented in the plasma EVs and EV-treated H9C2 cells. Findings from this study support the hypothesis that EVs from β-thal patients carry iron-load proteins that leads to the induction of cardiac cell proliferation., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Enterocyte damage and gut dysbiosis are caused by iron-overload in thalassemia (Thl), possibly making the gut vulnerable to additional injury. Hence, iron-overload in the heterozygous β-globin deficient (Hbbth3/+) mice were tested with 3% dextran sulfate solution (DSS). With 4 months of iron-gavage, iron accumulation, gut-leakage (fluorescein isothiocyanate dextran (FITC-dextran), endotoxemia, and tight junction injury) in Thl mice were more prominent than WT mice. Additionally, DSS-induced mucositis in iron-overloaded mice from Thl group was also more severe than the WT group as indicated by mortality, liver enzyme, colon injury (histology and tissue cytokines), serum cytokines, and gut-leakage (FITC-dextran, endotoxemia, bacteremia, and the detection of Green-Fluorescent Producing Escherichia coli in the internal organs after an oral administration). However, Lactobacillus rhamnosus GG attenuated the disease severity of DSS in iron-overloaded Thl mice as indicated by mortality, cytokines (colon tissue and serum), gut-leakage (FITC-dextran, endotoxemia, and bacteremia) and fecal dysbiosis (microbiome analysis). Likewise, Lactobacillus conditioned media (LCM) decreased inflammation (supernatant IL-8 and cell expression of TLR-4, nuclear factor κB (NFκB), and cyclooxygenase-2 (COX-2)) and increased transepithelial electrical resistance (TEER) in enterocytes (Caco-2 cells) stimulated by lipopolysaccharide (LPS) and LPS plus ferric ion. In conclusion, in the case of iron-overloaded Thl, there was a pre-existing intestinal injury that wask more vulnerable to DSS-induced bacteremia (gut translocation). Hence, the prevention of gut-derived bacteremia and the monitoring on gut-leakage might be beneficial in patients with thalassemia., (© 2021 The Author(s).)

Resistance to endocrine treatment occurs in ~30% of ER + breast cancer patients resulting in ~40,000 deaths/year in the USA. Preclinical studies strongly implicate activation of growth factor receptor, HER2 in endocrine treatment resistance. However, clinical trials of pan-HER inhibitors in ER + /HER2 - patients have disappointed, likely due to a lack of predictive biomarkers. Here we demonstrate that loss of mismatch repair activates HER2 after endocrine treatment in ER + /HER2 - breast cancer cells by protecting HER2 from protein trafficking. Additionally, HER2 activation is indispensable for endocrine treatment resistance in MutL - cells. Consequently, inhibiting HER2 restores sensitivity to endocrine treatment. Patient data from multiple clinical datasets supports an association between MutL loss, HER2 upregulation, and sensitivity to HER inhibitors in ER + /HER2 - patients. These results provide strong rationale for MutL loss as a first-in-class predictive marker of sensitivity to combinatorial treatment with endocrine intervention and HER inhibitors in endocrine treatment-resistant ER + /HER2 - breast cancer patients.

The clinical outcome of patients with a diagnosis of hormone receptor (HR)+ breast cancer has improved remarkably since the arrival of endocrine therapy. Yet, resistance to standard treatments is a major clinical challenge for breast cancer specialists and a life-threatening condition for the patients. In breast cancer, mismatch repair (MMR) status assessment has been demonstrated to be clinically relevant not only in terms of screening for inherited conditions such as Lynch syndrome, but also for prognostication, selection for immunotherapy, and early identification of therapy resistance. Peculiar traits characterize the MMR biology in HR+ breast cancers compared to other cancer types. In these tumors, MMR genetic alterations are relatively rare, occurring in ~3 % of cases. On the other hand, modifications at the protein level can be observed also in the absence of gene alterations and vice versa. In HR+ breast cancers, the prognostic role of MMR deficiency has been confirmed by several studies, but its predictive value remains a matter of controversy. The characterization of MMR status in these patients is troubled by the lack of tumor-specific guidelines and/or companion diagnostic tests. For this reason, precise identification of MMR-deficient breast cancers can be problematic. A deeper understanding of the MMR biology and clinical actionability in HR+ breast cancer may light the path to effective tumor-specific diagnostic tools. For a precise MMR status profiling, the specific strengths and limitations of the available technologies should be taken into consideration. This article aims at providing a comprehensive overview of the current state of knowledge of MMR alterations in HR+ breast cancer. The available armamentarium for MMR testing in these tumors is also examined along with possible strategies for a tailored pathological characterization.

β-Thalassemia/HbE disease has a wide spectrum of clinical phenotypes ranging from asymptomatic to dependent on regular blood transfusions. Ability to predict disease severity is helpful for clinical management and treatment decision making. A thalassemia severity score has been developed from Mediterranean β-thalassemia patients. However, different ethnic groups may have different allele frequency and linkage disequilibrium structures. Here, Thai β 0 -thalassemia/HbE disease genome-wild association studies (GWAS) data of 487 patients were analyzed by SNP interaction prioritization algorithm, interacting Loci (iLoci), to find predictive SNPs for disease severity. Three SNPs from two SNP interaction pairs associated with disease severity were identifies. The three-SNP disease severity risk score composed of rs766432 in BCL11A, rs9399137 in HBS1L-MYB and rs72872548 in HBE1 showed more than 85% specificity and 75% accuracy. The three-SNP predictive score was then validated in two independent cohorts of Thai and Malaysian β 0 -thalassemia/HbE patients with comparable specificity and accuracy. The SNP risk score could be used for prediction of clinical severity for Southeast Asia β 0 -thalassemia/HbE population.

Restoration of correct splicing of β IVS2-654 -globin pre-mRNA was previously accomplished in erythroid cells from β-thalassemia/HbE patients by an engineered U7 small nuclear RNA (snRNA) that carried a sequence targeted to the cryptic branch point and an exonic splicing enhancer, U7.BP+623 snRNA. In this study, this approach was tested in thalassemic mice carrying the β IVS2-654 mutation. While correction of β IVS2-654 pre-mRNA splicing was achieved in erythroid progenitors transduced with a lentiviral vector carrying the U7.BP+623 snRNA, a high level of truncated U7.BP+623 snRNA was also observed. The discrepancy of processing of the modified U7 snRNA in human and mouse constructs hamper the evaluation of pathologic improvement in mouse model.

Background: Hepcidin controls iron homeostasis by inducing the degradation of the iron efflux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and inflammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic ( Hbb th3 /+ ) mice with/without parenteral iron loading with iron dextran., Methods: Wild type and Hbb th3 mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and /+ mice were intramuscularly injected with 5 mg of iron dextran once daily for two consecutive days. After a 2-week equilibration, acute inflammation was induced by an intraperitoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inflammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbb th3 /+ mice were analyzed and compared by Kruskal-Wallis test with pairwise Mann-Whitney U test., Results: We found the inductive effects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory effect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbb th3 /+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbb th3 /+ mice with parenteral iron loading., Conclusion: Our study suggests that a hypoferremic response to LPS-induced acute inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner., Competing Interests: The authors declare that they have no competing interests., (© 2021 Sanyear et al.)

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG - 53, - 50 and + 6 of G γ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of G γ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at G γ-globin promoter region, without effect on A γ-globin promoter region.

Although an increased fibroblast growth factor 21 (FGF21) level was related to mild cognitive impairment (MCI) in metabolic syndrome patients, any association regarding FGF21 and MCI in thalassemia patients as well as mechanistic insight are questionable. Therefore, the objectives of this study were: (1) to investigate the prevalence and associative risk factors of MCI in thalassemia patients, (2) to evaluate the association between levels of FGF21 and MCI in thalassemia patients, and (3) to investigate brain FGF21 signaling in iron-overload thalassemia. Thalassemia patients were enrolled onto the study (n = 131). Montreal cognitive assessment (MoCA) was used to determine cognitive performance. Plasma FGF21 level was determined in all patients. Iron-overload β-thalassemic (HT) mice were used to investigate brain FGF21 level and signaling, the expression of synaptic proteins, and Alzheimer's like pathology. We found that 70% of thalassemia patients developed MCI. FGF21 level was positively correlated with the MCI. Interestingly, brain FGF21 resistance, as indicated by increased brain FGF21 levels with impaired FGF21 signaling, was found in iron-overload HT mice. The reduced synaptic protein expression and increased Alzheimer's like pathology were also observed. These suggest that FGF21 may play a role in MCI in thalassemia patients.

Introduction: Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of β-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe β-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for β-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly., Methods: Four recombinant β-globin plasmids, including a normal sequence and three different mutant panels covering 10 common β-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis., Results: We demonstrated the applicability of established positive DNA controls for β-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant β-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant β-globin plasmids, are required for detecting 10 common β-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis., Conclusion: The β-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of β-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches., (© 2020 John Wiley & Sons Ltd.)