[Effect of Staphylococcal Nuclease and Tudor Domain Containing 1/SLC7A11 on the Occurrence and Development of Osteosarcoma by Inhibiting Ferroptosis].

Objective To investigate the effect of staphylococcal nuclease and tudor domain containing 1(SND1) on the biological function of osteosarcoma cells and decipher the mechanism of SND1 in regulating ferroptosis in osteosarcoma cells via SLC7A11. Methods Human osteoblasts hFOB1.19 and osteosarcoma cell lines Saos-2,U2OS,HOS,and 143B were cultured,in which the expression level of SND1 was determined.Small interfering RNA was employed to knock down the expression of SND1(si-SND1) in the osteosarcoma cell line HOS and 143B.The CCK8 assay kit,colony formation assay,and Transwell assay were employed to examine the effect of SND1 expression on the biological function of osteosarcoma cells.Furthermore,we altered the expression of SND1 and SLC7A11 in osteosarcoma cells to investigate the effect of SND1 on osteosarcoma ferroptosis via SLC7A11. Results The mRNA and protein levels of SND1 in Saos-2,U2OS,HOS,and 143B cells were higher than those in hFOB1.19 cells(all P<0.01).Compared with the control group,transfection with si-SND1 down-regulated the expression level of SND1 in HOS and 143B cells(all P<0.01),decreased the viability of HOS and 143B cells,reduced the number of colony formation,and inhibited cell invasion and migration(all P<0.001).The ferroptosis inducer Erastin promoted the apoptosis of HOS and 143B cells,while the ferroptosis inhibitor Ferrostatin-1 improved the viability of HOS and 143B cells(all P<0.001).After SND-1 knockdown,Erastin reduced the viability of HOS and 143B cells,while Ferrostatin-1 restored the cell viability(all P<0.001).After treatment with Erastin in the si-SND1 group,the levels of iron and malondialdehyde were elevated,and the level of glutathione was lowered(all P<0.001).The results of in vivo experiments showed that SND1 knockdown inhibited the mass of the transplanted tumor in 143B tumor-bearing nude mice(P<0.001).Knocking down the expression of SND1 resulted in down-regulated SLC7A11 expression(all P<0.001) and increased ferroptosis in HOS and 143B cells(P<0.001,P=0.020). Conclusions SND1 presents up-regulated expression in osteosarcoma cells.It may inhibit ferroptosis by up-regulating the expression of SLC7A11,thereby improving the viability of osteosarcoma cells.

In vitro cytotoxicity of magnetic-fluorescent bioactive glasses on SaOS-2, MC3T3-E1, BJ fibroblast cells, their hemolytic activity, and sorafenib release behavior.

In the study, the fabrication of superparamagnetic-fluorescent bioactive glasses in the form of the particle, nanofiber, and 3D scaffolds was performed by including maghemite (γ-Fe2O3) nanoparticles and photoluminescent rare earth element ions (Eu3+, Gd3+, and Yb3+) using sol-gel, electrospinning, and robocasting techniques, respectively. The in vitro cytotoxicity of the magnetic-fluorescent bioactive glasses on osteosarcoma SaOS-2, pre-osteoblast MC3T3-E1, and BJ fibroblast cells, as well as their hemolytic activity and sorafenib tosylate loading and release behavior, were investigated. The cytotoxicity of the bioactive glass samples was tested using the MTT assay. Additionally, the alkaline phosphatase activity of the studied glasses was examined as a function of time. The mineralization behavior of the pre-osteoblast cell-seeded glass samples was analyzed using Alizarin red S staining. Results revealed that the in vitro cytotoxicity of the studied bioactive glasses in the form of particles and nanofibers depended on the sample concentration, whereas in the case of the 3D scaffolds, no cytotoxic response was observed on the osteosarcoma, pre-osteoblast, and fibroblast cells. Similarly, particle and nanofiber-based glass samples induced dose-dependent hemolysis on red blood cells. Drug loading rates were much lower for the 3D scaffolds compared to the particle and nanofiber-based samples. Drug release rates ranged from 25 % to 90 %, depending on the bioactive glass morphology and the pH of the release medium. It was concluded that the studied bioactive glasses have the potential to be used in tissue engineering applications and cancer therapy.

Effects of Daidzein, Tempeh, and a Probiotic Digested in an Artificial Gastrointestinal Tract on Calcium Deposition in Human Osteoblast-like Saos-2 Cells.

Adequate calcium intake is crucial for the prevention and treatment of bone-related issues. Developing a nutritional source of readily bioavailable calcium is particularly significant for individuals deficient in this essential element and at risk of developing osteoporosis. This research aimed to evaluate the impact of tempeh (T), daidzein (D), and Lactobacillus acidophilus (LA) within a simulated intestinal environment consisting of Caco-2 epithelial and Saos-2 cells, focusing on their implications for bone mineralization mechanisms. In the initial phase, calcium bioaccessibility from calcium citrate (CaCt), LA, D, the daidzein combination D-CaCt-LA (D1:1:1), and the tempeh combination T-CaCt-LA (T1:1:1) was assessed through digestion simulation. The calcium content of both untreated and digested samples was determined using atomic absorption spectrometry (AAS). In the subsequent stage, the digested samples were used to induce intestinal absorption in differentiated enterocyte-like Caco-2 cells. The permeable fractions were then evaluated in a culture of osteoblast-like Saos-2 cells. Preliminary cellular experiments employed the MTT assay to assess cytotoxicity. The results indicated that the analyzed products did not influence the deposition of extracellular calcium in Saos-2 cells cultured without mineralization stimulators. The combined formulations of permeable fractions of digested CaCt, LA, D, and T demonstrated the capacity to enhance the proliferation of Saos-2 cells. In Saos-2 cells, D, D1:1:1, and LA showed no discernible impact on intracellular calcium accumulation, whereas T and T1:1:1 reduced the calcium deposits. Additionally, mRNA transcripts and alkaline phosphatase (ALP) activity levels in Saos-2 cells cultured without mineralization induction were unaffected by the analyzed products. An examination of the products revealed no discernible effect on ALP activity or mRNA expression during Saos-2 cell differentiation. Our findings suggest that tempeh, daidzein, and L. acidophilus did not positively impact cellular calcium deposition in Saos-2 cells. However, tempeh, daidzein and its combination, and L. acidophilus might enhance the process of osteogenic differentiation in Saos-2 cells. Nevertheless, this study did not identify any synergistic impact on calcium deposition and the process of osteogenic differentiation in Saos-2 cells of isoflavones and probiotics.

Molecular characteristics of hereditary red blood cell membrane disorders in Thailand: a multi-center registry.

Red blood cell (RBC) membrane disorders represent a significant category of hereditary hemolytic anemia; however, information from Southeast Asia is limited. We established a national registry aiming to characterize RBC membrane disorders and their molecular features in Thailand. A total of 100 patients (99 kindreds) diagnosed with RBC membrane disorders between 2011 and 2020 from seven university hospitals were enrolled. The most prevalent disorders observed were hereditary elliptocytosis (HE; n=33), hereditary pyropoikilocytosis (HPP; n=28), hereditary spherocytosis (HS; n=19), Southeast Asian ovalocytosis (SAO; n=10 of 9 kindreds), and two cases of homozygous SAO. The remaining cases were grouped as unclassified membrane disorder. Seventy-six patients (76%) were molecularly confirmed by PCR, direct DNA sequencing, or hi-throughput sequencing. The primary causative gene for HE and HPP was SPTB, accounting for 28 out of 29 studied alleles for HE and 56 of 56 studied alleles for HPP. In the case of HS, dominant sporadic mutations in the ANK1 gene (n=4) and SPTB gene (n=3) were identified as the underlying cause. Notably, the four most common variants causing HE and HPP were SPTB Providence (c.6055 T>C), SPTB Buffalo (c.6074 T>G), SPTB Chiang Mai (c.6224 A>G), and SPTB c.6171__82delins TGCCCAGCT. These recurrent SPTB mutations accounted for 79 out of 84 mutated SPTB alleles (94%). In summary, HE and hereditary HPP associated with recurrent SPTB mutations are the predominant types of RBC membrane disorders observed in Thailand. These findings have significant implications for the clinical management and future research of RBC membrane disorders in the region.

Homozygous SPTA1-associated hereditary pyropoikilocytosis presenting as hydrops fetalis.

INTRODUCTION: Hereditary pyropoikilocytosis (HPP) is a heterogeneous inherited disorder of red blood cell (RBC) membrane and cytoskeletal proteins that leads to hemolytic anemia. HPP is characterized by marked poikilocytosis, microspherocytes, RBC fragmentation, and elliptocytes on peripheral blood smear. Mutations in SPTA1 can cause HPP due to a quantitative defect in α-spectrin and can lead to profound fetal anemia and nonimmune hydrops fetalis, which can be managed with intrauterine transfusion. CASE PRESENTATION: We present a case of a 26-year-old G4P2102 woman of Amish-Mennonite ancestry with a pregnancy complicated by fetal homozygosity for an SPTA1 gene variant (SPTA1c.6154delG) as well as severe fetal anemia and hydrops fetalis, which was managed with four intrauterine transfusions between 26 and 30 weeks gestation. Pre-transfusion peripheral smears from fetal blood samples showed RBC morphology consistent with HPP. The neonate had severe hyperbilirubinemia at birth, which has resolved, but remains transfusion-dependent at 6 months of life. DISCUSSION/CONCLUSION: To our knowledge, this is the first report that correlates homozygosity of the SPTA1c.6154delG gene variant with RBC dysmorphology and establishes the diagnosis of HPP.

Molecular insights into hereditary elliptocytosis and pyropoikilocytosis: NGS uncovers multiple potential candidate genes.

Hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP) are considered a group of hemolytic anemias (HE/HPP) due to inherited abnormalities of erythrocyte membrane proteins with a worldwide distribution. Most cases are associated with molecular abnormalities linked to spectrin, band 4.1, and ankyrin. The present study aimed to identify significant molecular signatures on a target panel of 8 genes using whole exome sequencing (WES) in 9 Bahraini patients with elliptocytosis. Case selection was based on presence of anemia not associated with iron deficiency or hemoglobinopathy and demonstrating > 50% elliptocytes in blood smears. The c.779 T > C mutation of SPTA1 (Spectrin alpha), which is a known deleterious missense mutation that inhibits normal association of spectrin molecules to form tetramers, was seen in 4 patients in homozygous (n = 1) and heterozygous (n = 3) states. The αLELY abnormality in association with compound heterozygous mutations in SPTA1 was present in 5 patients (2 associated with the SPTA1 c.779 T > C variant; 3 with c.3487 T > G and various other SPTA1 mutations of uncertain/unknown significance). Seven patients had SPTB (Spectrin beta) mutations, predicted as likely benign by in silico analysis. A novel EPB41 (Erythrocyte Membrane Protein Band 4.1) mutation with potential deleterious impact was also seen. Finally, 2 cases showed an InDel (insertion-deletion mutations) abnormality in the gene that codes for the mechanosensitive ion-channel PIEZO (Piezo Type Mechanosensitive Ion Channel Component 1). PIEZO mutations are reported to cause red cell dehydration but have not been previously described in HE/HPP. Results of this study confirm the involvement of previously reported abnormalities in SPTA1 and suggest possible involvement of other candidate genes in a disorder involving polygenic interactions.

Acquired elliptocytosis in chronic myeloid neoplasms: An enigmatic relationship to acquired red cell membrane protein and genetic abnormalities.

Nineteen reports of 41 cases of acquired red cell elliptocytosis associated with a chronic myeloid neoplasm are described. Although the majority of cases have an abnormality of the long arm of chromosome 20, del(q20), several cases do not. Moreover, in one case a specific qualitative abnormality of red cell protein band 4.1(4.1R) was reported; however, several subsequent cases could find no abnormality of a red cell membrane protein or found a different abnormality, usually quantitative. Thus, this striking red cell phenotypic feature, acquired elliptocytosis, seen in myelodysplastic syndrome and other chronic myeloproliferative diseases, closely simulating the red cell phenotype of hereditary elliptocytosis, has an unexplained genetic basis, presumably as the result of an acquired mutation(s) in some chronic myeloid neoplasms.

[Clinical and gene mutation characteristics of patients with hereditary ellipsocytosis: nine cases report and literature review].

Objective: To report gene mutations in nine patients with hereditary elliptocytosis (HE) and analyze the characteristics of pathogenic gene mutations in HE. Methods: The clinical and gene mutations of nine patients clinically diagnosed with HE at Institute of Hematology & Blood Diseases Hospital from June 2018 to February 2022 were reported and verified by next-generation sequencing to analyze the relationship between gene mutations and clinical phenotypes. Results: Erythrocyte membrane protein gene mutations were detected among nine patients with HE, including six with SPTA1 mutation, one with SPTB mutation, one with EPB41 mutation, and one with chromosome 20 copy deletion. A total of 11 gene mutation sites were involved, including 6 known mutations and 5 novel mutations. The five novel mutations included SPTA1: c.1247A>C (p. K416T) in exon 9, c.1891delG (p. A631fs*17) in exon 15, E6-E12 Del; SPTB: c.154C>T (p. R52W) ; and EPB41: c.1636A>G (p. I546V) . Three of the six patients with the SPTA1 mutation were SPTA1 exon 9 mutation. Conclusion: SPTA1 is the most common mutant gene in patients with HE.

Association between ovalocytosis and Plasmodium infection: a systematic review and meta-analysis.

Reports of an association between ovalocytosis and protection against Plasmodium infection are inconsistent. Therefore, we aimed to synthesise the overall evidence of the association between ovalocytosis and malaria infection using a meta-analysis approach. The systematic review protocol was registered with PROSPERO (CRD42023393778). A systematic literature search of the MEDLINE, Embase, Scopus, PubMed, Ovid, and ProQuest databases, from inception to 30 December 2022, was performed to retrieve studies documenting the association between ovalocytosis and Plasmodium infection. The quality of the included studies was assessed using the Newcastle-Ottawa Scale. Data synthesis included a narrative synthesis and a meta-analysis to calculate the pooled effect estimate (log odds ratios [ORs]) and 95% confidence intervals (CIs) using the random-effects model. Our database search retrieved 905 articles, 16 of which were included for data synthesis. Qualitative synthesis revealed that over half of the studies showed no association between ovalocytosis and malaria infections or severity. Furthermore, our meta-analysis demonstrated no association between ovalocytosis and Plasmodium infection (P = 0.81, log OR = 0.06, 95% CI - 0.44 to 0.19, I2: 86.20%; 11 studies). In conclusion, the meta-analysis results demonstrated no association between ovalocytosis and Plasmodium infection. Hence, the role of ovalocytosis in relation to protection against Plasmodium infection or disease severity should be further investigated in larger prospective studies.

Clinical utility of targeted next-generation sequencing panel in routine diagnosis of hereditary hemolytic anemia: A national reference laboratory experience.

INTRODUCTION: Hereditary hemolytic anemias (HHA) comprise a heterogeneous group of disorders resulting from defective red blood cell (RBC) cytoskeleton, RBC enzyme deficiencies, and hemoglobin (Hb) synthesis disorders such as thalassemia or sideroblastic anemia. MATERIALS AND METHODS: Our hemolytic anemia diagnostic next-generation sequencing (NGS) panel includes 28 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The panel covers the complete coding region of these genes, splice junctions, and, wherever appropriate, deep intronic or regulatory regions are also included. Four hundred fifty-six patients with unexplained hemolytic anemia were evaluated using our NGS panel between 2015 and 2019. RESULTS: We identified pathogenic/likely pathogenic variants in 111/456 (24%) patients that were responsible for the disease phenotype (e.g., moderate to severe hemolytic anemia and hyperbilirubinemia). Approximately 40% of the mutations were novel. As expected, 45/456 (10%) patients were homozygous for the promoter polymorphism in the UGT1A1 gene, A(TA)7 TAA (UGT1A1*28). 8/45 homozygous UGT1A1*28 cases were associated with additional pathogenic mutations causing hemolytic anemia, likely exacerbating hyperbilirubinemia. The most common mutated genes were membrane cytoskeleton genes SPTA1, and SPTB, followed by PKLR. Complex interactions between SPTA1 low expression alleles, alpha-LELY and alpha-LEPRA alleles, and intragenic SPTA1 variants were associated with hereditary pyropoikilocytosis and autosomal recessive hereditary spherocytosis in 23/111 patients. CONCLUSIONS: Our results demonstrate that hemolytic anemia is underscored by complex molecular interactions of previously known and novel mutations in RBC cytoskeleton/enzyme genes, and therefore, NGS should be considered in all patients with clinically unexplained hemolytic anemia and in neonates with hyperbilirubinemia. Moreover, low expression alleles alpha-LELY and alpha-LEPRA should be included in all targeted HHA panels.

A large family of hereditary spherocytosis and a rare case of hereditary elliptocytosis with a novel SPTA1 mutation underdiagnosed in Taiwan: A case report and literature review.

RATIONALE: Hereditary spherocytosis (HS) has a defect in the vertically connected proteins on the cell membrane of red blood cells (RBC). Hereditary elliptocytosis (HE) has a defect in proteins that connect the cell membrane horizontally. We reported two families of RBC membrane disorders in Taiwanese, one was HS and the other was HE. PATIENT CONCERNS: Case 1. A 19-year-old male student with chronic jaundice and splenomegaly. His mother, maternal uncle, grandmother, and many members of older generations also had splenomegaly and underwent splenectomy. Case 2. A 40-year-old man has experienced pallor and jaundice since the age of 20 and was found to have splenomegaly, and gall bladder stones in the older age. His younger sister also had pallor and jaundice for a long time. DIAGNOSES: In case 1, a peripheral blood smear showed 20% spherocytes. Eosin-5-maleimide labeled RBC by flow cytometry showed a result of 30.6 MCF (cutoff value: 45.5 MCF). He was diagnosed with HS. The gene analysis identified a heterozygous mutation with c.166A > G (p.Lys56Glu) in the SLC4A1 gene in this proband, his mother, and maternal uncle. In case 2, more than 40% of ellipsoid RBC present in the peripheral blood smear. He was diagnosed with HE. Genetic analysis of the SPTA1 gene identified a novel heterozygous exon2, c.86A > C, p.Gln29Prol mutation. INTERVENTIONS: The two patients had compensated anemia, clinical follow-up instead of splenectomy was done. OUTCOMES: The two patients had normal daily activities and lives. LESSONS: We reported two Taiwanese families, one was hereditary spherocytosis affected by a heterozygous mutation with c.166A > G (p.Lys56Glu) in SLC4A1, and the other was hereditary elliptocytosis caused by a novel heterozygous SPTA1 gene mutation, c. 86A > C, p.Gln29Prol. These 2 seemingly common hereditary red blood cell membrane protein defects induced by hemolysis are usually underdiagnosed or misdiagnosed.

Band 3-mediated Plasmodium vivax invasion is associated with transcriptional variation in PvTRAg genes.

The Plasmodium vivax reticulocyte invasion process is still poorly understood, with only a few receptor-ligand interactions identified to date. Individuals with the Southeast Asian ovalocytosis (SAO) phenotype have a deletion in the band 3 protein on the surface of erythrocytes, and are reported to have a lower incidence of clinical P. vivax malaria. Based on this observation, band 3 has been put forward as a receptor for P. vivax invasion, although direct proof is still lacking. In this study, we combined functional ex vivo invasion assays and transcriptome sequencing to uncover a band 3-mediated invasion pathway in P. vivax and potential band 3 ligands. Invasion by P. vivax field isolates was 67%-71% lower in SAO reticulocytes compared with non-SAO reticulocytes. Reticulocyte invasion was decreased by 40% and 27%-31% when blocking with an anti-band 3 polyclonal antibody and a PvTRAg38 peptide, respectively. To identify new band 3 receptor candidates, we mRNA-sequenced schizont-stage isolates used in the invasion assays, and observed high transcriptional variability in multigene and invasion-related families. Transcriptomes of isolates with low or high dependency on band 3 for invasion were compared by differential expression analysis, which produced a list of band 3 ligand candidates with high representation of PvTRAg genes. Our ex vivo invasion assays have demonstrated that band 3 is a P. vivax invasion receptor and confirm previous in vitro studies showing binding between PvTRAg38 and band 3, although the lower and variable inhibition levels observed suggest the involvement of other ligands. By coupling transcriptomes and invasion phenotypes from the same isolates, we identified a list of band 3 ligand candidates, of which the overrepresented PvTRAg genes are the most promising for future research.

Unravelling the genetic and phenotypic heterogeneity of SPTA1 gene variants in Hereditary Elliptocytosis and Hereditary Pyropoikilocytosis patients using next-generation sequencing.

Hereditary Elliptocytosis (HE) and Hereditary Pyropoikilocytosis (HPP) are clinically and genetically heterogeneous red cell membranopathies that result from the defects in the horizontal linkage between RBC (red blood cell) membrane and cytoskeletal proteins affecting its mechanical stability and deformability thereby reducing its lifespan. The principal defect in HE and HPP is due to dysfunction or deficiency of RBC cytoskeletal proteins namely, α-spectrin (SPTA1), ß-spectrin (SPTB) and protein 4.1R (EPB41R). This study reports the genetic and phenotypic heterogeneity of 10 Indian patients (5 with HE and 5 with HPP)harboringSPTA1 gene variants. We used targeted next-generation sequencing (t-NGS) to characterize the causative genetic variants in 10 HE/HPP suspected patients and studied the correlation between the identified variants with their corresponding phenotypic features.t-NGS detected 12 SPTA1 variants, out of which 8 are novel. Nearly all of the detected variants have a damaging effect on the protein stability and function, as shown by the insilico analysis. The possible effect of the detected variants on the protein structure was studied using the HOPE software and DynaMut tools wherever possible. To the best of our knowledge, this is the first report on HE/HPP cases confirmed by a genetic study from India. To conclude, HE is caused by monoallelic mutations while HPP, the more severe form, is typically caused by biallelic (homozygous or compound heterozygous) mutations justifying the phenotypic heterogeneity associated with patients. Moreover, analysis at the molecular level by NGS permits diagnosis in these disorders with highly variable heterogeneity requiring regular transfusions and may facilitate prognostic contemplations.