SF3B1 mutation and ATM deletion codrive leukemogenesis via centromeric R-loop dysregulation.

RNA splicing factor SF3B1 is recurrently mutated in various cancers, particularly in hematologic malignancies. We previously reported that coexpression of Sf3b1 mutation and Atm deletion in B cells, but not either lesion alone, leads to the onset of chronic lymphocytic leukemia (CLL) with CLL cells harboring chromosome amplification. However, the exact role of Sf3b1 mutation and Atm deletion in chromosomal instability (CIN) remains unclear. Here, we demonstrated that SF3B1 mutation promotes centromeric R-loop (cen-R-loop) accumulation, leading to increased chromosome oscillation, impaired chromosome segregation, altered spindle architecture, and aneuploidy, which could be alleviated by removal of cen-R-loop and exaggerated by deletion of ATM. Aberrant splicing of key genes involved in R-loop processing underlay augmentation of cen-R-loop, as overexpression of the normal isoform, but not the altered form, mitigated mitotic stress in SF3B1-mutant cells. Our study identifies a critical role of splice variants in linking RNA splicing dysregulation and CIN and highlights cen-R-loop augmentation as a key mechanism for leukemogenesis.

Knowledge of hepatitis C and awareness of reinfection risk among people who successfully completed direct acting antiviral therapy.

BACKGROUND: Hepatitis C virus (HCV) education may be changing following the simplification of HCV treatment and emergence of direct acting antiviral (DAA). We aimed to characterize HCV knowledge among people who recently completed DAA therapy. METHODS: The Per-SVR (Preservation of Sustained Virologic Response) is a prospective cohort of patients who achieved a sustained virologic response upon successful completion of DAA therapy. The per-SVR study provided the sampling frame of participants who completed a psychometrically validated 19-item HCV knowledge scale at cohort entry (n = 227). To score the questionnaire, for each correct response one point was awarded, with no point for incorrect response. We assessed mean HCV knowledge score in the overall sample and mutually exclusive populations: people who inject drug (PWID) (n = 71); people with co-occurring HIV (n = 23); PWID and co-occurring HIV (n = 29), and others (n = 104) Using a latent class analysis based on distal outcome, we identified unobserved subgroups and assessed HCV knowledge amongst them. RESULTS: Total mean (SD) percent of correct responses were 83 (11) in the overall sample; 83 (10) in PWID; 79 (12) in people with co-occurring HIV; 81 (10) in PWID and co-occurring HIV, and 84 (11) in rest of the sample Three latent groups were identified: baby boomers who ever experienced homelessness (n = 126); women sex workers who ever experienced homelessness (n = 68); men who inject drug, ever experienced homelessness and had ever diagnosis of mental health disorders (n = 18). Mean percent of correct responses were 85 (8), 82 (11), 85 (10), in latent class 1, 2, and 3, respectively. CONCLUSION: Patients successfully treated with DAAs had a high HCV knowledge. High knowledge and awareness of reinfection among complex patient groups often facing barriers to HCV care is encouraging and emphasizes the positive outcomes of universal access to treatment.

Evaluation the Effects of Alpha-tocopherol in Comparison with N-acetylcystein for Prevention of Contrast Induced Nephropathy (CIN) in CKD Patients.

INTRODUCTION: Contrast induced nephropathy (CIN), a well-known complication of using radio contrast media, dramatically increases the likelihood of patient morbidity and mortality following coronary angiography. As there is no specific treatment for CIN, prevention could be the best strategy to address this issue. Since now, the only approved preventing strategy was hydration with normal saline while antioxidant agents as a new yet unapproved remedy for this purpose could be applied .The present study was conducted to examine the effect of alpha tocopherol in CIN prevention. METHODS: This prospective controlled trial was carried out on 201 patients with chronic kidney disease (eGFR < 60 cc/min) underwent coronary angiography. We assigned three groups of CKD patients: 72 patients who received prophylaxis administration with isotonic saline (Group A), 66 patients with isotonic saline plus N-acetylcysteine (1200mg twice a day) for 2 days (Group B) and 63 patients who received isotonic saline plus daily alpha tocopherol (600 IU once daily from one day before till 2 days after angiography) for 4 days (Group C). The contrast media in all three groups was nonionic iso-osmolal agent, Visipaque. RESULTS: Even though CIN didn't developed in any of the three aforementioned groups but there was statistically significant reduction in eGFR from baseline in all three groups (P < .001). Moreover, We found no statistically significant difference in GFR reduction between three studied groups. CONCLUSION: Administration of alpha tocopherol has no additive beneficial effect over isotonic saline in CIN prevention in CKD patients.

The splicing factor Sf3b1 regulates erythroid maturation and proliferation via TGFß signaling in zebrafish.

The spliceosomal component Splicing Factor 3B, subunit 1 (SF3B1) is one of the most prevalently mutated factors in the bone marrow failure disorder myelodysplastic syndrome. There is a strong clinical correlation between SF3B1 mutations and erythroid defects, such as refractory anemia with ringed sideroblasts, but the role of SF3B1 in normal erythroid development is largely unknown. Loss-of-function zebrafish mutants for sf3b1 develop a macrocytic anemia. Here, we explore the underlying mechanism for anemia associated with sf3b1 deficiency in vivo. We found that sf3b1 mutant erythroid progenitors display a G0/G1 cell-cycle arrest with mutant erythrocytes showing signs of immaturity. RNA-sequencing analysis of sf3b1 mutant erythroid progenitors revealed normal expression of red blood cell regulators such as gata1, globin genes, and heme biosynthetic factors, but upregulation of genes in the transforming growth factor ß (TGFß) pathway. As TGFß signaling is a known inducer of quiescence, the data suggest that activation of the pathway could trigger sf3b1 deficiency-induced anemia via cell-cycle arrest. Indeed, we found that inhibition of TGFß signaling released the G0/G1 block in erythroid progenitors. Surprisingly, removal of this checkpoint enhanced rather than suppressed the anemia, indicating that the TGFß-mediated cell-cycle arrest is protective for sf3b1-mutant erythrocytes. Together, these data suggest that macrocytic anemia arising from Sf3b1 deficiency is likely due to pleiotropic and distinct effects on cell-cycle progression and maturation.

Splicing and neurodegeneration: Insights and mechanisms.

Splicing is the global cellular process whereby intervening sequences (introns) in precursor messenger RNA (pre-mRNA) are removed and expressed regions (exons) are ligated together, resulting in a mature mRNA transcript that is exported and translated in the cytoplasm. The tightly regulated splicing cycle is also flexible allowing for the inclusion or exclusion of some sequences depending on the specific cellular context. Alternative splicing allows for the generation of many transcripts from a single gene, thereby expanding the proteome. Although all cells require the function of the spliceosome, neurons are highly sensitive to splicing perturbations with numerous neurological diseases linked to splicing defects. The sensitivity of neurons to splicing alterations is largely due to the complex neuronal cell types and functions in the nervous system that require specific splice isoforms to maintain cellular homeostasis. In the past several years, the relationship between RNA splicing and the nervous system has been the source of significant investigation. Here, we review the current knowledge on RNA splicing in neurobiology and discuss its potential role and impact in neurodegenerative diseases. We will examine the impact of alternative splicing and the role of splicing regulatory proteins on neurodegeneration, highlighting novel animal models including mouse and zebrafish. We will also examine emerging technologies and therapeutic interventions that aim to "drug" the spliceosome. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Development.

Spliceosomal components protect embryonic neurons from R-loop-mediated DNA damage and apoptosis.

RNA splicing factors are essential for the viability of all eukaryotic cells; however, in metazoans some cell types are exquisitely sensitive to disruption of splicing factors. Neuronal cells represent one such cell type, and defects in RNA splicing factors can lead to neurodegenerative diseases. The basis for this tissue selectivity is not well understood owing to difficulties in analyzing the consequences of splicing factor defects in whole-animal systems. Here, we use zebrafish mutants to show that loss of spliceosomal components, including splicing factor 3b, subunit 1 (sf3b1), causes increased DNA double-strand breaks and apoptosis in embryonic neurons. Moreover, these mutants show a concomitant accumulation of R-loops, which are non-canonical nucleic acid structures that promote genomic instability. Dampening R-loop formation by conditional induction of ribonuclease H1 in sf3b1 mutants reduced neuronal DNA damage and apoptosis. These findings show that splicing factor dysfunction leads to R-loop accumulation and DNA damage that sensitizes embryonic neurons to apoptosis. Our results suggest that diseases associated with splicing factor mutations could be susceptible to treatments that modulate R-loop levels.

Developmental HSC Microenvironments: Lessons from Zebrafish.

Hematopoietic stem cells (HSCs) posses the ability to maintain the blood system of an organism from birth to adulthood. The behavior of HSCs is modulated by its microenvironment. During development, HSCs acquire the instructions to self-renew and differentiate into all blood cell fates by passing through several developmental microenvironments. In this chapter, we discuss the signals and cell types that inform HSC decisions throughout ontogeny with a focus on HSC specification, mobilization, migration, and engraftment.

Effect of Chlorhexidine and Ethanol on Microleakage of Composite Resin Restoration to Dentine.

OBJECTIVE: To evaluate the effect of chlorhexidine and ethanol on microleakage of composite resin restoration to dentine. METHODS: Class II cavities with dentinal margin were prepared on 96 premolar teeth. All specimens were acid-etched, rinsed and dried. Then the samples were randomly divided into four groups according to pre-treatment of the dentine: no treatment (control group); treatment with 100% ethanol for 60 s (group 2); treatment with 2% chlorhexidine for 60 s (group 3); 100% ethanol for 60 s and then 2% chlorhexidine for 60 s (group 4). After dentine treatment, each group was bonded and restored with a universal micro hybrid composite resin, according to the manufacturers' recommendation. Microleakage was evaluated by dye extraction method in two subgroups, immediately (24 h) and after 6 months in storage. Scan electronic microscope analyses for two samples of each group were also conducted. Data were analysed by two-way analysis of variance and Tukey test. RESULTS: The lowest and the highest amount of microleakage were observed in the ethanol group and in the control group, respectively. There were significant differences in microleakage among the groups (P = 0.003) and between measurement times (P = 0.001). For each storage time, the control group showed significant differences from the other groups and there were no differences between the other groups. CONCLUSION: Ethanol-wet bonding and chlorhexidine application may have potential benefits in lowering the occurrence of microleakage in the long term.

Spliceosomal component Sf3b1 is essential for hematopoietic differentiation in zebrafish.

SF3B1 (Splicing factor 3b, subunit 1) is one of the most commonly mutated factors in myelodysplastic syndrome (MDS). Although the genetic correlation between SF3B1 mutations and MDS etiology are quite strong, no in vivo model currently exists to explore how SF3B1 loss alters blood cell development. Using zebrafish mutants, we show here that proper function of Sf3b1 is required for all hematopoietic lineages. As in MDS patients, zebrafish sf3b1 mutants develop a macrocytic-anemia-like phenotype due to a block in maturation at a late progenitor stage. The mutant embryos also develop neutropenia, because their primitive myeloid cells fail to mature and turn on differentiation markers such as l-plastin and myeloperoxidase. In contrast, production of definitive hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelial cells within the dorsal aorta is greatly diminished, whereas arterial endothelial cells are correctly fated. Notch signaling, imperative for the endothelial-to-hematopoietic transition, is also normal, indicating that HSPC induction is blocked in sf3b1 mutants downstream or independent of Notch signaling. The data demonstrate that Sf3b1 function is necessary during key differentiation fate decisions in multiple blood cell types. Zebrafish sf3b1 mutants offer a novel animal model with which to explore the role of splicing in hematopoietic development and provide an excellent in vivo system with which to delve into the question of why and how Sf3b1 dysfunction is detrimental to hematopoietic differentiation, which could improve MDS diagnosis and treatment.

An mDia1-INF2 formin activation cascade facilitated by IQGAP1 regulates stable microtubules in migrating cells.

Multiple formins regulate microtubule (MT) arrays, but whether they function individually or in a common pathway is unknown. Lysophosphatidic acid (LPA) stimulates the formation of stabilized detyrosinated MTs (Glu MTs) in NIH3T3 fibroblasts through RhoA and the formin mDia1. Here we show that another formin, INF2, is necessary for mDia1-mediated induction of Glu MTs and regulation of MT dynamics and that mDia1 can be bypassed by activating INF2. INF2 localized to MTs after LPA treatment in an mDia1-dependent manner, suggesting that mDia1 regulates INF2. Mutants of either formin that disrupt their interaction failed to rescue MT stability in cells depleted of the respective formin, and the mDia1-interacting protein IQGAP1 regulated INF2's localization to MTs and the induction of Glu MTs by either formin. The N-terminus of IQGAP1 associated with the C-terminus of INF2 directly, suggesting the possibility of a tripartite complex stimulated by LPA. Supporting this, the interaction of mDia1 and INF2 was induced by LPA and dependent on IQGAP1. Our data highlight a unique mechanism of formin action in which mDia1 and INF2 function in series to stabilize MTs and point to IQGAP1 as a scaffold that facilitates the activation of one formin by another.

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures­as typified by DNA methylation or histone modification at silent pluripotency loci­and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

Hnrpab regulates neural development and neuron cell survival after glutamate stimulation.

The molecular mechanisms that govern the timing and fate of neural stem-cell differentiation toward the distinct neural lineages of the nervous system are not well defined. The contribution of post-transcriptional regulation of gene expression to neural stem-cell maintenance and differentiation, in particular, remains inadequately characterized. The RNA-binding protein Hnrpab is highly expressed in developing nervous tissue and in neurogenic regions of the adult brain, but its role in neural development and function is unknown. We raised a mouse that lacks Hnrpab expression to define what role, if any, Hnrpab plays during mouse neural development. We performed a genome-wide quantitative analysis of protein expression within the hippocampus of newborn mice to demonstrate significantly altered gene expression in mice lacking Hnrpab relative to Hnrpab-expressing littermates. The proteins affected suggested an altered pattern of neural development and also unexpectedly indicated altered glutamate signaling. We demonstrate that Hnrpab(-/-) neural stem and progenitor cells undergo altered differentiation patterns in culture, and mature Hnrpab(-/-) neurons demonstrate increased sensitivity to glutamate-induced excitotoxicity. We also demonstrate that Hnrpab nucleocytoplasmic distribution in primary neurons is regulated by developmental stage.

Identification of a stem-like cell population by exposing metastatic breast cancer cell lines to repetitive cycles of hypoxia and reoxygenation.

INTRODUCTION: The irregular vasculature of solid tumors creates hypoxic regions, which are characterized by cyclic periods of hypoxia and reoxygenation. Accumulated evidence suggests that chronic and repetitive exposure to hypoxia and reoxygenation seem to provide an advantage to tumor growth. Although the development of hypoxia tolerance in tumors predicts poor prognosis, mechanisms contributing to hypoxia tolerance remain to be elucidated. Recent studies have described a subpopulation of cancer stem cells (CSC) within tumors, which have stem-like properties such as self-renewal and the ability to differentiate into multiple cell types. The cancer stem cell theory suggests CSCs persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Since hypoxia is considered to be one of the critical niche factors to promote invasive growth of tumors, we hypothesize that repetitive cycles of hypoxia/reoxygenation also play a role in the enrichment of breast CSCs. METHODS: Two metastatic human breast cancer cell lines (MDA-MB 231 and BCM2) were used to optimize the conditions of hypoxia and reoxygenation cycles. The percentage of CSCs in the cycling hypoxia selected subpopulation was analyzed based on the CD44, CD24, ESA, and E-cadherin expression by three-color flow cytometry. Colony formation assays were used to assess the ability of this subpopulation to self-renew. Limiting dilution assays were performed to evaluate the tumor-initiating and metastatic ability of this subpopulation. Induction of EMT was examined by the expression of EMT-associated markers and EMT-associated microRNAs. RESULTS: Using an optimized hypoxia and reoxygenation regimen, we identified a novel cycling hypoxia-selected subpopulation from human breast cancer cell lines and demonstrated that a stem-like breast cancer cell subpopulation could be expanded through repetitive hypoxia/reoxygenation cycles without genetic manipulation. We also found that cells derived from this novel subpopulation form colonies readily, are highly tumorigenic in immune-deficient mice, and exhibit both stem-like and EMT phenotypes. CONCLUSIONS: These results provide the validity to the newly developed hypoxia/reoxygenation culture system for examining the regulation of CSCs in breast cancer cell lines by niche factors in the tumor microenvironment and developing differential targeting strategies to eradicate breast CSCs.