Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1.

SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.

SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes.

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

U2AF35(S34F) Promotes Transformation by Directing Aberrant ATG7 Pre-mRNA 3' End Formation.

Recurrent mutations in the splicing factor U2AF35 are found in several cancers and myelodysplastic syndrome (MDS). How oncogenic U2AF35 mutants promote transformation remains to be determined. Here we derive cell lines transformed by the oncogenic U2AF35(S34F) mutant and identify aberrantly processed pre-mRNAs by deep sequencing. We find that in U2AF35(S34F)-transformed cells the autophagy-related factor 7 (Atg7) pre-mRNA is abnormally processed, which unexpectedly is not due to altered splicing but rather selection of a distal cleavage and polyadenylation (CP) site. This longer Atg7 mRNA is translated inefficiently, leading to decreased ATG7 levels and an autophagy defect that predisposes cells to secondary mutations, resulting in transformation. MDS and acute myeloid leukemia patient samples harboring U2AF35(S34F) have a similar increased use of the ATG7 distal CP site, and previous studies have shown that mice with hematopoietic cells lacking Atg7 develop an MDS-like syndrome. Collectively, our results reveal a basis for U2AF35(S34F) oncogenic activity.

The prevalence of probable overactive bladder and associated risk factors among medical students in Jordan: a cross-sectional study.

BACKGROUND: To discuss the impact of overactive bladder (OAB) on medical students. overactive bladder. is a chronic condition that causes sudden and intense urges to urinate, which can have significant physical and psychological effects on patients' lives. The prevalence of OAB among medical students is relatively high, with some studies reporting rates as high as 35.4%. This research aims to shed light on the prevalence rates and risk factors associated with OAB among medical students in Jordan. METHODS: A cross-sectional study was conducted using an online self-reported questionnaire as the study tool. The questionnaire collected the sociodemographic, health, and academic characteristics of medical students, as well as the new 7-item OABSS score. RESULTS: Out of the total sample of medical students surveyed (n = 525), 44.5% reported experiencing symptoms of OAB. Furthermore, the analysis also revealed that there was a significant difference in the prevalence of OAB between the ages of medical students. In addition, the study also found that there was a significant association between OAB symptoms and basic years, positive history of diagnostic UTI, positive history of recent trauma, high stress, and taking certain medications. CONCLUSIONS: The study highlights the need for further research in this area and emphasizes the possible implications of OAB for medical students, including the need for additional support and resources to manage the condition.

The nuclear DEK interactome supports multi-functionality.

DEK is an oncoprotein that is overexpressed in many forms of cancer and participates in numerous cellular pathways. Of these different pathways, relevant interacting partners and functions of DEK are well described in regard to the regulation of chromatin structure, epigenetic marks, and transcription. Most of this understanding was derived by investigating DNA-binding and chromatin processing capabilities of the oncoprotein. To facilitate the generation of mechanism-driven hypotheses regarding DEK activities in underexplored areas, we have developed the first DEK interactome model using tandem-affinity purification and mass spectrometry. With this approach, we identify IMPDH2, DDX21, and RPL7a as novel DEK binding partners, hinting at new roles for the oncogene in de novo nucleotide biosynthesis and ribosome formation. Additionally, a hydroxyurea-specific interaction with replication protein A (RPA) was observed, suggesting that a DEK-RPA complex may form in response to DNA replication fork stalling. Taken together, these findings highlight diverse activities for DEK across cellular pathways and support a model wherein this molecule performs a plethora of functions.

Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

Median effective dose (ED50) of paracetamol and morphine for postoperative pain: a study of interaction.

BACKGROUND: Paracetamol is widely used to treat postoperative pain and is well known for its morphine-sparing effect. Therefore, the effect of morphine-paracetamol combination can be synergistic, additive, or infra-additive. The primary aim of our study is to define the median effective analgesic doses (ED50s) of paracetamol, morphine, and the combination of both. Also, the nature of the interaction for postoperative pain after moderately painful surgery using an up-and-down method and isobolographic analysis was determined. METHODS: Ninety patients, undergoing moderately painful surgery, were included in one of the three groups. Determination of the median ED50s was performed by the Dixon and Mood up-and-down method. Initial doses were 1.5 g and 5 mg, with dose adjustment intervals of 0.5 g and 1 mg, in the paracetamol and morphine groups, respectively. The initial doses of paracetamol and morphine were 1.5 g and 3 mg, in the paracetamol-morphine combination group with dose adjustment intervals of 0.25 g for paracetamol and 0.5 mg for morphine. Analgesic efficacy was defined as a reduction to or <3 on a 0-10 numeric rating scale, 45 min after the beginning of drug administration. Isobolographic analysis was used to define the nature of their interaction. RESULTS: The median ED50s of paracetamol and morphine were 2.1 g and 5 mg, respectively. The median ED50 of the combination was 1.3 g for paracetamol and 2.7 mg for morphine. CONCLUSIONS: Our study showed that the combination of the paracetamol and morphine produces an additive analgesic effect. Clinical trial registration NCT01366313.

Gene expression in notochord and nuclei pulposi: a study of gene families across the chordate phylum.

The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.

Loss of Grem1-lineage chondrogenic progenitor cells causes osteoarthritis.

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.

Rewriting the rules for care of MDS and AML patients in the time of COVID-19.

The care of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has been radically altered by COVID-19, especially in New York City, the epicenter of the pandemic. Here we summarize how telemedicine, virtual visits, delayed transfusions, and chemotherapy, preferably selecting self-administered medications and visits by home healthcare workers, are employed to minimize exposure of our high-risk population of patients to the virus. The unique challenges of transplants during the pandemic and the consequences of an abrupt halt in all non-essential research activities are described. Not all the changes forced by COVID-19 are detrimental.

Prevention and management of transcatheter balloon-expandable aortic valve malposition.

OBJECTIVES: Early clinical outcomes in selected high-risk patients undergoing catheter-based aortic valve replacement (AVR) compare favorably with conventional surgical AVR. Improved understanding of the mechanisms of success and failure of transcatheter AVR will likely improve outcomes further. To this end, we examined our experience during the developmental phases of transcatheter AVR and describe the causes and management of prosthetic valve malposition. METHODS: Transcatheter balloon-expandable AVR was performed in 170 patients at two centers. Malposition was defined as prosthetic valve implantation in a location other than within the native valve. Patients were prospectively identified and followed as part of an ongoing database. RESULTS: Valve malposition occurred in 9 of 170 patients (5.3%). Final position was supravalvular in eight of nine cases. In all cases, embolization to the ascending aorta occurred within a few cardiac cycles following deployment. Importantly, late embolization was not observed. In most cases, the prosthesis was uneventfully repositioned in the more distal aorta. Positioning was subvalvular in one patient (0.6%), resulting in a severe regurgitation with residual native valve stenosis. Implantation of a second transcatheter valve was attempted in six patients and was successful in all. Conventional AVR was performed in two patients, with early mortality in one. At late follow-up (mean 412 days), seven of nine patients remain alive (78%) with a functioning prosthesis and relief of aortic stenosis. CONCLUSIONS: Malposition of current balloon-expandable aortic valves is a largely preventable complication. An improved understanding of the procedure will likely minimize this possibility and mitigate the consequences should malposition occur.

Survey and evaluation of mutations in the human KLF1 transcription unit.

Erythroid Krüppel-like Factor (EKLF/KLF1) is an erythroid-enriched transcription factor that plays a global role in all aspects of erythropoiesis, including cell cycle control and differentiation. We queried whether its mutation might play a role in red cell malignancies by genomic sequencing of the KLF1 transcription unit in cell lines, erythroid neoplasms, dysplastic disorders, and leukemia. In addition, we queried published databases from a number of varied sources. In all cases we only found changes in commonly notated SNPs. Our results suggest that if there are mutations in KLF1 associated with erythroid malignancies, they are exceedingly rare.

DEK is required for homologous recombination repair of DNA breaks.

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

Eosinophilic myocarditis: case series and review of literature.

Although the etiology of eosinophilic myocarditis (EM) is not always apparent, several causes are identified, including hypersensitivity to a drug or substance, with the heart as the target organ. However, symptoms and signs of hypersensitivity are not found in all patients. EM can lead to progressive myocardial damage with destruction of the conduction system and refractory heart failure. The present report describes three cases of biopsy-proven EM with different presentations, including acute coronary syndrome, cardiogenic shock and newly diagnosed heart failure. In one patient, hypersensitivity to sumatriptan was suspected to be the underlying cause. All patients responded well to treatment with steroids, angiotensin-converting enzyme inhibitors and beta-blockers. There was a complete recovery of the ventricular function in all cases.

Two different "tales" of ATG7: Clinical relevance to myelodysplastic syndromes.

Somatic mutations in U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) are associated with various cancers including myelodysplastic syndrome (MDS). Mutant U2AF1 promotes malignant transformation by inhibiting autophagy, partly as a result of alterations in the 3' tail of ATG7. This results in altered mitochondrial function, increased reactive oxygen species production, and genomic instability.

Physiologic Expression of Sf3b1(K700E) Causes Impaired Erythropoiesis, Aberrant Splicing, and Sensitivity to Therapeutic Spliceosome Modulation.

More than 80% of patients with the refractory anemia with ring sideroblasts subtype of myelodysplastic syndrome (MDS) have mutations in Splicing Factor 3B, Subunit 1 (SF3B1). We generated a conditional knockin mouse model of the most common SF3B1 mutation, Sf3b1(K700E). Sf3b1(K700E) mice develop macrocytic anemia due to a terminal erythroid maturation defect, erythroid dysplasia, and long-term hematopoietic stem cell (LT-HSC) expansion. Sf3b1(K700E) myeloid progenitors and SF3B1-mutant MDS patient samples demonstrate aberrant 3' splice-site selection associated with increased nonsense-mediated decay. Tet2 loss cooperates with Sf3b1(K700E) to cause a more severe erythroid and LT-HSC phenotype. Furthermore, the spliceosome modulator, E7017, selectively kills SF3B1(K700E)-expressing cells. Thus, SF3B1(K700E) expression reflects the phenotype of the mutation in MDS and may be a therapeutic target in MDS.

Human MutS and FANCM complexes function as redundant DNA damage sensors in the Fanconi Anemia pathway.

The Fanconi Anemia (FA) pathway encodes a DNA damage response activated by DNA damage-stalled replication forks. Current evidence suggests that the FA pathway initiates with DNA damage recognition by the FANCM complex (FANCM/FAAP24/MHF). However, genetic inactivation of FANCM in mouse and DT40 cells causes only a partial defect in the FA pathway activation, suggesting the existence of redundant DNA damage sensors. Here we show that the MutS homologs function in this capacity. A RNAi screen revealed that MSH2 silencing caused defective FA pathway activation, as assessed by damage-induced FANCD2 mono-ubiquitination. A similar FA pathway defect was observed with MSH3 or MSH6 silencing. MSH2 depletion caused cellular phenotypes associated with defective FA pathway, including mitomycin C hypersensitivity and chromosomal instability. Further, silencing of FANCM in MSH2 deficient HEC59 cells caused a more severe FA defect relative to comparable silencing in MSH2 complemented HEC59+Chr2 cells, suggesting redundant functions between MSH2 and FANCM. Consistent with this hypothesis, depletion of MSH2 resulted in defective chromatin localization of the FA core complex upon DNA damage. Further, MSH2 was co-purified and co-immunoprecipitated with FA core complex components. Taken together, our results suggest that human MutS homologs and FANCM complexes function as redundant DNA damage sensors of the FA pathway.

Hepatic lipase deficiency in a Middle-Eastern-Arabic male.

Hepatic lipase (HL) deficiency is a rare genetic disorder that has been associated with premature atherosclerosis despite high plasma high-density lipoprotein (HDL) cholesterol concentrations in the affected individuals. The authors describe the clinical and biochemical features of HL deficiency in a young male of Middle-Eastern-Arabic origin. This is the first report of cholesterol ester transfer protein (CETP) activity and mass in HL deficiency in a patient from this ethnic group. While the CETP mass was high, its activity was low, a discrepancy likely due to the abnormal composition of patient's HDL particles.