RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by aggressive local invasion and metastatic spread, leading to high lethality. Although driver gene mutations during PDA progression are conserved, no specific mutation is correlated with the dissemination of metastases1-3. Here we analysed RNA splicing data of a large cohort of primary and metastatic PDA tumours to identify differentially spliced events that correlate with PDA progression. De novo motif analysis of these events detected enrichment of motifs with high similarity to the RBFOX2 motif. Overexpression of RBFOX2 in a patient-derived xenograft (PDX) metastatic PDA cell line drastically reduced the metastatic potential of these cells in vitro and in vivo, whereas depletion of RBFOX2 in primary pancreatic tumour cell lines increased the metastatic potential of these cells. These findings support the role of RBFOX2 as a potent metastatic suppressor in PDA. RNA-sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. Modulation of RBFOX2-regulated splicing events, such as via myosin phosphatase RHO-interacting protein (MPRIP), is associated with PDA metastases, altered cytoskeletal organization and the induction of focal adhesion formation. Our results implicate the splicing-regulatory function of RBFOX2 as a tumour suppressor in PDA and suggest a therapeutic approach for metastatic PDA.

Inhibition of nonsense-mediated mRNA decay may improve stop codon read-through therapy for Duchenne muscular dystrophy.

Duchene muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are genetic neuromuscular disorders that affect skeletal and cardiac muscle resulting from mutations in the dystrophin gene (DMD), coding for dystrophin protein. Read-through therapies hold great promise for the treatment of genetic diseases harboring nonsense mutations, such as DMD/BMD, as they enable a complete translation of the affected mRNA. However, to date, most read-through drugs have not achieved a cure for patients. One possible explanation for the limitation of these therapies for DMD/BMD is that they rely on the presence of mutant dystrophin mRNAs. However, the mutant mRNAs containing premature termination codons are identified by the cellular surveillance mechanism, the nonsense-mediated mRNA decay (NMD) process, and are degraded. Here, we show that the combination of read-through drugs together with known NMD inhibitors have a synergistic effect on the levels of nonsense-containing mRNAs, among them the mutant dystrophin mRNA. This synergistic effect may enhance read-through therapies' efficacy and improve the current treatment for patients.

The efficiency of cabergoline vs pyridoxine for lactation inhibition-a randomized controlled trial.

BACKGROUND: Some mothers may seek lactation inhibition on personal, social, or medical grounds. The common drug used for lactation inhibition is cabergoline. Several adverse effects and contraindications are known for this drug. Its use is contraindicated for patients with hypertensive disorders and fibrotic, cardiac, or hepatic diseases. In addition, pyridoxine (vitamin B6) has been used for this indication, with no significant adverse effect, following studies that demonstrated its efficacy. OBJECTIVE: This study aimed to compare the efficiency of cabergoline vs pyridoxine for lactation inhibition. STUDY DESIGN: A randomized controlled trial was conducted. Postpartum patients who requested lactation inhibition were randomly allocated to receive either cabergoline (1 mg once on postpartum day 1 or divided to 0.25 mg twice a day for 2 days thereafter, according to the departmental protocol, which is in line with the manufacturer recommendations) or pyridoxine (200 mg 3 times a day for 7 days). The patients enrolled were free of diseases in which contraindications to cabergoline are present. All patients completed a questionnaire for assessing breast engorgement, breast pain, and milk leakage on a scale of 0 (no symptom) to 5 (severe symptom) on days 0, 2, 7, and 14. The primary outcome was lactation inhibition success, defined as a score of 0 for both engorgement and pain on day 7. The secondary outcomes included the assessment of milk leakage, adverse effects, fever, mastitis, and treatment discontinuation or alteration. RESULTS: Of note, 45 and 43 patients received cabergoline or pyridoxine, respectively, and were included in the analysis following the intention-to-treat principle. Cabergoline was superior to pyridoxine in inhibiting lactation at day 7 (78% vs 35%, respectively; P<.0001). Mild symptoms, defined as a score of 0 to 2 for breast engorgement and pain, at day 7 were 40 (89%) in the cabergoline group and 29 (67%) in the pyridoxine group (P=.01). The incidence of milk leakage was lower in the cabergoline group after 7 and 14 days than in the pyridoxine group (9% vs 42% [P=.0003] and 11% vs 31% [P=.02], respectively). Cabergoline had more adverse effects than pyridoxine (31% vs 9%, respectively; P=.01), but all adverse effects were mild. The rates of mastitis and fever that were related to engorgement were similar in the cabergoline and pyridoxine groups (4 [9%] vs 2 [5%], respectively; P=.67). Furthermore, 9 patients (21%) in the pyridoxine group switched to or added cabergoline because of treatment failure. Accordingly, on day 7, the pyridoxine success rate was reduced from 35% (15 women) to 28% (12 women) and from 67% (29 women) to 53% (23 women) for a score of 0 and 0 to 2 for both engorgement and pain, respectively. CONCLUSION: Cabergoline was superior to pyridoxine in inhibiting lactation. Cabergoline had more adverse effects, but no major adverse effect was documented in either treatment group. As pyridoxine inhibited lactation successfully in previous studies and in 67% of patients in this study, its use should be considered in women with contraindications for cabergoline.

Bleeding Risk Assessment and the Role of Primary Hemostasis Screening in Patients Undergoing Kidney Biopsy.

BACKGROUND: Risk factors for bleeding complications after percutaneous kidney biopsy (PKB) and the role of primary hemostasis screening are not well established. OBJECTIVES: To determine the role of primary hemostasis screening and complication outcomes among individuals who underwent PKB. METHODS: We reviewed data of 456 patients who underwent PKB from 2010 to 2016 in a large university hospital. In 2015, bleeding time (BT) testing was replaced by light transmission aggregometry (LTA) as a pre-PKB screening test. RESULTS: Of the 370 patients who underwent pre-PKB hemostasis screening by BT testing, prolonged BT was observed in 42 (11.3%). Of the 86 who underwent LTA, an abnormal response was observed in 14 (16.3%). Overall, 155 (34.0%) patients experienced bleeding: 145 (31.8%) had minor events (hemoglobin fall of 1-2 g/dl, macroscopic hematuria, perinephric hematoma without the need for transfusion or intervention) and 17 (3.7%) had major events (hemoglobin fall > 2 g/dl, blood transfusion or further intervention). Abnormal LTA response did not correlate with bleeding (P = 0.80). In multivariate analysis, only prolonged BT (P = 0.0001) and larger needle size (P = 0.005) were identified as independent predictors of bleeding. CONCLUSIONS: Bleeding complications following PKB were common and mostly minor, and the risk of major bleeding was low. Larger needle size and prolonged BT were associated with a higher bleeding risk. Due to the relatively low risk of major bleeding and lack of benefit of prophylactic intervention, the use of pre-PKB hemostasis screening remains unestablished.

The role of RNA alternative splicing in regulating cancer metabolism.

Tumor cells alter their metabolism by a wide array of mechanisms to promote growth and proliferation. Dysregulated expression and/or somatic mutations of key components of the glycolytic pathway/TCA cycle as well as other metabolic pathways allow tumor cells to improve their ability to survive harsh conditions such as hypoxia and the presence of reactive oxygen species, as well as the ability to obtain nutrients to increase lipids, protein, and nucleic acids biogenesis. Approximately 95% of the human protein encoding genes undergo alternative splicing (AS), a regulated process of gene expression that greatly diversifies the proteome by creating multiple proteins from a single gene. In recent years, a growing body of evidence suggests that unbalanced AS, the formation of certain pro-tumorigenic isoforms and the reduction of anti-tumorigenic isoforms, is implicated in a variety of cancers. It is becoming increasingly clear that cancer-associated AS contributes to increased growth and proliferation, partially due to effects on metabolic reprogramming. Here, we summarize the known roles of AS in regulating cancer metabolism. We present evidence supporting the idea that AS, in many types of cancer, acts as a molecular switch that alters metabolism to drive tumorigenesis. We propose that the elucidation of misregulated AS and its downstream effects on cancer metabolism emphasizes the need for new therapeutic approaches aiming to modulate the splicing machinery to selectively target cancer cells.

Pharmacological treatment of ectopic pregnancy: Accuracy, safety and cost-effectiveness of day 1-7 ß-hCG measurements.

OBJECTIVE: The aim of the present study was to compare accuracy, safety and cost-effectiveness of three ß-hCG measurements protocols, applied in managing ectopic pregnancies (EP) with methotrexate (MTX): (1) day 1 to 7 ß-hCG levels, (2) day 1 to 4 ß-hCG levels and (3) day 4 to 7 ß-hCG levels. METHODS: Cost-minimization analysis (CMA) based on a retrospective study of patients treated with single-dose MTX for EP, was evaluated at a single institution between January 2001 to May 2021. Successful MTX treatment was defined as no surgical intervention. We evaluated safety by analyzing cases of day 4 interventions and cases of inconsistency between the different protocols. Predicting accuracy was assessed by the area under the receiver operating characteristic (AUC) curve. RESULTS: A total of 229 patients with single dose MTX treatment were included. Overall, 184 (80.3%) patients were treated successfully with a single dose of MTX. For days 1 and 7 the optimal cutoff point was 7% reduction in ß-hCG levels with sensitivity, specificity and PPV of 76.6% (69.9-82.5, 95% CI), 75.5% (60.5-87.1, 95% CI) and 92.8% (88.4-95.6, 95% CI), respectively. There was no significant difference between the protocols' AUC. None of the patients had any change of management during their day 4 visit in our 20 years of records. The cost for each visit day (day 4 and 7) was calculated with a total cost of 251 USD per patient. CONCLUSION: Patients treated with MTX for EP, measurement of day 1 and day 7 ß-hCG serum levels has a cost minimization advantage and is not inferior to the traditional protocol for predictive accuracy and safety.

Postpartum hemorrhage among twin pregnancies - Medically assisted versus spontaneously conceived.

INTRODUCTION: In singleton pregnancies, an increased risk of Postpartum hemorrhage (PPH) have been linked with assisted reproductive technology (ART) and abnormal placentation. It is unknown wheather such association exists in twin pregnancies conceived by Medically assisted reproduction (MAR). The aim of the current study was to compare maternal blood loss among twin pregnancies conceived by different types of MAR treatments to spontaneously conceived twins and to identify the cycle characteristics if an association exits. METHODS: Retrospective study conducted on data collected between 2011 and 2020. The study cohort included all twin pregnancies conceived by MAR and born at our institution. Controls were spontaneously conceived twins matched for maternal age on a 1:2 (study: controls) ratio. RESULTS: Overall 113 MAR twin births categorized into three groups; 25 ovulation induction, 59 fresh ART, 29 frozen-thawed ART cycles, and 226 controls were included. The incidence of PPH was higher among MAR twin pregnancies (5.3%) compared to the controls (4%). The highest incidence was observed among women in the frozen-thawed group (13.8%) which differed significantly compared with the controls (p = 0.024). A significant difference was also observed in the mean decrease of postpartum hemoglobin levels between these two groups (2.13 g/dL versus 1.3 g/dL respectively, p = 0.002). Blood transfusion was nearly 2.5 times more common in the frozen-thawed group (3.4%) compared to the control group (1.3%). DISCUSSION: The present study demonstrates that frozen embryo transfer (FET) ART-conceived twin pregnancies are associated with a markedly increased rate of PPH compared to spontaneously conceived twins.

Comparison of Different Finishing and Polishing Systems on Surface Roughness and Bacterial Adhesion of Resin Composite.

Insufficient dental restoration finishing and polishing may lead to plaque accumulation, gingival inflammation, staining, caries, and esthetic impairment. Here, the effect of two finishing and polishing systems on surface roughness and bacterial adhesion were evaluated. Two finishing and polishing kits were evaluated: diamond burs (Shine 1-2, Strauss & Co, Raanana, Israel) and paper discs (Sof-Lex 3M ESPE, Seefeld, Germany) (n = 30 each). For each group surface roughness was evaluated using an optical profilometer (Contour GT-K1, Bruker, Billerica, MA, USA) (n = 10). Surface bacteria were evaluated for biofilm biomass using crystal violet (CV) staining (absorbance measured at 538 nm) and viable counts (CFU/mL) (n = 20). The control group included polymerized discs against a Mylar strip (n = 30). Student's t test and one-way ANOVA were used for statistical evaluation. Diamond burs, paper discs, and control average surface RA were 169.4 ± 45.2 µ, 364 ± 77.7 µ, and 121.2 ± 18.1 µ, respectively. There was a significant difference found between all groups (p < 0.00001). Bacterial biomass on diamond burs, paper discs, and control samples were 0.458 ± 0.161, 0.507 ± 0.139, and 0.446 ± 0.142, respectively (p = 0.257). Viable bacterial counts (CFU/mL) on diamond burs, paper discs, and control samples were 2.25 × 104, 2.95 × 104, and 2.75 × 104, respectively (p = 0.856). A comparison between two finishing and polishing kits showed that the shine 1−2 diamond bur kit produced a smoother surface than the polishing disc kit. No differences were found in the biofilm biomass quantification and bacterial viable count between the groups.

S6K1 phosphorylates Cdk1 and MSH6 to regulate DNA repair.

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.

Damage to the DNA in our cells can cause harmful changes that, if unchecked, can lead to the development of cancer. To help prevent this, cellular mechanisms are in place to repair defects in the DNA. A particular process, known as the mTORC1-S6K1 pathway is suspected to be important for repair because when this pathway is blocked, cells become more sensitive to DNA damage. It is still unknown how the various proteins involved in the mTORC1-S6K1 pathway contribute to repairing DNA. One of these proteins, S6K1, is an enzyme involved in coordinating cell growth and survival. The tumor cells in some forms of breast cancer produce more of this protein than normal, suggesting that S6K1 benefits these cells' survival. However, it is unclear exactly how the enzyme does this. Amar-Schwartz, Ben-Hur, Jbara et al. studied the role of S6K1 using genetically manipulated mouse cells and human cancer cells. These experiments showed that the protein interacts with two other proteins involved in DNA repair and activates them, regulating two different repair mechanisms and protecting cells against damage. These results might explain why some breast cancer tumors are resistant to radiotherapy and chemotherapy treatments, which aim to kill tumor cells by damaging their DNA. If this is the case, these findings could help clinicians choose more effective treatment options for people with cancers that produce additional S6K1. In the future, drugs that block the activity of the enzyme could make cancer cells more susceptible to chemotherapy.

Induction and blockage of oligodendrogenesis by differently activated microglia in an animal model of multiple sclerosis.

The role of activated microglia (MG) in demyelinating neurodegenerative diseases such as multiple sclerosis is controversial. Here we show that high, but not low, levels of IFN-gamma (a cytokine associated with inflammatory autoimmune diseases) conferred on rodent MG a phenotype that impeded oligodendrogenesis from adult neural stem/progenitor cells. IL-4 reversed the impediment, attenuated TNF-alpha production, and overcame blockage of IGF-I production caused by IFN-gamma. In rodents with acute or chronic EAE, injection of IL-4-activated MG into the cerebrospinal fluid resulted in increased oligodendrogenesis in the spinal cord and improved clinical symptoms. The newly formed oligodendrocytes were spatially associated with MG expressing MHC class II proteins and IGF-I. These results point to what we believe to be a novel role for MG in oligodendrogenesis from the endogenous stem cell pool.

Microglia activated by IL-4 or IFN-gamma differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells.

Cell renewal in the adult central nervous system (CNS) is limited, and is blocked in inflammatory brain conditions. We show that both neurogenesis and oligodendrogenesis of adult neural progenitor cells in mice are blocked by inflammation-associated (endotoxin-activated) microglia, but induced by microglia activated by cytokines (IL-4 or low level of IFN-gamma) associated with T-helper cells. Blockage was correlated with up-regulation of microglial production of tumor necrosis factor-alpha. The effect induced by IL-4-activated microglia was mediated, at least in part, by insulin-like growth factor-I. The IL-4-activated microglia showed a bias towards oligodendrogenesis whereas the IFN-gamma-activated microglia showed a bias towards neurogenesis. It thus appears that microglial phenotype critically affects their ability to support or impair cell renewal from adult stem cell.