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Summary The study aims at assessing wound healing and safety of single-stage two-layers continuous closure in patients with unilateral cleft lip and palate (UCLP). Patients and methods In this retrospective, descriptive cohort study, we assessed wound healing without fistula formation at 1, 3, and 6 months after a single-stage two-layer UCLP repair, in which the midline suture is continuously circular all along the oral and nasal sides. We examined lengths of hospital stay and the incidence of intra- and postoperative adverse events. Furthermore, we compared the cleft width at birth and on the day of surgery, after presurgical orthopaedics. Results Eleven UCLP patients underwent one cleft surgery between July 2016 and June 2018 at the age of 8 to 9 months. Full primary healing occurred in all patients without fistulas. Median length of post-operative hospital stay was 5 days (range = 4–9 days). No intra- or postoperative adverse events above Grade I (according to ClassIntra and Clavien-Dindo, respectively) occurred. Median and interquartile range (IQR) of the palatal cleft width decreased significantly from birth to surgery, i.e., from 12.0 mm (10.8–13.6 mm) to 5.0 mm (4.0–7.5 mm) anteriorly and from 14.0 mm (11.5-15.0 mm) to 7.3 mm (6.0-8.5 mm) posteriorly (p=0.0033 in both cases). Conclusion Given these preliminary results, the concept of single-stage continuous circular closure in UCLP has potential for further investigation. In particular, it remains to be proven that there are no relevant adverse effects such as inhibition of maxillary growth. Registered in clinicaltrials.gov:NCT04108416.

Genotoxic stress induces alternative splicing of the oncogene MDM2 generating MDM2-ALT1, an isoform attributed with tumorigenic properties. However, the mechanisms underlying this event remain unclear. Here we explore MDM2 splicing regulation by utilizing a novel minigene that mimics endogenous MDM2 splicing in response to UV and cisplatinum-induced DNA damage. We report that exon 11 is necessary and sufficient for the damage-specific alternative splicing of the MDM2 minigene and that the splicing factor SRSF1 binds exon 11 at evolutionarily conserved sites. Interestingly, mutations disrupting this interaction proved sufficient to abolish the stress-induced alternative splicing of the MDM2 minigene. Furthermore, SRSF1 overexpression promoted exclusion of exon 11, while its siRNA-mediated knockdown prevented the stress-induced alternative splicing of endogenous MDM2. Additionally, we observed elevated SRSF1 levels under stress and in tumors correlating with the expression of MDM2-ALT1. Notably, we demonstrate that MDM2-ALT1 splicing can be blocked by targeting SRSF1 sites on exon 11 using antisense oligonucleotides. These results present conclusive evidence supporting a negative role for SRSF1 in MDM2 alternative splicing. Importantly, we define for the first time, a clear-cut mechanism for the regulation of damage-induced MDM2 splicing and present potential strategies for manipulating MDM2 expression via splicing modulation.

The Rbfox family of RNA-binding proteins is highly conserved with established roles in alternative splicing (AS) regulation. High-throughput studies aimed at understanding transcriptome remodeling have revealed skeletal muscle as displaying one of the largest number of AS events. This finding is consistent with requirements for tissue-specific protein isoforms needed to sustain muscle-specific functions. Rbfox1 is abundant in vertebrate brain, heart and skeletal muscle. Genome-wide genetic approaches have linked the Rbfox1 gene to autism, and a brain-specific knockout mouse revealed a critical role for this splicing regulator in neuronal function. Moreover, a Caenorhabditis elegans Rbfox1 homolog regulates muscle-specific splicing. To determine the role of Rbfox1 in muscle function, we developed a conditional knockout mouse model to specifically delete Rbfox1 in adult tissue. We show that Rbfox1 is required for muscle function but a >70% loss of Rbfox1 in satellite cells does not disrupt muscle regeneration. Deep sequencing identified aberrant splicing of multiple genes including those encoding myofibrillar and cytoskeletal proteins, and proteins that regulate calcium handling. Ultrastructure analysis of Rbfox1(-/-) muscle by electron microscopy revealed abundant tubular aggregates. Immunostaining showed mislocalization of the sarcoplasmic reticulum proteins Serca1 and Ryr1 in a pattern indicative of colocalization with the tubular aggregates. Consistent with mislocalization of Serca1 and Ryr1, calcium handling was drastically altered in Rbfox1(-/-) muscle. Moreover, muscle function was significantly impaired in Rbfox1(-/-) muscle as indicated by decreased force generation. These results demonstrate that Rbfox1 regulates a network of AS events required to maintain multiple aspects of muscle physiology.

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition.

Alternative splicing of the oncogene MDM2 is a phenomenon that occurs in cells in response to genotoxic stress and is also a hallmark of several cancer types with important implications in carcinogenesis. However, the mechanisms regulating this splicing event remain unclear. Previously, we uncovered the importance of intron 11 in MDM2 that affects the splicing of a damage-responsive MDM2 minigene. Here, we have identified discrete cis regulatory elements within intron 11 and report the binding of FUBP1 (Far Upstream element-Binding Protein 1) to these elements and the role it plays in MDM2 splicing. Best known for its oncogenic role as a transcription factor in the context of c-MYC, FUBP1 was recently described as a splicing regulator with splicing repressive functions. In the case of MDM2, we describe FUBP1 as a positive splicing regulatory factor. We observed that blocking the function of FUBP1 in in vitro splicing reactions caused a decrease in splicing efficiency of the introns of the MDM2 minigene. Moreover, knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions. These results indicate that FUBP1 is also a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1.

SummaryCardiac dysfunction is the second leading cause of death in myotonic dystrophy type 1 (DM1), primarily because of arrhythmias and cardiac conduction defects. A screen of more than 500 microRNAs (miRNAs) in a DM1 mouse model identified 54 miRNAs that were differentially expressed in heart. More than 80% exhibited downregulation toward the embryonic expression pattern and showed a DM1-specific response. A total of 20 of 22 miRNAs tested were also significantly downregulated in human DM1 heart tissue. We demonstrate that many of these miRNAs are direct MEF2 transcriptional targets, including miRNAs for which depletion is associated with arrhythmias or fibrosis. MEF2 protein is significantly reduced in both DM1 and mouse model heart samples, and exogenous MEF2C restores normal levels of MEF2 target miRNAs and mRNAs in a DM1 cardiac cell culture model. We conclude that loss of MEF2 in DM1 heart causes pathogenic features through aberrant expression of both miRNA and mRNA targets.

Pediatric rhabdomyosarcoma (RMS) is a morphologically and genetically heterogeneous malignancy commonly classified into three histologic subtypes, namely, alveolar, embryonal, and anaplastic. An issue that continues to challenge effective RMS patient prognosis is the dearth of molecular markers predictive of disease stage irrespective of tumor subtype. Our study involving a panel of 70 RMS tumors has identified specific alternative splice variants of the oncogenes Murine Double Minute 2 (MDM2) and MDM4 as potential biomarkers for RMS. Our results have demonstrated the strong association of genotoxic-stress inducible splice forms MDM2-ALT1 (91.6% Intergroup Rhabdomyosarcoma Study Group stage 4 tumors) and MDM4-ALT2 (90.9% MDM4-ALT2-positive T2 stage tumors) with high-risk metastatic RMS. Moreover, MDM2-ALT1-positive metastatic tumors belonged to both the alveolar (50%) and embryonal (41.6%) subtypes, making this the first known molecular marker for high-grade metastatic disease across the most common RMS subtypes. Furthermore, our results show that MDM2-ALT1 expression can function by directly contribute to metastatic behavior and promote the invasion of RMS cells through a matrigel-coated membrane. Additionally, expression of both MDM2-ALT1 and MDM4-ALT2 increased anchorage-independent cell-growth in soft agar assays. Intriguingly, we observed a unique coordination in the splicing of MDM2-ALT1 and MDM4-ALT2 in approximately 24% of tumor samples in a manner similar to genotoxic stress response in cell lines. To further explore splicing network alterations with possible relevance to RMS disease, we used an exon microarray approach to examine stress-inducible splicing in an RMS cell line (Rh30) and observed striking parallels between stress-responsive alternative splicing and constitutive splicing in RMS tumors.

Novel ALK fusion in anaplastic large cell lymphoma involving EEF1G, a subunit of the eukaryotic elongation factor-1 complex

Studies support the importance of microRNAs in physiological and pathological processes. Here we describe the regulation and function of miR-29 in myogenesis and rhabdomyosarcoma (RMS). Results demonstrate that in myoblasts, miR-29 is repressed by NF-kappaB acting through YY1 and the Polycomb group. During myogenesis, NF-kappaB and YY1 downregulation causes derepression of miR-29, which in turn accelerates differentiation by targeting its repressor YY1. However, in RMS cells and primary tumors that possess impaired differentiation, miR-29 is epigenetically silenced by an activated NF-kappaB-YY1 pathway. Reconstitution of miR-29 in RMS in mice inhibits tumor growth and stimulates differentiation, suggesting that miR-29 acts as a tumor suppressor through its promyogenic function. Together, these results identify a NF-kappaB-YY1-miR-29 regulatory circuit whose disruption may contribute to RMS.

Myocardial infarction (MI) is currently among the leading causes of death in the developed world. A functional SNP (rs7291467) in galectin-2 (LGALS2), a protein involved in the LTA cascade, has been associated with susceptibility to MI in the Japanese population. We explored for the first time the hypothesis that the same SNP could be associated with the risk of MI in the British population. We conducted a case-control association study on a cohort of 752 British MI patients and 705 population controls. Power calculations showed that our resource had 98% of power to detect a significant association at OR of 1.57, and 80% power to detect an association with an OR of 1.35 (recessive model). Despite this, we found no significant association of allele frequency with risk of MI. Stratification for age, gender and other cardiovascular risk factors also failed to reveal an association of this polymorphism with MI.

To assess the feasibility of using a two-dimensional partial Fourier (PF) reconstruction scheme to reduce the acquisition time of magnetic resonance imaging (MRI) of coronary arteries.Symmetric k-space data sets of coronary arteries were collected in seven volunteers using a three-dimensional breath-hold steady-state free precession (SSFP) sequence. Partial, asymmetric k-space data sets were generated by removing 25% of the data in the readout direction and 25% of the data in the phase encoding direction. The missing data were then estimated using a two-dimensional projection-onto-convex-sets (POCS) algorithm or filled with zeroes. Images were reconstructed from the full data set, the PF data set, and the zero-filled (ZF) data set, respectively. Coronary artery sharpness was evaluated quantitatively and qualitatively.Coronary artery sharpness in PF images was comparable to that in full k-space images and significantly better than that in ZF images.Two-dimensional POCS PF reconstruction is a potentially useful technique for reducing acquisition time or improving spatial resolution for breath-hold coronary MR angiography.

Seroconversion of immunoglobulin A (IgA) and immunoglobulin G (IgG) (> or =4-fold rise) to rotavirus nonstructural protein 4 (NSP4) was determined, by use of enzyme-linked immunosorbent assay with fusion proteins glutathione S-transferase (GST)-NSP4 from strains SA11 (A), 116E (B), and RRV (C), in 40 children with acute rotavirus gastroenteritis and in 30 with the same disease due to other pathogens. The IgG seroconversion rates in the rotavirus group were 67.5%, 70%, and 60% when recombinant (r) NSP4A, -B, and -C, respectively, were used as antigen in the assay, and, for rotavirus-uninfected children, rates were 10%, 13%, and 7%. IgA seroconversion occurred in 57%, 70%, and 50%, respectively, of children with rotavirus gastroenteritis; in rotavirus-uninfected children, 1 child each seroconverted to the different rNSP4s. Among 9 children infected with strain NSP4A, 7, 6, and 5 children showed IgG seroconversion, and, among 18 infected with NSP4A, -B, and -C, 16, 17, and 15, respectively, showed IgG seroconversion. Between NSP4A-infected and NSP4B-infected children, IgA responses were similar to IgG responses. In conclusion, significant NSP4-specific antibody response occurs in natural rotavirus infection, and the antibody response appears to be broad and heterotypic in nature.

Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1, this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1(+/-) mice to SCA1 mice (Atxn1(154Q/+)) exacerbated disease progression, whereas breeding them to Atxn1(+/-) mice normalized Ataxin1 levels and largely rescued the Pum1(+/-) phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.

MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

Right ventricular (RV) function often determines clinical outcome in patients with valvular heart disease. Though difficult to assess by echocardiography, Tei index is useful in its assessment. The aims of the study were to evaluate global RV function using the Tei index in patients with rheumatic heart disease and to observe if such abnormalities in RV function were reversible post-operatively.The study included patients with atrial septal defect (ASD, Group I, n = 15) and rheumatic valvular heart disease (RVHD, Group II, n = 18). Patients with atrial fibrillation were excluded from the study. Conventional 2-D echocardiography was performed preoperatively, immediate postoperative and in last follow-up.ASD group had lower left LVES and LVED dimensions as compared to RHVD (p = 0.001) and better ejection fraction (EF) than RVHD group (p = 0.02). LV Tei in the ASD group was above the normal limit (0.5), while RV Tei was increased in the RHVD group. The median RVSP was similar in two groups (p = 0.9). The impaired LVMPI in the ASD group improved as early as 2 weeks following surgery (p = 0.09) while in patients with RHVD it deteriorated which mirrored the reduction in median LVEF (p = 0.04). Group II that had an abnormal RV Tei pre-operatively demonstrated improvement following surgery (p = 0.03).RVHD is associated with impairment of RV function. Volume overload of RV in patients of ASD is associated with normal MPI. The abnormalities in RVMPI improved as early as 2 weeks after valve surgery with sustained improvement noted at follow up.

Acute mitral regurgitation (MR) may cause adverse hemodynamics following mitral balloon valvotomy (MBV). Some patients become severely hemodynamically unstable and require emergency mitral valve replacement (MVR), while others remain relatively stable with medical management. The study aim was to identify factors that would predict severe acute MR leading to severe hemodynamic compromise requiring emergency MVR.Between January 2001 and July 2009, a total of 46 patients developed acute severe MR following MBV at the authors' institution. Of these patients, 11 developed severe hemodynamic compromise and required emergency MVR within 6 h of the procedure (group I), while 35 were relatively stable, improved with time, and were discharged with advice to undergo an early MVR (group II).The demographic profile and routine echocardiographic parameters were comparable between the two groups. In group I, the right ventricular systolic pressure (RVSP) before and after MBV was significantly higher, and a significantly higher level of calcium was present in the mitral valve leaflets. Univariate analysis of the RVSP before and after MBV predicted the occurrence of hemodynamic instability leading to emergency MVR. The receiver operating characteristic (ROC) curve for RVSP before and after MBV had a significant area under the curve (0.944, p0.005 and 0.940, p0.005, respectively). Based on the ROC data, the pre- and post-MBV RVSPs of 76 mmHg and 77 mmHg, respectively, predicted the possibility of emergency MVR, with sensitivities and specificities of 72% and 63%, and 100% and 90%, respectively.Patients undergoing MBV with an RVSP76 mmHg and the presence of non-commissural calcium on the mitral valve leaflet, or those who develop an RVSP of 77 mmHg following the procedure will very likely require emergency MVR.

In metazoans, alternative splicing of genes is essential for regulating gene expression and contributing to functional complexity. Computational predictions, comparative genomics, and transcriptome profiling of normal and diseased tissues indicate that an unexpectedly high fraction of diseases are caused by mutations that alter splicing. Mutations in cis elements cause missplicing of genes that alter gene function and contribute to disease pathology. Mutations of core spliceosomal factors are associated with hematolymphoid neoplasias, retinitis pigmentosa, and microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Mutations in the trans regulatory factors that control alternative splicing are associated with autism spectrum disorder, amyotrophic lateral sclerosis (ALS), and various cancers. In addition to discussing the disorders caused by these mutations, this review summarizes therapeutic approaches that have emerged to correct splicing of individual genes or target the splicing machinery.

The tumor suppressor protein p53 is a transcription factor that induces G1 arrest of the cell cycle and/or apoptosis. The murine double-minute protein MDM2 and its homologue MDM4 (also known as MDMX) are critical regulators of p53. Altered transcripts of the human homologue of mdm2, MDM2, have been identified in human tumors, such as invasive carcinoma of the breast, lung carcinoma, and liposarcoma. MDM2 alternate forms act to negatively regulate the normal MDM2 gene product, thus activating p53. Although many reports have documented a plethora of tumor types characterized by MDM2 alternative transcripts, few have investigated the signals that might initiate alternative splicing. We have identified a novel role of these alternative MDM2 transcripts in the normal surveillance mechanism of the cell and in DNA damage response. We report that alternate forms of MDM2 are detected after UV irradiation. Furthermore, we show that mouse cells treated with UV are also characterized by alternative transcripts of mdm2, suggesting that this is an important and evolutionarily conserved mechanism for regulating the expression of MDM2/mdm2. An additional p53 regulator and mdm2 family member, MDM4, is likewise alternatively spliced following UV irradiation. By activating alternative splicing of both MDM2 and MDM4, yet another layer of p53 regulation is initiated by the cells in response to damage. A stepwise model for malignant conversion by which alternate forms of MDM2 and MDM4 place selective pressure on the cells to acquire additional alterations in the p53 pathway is herein proposed. (Cancer Res 2006; 66(19): 9502-8)

Objective— The platelet ADP receptors P2Y1 and P2Y12 play a pivotal role in platelet aggregation. There is marked interindividual variation in platelet response to ADP. We studied whether genetic variants in the P2Y1 or P2Y12 genes affect platelet response to ADP. Methods and Results— The P2Y1 and P2Y12 genes were screened for polymorphisms. Associations between selected polymorphisms and the platelet response to ADP (0.1, 1.0, and 10 μmol/L), assessed by whole blood flow cytometric measurement of fibrinogen binding to activated glycoprotein IIb-IIIa, were then determined in 200 subjects. Five polymorphisms were found in the P2Y1 gene and 11 in the P2Y12 gene. All polymorphisms were silent. A P2Y1 gene dimorphism, 1622A〉G, was associated with a significant ( P =0.007) effect on platelet ADP response, with a greater response in carriers of the G allele (frequency 0.15). The effect was seen at all concentrations of ADP but greatest at 0.1 μmol/L ADP, where the response in GG homozygotes was on average 130% higher than that seen in AA homozygotes ( P =0.006). Conclusions— A common genetic variant at the P2Y1 locus is associated with platelet reactivity to ADP. This genotype effect partly explains the interindividual variation in platelet response to ADP and may have clinical implications with regard to thrombotic risk.

Objective: To see whether it was possible to replicate in a prospective study the association recently reported between infection with the more virulent (type 1) cytotoxin associated gene A (CagA) antigen carrying strains of Helicobacter pylori and increased risk of coronary heart disease. Design and setting: Nested case–control study in a clinical outcomes trial. Subjects: Participants in the West of Scotland coronary prevention study. Methods: H pylori CagA serological status was determined in plasma samples of 201 subjects (cases) who subsequently had a coronary event during follow up and in 414 subjects (controls) matched for age and smoking who remained event-free, using a semiquantitative commercial enzyme linked immunosorbent assay (ELISA) kit against the p120 antigen of CagA. Results: 105 (52%) in the case group and 176 (43%) in the control group were seropositive (odds ratio (OR) 1.49, 95% confidence interval (CI) 1.06 to 2.10, p = 0.022). The association remained significant after adjustment for blood pressure, body mass index, plasma concentrations of low density lipoprotein and high density lipoprotein cholesterol, history of hypertension and diabetes, statin treatment, and socioeconomic status (OR 1.51, 95% CI 1.06 to 2.16, p = 0.023). Baseline inflammatory markers (white cell count, C reactive protein, fibrinogen) were not significantly increased in either H pylori CagA positive cases or controls. Conclusions: The findings provide support from a prospective study for the hypothesis that there is an association between infection with CagA bearing strains of H pylori and coronary heart disease. The mechanism(s) underlying the association remain to be elucidated.