Continuous circular closure in unilateral cleft lip and plate repair in one surgery.

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). 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. Eleven UCLP patients underwent one cleft surgery between July 2016 and June 2018 at the age of 8-9 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). Given these preliminary results, the concept of single-stage continuous circular closure in UCLP has potential for further investigation. However, it remains to be proven that there are no relevant adverse effects such as inhibition of maxillary growth. Registered in clinicaltrials.gov:NCT04108416.

Mechanical Anastomotic Coupling Device versus Hand-sewn Venous Anastomosis in Head and Neck Reconstruction-An Analysis of 1694 Venous Anastomoses.

Background There is a steep learning curve to attain a consistently good result in microvascular surgery. The venous anastomosis is a critical step in free-tissue transfer. The margin of error is less and the outcome depends on the surgeon's skill and technique. Mechanical anastomotic coupling device (MACD) has been proven to be an effective alternative to hand-sewn (HS) technique for venous anastomosis, as it requires lesser skill. However, its feasibility of application in emerging economy countries is yet to be established. Material and Method We retrospectively analyzed the data of patients who underwent free-tissue transfer for head and neck reconstruction between July 2015 and October 2020. Based on the technique used for the venous anastomosis, the patients were divided into an HS technique and MACD group. Patient characteristics and outcomes were measured. Result A total of 1694 venous anastomoses were performed during the study period. There were 966 patients in the HS technique group and 719 in the MACD group. There was no statistically significant difference between the two groups in terms of age, sex, prior radiotherapy, prior surgery, and comorbidities. Venous thrombosis was noted in 62 (6.4%) patients in the HS technique group and 7 (0.97%) in the MACD group ( p = 0.000). The mean time taken for venous anastomosis in the HS group was 17 ± 4 minutes, and in the MACD group, it was 5 ± 2 minutes ( p = 0.0001). Twenty-five (2.56%) patients in the HS group and 4 (0.55%) patients in MACD group had flap loss ( p = 0.001). Conclusion MACD is an effective alternative for HS technique for venous anastomosis. There is a significant reduction in anastomosis time, flap loss, and return to operation theater due to venous thrombosis. MACD reduces the surgeon's strain, especially in a high-volume center. Prospective randomized studies including economic analysis are required to prove the cost-effectiveness of coupler devices.

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.

MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of the alternatively spliced transcripts predominantly produced in response to genotoxic stress, and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 under genotoxic stress, whereas SRSF2 knockdown induced the expression of MDM2-ALT1 in the absence of genotoxic stress. Blocking the exon 11 SRSF2-binding sites using oligonucleotides promoted MDM2-ALT1 splicing and induced p53 protein expression, and apoptosis in p53 wild-type cells. The regulation of MDM2 splicing by SRSF2 is also conserved in mice, as mutation of one SRSF2-binding site in Mdm2 exon 11, using CRISPR-Cas9, increased the expression of the MDM2-ALT1 homolog Mdm2-MS2. IMPLICATIONS: Taken together, the data indicate that modulating MDM2 splicing may be a useful tool for fine-tuning p53 activity in response to genotoxic stress.

Argonaute 8 (AGO8) Mediates the Elicitation of Direct Defenses against Herbivory.

In Nicotiana attenuata, specific RNA-directed RNA polymerase (RdR1) and the Dicer-like (DCL3 and DCL4) proteins are recruited during herbivore attack to mediate the regulation of defense responses. However, the identity and role(s) of Argonautes (AGOs) involved in herbivory remain unknown. Of the 11 AGOs in the N. attenuata genome, we silenced the expression of 10. Plants silenced in NaAGO8 expression grew normally but were highly susceptible to herbivore attack. Larvae of Manduca sexta grew faster when consuming inverted-repeat stable transformants (irAGO8) plants but did not differ from the wild type when consuming plants silenced in AGO1 (a, b, and c), AGO2, AGO4 (a and b), AGO7, or AGO10 expression. irAGO8 plants were significantly compromised in herbivore-induced levels of defense metabolites such as nicotine, phenolamides, and diterpenoid glycosides. Time-course analyses revealed extensively altered microRNA profiles and the reduced accumulation of MYB8 transcripts and of the associated genes of the phenolamide and phenylpropanoid pathways as well as the nicotine biosynthetic pathway. A possible AGO8-modulated microRNA-messenger RNA target network was inferred. Furthermore, comparative analysis of domains revealed the diversity of AGO conformations, particularly in the small RNA-binding pocket, which may influence substrate recognition/binding and functional specificity. We infer that AGO8 plays a central role in the induction of direct defenses by modulating several regulatory nodes in the defense signaling network during herbivore response. Thus, our study identifies the effector AGO of the herbivore-induced small RNA machinery, which in N. attenuata now comprises RdR1, DCL3/4, and AGO8.

Splicing factor SRSF1 negatively regulates alternative splicing of MDM2 under damage.

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.

Molecular evolution and diversification of the Argonaute family of proteins in plants.

BACKGROUND: Argonaute (AGO) proteins form the core of the RNA-induced silencing complex, a central component of the smRNA machinery. Although reported from several plant species, little is known about their evolution. Moreover, these genes have not yet been cloned from the ecological model plant, Nicotiana attenuata, in which the smRNA machinery is known to mediate important ecological traits. RESULTS: Here, we not only identify 11 AGOs in N. attenuata, we further annotate 133 genes in 17 plant species, previously not annotated in the Phytozome database, to increase the number of plant AGOs to 263 genes from 37 plant species. We report the phylogenetic classification, expansion, and diversification of AGOs in the plant kingdom, which resulted in the following hypothesis about their evolutionary history: an ancestral AGO underwent duplication events after the divergence of unicellular green algae, giving rise to four major classes with subsequent gains/losses during the radiation of higher plants, resulting in the large number of extant AGOs. Class-specific signatures in the RNA-binding and catalytic domains, which may contribute to the functional diversity of plant AGOs, as well as context-dependent changes in sequence and domain architecture that may have consequences for gene function were found. CONCLUSIONS: Together, the results demonstrate that the evolution of AGOs has been a dynamic process producing the signatures of functional diversification in the smRNA pathways of higher plants.

Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

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.

The splicing factor FUBP1 is required for the efficient splicing of oncogene MDM2 pre-mRNA.

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.

Stress-induced isoforms of MDM2 and MDM4 correlate with high-grade disease and an altered splicing network in pediatric rhabdomyosarcoma.

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.

Factors predicting emergency surgery in severe mitral regurgitation following mitral balloon valvotomy.

BACKGROUND AND AIM OF THE STUDY: 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. METHODS: 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). RESULTS: 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, p < 0.005 and 0.940, p < 0.005, 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. CONCLUSION: Patients undergoing MBV with an RVSP >76 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.

Pre-mRNA splicing in disease and therapeutics.

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.

Does rheumatic valvular heart disease affect right ventricular performance?

AIM: 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. METHOD: 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. RESULT: 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). CONCLUSION: 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.

Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress.

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.

Genotoxic stress induces coordinately regulated alternative splicing of the p53 modulators MDM2 and MDM4.

The tumor suppressor protein p53 is a transcription factor that induces G(1) 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.

Dimorphism in the P2Y1 ADP receptor gene is associated with increased platelet activation response to ADP.

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 micromol/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, 1622AG, 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 mumol/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.