SpliceWiz: interactive analysis and visualization of alternative splicing in R.

Alternative splicing (AS) is a crucial mechanism for regulating gene expression and isoform diversity in eukaryotes. However, the analysis and visualization of AS events from RNA sequencing data remains challenging. Most tools require a certain level of computer literacy and the available means of visualizing AS events, such as coverage and sashimi plots, have limitations and can be misleading. To address these issues, we present SpliceWiz, an R package with an interactive Shiny interface that allows easy and efficient AS analysis and visualization at scale. A novel normalization algorithm is implemented to aggregate splicing levels within sample groups, thereby allowing group differences in splicing levels to be accurately visualized. The tool also offers downstream gene ontology enrichment analysis, highlighting ASEs belonging to functional pathways of interest. SpliceWiz is optimized for speed and efficiency and introduces a new file format for coverage data storage that is more efficient than BigWig. Alignment files are processed orders of magnitude faster than other R-based AS analysis tools and on par with command-line tools. Overall, SpliceWiz streamlines AS analysis, enabling reliable identification of functionally relevant AS events for further characterization. SpliceWiz is a Bioconductor package and is also available on GitHub (https://github.com/alexchwong/SpliceWiz).

Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.

Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15-20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.

Splice and Dice: Intronic microRNAs, Splicing and Cancer.

Introns span only a quarter of the human genome, yet they host around 60% of all known microRNAs. Emerging evidence indicates the adaptive advantage of microRNAs residing within introns is attributed to their complex co-regulation with transcription and alternative splicing of their host genes. Intronic microRNAs are often co-expressed with their host genes, thereby providing functional synergism or antagonism that is exploited or decoupled in cancer. Additionally, intronic microRNA biogenesis and the alternative splicing of host transcript are co-regulated and intertwined. The importance of intronic microRNAs is under-recognized in relation to the pathogenesis of cancer.

Macrophage development and activation involve coordinated intron retention in key inflammatory regulators.

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

We skip to work: alternative splicing in normal and malignant myelopoiesis.

Alternative splicing expands the transcriptome thereby promoting protein diversity. It governs critical cellular processes such as differentiation, proliferation and apoptosis in a tissue-specific manner. Aberrant splicing consequent to mutations in splicing factors and disruption of isoform ratios in key regulatory genes provides an important contribution to the pathogenesis of the myelodysplastic syndromes and myeloid leukemia. We review here the central role of alternative splicing in regulating myelopoiesis, and provide clear examples of how global splicing disruption or specific aberrant splicing events might promote leukemogenesis. We discuss the growing number of mechanistic links between epigenetic factors and alternative splicing. Finally, we address the potential utility of alternatively spliced isoforms as biomarkers and the development of novel therapies that modulate alternative splicing in myeloid and other malignancies.

Primary vs Secondary Endoscopic Dacryocystorhinostomy for Acute Dacryocystitis With Lacrimal Sac Abscess Formation: A Randomized Clinical Trial.

Importance: Endoscopic dacryocystorhinostomy (EN-DCR) is emerging as the preferred procedure in the management of nasolacrimal duct obstructions. However, its safety and long-term efficacy in the setting of acute dacryocystitis with lacrimal sac abscess have not been well studied. Objective: To compare outcomes of EN-DCR as primary treatment with EN-DCR as a secondary treatment after percutaneous drainage of lacrimal sac abscess in acute dacryocystitis. Design, Setting, and Participants: This randomized clinical trial was conducted from October 1, 2012, to October 31, 2015, at a tertiary ophthalmic center. The assessors of success at postoperative year 1 were masked to the procedures received by the participants. All surgical procedures were performed by 2 oculoplastic surgeons with different levels of EN-DCR experience. Eligible participants had acute dacryocystitis and lacrimal sac abscess presenting within 2 weeks of onset, who were 18 to 90 years of age. Analysis was of the intention-to-treat population. Interventions: Patients were allocated by block randomization to receive either percutaneous drainage of lacrimal sac abscess followed by EN-DCR after the acute episode subsided (control group) or primary EN-DCR within 2 weeks of presentation (intervention group). Both groups received a course of empirical systemic antibiotics (amoxicillin and clavulanic acid, 375 mg, to be taken 3 times a day for 1 week). Main Outcomes and Measures: Primary outcomes were time from presentation to documentation of symptom resolution and recurrence within 3 months. Results: Thirty-two patients were randomized equally into 2 treatment arms (control and intervention). The mean (SD) age of patients was 61 (13) years, and there was a predominance of women (27 [84%]). The mean (SD) time to symptom resolution was 13.8 (5.8) days in the intervention group compared with 31.7 (27.1) days in the control group (mean difference, 17.9; 95% CI, 3.71-32.01; P = .02). The mean (SD) time to surgery in the intervention group was shorter at 11.9 (6.3) days compared with 45.6 (30.1) days in the control group (mean difference, 33.6; 95% CI, 17.92-49.33; P < .001). Recurrences occurred once in the control group and did not occur in the intervention group. No differences in operation time and complications between the 2 groups were identified. The anatomical and functional success was 87.5% (14 of 16 cases) in both groups at postoperative year 1. Conclusions and Relevance: Primary EN-DCR in acute dacryocystitis with lacrimal sac abscess results in faster resolution compared with secondary treatment. No differences in recurrence, safety, or outcomes at postoperative year 1 were noted between the 2 treatment groups.

Safety and efficacy of adjunctive intranasal mitomycin C and triamcinolone in endonasal endoscopic dacryocystorhinostomy.

One of the common causes of failure in dacryocystorhinostomy for nasolacrimal duct obstruction (NLDO) is mucosal scarring and fibrosis around the ostium. Steroid and mitomycin C (MMC) can potentially reduce scarring by their action on the inflammatory and proliferative phase of wound healing, respectively. The purpose of this study is to evaluate the safety and efficacy of combined usage of adjunctive MMC and intranasal triamcinolone (TA) in endonasal endoscopic dacryocystorhinostomy (EE-DCR). This is a retrospective interventional case series. All patients underwent mechanical EE-DCR in two regional hospitals in Hong Kong from January 2005 to December 2006 were included. All received intraoperative MMC application for 5 min and gelfoam soaked with TA onto the ostium. Main outcome measures include the anatomical and functional success rate at follow-up at least 6 months after operation. Other outcomes include complications occurred during and after operation. A total of 73 EE-DCR were performed in 69 patients. Three patients had simultaneous bilateral DCR; one had sequential DCRs for both sides. At the last follow-up, anatomical success was achieved in 68 cases (93 %) and both anatomical with functional success in 67 cases (92 %). No major complication was observed. Minor complications included asymptomatic mucosal adhesion between the nasal septum and lateral nasal wall in one patient and moderate secondary hemorrhage in another. EE-DCR with adjunctive MMC and TA is a safe and successful procedure for the treatment of NLDO.

Fat-removal orbital decompression for disfiguring proptosis associated with Graves' ophthalmopathy: safety, efficacy and predictability of outcomes.

The objective of this study was to analyse clinical outcomes of patient who underwent fat-removal orbital decompression (FROD) for disfiguring proptosis associated with Graves' ophthalmopathy. This is a retrospective review of 21 eyes of 11 patients who have received transforniceal FROD for disfiguring Graves' exophthalmos at the Hong Kong Eye Hospital from January 2009 to March 2012. The amount of orbital fat removed and proptosis reduction in terms of Hertel value and complications were evaluated. The mean volume of orbital fat removed was 4.0 ± 1.1 ml (range 1.6-5.5 ml), and the mean change of Hertel value was 4.2 ± 1.3 (range 1-6, p < 0.000). None has visual loss or new-onset diplopia at primary gaze after FROD. No complications such as retrobulbar haemorrhage, meningitis, sinusitis, tissue necrosis, infraorbital paraesthesia or unsightly scar were observed. FROD could achieve reasonable proptosis reduction. It had a good safety profile, and results were predictable.

Limited nasal septoplasty by ophthalmologists during endonasal dacryocystorhinostomy: is it safe?

PURPOSE: Significant deviated nasal septum (DNS) may preclude the performance of endonasal dacryocystorhinostomy (DCR). The purpose of this study was to investigate the outcomes of limited nasal septoplasty in tackling signficant DNS performed by trained ophthalmologists during endonasal DCR. METHODS: In this retrospective interventional case series, the records of all patients requiring limited nasal septoplasty for significant DNS during endonasal DCR at 2 tertiary ophthalmic centers in Hong Kong during January 2006 to December 2007 were reviewed. Surgical outcomes, demographic factors, and intraoperative and postoperative details were recorded and analyzed. RESULTS: A total of 25 septoplasties were performed in 25 patients (total of 28 DCR). After the nasal septoplasty, all nasal cavities had better exposure of the surgical site allowing completion of the endonasal DCR. At the latest follow-up, the anatomical and functional success rates of the endonasal DCR were both 96.4% (27/28). In one patient, septoplasty was complicated by asymptomatic mucosal adhesion between the lateral nasal wall and the nasal septum. CONCLUSIONS: In the hands of trained ophthalmic lacrimal surgeons, limited nasal septoplasty is an effective and safe procedure during endonasal DCR, allowing better exposure of the surgical field in patients with significant DNS.

Differential rates of apoptosis and recruitment limit eosinophil accumulation in the lungs of asthma-resistant CBA/Ca mice.

The accumulation of eosinophils is a common feature of allergic airway inflammation and correlates with disease severity. In an ovalbumin (OVA)-induced murine model of allergic lung disease, CBA/Ca mice develop much lower levels of lung eosinophilia, lung oedema, mucus hypersecretion and airways obstruction than BALB/c and C57BL/6 strains. In this study these strains have been examined to identify mechanisms that control the recruitment and survival of eosinophils in the allergic lung. Following immunization with OVA, CBA/Ca mice developed a robust systemic allergic response, with high levels of total and OVA-specific IgE and increases in peripheral blood eosinophils. Lung eotaxin-1 levels and expression of CD18 on eosinophils recovered by bronchoalveolar lavage (BAL) were least pronounced in CBA/Ca mice, whereas mRNA for L-selectin was highest in eosinophils from C57BL/6 mice. Apoptosis of BAL eosinophils ex vivo was most pronounced in the CBA/Ca strain. BALB/c mice expressed the highest levels of the eosinophil growth and survival factor interleukin (IL)-5 in the lungs and BAL eosinophils from these animals expressed more of the anti-apoptotic proteins Bcl-xL and Bcl-2 than cells from the other strains. A combination of lower levels of recruitment and rapid apoptosis may therefore limit the accumulation of eosinophils and pathology in the lungs of CBA/Ca mice. In addition, although the level of pathology that developed in C57BL/6 and BALB/c mice was similar, some of the underlying mechanisms are likely to differ.

Cholesteatoma palpebrae: an unusual cholesterol-filled cyst in the eyelid.

Cholesteatomas, also known as epidermal cholesterol cysts, are unusual cystic lesions containing numerous cholesterol clefts that are usually found in the orbits in the ophthalmic setting. This report describes the clinical and microscopic features of an unusual cholesteatoma in the eyelid.