A systematic analysis of intronic sequences downstream of 5' splice sites reveals a widespread role for U-rich motifs and TIA1/TIAL1 proteins in alternative splicing regulation

To identify human intronic sequences associated with 5' splice site recognition, we performed a systematic search for motifs enriched in introns downstream of both constitutive and alternative cassette exons. Significant enrichment was observed for U-rich motifs within 100 nucleotides downstream of 5' splice sites of both classes of exons, with the highest enrichment between positions +6 and +30. Exons adjacent to U-rich intronic motifs contain lower frequencies of exonic splicing enhancers and higher frequencies of exonic splicing silencers, compared with exons not followed by U-rich intronic motifs. These findings motivated us to explore the possibility of a widespread role for U-rich motifs in promoting exon inclusion. Since cytotoxic granule-associated RNA binding protein (TIA1) and TIA1-like 1 (TIAL1; also known as TIAR) were previously shown in vitro to bind to U-rich motifs downstream of 5' splice sites, and to facilitate 5' splice site recognition in vitro and in vivo, we investigated whether these factors function more generally in the regulation of splicing of exons followed by U-rich intronic motifs. Simultaneous knockdown of TIA1 and TIAL1 resulted in increased skipping of 36/41 (88%) of alternatively spliced exons associated with U-rich motifs, but did not affect 32/33 (97%) alternatively spliced exons that are not associated with U-rich motifs. The increase in exon skipping correlated with the proximity of the first U-rich motif and the overall "U-richness" of the adjacent intronic region. The majority of the alternative splicing events regulated by TIA1/TIAL1 are conserved in mouse, and the corresponding genes are associated with diverse cellular functions. Based on our results, we estimate that approximately 15% of alternative cassette exons are regulated by TIA1/TIAL1 via U-rich intronic elements.     

Standards of Outcome Reporting in Surgical Oncology: A Case Study in Esophageal Cancer

Background

Multimodal strategies before surgery are often used to improve outcomes, but disease progression (precluding surgical resection) and inoperability at planned surgery still occur following neoadjuvant treatment. The standards of reporting of these outcomes have not previously been considered. This study examined reporting of rates of progression to surgical resection and inoperability at planned surgery following neoadjuvant treatment in surgical oncology, using esophageal cancer as a case study.

Methods

A systematic review identified randomized trials and prospective nonrandomized studies reporting short-term outcomes of neoadjuvant treatment and surgery for esophageal cancer.

Results

Of 4,763 abstracts, 224 papers were retrieved and 76 studies included (8 randomized trials and 68 cohort studies of 19,441 esophagectomies). Articles reported outcomes of preoperative chemotherapy (n?=?33, 43.4?%), chemoradiotherapy (n?=?13, 17.1?%), or both within one paper (n?=?18, 23.7?%) and 12 (15.8?%) did not specify the type of neoadjuvant treatment. Also, 20 papers (26.3?%) reported numbers of patients not progressing to surgery after neoadjuvant treatment (with rates of nonprogression ranging between 2.0 and 35.3?%). In addition, 24 papers (31.6?%) reported rates of inoperability at planned surgery (with inoperability rates ranging between 0 and 26.2?%). More randomized controlled trials (RCTs) than observational studies reported nonprogression (4 randomized and 16 nonrandomized studies, 95?% CI ?9.6 to 62.6?%, p?=?0.108) and inoperability (6 randomized trials and 18 observational studies, 95?% CI 16.8?C80.3?%, p?=?0.005). Some 17 and 10 articles provided reasons for the observed rates of nonprogression and inoperability, respectively.

Conclusions

Reporting rates of progression to surgery after neoadjuvant treatment and inoperability at planned surgery for esophageal cancer were poor, limiting data synthesis and comparisons. It is suggested that core outcome sets for trials in surgical oncology are developed with inclusion of these important endpoints. Collaboration between medical and surgical oncologists is necessary to achieve this.     

REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNP-type RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP/Mex67p, and it has been suggested that they facilitate the recruitment of TAP/Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP/Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm.     

Colonic diverticulitis in the neck: a late complication of laryngopharyngectomy surgery

We report a case of diverticulitis affecting a colonic segment used as an interposition graft following laryngopharyngectomy. The patient presented as an emergency to our department with a history of a red, swollen and painful neck. She had undergone laryngopharyngectomy for laryngeal cancer in 1967. Computed tomography imaging revealed several diverticula in the colonic graft and associated abscess formation. The patient’s clinical condition behaved similarly to that of conventional colonic diverticulitis. The difficulties in reaching a definite diagnosis and management of this unusual complication following laryngopharyngectomy are discussed.     

CDKN1C mutation in Wiedemann-Beckwith syndrome patients reduces RNA splicing efficiency and identifies a splicing enhancer

Wiedemann-Beckwith syndrome (WBS) is a human overgrowth disorder that is accompanied by an increased risk of embryonal tumors and is associated with dsyregulation of the imprinting of genes in chromosome 11p15.5. Maternally inherited mutations in the imprinted CDKN1C gene are known to be associated with WBS. We have identified a novel mutation in several members of a large family affected by WBS. The mutation is a G --> T change in a run of seven G's near the 5' splice site of intron 3. All obligate carriers and affected individuals carry the mutation, and in each affected case, the allele was inherited maternally, strongly suggesting a role in causing WBS. The mutation is located in a poly-G tract in the intron; intronic G-rich sequences in other genes have been shown to have a role in promoting splicing. In transfected 293HEK cells, we found that the G --> T mutation reduced splicing efficiency. Mutation of all seven G's in the poly-G tract further reduced splicing efficiency, supporting a role for the G-tract as a splicing enhancer. The fibroblasts of one affected patient showed a similar reduction in splicing efficiency. Maternal monoallelic expression of CDKN1C was verified in this patient cell line. However, the total amount of spliced message was not reduced by the mutation in spite of the reduced efficiency of splicing. We discuss the possible role of the splicing defect in the pathogenesis of WBS in this pedigree.     

Inpatient care of small and sick newborns: a multi-country analysis of health system bottlenecks and potential solutions

Background

Preterm birth is the leading cause of child death worldwide. Small and sick newborns require timely, high-quality inpatient care to survive. This includes provision of warmth, feeding support, safe oxygen therapy and effective phototherapy with prevention and treatment of infections. Inpatient care for newborns requires dedicated ward space, staffed by health workers with specialist training and skills. Many of the estimated 2.8 million newborns that die every year do not have access to such specialised care.

Methods

The bottleneck analysis tool was applied in 12 countries in Africa and Asia as part of the Every Newborn Action Plan process. Country workshops involved technical experts to complete the survey tool, which is designed to synthesise and grade health system "bottlenecks" (or factors that hinder the scale up) of maternal-newborn intervention packages. For this paper, we used quantitative and qualitative methods to analyse the bottleneck data, and combined these with literature review, to present priority bottlenecks and actions relevant to different health system building blocks for inpatient care of small and sick newborns.

Results

Inpatient care of small and sick newborns is an intervention package highlighted by all country workshop participants as having critical health system challenges. Health system building blocks with the highest graded (significant or major) bottlenecks were health workforce (10 out of 12 countries) and health financing (10 out of 12 countries), followed by community ownership and partnership (9 out of 12 countries). Priority actions based on solution themes for these bottlenecks are discussed.

Conclusions

Whilst major bottlenecks to the scale-up of quality inpatient newborn care are present, effective solutions exist. For all countries included, there is a critical need for a neonatal nursing cadre. Small and sick newborns require increased, sustained funding with specific insurance schemes to cover inpatient care and avoid catastrophic out-of-pocket payments. Core competencies, by level of care, should be defined for monitoring of newborn inpatient care, as with emergency obstetric care. Rather than fatalism that small and sick newborns will die, community interventions need to create demand for accessible, high-quality, family-centred inpatient care, including kangaroo mother care, so that every newborn can survive and thrive.

     

Evaluating the efficacy of functional electrical stimulation therapy assisted walking after chronic motor incomplete spinal cord injury: effects on bone biomarkers and bone strength

Objectives: To determine the efficacy of functional electrical stimulation therapy assisted walking (FES-T) compared to a conventional aerobic and resistance training (CONV) with respect to bone biomarkers and lower extremity bone strength outcomes among adults with chronic motor incomplete spinal cord injury (SCI).

Design: Parallel group randomized controlled trial (www.clinicaltrials.gov - NCT0020196819).

Site: Tertiary academic rehabilitation centre in Canada.

Methods: Adults with chronic (≥18 months) motor incomplete SCI (C2-T12 AIS C-D) were consented and randomized to FES-T or CONV training for 45 minutes thrice-weekly for 4 months. Osteocalcin (OC), β-cross laps (CTX) and sclerostin were assessed at baseline, and 4 months. Similarly, total hip, distal femur and proximal tibia region bone mineral density (BMD) via DXA (4500A, Hologic Inc. Waltham, MA, USA) and tibia bone quality via pQCT (Stratec XCT-2000, Mezintecknik, Pforzheim, Germany) were assessed at baseline, 4, and 12 months. Between group differences were analyzed using repeated measures general linear models.

Results: Thirty-four participants (17 FES-T, 17 CONV) consented and were randomized, 27 participants completed the 4-month intervention and 12-month outcome assessments. Participants in the FES-T arm had a decrease in CTX and a significant increase in OC at intervention completion (P<0.05). Significant biomarker changes were not observed in the CONV group. No within or between group differences from baseline were observed in sclerostin or bone strength.

Conclusions: Four months of FES-T improved bone turnover (increase in OC and decrease in CTX) but not bone strength among individuals with chronic SCI. Future, long term FES-T may augment lower extremity bone strength.