The Splicing Efficiency of Activating HRAS Mutations Can Determine Costello Syndrome Phenotype and Frequency in Cancer

Costello syndrome (CS) may be caused by activating mutations in codon 12/13 of the HRAS proto-oncogene. HRAS p.Gly12Val mutations have the highest transforming activity, are very frequent in cancers, but very rare in CS, where they are reported to cause a severe, early lethal, phenotype. We identified an unusual, new germline p.Gly12Val mutation, c.35_36GC>TG, in a 12-year-old boy with attenuated CS. Analysis of his HRAS cDNA showed high levels of exon 2 skipping. Using wild type and mutant HRAS minigenes, we confirmed that c.35_36GC>TG results in exon 2 skipping by simultaneously disrupting the function of a critical Exonic Splicing Enhancer (ESE) and creation of an Exonic Splicing Silencer (ESS). We show that this vulnerability of HRAS exon 2 is caused by a weak 3’ splice site, which makes exon 2 inclusion dependent on binding of splicing stimulatory proteins, like SRSF2, to the critical ESE. Because the majority of cancer- and CS- causing mutations are located here, they affect splicing differently. Therefore, our results also demonstrate that the phenotype in CS and somatic cancers is not only determined by the different transforming potentials of mutant HRAS proteins, but also by the efficiency of exon 2 inclusion resulting from the different HRAS mutations. Finally, we show that a splice switching oligonucleotide (SSO) that blocks access to the critical ESE causes exon 2 skipping and halts proliferation of cancer cells. This unravels a potential for development of new anti-cancer therapies based on SSO-mediated HRAS exon 2 skipping.     

Remote Control of Intestinal Stem Cell Activity by Haemocytes in Drosophila

The JAK/STAT pathway is a key signaling pathway in the regulation of development and immunity in metazoans. In contrast to the multiple combinatorial JAK/STAT pathways in mammals, only one canonical JAK/STAT pathway exists in Drosophila. It is activated by three secreted proteins of the Unpaired family (Upd): Upd1, Upd2 and Upd3. Although many studies have established a link between JAK/STAT activation and tissue damage, the mode of activation and the precise function of this pathway in the Drosophila systemic immune response remain unclear. In this study, we used mutations in upd2 and upd3 to investigate the role of the JAK/STAT pathway in the systemic immune response. Our study shows that haemocytes express the three upd genes and that injury markedly induces the expression of upd3 by the JNK pathway in haemocytes, which in turn activates the JAK/STAT pathway in the fat body and the gut. Surprisingly, release of Upd3 from haemocytes upon injury can remotely stimulate stem cell proliferation and the expression of Drosomycin-like genes in the intestine. Our results also suggest that a certain level of intestinal epithelium renewal is required for optimal survival to septic injury. While haemocyte-derived Upd promotes intestinal stem cell activation and survival upon septic injury, haemocytes are dispensable for epithelium renewal upon oral bacterial infection. Our study also indicates that intestinal epithelium renewal is sensitive to insults from both the lumen and the haemocoel. It also reveals that release of Upds by haemocytes coordinates the wound-healing program in multiple tissues, including the gut, an organ whose integrity is critical to fly survival.     

Measurement of Heart Rate Variability to Assess Pain in Sedated Critically Ill Patients: A Prospective Observational Study

Introduction

The analgesia nociception index (ANI) assesses the relative parasympathetic tone as a surrogate for antinociception/nociception balance in sedated patients. The aim of this study is to determine the effectiveness of ANI in detecting pain in deeply sedated critically ill patients.

Methods

This prospective observational study was performed in two medical ICUs. All patients receiving invasive mechanical ventilation and deep sedation were eligible. In all patients, heart rate and ANI were continuously recorded using the Physiodoloris^® device during 5 minutes at rest (T1), during a painful stimulus (T2), and during 5 minutes after the end of the painful stimulus (T3). The chosen painful stimulus was patient turning for washstand. Pain was evaluated at T2, using the behavioral pain scale (BPS). The primary objective was to determine the effectiveness of ANI in detecting pain. Secondary objectives included the impact of norepinephrine on the effectiveness of ANI in detecting pain, and the correlation between ANI and BPS.

Results

Forty-one patients were included. ANI was significantly lower at T2 (Med (IQR) 69(55–78)) compared with T1 (85(67–96), p<0.0001), or T3 (81(63–89), p<0.0001). Similar results were found in the subgroups of patients with (n = 21) or without (n = 20) norepinephrine. ANI values were significantly higher in patients with norepinephrine compared with those without norepinephrine at T1, and T2. No significant correlation was found between ANI and BPS at T2.

Conclusions

ANI is effective in detecting pain in deeply sedated critically ill patients, including those patients treated with norepinephrine. No significant correlation was found between ANI and BPS.     

Cognitive behavioural therapy for ADHD in adults: systematic review and meta-analyses

Systematically review and analyse the efficacy of CBT versus treatment as usual in adults with ADHD. The literature was systematically searched ending the 28 March 2014. Standardised mean differences (SMD) and 95 % confidence intervals were calculated. CBT was efficacious in reducing symptoms of ADHD (SDM ?1.0, 95 % CI ?1.5 to ?0.5) when evaluated by the patients, but not when evaluated by a clinician. Symptoms of depression and anxiety were significantly reduced when self-reported (SMD ?1.0, 95 % CI ?1.6 to ?0.5 and ?1.0, 95 % CI ?1.3 to ?0.3, respectively) and evaluated by a clinician (SMD ?0.9, 95 % CI ?1.7 to ?0.2 and ?0.9, 95 % CI ?1.6 to ?0.1). The clinical global impression scores improved more in the group randomised to CBT (?1.0; 95 % CI ?1.6 to ?0.4). CBT seems efficacious in some domains affecting adult patients with ADHD, but needs further evaluation.     

Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

The More the Merrier: Swarming as an Antipredator Strategy in the Mysid <Emphasis Type="Italic">Neomysis Integer</Emphasis>

The swarming behaviour of a Baltic littoral mysid shrimp, Neomysis integer, was studied both in the presence and absence of a predator (European perch, Perca fluviatilis L.). I performed two kinds of laboratory experiments. First, the swarming tendency of mysids and the effect of swarm size on swarm choice were studied. Second, the ingestion rate of mysids was measured when feeding alone versus in a swarm. The results indicate that N. integer actively join swarms. The avoidance of the perch by N. integer individuals was stronger when there was a swarm present. Larger swarms were preferred over smaller ones regardless of presence or absence of the predator. The overall feeding rate was similar when feeding alone and in swarm, but predator cues reduced feeding rate only when the mysids were feeding alone. This study demonstrates the capability of N. integer to assess predation risk and social context and alter their behaviour accordingly.