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The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti–K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants. Significance The KRAS oncogene is mutated more frequently in human cancer than any other. The KRAS transcript is alternatively spliced to give rise to two products, K-Ras4A and K-Ras4B, both of which are oncogenic when KRAS is mutated. We detected significant amounts of each transcript in human tumor cells and colorectal carcinomas. We found that K-Ras4A is targeted to the plasma membrane by dual targeting motifs distinct from those of K-Ras4B. Because interfering with membrane association of Ras proteins remains one of the most attractive approaches to anti-Ras therapy, efforts in this direction will have to disrupt both the K-Ras4A and the K-Ras4B membrane-targeting pathways.

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho–K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL–dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP ₃ regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.

Aims To evaluate the long-term effectiveness of the community-based Low Vision Service Wales (LVSW). Methods A long-term observational study of the Government-funded, community-based, low-vision rehabilitation service which operates in over 180 optometry practices in Wales. Participants were recruited from the LVSW (n=342; 246 women; median age 82 years) at baseline (before the Low Vision intervention). The primary outcome measure was change in visual disability as evaluated by the seven-item National Eye Institute Visual Function Questionnaire (NEI-VFQ). Change was measured on the same cohort at three separate time points, and comparisons were made between these: baseline–3 months; 3–18 months; baseline–18 months. Secondary outcome measures included: use of low-vision aids (LVAs) and satisfaction with the service provided. Results Questionnaires were sent to 281 participants (whose visual disability had been measured at baseline and 3 months) at 18 months postintervention. Responses were received from 190 (67.6%) people; 24 were deceased. Self-reported visual disability was significantly reduced (Wilcoxon Signed Rank (WSR) test: p<0.001) between baseline and 18 months by −0.28 logits (−1.24 to 0.52). This was less than that found between baseline and 3 months; −0.61 logits (−1.81 to 0.02). At 18 months, 79% patients used their LVAs at least once a week which was not significantly different to that found at 3 months (WSR: p=0.127). Conclusions This study provides evidence that the effect of the LVSW persists over a period of 18 months; disability is reduced from baseline, and use of LVAs remains high.

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.

AimsTo determine if there was a significant difference between user-centred and clinical outcomes in people with low vision who attended a new community-based low vision service (CLVS) or the hospital-based low vision service (HBLV).MethodsA prospective controlled before and after study. Participants were recruited from the CLVS (n=343; 96 male, 247 female; median age 82 years) and from the HLVS (n=145; 55 male, 90 female; median age 80 years). The primary outcome measure was change (baseline to 3 months) in visual disability as evaluated by the seven-item National Eye Institute-Visual Function Questionnaire (NEI-VFQ). Secondary outcome measures included: use of low vision aids, satisfaction with the service provided and change in near visual acuity before and after the provision of low vision aids.ResultsThere were no significant differences in user-centred and clinical outcome measures between the CLVS and HLVS. Self-reported visual disability was significantly reduced after low vision service intervention for participants in both groups by 0.46 and 0.57 logits in the HLVS and CLVS, respectively.ConclusionThis study provides strong evidence that CLVS and HLVS are effective methods of service provision in Wales.

Background Visual impairment is common in older people and the presence of additional health conditions can compromise health and rehabilitation outcomes. A small number of studies have suggested that comorbities are common in visual impairment; however, those studies have relied on self-report and have assessed a relatively limited number of comorbid conditions. Methods We conducted a cross-sectional analysis of a dataset of 291,169 registered patients (65-years-old and over) within 314 primary care practices in Scotland, UK. Visual impairment was identified using Read Code ever recorded for blindness and/or low vision (within electronic medical records). Prevalence, odds ratios (from prevalence rates standardised by stratifying individuals by age groups (65 to 69 years; 70 to 74; 75 to 79; 80 to 84; and 85 and over), gender and deprivation quintiles) and 95% confidence intervals (95% CI) of 37 individual chronic physical/mental health conditions and total number of conditions were calculated and compared for those with visual impairment to those without. Results Twenty seven of the 29 physical health conditions and all eight mental health conditions were significantly more likely to be recorded for individuals with visual impairment compared to individuals without visual impairment, after standardising for age, gender and social deprivation. Individuals with visual impairment were also significantly more likely to have more comorbidities (for example, five or more conditions (odds ratio (OR) 2.05 95% CI 1.94 to 2.18)). Conclusions Patients aged 65 years and older with visual impairment have a broad range of physical and mental health comorbidities compared to those of the same age without visual impairment, and are more likely to have multiple comorbidities. This has important implications for clinical practice and for the future design of integrated services to meet the complex needs of patients with visual impairment, for example, embedding depression and hearing screening within eye care services.

The most frequently mutated oncogene in cancer is KRAS , which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region 1 . Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins—each capable of transforming cells—are encoded when KRAS is activated by mutation 2 . No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells 3 in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen 4 (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes 3 – 5 , it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation–depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically. KRAS4A interacts directly with hexokinase 1 in a GTP-dependent manner at the outer mitochondrial membrane, leading to kinase activation and an increase in glucose uptake and glycolysis in tumour cells.

AimsTo develop and validate a short questionnaire to assess self-reported visual ability in children and young people with a visual impairment.MethodsA list of 121 items was generated from 13 focus groups with children and young people with and without a visual impairment. A long 89-item questionnaire was piloted with 45 visually impaired children and young people using face-to-face interviews. Rasch analysis was used to analyse the response category function and to facilitate item removal ensuring a valid unidimensional scale. The validity and reliability of the short questionnaire were assessed on a group of 109 visually impaired children (58.7% boys; median age 13 years) using Rasch analysis and intraclass correlation coefficient (ICC).ResultsThe final 25-item questionnaire has good validity and reliability as demonstrated by a person separation index of 2.28 and reliability coefficient of 0.84. The items are well targeted to the subjects with a mean difference of −0.40 logit between item and person means, and an ICC of 0.89 demonstrates good temporal stability.ConclusionThe Cardiff Visual Ability Questionnaire for Children (CVAQC) is a short, psychometrically robust and a self-reported instrument that works to form a unidimensional scale for the assessment of the visual ability in children and young people with a visual impairment.

RAS is the most frequently mutated oncogene in human cancers. Despite decades of effort, anti-RAS therapies have remained elusive. Isoprenylcysteine carboxylmethyltransferase (ICMT) methylates RAS and other CaaX-containing proteins, but its potential as a target for cancer therapy has not been fully evaluated. We crossed a Pdx1-Cre;LSL-KrasG12D mouse, which is a model of pancreatic ductal adenocarcinoma (PDA), with a mouse harboring a floxed allele of Icmt. Surprisingly, we found that ICMT deficiency dramatically accelerated the development and progression of neoplasia. ICMT-deficient pancreatic ductal epithelial cells had a slight growth advantage and were resistant to premature senescence by a mechanism that involved suppression of cyclin-dependent kinase inhibitor 2A (p16INK4A) expression. ICMT deficiency precisely phenocopied Notch1 deficiency in the Pdx1-Cre;LSL-KrasG12D model by exacerbating pancreatic intraepithelial neoplasias, promoting facial papillomas, and derepressing Wnt signaling. Silencing ICMT in human osteosarcoma cells decreased Notch1 signaling in response to stimulation with cell-surface ligands. Additionally, targeted silencing of Ste14, the Drosophila homolog of Icmt, resulted in defects in wing development, consistent with Notch loss of function. Our data suggest that ICMT behaves like a tumor suppressor in PDA because it is required for Notch1 signaling.