LINC01013 Is a Determinant of Fibroblast Activation and Encodes a Novel Fibroblast-Activating Micropeptide.

Myocardial fibrosis confers an almost threefold mortality risk in heart disease. There are no prognostic therapies and novel therapeutic targets are needed. Many thousands of unannotated small open reading frames (smORFs) have been identified across the genome with potential to produce micropeptides (< 100 amino acids). We sought to investigate the role of smORFs in myocardial fibroblast activation.Analysis of human cardiac atrial fibroblasts (HCFs) stimulated with profibrotic TGFß1 using RNA sequencing (RNA-Seq) and ribosome profiling (Ribo-Seq) identified long intergenic non-coding RNA LINC01013 as TGFß1 responsive and containing an actively translated smORF. Knockdown of LINC01013 using siRNA reduced expression of profibrotic markers at baseline and blunted their response to TGFß1. In contrast, overexpression of a codon-optimised smORF invoked a profibrotic response comparable to that seen with TGFß1 treatment, whilst FLAG-tagged peptide associated with the mitochondria.Together, these data support a novel LINC01013 smORF micropeptide-mediated mechanism of fibroblast activation. TGFß1 stimulation of atrial fibroblasts induces expression of LINC01013, whose knockdown reduces fibroblast activation. Overexpression of a smORF contained within LINC01013 localises to mitochondria and activates fibroblasts.

Identifying the optimal regional predictor of right ventricular global function: a high-resolution three-dimensional cardiac magnetic resonance study.

Right ventricular (RV) function has prognostic value in acute, chronic and peri-operative disease, although the complex RV contractile pattern makes rapid assessment difficult. Several two-dimensional (2D) regional measures estimate RV function, however the optimal measure is not known. High-resolution three-dimensional (3D) cardiac magnetic resonance cine imaging was acquired in 300 healthy volunteers and a computational model of RV motion created. Points where regional function was significantly associated with global function were identified and a 2D, optimised single-point marker (SPM-O) of global function developed. This marker was prospectively compared with tricuspid annular plane systolic excursion (TAPSE), septum-freewall displacement (SFD) and their fractional change (TAPSE-F, SFD-F) in a test cohort of 300 patients in the prediction of RV ejection fraction. RV ejection fraction was significantly associated with systolic function in a contiguous 7.3 cm2 patch of the basal RV freewall combining transverse (38%), longitudinal (35%) and circumferential (27%) contraction and coinciding with the four-chamber view. In the test cohort, all single-point surrogates correlated with RV ejection fraction (p < 0.010), but correlation (R) was higher for SPM-O (R = 0.44, p < 0.001) than TAPSE (R = 0.24, p < 0.001) and SFD (R = 0.22, p < 0.001), and non-significantly higher than TAPSE-F (R = 0.40, p < 0.001) and SFD-F (R = 0.43, p < 0.001). SPM-O explained more of the observed variance in RV ejection fraction (19%) and predicted it more accurately than any other 2D marker (median error 2.8 ml vs 3.6 ml, p < 0.001). We conclude that systolic motion of the basal RV freewall predicts global function more accurately than other 2D estimators. However, no markers summarise 3D contractile patterns, limiting their predictive accuracy.

Acute myocardial infarction: susceptibility-weighted cardiac MRI for the detection of reperfusion haemorrhage at 1.5 T.

AIM: To assess whether susceptibility-weighted imaging (SWI) provides better image contrast for the detection of haemorrhagic ischaemia-reperfusion injury in the heart. MATERIALS AND METHODS: Thirty patients (all men; mean age 53 years) underwent cardiac magnetic resonance imaging (MRI) within 7 days of primary percutaneous intervention for acute ST elevation myocardial infarction (STEMI). Multiple gradient-echo T2* sequences with magnitude and phase reconstructions were acquired. A high-pass filtered phase map was used to create a mask for the SWI reconstructions. The difference in image contrast was assessed in those patients with microvascular obstruction. A mixed effects regression model was used to test the effect of echo time and reconstruction method on phase and contrast-to-noise ratio (CNR). Medians and interquartile ranges (IQR) are reported. RESULTS: T2* in haemorrhagic infarcts was shorter than in non-haemorrhagic infarcts (33.5 ms [24.9-43] versus 49.9 ms [44.6-67.6]; p=0.0007). The effect of echo time on phase was significant (p<0.0001), as was the effect of haemorrhage on phase (p=0.0016). SWI reconstruction had a significant effect on the CNR at all echo times (echoes 1-5, p<0.0001; echo 6, p=0.01; echo 7, p=0.02). The median echo number at which haemorrhage was first visible was less for SWI compared to source images (echo 2 versus echo 5, p=0.0002). CONCLUSION: Cardiac SWI improves the contrast between myocardial haemorrhage and the surrounding tissue following STEMI and has potential as a new tool for identifying patients with ischaemia-reperfusion injury.

Psychological aspects of cytogenetic testing of uveal melanoma: preliminary findings and directions for future research.

PURPOSE: To determine the proportion of uveal melanoma patients who accept cytogenetic prognostication and to understand the reasons for their decision and the psychological impact of an adverse prognosis. METHODS: Patients treated by enucleation or local resection for uveal melanoma between 01 January 2003 and 31 December 2006 were identified and the proportion undergoing cytogenetic studies was determined. In-depth interviews of fourteen patients living near our centre were conducted to determine their reasons for accepting cytogenetic testing and their reactions to any results received. RESULTS: In total 97% of 298 eligible patients with uveal melanoma treated by enucleation or local resection accepted an offer of cytogenetic prognostication. None of the patients interviewed in detail expressed any regret about having this test and there was no evidence of any harm. The main benefit perceived by patients was that they would have greater control and that screening for metastatic disease and early treatment might enhance chances of survival. This was despite counselling that prognostication, screening, and treatment are unlikely to prolong life and that the main purpose of cytogenetic studies is to allow for life-planning. CONCLUSIONS: Almost all patients with uveal melanoma desire cytogenetic prognostication, although not for the reasons intended by their medical practitioners. Further studies are needed to understand patients' reactions to cytogenetic testing, so that care can be optimised.

Two novel alleles of tottering with distinct Ca(v)2.1 calcium channel neuropathologies.

The calcium channel CACNA1A gene encodes the pore-forming, voltage-sensitive subunit of the voltage-dependent calcium Ca(v)2.1 type channel. Mutations in this gene have been linked to several human disorders, including familial hemiplegic migraine, episodic ataxia 2 and spinocerebellar ataxia type 6. The mouse homologue, Cacna1a, is associated with the tottering, Cacna1a(tg), mutant series. Here we describe two new missense mutant alleles, Cacna1a(tg-4J) and Cacna1a(Tg-5J). The Cacna1a(tg-4J) mutation is a valine to alanine mutation at amino acid 581, in segment S5 of domain II. The recessive Cacna1a(tg-4J) mutant exhibited the ataxia, paroxysmal dyskinesia and absence seizures reminiscent of the original tottering mouse. The Cacna1a(tg-4J) mutant also showed altered activation and inactivation kinetics of the Ca(v)2.1 channel, not previously reported for other tottering alleles. The semi-dominant Cacna1a(Tg-5J) mutation changed a conserved arginine residue to glutamine at amino acid 1252 within segment S4 of domain III. The heterozygous mouse was ataxic and homozygotes rarely survived. The Cacna1a(Tg-5J) mutation caused a shift in both voltage activation and inactivation to lower voltages, showing that this arginine residue is critical for sensing Ca(v)2.1 voltage changes. These two tottering mouse models illustrate how novel allelic variants can contribute to functional studies of the Ca(v)2.1 calcium channel.

A new instrument for assessing quality of life in atopic dermatitis: international development of the Quality of Life Index for Atopic Dermatitis (QoLIAD).

BACKGROUND: Atopic dermatitis (AD) is a chronic or chronically relapsing inflammatory skin condition that can have a considerable impact on those affected. There are a number of instruments available to measure outcome in dermatological conditions but none have been developed specifically for AD. In addition, most measure symptoms and/or daily functioning, which are potential influences on quality of life (QoL) rather than assessments of the construct itself. OBJECTIVES: The aim of the current study was to develop a new instrument specifically designed to measure QoL in adults with AD-the Quality of Life Index for Atopic Dermatitis (QoLIAD). METHODS: The instrument was developed based on the needs-based model of QoL and was produced in several different countries simultaneously. Its content was derived from 65 in-depth interviews with relevant patients in the U.K., Italy and the Netherlands. The initial version of the measure was produced in U.K. English and translations were produced for the Netherlands, Italy, Germany, France and the U.S.A. using a dual translation panel methodology. A Spanish version was developed using the same adaptation process after the instrument was finalized. Field-test interviews were conducted with approximately 20 patients in each country to assess face and content validity. The instrument [in addition to the Dermatology Life Quality Index (DLQI) and the Psychological General Well-Being Schedule (PGWB)] was then administered to up to 300 AD patients in each country at two time points to finalize the instrument and test its psychometric properties. RESULTS: The initial version of the QoLIAD had 56 items that reflected the areas of need fulfillment identified in the qualitative interviews as having been affected by AD: mental and emotional stimulation, physical and emotional stability, security, sharing and belonging, self-esteem, personal development and fulfillment. Comments from patients in field-test interviews resulted in the removal of 14 items, to leave a 42-item instrument that was considered relevant and acceptable. The number of patients participating in the survey were 286 in the U.K., 46 in the Netherlands, 213 in France, 187 in Germany, 178 in the U.S.A. and 83 in Spain. Application of the Rasch model to these data identified the final 25-item QoLIAD. Unidimensionality was confirmed, with deviation of the total scale from the Rasch model evident at a single time point in one country only (the U.K.). All language versions, with the exception of the Dutch measure, had test-retest reliability coefficients in excess of 0.85. The test-retest in the Netherlands was 0.80. However, this country had the smallest sample size and the corresponding reliability for the DLQI was only 0.40. The QoLIAD had adequate internal consistency and the initial indications of construct validity were good. The levels of association with the DLQI indicated that the two instruments measure related but distinct constructs. CONCLUSIONS: The QoLIAD is a practical, reliable, valid and culturally applicable instrument for measuring the impact of AD and its treatment on QoL in clinical trials or in routine clinical practice.

Development of the PSORIQoL, a psoriasis-specific measure of quality of life designed for use in clinical practice and trials.

BACKGROUND: Patients with psoriasis have significant impairment in their quality of life (QoL). Several patient-completed instruments are available to measure outcome in dermatological conditions but these primarily focus on severity of disease rather than on QoL. OBJECTIVES: To develop a new instrument specifically designed to measure QoL in psoriasis-the Psoriasis Index of Quality of Life (PSORIQoL). METHODS: The content of the instrument was derived from qualitative interviews with 62 psoriasis patients in the U.K., Italy and the Netherlands. Content analysis of the interview transcripts from the three countries identified potential items for the questionnaire. All further stages of the project were conducted in the U.K. only. Face and content validity were determined by means of a focus group held with seven psoriasis patients and face-to-face interviews with a further 21 patients. A new draft version of the PSORIQoL, together with the Dermatology Life Quality Index (DLQI) and the General Well-Being Index (GWBI), were completed by a new sample of psoriasis patients contacted by mail, in order to determine construct validity. A subsample of the respondents completed a similar package 2 weeks later, to assess reproducibility (reliability). RESULTS: The first version of the PSORIQoL consisted of 61 items covering the needs identified in the interviews as having been affected by psoriasis or its treatment. These included fear of negative reactions from others, self-consciousness and poor self-confidence, problems with socialization, physical contact and intimacy, limitations on personal freedom and impaired relaxation, sleep and emotional stability. Following the focus group and field-test interviews, a new draft measure consisting of 45 items was completed by 148 patients, 88 of whom completed and returned a similar package 2 weeks later. Application of Rasch analysis identified a final 25-item version of the PSORIQoL. This version had a test-retest reliability coefficient of 0.89, indicating good reproducibility. The levels of association with the DLQI and PGWB indicated that the PSORIQoL measures a related but distinct construct. CONCLUSIONS: The PSORIQoL appears to be a practical, reliable and valid instrument for measuring the impact of psoriasis on QoL. It remains necessary to establish the instrument's responsiveness to changes in QoL associated with treatment.

Clinical decision guidelines for NHS cosmetic surgery: analysis of current limitations and recommendations for future development.

Because of increasing demand for publicly funded elective cosmetic surgery, clinical decision guidelines have been developed to select those patients who should receive it. The aims of this study were to identify: the main characteristics of such guidelines; whether and how they influence clinical decision making; and ways in which they should be improved. UK health authorities were asked for their current guidelines for elective cosmetic surgery and, in a single plastic surgery unit, we examined the impact of its guidelines by observing consultations and interviewing surgeons and managers. Of 115 authorities approached, 32 reported using guidelines and provided sufficient information for analysis. Guidelines mostly concerned arbitrary sets of cosmetic procedures and lacked reference to an evidence base. They allowed surgery for specified anatomical, functional or symptomatic reasons, but these indications varied between guidelines. Most guidelines also permitted surgery 'exceptionally' for psychological reasons. The guidelines that were studied in detail did not appreciably influence surgeons' decisions, which reflected criteria that were not cited in the guidelines, including cost of the procedure and whether patients sought restoration or improvement of their appearance. Decision guidelines in this area have several limitations. Future guidelines should: include all cosmetic procedures; be informed by a broad range of evidence; and, arguably, include several nonclinical criteria that currently inform surgeons' decision-making.

Development of the ASQoL: a quality of life instrument specific to ankylosing spondylitis.

BACKGROUND: Although disease-specific health status measures are available for ankylosing spondylitis (AS), no instrument exists for assessing quality of life (QoL) in the condition. OBJECTIVE: To produce an AS-specific QoL measure that would be relevant and acceptable to respondents, valid, and reliable. METHODS: The ASQoL employs the needs-based model of QoL and was developed in parallel in the UK and the Netherlands (NL). Content was derived from interviews with patients in each country. Face and content validity were assessed through patient field test interviews (UK and NL). A postal survey in the UK produced a more efficient version of the ASQoL, which was tested for scaling properties, reliability, internal consistency, and validity in a further postal survey in each country. RESULTS: A 41 item questionnaire was derived from interview transcripts. Field testing interviews confirmed acceptability. Rasch analysis of data from the first survey (n=121) produced a 26 item questionnaire. Rasch analysis of data from the second survey (UK: n=164; NL: n=154) showed some item misfit, but showed that items formed a hierarchical order and were stable over time. Problematic items were removed giving an 18 item scale. Both language versions had excellent internal consistency (alpha=0.89-0.91), test-retest reliability (r(s)=0.92 UK and r(s)=0.91 NL), and validity. CONCLUSIONS: The ASQoL provides a valuable tool for assessing the impact of interventions for AS and for evaluating models of service delivery. It is well accepted by patients, taking about four minutes to complete, and has excellent scaling and psychometric properties.

Mouse paracentric inversion In(3)55Rk mutates the urate oxidase gene.

The paracentric inversion In(3)55Rk on mouse Chromosome 3 (Chr 3) was induced by cesium irradiation. Genetic crosses indicate the proximal breakpoint cosegregates with D3Mit324 and D3Mit92; the distal breakpoint cosegregates with D3Mit127, D3Mit160, and D3Mit200. Giemsa-banded chromosomes show the inversion spans approximately 80% of Chr 3. The proximal breakpoint occurs within band 3A2, not 3B as reported previously; the distal breakpoint occurs within band 3H3. Mice homozygous for the inversion exhibit nephropathy indicative of uricase deficiency. Southern blot analyses of urate oxidase, Uox, show two RFLPs of genomic mutant DNA: an EcoRI site between exons 4-8 and a BamHI site 3' to exon 6. Mutant cDNA fails to amplify downstream of base 844 at the 3' end of exon 7. FISH analysis of chromosomes from inversion heterozygotes, using a cosmid clone containing genomic wild-type DNA for Uox exons 2-4, shows that a 5' segment of the mutated Uox allele on the inverted chromosome has been transposed from the distal breakpoint region to the proximal breakpoint region. Clinical, histopathological, and Northern analyses indicate that our radiation-induced mutation, uox(In), is a putative null.

Outbreak of tuberculosis associated with a church.

Investigation of an outbreak of tuberculosis (TB) in a West Midlands health district in 1999 revealed spread in an extended family network and to church contacts. Within the family four cases of smear positive TB, four cases of smear negative infection, and 14 cases requiring chemoprophylaxis were identified. One of the infectious cases visited a local church on two occasions, which resulted in a further 16 cases of infection including one case of tuberculous meningitis. DNA fingerprinting of isolates from five culture positive cases indicated that the same strain of Mycobacterium tuberculosis was responsible. This outbreak is a reminder that while outbreaks of TB usually arise within households or family networks, where close contact over extended periods provides more opportunity for exposure, community outbreaks of TB can occur after only causal contact.

Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential.

Cardiac myocyte apoptosis is potentially important in many cardiac disorders. In other cells, Bcl-2 family proteins and mitochondrial dysfunction are probably key regulators of the apoptotic response. In the present study, we characterized the regulation of antiapoptotic (Bcl-2, Bcl-xL) and proapoptotic (Bad, Bax) Bcl-2 family proteins in the rat heart during development and in oxidative stress-induced apoptosis. Bcl-2 and Bcl-xL were expressed at high levels in the neonate, and their expression was sustained during development. In contrast, although Bad and Bax were present at high levels in neonatal hearts, they were barely detectable in adult hearts. We confirmed that H(2)O(2) induced cardiac myocyte cell death, stimulating poly(ADP-ribose) polymerase proteolysis (from 2 hours), caspase-3 proteolysis (from 2 hours), and DNA fragmentation (from 8 hours). In unstimulated neonatal cardiac myocytes, Bcl-2 and Bcl-xL were associated with the mitochondria, but Bad and Bax were predominantly present in a crude cytosolic fraction. Exposure of myocytes to H(2)O(2) stimulated rapid translocation of Bad (<5 minutes) to the mitochondria. This was followed by the subsequent degradation of Bad and Bcl-2 (from approximately 30 minutes). The levels of the mitochondrial membrane marker cytochrome oxidase remained unchanged. H(2)O(2) also induced translocation of cytochrome c from the mitochondria to the cytosol within 15 to 30 minutes, which was indicative of mitochondrial dysfunction. Myocytes exposed to H(2)O(2) showed an early loss of mitochondrial membrane potential (assessed by fluorescence-activated cell sorter analysis) from 15 to 30 minutes, which was partially restored by approximately 1 hour. However, a subsequent irreversible loss of mitochondrial membrane potential occurred that correlated with cell death. These data suggest that the regulation of Bcl-2 and mitochondrial function are important factors in oxidative stress-induced cardiac myocyte apoptosis.

Activation of c-Jun N-terminal kinases and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease.

Three well-characterized mitogen-activated protein kinase (MAPK) subfamilies are expressed in rodent and rabbit hearts, and are activated by pathophysiological stimuli. We have determined and compared the expression and activation of these MAPKs in donor and failing human hearts. The amount and activation of MAPKs was assessed in samples from the left ventricles of 4 unused donor hearts and 12 explanted hearts from patients with heart failure secondary to ischaemic heart disease. Total MAPKs or dually phosphorylated (activated) MAPKs were detected by Western blotting and MAPK activities were measured by in gel kinase assays. As in rat heart, c-Jun N-terminal kinases (JNKs) were detected in human hearts as bands corresponding to 46 and 54 kDa; p38-MAPK(s) was detected as a band corresponding to approximately 40 kDa, and extracellularly regulated kinases, ERK1 and ERK2, were detected as 44- and 42-kDa bands respectively. The total amounts of 54 kDa JNK, p38-MAPK and ERK2 were similar in all samples, although 46-kDa JNK was reduced in the failing hearts. However, the mean activities of JNKs and p38-MAPK(s) were significantly higher in failing heart samples than in those from donor hearts (P<0.05). There was no significant difference in phosphorylated (activated) ERKs between the two groups. In conclusion, JNKs, p38-MAPK(s) and ERKs are expressed in the human heart and the activities of JNKs and p38-MAPK(s) were increased in heart failure secondary to ischaemic heart disease. These data indicate that JNKs and p38-MAPKs may be important in human cardiac pathology.

Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eya1 gene in a mouse model of BOR syndrome.

A spontaneous mutation causing deafness and circling behavior was discovered in a C3H/HeJ colony of mice at the Jackson Laboratory. Pathological analysis of mutant mice revealed gross morphological abnormalities of the inner ear, and also dysmorphic or missing kidneys. The deafness and abnormal behavior were shown to be inherited as an autosomal recessive trait and mapped to mouse chromosome 1 near the position of the Eya1 gene. The human homolog of this gene, EYA1, has been shown to underly branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by hearing loss with associated branchial and renal anomalies. Molecular analysis of the Eya1 gene in mutant mice revealed the insertion of an intracisternal A particle (IAP) element in intron 7. The presence of the IAP insertion was associated with reduced expression of the normal Eya1 message and formation of additional aberrant transcripts. The hypomorphic nature of the mutation may explain its recessive inheritance, if protein levels in homozygotes, but not heterozygotes, are below a critical threshold needed for normal developmental function. The new mouse mutation is designated Eya1(bor) to denote its similarity to human BOR syndrome, and will provide a valuable model for studying mutant gene expression and etiology.

A new allelic series for the underwhite gene on mouse chromosome 15.

A new allelic series at the underwhite gene is described. Three of the alleles in the series--uw, uwd, and Uwdbr--arose as spontaneous mutations on different genetic backgrounds at The Jackson Laboratory. We report here the visible phenotypes and dominance hierarchy of these alleles, all of which are defined by a reduction of pigmentation in both eye and coat color. Electron microscopic analysis of retinal epithelium suggests that the primary defect is in the melanosome. The degree of severity of melanosome anomalies in the retina correlates with the degree of hypopigmentation in the coat. The perturbed gene and its gene product are unknown. We show that the uw locus is genetically distinct from Myo10, a suggested candidate gene for this mutation.

The original shaker-with-syndactylism mutation (sy) is a contiguous gene deletion syndrome.

Tests for allelism among mice with four different mutant alleles at the shaker-with-syndactylism locus on mouse Chromosome (Chr) 18 provide evidence that the original radiation-induced mutation, sy, is a deletion including at least two genes associated with distinct phenotypes. Mice homozygous for sy have syndactylous feet and other skeletal malformations, are deaf, and exhibit abnormal behavior characteristic of vestibular dysfunction. Two less severe spontaneous mutations, shown to be allelic with sy, cause syndactylism when homozygous (hence named fused phalanges, sy(fp) and sy(fp-2J)), but do not affect hearing and behavior. Here we describe a third spontaneous mutation allelic with sy that does not affect foot morphology (hence named no syndactylism, sy(ns)), but that does cause deafness and balance defects when homozygous. Complementation test results indicate that sy(fp) and sy(fp-2J) are alleles of the same gene, but that sy(ns) is an allele of a different gene. The original sy mutation, therefore, includes both of the genes defined by these three spontaneous mutations. Typing of DNA markers in sy/sy mice revealed a deletion of approximately 1 cM in the sy region of Chr 18, including D18Mit52, D18Mit124, D18Mit181, and D18Mit205. The genetic relationships described here will aid in positional cloning efforts to identify the genes responsible for the disparate phenotypes associated with the sy locus.

Juvenile bare: a new hair loss mutation on chromosome 7 of the mouse.

We describe a new juvenile hair loss mutant in the mouse in which the hair follicle follows irregular pathways to the surface and generally becomes dystrophic when the mouse is about 23 days of age. Skin from mutant mice older than 1 month of age is histologically normal, although adult mutant mice show a slightly more sparse coat than normal. Grafts of mutant littermates skin to SCID hosts indicate that the condition is probably a systemic response rather than one of the follicle per se. The hair loss is caused by a recessive mutation, which we have named juvenile bare (jb), located on proximal chromosome 7.