Well-being, physical activity and long-term conditions: cross-sectional analysis of Health Survey for England 2016.

OBJECTIVES: We investigated whether physical activity is associated with greater well-being in people with multiple long-term conditions or limiting long-term illness (LLI). STUDY DESIGN: Cross-sectional analysis of data from the Health Survey for England 2016. METHODS: The Warwick-Edinburgh mental well-being score (WEMWBS) was evaluated according to number of days per week with >30 min moderate or vigorous activity. LLI and number of long-term conditions were evaluated as effect modifiers, adjusting for age, sex, smoking, body mass index and education. Marginal effects were estimated for female non-smokers, aged 45-54 years. RESULTS: Data were analyzed for 5952 adults (female, 3275; male, 2677) including 1104 (19%) with non-limiting long-term illness and 1486 (25%) with LLI. There were 2065 (35%) with 1-2 long-term conditions, 461 (8%) with 3-4 and 58 (1%) with 5-6 long-term conditions. Participants with LLI were less likely to engage in physical activity on 5 or more days per week (LLI, 24%; No LLI, 47%) and more likely to be inactive (LLI, 41%; No LLI 13%). The adjusted marginal mean WEMWBS for inactive participants with no long-term illness was 49.0 (95% confidence interval 48.1 to 50.0), compared with 51.1 (50.4-51.8) if active on 5+ days per week. In LLI, the adjusted marginal mean WEMWBS was 41.6 (40.7-42.5) if inactive but 47.6 (46.6-48.6) if active on 5+ days per week. Similar associations were observed for the number of long-term conditions. CONCLUSIONS: Physical activity may be associated with greater increments in well-being among people with multiple long-term conditions or LLI than those without.

Reliability of OperaVOX against Multidimensional Voice Program (MDVP).

OBJECTIVE: To evaluate the agreement between OperaVOX and MDVP. DESIGN: Cross sectional reliability study. SETTING: University teaching hospital. METHODS: Fifty healthy volunteers and 50 voice disorder patients had supervised recordings in a quiet room using OperaVOX by the iPod's internal microphone with sampling rate of 45 kHz. A five-seconds recording of vowel/a/was used to measure fundamental frequency (F0), jitter, shimmer and noise-to-harmonic ratio (NHR). All healthy volunteers and 21 patients had a second recording. The recorded voices were also analysed using the MDVP. The inter- and intrasoftware reliability was analysed using intraclass correlation (ICC) test and Bland-Altman (BA) method. Mann-Whitney test was used to compare the acoustic parameters between healthy volunteers and patients. RESULTS: Nine of 50 patients had severe aperiodic voice. The ICC was high with a confidence interval of >0.75 for the inter- and intrasoftware reliability except for the NHR. For the intersoftware BA analysis, excluding the severe aperiodic voice data sets, the bias (95% LOA) of F0, jitter, shimmer and NHR was 0.81 (11.32, -9.71); -0.13 (1.26, -1.52); -0.52 (1.68, -2.72); and 0.08 (0.27, -0.10). For the intrasoftware reliability, it was -1.48 (18.43, -21.39); 0.05 (1.31, -1.21); -0.01 (2.87, -2.89); and 0.005 (0.20, -0.18), respectively. Normative data from the healthy volunteers were obtained. There was a significant difference in all acoustic parameters between volunteers and patients measured by the Opera-VOX (P < 0.001) except for F0 in females (P = 0.87). CONCLUSION: OperaVOX is comparable to MDVP and has high internal consistency for measuring the F0, jitter and shimmer of voice except for the NHR.

Targeting gene expression to hypoxic tumor cells.

Solid tumors with areas of low oxygen tension (hypoxia) have a poor prognosis, as cells in this environment often survive radiation and chemotherapy. In this report we describe how this hypoxic environment can be used to activate heterologous gene expression driven by a hypoxia-responsive element (HRE), which interacts with the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Our results demonstrate that the HIF-1/HRE system of gene regulation is active in hypoxic tumor cells and show the potential of exploiting tumor-specific conditions for the targeted expression of diagnostic or therapeutic genes in cancer therapy.

Oxygen-dependent modulation of erythropoietin mRNA levels in isolated rat kidneys studied by RNase protection.

Using oligonucleotide primers complementary to conserved regions in the mouse erythropoietin (Epo) gene, a portion of the rat Epo gene was amplified by the polymerase chain reaction to produce a probe suitable for assay of rat Epo mRNA by RNAse protection. The assay, which has sufficient sensitivity to measure to Epo mRNA in unstimulated rat kidneys, was used to demonstrate high amplitude in vitro modulation of Epo mRNA levels in response to changes in perfusate flow rate and oxygen tension in isolated kidneys, thus providing clear evidence that all the necessary events linking changes in oxygen delivery to the modulation of Epo mRNA levels can occur intrarenally.

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Activation of the HIF pathway in cancer.

The maintenance of oxygen homeostasis is required both in physiological development and tumour growth. Hypoxia inducible factor (HIF) plays a central role in both processes. Reliable methods for visualising HIF alpha subunits have established that HIF activation occurs in the majority of common cancers. This occurs both by genetic mechanisms and through microenvironmental hypoxia. Activation of the HIF pathway has important effects on patterns of gene expression in tumours.

Mutation of the von Hippel-Lindau gene alters human cardiopulmonary physiology.

Intracellular responses to hypoxia are coordinated by the von Hippel-Lindau--hypoxia-inducible factor (VHL-HIF) transcriptional system. This study investigated the potential role of the VHL-HIF pathway in human systems-level physiology. Patients diagnosed with Chuvash polycythaemia, a rare disorder in which VHL signalling is specifically impaired, were studied during acute hypoxia and hypercapnia. Subjects breathed through a mouthpiece and ventilation was measured while pulmonary vascular tone was assessed echocardiographically. The patients were found to have elevated basal ventilation and pulmonary vascular tone, and ventilatory, pulmonary vasoconstrictive and heart rate responses to acute hypoxia were greatly increased, as were heart rate responses to hypercapnia. The patients also had abnormal pulmonary function on spirometry. This study's findings demonstrate that the VHL-HIF signalling pathway, which is so central to intracellular oxygen sensing, also regulates the organ systems upon which cellular oxygen delivery ultimately depends.

Organ distribution of the three rat endothelin messenger RNAs and the effects of ischemia on renal gene expression.

To determine the organ distribution of production of the three endothelin (ET) isopeptides, we have developed three ribonuclease protection assays specific for the messenger RNAs (mRNAs) of rat ETs 1, 2, and 3.12 organs from adult Sprague-Dawley rats were examined: heart, lung, liver, spleen, kidney, stomach, small intestine, large intestine, testis, muscle, salivary gland, and brain. The mRNA for ET1 was five times more abundant in the lung than in any other organ studied, moderate expression was seen in the large intestine, and lower levels of mRNA were detected in each of the other organs examined. ET2 was expressed at high level in both large and small intestine and at low level in stomach, muscle, and heart, but ET2 mRNA could not be detected elsewhere. ET3 mRNA was found in all organs, particularly in small intestine, lung, kidney, and large intestine. Because of reports suggesting that ETs might be involved in the hypoperfusion and hypofiltration observed in postischemic kidneys, we have also studied levels of mRNA in kidneys that had previously been subjected to 25 or 45 min of clamping of the renal pedicle. At 6 h after 45 min of ischemia, ET1 mRNA increased to a peak of 421 +/- 69% (mean +/- SEM, n = 3) of that in a standard renal RNA preparation. By contrast, ET3 mRNA decreased in the postischemic organ, falling to a value of 19 +/- 2% of standard at the same time point. The effects of ischemia on ET1 and ET3 mRNAs were long-lasting, with elevation of ET1 and depression of ET3 persisting for days. ET2 mRNA remained undetectable throughout. These findings (a) support a role for ET1 in postischemic renal vascular phenomena and (b) demonstrate a situation in which the expression of ET isoforms is clearly subject to differential regulation.

Age-dependent expression of the erythropoietin gene in rat liver and kidneys.

Using RNAse protection, we have made quantitative measurements of erythropoietin (EPO) mRNA in liver and kidneys of developing rats (days 1-54), to determine the relative contribution of both organs to the total EPO mRNA, to monitor changes which occur with development, and to compare the hypoxia-induced accumulation of EPO mRNA with the changes in serum EPO concentrations. To determine whether developmental and organ-specific responsiveness is related to the type of hypoxic stimulus, normobaric hypoxia was compared with exposure to carbon monoxide (functional anemia). Under both stimuli EPO mRNA concentration in liver was maximal on day 7 and declined during development. In contrast, EPO mRNA concentration in kidney increased during development from day 1 when it was 30-65% the hepatic concentration to day 54 when it was 12-fold higher than in liver. When organ weight was considered the liver was found to contain the majority of EPO mRNA in the first three to four weeks of life, and although, in stimulated animals, the hepatic proportion declined from 85-91% on day 1, it remained approximately 33% at day 54 and was similar for the two types of stimuli. When normalized for body weight the sum of renal and hepatic EPO mRNA in animals of a particular age was related linearly to serum hormone concentrations. However, the slope of this regression increased progressively with development, suggesting age-dependent alterations in translational efficiency or EPO metabolism.

Increased plasma viscosity as a reason for inappropriate erythropoietin formation.

The aim of this study was to examine whether altered plasma viscosity could contribute to the inappropriately low production rate of erythropoietin (EPO) observed in patients suffering from hypergammaglobulinemias associated with multiple myeloma or Waldenström's disease. We found that the EPO formation in response to anemia in these patients was inversely related to plasma viscosity. A similar inverse relationship between plasma viscosity and EPO production was seen in rats in which EPO formation had been stimulated by exchange transfusion and the plasma viscosity of which was thereby altered by using exchange solutions of different composition to alter plasma viscosity and thus whole blood viscosity independently from hematocrit. Raising the gammaglobulin concentration to approximately 40 mg/ml plasma in the rats almost totally blunted the rise in serum EPO levels despite a fall of the hematocrit to 20%. Determination of renal EPO mRNA levels by RNase protection revealed that the reductions in serum EPO levels at higher plasma viscosities were paralleled by reductions in renal EPO mRNA levels. Taken together, our findings suggest that plasma viscosity may be a significant inhibitory modulator of anemia-induced EPO formation. The increased plasma viscosity in patients with hypergammaglobulinemias may therefore contribute to the inappropriate EPO production, which is a major reason for the anemia developing in these patients.

HIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumors.

Solid tumors contain regions of hypoxia, a physiological stress that can activate cell death pathways and, thus, result in the selection of cells resistant to death signals and anticancer therapy. Bcl2/adenovirus EIB 19kD-interacting protein 3 (BNIP3) is a cell death factor that is a member of the Bcl-2 proapoptotic family recently shown to induce necrosis rather than apoptosis. Using cDNA arrays and serial analysis of gene expression, we found that hypoxia induces up-regulation of BNIP3 and its homologue, Nip3-like protein X. Analysis of human carcinoma cell lines showed that they are hypoxically regulated in many tumor types, as well as in endothelial cells and macrophages. Regulation was hypoxia inducible factor-1-dependent, and hypoxia inducible factor-1 expression was suppressed by von Hippel-Lindau protein in normoxic cells. Northern blotting and in situ hybridization analysis has revealed that these factors are highly expressed in human tumors compared with normal tissue and that BNIP3 is up-regulated in perinecrotic regions of the tumor. This study shows that genes regulating cell death can be hypoxically induced and are overexpressed in clinical tumors.

Carbonic anhydrase IX, an endogenous hypoxia marker, expression in head and neck squamous cell carcinoma and its relationship to hypoxia, necrosis, and microvessel density.

Carbonic anhydrase IX (CA IX) is a transmembrane glycoprotein with an active extracellular enzyme site. We have shown previously that it was hypoxia inducible and may therefore be an endogenous marker of hypoxia. It is overexpressed in some tumors, particularly renal cell carcinoma. The aim of this study was to examine the expression and localization of CA IX in head and neck squamous cell carcinoma (HNSCC) and relate this to the location of tumor microvessels, angiogenesis, necrosis, and stage. Expression of CA IX was determined by immunoblotting in three HNSCC cell lines grown in normoxia and hypoxia (pO(2) 0.1%) and three paired tumor and normal tissue samples of HNSCC. Archived paraffin sections (79) of HNSCC were immunostained with antibodies to CA IX and CD34 to determine microvessel density (MVD). By double staining sections with CA IX and CD34, the distance between blood vessels and the start of CA IX expression and necrosis was calculated. CA IX was induced by hypoxia in all three HNSCC cell lines and overexpressed in HNSCC tumor tissue. Overexpression was localized to the perinecrotic area of the tumor on immunostaining, and the percentage area of the tumor expressing CA IX was significantly higher with more tumor necrosis (P = 0.001), a high MVD (P = 0.02), and advanced stage (P = 0.033) on univariate analysis and necrosis (P = 0.0003) and MVD (P = 0.0019) on multivariate analysis. The median distance between a blood vessel and the start of CA IX expression was 80 microm (range, 40-140 microm). CA IX is overexpressed in HNSCC because of hypoxia and is a potential biomarker for hypoxia in this tumor. Overexpression may help to maintain the intracellular pH, giving tumor cells a survival advantage and enhancing resistance to radiotherapy and chemotherapy. CA IX is a potential target for future therapy in HNSCC.

Hypoxia-inducible expression of tumor-associated carbonic anhydrases.

The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.

Carbonic anhydrase (CA IX) expression, a potential new intrinsic marker of hypoxia: correlations with tumor oxygen measurements and prognosis in locally advanced carcinoma of the cervix.

There is increasing evidence that hypoxia-regulated gene expression influences tumor aggressiveness, contributing to the poorer outcome of patients with hypoxic tumors. The role of the transcriptional complex hypoxia-inducible factor-1 as an important mediator of hypoxia-regulated gene expression is one of the best documented pathways. Recently, it has emerged that certain tumor-associated carbonic anhydrases (CAs) can be added to the list of known hypoxia-inducible factor-responsive genes. Here we show that the immunohistochemical expression of the tumor-associated CA IX is correlated with the level of hypoxia in human cervical tumors. We performed a prospective study in 68 patients where needle electrodes were used to make direct measurements of tumor oxygenation levels. CA IX expression was evaluated immunohistochemically in pretreatment tumor biopsies. There was a significant positive correlation between the level of tumor hypoxia (HP5) and the extent of CA IX expression. A retrospective study of 130 squamous cell cervical carcinomas demonstrated that a semiquantitative immunohistochemical analysis of CA IX expression in tumor biopsies is a significant and independent prognostic indicator of overall survival and metastasis-free survival after radiation therapy. These studies provide clinical evidence that CA IX expression is up-regulated in hypoxic human cervical tumors and is associated with a poor prognosis. CA IX may act as an intrinsic marker of tumor hypoxia and poor outcome after radiation therapy. The level of CA IX expression may be used to aid in the selection of patients who would benefit most from hypoxia-modification therapies or bio-reductive drugs.

Identification of novel hypoxia dependent and independent target genes of the von Hippel-Lindau (VHL) tumour suppressor by mRNA differential expression profiling.

The von Hippel-Lindau tumour suppressor gene (VHL) targets hypoxia inducible factor (HIF)-alpha subunits for ubiquitin dependent proteolysis. To better understand the role of this and other putative pathways of gene regulation in VHL function we subjected mRNA from VHL defective renal carcinoma cells and transfectants re-expressing a wild type VHL allele to differential expression profiling, and analysed VHL target genes for oxygen regulated expression. Among a group of newly identified VHL target genes the majority but not all were regulated by oxygen, indicating that whilst dysregulation of the HIF system makes a dominant contribution to alterations in transcription, VHL has other influences on patterns of gene expression. Genes newly defined as targets of the VHL/hypoxia pathway (conditionally downregulated by VHL in normoxic cells) include aminopeptidase A, collagen type V, alpha 1, cyclin G2, DEC1/Stra13, endothelin 1, low density lipoprotein receptor-related protein 1, MIC2/CD99, and transglutaminase 2. These genes have a variety of functions relevant to tumour biology. However, not all are connected with the promotion of tumour growth, some being pro-apoptotic or growth inhibitory. We postulate that co-ordinate regulation as part of the HIF pathway may explain this paradox, and that evolution of anti-apoptotic pathways may be required for tumour growth under VHL-dysregulation. Our results indicate that it will be necessary to consider the effects of abnormal activity in integral regulatory pathways, as well as the effects of individual genes to understand the role of abnormal patterns of gene expression in cancer.

The pVHL-associated SCF ubiquitin ligase complex: molecular genetic analysis of elongin B and C, Rbx1 and HIF-1alpha in renal cell carcinoma.

The VHL gene product (pVHL) forms a multimeric complex with the elongin B and C, Cul2 and Rbx1 proteins (VCBCR complex), which is homologous to the SCF family of ubiquitin ligase complexes. The VCBCR complex binds HIF-1alpha and HIF-2alpha, transcription factors critically involved in cellular responses to hypoxia, and targets them for ubiquitin-mediated proteolysis. Germline mutations in the VHL gene cause susceptibility to haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and other tumours. In addition somatic inactivation of the VHL gene occurs in most sporadic clear cell RCC (CC-RCC). However, the absence of somatic VHL inactivation in 30-40% of CC-RCC implies the involvement of other gatekeeper genes in CC-RCC development. We reasoned that in CC-RCC without VHL inactivation, other pVHL-interacting proteins might be defective. To assess the role of elongin B/C, Rbx1 and HIF-1alpha in RCC tumorigenesis we (a) mapped the genes to chromosomes 8q(cen) (elongin C), 16p13.3 (elongin B) and 22q11.2 (Rbx1) by FISH, monochromosomal somatic cell hybrid panel screening and in silico GenBank homology searching; (b) determined the genomic organisation of elongin C (by direct sequencing of PAC clones), Rbx1 and elongin B (by GenBank homology searching); and (c) performed mutation analysis of exons comprising the coding regions of elongins B, C and Rbx1 and the oxygen-dependent degradation domain of HIF-1alpha by SSCP screening and direct sequencing in 35 sporadic clear cell RCC samples without VHL gene inactivation and in 13 individuals with familial non-VHL clear cell RCC. No coding region sequence variations were detected for the elongin B, elongin C or Rbx1 genes. Two amino acid substitutions (Pro582Ser and Ala588Thr) were identified in the oxygen-dependent degradation/pVHL binding domain of HIF-1alpha, however neither substitution was observed exclusively in tumour samples. Association analysis in panels of CC-RCC and non-neoplastic samples using the RFLPs generated by each variant did not reveal allelic frequency differences between RCC patients and controls (P>0.32 by chi-squared analysis). Nevertheless, the significance of these variations and their potential for modulation of HIF-1alpha function merits further investigation in both other tumour types and in non-neoplastic disease. Taken together with our previous Cul2 mutation analysis these data suggest that development of sporadic and familial RCC is not commonly contributed to by genetic events altering the destruction domain of HIF-1alpha, or components of the HIF-alpha destruction complex other than VHL itself. Although (a) activation of HIF could occur through mutation of another region of HIF-a, and (b) epigenetic silencing of elongin B/C, Cul2 or Rbx1 cannot be excluded, these findings suggest that pVHL may represent the sole mutational target through which the VCBR complex is disrupted in CC-RCC. HIF response is activated in CC-RCC tumorigenesis.

Characterisation of functional domains within the mouse erythropoietin 3' enhancer conveying oxygen-regulated responses in different cell lines.

We have analysed sequences within the mouse erythropoietin enhancer which are required for oxygen regulated operation in the erythropoietin producing cell line, HepG2, and in two non-erythropoietin producing cell lines; the lung fibroblastoid cell line a23, and mouse erythroleukaemia (MEL) cells. At least three critical sites were demonstrated within a 96 nucleotide sequence. Oxygen regulated operation was dependent on sites within the first 26 nucleotides. Sequences lying 3' to this region modulated enhancer function but did not themselves convey oxygen regulated operation. In HepG2 cells these 3' sequences co-operated to permit operation of the inducible element at a distance from a promoter, but in MEL cells 3' sequences repressed activity of the inducible element. Though operation of this 3' sequence differed according to the cell type, oxygen regulated operation was dependent on the same two critical sites in the 5' region in both erythropoietin producing and non-erythropoietin producing cells. These findings support the existence of a widespread oxygen sensing system in mammalian cells which is similar to that operating in specific cells to regulate erythropoietin production, and they indicate that the system activates factors with similar DNA sequence specificity in different cells.

Prognostic significance of a novel hypoxia-regulated marker, carbonic anhydrase IX, in invasive breast carcinoma.

PURPOSE: To assess the frequency of expression and the prognostic significance of a hypoxia-regulated marker, carbonic anhydrase IX (CA IX), in a cohort of patients with invasive breast cancer. PATIENTS AND METHODS: CA IX expression was evaluated by immunohistochemistry with a murine monoclonal antibody, M75, in a series of 103 women treated surgically for invasive breast cancer. The majority of patients were treated with adjuvant hormonal or chemotherapy. The frequency of CA IX expression, its association with recognized prognostic factors, and the relationship with outcome was evaluated by univariate and multivariate statistical analyses. RESULTS: CA IX expression was present in 49 (48%) of 103 cases. The level of CA IX expression was found to be significantly associated with tumor necrosis (P <.001), higher grade (P =.02), and negative estrogen receptor status (P <.001). Furthermore, CA IX expression was associated with a higher relapse rate (P =.004) and a worse overall survival (P =.001). By multivariate analysis, CA IX was also shown to be an independent predictive factor for overall survival (hazard ratio, 2.61; 95% confidence interval, 1.01 to 6.75, P =.05). CONCLUSION: CA IX expression was associated with worse relapse-free survival and overall survival in an unselected cohort of patients with invasive breast carcinoma. The potential role of CA IX as a marker of hypoxia within breast carcinomas was also indicated by a significant association with necrosis. Further work assessing its prognostic significance in breast cancer is warranted, particularly interactions with radiotherapy and chemotherapy resistance.

Tumor hypoxia induces nuclear paraspeckle formation through HIF-2α dependent transcriptional activation of NEAT1 leading to cancer cell survival.

Correction to: Oncogene (2015) 34, 4482­4490; doi:10.1038/onc.2014.378; published online 24 November 2014. Following the online publication of this article, the authors have noticed a misspelt surname: S Hider should read S Haider. There is also an addition to the acknowledgements to read 'This study makes use of data generated by the Molecular Taxonomy of Breast Cancer International Consortium, which was funded by Cancer Research UK and the British Columbia Cancer Agency Branch'. The corrected article appears in this issue. The authors would like to apologise for any inconvenience this may cause.