Tablet and web-based audiometry to screen for hearing loss in adults with cystic fibrosis.

INTRODUCTION: Individuals with chronic lung disease (eg, cystic fibrosis (CF)) often receive antimicrobial therapy including aminoglycosides resulting in ototoxicity. Extended high-frequency audiometry has increased sensitivity for ototoxicity detection, but diagnostic audiometry in a sound-booth is costly, time-consuming and requires a trained audiologist. This cross-sectional study analysed tablet-based audiometry (Shoebox MD) performed by non-audiologists in an outpatient setting, alongside home web-based audiometry (3D Tune-In) to screen for hearing loss in adults with CF. METHODS: Hearing was analysed in 126 CF adults using validated questionnaires, a web self-hearing test (0.5 to 4 kHz), tablet (0.25 to 12 kHz) and sound-booth audiometry (0.25 to 12 kHz). A threshold of ≥25 dB hearing loss at ≥1 audiometric frequency was considered abnormal. Demographics and mitochondrial DNA sequencing were used to analyse risk factors, and accuracy and usability of hearing tests determined. RESULTS: Prevalence of hearing loss within any frequency band tested was 48%. Multivariate analysis showed age (OR 1.127; (95% CI: 1.07 to 1.18; p value<0.0001) per year older) and total intravenous antibiotic days over 10 years (OR 1.006; (95% CI: 1.002 to 1.010; p value=0.004) per further intravenous day) were significantly associated with increased risk of hearing loss. Tablet audiometry had good usability, was 93% sensitive, 88% specific with 94% negative predictive value to screen for hearing loss compared with web self-test audiometry and questionnaires which had poor sensitivity (17% and 13%, respectively). Intraclass correlation (ICC) of tablet versus sound-booth audiometry showed high correlation (ICC >0.9) at all frequencies ≥4 kHz. CONCLUSIONS: Adults with CF have a high prevalence of drug-related hearing loss and tablet-based audiometry can be a practical, accurate screening tool within integrated ototoxicity monitoring programmes for early detection.

Challenges of emerging adulthood-transition from paediatric to adult diabetes.

Diabetes mellitus is a complex condition with far reaching physical, psychological and psychosocial effects. These outcomes can be significant when considering the care of a youth transferring from paediatric through to adult diabetes services. The art of mastering a smooth care transfer is crucial if not pivotal to optimising overall diabetic control. Quite often the nature of consultation varies between the two service providers and the objectives and outcomes will mirror this. The purpose of this review is to analyse the particular challenges and barriers one might expect to encounter when transferring these services over to an adult care provider. Particular emphasis is paid towards the psychological aspects of this delicate period, which needs to be recognised and appreciated appropriately in order to understand the particular plights a young diabetic child will be challenged with. We explore the approaches that can be positively adopted in order to improve the experience for child, parents and also the multi- disciplinary team concerned with the overall delivery of this care. Finally we will close with reflection on the potential areas for future development that will ultimately aim to improve long-term outcomes and experiences of the young adolescent confronted with diabetes as well as the burden of disease and burden of cost of disease.

Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling.

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer.

p53 mutation and loss have different effects on tumourigenesis in a novel mouse model of pleomorphic rhabdomyosarcoma.

Pleomorphic rhabdomyosarcoma is the most common variant of this tumour in adults and has a very poor outcome. Two genes which are known to play a role in rhabdomyosarcoma development are KRas and p53. In the majority of human tumours, p53 abnormalities are point mutations that result in the expression of a mutant form of the protein. It is now hypothesized that these mutant forms of p53 may be playing an oncogenic role, over and above simple loss of the wild-type function. In this study, we use Cre-LoxP technology to develop a novel mouse model of rhabdomyosarcoma, crossing mice expressing a common KRas mutation (G12V) with mice that either lose p53 expression or express a mutant form of p53. We use this model to explore the different effects of p53 loss and mutation in the setting of an activating KRas mutation. We found that either complete loss of p53 (p53(fl/fl)) or the expression of one mutant p53 allele with concomitant loss of the second allele (p53(R172H/+)) resulted in the rapid development of rhabdomyosarcoma in 15/16 and 19/19 mice, respectively. In contrast, there was a marked difference between mice which lose a single copy of p53 (p53(fl/+)) and mice expressing a single copy of mutant p53 (p53(172H/+)). Fourteen out of 16 p53(R172H/) mice developed rhabdomyosarcoma, compared with two out of 31 p53(fl/+) mice. As a consequence of this, p53(fl/+) mice had a median lifespan nearly double that of the p53(R172H/+) mice. To underline the enhanced effect of p53 mutation in tumour progression, metastases were seen only in those mice which expressed the mutant form. These data demonstrate that mutant p53 can co-operate with activated, mutant KRas to influence tumourigenesis and metastatic potential, over and above simple loss of normal protein function.

B-catenin deficiency, but not Myc deletion, suppresses the immediate phenotypes of APC loss in the liver.

Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear beta-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors beta-catenin and c-Myc. beta-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being beta-catenin-dependent but c-Myc-independent in the liver.

C-Myc is a critical mediator of the phenotypes of Apc loss in the intestine.

The Adenomatous polyposis coli (Apc) gene is mutated in up to 80% of sporadic colorectal cancers. After Apc loss, there is deregulation of the Wnt signaling pathway and transactivation of T-cell factor/leukemia enhancing factor target genes such as C-Myc. This review focuses on recent data highlighting the importance of the C-Myc oncogene and its transcriptional targets in establishing all of the phenotypes caused by the deletion of the Apc tumor suppressor gene within the intestinal epithelium. The importance of investigating Apc and C-Myc gene function in the correct tissue context is also discussed.

Myc deletion rescues Apc deficiency in the small intestine.

The APC gene encodes the adenomatous polyposis coli tumour suppressor protein, germline mutation of which characterizes familial adenomatous polyposis (FAP), an autosomal intestinal cancer syndrome. Inactivation of APC is also recognized as the key early event in the development of sporadic colorectal cancers, and its loss results in constitutive activity of the beta-catenin-Tcf4 transcription complex. The proto-oncogene c-MYC has been identified as a target of the Wnt pathway in colorectal cancer cells in vitro, in normal crypts in vivo and in intestinal epithelial cells acutely transformed on in vivo deletion of the APC gene; however, the significance of this is unclear. Therefore, to elucidate the role Myc has in the intestine after Apc loss, we have simultaneously deleted both Apc and Myc in the adult murine small intestine. Here we show that loss of Myc rescued the phenotypes of perturbed differentiation, migration, proliferation and apoptosis, which occur on deletion of Apc. Remarkably, this rescue occurred in the presence of high levels of nuclear beta-catenin. Array analysis revealed that Myc is required for the majority of Wnt target gene activation following Apc loss. These data establish Myc as the critical mediator of the early stages of neoplasia following Apc loss.

Deficiency of SPARC suppresses intestinal tumorigenesis in APCMin/+ mice.

BACKGROUND AND AIMS: SPARC (secreted protein acidic, rich in cysteine) is a matricellular protein that has been found to be activated in a number of human cancers. More recently, it has been shown to be upregulated in human gastric and colorectal cancer. We therefore wished to address the functional importance of SPARC upregulation to intestinal tumorigenesis in vivo. METHODS: SPARC upregulation was determined in intestinal adenomas of tumour-prone Apc(Min/+) mice at both the RNA and the protein level. To determine the functional importance of SPARC for intestinal tumorigenesis we then intercrossed Sparc knockout mice with Apc(Min/+) mice (n = 20). Intestinal enterocyte migration was examined using bromodeoxyuridine labelling studies. RESULTS: Levels of murine Sparc and several related proteins were upregulated in adenomas arising in Apc(Min/+) mice. A deficiency of Sparc strongly suppressed adenoma formation in Apc(Min/+) mice (p>or=0.0001). Importantly, a deficiency of Sparc also accelerated enterocyte migration (p = 0.01), as perturbed slow epithelial migration may underpin adenoma formation in the intestine. CONCLUSIONS: These data implicate Sparc in both cell migration and tumour formation, and identify Sparc as a potential therapeutic target for colorectal cancer.

Loss of Apc allows phenotypic manifestation of the transforming properties of an endogenous K-ras oncogene in vivo.

Oncogenic mutations in the K-ras gene occur in approximately 50% of human colorectal cancers. However, the precise role that K-ras oncogenes play in tumor formation is still unclear. To address this issue, we have conditionally expressed an oncogenic K-ras(V12) allele in the small intestine of adult mice either alone or in the context of Apc deficiency. We found that expression of K-ras(V12) does not affect normal intestinal homeostasis or the immediate phenotypes associated with Apc deficiency. Mechanistically we failed to find activation of the Raf/MEK/ERK pathway, which may be a consequence of the up-regulation of a number of negative feedback loops. However, K-ras(V12) expression accelerates intestinal tumorigenesis and confers invasive properties after Apc loss over the long term. In renal epithelium, expression of the oncogenic K-ras(V12) allele in the absence of Apc induces the rapid development of renal carcinoma. These tumors, unlike those of intestinal origin, display activation of the Raf/MEK/ERK and Akt signaling pathways. Taken together, these data indicate that normal intestinal and kidney epithelium are resistant to malignant transformation by an endogenous K-ras oncogene. However, activation of K-ras(V12) after Apc loss results in increased tumorigenesis with distinct kinetics. Whereas the effect of K-ras oncogenes in the intestine can been observed only after long latencies, they result in rapid carcinogenesis in the kidney epithelium. These data imply a window of opportunity for anti-K-ras therapies after tumor initiation in preventing tumor growth and invasion.

Utilization of microarrayed compound screening (microARCS) to identify inhibitors of p56lck tyrosine kinase.

Protein tyrosine kinases play critical roles in cell signaling and are considered attractive targets for drug discovery. The authors have applied microARCS (microarrayed compound screening) technology to develop a high-throughput screen for finding inhibitors of the p56lck tyrosine kinase. Initial assay development was performed in a homogeneous time-resolved (LANCE) format in 96-well microplates and then converted into the gel-based microARCS format. The microARCS methodology is a well-less screening format in which 8640 compounds are arrayed on a microplate-sized piece of polystyene and subsequently assayed by placing reagents cast in agarose gels in contact with these compound sheets. A blotting paper soaked with adenosine triphosphate is applied on the gel to initiate the kinase reaction in the gel. Using this screening methodology, 300,000 compounds were screened in less than 40 h. Substantial reagent reduction was achieved by converting this tyrosine kinase assay from a 96-well plate assay to microARCS, resulting in significant cost savings.

High throughput electrophysiology using a fully automated, multiplexed recording system.

The drug discovery process centers around finding and optimizing novel compounds active at therapeutic targets. This process involves direct and indirect measures of how compounds affect the behavior of the target in question. The sheer number of compounds that must be tested poses problems for classes of ion channel targets for which direct functional measurements (e.g., traditional patch-clamping) are too cumbersome and indirect measurements (e.g., Ca(2+)-sensitive dyes) lack sufficient sensitivity or require unacceptable compromises. We present an optimized process for obtaining large numbers of direct electrophysiological measurements (two-electrode voltage-clamp) from Xenopus oocytes using a combination of automated oocyte handling, efficient and flexible liquid delivery, parallel operation, and powerful integrated data analysis. These improvements have had a marked impact, increasing the contribution electrophysiology makes in optimizing lead compound series and the discovery of new ones. The design of the system is detailed along with examples of data generated in support of lead optimization and discovery.

TH1 and TH2 cytokine inhibition by 3,5-bis(trifluoromethyl)pyrazoles, a novel class of immunomodulators.

In order to discover novel immunomodulators for application in treating autoimmune diseases, a stable Jurkat transfectant was constructed in which luciferase reporter gene is driven by a full-length IL-2 promotor. A chemical library was screened to identify compounds that inhibited luciferase expression in Jurkat transfectants stimulated with PMA and ionomycin. A class of compounds (bis-trifluoromethyl pyrazole, BTPs) was identified from this screen. BTPs were shown to inhibit anti-CD3 and anti-CD28 antibody-induced IL-2 secretion, mixed lymphocyte reaction, and Con A-induced T cell proliferation in normal human peripheral blood T cells. In addition, mRNA levels of IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-gamma were markedly inhibited by BTPs in peripheral blood mononuclear cells stimulated by Con A as determined by multi-probe RNA protection assay. Furthermore, IL-2, IL-4, IL-5, and IFN-gamma secretion by Hut 78 cells or CD3(+) T cells stimulated with PMA plus ionomycin or anti-CD3 antibody plus PMA were inhibited in a concentration-dependent manner by BTPs. Therefore, BTPs inhibit a wide spectrum of cytokine production including TH1 and TH2 type cytokines. Taken together, these compounds may be useful for treating autoimmune diseases and organ transplant rejection.