De novo individualized disease modules reveal the synthetic penetrance of genes and inform personalized treatment regimens.

Current understandings of individual disease etiology and therapeutics are limited despite great need. To fill the gap, we propose a novel computational pipeline that collects potent disease gene cooperative pathways to envision individualized disease etiology and therapies. Our algorithm constructs individualized disease modules de novo, which enables us to elucidate the importance of mutated genes in specific patients and to understand the synthetic penetrance of these genes across patients. We reveal that importance of the notorious cancer drivers TP53 and PIK3CA fluctuate widely across breast cancers and peak in tumors with distinct numbers of mutations and that rarely mutated genes such as XPO1 and PLEKHA1 have high disease module importance in specific individuals. Furthermore, individualized module disruption enables us to devise customized singular and combinatorial target therapies that were highly varied across patients, showing the need for precision therapeutics pipelines. As the first analysis of de novo individualized disease modules, we illustrate the power of individualized disease modules for precision medicine by providing deep novel insights on the activity of diseased genes in individuals.

The combined diabetes and renal control trial (C-DIRECT) - a feasibility randomised controlled trial to evaluate outcomes in multi-morbid patients with diabetes and on dialysis using a mixed methods approach.

BACKGROUND: This cluster randomised controlled trial set out to investigate the feasibility and acceptability of the "Combined Diabetes and Renal Control Trial" (C-DIRECT) intervention, a nurse-led intervention based on motivational interviewing and self-management in patients with coexisting end stage renal diseases and diabetes mellitus (DM ESRD). Its efficacy to improve glycaemic control, as well as psychosocial and self-care outcomes were also evaluated as secondary outcomes. METHODS: An assessor-blinded, clustered randomised-controlled trial was conducted with 44 haemodialysis patients with DM ESRD and ≥ 8% glycated haemoglobin (HbA1c), in dialysis centres across Singapore. Patients were randomised according to dialysis shifts. 20 patients were assigned to intervention and 24 were in usual care. The C-DIRECT intervention consisted of three weekly chair-side sessions delivered by diabetes specialist nurses. Data on recruitment, randomisation, and retention, and secondary outcomes such as clinical endpoints, emotional distress, adherence, and self-management skills measures were obtained at baseline and at 12 weeks follow-up. A qualitative evaluation using interviews was conducted at the end of the trial. RESULTS: Of the 44 recruited at baseline, 42 patients were evaluated at follow-up. One patient died, and one discontinued the study due to deteriorating health. Recruitment, retention, and acceptability rates of C-DIRECT were generally satisfactory HbA1c levels decreased in both groups, but C-DIRECT had more participants with HbA1c < 8% at follow up compared to usual care. Significant improvements in role limitations due to physical health were noted for C-DIRECT whereas levels remained stable in usual care. No statistically significant differences between groups were observed for other clinical markers and other patient-reported outcomes. There were no adverse effects. CONCLUSIONS: The trial demonstrated satisfactory feasibility. A brief intervention delivered on bedside as part of routine dialysis care showed some benefits in glycaemic control and on QOL domain compared with usual care, although no effect was observed in other secondary outcomes. Further research is needed to design and assess interventions to promote diabetes self-management in socially vulnerable patients. TRIAL REGISTRATION NUMBER: Trial registered with the International Standard Randomised Controlled Trial (ISRCTN10546597). Registered 12 September 2016 (Retrospectively registered).

Functional and neurochemical characterization of angiotensin type 1A receptor-expressing neurons in the nucleus of the solitary tract of the mouse.

Angiotensin II acts via two main receptors within the central nervous system, with the type 1A receptor (AT1AR) most widely expressed in adult neurons. Activation of the AT1R in the nucleus of the solitary tract (NTS), the principal nucleus receiving central synapses of viscerosensory afferents, modulates cardiovascular reflexes. Expression of the AT1R occurs in high density within the NTS of most mammals, including humans, but the fundamental electrophysiological and neurochemical characteristics of the AT1AR-expressing NTS neurons are not known. To address this, we have used a transgenic mouse, in which the AT1AR promoter drives expression of green fluorescent protein (GFP). Approximately one-third of AT1AR-expressing neurons express the catecholamine-synthetic enzyme tyrosine hydroxylase (TH), and a subpopulation of these stained for the transcription factor paired-like homeobox 2b (Phox2b). A third group, comprising approximately two-thirds of the AT1AR-expressing NTS neurons, showed Phox2b immunoreactivity alone. A fourth group in the ventral subnucleus expressed neither TH nor Phox2b. In whole cell recordings from slices in vitro, AT1AR-GFP neurons exhibited voltage-activated potassium currents, including the transient outward current and the M-type potassium current. In two different mouse strains, both AT1AR-GFP neurons and TH-GFP neurons showed similar AT1AR-mediated depolarizing responses to superfusion with angiotensin II. These data provide a comprehensive description of AT1AR-expressing neurons in the NTS and increase our understanding of the complex actions of this neuropeptide in the modulation of viscerosensory processing.

Gene utility recapitulates chromosomal aberrancies in advanced stage neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Although only a few recurrent somatic mutations have been identified, chromosomal abnormalities, including the loss of heterozygosity (LOH) at the chromosome 1p and gains of chromosome 17q, are often seen in the high-risk cases. The biological basis and evolutionary forces that drive such genetic abnormalities remain enigmatic. Here, we conceptualize the Gene Utility Model (GUM) that seeks to identify genes driving biological signaling via their collective gene utilities and apply it to understand the impact of those differentially utilized genes on constraining the evolution of NB karyotypes. By employing a computational process-guided flow algorithm to model gene utility in protein-protein networks that built based on transcriptomic data, we conducted several pairwise comparative analyses to uncover genes with differential utilities in stage 4 NBs with distinct classification. We then constructed a utility karyotype by mapping these differentially utilized genes to their respective chromosomal loci. Intriguingly, hotspots of the utility karyotype, to certain extent, can consistently recapitulate the major chromosomal abnormalities of NBs and also provides clues to yet identified predisposition sites. Hence, our study not only provides a new look, from a gene utility perspective, into the known chromosomal abnormalities detected by integrative genomic sequencing efforts, but also offers new insights into the etiology of NB and provides a framework to facilitate the identification of novel therapeutic targets for this devastating childhood cancer.

Bridging the communication gap between radiographers and patients to improve chest radiography image acquisition: A multilingual solution in the COVID-19 pandemic.

INTRODUCTION: A chest X-ray (CXR), taken in full inspiration, is important to ensure pathology in the lungs will not be missed. To achieve this, effective communication on breathing instructions for patients is crucial. During the COVID-19 pandemic, radiographers in Sengkang General Hospital (SKH) were challenged when performing CXR for the patients whose native language is not English. Most of these patients were foreign workers living in the same dormitory which had formed the largest COVID-19 cluster in Singapore. These dormitory residents found it difficult to understand and adhere to breathing instructions, resulting in a suboptimal degree of inspiration when the CXRs were taken. This may ultimately affect the diagnostic value of the radiographs. This paper aims to share and evaluate how radiographers tackled this issue and continued to acquire fully-inspired CXR for the dormitory residents despite the language barrier. METHODS: Using a combination of online survey and retrospective analysis of the rejection rates of CXR done over the period of early April to early June, a team of radiographers evaluated the effectiveness of using audio recordings in managing the issue of not achieving a fully inspired CXR for patients due to language barrier. RESULTS: The rejection rate for CXR due to suboptimal inspiration decreased from 26% to 9% upon implementation of the audio recordings. 92.3% of the CXRs taken within this period also fulfilled the criteria of a fully-inspired CXR, as evidenced by having at least 9 posterior ribs seen above the right hemi-diaphragm. Survey results found a fairly balanced number of radiographers who agreed and disagreed that a fully-inspired CXR was achieved for most of their patients after utilisation of translation manuals and audio recordings. CONCLUSION: After the implementation of audio recordings, the decrease in rejection rate of CXR and an audit which demonstrated that CXR quality was upheld had proven that the radiographers successfully achieved fully-inspired CXR for suspected COVID-19 patients. This confirmed that using pre-recorded audio instructions was an efficient intervention albeit being a one-way communication, leads to more accurate imaging results, aligning with existing literature on communication experiences between radiographers and patients. Moreover, the decreased rejection rate of CXRs had increased department efficiency consequently reducing departmental expenses in the long run. IMPLICATIONS OF PRACTICE: Given that we have an ageing population and the vast majority of the elderly converse in their various dialects, positive feedback from radiographers presented opportunities to expand the translation manual and audio recordings to include local dialects. These can be seamlessly integrated in CXR and other procedures in the hospital setting. To ensure that the translations are culturally sensitive, attention should be paid to the translation process of instructions into other languages and local dialects by enlisting the help of native speakers.

Cerebral palsy in siblings caused by compound heterozygous mutations in the gene encoding protein C.

We report two sisters with extensive bilateral periventricular haemorrhagic infarction (PVHI) causing cerebral palsy (CP). The older sister presented at 20 months with cortical visual blindness, spastic diplegia, and purpura fulminans. The younger sister presented aged 3 days old with apnoeas and multifocal seizures. She subsequently had global developmental delay, cortical visual blindness, spastic quadriplegia, epilepsy, and purpura fulminans at age 2 years. Neuroimaging of both siblings showed bilateral PVHI consistent with bilateral cerebral intramedullary venous thrombosis occurring at under 28 weeks' gestation for the older sister and around time of birth for the younger sister. At latest follow-up, the older sister (13y) has spastic diplegia at Gross Motor Function Classification System (GMFCS) level II, and the younger sister (10y) has spastic quadriplegia at GMFCS level IV. Both sisters showed partial quantitative reduction in plasma protein C antigen and severe qualitative reduction in plasma protein C anticoagulant activity. They were heterozygous for two independent mutations in the protein C gene (PROC). There was no other risk factor for CP. To our knowledge, this is the first family reported with compound heterozygous PROC mutations as the likely genetic cause of familial CP. This report adds to the list of known monogenic causes of CP.

Pharmacokinetic parameters of chlorzoxazone and its main metabolite, 6-hydroxychlorzoxazone, after intravenous and oral administration of chlorzoxazone to liver cirrhotic rats with diabetes mellitus.

Protein expression of the hepatic CYP2E1 has been reported to be increased in diabetic rats. This enzyme is the primary metabolizer of chlorzoxazone (CZX) to 6-hydroxychlorzoxazone (OH-CZX). Although patients with liver cirrhosis have a higher prevalence of diabetes mellitus, there have been no reported studies on the protein expression of CYP2E1 in rats induced to have liver cirrhosis and diabetes mellitus by injection of N-dimethylnitrosamine followed by streptozotocin [liver cirrhosis with diabetes mellitus (LCD) rats]. Thus, in the present study, the pharmacokinetics of CZX and OH-CZX were evaluated in LCD rats. Compared with control rats, LCD rats had significantly decreased (by 62%) total liver protein and significantly increased (by 124%) protein expression of CYP2E1, but the intrinsic clearance (Cl(int); formation of OH-CZX per milligram protein) was comparable in both groups of rats. As a result, the relative Cl(int) was also comparable for the two groups. Thus, OH-CZX formation in LCD and control rats was expected to be similar. As expected, after i.v. (20 mg/kg) and p.o. (50 mg/kg) administration of CZX, the area under the curve (AUC) of OH-CZX was comparable in control and LCD rats (i.v., 571 +/- 85.8 and 578 +/- 413 microg x min/ml, respectively; p.o., 1540 +/- 338 and 2170 +/- 1070 microg x min/ml, respectively). In LCD rats, the AUC(OH-CZX)/AUC(CZX) ratio was similar to the value in control rats after i.v. and p.o. administration. These results indicate that OH-CZX can be used as a chemical probe to assess the activity of CYP2E1 in LCD rats.

Reducing the RNA binding protein TIA1 protects against tau-mediated neurodegeneration in vivo.

Emerging studies suggest a role for tau in regulating the biology of RNA binding proteins (RBPs). We now show that reducing the RBP T-cell intracellular antigen 1 (TIA1) in vivo protects against neurodegeneration and prolongs survival in transgenic P301S Tau mice. Biochemical fractionation shows co-enrichment and co-localization of tau oligomers and RBPs in transgenic P301S Tau mice. Reducing TIA1 decreased the number and size of granules co-localizing with stress granule markers. Decreasing TIA1 also inhibited the accumulation of tau oligomers at the expense of increasing neurofibrillary tangles. Despite the increase in neurofibrillary tangles, TIA1 reduction increased neuronal survival and rescued behavioral deficits and lifespan. These data provide in vivo evidence that TIA1 plays a key role in mediating toxicity and further suggest that RBPs direct the pathway of tau aggregation and the resulting neurodegeneration. We propose a model in which dysfunction of the translational stress response leads to tau-mediated pathology.

Knowledge and characteristics of herbal supplement usage among community pharmacy customers in a Malaysian population.

OBJECTIVES: We investigated the knowledge and characteristics of herbal supplement usage of the customers of community pharmacies in a Malaysian population. DESIGN AND SETTING: Self-administered questionnaires (in English, Malay, or Chinese) were provided to customers at three community pharmacies in Malaysia (Ipoh, Perak). Questionnaire validation and translation validation were performed. A pilot study was conducted before actual questionnaire distribution. Informed consent was obtained from all participants. RESULTS: Total number of participants was 270 (99 males and 171 females) with majority from the 31-50 age group (41.5%). Among the participants, 45.6% were herbal users. The most commonly used herbal supplements were evening primrose oil (17.9%), ginkgo biloba (13.0%), and milk thistle (8.5%). The participants seemed to have sufficient knowledge regarding herbal supplements including safety, quality, and indication of use from medical literature. Participants obtained information about herbal supplements from pharmacists (26.9%), package inserts (25.2%), friends (20.5%), and the Internet (13.3%) more often than from their doctors (9.8%). Most herbal users did not inform their doctors about their usage of herbal supplements (68.3%) or the side effects (61.5%). Herbal supplement users also tended to be women, >50-year-old, and those with higher monthly household incomes. CONCLUSIONS: Community pharmacists have a vital role in educating their customers about the safe use of herbal supplements. The participants had sufficient knowledge about herbal supplement usage; therefore, customers of these community pharmacies may have benefitted from the advice of the pharmacists. Further studies could be carried out in future on the knowledge, skills and roles of community pharmacists in the safe use of herbal supplements.

Effect of high-fat meals and fatty acid saturation on postprandial levels of the hormones ghrelin and leptin in healthy men.

OBJECTIVE: Ghrelin and leptin play a role in control of food intake and adiposity but mechanisms regulating these hormones in man are poorly defined and evidence that dietary fats may have adverse effects is inconclusive. We investigated whether high-fat meals, which differed in saturated fatty acid (SFA) content acutely modified these hormones. DESIGN: Randomised, double-blind, crossover trial. A high-fat (HF) test meal (59 +/- 4 g fat; 71% of energy as fat) was given for breakfast on two occasions. Meals comprised either high (approximately 70:30) or low (approximately 55:45) saturated:unsaturated fatty acid (SFA:USFA) ratio. Fasting and postprandial measurements of serum total ghrelin (RIA), leptin (enzyme-linked immunosorbent assay (ELISA)) and insulin (RIA) were made over 6 h. Postprandial measurements were also made at 10 and 24 h following a fat-exclusion lunch, snack and dinner. SUBJECTS: A total of 18 lean, healthy men. RESULTS: There was no significant effect of the fatty meal (time, P > 0.05), nor a differential effect of SFA:USFA ratio (treatment*time, P > 0.05) on ghrelin over 6h. Leptin decreased in response to both HF treatments (time, P < 0.001) but increased SFA content did not further inhibit hormone secretion (treatment*time, P > 0.05). There was no significant correlation between ghrelin or leptin and circulating insulin (P>0.05). CONCLUSION: We conclude that HF diets may adversely effect serum leptin, although the circadian decrease may account in part for this response. Increasing dietary SFAs had no deleterious effects on leptin or total ghrelin.

Digital imaging of the optic nerve head: monoscopic and stereoscopic analysis.

AIMS: To compare monoscopic and stereoscopic assessment of the optic disc using novel software for the digital stereoscopic analysis of optic disc stereopairs. METHODS: Software was developed for the stereoscopic display of digital optic disc images using an interlaced display method. Neuroretinal rim width was determined at 10 degree intervals around the optic disc using a custom (stereoscopic) cursor whose depth was adjusted to that of Elschnig's rim. Measurements were taken, first viewing the disc monoscopically and at a separate sitting, stereoscopically. RESULTS: Measurements were made in 35 eyes from 35 patients (1260 estimates for each observer) using three observers. The mean cup to disc ratio (CDR) ranged from 0.57 to 0.66 (SD 0.13-0.14) for monoscopic viewing compared with 0.64 to 0.69 (SD 0.12-0.14) for stereoscopic viewing. Stereoscopic assessments gave higher CDRs in temporal, superior, nasal, and inferior aspects of the optic disc (p<0.001, Mann-Whitney U test). Agreement between observers in estimating CDR was high for monoscopic assessment (intraclass correlation coefficient 0.74 (CI 0.72 to 0.76) increasing to 0.80 (0.78 to 0.82) for stereoscopic assessment. CONCLUSION: Digital stereoscopic optic disc assessment provides lower estimates of neuroretinal rim width and higher levels of interobserver agreement compared with monoscopic assessments.

Influence of storage volume on functional recovery and metabolism of explanted hearts following cold cardioplegia: studies in the rat.

STUDY OBJECTIVE: The aim was to characterise the metabolic and functional recovery after extended periods of hypothermic storage of hearts in varying volume of oxygenated cardioplegic solution. DESIGN: Explanted rat hearts were arrested with and stored in 5, 10, or 100 ml of oxygenated (95% O2: 5% CO2) St Thomas's Hospital cardioplegic solution No 2 (STH) at 4 degrees C for 5 or 10 h. The isolated working heart model was used to determine the cardiac functions and the degree of functional recovery was expressed as a percentage of the prearrest value. Groups of hearts (n = 6) were rapidly freeze-clamped immediately after arrest, at end of storage, or after 30 min reperfusion for quantitation of the high energy phosphate content. SUBJECTS: 84 Male albino Wistar rats weighing 280 to 320 g (heart weight = 1.25 g) were used. MEASUREMENTS AND MAIN RESULTS: All hearts in the three different volumes of STH recovered 80% or more [mean 90 (SEM 3)%] of their prearrest aortic output after 5 h storage but after 10 h, recovery was significantly reduced [23(7)%] with no significant difference among groups. During 5 h storage the phosphocreatine content of the hearts rapidly declined from 33.2(3.1) to 5.0(0.2) mumol.g-1 dry wt in the 5 ml group but only to 16.7(1.2) mumol.g-1 dry wt in the 100 ml group. Similarly ATP preservation was improved in the latter group (81% v 63%, p less than 0.02). Extending the storage time to 10 h resulted in a further decline in high energy phosphates (ATP + phosphocreatine) to 30% of normal values in the 100 ml group and to 9% in the 5 ml group (p less than 0.01). While the total nucleotide pool did not decrease the major catabolite was inosine monophosphate (IMP) which comprised 46% of the nucleotide pool in all groups. The extent of cardiac function recovered was correlated inversely with tissue IMP (R = 0.859) and positively with ATP (R = 0.835) but showed no significant correlations with the postischaemic ADP, AMP, or phosphocreatine. CONCLUSIONS: Increased storage volume of oxygenated cardioplegic solution is superior in the preservation of ATP and phosophocreatine content in donor hearts with full recovery of cardiac function after the first 5 h of hypothermic (4 degrees C) storage.

L-aspartate improves the functional recovery of explanted hearts stored in St. Thomas' Hospital cardioplegic solution at 4 degrees C.

Explanted rat hearts were subjected to cardioplegic arrest by 3 minutes' perfusion with oxygenated St. Thomas' Hospital solution no. 2 and then were stored by immersion in the same solution at 4 degrees C. Prearrest and postischemic left ventricular functions were compared by means of an isolated working heart apparatus. Hearts (n = 8 per group) arrested and stored for up to 8 hours all resumed the spontaneous rhythm of contraction during reperfusion for 30 minutes at 37 degrees C. There was good recovery of aortic flow rate (105% +/- 3%) against a pressure of 100 cm H2O, of heart rate (102% +/- 2%), and of aortic pressure (86% +/- 5% of prearrest values). Hearts stored for 10 and 20 hours showed poor or no postischemic recovery of cardiac pump function (aortic flow, 16% +/- 11% and 0%, respectively). Enrichment of St. Thomas' Hospital solution with L-glutamate (20 mmol/L) also failed to improve functional recovery of hearts subjected to 10 hours of storage, but hearts treated with St. Thomas' Hospital solution containing L-aspartate (20 mmol/L) or L-aspartate plus L-glutamate (20 mmol/L each) reestablished aortic flow rates of 99% +/- 5% and 93% +/- 4%, respectively. These results indicate that the addition of L-aspartate to St. Thomas' Hospital solution improves the functional recovery and extends the safe preservation of explanted hearts stored at 4 degrees C.

Long-term preservation of explanted hearts perfused with L-aspartate-enriched cardioplegic solution. Improved function, metabolism, and ultrastructure.

The effects of supplementing oxygenated St. Thomas' Hospital cardioplegic solution No. 2 with L-aspartate and/or D-glucose for the long-term preservation of excised rat hearts were determined with isolated working heart preparations. Left ventricular function was assessed at 37 degrees C with a crystalloid perfusate, before cardioplegic arrest and after 20 hours of low-flow perfusion (1.5 ml/min) with continuing arrest at 4 degrees C, and after this period, again at 37 degrees C with a crystalloid perfusate. Four groups (n = 8/group) of hearts were studied with four cardioplegic solutions: St. Thomas' Hospital solution alone, St. Thomas' Hospital solution with aspartate 20 mmol/L, St. Thomas' Hospital solution with glucose 20 mmol/L, and St. Thomas' Hospital solution plus both aspartate and glucose (20 mmol/L each). The addition of glucose to St. Thomas' Hospital solution made no significant difference in the recovery of aortic flow rates (17.7% +/- 8.6% and 21.6% +/- 7.8% of prearrest values), but when aspartate or aspartate and glucose were present, hearts showed significant improvements (89.8% +/- 5.2% and 85.0% +/- 6.2%, respectively). These improvements were associated with a reduction in the decline of myocardial high-energy phosphates during reperfusion, a reduction in cellular uptake of Na+ and Ca++, and a reduction in ultrastructural damage. These results indicate that low-flow perfusion with St. Thomas' Hospital solution plus aspartate can considerably extend the duration of safe storage of explanted hearts.

Functional recovery of hearts after cardioplegia and storage in University of Wisconsin and in St. Thomas' Hospital solutions.

There are conflicting reports of the beneficial effects of University of Wisconsin (UW) cardioplegic solution used in heart preservation techniques. Therefore we investigated the efficacy of myocardial protection in adult rat hearts subjected to single-dose infusion (3 minutes) of nonoxygenated cardioplegic solutions (UW or St. Thomas' Hospital solution No. 2 [STH]) and stored at 4 degrees C by immersion in the same solution or in saline solution. Isolated working-heart preparations (n = 8 per group) were used to assess the prearrest (20 minutes' normothermic perfusion) and postischemic left ventricular functions. Four groups of hearts underwent 5, 8, 10, and 20 hours of cold ischemia (4 degrees C) in UW solution. Hearts stored for 8 to 20 hours showed no postischemic recovery of cardiac pump function (aortic flow, 0%), had decreased levels of myocardial high-energy phosphates, and were highly edematous (50% to 70% increased). After 5 hours of storage there was also poor recovery of aortic flow, coronary flow, and aortic pressure (55.0% +/- 19.4%, 67.1% +/- 5.1%, and 58.1% +/- 11.7%, respectively) but good recovery of adenosine triphosphate, creatine phosphate, and guanosine triphosphate (18.54 +/- 1.42, 29.99 +/- 2.05, and 1.64 +/- 0.14 mumol/gm dry weight, respectively). In contrast, hearts arrested and stored in STH solution for 5 hours rapidly established normal left ventricular functions (aortic flow, 111.5% +/- 2.5%; cardiac output, 99.1% +/- 1.2%; coronary flow, 85.0% +/- 3.4%; heart rate, 95.8% +/- 2.7%; and aortic pressure, 94.6%). A group of hearts arrested with STH solution but stored in saline solution recovered more slowly, had only partial return of function (aortic flow, 73.6% +/- 14.8%; p less than 0.01 vs STH/STH group), and had significantly greater tissue water content (8.020 +/- 0.080 vs 6.870 +/- 0.126 ml/gm dry wt; p less than 0.01). These results demonstrate the superior preservation of explanted hearts at 4 degrees C obtained by STH cardioplegic solution compared with UW solution under conditions used for transplantation.

The degradation of 5'-deoxy-5-fluorouridine by pyrimidine nucleoside phosphorylase in normal and cancer tissues.

We describe an assay procedure for the quantification of pyrimidine nucleoside phosphorylase, the enzyme thought to degrade the pro-drug 5'-deoxy-5-fluorouridine to 5-fluorouracil. The method is based on the known differences in the ultraviolet absorption spectra in alkaline medium between pyrimidines and their nucleosides. Analogous to the cleavage of uridine by uridine phosphorylase, the enzymatic degradation of 5'-deoxy-5-fluorouridine in the presence of inorganic phosphate yields 5-fluorouracil and ribose-1-phosphate. We have shown that the rate of phosphorolysis of this pyrimidine nucleoside could be readily measured by spectrophotometry. The method described is simple, specific for the 'pro-drug' as well as reproducible. We have also applied this method to define tissue enzyme activities in extracts of human tumours, normal tissues of the same organ and tissues of mice.

Acutance, an objective measure of retinal nerve fibre image clarity.

BACKGROUND/AIMS: The interpretation of high contrast retinal nerve fibre layer (RNFL) images in glaucoma can be confounded by the presence of image blur; it can be difficult to discern diffuse axon loss in a poor quality image. One solution is to provide an objective measure of the image quality based on features in the image other than the RNFL. In this study the authors have developed an objective method to quantify the clarity of RNFL images, comparing it with a subjective image grading system. METHODS: Digitally acquired, monochrome retinal images were taken from 58 eyes (one image per eye) with a Topcon 50 IX retinal camera. Image resolution was 1320 x 1032 pixels at 8 bits per pixel. Image sharpness was subjectively graded by two masked experienced observers on a scale 1 to 5 relative to a reference set of RNFL images. Software algorithms were developed using Matlab (5.2) to calculate the acutance, an objective measure of the physical characteristics that underlie the subjective impression of sharpness in an image. RESULTS: Acutance values could be calculated for all the images. The Pearson correlation coefficients of the log of the acutance for each image and the subjective grades of observer 1 and observer 2 were 0.90 (p<0.001, n=58) and 0.84 (p<0.001, n=58) respectively. CONCLUSIONS: These data suggest that acutance may provide a useful objective measure of image quality, which correlates well with the subjective impression of the digital retinal image sharpness. Objective measures of image quality should help in the discrimination of diffuse retinal nerve fibre loss from image blur in patients with diffuse glaucomatous damage.

Effects of cysteine on the pharmacokinetics of intravenous clarithromycin in rats with protein-calorie malnutrition.

Effects of cysteine on the pharmacokinetics of clarithromycin were investigated after intravenous administration of the drug at a dose of 20 mg/kg to control rats (4-week fed on 23% casein diet) and rats with PCM (protein-calorie malnutrition, 4-week fed on 5% casein diet) and PCMC (PCM treated with 250 mg/kg for oral cysteine twice daily during the fourth week). Clarithromycin has been reported to be metabolized via hepatic microsomal cytochrome P450 (CYP) 3A4 to 14-hydroxyclarithromycin (primary metabolite of clarithromycin) in human subjects. It has also been reported that in rats with PCM, CYP3A23 level decreased to 40-50% of control level, but decreased CYP3A23 level in rats with PCM completely returned to control level by oral cysteine supplementation (rats with PCMC). Human CYP3A4 and rat CYP3A23 proteins have 73% homology. In rats with PCM, the area under the plasma concentration-time curve from time zero to time infinity, AUC (567, 853 and 558 microg min/ml for control rats and rats with PCM and PCMC, respectively) and percentage of clarithromycin remaining after incubation with liver homogenate (69.6, 83.9 and 71.7%) were significantly greater than those in control rats and rats with PCMC. Moreover, in rats with PCM, the total body clearance, CL (35.3, 23.4 and 35.8 ml/min/kg), nonrenal clearance, CL(NR) (21.3, 15.2 and 24.1 ml/min/kg) and maximum velocity for the disappearance of clarithromycin after incubation with hepatic microsomal fraction, V(max) (351, 211 and 372 pmol/min/mg protein) were significantly slower than those in control rats and rats with PCMC. However, above mentioned each parameter was not significantly different between control rats and rats with PCMC. The above data suggested that metabolism of clarithromycin decreased significantly in rats with PCM as compared to control due to significantly decreased level of CYP3A23 in the rats. By cysteine supplementation (rats with PCMC), some pharmacokinetic parameters of clarithromycin (AUC, CL, CL(NR) and V(max)) were restored fully to control levels because CYP3A23 level was completely returned to control level in rats with PCMC.

Effects of glucose on the pharmacokinetics of intravenous chlorzoxazone in rats with acute renal failure induced by uranyl nitrate.

The effects of glucose on CYP2E1 expression in rats with acute renal failure induced by uranyl nitrate (U-ARF) have been reported. CYP2E1 was significantly induced (2.3-fold) in rats with U-ARF compared with that in control rats. In contrast, CYP2E1 expression was significantly decreased in rats with U-ARF supplied with glucose (dissolved in tap water to make 10%, w/v) in their drinking water for 5 days (U-ARFG) compared with that in rats with U-ARF. However, CYP2E1 in rats with U-ARFG was significantly greater than that in control rats. Chlorzoxazone (CZX) primarily undergoes hydroxylation, catalyzed mainly by CYP2E1, to form 6-hydroxychlorzoxazone (OH-CZX) rats. Hence, it could be expected that in rats with U-ARFG, formation of OH-CZX could significantly decrease and increase compared with those in rats with U-ARF and control rats, respectively. This expectation is proven by the following results of a study of intravenous administration of CZX at a dose 20 mg/kg to control rats and rats with U-ARF and U-ARFG. First, the total area under the plasma concentration-time curve from time zero to 8 h (AUC(0-8 h)) of OH-CZX in rats with U-ARFG (8730 microg x min/mL) was significantly greater than that in control rats (414 microg x min/mL) and significantly smaller than that in rats with U-ARF (11500 microg x min/mL). Second, the AUC(0-8 h, OH-CZX)/AUC(CZX) ratio in rats with U-ARFG (10.0) was significantly greater than that in control rats (0.252) and significantly smaller than that in rats with U-ARF (17.5). Finally, the in vitro intrinsic OH-CZX formation clearance (CL(int)) in rats with U-ARFG (27.9 mL/min/mg protein) was significantly slower than that in rats with U-ARF (36.7 mL/min/mg protein) and significantly faster than that in control rats (17.7 mL/min/mg protein).

Functional, metabolic and ultrastructure evidence for improved myocardial protection during severe ischaemic stress with MBS, a new crystalloid cardioplegic solution.

The duration of aortic clamping and the temperature of the arrested heart are two important factors in the overall strategy of myocardial protection with cardioplegic solutions. The isolated working rat heart was used to compare the cardioprotection effects (function, metabolism and ultrastructure) of the new "extracellular" crystalloid solution, MBS (containing glucose, aspartate and lactobionate) and St. Thomas' Hospital No. 2 (STH) during prolonged moderate hypothermic ischaemia (30 degrees C, 2 hours and 4 hours) with multidose reinfusion (2 min every 30 min interval). All MBS treated hearts (n = 9 per group) rapidly resumed spontaneous regular sinus rhythm (0.8 +/- 0.2 min) and had similar high degree of functional recovery (cardiac output: 90.2 +/- 4.5% & 80.9 +/- 3.5%, stroke volume: 89.1 +/- 4.7% & 81.9 +/- 3.4% and aortic pressure: 102.0 +/- 4.0% & 100.0 +/- 7.3% of pre-arrest values for 2 hours and 4 hours groups, respectively) during 30 min post-ischaemic reperfusion. In contrast, hearts protected with STH showed significantly (p<0.01) less recovery of left ventricular function (cardiac output: 64.3 +/- 2.9% & 5.5 +/- 3.9%, respectively) with two of the nine hearts failing to regain any cardiac pump function after 4 hours. MBS increased lactate efflux (glycolysis) and completely abolished the progressive increase in the coronary vascular resistance during 4 hours ischaemic arrest. These improvements were directly related to the significantly (p<0.01) reduced depletion of the myocardial adenosine triphosphate (13.32 +/- 1.65 vs 2.42 +/- 0.09 micromol/g dry wt) and guanosine triphosphate (1.56 +/- 0.08 vs 0.74 +/- 0.04 micromol/g dry wt) during arrest; to their enhanced repletion after reperfusion (ATP: 96% vs 36%, TAN: 90% vs 40% and GTP: 69% vs 48%); and to the absence of ultrastructural injury to cardiac myocytes and the microvasculature. We conclude that the new crystalloid cardioplegic solution MBS provides markedly improved myocardial protection particularly during severe ischaemic stress.