Evaluation of variable new antigen receptors (vNARs) as a novel cathepsin S (CTSS) targeting strategy.

Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.

Disability progression in multiple sclerosis is associated with plasma neuroactive steroid profile.

BACKGROUND: Neuroactive steroids (NASs) exert multiple biological effects on development and inflammation. The effects of NASs on disease progression in multiple sclerosis (MS) are uncertain, prompting analyses of NAS profiles during the transition from clinically isolated syndrome (CIS) to relapsing-remitting (RR) MS. METHODS: Subjects with CIS or RRMS and healthy controls (HCs) were recruited; demographic and clinical data as well as disability scores measured by the Expanded Disability Status Scale (EDSS) were recorded. Matched plasma NAS and amino acid (AA) concentrations were measured. RESULTS: HC (n = 17), CIS (n = 31), and RRMS (n = 33) groups showed similar ages and sex distribution although disability scores were higher in the RRMS group. The conversion rate of CIS to RRMS group was 51.6% (n = 16) during a mean follow-up period of 1.85 years. The RRMS group showed significantly higher mean allopregnanolone, aspartate, and taurine concentrations with lower epiallopregnanolone concentrations than CIS patients, and higher L-serine-O-phosphate and lower alanine, arginine, and glutamine concentrations than the HC group. Among CIS and RRMS groups, multivariate hierarchical regressions revealed that higher concentrations of plasma tetrahydrodeoxycorticosterone (THDOC) may predict disability worsening. CONCLUSIONS: RRMS and CIS patients exhibited differing concentrations of both NASs and AAs in plasma while both THDOC and pregnanolone might serve as biomarkers of disability worsening.

Selenium status in a Northern Irish pregnant cohort with iodine deficiency.

Selenium and iodine are trace elements that are maximally concentrated in the thyroid. Iodine is a substrate for thyroid hormone synthesis, while the selenoproteins protect the thyroid from the oxidative stress incurred. We measured plasma selenium concentration in 241 pregnant women in 1st trimester, previously reported to have iodine deficiency. Mean age was 30.3 years (SD 5.4), BMI 26.2 kg/m2 (SD 4.9) and 53% reported taking supplements. Median urinary iodine concentration was 73 µg/L (IQR 37-122) (WHO recommendation, ≥150 µg/L). Mean plasma selenium concentration was 75 µg/L (SD 7.7) which is below the 80-125 µg/L reported to be optimal. Four-day food diaries revealed a selenium intake of 43 µg/day (SD 15.9), also below the 55-70 µg/day reported to be optimal. This is the first report of selenium status in pregnancy on the island of Ireland. The possible combined effects of iodine and selenium deficiencies in pregnancy merit further investigation.

Peripheral blood microRNA and VEGFA mRNA changes following electroconvulsive therapy: implications for psychotic depression.

OBJECTIVE: MicroRNAs are short, non-coding molecules that regulate gene expression. Here, we investigate the role of microRNAs in depression and electroconvulsive therapy (ECT). METHODS: We performed three studies: a deep sequencing discovery-phase study of miRNA changes in whole blood following ECT (n = 16), followed by a validation study in a separate cohort of patients pre-/post-ECT (n = 37) and matched healthy controls (n = 34). Changes in an experimentally validated gene target (VEGFA) were then analysed in patients pre-/post-ECT (n = 97) and in matched healthy controls (n = 53). RESULTS: In the discovery-phase study, we found no statistically significant differences in miRNA expression from baseline to end of treatment in the group as a whole, but post hoc analysis indicated a difference in patients with psychotic depression (n = 3). In a follow-up validation study, patients with psychotic depression (n = 7) had elevated baseline levels of miR-126-3p (t = 3.015, P = 0.006) and miR-106a-5p (t = 2.598, P = 0.025) compared to healthy controls. Following ECT, these differences disappeared. Baseline VEGFA levels were significantly higher in depressed patients compared to healthy controls (F(1,144) = 27.688, P = <0.001). Following ECT, there was a significant change in VEGFA levels in the psychotic group only (t = 2.915, P = 0.010). CONCLUSION: Molecular differences (miRNA and VEGFA) may exist between psychotic and non-psychotic depression treated with ECT.

Physicians' attentional performance following a 24-hour observation period: do we need to regulate sleep prior to work?

BACKGROUND: The tradition of physicians working while sleep deprived is increasingly criticised. Medical regulatory bodies have restricted resident physician duty-hours, not addressing the greater population of physicians. We aimed to assess factors such as sleep duration prior to a 24-hour observation period on physicians' attention. METHODS: We studied 70 physicians (mean age 38 years old (SD 10.8 years)): 36 residents and 34 faculty from call rosters at the University of Alberta. Among 70 physicians, 52 (74%) performed overnight call; 18 did not perform overnight call and were recruited to control for the learning effect of repetitive neuropsychological testing. Attentional Network Test (ANT) measured physicians' attention at the beginning and end of the 24-hour observation period. Participants self-reported ideal sleep needs, sleep duration in the 24 hours prior to (ie, baseline) and during the 24-hour observation period (ie, follow-up). Median regression models examined effects on ANT parameters. RESULTS: Sleep deprivation at follow-up was associated with reduced attentional accuracy following the 24-hour observation period, but only for physicians more sleep deprived at baseline. Other components of attention were not associated with sleep deprivation after adjusting for repetitive testing. Age, years since medical school and caffeine use did not impact changes in ANT parameters. CONCLUSIONS: Our study suggests that baseline sleep before 24 hours of observation impacts the accuracy of physicians' attentional testing at 24 hours. Further study is required to determine if optimising physician sleep prior to overnight call shifts is a sustainable strategy to mitigate the effects of sleep deprivation.

Iodine Status over Two Decades: Influence of Seaweed Exposure.

Iodine deficiency is known to result in deficits in neuropsychological development in children born to iodine deficient mothers. However, a remedy in terms of iodisation of table salt as is the norm in most countries, has not been embraced by Ireland. The borderline iodine status of the Irish population persists unchanged over the past 20 years with the annual median urinary iodine (UI) varying from 62.9 to 105 µg/L; overall median 72.4 µg/L (WHO recommended 100 µg/L). Exposure to a seaweed rich environment allowing for intake by respiration of seaweed derived gaseous iodine, rather than simple proximity to the sea, confers advantages in terms of iodine intake with 21/46 (45.6%) of schoolchildren in seaweed rich coastal areas having UI values > 150 µg/L compared to 1/28 (3.6 %) and 2/93 (2.2%) in low seaweed abundant coastal and inland areas respectively. The corresponding values for adult females were 31/72 (43%), 7/60 (11.6%) and 21/132 (16.0%). The findings support introduction of iodine prophylaxis through Universal Salt Iodisation (USI), to ensure that women of child bearing age have access to sufficient dietary iodine.

Prevalence of human papillomavirus in men who have sex with men in the era of an effective vaccine; a call to act.

OBJECTIVES: The incidence of human papillomavirus (HPV)-associated anal cancer is increasing. Men who have sex with men (MSM), particularly those coinfected with HIV, are disproportionately affected. Documenting the molecular epidemiology of HPV infection is important in guiding policy makers in formulating universal and/or targeted vaccine guidelines. METHODS: A prospective cohort study was conducted. HIV-positive and HIV-negative MSM > 18 years old were invited to participate. Provider-performed anal swabs were collected and anal HPV infection was detected using consensus primer solution phase polymerase chain reaction (PCR) followed by type-specific PCR for high-risk (HR)-HPV types 16, 18 and 31. Between-group differences were analysed using χ(2) tests and Wilcoxon rank tests. RESULTS: One hundred and ninety-four MSM [mean (standard deviation (SD)) age 36 (10) years; 51% HIV-positive) were recruited. The median number of sexual contacts in the preceding 12 months was 4 (interquartile range 2-10). HIV-positive subjects had a mean (SD) CD4 count of 557 (217) cells/µL, and 84% were on highly active antiretroviral therapy (HAART). Thirty-one samples were B-globin negative and thus excluded from further analysis. A total of 113 subjects (69%) had detectable HPV DNA. Sixty-eight subjects (42%) had an HR-HPV type detected. HR HPV type 16 was detected in 44 samples (27%), HR-HPV type 18 in 26 samples (16%) and HR-HPV type 31 in 14 samples (23%). Twenty-eight subjects (17%) had more than one type of HR-HPV type detected. When HPV and HR-HPV were stratified by age, those > 35 years had a higher prevalence (P = 0.001 and P = 0.028, respectively). HIV-positive subjects were more likely than HIV-negative subjects to have any detectable HPV (77% vs. 61%, respectively; P = 0.04), to have HR-HPV type 18 or 31 (P = 0.05 and P = 0.006, respectively) and to be infected with more than one HR-HPV type (31% vs. 3%, respectively; P < 0.001). Within the HIV-positive group, the prevalence of HPV was higher in those not on HAART (P = 0.041), although it did not differ when stratified by CD4 count. CONCLUSIONS: The identified prevalence of anal HPV infection was high. Emerging patterns of HPV-related disease strengthen the call for universal vaccination of boys and girls with consideration of catch-up and targeted vaccination of high-risk groups such as MSM and those with HIV infection.

The average roughness and fractal dimension of articular cartilage during drying.

Cartilage is a unique material in part because of it biphasic properties. The structure of cartilage is a porous matrix of collagen fibers, permeated with synovial fluid which creates a gliding and near frictionless motion in articulating joints. However, during in vitro testing or surgery, there exists potential for cartilage to dehydrate, or dry out. The effects of this drying can influence experimental results. It is likely that drying also changes joint performance in vivo. In in vitro testing of equine cartilage explants exposed to open air, the average roughness of cartilage changes from 1.85 ± 0.78 to 3.66 ± 1.41 µm SD in a 5-h period. Significant changes in roughness in individual samples are seen within 10 min of exposure to open air. However, the multi-scale nature of cartilage, characterized by the fractal dimension, does not significantly change during the same period. The current study attempts to quantify the magnitude of error that is introduced when cartilage is removed from its native environment.

Regulation of iodide uptake in placental primary cultures.

BACKGROUND: Maintenance of adequate iodide supply to the developing fetus is dependent not only on maternal dietary iodine intake but also on placental iodide transport. The objective of this study was to examine the effects of different pregnancy-associated hormones on the uptake of radioiodide by the placenta and to determine if iodide transporter expression is affected by hormone incubation. METHODS: Primary cultures of placental trophoblast cells were established from placentas obtained at term from pre-labor caesarean sections. They were pre-incubated with 17ß-estradiol, prolactin, oxytocin, human chorionic gonadotropin (hCG) and progesterone either singly or in combination over 12 h with (125)I uptake being measured after 6 h. RNA was isolated from placental trophoblasts and real-time RT-PCR performed using sodium iodide symporter (NIS) and pendrin (PDS) probes. RESULTS: Significant dose response increments in (125)I uptake by trophoblast cells (p < 0.01) were observed following incubation with hCG (60% increase), oxytocin (45% increase) and prolactin (32% increase). Although progesterone (50-200 ng/ml) and 17ß-estradiol (1,000-15,000 pg/ml) alone produced no significant differences in uptake, they facilitated increased uptake when combined with prolactin or oxytocin, with a combination of all four hormones producing the greatest increase (82%). Increased (125)I uptake was accompanied by corresponding increments in NIS mRNA (ratio 1.52) compared to untreated control cells. No significantly increased expression levels of PDS were observed. CONCLUSIONS: Pregnancy-associated hormones, particularly oxytocin and hCG, have a role in promoting placental iodide uptake which may protect the fetus against iodine deficiency.

Predictive modeling of cardiovascular complications in incident hemodialysis patients.

The administration of hemodialysis (HD) treatment leads to the continuous collection of a vast quantity of medical data. Many variables related to the patient health status, to the treatment, and to dialyzer settings can be recorded and stored at each treatment session. In this study a dataset of 42 variables and 1526 patients extracted from the Fresenius Medical Care database EuCliD was used to develop and apply a random forest predictive model for the prediction of cardiovascular events in the first year of HD treatment. A ridge-lasso logistic regression algorithm was then applied to the subset of variables mostly involved in the prediction model to get insights in the mechanisms underlying the incidence of cardiovascular complications in this high risk population of patients.

Is placental iodine content related to dietary iodine intake?

OBJECTIVE: Delivery of iodine to the foetus depends not only on maternal dietary iodine intake but also on the presence of a functioning placental transport system. A role for the placenta as an iodine storage organ has been suggested, and this study compares the iodine content of placentas from women giving birth at term in Ireland and Iran, areas with median urinary iodine of 79 and 206 µg/l respectively. DESIGN: Placental cotyledon iodine was measured using an alkaline ashing technique with Sandell-Kolthoff kinetic colorimetry. Samples were taken from six sites from the centre and periphery of each cotyledon. Placentas (Ireland n = 58; Iran n = 45) were obtained from consecutive euthyroid women delivering at term. RESULTS: The median placental iodine (µg/g wet weight) was significantly higher in Iranian than in Irish women (187·2 µg/g vs 34·3 µg/g; P < 0·001). The distribution of individual placental iodine values showed that values >50µg/g were found in 71·0% of Iranian and in only 21·0% of Irish samples. In Irish subjects, the relationship of placental iodine to pregnant population urinary iodine (UI) (ng/g:µg/l) was 1:2 (40:79), while in Iranians this ratio is closer to 1:1 (211:206). CONCLUSIONS: These findings, by demonstrating an apparent ability of the placenta to store iodine in a concentration-dependent manner, suggest a hitherto undetected role for the placenta. Whether placental iodine has a role in protecting the foetus from inadequacies in maternal dietary iodine intake is as yet unknown.

Does iodine gas released from seaweed contribute to dietary iodine intake?

Thyroid hormone levels sufficient for brain development and normal metabolism require a minimal supply of iodine, mainly dietary. Living near the sea may confer advantages for iodine intake. Iodine (I(2)) gas released from seaweeds may, through respiration, supply a significant fraction of daily iodine requirements. Gaseous iodine released over seaweed beds was measured by a new gas chromatography-mass spectrometry (GC-MS)-based method and iodine intake assessed by measuring urinary iodine (UI) excretion. Urine samples were obtained from female schoolchildren living in coastal seaweed rich and low seaweed abundance and inland areas of Ireland. Median I(2) ranged 154-905 pg/L (daytime downwind), with higher values (~1,287 pg/L) on still nights, 1,145-3,132 pg/L (over seaweed). A rough estimate of daily gaseous iodine intake in coastal areas, based upon an arbitrary respiration of 10,000L, ranged from 1 to 20 µg/day. Despite this relatively low potential I(2) intake, UI in populations living near a seaweed hotspot were much higher than in lower abundance seaweed coastal or inland areas (158, 71 and 58 µg/L, respectively). Higher values >150 µg/L were observed in 45.6% of (seaweed rich), 3.6% (lower seaweed), 2.3% (inland)) supporting the hypothesis that iodine intake in coastal regions may be dependent on seaweed abundance rather than proximity to the sea. The findings do not exclude the possibility of a significant role for iodine inhalation in influencing iodine status. Despite lacking iodized salt, coastal communities in seaweed-rich areas can maintain an adequate iodine supply. This observation brings new meaning to the expression "Sea air is good for you!"

Interns as teachers of medical students: a pilot programme.

BACKGROUND: In recent years, rising numbers of medical students and an increasingly demanding clinical workload has put pressures on the educational systems for medical students in the hospital. Bedside teaching remains central to education, but tutorial delivery by registrars, tutors and consultants has proven to be increasingly difficult with the greater numbers of students now in the undergraduate system. AIMS: We have performed a pilot study to determine the feasibility of developing a Junior Tutor Programme, to assist in the delivery of tutorials to undergraduate medical students. METHODS: This was designed and delivered by interns under the supervision of the academic staff in the Departments of Medicine and Surgery in Connolly Hospital. The programme was evaluated by a questionnaire filled in by the students anonymously. RESULTS: A supervised programme of tutorials delivered by interns is a potentially useful way to ensure delivery of clinical teaching to undergraduate medical students.

Gun shot-101: an 8-year review of gunshot injuries in an Irish teaching hospital from a general surgical perspective.

BACKGROUND: Gun-related crime offences have increased in the Republic of Ireland steadily over the past number of years. Regional trauma units are witnessing unprecedented numbers of injuries in the Republic of Ireland with limited prior experience. AIMS: Eight-year retrospective study analysing demographic data, management and outcome of firearm-related injuries. RESULTS: Patients who experience gunshot injuries in this region are statistically likely to be young, male and unemployed with a single shotgun injury to an extremity. Post-operative survival rates of 100% for those who undergo an exploratory laparotomy. CONCLUSION: Ireland has comparable survival outcomes to other international centres with similar patient demographics due to timely and appropriate operative intervention. These results serve to provide a template for further patient management.

Changing patterns of thyroid carcinoma.

BACKGROUND: To assess changing trends in histological types of thyroid cancer in an Irish hospital over the past 30 years. METHODS: Biographical data, tumour characteristics, treatment and outcome from 190 patients with thyroid carcinoma from 1970 to 2000 were reviewed retrospectively. RESULTS: Detailed records of 190 patients with thyroid cancer were identified with a mean age at presentation of 50 years. From 1970 to 1979 the distribution of histological types was: papillary carcinoma; 9 patients (4.7%), follicular; 17 patients (8.9%), anaplastic; 9 patients (4.7%), medullary; 1 patient (0.5%) and lymphoma; 1 patient (0.5%). From 1980 to 1989 papillary carcinoma accounted for 32 patients (16.8%), follicular; 14 patients (7.3%), anaplastic; 13 patients (6.8%), medullary; 7 patients (3.7%) and lymphoma; 5 patients (2.6%). From 1990 to 1999 papillary cancer accounted for 48 patients (25.2%), follicular; 14 patients (7.3%), anaplastic; 8 patients (4.2%), medullary; 7 patients (3.7%) and lymphoma; 5 patients (2.6%). Survival rates were significantly better for those aged less than 45 years (P < 0.0001), female sex (P < 0.01) and those with papillary carcinoma (P < 0.01). CONCLUSIONS: This study demonstrated a significant increase in the incidence of papillary carcinoma. This may be related to increasing dietary iodine intake and may be significant as papillary carcinoma is associated with a more favourable prognosis.

Short-term changes in maternal and neonatal urinary iodine excretion.

Investigation of maternal urinary iodine (UI) excretion in the immediate antenatal and early postpartum periods showed a precipitous fall in median values from 93 microg/L antenatally to 36 microg/L at delivery subsequently rising to 49 microg/L and 63 microg/L at days 3 and 10 postpartum respectively. The fate of ingested iodine not appearing in the maternal urine is unknown but measurement of UI in babies born to nursing mothers suggested transfer from the mother with median neonatal values of 117 and 159 microg/L being recorded at days 3 and 10. While maternal UI seemed to relatively unaffected by breast feeding, median UI from breast feeding babies (148 microg/L) was significantly greater than in those bottle feeding (50 microg/L). This was also reflected by the finding that no breast feeding baby had a UI values < 50 microg/L in comparison to 50% of bottle feeders. The depressed values in mothers and relatively high values in their infants could present a false picture and suggest the need to defer any investigations of iodine status at this time. The findings do however suggest a need for further investigations aimed at determining the fate of iodine ingested perinatally and its possible physiological significance in maintaining thyroid status in the mother and neonate.

Expression microarray analysis of papillary thyroid carcinoma and benign thyroid tissue: emphasis on the follicular variant and potential markers of malignancy.

The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan(R) reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.

Iodine intake in pregnancy in Ireland--a cause for concern?

BACKGROUND: Adequate dietary iodine intake is necessary to maintain maternal thyroid function at a level permitting normal neuropsychological development of the foetus. AIMS AND METHODS: To determine dietary iodine status by measuring urinary iodine excretion (UIE), proportional to dietary intake, in Irish mothers during the first trimester of pregnancy. RESULTS: Median UIE showed seasonal variations, being lower in summer than in winter. The median values in pregnant women were, summer 45microg/l, winter 68microg/l. Equivalent values for controls were 43 and 91microg/l respectively. UIE required to achieve WHO recommended daily iodine intakes would be 120-180microg/l. In the Irish subjects UIE values suggestive of iodine deficiency (<50microg/l) were observed in 55% of pregnant women tested in summer and 23% in winter. Dairy milk iodine, a major dietary iodine source, showed similar variation. CONCLUSIONS: While there is as yet no available evidence of widespread thyroid hypofunction in the Irish obstetric population, the findings are a cause of concern, which if confirmed by a more comprehensive investigation, may indicate the need for iodine prophylaxis.

Genome-wide analysis of deoxyribonucleic acid in endometrial cancer using comparative genomic hybridization microarrays.

The aim of this study was to identify amplified oncogenes in endometrial cancer using array-based comparative genomic hybridization (array CGH). Despite its prevalence, the molecular mechanisms of endometrial carcinogenesis are still poorly understood. The selected array CGH allows the simultaneous examination of 58 oncogenes commonly amplified in human cancers and is capable of achieving increased mapping resolution compared with conventional CGH. A subset of 8 specimens from a bank of 60 malignant and normal specimens was selected for array analysis to identify potential genes of interest. TaqMan polymerase chain reaction was carried out on the 60 specimens to examine if aberrations at the genomic level correlated with gene expression and to compare expression in normal and malignant samples. Oncogenes amplified in the endometrial cancers included AR, PIK3CA, MET, HRAS, NRAS, D17S1670, FGFR1, CTSB, RPS6KB1, LAMC2, MYC, PDGFRA, FGF4/FGF3, PAKI, and FGR. Three genes were examined at the messenger RNA level. AR and PIK3CA were higher in normal specimens, and MET was higher in malignant samples, suggesting a role for MET in endometrial cancer. Newer arrays examining more genes and larger sample numbers are necessary to elucidate the carcinogenic pathway in endometrial cancer.