The generation and validation of two NKX2-5 fluorescent reporter human embryonic stem cell lines: UMANe002-A-1 and UMANe002-A-2.

The transcription factor NKX2-5 is a highly conserved master regulator of heart development which is widely expressed in cardiac progenitors and cardiomyocytes. Fluorescent reporters of NKX2-5 that minimally perturb normal protein expression can enable the identification, quantification and isolation of NKX2-5-expressing cells in a normal physiological state. Here we report the generation of two new hESC lines with eGFP inserted upstream (5') or downstream (3') of NKX2-5, linked by a cleavable T2A peptide. These complementary reporters produce a robust fluorescent signal in cardiac cells and have wide utility particularly for research on developmental biology and disease modelling.

Dementia palliative care: A multi-site survey of long term care STAFF'S education needs and readiness to change.

Many people with dementia reside in long-term care, where limited staff knowledge of dementia palliative care has been identified, along with poor awareness that a palliative approach can assist in identifying unmet care needs. Evidence-based guidance in palliative care for people with dementia is available however, implementing this guidance requires staff engagement and a tailored educational approach. This pre-implementation situational analysis informed a tailored staff education intervention to support the implementation of national guidance on dementia palliative care in long term care. Using a cross-sectional study design, underpinned by the Consolidated Framework for Implementation Research, survey data were collected on site profile, staff demographics, learning needs, and readiness-to change at three residential care sites for older people in Ireland. In total, 69 staff (predominantly nurses and healthcare attendants) completed the surveys. Medication management and management of pain were the most frequently identified learning needs. Staff were confident in their ability to implement change but de-motivation and powerlessness were substantial factors as only one-third of staff were "ready for change". Staffing levels, managing risk during change and perceived reluctance in others were common barriers. These results informed an educational intervention to address the specific care context, staff learning needs and barriers to change prior to implementation.

Low-flow apnoeic oxygenation for laryngeal surgery: a prospective observational study.

Laryngeal surgery requires a shared airway and close collaboration between surgeon and anaesthetist in order to optimise operating conditions. Apnoeic oxygenation uses the principle of aventilatory mass flow to maintain oxygenation of pulmonary capillary blood under apnoeic conditions while minimising laryngeal movement. Concerns regarding accumulation of carbon dioxide and resultant acidaemia have limited the use of the technique. We performed a prospective study of low-flow apnoeic oxygenation for patients undergoing microlaryngoscopy under general anaesthesia in order to evaluate the ability of the technique to maintain oxygenation and determine the resultant rate of carbon dioxide accumulation. Sixty-four patients undergoing microlaryngoscopy under general anaesthesia were studied between November 2016 and December 2018. Intra-operative oxygenation was provided via a 10-French oxygen catheter placed into the trachea delivering oxygen at 0.5-1.0 l.min-1 . Data regarding apnoea time, peripheral oxygen saturation and venous blood gas concentrations were recorded. The mean (SD) duration of apnoea was 18.7 (7.2) min. Apnoeic oxygenation allowed successful completion of the surgical procedure in 62/64 patients. Mean (SD) rate of rise of the venous partial pressure of carbon dioxide was 0.15 (0.10) kPa.min-1 . Operating conditions were recorded qualitatively as being adequate in all cases. No adverse effects were reported. Low-flow intra-tracheal apnoeic oxygenation is a simple, effective and inexpensive technique to maintain oxygenation for laryngeal surgery.

Multiple intra-hospital transports during relocation to a new critical care unit.

OBJECTIVE: Intra-hospital transport (IHT) of critically ill patients is associated with morbidity and mortality. Mass transfer of patients, as happens with unit relocation, is poorly described. We outline the process and adverse events associated with the relocation of a critical care unit. DESIGN: Extensive planning of the relocation targeted patient and equipment transfer, reduction in clinical pressure prior to the event and patient care during the relocation phase. SETTING: The setting was a 30-bed, tertiary referral, combined medical and surgical critical care unit, located in a 570-bed hospital that serves as the national referral centre for cardiothoracic surgery and spinal injuries. PARTICIPANTS: All stakeholders relevant to the critical care unit relocation were involved, including nursing and medical staff, porters, information technology services, laboratory staff, project development managers, pharmacy staff and building contractors. MAIN OUTCOME MEASURES: Mortality at discharge from critical care unit and discharge from hospital were the main outcome measures. A wide range of adverse events were prospectively recorded, as were transfer times. RESULTS: Twenty-one patients underwent IHT, with a median transfer time of 10 min. Two transfers were complicated by equipment failure and three patients experienced an episode of hypotension requiring intervention. There were no cases of central venous or arterial catheter or endotracheal tube dislodgement, and hospital mortality at 30 days was 14%. CONCLUSION: Although IHT is associated with morbidity and mortality, careful logistical planning allows for efficient transfer with low complication rates.

Profiling B-type natriuretic peptide in a stable heart failure population: a valuable adjunct to care.

AIM: To examine the prognostic importance of absolute values and change in values of BNP in patients with stable heart failure (HF). METHODS: Five-hundred and fifty-nine patients attending a disease management programme were categorized into tertiles of BNP (group 1; ≤ 95 pg/ml, group 2; 96-249 pg/ml and group 3; ≥ 250 pg/ml). A change in BNP between two stable visits was recorded. Patients were followed up for 1 year for death and a composite morbidity measure of HF hospitalization, all-cause hospitalization, unscheduled visits for clinical deterioration(UC) of HF using survival analysis. RESULTS: The risk of the combined morbidity outcome increased with increasing tertiles of BNP (Log rank = 17.8 (2), p < 0.001). Furthermore, a 50 and 25% increase in BNP predicted morbidity in stable HF patients with an initial BNP > 200 pg/ml (p = 0.02) and > 450 pg/ml (p = 0.03), respectively. CONCLUSION: In a stable community HF population, an elevated BNP or an increase in BNP predicts an adverse prognosis thereby potentially identifying a population in need of closer clinical follow-up.

Inappropriate use of proton pump inhibitors.

BACKGROUND: Proton pump inhibitors (PPIs) are widely used expensive medications. AIMS: We performed a cross-sectional study to determine the extent and indication of PPI use in Irish acute medical wards. METHODS: Fifty-five medical charts were reviewed at the beginning and end of 1 month. RESULTS AND CONCLUSIONS: Thirty-three patients were prescribed PPIs; 26 prior to admission. The prescribing of PPIs was concordant with guideline recommendations in only 30% of cases. Two-thirds of PPI use was unlicensed.

Can we reduce preventable heart failure readmissions in patients enrolled in a Disease Management Programme?

BACKGROUND: Disease Management Programmes (DMPs) are successful in reducing hospital readmissions in heart failure (HF). However, there remain a number of patients enrolled in a DMP who are readmitted with HF. The primary aim of the study was to determine the proportion of preventable readmissions (PR). The secondary aim was to recognise patient characteristics which would identify certain patients at risk of having a PR. METHODS: A retrospective chart search was performed on patients readmitted over a 1-year period. RESULTS: 38.5% of readmissions were classified as PR. None of these patients made prior contact with the DMP. Admission levels of BNP, potassium, urea and creatinine were significantly lower in the PR group. CONCLUSION: DMP have proven benefits in reducing hospital readmission nonetheless a significant proportion of these readmissions are preventable. Further work is required to prospectively analyse why these patients fail to contact the DMP.

Diagnosis of new onset heart failure in the community: the importance of a shared-care approach and judicious use of BNP.

BACKGROUND: Brain natriuretic peptide (BNP) may help general practitioners (GPs) to "rule-out" heart failure (HF) and reduce referral burden on specialist assessment clinics. AIMS: To determine the diagnostic value of BNP in HF referrals by GPs to a specialist unit. METHODS: From 2003 to 2007, 327 GP referrals were made to a HF new patient diagnostic clinic (NDC) with a provisional diagnosis of HF. The NDC provides rapid assessment of potential HF patients and ensures appropriate therapy and follow-up for those with a confirmed diagnosis. HF diagnosis was confirmed by the Framingham criteria. RESULTS: HF was present in 39% of cases referred (mean age 75 +/- 10 years, 49% male). The inclusion of BNP as a "rule-out" test with a cut-off value of 100 pg/mL would have reduced the number of patients originally referred to the NDC by 175. However, this would have resulted in delayed diagnosis and treatment of 20 (16%) "false-negative" patients. CONCLUSIONS: Availability of BNP to GPs would improve referral patterns but with high risk of delayed diagnosis. The data underline the need for a shared-care approach to the new diagnosis of HF.

Elevated BNP with normal systolic function in asymptomatic individuals at-risk for heart failure: a marker of diastolic dysfunction and clinical risk.

BACKGROUND: B-type natriuretic peptide (BNP) is widely accepted in the evaluation of left ventricular systolic dysfunction and heart failure. However, little is known of the implications of elevated BNP levels in individuals with preserved systolic function (PSF). AIMS: To investigate the drivers and clinical implications of elevated BNP levels in asymptomatic individuals with established PSF. METHODS: We enrolled 154 individuals who all underwent physical examination, BNP evaluation and Doppler-echocardiographic studies. They were divided into those above and below the median BNP level (50 pg/ml). RESULTS: Independent predictors of higher BNP were older age, more severe left ventricular hypertrophy (LVH), reduced E/A ratio and ischaemic heart disease. Survival and multivariable analysis demonstrated more death and/or admission in those above the median BNP (HR: 4.79, p=0.007). CONCLUSIONS: Elevated BNP is the strongest, independent predictor of serious adverse cardiovascular outcomes in this population and requires closer clinical follow-up.

Cheddar cheese cooking temperature induces differential lactococcal cell permeabilization and autolytic responses as detected by flow cytometry: implications for intracellular enzyme accessibility.

AIMS: To determine the influence of cheese cooking temperature on autolysis and permeabilization of two lactococcal starter strains in broth and in Cheddar cheese juice during ripening. METHODS AND RESULTS: Flow cytometry (FCM) was used to identify and enumerate intact and permeabilized cells in broth and in Cheddar cheese juice. Levels of intracellular enzyme activities were quantified concurrently. Permeabilized cell numbers increased for both strains in broth following a temperature shift from 32 to 38 degrees C and was accompanied by an increase in the level of accessible intracellular enzyme activities. The relative proportions of intact and permeabilized cell populations, as detected by FCM in cheese juice, changed during 42-day ripening. Permeabilized cell populations increased during ripening for both strains; however, an increase in accessible intracellular enzyme activity was observed only for the highly autolytic strain Lactococcus lactis AM2. CONCLUSIONS: Differences in the autolytic and permeabilization response induced by cooking temperature in two lactococcal strains affects intracellular enzyme accessibility in Cheddar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the importance of the autolytic and permeabilization properties of lactic acid bacteria starter strains and their impact on cheese ripening.

Radiation induced small bowel "web" formation is associated with acquired microvascular dysfunction.

BACKGROUND AND AIMS: Radiation therapy of abdominal and pelvic solid tumours results in late intestinal toxicity of a severe nature in approximately 5% of cases. These manifestations may include ischaemia and stricture formation, which may present as "webs". These webs are likely to play a role in the pathogenesis of recurrent bowel obstruction. The mechanisms of microvascular injury to the bowel in the setting of radiation have not been defined. We hypothesised that microvascular dysfunction with impaired vasodilation to acetylcholine (Ach) would be an acquired pathophysiological abnormality in radiation and "web" formation. METHODS: A 40 year old patient treated with radiation, two years previously, for an anal squamous cell cancer presented with recurrent small bowel obstruction. "Webs" in the distal ileum were detected using wireless capsule endoscopy, after small bowel barium radiographs failed to demonstrate a lesion. Following resection, freshly isolated 50-150 mum diameter arterioles from the "web" and adjacent normal calibre bowel were analysed with histology and microvessel physiological studies. RESULTS: After constriction (30-50%) with endothelin, dilation to graded doses of Ach (10(-9)-10(-4) M) was observed in vessels dissected from the stricture and the adjacent normal calibre area. Ach dilation was reduced in vessels from "web" (mean diameter 7 (2)%; n = 3, p < 0.01) compared with the adjacent unaffected bowel (mean diameter 85 (5)%). Dihydroethidine and dichlorofluorescein diacetate intravital staining demonstrated increased reactive oxygen species production in microvessels from "web" compared with adjacent normal calibre bowel. Histology from the strictured bowel demonstrated narrowing of the arterial lumen due to intimal and muscularis propria fibrosis, with endothelial preservation. CONCLUSIONS: External radiation is associated with acquired microvascular endothelial dysfunction and "web" formation in the small bowel.

Ketamine disrupts frontal and hippocampal contribution to encoding and retrieval of episodic memory: an fMRI study.

The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine produces episodic memory deficits. We used functional magnetic resonance imaging to characterize the effects of ketamine on frontal and hippocampal responses to memory encoding and retrieval in healthy volunteers using a double-blind, placebo-controlled, randomized, within-subjects comparison of two doses of intravenous ketamine. Dissociation of the effects of ketamine on encoding and retrieval processes was achieved using two study-test cycles: in the first, items were encoded prior to drug infusion and retrieval tested, during scanning, on drug; in the second, encoding was scanned on drug, and retrieval tested once ketamine plasma levels had declined. We additionally determined the interaction of ketamine with the depth of processing that occurred at encoding. A number of effects upon task-dependent activations were seen. Overall, our results suggest that left frontal activation is augmented by ketamine when elaborative semantic processing is required at encoding. In addition, successful encoding on ketamine is supplemented by additional non-verbal processing that is incidental to task demands. The effects of ketamine at retrieval are consistent with impaired access to accompanying contextual features of studied items. Our findings show that, even when overt behaviour is unimpaired, ketamine has an impact upon the recruitment of key regions in episodic memory task performance.

Acute ketamine administration alters the brain responses to executive demands in a verbal working memory task: an FMRI study.

We have used functional MRI to determine the effects of ketamine on brain systems activated in association with a working memory task. Healthy volunteers received intravenous infusions of placebo, ketamine at 50 ng/ml plasma concentration, and ketamine at 100 ng/ml. They were scanned while carrying out a verbal working memory task in which we varied the executive requirements (manipulation vs maintenance processes) and the mnemonic load (three vs five presented letters). We previously showed that ketamine produces a specific behavioral impairment in the manipulation task. In the current study, we modified tasks in order to match performance across drug and placebo conditions, and used an event-related fMRI design, allowing us to remove unsuccessful trials from the analysis. Our results suggest a task-specific effect of ketamine on working memory in a brain system comprising frontal cortex, parietal cortex, and putamen. When subjects are required to manipulate presented letters into alphabetical order, as opposed to maintaining them in the order in which they were presented, ketamine is associated with significantly greater activity in this system, even under these performance-matched conditions. No significant effect of ketamine was seen in association with increasing load. This suggests that our findings are not explicable in terms of a nonspecific effect of ketamine when task difficulty is increased. Rather, our findings provide evidence that the predominant effects of low, subdissociative doses of ketamine are upon the control processes engaged by the manipulation task. Furthermore, we have shown that ketamine's effects may be elucidated by fMRI even when overt behavioral measures show no evidence of impairment.

The effects of concurrent atorvastatin therapy on the pharmacokinetics of intravenous midazolam.

Midazolam is a commonly used anaesthetic agent and is metabolised by the 3A4 isoform of the cytochrome P450 enzyme system. Atorvastatin is also metabolised by cytochrome P450 3A4 and, in vitro, atorvastatin inhibits the cytochrome P450 3A4-mediated metabolism of mexazolam. We hypothesised that concurrent administration of atorvastatin and midazolam would result in altered midazolam pharmacokinetics. Fourteen patients scheduled to undergo general anaesthesia for elective surgery were recruited in a matched pair design to receive intravenous midazolam (0.15 mg.kg-1). Of these patients, seven were taking long-term atorvastatin. Atorvastatin patients demonstrated a greater area under the curve (889.4 (standard deviation 388.6) ng-h.ml-1) vs. control patients (629.1 (standard deviation 197.2) ng-h.ml-1) (p < 0.05). Patients taking atorvastatin also demonstrated a decreased clearance (0.18 (standard deviation 0.08) l-kg. h-1) vs. control patients (0.27 (standard deviation 0.08) l-kg.h-1) (p < 0.05). This study suggests that chronically administered atorvastatin decreases the clearance of intravenously administered midazolam.

IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived non-dividing CD5(+) B cells. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells may be dependent on the autocrine production of nitric oxide by inducible nitric oxide synthase (NOS2). We performed this study to determine whether cytokine factors that prevent spontaneous in vitroapoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity. B-CLL cells expressed NOS enzyme activity and NOS2 protein and mRNA. IL-4 and IFN-gamma increased B-CLL cell NOS2 enzyme activity and protein expression during in vitro culture. IFN-gamma, but not IL-4, increased NOS2 mRNA expression in cultured B-CLL cells suggesting that IL-4-mediated changes of NOS2 protein expression occurred at the post-transcriptional level. We were unable to detect increased concentrations of nitrite or nitrate (NO(x)) as surrogate markers of NO production in B-CLL cell cultures treated with IL-4 or IFN-gamma. IL-4 and IFN-gamma diminished NOS inhibitor-induced B-CLL cell death. In summary, we found that B-CLL cells expressed NOS2 and that IL-4 and IFN-gamma increased B-CLL NOS2 expression. Cytokine-mediated expression of NOS2 by B-CLL cells may promote their survival, and therapeutic strategies that target NOS2 or quench NO may be beneficial in patients with B-CLL.

Altered cell cycle response of drug-resistant lung carcinoma cells to doxorubicin.

The effect of doxorubicin treatment on cell cycle parameters in asynchronous populations of multidrug-resistant human lung carcinoma cell lines was investigated. A sensitive (DLKP-SQ) and three resistant (DLKP-SQ A250 10p#7, DLKP-A2B and DLKP-A5F) variants of a human lung carcinoma cell line DLKP were exposed to equitoxic concentrations of doxorubicin. The latter three were 8-fold, 30-fold and 300-fold resistant to doxorubicin, respectively. Irreversible G2/M arrest in sensitive (DLKP-SQ) cells was observed 24 h after initiation of doxorubicin treatment. In resistant variants, G2/M arrest occurred at 12-16 h with a subsequent bypass of the G2/M arrest to re-emerge and accumulate in G1. This transient G2/M arrest and subsequent progression into G1 indicated an inefficient checkpoint for monitoring DNA damage induced by doxorubicin treatment. Caffeine treatment could bypass the G2/M block in DLKP-SQ cells. Doxorubicin treatment did not alter cyclin B or cdc2 protein levels, the ability of cdc2 to form complexes with cyclin B or the levels of cyclin B bound to cdc2. The G2/M arrest seen in sensitive cells was associated with an increase in inhibitory phosphorylation of Tyr15 on cdc2. In contrast, tyrosine 15 phosphorylation did not change in resistant variants after drug treatment and a general increase in cdc2 kinase activity was seen. Cdc25C levels were not altered following drug treatment.