Quantification of mitochondrial sublimons in human fibrillating atria

Supraventricular tachycardias, including AF (atrial fibrillation), and mtDNA (mitochondrial DNA) deletions may lead to dilated cardiomyopathy. It is unknown whether mtDNA function is impaired in the human atrium in AF. In the present study, we investigated the role of rearranged mtDNA 'sublimons' in the pathogenesis of AF. Right atrial biopsies were collected from 38 patients in AF and 35 patients with SR (sinus rhythm) undergoing elective cardiac surgery. Total DNA was extracted by standard methods. The break-point regions of the two most prevalent classes of sublimon were amplified by PCR using fluorescent oligonucleotides for the 3.75 kb partial duplication and the 2.83 kb deletion. Multiplex reactions included additional primers to amplify an internal genomic standard for semi-quantitative analysis. Reaction products were quantified as peak areas in the electrophoretogram and ratios computed of the sublimon abundance relative to the genomic standard. There was no difference in SCN (sublimon copy number) between AF and SR patients [19.09+/-28.29 compared with 10.25+/-24.68, the difference was 0.28 (95% confidence interval, -0.04 and +0.61; P =0.08)]. SCN did not increase with age ( P =0.207) and was unrelated to AF duration ( P =0.661), left atrial diameter ( P =0.560), post-operative AF ( P =0.52), underlying disease ( P =0.94), medication and gender (2.84+/-0.72 in females vs 2.97+/-0.67 in males; P =0.431). In conclusion, our findings do not indicate any role of mtDNA in the pathophysiology of AF.     

Intravenous antidepressants: a review

Antidepressant medications have an onset of action of several weeks and have moderate efficacy. Their mode of administration is oral (p.o.). Some clinicians wondered whether intravenous (i.v.) administration would speed onset of action and increase efficacy. In this article we review controlled studies on i.v. administration of antidepressants. These include clomipramine, citalopram, and other antidepressants. Overall these studies do not support increased efficacy of i.v. over p.o.administration but there are suggestions of a faster onset of action. In one study i.v. citalopram showed superior response rates over p.o. citalopram (79% vs. 63%) in severely depressed patients at 8 weeks.     

Surgery versus pharmacotherapy of benign thyroid diseases

Surgical management of benign thyroid diseases (BTDs) has been a topic of interest and confusion for many years. Almost 80% of thyroidectomies at an average endocrine surgical unit are carried out for BTDs. Resistance to surgical intervention in BTDs has been based on the belief that increased complication rate is inherent in its use, this is despite the potential advantages in terms of confirming the benign nature of the lesion, controlling the disease, and relieving local symptoms of large neck mass. Benign thyroid diseases are more likely to occur in middle-aged women living in iodine deficient areas, or have a family history of goiter, or in patients taking iodine-containing drugs, like amoidarone, or in patients with previous history of x-ray exposure. However, the physician must be careful in making the diagnosis of BTDs in patients at the extremes of age or in the presence of positive history of radiation, or in patients with family history of thyroid or colon cancer. In this article we will review the etiology, epidemiology, diagnostic methodologies and the recent trends in the surgical and medical management of BTDs.     

Clinical and therapeutic variables influencing hospitalisation for bronchiolitis in a community-based paediatric group practice.

AIM: To examine the effect of different clinical characteristics and different treatments on the hospitalisation of infants with bronchiolitis seen in an outpatient clinic setting. METHODS: The medical records of infants under 2 years of age who presented with a first episode of wheezing over a two-year period were reviewed retrospectively. Hospitalisation within ten days of evaluation was used as the primary outcome measure. Results: Data from 320 patients were included. 17% were hospitalised. Age was lower in the hospitalised patients (4.9 months vs. 7.1, p<0.001). Hospitalisation was higher in RSV-positive versus RSV-negative patients (38% vs. 10%, p<0.001) and was higher in those children who had been exposed to tobacco smoke versus those who hadn't (24% vs. 12%, p<0.01). Treatment with oral corticosteroids was associated with fewer hospitalisations in those patients with a family history of asthma or allergic rhinitis (9.7% vs. 24%, p=0.02) and in RSV-negative patients (2.5% vs. 16.7%, p<0.05). CONCLUSION: Early treatment of bronchiolitis with oral corticosteroid in an outpatient clinic setting was associated with lower hospitalisation rates in patients with a family history of asthma or allergic rhinitis and in RSV-negative patients.     

Assessment of systolic left ventricular function: a multi-centre comparison of cineventriculography, cardiac magnetic resonance imaging, unenhanced and contrast-enhanced echocardiography.

AIMS: To assess the agreement of left ventricular ejection fraction (LVEF) determinations from unenhanced echocardiography, contrast-enhanced echocardiography, magnetic resonance imaging (MRI), and cineventriculography as well as the inter-observer agreement for each method. METHODS AND RESULTS: In 120 patients, with evenly distributed EF-groups (> 55, 35-55, < 35%), cineventriculography, unenhanced echocardiography with second harmonic imaging, and contrast echocardiography at low mechanical index with iv administration of SonoVue were performed. In addition, cardiac MRI at 1.5 T using a steady-state free precession sequence was performed in a subset of 55 patients. On-site, and two blinded off-site assessments were performed for unenhanced and contrast echocardiography, cineventriculography, and MRI according to pre-defined standards. Intra-class correlation coefficients (ICCs) were determined to assess inter-observer reliability between all three readers (i.e. one on-site and two off-site). EF was 56.2 +/- 18.3% by cineventriculography, 54.1 +/- 12.9% by MRI, 50.9 +/- 15.3% by unenhanced echocardiography, and 54.6 +/- 16.8% by contrast echocardiography. Correlation on EF between cineventriculography and echocardiography increased from 0.72 with unenhanced echocardiography to 0.83 with contrast echocardiography (P < 0.05). Similarly, correlation on EF between MRI and echocardiography increased from 0.60 with unenhanced echocardiography to 0.77 with contrast echocardiography (P < 0.05). The inter-observer reliability ICC was 0.91 (95% CI 0.88-0.94) in contrast echocardiography, followed by cardiac MRI (0.86; 95% CI 0.80-0.92), cineventriculography (0.80; 95% CI 0.74-0.85), and unenhanced echocardiography (0.79; 95% CI 0.74-0.85). CONCLUSIONS: Unenhanced echocardiography resulted in slight underestimation of EF and only moderate correlation compared with cineventriculography and MRI. Contrast echocardiography resulted in more accurate EF and significantly improved correlation with cineventriculography and MRI. Contrast echocardiography significantly improved inter-observer agreement on EF compared with unenhanced echocardiography. Inter-observer reliability on EF using contrast echocardiography reaches a level comparable to MRI and is better than those obtained by cineventriculography.     

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.The ability to reprogram cellular metabolism, a hallmark of cancer first noted long ago (Warburg et al., 1927) and recently reappreciated, is essential for tumor progression (Hanahan and Weinberg, 2011). Although cancer-initiated metabolic reprogramming processes are promising therapeutic targets (Vander Heiden, 2011), the existence of alternative, compensatory biosynthetic pathways presents a significant challenge for developing such therapies. For example, in lipid metabolism, cancer cells scavenge extracellular lipids as an alternative to energy-requiring de novo fatty acid biosynthesis (Kamphorst et al., 2011). In amino acid metabolism, glycine and serine required for tumor growth can be produced de novo and can also be scavenged from the extracellular environment (Jain et al., 2012; Maddocks et al., 2013).Nucleotide metabolism also involves redundant and convergent biosynthetic pathways. Deoxyribonucleotide triphosphate (dNTP) pools required for DNA replication and repair can be produced by the de novo pathway (DNP) or by the nucleoside salvage pathway (NSP; Fig. 1 A; Reichard, 1988). The DNP uses glucose and amino acids to generate ribonucleotide diphosphates (NDPs), which are converted to deoxyribonucleotide diphosphates (dNDPs) by ribonucleotide reductase (RNR). The same dNDPs can also be produced via the NSP (Reichard, 1988), starting with extracellular deoxyribonucleosides (dNs) which are imported in the cell via specialized transporters. The first enzymatic steps in the cytosolic NSP are catalyzed by two kinases: thymidine kinase 1 (TK1) phosphorylates thymidine (dT), while deoxycytidine (dC) kinase (dCK) phosphorylates dC, deoxyadenosine (dA), and deoxyguanosine (dG; Reichard, 1988). The relevance of these two NSP kinases for dNTP production in normal and malignant cells is yet to be defined. Because dN substrates for the NSP kinases are absent from most cell culture media, it has been assumed that the NSP is dispensable for DNA replication (Xu et al., 1995). However, recent in vivo findings have challenged this assumption. For example, we reported impaired hematopoiesis in dCK^−/− mice due to dCTP pool deficiency, resulting in replication stress (RS), S-phase arrest, and DNA damage in hematopoietic progenitors (Toy et al., 2010; Austin et al., 2012). Analyses of dCK/TK1 double-knockout mice showed that NSP-derived dCTP synthesis is required to compensate for the inhibition of de novo dCTP production (Austin et al., 2012; Fig. 1 A). The mechanism of DNP inhibition involves allosteric regulation of RNR-mediated reduction of cytidine diphosphate (CDP) to dC diphosphate (dCDP) by dT triphosphate (dTTP) produced via TK1 from endogenous dT (Austin et al., 2012; Fig. 1 A).Open in a separate windowFigure 1.dC salvage via dCK prevents dT-induced lethal RS in T-ALL cells. (A) Allosteric control of DNP dCTP production by dT via dTTP. (B) Effects of dT treatment (24 h) on dCTP and dTTP pools. Values represent mean ± SEM. (C) CEM cell cycle analysis after treatment with vehicle or 50 µM dT ± 2.5 µM dC for 24 h. (D) CEM cell cycle analysis after treatment with 50 µM hydroxyurea, 15 µM 5-fluorouracil, or 1.6 µM cisplatin for 24 h ± 2.5 µM dC. (E and F) Representative immunoblots of dCK and actin expression (E) and dCK kinase assay (F) in CEM dCK^wt (scrambled shRNA) cells and dCK^low (shRNA against dCK) cells. Values are mean ± SEM. ***, P < 0.001. (G) dCTP levels in CEM dCK^wt and dCK^low cells treated for 24 h with vehicle or 50 µM dT ± 2.5 µM dC. Values are mean ± SEM. ***, P < 0.001. (H) Cell cycle analysis of CEM dCK^low cells treated with vehicle or 50 µM dT ± 2.5 µM dC for 24 h. (I) Representative immunoblots detecting Chk1, pChk1 (Ser345), Chk2, pChk2 (Thr68), dCK, and actin in CEM dCK^wt and dCK^low cells treated with vehicle or 50 µM dT in the presence of 2.5 µM dC for 24, 48, and 72 h. (J) pH2A.X (Ser139) and DNA content (DAPI) in CEM dCK^wt and dCK^low cells treated with vehicle or 50 µM dT in the presence of 2.5 µM dC for 24 h. (K) Representative images and quantification of the COMET assay conducted on CEM dCK^wt and dCK^low cells 48 h after treatment with vehicle or 50 µM dT in the presence of 2.5 µM dC. Values represent the mean Olive Tail Moment ± SEM from 100 cells per image × 4 images/group; n = 2 independent experiments. ***, P < 0.001. Bars, 50 µm. (L) Annexin V staining of CEM dCK^wt and dCK^low cells after treatment with vehicle, 2.5 µM dC, 50 µM dT, or dC + dT for 72 h. All values are mean ± SEM from at least three replicates/data point. ***, P < 0.001. All data are representative of n = 3 independent experiments, unless indicated.Production of dNTPs by the NSP may be therapeutically relevant in cancer. For example, the ability of cancer cells to switch their dCTP synthesis from the DNP to the NSP may explain why dT given as a single dCTP-depleting agent showed limited efficacy in clinical trials (Chiuten et al., 1980; Kufe et al., 1980, 1981). If correct, this hypothesis suggests that a combination of dT (to inhibit DNP-mediated dCTP production) and a dCK inhibitor (to co-target dCTP production by the NSP) would be more efficacious in killing tumor cells than either treatment alone. Here, we investigate this possibility in the context of acute lymphoblastic leukemia (ALL). We demonstrate that co-targeting both de novo and salvage pathways for dCTP biosynthesis is well tolerated in mice and is efficacious in T-ALL and B-ALL models. We also describe a positron emission tomography (PET)–based assay to noninvasively monitor in vivo pharmacological targeting of dCTP biosynthesis in cancer cells.     

Influence of primary care practices on patients’ uptake of diabetic retinopathy screening: a qualitative case study

Background

The NHS Diabetic Eye Screening Programme aims to reduce the risk of sight loss among people with diabetes in England by enabling prompt diagnosis of sight-threatening retinopathy. However, the rate of screening uptake between practices can vary from 55% to 95%. Existing research focuses on the impact of patient demographics but little is known about GP practice-related factors that can make a difference.

Aim

To identify factors contributing to high or low patient uptake of retinopathy screening.

Design and setting

Qualitative case-based study; nine purposively selected GP practices (deprived/affluent; high/low screening uptake) in three retinopathy screening programme areas.

Methods

Semi-structured interviews were conducted with patients, primary care professionals, and screeners. A comparative case-based analysis was carried out to identify factors related to high or low screening uptake.

Results

Eight possible factors that influenced uptake were identified. Five modifiable factors related to service and staff interactions: communication with screening services; contacting patients; integration of screening with other care; focus on the newly diagnosed; and perception of non-attenders. Three factors were non-modifiable challenges related to practice location: level of deprivation; diversity of ethnicities and languages; and transport and access. All practices adopted strategies to improve uptake, but the presence of two or more major barriers made it very hard for practices to achieve higher uptake levels.

Conclusions

A range of service-level opportunities to improve screening attendance were identified that are available to practices and screening teams. More research is needed into the complex interfaces of care that make up retinopathy screening.     

Comparison of women versus men hospitalized with heart failure (from a 20-year registry in a middle-eastern country 1991-2010)

The aim of the present study was to compare the clinical characteristics, treatment, and outcomes of women and men hospitalized with heart failure (HF) in a Middle-Eastern country. A retrospective analysis of all patients hospitalized with HF in the State of Qatar from 1991 through 2010 was made. The clinical characteristics, management, and outcomes of the patients with HF were compared according to gender. A subset analysis according to ethnicity was also done (Middle Eastern Arabs vs South Asians). During the 20-year period, 2,379 women and 4,689 men were hospitalized for HF. The women were older and more likely to have diabetes mellitus, hypertension, and chronic renal impairment compared to the male patients. The women were less likely to be current smokers and to have ischemic heart disease compared to the men. Impaired left ventricular function was more common among men. The in-hospital mortality rates were comparable between the 2 groups (7.7% in women vs 8.2% in men; p = 0.4) and significantly improved with time in the 2 groups (p = 0.001). The mortality rates were comparable among the women, regardless of the ethnicity. In conclusion, overall improvement occurred in survival in patients hospitalized with HF in a Middle-Eastern country, regardless of gender. Women hospitalized with HF had mortality rates comparable to those of men.     

Marital Status and Outcome of Patients Presenting with Acute Coronary Syndrome: An Observational Report

Background & hypothesis:

Data on the clinical characteristics and outcome of patients presenting with acute coronary syndrome (ACS) according to their marital status is not clear.

Methods:

A total of 5334 patients presenting with ACS in 65 hospitals in 6 Middle East countries in the 2nd Gulf Registry of Acute Coronary Events (Gulf RACE‐2) were studied according to their marital status (5024 married, 100 single, and 210 widowed patients).

Result:

When compared to married patients, widowed patients were older and more likely to be female. Widowed patients were more likely to have diabetes mellitus, hypertension, history of heart failure, and peripheral vascular disease and were less likely to be tobacco users when compared to the other groups. Widowed patients were also more likely to present with atypical symptoms and have advanced Killip class. Widowed patients were more likely to present with non‐ST‐elevation myocardial infarction (NSTEMI) when compared to the other 2 groups. Widowed patients were more likely to have heart failure (P = 0.001), cardiogenic shock (P = 0.001), and major bleeding (P = 0.002) when compared to the other groups. No statistically significant difference was observed in regard to duration of hospital stay, door to needle time in STEMI patients, or cardiac arrhythmias between the various groups. Widowed patients had higher in‐hospital, 30‐day, and 1‐year mortality rates (P = 0.001). Marital status was an independent predictor for in‐hospital mortality.

Conclusion:

Widowed marital status was associated with worse cardiovascular risk profile, and worse in‐hospital and 1‐year outcome. Future work should be focused on whether the provision of psychosocial support will result in improved outcomes among this high‐risk group. Clin. Cardiol. 2011 DOI: 10.1002/clc.22034 Gulf RACE is a Gulf Heart Association (GHA) project and was financially supported by the GHA, Sanofi Aventis, and the College of Medicine Research Center at King Khalid University Hospital, King Saud University, and Riyadh, Saudi Arabia. The authors have no other funding, financial relationships, or conflicts of interest to disclose.