Mitochondrial DNAs provide insight into trypanosome phylogeny and molecular evolution.

BACKGROUND: Trypanosomes are single-celled eukaryotic parasites characterised by the unique biology of their mitochondrial DNA. African livestock trypanosomes impose a major burden on agriculture across sub-Saharan Africa, but are poorly understood compared to those that cause sleeping sickness and Chagas disease in humans. Here we explore the potential of the maxicircle, a component of trypanosome mitochondrial DNA to study the evolutionary history of trypanosomes. RESULTS: We used long-read sequencing to completely assemble maxicircle mitochondrial DNA from four previously uncharacterized African trypanosomes, and leveraged these assemblies to scaffold and assemble a further 103 trypanosome maxicircle gene coding regions from published short-read data. While synteny was largely conserved, there were repeated, independent losses of Complex I genes. Comparison of pre-edited and non-edited genes revealed the impact of RNA editing on nucleotide composition, with non-edited genes approaching the limits of GC loss. African tsetse-transmitted trypanosomes showed high levels of RNA editing compared to other trypanosomes. The gene coding regions of maxicircle mitochondrial DNAs were used to construct time-resolved phylogenetic trees, revealing deep divergence events among isolates of the pathogens Trypanosoma brucei and T. congolense. CONCLUSIONS: Our data represents a new resource for experimental and evolutionary analyses of trypanosome phylogeny, molecular evolution and function. Molecular clock analyses yielded a timescale for trypanosome evolution congruent with major biogeographical events in Africa and revealed the recent emergence of Trypanosoma brucei gambiense and T. equiperdum, major human and animal pathogens.

Transporter gene acquisition and innovation in the evolution of Microsporidia intracellular parasites.

The acquisition of genes by horizontal transfer can impart entirely new biological functions and provide an important route to major evolutionary innovation. Here we have used ancient gene reconstruction and functional assays to investigate the impact of a single horizontally transferred nucleotide transporter into the common ancestor of the Microsporidia, a major radiation of intracellular parasites of animals and humans. We show that this transporter provided early microsporidians with the ability to steal host ATP and to become energy parasites. Gene duplication enabled the diversification of nucleotide transporter function to transport new substrates, including GTP and NAD+, and to evolve the proton-energized net import of nucleotides for nucleic acid biosynthesis, growth and replication. These innovations have allowed the loss of pathways for mitochondrial and cytosolic energy generation and nucleotide biosynthesis that are otherwise essential for free-living eukaryotes, resulting in the highly unusual and reduced cells and genomes of contemporary Microsporidia.

Familial hyperaldosteronism type III.

Primary aldosteronism is the most common form of endocrine hypertension. This disorder comprises both sporadic and familial forms. Four familial forms of primary aldosteronism (FH-I to FH-IV) have been described. FH-III is caused by germline mutations in KCNJ5, encoding the potassium channel Kir3.4 (also called GIRK4). These mutations alter the selectivity filter of the channel and lead to abnormal ion currents with loss of potassium selectivity, sodium influx and consequent increased intracellular calcium that causes excessive aldosterone biosynthesis. To date, eleven families have been reported, carrying six different mutations. Although the clinical features are variable, FH-III patients often display severe hyperaldosteronism with an early onset, associated with hypokalemia and diabetes insipidus-like symptoms. In most cases FH-III patients are resistant to pharmacological therapy and require bilateral adrenalectomy to control symptoms. In the present manuscript, we review the genetics and pathological basis of FH-III, the diagnostic work-up, clinical features and therapeutic management. Finally, we will describe a new case of FH-III of an Italian patient carrying a Gly151Arg mutation.

Recent Developments in Primary Aldosteronism.

Primary aldosteronism (PA) is the most frequent endocrine cause of secondary arterial hypertension. Sporadic forms of PA caused mainly by an aldosterone producing adenoma (APA) or idiopathic adrenal hyperplasia (IAH) predominate; in contrast, familial forms (familial hyperaldosteronism types I, II and III) affect only a minor proportion of PA patients. Patient based registries and biobanks, international networks and next generation sequencing technologies have emerged over recent years. Somatic hot-spot mutations in the potassium channel GIRK4 (encoded by KCNJ5), in ATPases and a L-type voltage-gated calcium-channel correlate with the autonomous aldosterone production in approximately half of all APAs. The recently discovered form FH III is caused by different germline KCNJ5 mutations with variable clinical presentations and severity. Autoantibodies to the angiotensin II Type 1 receptor have been identified in patients with PA and possibly play a pathophysiological role in the development of PA. Adrenal vein sampling (AVS) represents the gold standard in differentiating unilateral and bilateral forms of PA. Recent consensus papers have tried to implement current guidelines in order to standardise the technique of AVS. New techniques like segmental AVS might allow a finer mapping of the aldosterone production within the adrenal gland. The measurement of the steroids 18-hydroxycortisol and 18-oxocortisol by liquid chromatography tandem mass spectrometry has been shown to be useful to distinguish between unilateral and bilateral forms of PA.

ARMC5 mutation analysis in patients with primary aldosteronism and bilateral adrenal lesions.

Idiopathic hyperaldosteronism (IHA) due to bilateral adrenal hyperplasia is the most common subtype of primary aldosteronism (PA). The pathogenesis of IHA is still unknown, but the bilateral disease suggests a potential predisposing genetic alteration. Heterozygous germline mutations of armadillo repeat containing 5 (ARMC5) have been shown to be associated with hypercortisolism due to sporadic primary bilateral macronodular adrenal hyperplasia and are also observed in African-American PA patients. We investigated the presence of germline ARMC5 mutations in a group of PA patients who had bilateral computed tomography-detectable adrenal alterations. We sequenced the entire coding region of ARMC5 and all intron/exon boundaries in 39 patients (37 Caucasians and 2 black Africans) with confirmed PA (8 unilateral, 27 bilateral and 4 undetermined subtype) and bilateral adrenal lesions. We identified 11 common variants, 5 rare variants with a minor allele frequency <1% and 2 new variants not previously reported in public databases. We did not detect by in silico analysis any ARMC5 sequence variations that were predicted to alter protein function. In conclusion, ARMC5 mutations are not present in a fairly large series of Caucasian patients with PA associated to bilateral adrenal disease. Further studies are required to definitively clarify the role of ARMC5 in the pathogenesis of adrenal nodules and aldosterone excess in patients with PA.

Steroid Hormone Production in Patients with Aldosterone Producing Adenomas.

Primary aldosteronism encompasses 2 major underlying causes: (1) aldosterone producing adenoma and (2) bilateral adrenal hyperplasia. In addition to the aldosterone excess, increased production of other compounds of the steroidogenic pathways may be involved. Until recently, most studies examined the production of steroids other than aldosterone in tumor tissue, urine, or peripheral plasma samples, but several new studies have also addressed steroid levels in adrenal venous blood samples using liquid chromatography tandem mass spectrometry. Plasma and tissue levels of several precursors of aldosterone with mineralocorticoid activity are higher in patients with aldosterone producing adenomas than in those with bilateral hyperplasia. These include corticosterone, deoxycorticosterone, and their 18-hydroxylated metabolites. Similarly, urinary, peripheral, and adrenal venous concentrations of the hybrid steroids 18-oxocortisol and 18-hydroxycortisol are higher in patients with aldosterone producing adenomas than in bilateral hyperplasia. Differences in the pathophysiology and in clinical and biochemical phenotypes caused by aldosterone producing adenomas and bilateral adrenal hyperplasia may be related to the differential expression of steroidogenic enzymes, and associated to specific underlying somatic mutations. Correct appreciation of differences in steroid profiling between aldosterone producing adenomas and bilateral adrenal hyperplasia may not only contribute to a better understanding of the pathogenesis of primary aldosteronism but may also be helpful for future subtyping of primary aldosteronism.

Subtype Diagnosis of Primary Aldosteronism: Approach to Different Clinical Scenarios.

Identification and management of patients with primary aldosteronism are of utmost importance because it is a frequent cause of endocrine hypertension, and affected patients display an increase of cardio- and cerebro-vascular events, compared to essential hypertensives. Distinction of primary aldosteronism subtypes is of particular relevance to allocate the patients to the appropriate treatment, represented by mineralocorticoid receptor antagonists for bilateral forms and unilateral adrenalectomy for patients with unilateral aldosterone secretion. Subtype differentiation of confirmed hyperaldosteronism comprises adrenal CT scanning and adrenal venous sampling. In this review, we will discuss different clinical scenarios where execution, interpretation of adrenal vein sampling and subsequent patient management might be challenging, providing the clinician with useful information to help the interpretation of controversial procedures.

KCNJ5 Mutations: Sex, Salt and Selection.

Somatic mutations have been identified in the KCNJ5 gene (encoding the potassium channel GIRK4) in aldosterone-producing adenomas (APA). Most of these mutations are located in or near the selectivity filter of the GIRK4 channel pore and several have been shown to lead to the constitutive overproduction of aldosterone. KCNJ5 mutations in APA are more frequent in women; however, this gender dimorphism is a reported phenomenon of Western but not East Asian populations. In this review we discuss some of the issues that could potentially underlie this observation.

Renin and aldosterone measurements in the management of arterial hypertension.

Renin-angiotensin-aldosterone system (RAAS) is recognized as the main regulatory system of hemodynamics in man, and its derangements have a key role in the development and maintenance of arterial hypertension. Classification of the hypertensive states according to different patterns of renin and aldosterone levels ("RAAS profiling") allows the diagnosis of specific forms of secondary hypertension and may identify distinct hemodynamic subsets in essential hypertension. In this review, we summarize the application of RAAS profiling for the diagnostic assessment of hypertensive patients and discuss how the pathophysiological framework provided by RAAS profiling may guide therapeutic decision-making, especially in the context of uncontrolled hypertension not responding to multi-therapy.

Changing cell behaviours during beetle embryogenesis correlates with slowing of segmentation.

Segmented animals are found in major clades as phylogenetically distant as vertebrates and arthropods. Typically, segments form sequentially in what has been thought to be a regular process, relying on a segmentation clock to pattern budding segments and posterior mitosis to generate axial elongation. Here we show that segmentation in Tribolium has phases of variable periodicity during which segments are added at different rates. Furthermore, elongation during a period of rapid posterior segment addition is driven by high rates of cell rearrangement, demonstrated by differential fates of marked anterior and posterior blastoderm cells. A computational model of this period successfully reproduces elongation through cell rearrangement in the absence of cell division. Unlike current models of steady-state sequential segmentation and elongation from a proliferative growth zone, our results indicate that cell behaviours are dynamic and variable, corresponding to differences in segmentation rate and giving rise to morphologically distinct regions of the embryo.

A comparison of outcomes among hospital survivors with and without severe comorbidity admitted to the intensive care unit.

Little is known about the experiences of patients with severe comorbidity discharged from Intensive Care Units (ICUs). This project aimed to determine the effects of an ICU stay for patients with severe comorbidity by comparing 1) quality of life (QOL), 2) the symptom profile of hospital survivors and 3) health service use after hospital discharge for patients admitted to ICU with and without severe comorbidity. A case-control study was used. Patients with severe comorbidity were matched to a contemporaneous cohort of ICU patients by age and severity of illness. Assessment tools were the Medical Outcome Study 36-item short-form and European Organisation for Research and Treatment of Cancer QLQ-C15-PAL questionnaires for QOL and the Symptom Assessment Scale for symptom distress. A proportional odds assumption was performed using an ordinal regression model. The difference in QOL outcome was the dependent variable for each pair. Health service use after discharge from ICU was monitored with patient diaries. Patients aged 18+ years admitted to an ICU in a metropolitan teaching hospital between 2011 and 2012 were included. We recruited 30 cases and 30 controls. QOL improved over the six months after hospital discharge for patients with and without severe comorbidity (P <0.01) within the groups but there was no difference found between the groups (P >0.3). There was no difference in symptoms or health service use between patients with and without severe comorbidity. ICU admission for people with severe comorbidity can be appropriate to stabilise the patient's condition and is likely to be followed by some overall improvement over the six months after hospital discharge.

The APOL1 genotype of African American kidney transplant recipients does not impact 5-year allograft survival.

Apolipoprotein L-1 (APOL1) gene variants are associated with end-stage renal disease in African Americans (AAs). Here we investigate the impact of recipient APOL1 gene distributions on kidney allograft outcomes. We conducted a retrospective analysis of 119 AA kidney transplant recipients, and found that 58 (48.7%) carried two APOL1 kidney disease risk variants. Contrary to the association seen in native kidney disease, there is no difference in allograft survival at 5-year posttransplant for recipients with high-risk APOL1 genotypes. Thus, we were able to conclude that APOL1 genotypes do not increase risk of allograft loss after kidney transplantations, and carrying 2 APOL1 risk alleles should not be an impediment to transplantation.

Aldosterone does not modify gene expression in human endothelial cells.

The toxic effects of aldosterone on the vasculature, and in particular on the endothelial layer, have been proposed as having an important role in the cardiovascular pathology observed in mineralocorticoid-excess states. In order to characterize the genomic molecular mechanisms driving the aldosterone-induced endothelial dysfunction, we performed an expression microarray on transcripts obtained from both human umbilical vein endothelial cells and human coronary artery endothelial cells stimulated with 10 - 7 M aldosterone for 18 h. The results were then subjected to qRT-PCR confirmation, also including a group of genes known to be involved in the control of the endothelial function or previously described as regulated by aldosterone. The state of activation of the mineralocorticoid receptor was investigated by means of a luciferase-reporter assay using a plasmid encoding a mineralocorticoid and glucocorticoid-sensitive promoter. Aldosterone did not determine any significant change in gene expression in either cell type both in the microarray and in the qRT-PCR analysis. The luciferase-reporter assay showed no activation of the mineralocorticoid receptor following aldosterone stimulation. The status of nonfunctionality of the mineralocorticoid receptor expressed in cultured human umbilical and coronary artery endothelial cells does not allow aldosterone to modify gene expression and provides evidence against either a beneficial or harmful genomic effect of aldosterone on healthy endothelial cells.

Outcome of ranibizumab treatment in neovascular age related macula degeneration in eyes with baseline visual acuity better than 6/12.

BACKGROUND: The beneficial effect of intravitreal ranibizumab in the treatment of neovascular age-related macula degeneration (nAMD) is well known. Outcome data for eyes presenting with visual acuity better than 6/12 is limited. AIMS: To assess the effect of baseline vision on outcome in ranibizumab-treated nAMD eyes, including a subgroup with baseline vision ≥6/12 (<0.30 logmar). DESIGN: Prospective, consecutive and interventional case series. METHODS: A consecutive cohort of patients treated with intravitreal ranibizumab for nAMD with 52-week follow-up were studied. Patients who had received previous treatment for nAMD were excluded. Eyes were stratified according to baseline logmar visual acuity into four groups: <0.30 (>6/12), 0.30-0.59 (6/12-6/24), 0.60-0.99 (6/24-6/60) and 1.00-1.20 (6/60-6/96). Intravitreal ranibizumab (0.5 mg in 0.05 ml) was administered in three loading monthly doses followed by PRN dosing according to optical coherence tomography (OCT) findings. RESULTS: A total of 615 eyes were studied including 88 eyes with baseline vision <0.30. The mean change in logmar letters at 52 weeks was +5.5 (entire study group), -0.5 (<0.30 subgroup), +2.2 (0.30-0.59 subgroup), +6.5 (0.60-0.99 subgroup) and +15.3 (1.00-1.20 subgroup). In the <0.30 subgroup, 60 of 88 eyes (68%) had best-corrected visual acuity (BCVA) equal to or better than baseline and 82 of 88 eyes (93%) lost <15 letters at 52 weeks. Within this subgroup 56 of 67 eyes (84%) maintained UK driving standard BCVA visual acuity over the study period. CONCLUSIONS: This study provides evidence that intravitreal ranibizumab treatment stabilises good vision in nAMD presenting with vision better than 6/12 over 52 weeks follow-up.

Changes in case-mix and outcomes of critically ill patients in an Australian tertiary intensive care unit.

Critical care service is expensive and the demand for such service is increasing in many developed countries. This study aimed to assess the changes in characteristics of critically ill patients and their effect on long-term outcome. This cohort study utilised linked data between the intensive care unit database and state-wide morbidity and mortality databases. Logistic and Cox regression was used to examine hospital survival and five-year survival of 22,298 intensive care unit patients, respectively. There was a significant increase in age, severity of illness and Charlson Comorbidity Index of the patients over a 16-year study period. Although hospital mortality and median length of intensive care unit and hospital stay remained unchanged, one- and five-year survival had significantly improved with time, after adjusting for age, gender; severity of illness, organ failure, comorbidity, 'new' cancer and diagnostic group. Stratified analyses showed that the improvement in five-year survival was particularly strong among patients admitted after cardiac surgery (P = 0.001). In conclusion, although critical care service is increasingly being provided to patients with a higher severity of acute and chronic illnesses, long-term survival outcome has improved with time suggesting that critical care service may still be cost-effectiveness despite the changes in case-mix.

Healthcare utilisation among patients discharged from hospital after intensive care.

Surviving critical illness can be life-changing and presents new healthcare challenges for patients after hospital discharge. This feasibility study aimed to examine healthcare service utilisation for patients discharged from hospital after intensive care unit stay. Following Ethics Committee approval, patients aged 18 years and older were recruited over three months. Those admitted after cardiac surgery, discharged to another facility or against medical advice were excluded. Patients were informed of the study by post and followed-up by telephone at two and six months after discharge. General practitioners were also contacted (44% responded). Among 187 patients discharged from hospital, 11 died, 25 declined to participate and 39 could not be contacted. For 112 patients (60%) who completed a survey, the majority (82%) went home from hospital and were cared for by their partner (53%). More than half of the patients (58%) reported taking the same number of medications after intensive care unit stay but 30% took more (P = 0.023). While there was no change in the number of visits to the general practitioner for 64% of patients, 29% reported an increase after intensive care unit stay. At six months, 40% of responders who were not retired were unemployed. Discharge summary surveys revealed 39 general practitioners (71%) were satisfied with details of ongoing healthcare needs. Twenty-one general practitioners wrote comments: 10 reported insufficient information about ongoing needs/rehabilitation and two reported no mention of intensive care unit stay. Survivors of critical illness had increased healthcare needs and despite most returning home, had a low workforce participation rate. This requires further investigation to maximise the benefits of survival from critical illness.

Effect of length of stay in intensive care unit on hospital and long-term mortality of critically ill adult patients.

BACKGROUND: Critical illness leading to prolonged length of stay (LOS) in an intensive care unit (ICU) is associated with significant mortality and resource utilization. This study assessed the independent effect of ICU LOS on in-hospital and long-term mortality after hospital discharge. METHODS: Clinical and mortality data of 22 298 patients, aged 16 yr and older, admitted to ICU between 1987 and 2002 were included in this linked-data cohort study. Cox's regression with restricted cubic spline function was used to model the effect of LOS on in-hospital and long-term mortality after adjusting for age, gender, acute physiology score (APS), maximum number of organ failures, era of admission, elective admission, Charlson's co-morbidity index, and diagnosis. The variability each predictor explained was calculated by the percentage of the chi(2) statistic contribution to the total chi(2) statistic. RESULTS: Most hospital deaths occurred within the first few days of ICU admission. Increasing LOS in ICU was not associated with an increased risk of in-hospital mortality after adjusting for other covariates, but was associated with an increased risk of long-term mortality after hospital discharge. The variability on the long-term mortality effect associated with ICU LOS (2.3%) appeared to reach a plateau after the first 10 days in ICU and was not as important as age (35.8%), co-morbidities (18.6%), diagnosis (10.9%), and APS (3.6%). CONCLUSIONS: LOS in ICU was not an independent risk factor for in-hospital mortality, but it had a small effect on long-term mortality after hospital discharge after adjustment for other risk factors.

Effect of an episode of critical illness on subsequent hospitalisation: a linked data study.

Healthcare utilisation can affect quality of life and is important in assessing the cost-effectiveness of medical interventions. A clinical database was linked to two Australian state administrative databases to assess the difference in incidence of healthcare utilisation of 19,921 patients who survived their first episode of critical illness. The number of hospital admissions and days of hospitalisation per patient-year was respectively 150% and 220% greater after than before an episode of critical illness (assessed over the same time period). This was the case regardless of age or type of surgery (i.e. cardiac vs non-cardiac). After adjusting for the ageing effect of the cohort as a whole, there was still an unexplained two to four-fold increase in hospital admissions per patient-year after an episode of critical illness. We conclude that an episode of critical illness is a robust predictor of subsequent healthcare utilisation.

Examining the occurrence of adverse events within 72 hours of discharge from the intensive care unit.

Adverse events have negative consequences for patients, including increased risk of death or permanent disability. Reports describe suboptimal patient care on hospital wards and reasons for re-admission to the intensive care unit (ICU) but limited data exists on the occurrence of adverse events, their characteristics and outcomes in patients recently discharged from the ICU to the ward. This prospective observational study describes the incidence and outcomes of adverse events within 72 hours of discharge from an Australian ICU over 12 weeks in 2006. Patients were excluded if they were admitted to ICU after booked surgery or uncomplicated drug overdose, were discharged from ICU to the high dependency unit or had a 'do-not-resuscitate' order Clinical antecedents and preventability were determined for each event. Seventeen (10%) of the 167 discharges that met the inclusion criteria were associated with an adverse event, with nine (52%) judged as probably preventable. Seven adverse events occurred from discharges between 1700 and 0700 hours and seven were on weekends. The most common adverse events were related to fluid management (47%). Outcomes included three ICU readmissions, two high dependency unit admissions and two required one-to-one ward nursing. Two adverse events resulted in temporary disability, seven resulted in prolonged hospital stays and two were associated with death. Delay in taking action for abnormal physiological signs and infrequent charting were evident. Whilst the adverse event rate compared favourably with other reports, 64% of the events were considered preventable. A review of support systems and processes is recommended to better target transition from the ICU.

Conceptual basis and methodology of the SOPHIA study.

To clarify the role of gene polymorphisms on the effect of losartan and losartan plus hydrochlorothiazide on blood pressure (primary end point) and on cardiac, vascular and metabolic phenotypes (secondary end point) after 4, 8, 12, 16 and 48 weeks treatment, an Italian collaborative study - The Study of the Pharmacogenomics in Italian hypertensive patients treated with the Angiotensin receptor blocker losartan (SOPHIA) - on never-treated essential hypertensives (n = 800) was planned. After an 8 week run-in, losartan 50 mg once daily will be given and doubled to 100 mg at week +4 if blood pressure is more than 140/90 mmHg. Hydroclorothiazide 25 mg once daily at week +8 and amlodipine 5 mg at week +16 will be added if blood pressure is more than 140/90 mmHg. Cardiac mass (echocardiography), carotid intima-media thickness, 24 h ambulatory blood pressure, homeostatic model assessment (HOMA) index, microalbuminuria, plasma renin activity and aldosterone, endogenous lithium clearance, brain natriuretic peptide and losartan metabolites will be evaluated. Genes of the renin-angiotensin-aldosterone system, salt sensitivity, the beta-adrenergic system and losartan metabolism will be studied (Illumina custom arrays). A whole-genome scan will also be performed in half of the study cohort (1M array, Illumina 500 GX beadstation).