Genome sequencing of C. elegans balancer strains reveals previously unappreciated complex genomic rearrangements.

Genetic balancers in Caenorhabditis elegans are complex variants that allow lethal or sterile mutations to be stably maintained in a heterozygous state by suppressing crossover events. Balancers constitute an invaluable tool in the C. elegans scientific community and have been widely used for decades. The first/traditional balancers were created by applying X-rays, UV, or gamma radiation on C. elegans strains, generating random genomic rearrangements. Their structures have been mostly explored with low-resolution genetic techniques (e.g., fluorescence in situ hybridization or PCR), before genomic mapping and molecular characterization through sequencing became feasible. As a result, the precise nature of most chromosomal rearrangements remains unknown, whereas, more recently, balancers have been engineered using the CRISPR-Cas9 technique for which the structure of the chromosomal rearrangement has been predesigned. Using short-read whole-genome sequencing (srWGS) and tailored bioinformatic analyses, we previously interpreted the structure of four chromosomal balancers randomly created by mutagenesis processes. Here, we have extended our analyses to five CRISPR-Cas9 balancers and 17 additional traditional balancing rearrangements. We detected and experimentally validated their breakpoints and have interpreted the balancer structures. Many of the balancers were found to be more intricate than previously described, being composed of complex genomic rearrangements (CGRs) such as chromoanagenesis-like events. Furthermore, srWGS revealed additional structural variants and CGRs not known to be part of the balancer genomes. Altogether, our study provides a comprehensive resource of complex genomic variations in C. elegans and highlights the power of srWGS to study the complexity of genomes by applying tailored analyses.

Whole genome sequencing facilitates intragenic variant interpretation following modifier screening in C. elegans.

BACKGROUND: Intragenic modifiers (in-phase, second-site variants) are known to have dramatic effects on clinical outcomes, affecting disease attributes such as severity or age of onset. However, despite their clinical importance, the focus of many genetic screens in model systems is on the discovery of extragenic variants, with many labs still relying upon more traditional methods to identify modifiers. However, traditional methods such as PCR and Sanger sequencing can be time-intensive and do not permit a thorough understanding of the intragenic modifier effects in the context of non-isogenic genomic backgrounds. RESULTS: Here, we apply high throughput approaches to identify and understand intragenic modifiers using Caenorhabditis elegans. Specifically, we applied whole genome sequencing (WGS) to a mutagen-induced forward genetic screen to identify intragenic suppressors of a temperature-sensitive zyg-1(it25) allele in C. elegans. ZYG-1 is a polo kinase that is important for centriole function and cell divisions, and mutations that truncate its human orthologue, PLK4, have been associated with microcephaly. Combining WGS and CRISPR/Cas9, we rapidly identify intragenic modifiers, show that these variants are distributed non-randomly throughout zyg-1 and that genomic context plays an important role on phenotypic outcomes. CONCLUSIONS: Ultimately, our work shows that WGS facilitates high-throughput identification of intragenic modifiers in clinically relevant genes by reducing hands-on research time and overall costs and by allowing thorough understanding of the intragenic phenotypic effects in the context of different genetic backgrounds.

Reducing the expression of the Numb adaptor protein in neurons increases the searching behavior of Drosophila larvae.

Drosophila larval crawling is easily-observable and relatively stereotyped. Crawling consists of linear locomotion interrupted by periods when the larvae pause, swing their heads, and change direction (a 'search'). Here we identify Numb, a peripheral membrane adaptor protein, as an important regulator of searching behavior. When Numb RNAi transgenes were expressed in all neurons, searching frequency increased while linear movement appeared normal. Numb's role in suppressing searching behavior was verified by rescuing this phenotype with a Numb homologue from mice. Such behavioral specificity suggests that further analysis of searching might help identify additional, evolutionarily-conserved interactors of the Numb protein.

Narrowing of T-cell receptor beta variable repertoire during symptomatic herpesvirus infection in transplant patients.

Primary infection or recrudescence of latent virus infection in transplant recipients can be manifested either as asymptomatic or symptomatic disease. Here we show that symptomatic human cytomegalovirus (HCMV) or Epstein-Barr virus (EBV) infection or recrudescence following solid organ transplantation (SOT) was coincident with a dramatic skewing of T-cell receptor beta variable (TRBV) repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no viral infection or recrudescence showed minimal or no skewing of the T-cell receptor repertoire to maintain peripheral blood repertoire diversity. More importantly, we show that large monoclonal/oligoclonal repertoire expansions are associated with the loss of HCMV-specific T-cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, whereas SOT recipients who maintain peripheral blood TRBV repertoire diversity and functional antigen-specific T-cell responses can resist clinical symptomatic disease in spite of high levels of viral load.

Familiarisation and reliability of sprint test indices during laboratory and field assessment.

The aim of the study was to assess the reliability of sprint performance in both field and laboratory conditions. Twenty-one male (mean ± s: 19 ± 1 years, 1.79 ± 0.07 m, 77.6 ± 7.1 kg) and seventeen female team sport players (mean ± s: 21 ± 4 years, 1.68 ± 0. 07 m, 62.7 ± 4.7 kg) performed a maximal 20-metre sprint running test on eight separate occasions. Four trials were conducted on a non-motorised treadmill in the laboratory; the other four were conducted outdoors on a hard-court training surface with time recorded by single-beam photocells. Trials were conducted in random order with no familiarisation prior to testing. There was a significant difference between times recorded during outdoor field trials (OFT) and indoor laboratory trials (ILT) using a non-motorised treadmill (3.47 ± 0.53 vs. 6.06 ±1.17s; p < 0.001). The coefficient of variation (CV) for time was 2.55-4.22% for OFT and 5.1-7.2% for ILT. During ILT peak force (420.9 ± 87.7N), mean force (147.2 ± 24.7N), peak power (1376.8 ± 451.9W) and mean power (514.8 ± 164.4W), and were measured. The CV for all ILT variables was highest during trial 1-2 comparison. The CV (95% confidence interval) for the trial 3-4 comparison yielded: 9.4% (7.7-12. 1%), 7.9% (6.4-10.2%), 10.1% (8.2-13.1%) and 6.2% (5.1-8.0%) for PF, MF, PP and MP and respectively. The results indicate that reliable data can be derived for single maximal sprint measures, using fixed distance protocols. However, significant differences in time/speed over 20-m exist between field and laboratory conditions. This is primarily due to the frictional resistance in the non- motorised treadmill. Measures of force and power during ILT require at least 3 familiarisations to reduce variability in test scores. Key pointsReliable data can be derived from single maximal sprint measures in both indoor and outdoor environments using fixed distance protocols.There may be significant time differences to complete fixed distance trials between the two environments.Measures of mean force, peak force and peak power during indoor trials may require multiple trials to reduce variability in test scores.

Effect of the rotor crank system on cycling performance.

The aim of this study was to evaluate the impact of a novel crank system on laboratory time-trial cycling performance. The Rotor system makes each pedal independent from the other so that the cranks are no longer fixed at 180°. Twelve male competitive but non-elite cyclists (mean ± s: 35 ± 7 yr, Wmax = 363 ± 38 W, VO2peak = 4.5 ± 0.3 L·min(-1)) completed 6-weeks of their normal training using either a conventional (CON) or the novel Rotor (ROT) pedal system. All participants then completed two 40.23-km time-trials on an air-braked ergometer, one using CON and one using ROT. Mean performance speeds were not different between trials (CON = 41.7 km·h(-1) vs. ROT = 41.6 km·h(-1), P > 0.05). Indeed, the pedal system used during the time-trials had no impact on any of the measured variables (power output, cadence, heart rate, VO2, RER, gross efficiency). Furthermore, the ANOVA identified no significant interaction effect between main effects (Time-trial crank system*Training crank system, P > 0.05). To the authors' knowledge, this is the first study to examine the effects of the Rotor system on endurance performance rather than endurance capacity. These results suggest that the Rotor system has no measurable impact on time-trial performance. However, further studies should examine the importance of the Rotor 'regulation point' and the suggestion that the Rotor system has acute ergogenic effects if used infrequently. Key pointsThe Rotor crank system does not improve gross efficiency in well-trained cyclists.The Rotor crank system has no measurable impact on laboratory 40.23-km time-trial performance.A 6-week period of familiarisation does not increase the effectiveness of the Rotor crank system.

Arg389Gly-beta1-adrenergic receptors determine improvement in left ventricular systolic function in nonischemic cardiomyopathy patients with heart failure after chronic treatment with carvedilol.

OBJECTIVE: Administration of the beta-adrenergic receptor blocker carvedilol to patients with chronic heart failure leads to clinically significant benefits, including improvement in left ventricular systolic function in some, but not all, patients. We sought to determine the basis of the variable effect obtained with carvedilol in patients with heart failure. Carvedilol blocks both beta1-adrenergic and beta2-adrenergic receptors, and both receptors exist as polymorphisms. We aimed to determine whether these polymorphisms contribute to variability in response to carvedilol in patients with chronic heart failure. METHODS: We retrospectively and prospectively investigated 135 patients with nonischemic cardiomyopathy and chronic stable heart failure (New York Heart Association class II, III) treated with carvedilol. Baseline echocardiography was obtained before introduction of carvedilol and repeated after stabilization of a maximally tolerated dose of carvedilol (50-100 mg/day) for at least 1 year. Polymerase chain reaction and restriction fragment length polymorphism analysis were used to genotype beta1-adrenergic and beta2-adrenergic receptor polymorphisms. RESULTS: When grouped according to receptor polymorphisms patients were well matched for severity of heart failure, comorbidity and treatment. No significant difference was observed in baseline left ventricular ejection fraction (LVEF) between groups (P>0.05). After 1.5 years of treatment with carvedilol patients with Arg389Arg-beta1-adrenergic receptors had a significantly greater improvement in LVEF compared with Gly389 carriers (Arg389Arg 18.8%; Arg389Gly 9.4%; Gly389Gly 6.0%; P<0.001) whereas there were no differences attributable to other beta1-adrenergic and beta2-adrenergic receptor polymorphisms (P>0.05). CONCLUSION: In patients with nonischemic dilated cardiomyopathy, carvedilol leads to a significantly greater improvement in LVEF in patients with the Arg389Arg-beta1 adrenergic receptor phenotype.

Symptomatic and asymptomatic viral recrudescence in solid-organ transplant recipients and its relationship with the antigen-specific CD8(+) T-cell response.

Using ex vivo antigen-specific T-cell analysis, we found that symptomatic cytomegalovirus recrudescence in transplant recipients was coincident with reduced expression of gamma interferon (IFN-gamma) by virus-specific CD8(+) T cells and an up-regulation of CD38 expression on these T cells, although there was no significant change in the absolute number of virus-specific cells (as assessed by major histocompatibility complex-peptide multimers). In contrast, HLA class I-matched transplant patients with asymptomatic viral recrudescence showed increased expansion of antigen-specific T cells and highly stable IFN-gamma expression by epitope-specific T cells. These studies suggest that a strong functional T-cell response plays a crucial role in defining the clinical outcome of acute viral recrudescence.

A complete mock circulation loop for the evaluation of left, right, and biventricular assist devices.

A new mock circulation loop was developed to replicate the necessary features of the systemic and pulmonic circulatory systems, including pulsatile left and right ventricles coupled with vascular compliances and resistances. A brief description of the mock loop construction is provided before results are presented confirming the recreation of perfusion rates and pressures found in the natural systemic and pulmonic vascular trees for a normal and failing heart at rest. This rig provides the ability to evaluate the hemodynamic effect of left, right, and biventricular assist devices in vitro. The small and compact mock circulation rig has the potential to reduce device evaluation costs by simulating the natural circulatory system, thus providing valuable device performance feedback prior to expensive in vivo animal trials.

Value of B-type natriuretic peptide for identifying significantly elevated pulmonary artery wedge pressure in patients treated for established chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy.

It has been suggested that plasma B-type natriuretic peptide (BNP) level may be used as a noninvasive biomarker of the adequacy of long-term heart failure therapy. The effect of contemporary therapy on the relation between BNP measured using modern commercially available assays and cardiac filling pressures has not been studied in detail, because most of the original studies predate these developments. The investigators sought to assess the diagnostic accuracy of BNP to identify significantly elevated pulmonary capillary wedge pressure (PCWP) in a group of patients with severe chronic heart failure on treatment. BNP correlated well with PCWP (r = 0.50, p <0.001), but the diagnostic accuracy of the test to identify patients with PCWP >15 mm Hg was only 74%, largely because of poor sensitivity or a large number of false-negative test results. Maximizing medical therapy irrespective of plasma BNP results remains the best approach to managing chronic heart failure.

The role of left and right ventricular early diastolic Doppler tissue echocardiographic indices in the evaluation of acute rejection in orthotopic heart transplant.

BACKGROUND: The aim was to evaluate whether Doppler tissue echocardiographic early diastolic indices of both the right and left ventricle (LV) may assist in the detection of acute heart transplant (HT) rejection. METHODS: In all, 44 consecutive patients with HT (mean age 52.0 +/- 9.6 years, 39 men) were divided into group 1 with no rejection (histopathology grade < or = 2) and group 2 with acute (severe) rejection (grade > or = 3A). In group 2, echocardiographic examinations were performed before (A), during (B), and after (C) acute rejection. RESULTS: Although patients with HT in group 2B compared with group 1 had lower early diastolic velocities at medial/septal (E Med ) and tricuspid/lateral (E Tric ) annulus, as a result of substantial data overlapping this finding did not allow for the detection of patients with acute rejection. In group 2B, both onsets of E Med and E Tric were delayed and LV early diastolic mitral/lateral annulus velocities (E Mitr ) markedly preceded E Tric (E Tric -E Mitr 68 +/- 45 milliseconds for group 2B vs 7 +/- 43 milliseconds for group 1 and 14 +/- 40 milliseconds for group 2A; P < .01). Additionally, patients with HT in group 2B had pathologically positive late isovolumic relaxation myocardial velocity gradient of LV posterior wall compared with group 1 or group 2A (1.5 +/- 1.4 s -1 vs -0.3 +/- 2.0 s -1 or 0.3 +/- 1.8, respectively; P < .01). Late isovolumic relaxation myocardial velocity gradient greater than 0.1 s -1 and timing differences between onsets of: (1) mitral early diastolic velocity (E wave) and E Med greater than -35 milliseconds; and (2) E Tric -E Mitr greater than 15 milliseconds allowed for the distinction of patients with acute HT rejection (group 2B vs 1) with sensitivity and specificity greater than 0.80. CONCLUSIONS: For patients with HT and acute rejection abnormal Doppler tissue echocardiographic indices may be caused by both: (1) altered early diastolic untwist of the oblique LV fibers; and (2) the delay in early diastolic right ventricular relaxation. Late isovolumic relaxation myocardial velocity gradient and early diastolic timing intervals (mitral E wave-E Med and E Tric -E Mitr ) are promising new echocardiographic markers that can be used in the surveillance for acute rejection in patients with HT.

The role of growth hormone replacement in a growth hormone deficient patient with underlying cardiomyopathy and severe congestive heart failure.

It has been reported that growth hormone (GH) deficiency induced cardiomyopathy responds to growth hormone replacement therapy. We describe the case of a middle-aged male with cardiomyopathic heart failure and growth hormone deficiency of the adult secondary to surgical panhypopituitarism. We demonstrate clinical and hemodynamic improvement of cardiac function with growth hormone replacement therapy despite underlying structural heart disease.

Sirolimus in de novo heart transplant recipients reduces acute rejection and prevents coronary artery disease at 2 years: a randomized clinical trial.

BACKGROUND: Sirolimus reduces acute rejection in renal transplant recipients and prevents vasculopathy in nonhuman primates and in-stent restenosis in humans. Its effects on rejection and transplant vasculopathy in human heart transplant recipients are unknown. METHODS AND RESULTS: In a randomized, open-label study, sirolimus was compared with azathioprine in combination with cyclosporine and steroids administered from the time of cardiac transplantation. We report 6-month rejection rates (primary end point), 12-month safety and efficacy data, and 6- and 24-month graft vasculopathy data in 136 cardiac allograft recipients randomly assigned (2:1) to sirolimus (n=92) or azathioprine (n=44). At 6 months, the proportion of patients with grade 3a or greater acute rejection was 32.4% for sirolimus 3 mg/d (P=0.027), 32.8% for sirolimus 5 mg/d (P=0.013), and 56.8% for azathioprine. Patient survival at 12 months was comparable among groups. Intracoronary ultrasound at 6 weeks, 6 months, and 2 years demonstrated highly significant progression of transplant vasculopathy in azathioprine-treated patients. At 6 months, a highly significant absence of progression in intimal plus medial proliferation and significant protection against luminal encroachment was evident in sirolimus-treated patients, and these effects were sustained at 2 years. CONCLUSIONS: Sirolimus use from the time of transplantation approximately halved the number of patients experiencing acute rejection. The measurable development of transplant vasculopathy at 6 months and 2 years in patients receiving azathioprine was not observed in patients receiving sirolimus.

Eosinophilic heart: marked left ventricular wall thickening and myocardial dysfunction improving with corticosteroid therapy.

We report a case of a 34-year-old male with acute severe heart failure associated with marked concentric left ventricular wall thickening and biopsy evidence of eosinophilic myocardial infiltrate. This appears to be an unusual description of this degree of concentric myocardial thickening in eosinophilic myocarditis coupled with Doppler tissue echocardiography. Following high-dose corticosteroid treatment, wall thickness, systolic and diastolic left ventricular function normalized and the patient experienced a dramatic clinical improvement.

Elevated plasma levels of human urotensin-II immunoreactivity in congestive heart failure.

Human urotensin-II (hU-II) is the most potent endogenous cardiostimulant identified to date. We therefore determined whether hU-II has a possible pathological role by investigating its levels in patients with congestive heart failure (CHF). Blood samples were obtained from the aortic root, femoral artery, femoral vein, and pulmonary artery from CHF patients undergoing cardiac catheterization and the aortic root from patients undergoing investigative angiography for chest pain who were not in heart failure. Immunoreactive hU-II (hU-II-ir) levels were determined with radioimmunoassay. hU-II-ir was elevated in the aortic root of CHF patients (230.9 +/- 68.7 pg/ml, n = 21; P < 0.001) vs. patients with nonfailing hearts (22.7 +/- 6.1 pg/ml, n = 18). This increase was attributed to cardiopulmonary production of hU-II-ir because levels were lower in the pulmonary artery (38.2 +/- 6.1 pg/ml, n = 21; P < 0.001) than in the aortic root. hU-II-ir was elevated in the aortic root of CHF patients with nonischemic cardiomyopathy (142.1 +/- 51.5 pg/ml, n = 10; P < 0.05) vs. patients with nonfailing hearts without coronary artery disease (27.3 +/- 12.4 pg/ml, n = 7) and CHF patients with ischemic cardiomyopathy (311.6 +/- 120.4 pg/ml, n = 11; P < 0.001) vs. patients with nonfailing hearts and coronary artery disease (19.8 +/- 6.6 pg/ml, n = 11). hU-II-ir was significantly higher in the aortic root than in the pulmonary artery and femoral vein, with a nonsignificant trend for higher levels in the aortic root than in the femoral artery. The findings indicated that hU-II-ir is elevated in the aortic root of CHF patients and that hU-II-ir is cleared at least in part from the microcirculation.

Reconstitution of the latent T-lymphocyte response to Epstein-Barr virus is coincident with long-term recovery from posttransplant lymphoma after adoptive immunotherapy.

BACKGROUND: Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been used to treat EBV-induced posttransplant lymphoproliferative disease (PTLD) in solid-organ recipients. This study defines, in detail, the temporal relationship between adoptive transfer and the clinical response, EBV DNA load, and CTL response to EBV latent and lytic antigens in a patient with a subcutaneous PTLD presentation treated with adoptive transfer of autologous CTL. METHODS: A heart transplant patient developed multiple subcutaneous PTLD deposits and was treated with a total of six doses (20 x 106 CTL per dose) of cultured autologous polyclonal EBV-specific CTL by adoptive transfer. RESULTS: Complete regression occurred after the sixth CTL dose, and the patient has remained disease-free from 47 weeks to the present (136 weeks). Real-time polymerase chain reaction analysis showed a reduction in viral load after therapy. Enzyme-linked immunospot analysis using defined EBV CTL epitopes showed that the CTL precursor frequency (pCTL) toward a lytic antigen epitope was elevated early in the course of disease but tended to decrease to lower levels after long-term regression of PTLD. The most dramatic result was seen in relation to three latent CTL epitopes studied. Long-term regression of PTLD was characterized by high pCTL toward the latent antigens. CONCLUSIONS: Increased pCTL reactivity to latent EBV CTL epitopes is coincident with recovery from disease after adoptive transfer of autologous CTL. Furthermore, the results are compatible with the belief that activation of a sustained CTL response to EBV latent epitopes is protective and may be a characteristic of patients in long-term remission from PTLD.

Acitretin for prophylaxis of cutaneous malignancies after cardiac transplantation.

BACKGROUND: Heart transplant recipients have an increased risk of developing actinic keratoses and non-melanotic skin cancers when compared with the general population. Systemic retinoids have been shown to be beneficial in the treatment of such lesions in recipients of other organs, but as of yet the heart transplant model has rarely been studied. In this investigation we describe our experience with the use of acitretin in a group of heart transplant patients. METHODS: Five heart transplant recipients with multiple new skin cancer presentations were treated with acitretin at doses of either 10 or 25 mg/day. Inclusion criteria were based on progressive actinic keratoses, recurrent skin malignancies or continuous lesions despite treatment with appropriate topical therapies. Patients were excluded if they were women of child-bearing age, had severe hepatic or renal impairment or were taking contraindicated medications. RESULTS: Over the treatment period all patients showed a reduction in the number of new non-melanotic skin cancers excised and histologically confirmed. Three patients had a very large reduction and 2 patients had a more moderate reduction in the number of new presentations. All patients had an objective decrease in the number of actinic keratoses. All patients tolerated the drug well with only 1 patient transiently discontinuing the Acitretin due to side effects. No significant alterations in the biochemical tests or serum lipids were reported. CONCLUSIONS: Over the treatment period, low-dose acitretin proved a valuable addition in the long-term strategy of reduction and treatment of non-melanotic skin cancers in heart transplant recipients with multiple skin cancers and actinic keratoses.