Conservation genomics of an endangered montane amphibian reveals low population structure, low genomic diversity and selection pressure from disease.

Wildlife diseases are a major global threat to biodiversity. Boreal toads (Anaxyrus [Bufo] boreas) are a state-endangered species in the southern Rocky Mountains of Colorado and New Mexico, and a species of concern in Wyoming, largely due to lethal skin infections caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We performed conservation and landscape genomic analyses using single nucleotide polymorphisms from double-digest, restriction site-associated DNA sequencing in combination with the development of the first boreal toad (and first North American toad) reference genome to investigate population structure, genomic diversity, landscape connectivity and adaptive divergence. Genomic diversity (π = 0.00034-0.00040) and effective population sizes (Ne = 8.9-38.4) were low, likely due to post-Pleistocene founder effects and Bd-related population crashes over the last three decades. Population structure was also low, likely due to formerly high connectivity among a higher density of geographically proximate populations. Boreal toad gene flow was facilitated by low precipitation, cold minimum temperatures, less tree canopy, low heat load and less urbanization. We found >8X more putatively adaptive loci related to Bd intensity than to all other environmental factors combined, and evidence for genes under selection related to immune response, heart development and regulation and skin function. These data suggest boreal toads in habitats with Bd have experienced stronger selection pressure from disease than from other, broad-scale environmental variations. These findings can be used by managers to conserve and recover the species through actions including reintroduction and supplementation of populations that have declined due to Bd.

Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE).

Gap junction channels formed by the connexin43 protein are considered to play crucial roles in development and function because they allow the direct cell-to-cell exchange of molecules that mediate multiple signaling events. Previous results have shown that connexin43 channels are intricately gated by phosphorylation and that disruption of this regulation gives rise to severe heart malformations and defects of laterality in human, chick and frog. Here we report the identification of connexin43 gene mutations that represent a minor population of connexin43 alleles, which could be reliably detected by using denaturing gradient gel electrophoresis (DGGE) to visualize normal and mutant DNAs that were separately sequenced. In contrast, sequencing of total PCR products without DGGE-pre-selection failed to consistently identify these mutations. Forty-six controls and 20 heart transplant recipients were examined in this study. In the latter group, 14 children had hypoplastic left heart syndrome (HLHS) in which connexin43 gene defects were detected in eight. The remaining six transplant patients with HLHS and all controls showed no defects. All eight HLHS children with gene defects had the same four substitutions: two that were silent polymorphisms, and two that were missense, replacing arginine codons at positions 362 and 376 with codons for glutamines. All four of these substitutions are identical to the nucleotide sequence of the connexin43 pseudogene, suggesting the possibility of an illicit recombination. A breakpoint region was identified 5' to the mutation site in a 63bp domain that is 100% identical in the gene and pseudogene. Results from in vitro phosphorylation indicate that the absence of arginines 362 and 376 completely abolishes phosphorylation in the connexin43 channel regulation domain suggesting a possible mechanism for the pathologies associated with HLHS.

Concomitant traumatic coronary artery and tricuspid valve injury: a heterogeneous presentation.

Blunt thoracic trauma resulting in both tricuspid valve rupture and coronary artery injury is uncommon, encompasses a large spectrum of presentations and, therefore, can be difficult to diagnose. This report illustrates the heterogeneous presentation and clinical course of two patients with such a combination of cardiac injuries. The patient with associated right coronary artery dissection developed progressive right ventricular failure over a 12-year period before successful surgical repair, whereas another patient with left anterior descending coronary artery thrombosis required urgent operation for acute right ventricular dysfunction and hemodynamic decompensation.

Pediatric cardiac retransplantation: intermediate-term results.

BACKGROUND: Cardiac retransplantation (re-CTx) in children is a controversial therapy, yet it remains the best treatment option to recipients with failing grafts. Our objective was to determine the incidence of re-CTx in a large pediatric population of recipients and evaluate the outcome of such therapy. METHODS: Between November 1985 and November 1999, 347 children underwent cardiac transplantation at the Loma Linda University Medical Center. Of these, 32 children were listed for re-CTx. Ten patients died while waiting, and 22 recipients underwent re-CTx. Median age at re-CTx was 7.1 years (range, 52 days to 20.1 years). RESULTS: Indications for re-CTx were allograft vasculopathy (n = 16), primary graft failure (n = 5), and acute rejection (n = 1). Two patients with primary graft failure underwent retransplantation within 24 hours of the first transplantation procedure while on extracorporeal membrane oxygenation support. Median time interval to re-CTx for the others was 7.2 years (range, 32 days to 9.4 years). Operative mortality for all cardiac re-CTx procedures was 13.6%. Causes of hospital mortality were pulmonary hypertension with graft failure (n = 2) and multiorgan failure (n = 1). Median hospital stay after re-CTx was 14.1 days (range, 6 to 45 days). There was one late death from severe rejection. Actuarial survival at 3 years for re-CTx was 81.9% +/- 8.9% compared with 77.3% +/- 2.6% for primary cardiac transplantation recipients (p = 0.70). CONCLUSIONS: Elective re-CTx can be performed with acceptable mortality. Although the number of patients undergoing retransplantation in this report is small and their long-term outcome is unknown, the intermediate-term survival after re-CTx is similar to that of children undergoing primary cardiac transplantation.

Late rejection is a predictor of transplant coronary artery disease in children.

OBJECTIVE: The study objectives were to determine posttransplant coronary artery disease (TxCAD) incidence, predisposing factors and optimal timing for retransplantation (re-Tx) in pediatric heart transplantation (Tx) recipients. BACKGROUND: The TxCAD limits long-term survival following heart Tx, with re-Tx being the primary therapy. Information on risk factors and timing of listing for re-Tx is limited in children. METHODS: The records of children who survived >1 year post-Tx at Loma Linda University were reviewed. Nonimmune and immune risk factors were analyzed. RESULTS: TxCAD was documented in 24 of 210 children. Freedom from TxCAD was 92 +/- 2% and 75 +/- 5% at 5 and 10 years' post-Tx, respectively. The TxCAD diagnosis was established at autopsy in 10 asymptomatic patients who died suddenly within nine months following the most recent negative angiograms. The remaining 14 children had angiographic diagnoses of TxCAD and had symptoms and/or graft dysfunction (n = 10) or positive stress studies (n = 4). Three of 14 died within three months after the diagnosis was made. Eleven patients underwent re-Tx within seven months of diagnosis; nine survived. Univariate and multivariate analyses showed that only late rejection (>1 year posttransplant) frequency (p = 0.025) and severity (hemodynamically compromising) (p < 0.01) were independent predictors of TxCAD development. Freedom from TxCAD after severe late rejection was 78 +/- 8% one year postevent and 55 +/- 10% by two years. CONCLUSIONS: Late rejection is an independent predictor of TxCAD. Patients suffering severe late rejection develop angiographically apparent TxCAD rapidly and must be monitored aggressively. Both TxCAD mortality and morbidity occur early; therefore, we recommend immediate listing for re-Tx upon diagnosis.

Electroejaculated baboon (Papio anubis) sperm requires a higher dosage of pentoxifylline to enhance motility.

PURPOSE: Sperm collected by electroejaculation often show poor motility. The objective was to determine whether the addition of the phosphodiesterase inhibitor, pentoxifylline, would stimulate electroejaculated baboon sperm motility. METHODS: Electroejaculation was performed on several occasions on a male baboon and sperm collected after familiarization. Pentoxifylline was tested at the standard concentration (1 mg/ml) and at twice the concentration. Sperm parameters were evaluated using a sperm motility analyzer, as well as acrosome and DNA integrity techniques. RESULTS: Sperm exposed to 2 mg/ml pentoxifylline had higher total motility when compared with the control and 1 mg/ml treatment. Rapid progression and velocities were higher after pentoxifylline. The acridine orange DNA normality test showed that over 90% of collected sperm had intact unfragmented DNA. About half the sperm population had normal morphology and intact acrosomes. A low percentage had cytoplasmic droplets. CONCLUSIONS: Sperm collected by rectal probe electroejaculation required a higher concentration (2 mg/ml) of pentoxifylline for enhanced total motility, rapid progression, and higher velocity. This suggested differences in membrane properties or phosphodisterase activity in electrojeaculated sperm. The electroejaculation procedure did not denature sperm DNA at the acridine orange assay level nor were the acrosomes disrupted. The present study also documented unique information on baboon kinematic parameters.

Outcome of infants with hypoplastic left heart syndrome who undergo atrial septostomy before heart transplantation.

This study evaluated the procedural and long-term outcome of infants who underwent atrial septostomy while awaiting transplant. The results suggest that septostomy improved outcome in these patients although infants needing a transseptal perforation were at higher risk.

Right ventricular outflow tract reconstruction using a Goretex membrane monocusp valve in infant animals.

We examined the feasibility of using a polytetrafluoroethylene membrane (goretex) valve and transannular patch (TAP) for right ventricular outflow tract (RVOT) reconstruction in growing animal models. Eleven infant goats (Group A) and 12 infant sheep (Group B) underwent RVOT reconstruction under cardiopulmonary bypass. In Group A, a monocusp valve was constructed of goretex, and the RVOT was roofed over utilizing a TAP of bovine pericardium. In Group B, both a monocusp valve and a TAP were constructed of goretex. Animals were sacrificed at 6 or 12 postoperative months. Two goats in Group A died at 9 days and 4 months postoperatively due to RVOT obstruction caused by thrombus formation. Seven goats in Group A and 11 in Group B showed no pressure gradient across the valve. All animals in Group B revealed mild to moderate valvular insufficiency, but no ventricular deterioration. In contrast to the bovine pericardium, which showed a prominent fibroinflammatory reaction with calcified areas causing TAP shrinkage and RVOT stenosis, there was minimal calcification and inflammatory reaction directed against the goretex valve and TAP. We conclude that goretex can be used as a material from which to create both a monocusp valve and TAP for long-term RVOT reconstruction.

Emergency implantation of a left ventricular assist device in adolescents with biventricular failure.

Adolescents with congestive cardiomyopathy who present with intractable arrhythmia or progressive ventricular failure have a very poor prognosis and often die awaiting cardiac transplantation (CTx). We present our recent experience with a pneumatically powered left ventricular assist device (LVAD) implanted emergently to salvage adolescents with severe biventricular failure. Four patients, aged 15-17 years, body surface areas of 1.5-1.7 m2, with dilated cardiomyopathy (LV diastolic dimension, 7.1-8.3 cm); two presented with cardiovascular collapse, one with refractory ventricular tachycardia, and one with cardiac arrest. Hemodynamic and biochemical data before and 1 week after LVAD placement are expressed as mean and range values. None of the patients required right ventricular assist, and all patients achieved functional recovery while on LVAD support (8-71 days). Currently, all four patients are alive (11-22 months) after successful CTx. We conclude that emergency implantation of an LVAD in adolescents with biventricular heart failure can be life saving. As has been shown in the adult population, such a ventricular assist system restores normal circulatory hemodynamics, reverses multi-organ dysfunction, and provides a "safe" bridge to transplantation.

Repair of traumatic aortic rupture: a 25-year experience.

BACKGROUND: Surgical management of traumatic aortic rupture (TAR) is controversial, specifically whether distal aortic perfusion modifies the outcome. HYPOTHESIS: The outcome of patients who undergo repair of TAR is not dependent on the technique of repair. DESIGN: Retrospective review. SETTING: Tertiary care teaching hospital, level I regional trauma center. PATIENTS: One hundred fifteen victims (aged 5-81 years) of blunt chest trauma with aortic tear, presenting between January 1, 1974, and June 30, 1999. METHODS: Medical records were reviewed for prehospital and emergency department data, operative findings, and outcome. Statistical comparison was made using a paired 2-tailed t test. INTERVENTION: Surgical repair of TAR with (group 1) or without (group 2) distal aortic perfusion. RESULTS: Thirty-two patients in group 1 had TAR repair using active bypass (n = 18) or Gott shunt (n = 14). The clamp-and-sew technique was used in 83 patients (group 2). Primary repair was possible in 14 patients (44%) in group 1 and 69 patients (83%) in group 2. The average aortic cross-clamp time was 48 minutes for group 1 (range, 25-113 minutes) and 20 minutes for group 2 (range, 5-40 minutes) (P<.03). There was no significant difference in hospital mortality (6 [18.7%] of 32 vs 15 [18.1%] of 83) or the incidence of paraplegia (2 [6%] of 32 vs 5 [6%] of 83) between groups 1 and 2. During the last 15 years, 78 patients (73 in group 2) had repair of TAR with an operative mortality rate of 19.2%. CONCLUSIONS: Acute TAR remains a highly lethal injury with no change in prognosis during the last 2(1/2) decades. Repair of TAR using simple aortic cross-clamping alone is feasible in the majority of patients without increased mortality or spinal cord injury.

The medium-term findings in coronary arteries by intravascular ultrasound in infants and children after heart transplantation.

OBJECTIVES: The study purposes were to determine 1) whether intravascular ultrasound (IVUS) was more sensitive than angiography for the detection of post-transplant coronary artery disease (PTCAD) in pediatric patients; and 2) whether those transplanted as neonates reacted differently than older patients. BACKGROUND: Experience with IVUS for the diagnosis of PTCAD in children is limited. METHODS: Patients were divided into two groups: those transplanted as neonates (early group) and those transplanted in infancy or childhood (late group). Morphometric analysis was performed, including maximal intimal thickness (MIT) and intimal index (II). Stanford classification was used to grade lesion severity. Acute rejection and cytomegalovirus (CMV) status were correlated with MIT and II. RESULTS: Thirty children were studied (early group, n = 13; late group, n = 17). All segments studied were angiographically normal. Mean MIT and mean II were significantly greater in the late group (0.26 +/- 0.14 vs. 0.13 +/- 0.04 mm, p < 0.001 and 0.11 +/- 0.07 vs. 0.07 +/- 0.03 mm, p = 0.04, respectively). There was a significant correlation between MIT and II in those who had acute rejection in the late group. Patients in the late group who were CMV-positive had a significantly higher MIT compared with those in the late group with negative serology (p = 0.04). CONCLUSIONS: Intravascular ultrasound was more sensitive than angiography in detecting PTCAD after pediatric heart transplantation. There is a possible role for acute rejection and CMV in the development of PTCAD.

Successful myocardial volume reduction in a 9-month-old infant.

Left ventricular reduction has shown promise as a treatment for end-stage dilated cardiomyopathy, with restoration of the physiologic ratio between myocardial mass and left ventricular diameter. We present a case of successful partial left ventriculectomy utilizing both lateral and septal wall excision as treatment of dilated cardiomyopathy in a 9-month-old patient.

Ultra fast track in elective congenital cardiac surgery.

BACKGROUND: Changes in healthcare delivery have affected the practice of congenital cardiac surgery. We recently developed a strategy of limited sternotomy, early extubation, and very early discharge, and reviewed the perioperative course of 198 pediatric patients undergoing elective cardiovascular surgical procedures, to assess the efficacy and safety of this approach. METHODS: One hundred ninety-eight patients aged 0 to 18 years (median 3.2 years) underwent 201 elective cardiovascular surgical procedures over a 1-year period. All patients were admitted on the day of surgery. Patients were divided into six diagnostic groups: group 1, complex left-to-right shunts (n = 14, 7.0%); group 2, simple left-to-right shunts (n = 83, 41.3%); group 3, right-to-left shunts with pulmonary obstruction (n = 33, 16.4%); group 4, isolated, nonvalvular obstructive lesions (n = 30, 14.9%); group 5, isolated valvular anomalies (n = 20, 10.0%); and group 6, miscellaneous (n = 21, 10.4%). RESULTS: After 201 procedures, 175 patients (87.1%) were extubated in the operating room and 188 (93.6%) within 4 hours from operation. Four patients (2.0%) were extubated more than 24 hours from completion of the procedure, and 2 (1.0%) died while on respiratory support (never weaned). Five patients (2.6%) failed early extubation (<4 hours). Early discharge was achieved for the vast majority of patients. Overall median length of stay (LOS, including day of surgery as day 1) was 2.0 days, with a median LOS of 3.0 days for those patients requiring circulatory arrest duration exceeding 20 minutes. Of 195 patients, 43 (24.6%), 121 (74.0%), and 159 (81.5%) were discharged, respectively, at <24, <48, <72 hours from admission. Longest and shortest mean postoperative LOS were in group 6 (9.9+/-14.5 days) and group 2 (1.6 = 0.7 days), respectively. Six patients (2.9%) died, and 11 (5.5%) suffered in-hospital complications. Thirty patients (15.4%) were either treated as outpatients (n = 11, 5.7%) or readmitted (n = 19, 9.7%) within 30 days from the time of surgery. Only 8 of 195 patients (4.1%) were readmitted with true surgical complications requiring invasive therapeutic procedures. CONCLUSIONS: Selected patients with a broad spectrum of congenital heart disease may enjoy same-day admission, limited sternotomy, immediate extubation, and very early discharge with excellent outcomes and acceptable morbidity.