Optimizing pediatric liver transplantation: Evaluating the impact of donor age and graft type on patient survival outcome.

BACKGROUND: We examined the combined effects of donor age and graft type on pediatric liver transplantation outcomes with an aim to offer insights into the strategic utilization of these donor and graft options. METHODS: A retrospective analysis was conducted using a national database on 0-2-year-old (N = 2714) and 3-17-year-old (N = 2263) pediatric recipients. These recipients were categorized based on donor age (≥40 vs <40 years) and graft type. Survival outcomes were analyzed using the Kaplan-Meier and Cox proportional hazards models, followed by an intention-to-treat (ITT) analysis to examine overall patient survival. RESULTS: Living and younger donors generally resulted in better outcomes compared to deceased and older donors, respectively. This difference was more significant among younger recipients (0-2 years compared to 3-17 years). Despite this finding, ITT survival analysis showed that donor age and graft type did not impact survival with the exception of 0-2-year-old recipients who had an improved survival with a younger living donor graft. CONCLUSIONS: Timely transplantation has the largest impact on survival in pediatric recipients. Improving waitlist mortality requires uniform surgical expertise at many transplant centers to provide technical variant graft (TVG) options and shed the conservative mindset of seeking only the "best" graft for pediatric recipients.

Liver transplant versus liver resection in patients with multifocal hepatocellular carcinoma.

BACKGROUND: The optimal surgical option in patients with multifocal hepatocellular carcinoma (MHCC) is an area of active research. The preference varies based on geographic variations and institutional policies. We sought to determine long-term outcomes in patients with MHCC based on surgical treatment-liver transplant (LT) vs resection (LR). METHODS: We performed a retrospective analysis of the National Cancer Database (2004-2015) and identified patients with MHCC within Milan criteria. Patients with α-fetoprotein ≥ 1000 ng/mL and those who underwent ablation were excluded. The primary outcome measure was long-term survival in patients undergoing LT vs LR. The secondary aim of our study was to determine clinicodemographic factors associated with the receipt of LT and LR. RESULTS: A total of 1546 patients were included, of whom 1211 received LT and 335 underwent LR. Patients who were non-Hispanic White (70.8% vs 54.9%; P < .01), privately insured (53.7% vs 36.7%; P < .01), and treated at academic centers (85.4% vs 71.6%; P < .01) were more likely to receive an LT. Multivariable Cox analysis revealed LT was associated with improved survival compared with LR (hazard ratio, 0.34; 95% CI, 0.28-0.42). CONCLUSION: We described clinical and sociodemographic differences in LT and LR patients and found LT to be associated with a decreased mortality risk compared with LR. The study's findings should be interpreted in the context of several limitations, including the selection of MHCC criteria within Milan criteria.

Liver resection is superior to tumor ablation in patients with multifocal hepatocellular carcinoma.

INTRODUCTION: The management of T2 multifocal hepatocellular carcinoma (MHCC) is controversial, and the comparative impact of liver resection (LR) versus tumor ablation (TA) on survival continues to be debated. The aim of our study was to examine short- and long-term survival for LR and TA in a nationally representative cohort. We hypothesized that patients who underwent LR would have improved survival. METHODS: We utilized the National Cancer Database (2004-2015) to identify patients diagnosed with non-metastatic T2 MHCC. Kaplan-Meier survival curves were generated to compare 10-year overall survival (OS) between LR and TA patients. Kaplan-Meier analysis with stratification was also performed based on lymphovascular invasion, resection margin status, and Charlson-Deyo score. Cox proportional hazard models were used in multivariable analyses. RESULTS: A total of 1225 patients met the inclusion criteria. 991 patients received LR, and 234 received TA. The majority of patients were male, White, and older than ≥60 years old. Clinicodemographic characteristics were generally similar between LR and TA patients. Among patients who underwent LR, 84% had negative margins, and 17% had lymphovascular invasion. Mortality at 30 days was significantly higher among LR patients compared to TA patients (5.4% vs 0.0%, p < 0.001), with those having a Charlson-Deyo score ≥2 facing the highest risk at 7.3%. Nevertheless, 10-year OS for the LR cohort was 27.5% (95% confidence interval [CI]: 24.4%-30.8%) versus 14.7% (95% CI: 9.8%-20.7%, p < 0.001) for TA patients. In stratified analysis, survival benefit was statistically significant only among those with negative resection margin, no lymphovascular invasion, and Charlson-Deyo score ≤1. In multivariable Cox analysis, LR was independently associated with improved survival compared to TA (hazard ratio: 0.80; 95% CI = 0.67-0.95). CONCLUSION: LR poses a higher long-term survival benefit than TA. Prospective studies are warranted to confirm these findings. Although our study patients are a highly selected group of multifocal T2 patients, it gives us a good insight into the fact that LR provides better outcomes if a transplant option is unavailable.

Small-for-size syndrome in a 9.7 kg pediatric recipient with a whole liver graft.

BACKGROUND: Small-for-size syndrome (SFSS) in pediatric liver transplant recipients, particularly those weighing less than 10 kg, is rare. This report describes a case of a 15-month-old whole liver transplant recipient who suffered SFSS, and systematic literature review was performed to identify outcomes of such cases and potential risk factors for SFSS. CASE PRESENTATION: A 15-month-old toddler with a history of biliary atresia underwent a deceased donor whole liver transplant. The graft weighed 160 g, resulting in a graft-to-recipient weight ratio (GRWR) of 1.6%. The post-operative course was complicated by SFSS, characterized by massive ascites causing hemodynamic instability and compromised hepatic artery flow. Pharmacological intervention with octreotide was initiated, and the patient eventually recovered. CONCLUSION: In small pediatric recipients, especially those weighing less than 10 kg, the native liver body weight ratio (LBWR) is significantly higher. When selecting an appropriately sized graft for these recipients, this higher ratio should be taken into consideration. The literature review suggests that a GRWR of less than 2% is associated with a higher incidence of small-for-size syndrome in small pediatric recipients weighing less than 10 kg.

Association Between Preoperative Angioembolization and Bleeding Complications in Patients With Benign Liver Tumors: Analysis of a National Database.

INTRODUCTION: The role of angioembolization (AE) in patients with benign liver diseases is an area of active research. This study aims to assess any difference in liver resection outcomes in patients with benign tumors dependent on utilization of preoperative AE. METHODS: A retrospective cohort study of patients undergoing elective liver resections for benign liver tumors was performed using the National Surgical Quality Improvement Program database (2014-2019). Only tumors of 5 cm in size or more were included in the analysis. We categorized the patients based on preoperative AE (AE + versus AE -). The primary outcome measured included bleeding complications within 72 h. The secondary outcomes were to determine predictors of bleeding. RESULTS: After propensity score matching, there were 103 patients in both groups. There was no difference in intraoperative or postoperative blood transfusions within 72 h of surgery (14.6% versus 12.6%; P = 0.68), reoperation (1.9% versus 1.9%; P = 1), or mortality (1.0% versus 0.0%; P = 1) between the two groups. Multivariate regression analysis revealed an open surgical approach (odds ratio [OR]: 4.59 confidence interval [CI]: 2.94-7.16), use of Pringle maneuver (OR: 1.7, CI: 1.26-2.310), preoperative anemia (OR: 2.79, CI: 2.05-3.80), and preoperative hypoalbuminemia (OR: 1.53 [1.14-2.05]) were associated with the need for intraoperative or postoperative blood transfusions within 72 h of surgery. CONCLUSIONS: Preoperative AE was not associated with reducing intraoperative or postoperative bleeding complications or blood transfusions within 72 h after surgery.

Outcomes in elderly patients undergoing hepatic resection compared to liver transplant for hepatocellular carcinoma.

BACKGROUND: Hepatic resection (HR) is an excellent option for patients with hepatocellular carcinoma (HCC). For patients meeting the Milan criteria, a liver transplant (LT) is also a viable option for patients with HCC, especially those with end-stage liver disease. With increasing rates of LTs amongst the elderly, we sought to determine long-term outcomes in patients who underwent HR compared to LTs in this patient population. METHODS: We queried the national cancer database for elderly patients (≥70 years) diagnosed with HCC between 2004 and 2020. The primary outcome was overall survival (OS) computed using the Kaplan-Meier method and Cox proportional hazard regression. One-to-one propensity score matching was conducted on the basis of clinicodemographic features to account for baseline differences between patients undergoing each procedure. RESULTS: Of the 5090 patients included, 4674 (91.8%) and 416 (8.2%) patients underwent HR and LT, respectively. Compared with HR patients, patients receiving LT had better OS (p < 0.001) and greater median survival time (65.6 months HR vs. 97.9 months LT, p < 0.001). On multivariable analysis, a LT was independently associated with improved survival (adjusted hazard ratio: 0.61, 95% confidence interval: 0.50-0.76, p < 0.001). CONCLUSIONS: LT is associated with improved survival for well-selected elderly patients with HCC. Age alone should not be used as the sole parameter for the candidacy of LT in elderly patients.

Living donor liver paired exchange between pediatric and adult recipients due to donor graft size mismatch.

Living donor liver transplantation is an effective means to decrease organ shortage. However, many potential living donors are currently being denied due to ABO incompatibility or inadequate donor liver volume. Liver paired exchange (LPE) provides a practical solution to overcome these obstacles, and yet the first case of LPE in the United States was only recently reported in 2020. Here, we report world's first case of LPE involving pediatric and adult recipients to avoid surgical complexity of the pediatric recipient and to increase the graft-to-recipient weight ratio of the adult recipient between 2 ABO compatible pairs. As living donor liver transplantation becomes more widely adopted, the need for pair exchange to improve surgical safety and postoperative outcomes between 2 ABO compatible pairs is likely to increase.

Critical Appraisal of the Quality of Publications in Hepatobiliary and Pancreatic Surgery Research Using the American College of Surgeons NSQIP Database.

BACKGROUND: The use of the American College of Surgeons (ACS) NSQIP has increased in hepatobiliary and pancreatic surgery (HPB) research as it provides access to high-quality surgical outcome data on a national scale. Using the ACS NSQIP database, this study examined the methodologic reporting of HPB publications. STUDY DESIGN: Web of Science core collection (all editions) was queried for all HPB studies using the ACS NSQIP database published between 2004 and 2022. In addition, a critical appraisal was performed using the JAMA Surgery checklist, Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement, and Reporting of Studies Conducted Using Observational Routinely-Collected Health Data (RECORD) statement. RESULTS: In total, 276 studies were included in the analysis. The median scores for the JAMA Surgery checklist, RECORD statement, and STROBE statement were 4 of 7 (interquartile range [IQR] 3 to 5), 3 of 10 (IQR 2 to 4), and 15 of 21 (IQR 13 to 17), respectively. The criteria with the highest rates of nonadherence were discussing competing risks, clear definitions of inclusion and exclusion criteria, unadjusted and adjusted outcomes, provision of supplementary data, and performing subgroup analyses. Additionally, when examining checklist fulfillment of hepatobiliary studies and pancreatic studies separately, pancreatic studies demonstrated significantly greater fulfillment of the STROBE statement checklist items. CONCLUSIONS: Satisfactory reporting of methodology is present among HPB studies utilizing the ACS NSQIP database, with multiple opportunities for improvement. Areas for improved adherence include discussing competing risks, providing supplementary information, and performing appropriate subgroup analysis. Given the increasing role of large-scale databases in surgical research, enhanced commitment to reporting guidelines may advance HPB research and ensure quality reporting.

Perceptions and Early Outcomes of the Acuity Circles Allocation Policy Among Liver Transplant Centers in the United States.

Recently, a new liver allocation policy called the acuity circles (AC) framework was implemented to decrease geographic disparities in transplant metrics across donor service areas. Early analyses have examined the changes in outcomes because of the AC policy. However, perceptions among transplant surgeons and staff regarding the new policy remain unknown. Methods: A 28-item survey was sent to division chiefs and surgical directors of liver transplantation across the United States. Questions assessed the respondents' perceptions regarding center-level metrics and staff satisfaction. We used Organ Procurement and Transplantation Network data to study differences in allocation between the pre-AC implementation period (2019) and the post-AC implementation period (2020-2021). Results: A total of 40 participants completed this ongoing survey study. Most responses were from region 8 (13%), region 10 (15%), and region 11 (13%). Sixty-three percent of respondents stated that the wait time for a suitable offer for recipients with model of end-stage liver disease score <30 has decreased, whereas 50% stated that wait time for a suitable offer for recipients with model of end-stage liver disease score >30 has increased. However, most respondents (75%) felt that the average cost per transplant had increased and that the rate of surgical complications and 1-y graft survival had remained the same. In most states, an observable decrease in in-state liver transplantations occurred each year between 2019 and 2021. In addition, most allocation regions reported an increase in donations after circulatory deaths between 2019 and 2021. Conclusions: Perceptions of the new AC policy among liver transplant surgeons in the United States remain mixed, highlighting the potential strengths and concerns regarding its future impact. Further studies should assess the effects of the AC policy on clinical outcomes and liver transplantation access.

Does Chronic Use of Steroids Affect Outcomes After Liver Resection? Analysis of a National Database.

INTRODUCTION: With the increasing age of patients, more patients on chronic preoperative steroids are undergoing liver resections. Our study aimed to assess the relationship between preoperative steroids and outcomes. METHODS: We performed a retrospective review of the 2014-2019 NSQIP database of all patients undergoing liver resections. Propensity score matching was utilized to match the two groups (chronic steroids vs. no steroids) based on demographics, preoperative laboratory data, and operative findings. The primary outcome measure was mortality. RESULTS: There were 712 patients in the chronic steroid group and 21,751 in the no steroid group. After propensity score matching; there were 420 patients in both groups. Post-match analysis again demonstrated that patients on chronic steroids were at higher risk of cardiac arrest than those not on steroids (OR 2.01, 95% CI 1.02-2.45, p = 0.04). In addition, rates of organ space wound infection (OR 2.66, CI 1.33-5.38, p = 0.03), superficial wound infection (OR 2.79, CI 1.08-5.41, p = 0.035), renal insufficiency (OR 1.25, CI 1.03-1.62, p = 0.02), postoperative sepsis (OR 1.28, CI 1.08-1.82, p = 0.04), DVT (OR 1.7, CI 1.01-2.82, p = 0.04), and bile leakage (OR 1.75, CI 1.24-3.36, p = 0.04) were also increased in patients on steroids. However, the matched cohorts were similar in postoperative mortality rates (OR 0.11, CI 0.6-1.17, p = 0.72). CONCLUSION: The study found higher morbidity rates in patients undergoing liver resections on chronic preoperative steroids but no differences in mortality.

Liver Transplantation as a Curative Approach for Patients With Nonresectable Colorectal Liver Metastases.

OBJECTIVES: Liver transplant is emerging as a potential treatment option for patients with isolated colorectal liver metastasis. In this review article, we analyzed the published literature on liver transplant outcomes in such patients. MATERIALS AND METHODS: Four prospective studies documenting the clinical outcomes in patients with colorectal liver metastasis who underwent liver transplant were analyzed to study the feasibility of liver transplant in such patients. RESULTS: The SECA-II trial demonstrated the highest overall survival of 100%, 83%, and 83% at 1, 3, and 5 years, respectively, and disease-free survival of 53%, 44%, and 35%, respectively, with a narrow inclusion criterion. Conversely, extended criteria for selection and donors in arm D of the same trial resulted in median overall survival and disease-free survival of 18 and 4 months, respectively. CONCLUSIONS: Liver transplant provided more prolonged overall survival compared with other therapeutic modalities. Patients with isolated colorectal liver metastasis of less aggressive biology, good performance status, at least 6 weeks of chemotherapy, low clinical risk scores, and negative nodal disease should be considered for patient selection. Moreover, exclusion criteria consisting of patients with the right-sided primary tumor, less than 3 years to liver transplant after diagnosis, and elevation of carbohydrate antigen (CA19-9) in the presence of BRAF mutation should be explored.

Living Donor Versus Deceased Donor Pediatric Liver Transplantation: A Systematic Review and Meta-analysis.

Reduced-size deceased donors and living donor liver transplantation (LDLT) can address the organ shortage for pediatric liver transplant candidates, but concerns regarding technical challenges and the risk of complications using these grafts have been raised. The aim of this study was to compare outcomes for pediatric LDLT and deceased donor liver transplantation (DDLT) via systematic review. METHODS: A systematic literature search was performed to identify studies reporting outcomes of pediatric (<18 y) LDLT and DDLT published between 2005 and 2019. A meta-analysis was conducted to examine peri- and postoperative outcomes using fixed- and random-effects models. RESULTS: Overall, 2518 abstracts were screened, and 10 studies met criteria for inclusion. In total, 1622 LDLT and 6326 DDLT pediatric patients from 4 continents were examined. LDLT resulted in superior patient survival when compared with DDLT at 1, 3, and 5 y post-LT (1-y hazard ratio: 0.58, 95% confidence interval [CI] 0.47-0.73, P < 0.0001). Similarly, LDLT resulted in superior graft survival at all time points post-LT when compared with DDLT (1-y hazard ratio: 0.56 [95% CI 0.46-0.68], P < 0.0001]. The OR for vascular complications was 0.73 (95% CI 0.39-1.39) and 1.31 (95% CI 0.92-1.86) for biliary complications in LDLT compared with DDLT, whereas LDLT was associated with lower rates of rejection (OR: 0.66 [95% CI 0.45-0.96], P = 0.03). CONCLUSIONS: This meta-analysis demonstrates that LDLT may offer many advantages when compared with DDLT in children and suggests that LDLT should continue to be expanded to optimize outcomes for pediatric LT candidates.

Effects of Coronavirus Disease 2019 on Solid Organ Transplantation.

BACKGROUND: As the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a viral pandemic, data on the clinical characteristics and outcomes of patients with SARS-CoV-2 infection undergoing solid organ transplant are emerging. The objective of this systematic review was to assess currently published literature relating to the management, clinical course, and outcome of SARS-CoV-2 infection in liver, kidney, and heart solid organ transplant recipients. METHODS: We conducted a systematic review to assess currently published literature relating to the management, clinical course, and outcome of SARS-CoV-2 infection in liver, kidney, and heart solid organ transplant recipients. Articles published through June 2020 were searched in the MEDLINE, ClinicalTrials.gov, and PubMed databases. We identified 49 eligible studies comprising a total of 403 solid organ transplant recipients. RESULTS: Older age, male sex, and preexisting comorbidities, including hypertension and/or diabetes, were the most common prevailing characteristics among the solid organ transplant recipients. Clinical presentation ranged from mild to severe disease, including multiorgan failure and death. We found an overall mortality rate of 21%. CONCLUSION: Our analysis suggests no increase in overall mortality or worse outcome in solid organ transplant recipients receiving immunosuppressive therapy compared with mortality in the general surgical population with SARS-CoV-2. Our findings suggest that transplant surgery and its immunosuppressive effects should not be a deterrent to proper surgical care for patients in the SARS-CoV-2 era.

Should living donor liver transplant selection be subject to the same restrictions as deceased donor transplant?

PURPOSE OF REVIEW: In the United States, most of the liver allografts come from deceased donors, and our current liver recipient selection process is heavily centered on the ethical principle of utility to maximize the net benefit to the liver recipient community as a group rather than individuals due to the organ scarcity. Although living donor liver transplantation contributes less than 5% of total liver transplant in the United States, these living donor recipients are being subjected to the same selection process designed to benefit the group as a whole rather than the individuals. We would like to examine if these recipients who have living donors should be subjected to the same selection process. RECENT FINDINGS: There are several disease processes where liver transplantation is the only curative option, and recent studies have shown clear survival benefits with liver transplantation. SUMMARY: For those who have living donors, different selection criteria based on their specific disease, not based on the principle of utilization should be used to evaluate their candidacy.

Clostridium paraputrificum septicemia and liver abscess.

We report the first case of a healthy 23-year-old female who underwent an interventional radiology-guided embolization of a hepatic adenoma, which resulted in a gas forming hepatic liver abscess and septicemia by Clostridium paraputrificum. A retrospective review of Clostridial liver abscesses was performed using a PubMed literature search, and we found 57 clostridial hepatic abscess cases. The two most commonly reported clostridial species are C. perfringens and C. septicum (64.9% and 17.5% respectively). C. perfringens cases carried a mortality of 67.6% with median survival of 11 h, and 70.2% of the C. perfringens cases experienced hemolysis. All C. septicum cases were found to have underlying liver malignancy at the time of the presentation with a mortality of only 30%. The remaining cases were caused by various Clostridium species, and this cohort's clinical course was significantly milder when compared to the above C. perfringens and C. septicum cohorts.

The use of vascular homografts in pediatric small bowel transplantation: Single-center experience over a decade.

Intestinal transplantation in children has evolved with more isolated small intestine transplants being performed compared to combined liver-intestine transplants. Consequently, surgical techniques have changed, frequently requiring the use of vascular homografts of small caliber to revascularize the isolated small intestine, the impact of which on outcomes is unknown. Among 106 pediatric intestine and multivisceral transplants performed at our center since 2003, 33 recipients of an isolated small intestine graft were included in this study. Outcome parameters were thrombotic complications, graft, and patient survival. A total of 29 of 33 (87.9%) patients required arterial and/or venous homografts from the same donor, mainly iliac or carotid artery and iliac or innominate vein, respectively (donor's median age 1.1 years [2 months to 23 years], median weight 10 kg [14.7-48.5]). Post-transplant, there were three acute arterial homograft thromboses and one venous thrombosis resulting in two peri-operative graft salvages and two graft losses. Three of four thromboses occurred in patients with primary hypercoagulable state, including the two graft losses. Overall, at a median of 4.1 years (1-10.2) from transplant, 29 of 33 (88%) patients are alive with 26 of 33 (79%) functioning grafts. The procurement of intact, size-matched donor vessels and the management of effective post-transplant anticoagulation are critical.

Multilocus variable-number tandem-repeat analysis for subtyping Salmonella enterica serovar Gallinarum.

Salmonella enterica serovar Gallinarum causes a severe systemic disease, fowl typhoid, primarily in chickens and turkeys, and it remains a disease of worldwide significance. Multilocus variable-number tandem-repeat analysis (MLVA) has proved to be very useful for subtyping other Salmonella serovars. We describe the development of a simple MLVA assay for S. enterica serovar Gallinarum that is comparable with pulsed-field gel electrophoresis (PFGE) in resolution. The genome sequence of S. enterica serovar Gallinarum strain 287/91 was analysed for potential variable-number tandem repeats (VNTRs) and then polymerase chain reaction assays were developed to assess the variability of the loci. Four VNTR markers were selected and used in a multiplex fragment analysis assay. The stability of the VNTR markers was assessed by conducting in vitro passage experiments with two strains (95 clones per strain) over a 30-day period. A MLVA of 68 strains of S. enterica serovar Gallinarum based on the four VNTR loci distinguished 26 allelic profiles. The MLVA assay showed a Simpson's diversity index of 0.918, whereas PFGE analysis produced 23 patterns and had a diversity index of 0.874. Most importantly, the MLVA further discriminated strains having the same PFGE pattern. The MLVA assay is a highly discriminatory genotyping method for S. enterica serovar Gallinarum. Therefore, MLVA can be a useful addition to routine PFGE analysis for molecular epidemiological investigation of fowl typhoid.

Characterization of antimicrobial resistance of recent Salmonella enterica serovar Gallinarum isolates from chickens in South Korea.

Salmonella enterica serovar Gallinarum isolates (n=105) from chickens in South Korea between 2002 and 2007 were tested for antimicrobial susceptibility by determining minimum inhibitory concentrations of 16 antimicrobials, and their predominant resistance profiles were genetically characterized. Most isolates (99/105; 94.3%) were resistant to nalidixic acid and resistant/intermediately resistant to fluoroquinolones, and 63.8% (67/105) of the isolates were resistant to three or more antimicrobials. Forty-two quinolone-resistant isolates, of which the quinolone resistance-determining regions of the gyrA genes were sequenced, contained a substitution of a Ser to a Phe or Tyr at position 83 (71.4%), or a substitution of an Asp to an Asn, Gly, or Tyr at position 87 (28.6%). Fifty-seven sulphamethoxazole-resistant isolates were tested for the presence of class 1 integrons by polymerase chain reaction, and their resistance gene cassettes were analysed by sequencing. Three different class 1 integrons containing the resistance-gene insert aadA (52.6%; n=30), aadB (12.3%; n=7), or aadB-aadA (12.3%; n=7) were identified. Most isolates harbouring the integron containing aadB-aadA displayed resistance to all three aminoglycosides tested and also showed increased resistance to fluoroquinolones. These findings suggest that fluoroquinolone resistance may be epidemiologically linked to multiple aminoglycoside resistance.

In vitro analysis of the relationship between endonuclease and maturase activities in the bi-functional group I intron-encoded protein, I-AniI.

The AnCOB group I intron from Aspergillus nidulans encodes a homing DNA endonuclease called I-AniI which also functions as a maturase, assisting in AnCOB intron RNA splicing. In this investigation we biochemically characterized the endonuclease activity of I-AniI in vitro and utilized competition assays to probe the relationship between the RNA- and DNA-binding sites. Despite functioning as an RNA maturase, I-AniI still retains several characteristic properties of homing endonucleases including relaxed substrate specificity, DNA cleavage product retention and instability in the reaction buffer, which suggest that the protein has not undergone dramatic structural adaptations to function as an RNA-binding protein. Nitrocellulose filter binding and kinetic burst assays showed that both nucleic acids bind I-AniI with the same 1 : 1 stoichiometry. Furthermore, in vitro competition activity assays revealed that the RNA substrate, when prebound to I-AniI, stoichiometrically inhibits DNA cleavage activity, yet in reciprocal experiments, saturating amounts of prebound DNA substrate fails to inhibit RNA splicing activity. The data suggest therefore that both nucleic acids do not bind the same single binding site, rather that I-AniI appears to contain two binding sites.