Genotoxic effects of the major alkylation damage N7-methylguanine and methyl formamidopyrimidine.

Various alkylating agents are known to preferentially modify guanine in DNA, resulting in the formation of N7-alkylguanine (N7-alkylG) and the imidazole ring opened alkyl-formamidopyrimidine (alkyl-FapyG) lesions. Evaluating the mutagenic effects of N7-alkylG has been challenging due to the instability of the positively charged N7-alkylG. To address this issue, we developed a 2'-fluorine-mediated transition-state destabilization approach, which stabilizes N7-alkylG and prevents spontaneous depurination. We also developed a postsynthetic conversion of 2'-F-N7-alkylG DNA into 2'-F-alkyl-FapyG DNA. Using these methods, we incorporated site-specific N7-methylG and methyl-FapyG into pSP189 plasmid and determined their mutagenic properties in bacterial cells using the supF-based colony screening assay. The mutation frequency of N7-methylG was found to be less than 0.5%. Our crystal structure analysis revealed that N7-methylation did not significantly alter base pairing properties, as evidenced by a correct base pairing between 2'-F-N7-methylG and dCTP in Dpo4 polymerase catalytic site. In contrast, the mutation frequency of methyl-FapyG was 6.3%, highlighting the mutagenic nature of this secondary lesion. Interestingly, all mutations arising from methyl-FapyG in the 5'-GGT(methyl-FapyG)G-3' context were single nucleotide deletions at the 5'-G of the lesion. Overall, our results demonstrate that 2'-fluorination technology is a useful tool for studying the chemically labile N7-alkylG and alkyl-FapyG lesions.

Machine learning for enumeration of cell colony forming units.

As one of the most widely used assays in biological research, an enumeration of the bacterial cell colonies is an important but time-consuming and labor-intensive process. To speed up the colony counting, a machine learning method is presented for counting the colony forming units (CFUs), which is referred to as CFUCounter. This cell-counting program processes digital images and segments bacterial colonies. The algorithm combines unsupervised machine learning, iterative adaptive thresholding, and local-minima-based watershed segmentation to enable an accurate and robust cell counting. Compared to a manual counting method, CFUCounter supports color-based CFU classification, allows plates containing heterologous colonies to be counted individually, and demonstrates overall performance (slope 0.996, SD 0.013, 95%CI: 0.97-1.02, p value < 1e-11, r = 0.999) indistinguishable from the gold standard of point-and-click counting. This CFUCounter application is open-source and easy to use as a unique addition to the arsenal of colony-counting tools.

Early Results and Technical Tips of Combining Iliac Branch Endoprostheses with Fenestrated Aortic Stent Grafts during Endovascular Repair of Complex Abdominal and Thoracoabdominal Aortic Aneurysms.

BACKGROUND: Concomitant iliac artery aneurysms can pose challenges during repair of complex abdominal and thoracoabdominal aortic aneurysms. In fenestrated aortic aneurysm repairs (FEVAR), preservation of internal iliac perfusion is important to minimize risk of spinal cord ischemia. Currently, most commonly used fenestrated stent grafts and the only approved iliac branch devices are manufactured by different companies in the United States. We report our experience with combining Iliac Branch Endoprosthesis (IBE) (W.L. Gore and Associates, Flagstaff, AZ) and fenestrated stent grafts, using the Zenith platform (Cook Medical, Bloomington, IN). METHODS: Retrospective review of consecutive patients who underwent FEVAR at a single institution from September, 2015 to June, 2020 was performed. Patients were deemed high-risk for open repair. Fenestrated aortic components implanted were either physician-modified or custom manufactured. Cases in which IBEs were deployed during FEVAR were specifically reviewed. Anatomic details were obtained from preoperative CT scans. Postoperative outcomes such as mortality, technical success, major adverse events, limb patency, limb-related endoleaks and re-intervention rates were assessed. RESULTS: During the study period, 171 patients underwent FEVAR at our institution. Among those, 15 patients had unilateral IBE implantation during FEVAR, while one received bilateral IBE implantation. Fourteen cases involved physician-modified fenestrated endograft, and Zenith Fenestrated (Cook Medical, Bloomington, IN) in combination with Excluder bifurcated main body and IBE (W.L. Gore and Associates, Flagstaff, AZ). Mean operative, and fluoroscopy times were 340.2 minutes, and 65.4 minutes respectively. A total of 67 viscerorenal target vessels (mean = 3.9, range =_3-5) and 15 internal iliac arteries were incorporated, with a mean of 160 cc contrast used. Completion angiograms were free of type 1 and type 3 endoleaks. Technical success was 100%. There was no perioperative mortality. One patient developed spinal cord ischemia post-operative day two with neurological recovery. At mean follow-up of 430 days, overall survival was 100% with no aneurysm-related mortalities. Limb patency remained 100%. There were no type 3 endoleaks while one patient had a type 1B endoleak that is currently being monitored. There was one re-intervention for type 1C renal branch graft endoleak. CONCLUSION: Combining IBE with FEVAR allows internal iliac preservation during endovascular repair of complex abdominal aortic aneurysms, with encouraging early results.

Use of Inner Branches During Physician-Modified Endografting for Complex Abdominal and Thoracoabdominal Aortic Aneurysms.

OBJECTIVES: Endovascular repair of complex abdominal and thoracoabdominal aortic aneurysms have been performed widely in an increasing number of centers, utilizing custom-manufactured or physician-modified stent grafts containing fenestrations and side-arm branches for visceral and renal artery incorporation. Alternatively, inner branch configurations may be useful in complex anatomy, where application of fenestrations or side-arm branches can be challenging. Our study aims to evaluate the incidence of target vessel instability when incorporated with inner branch configurations, and report clinical outcomes of patients who underwent fenestrated/branched endovascular aortic repairs (F-BEVAR) containing one or more inner branches. METHODS: We reviewed patients who underwent F-BEVAR with at least one inner branch configuration for complex abdominal or thoracoabdominal aortic aneurysms at Keck Hospital of University of Southern California from 2014 to 2020. Endpoints were mortality, major adverse events (MAE), technical success, and target vessel instability. Target vessel instability was assessed using follow-up computed tomography (CT) and duplex imaging. RESULTS: Out of the 175 patients who underwent F-BEVAR for complex abdominal and TAAA during the study period, 17 patients had at least one inner branch configuration. All were deemed high-risk for open repair with multiple cardiovascular and/or pulmonary comorbidities. Eight (47%) patients had extent I, II, III thoracoabdominal aortic aneurysms, and 10 (59%) had prior aortic repairs. A total of 68 target vessels were incorporated (mean = 4 vessels/patient, range=1~6), of which 40% were inner branch configurations, most commonly for renal arteries. Technical success was 94.1%. There was one perioperative mortality due to massive myocardial infarction, as well as one patient who needed temporary hemodialysis. No device-related mortalities were observed. At 30 days, primary inner branch patency was 100% with no target vessel instability or reintervention. At mean follow-up of 5.8 months, the overall survival was 94% with one patient who expired from unknown cause. Overall primary inner branch patency was 96.3%, due to occlusion of a long lumbar artery branch with no clinical sequelae. CONCLUSION: Inner branch configurations can provide a safe alternative technique of branch incorporation during complex endovascular aortic repair.

Periscope sandwich stenting as an alternative to open cervical revascularization of left subclavian artery during zone 2 thoracic endovascular aortic repair.

OBJECTIVE: Revascularization of the left subclavian artery (LSA) during zone 2 thoracic endovascular aortic repair (TEVAR) maintains collateral circulation to decrease ischemic complications, including stroke, spinal cord ischemia, and upper extremity ischemia. Both open surgical and endovascular LSA revascularization techniques have been described, each with unique risks and benefits. We describe our "periscope sandwich" technique for the LSA during zone 2 TEVAR, which maintains antegrade access to the distal abdominal aorta if subsequent interventions are necessary. Technical results and short-term outcomes are compared with LSA open surgical debranching. METHODS: A single-institution retrospective review was performed for patients requiring zone 2 TEVAR with LSA revascularization by periscope sandwich technique or open surgical debranching with subclavian to carotid transposition (SCT) or carotid-subclavian bypass (CSB). The presenting aortic disease and perioperative details were recorded. Intraoperative angiography and postoperative computed tomography images were reviewed for occurrence of endoleak and branch patency. RESULTS: Between January 2013 and December 2018, the LSA was revascularized by periscope sandwich in 18 patients, SCT in 22 patients, and CSB in 13 patients. Compared with open surgical debranching, periscope sandwich had a lower median estimated blood loss (100 mL vs 200 mL for pooled SCT and CSB; P = .03) and lower median case duration (133.5 minutes vs 226 minutes; P < .001). Contrast material volume (120 mL vs 120 mL; P = .98) and fluoroscopy time (13.1 minutes vs 13.3 minutes; P = .92) did not differ significantly between the groups. There was no difference in aorta-related mortality (P = .14), and LSA patency was 100%. Median follow-up for the periscope sandwich group was 11 months, with an overall estimated 91% freedom from gutter leak at 6 months. CONCLUSIONS: LSA periscope sandwich technique provides safe and effective LSA revascularization during zone 2 TEVAR. LSA periscope sandwich can be used emergently with off-the-shelf endovascular components and facilitates future branched-fenestrated endovascular repair of thoracoabdominal aortic diseases.

Physician-modified fenestrated endovascular repair of type 1A endoleaks from polymer-based low-profile endografts.

PURPOSE: There have been increasing number of endovascular aortic aneurysm repair performed in hostile necks using newer generation technology including polymer-based proximal sealing devices such as the Ovation system. Unique design features of the device can pose challenges during endovascular salvage of type 1A endoleak. We describe two cases of successful application of physician-modified fenestrated endografting, in order to repair type 1A endoleaks following endovascular aortic aneurysm repair with ovation system. TECHNIQUE: In both cases, multi-fenestrated endografts were custom-modified using preloaded wire technique on Cook Zenith Alpha thoracic stent grafts at the back table. Under general anesthesia, left brachial cut down and a single percutaneous femoral access were performed. Staggered deployment of fenestrated endograft, accompanied by sequential catheterization of target vessels, facilitated correct alignment of fenestrated endograft. Infolding of fenestrated endograft inside the Ovation main body resulted in leg claudication, and repaired with balloon expandable covered tent. Prophylactic deployment of balloon expandable covered stent was performed in the second case. Both cases showed resolution of type 1A endoleak. CONCLUSION: Fenestrated endovascular repair is feasible for proximal failure of Ovation endografts. Careful planning and advanced skill set in complex endovascular aortic repair are required, as well as detailed knowledge of the failed endografts.

Antegrade in situ fenestrated endovascular repair of a ruptured thoracoabdominal aortic aneurysm.

We describe a technique for antegrade in situ laser fenestration that has several advantages in the setting of ruptured thoracoabdominal aortic aneurysms. This technique involves rapid aneurysm sealing by deployment of aortic stent graft, followed by sequential incorporation of branch vessels using a laser probe through steerable sheath. The advantages of this technique include (1) rapid seal of the ruptured aneurysm, (2) preservation of the visceral and renal branch perfusion, (3) use of an off-the-shelf device, and (4) the ability to be performed without general anesthesia.

Low-profile Zenith Alpha™ Thoracic Stent Graft Modification Using Preloaded Wires for Urgent Repair of Thoracoabdominal and Pararenal Abdominal Aortic Aneurysms.

BACKGROUND: The aim of this study is to describe a modification technique using the low-profile Cook Zenith Alpha™ thoracic stent graft, and addition of a preloaded wire system, for urgent repair of pararenal (PRA) and thoracoabdominal (TAAA) aortic aneurysms. METHODS: We analyzed 20 consecutive patients who underwent urgent physician modified endograft repair (PMEG) of PRA and TAAA at 2 institutions. The low-profile Cook Zenith Alpha Thoracic stent graft was modified in accordance with each specific patient anatomic characteristics. End points were technical success, 30-day mortality, and major adverse events (MAEs). RESULTS: Technical success was achieved in all patients (100%). A total of 76 renal-mesenteric arteries were incorporated by fenestrations (70%) or directional branches (30%) with an average of 3.7 ± 0.6 vessels per patient. There were 6 different types of stent configuration. The most common design consisted of 4 fenestrations (9 patients, 45%). The average of modification time was 110 ± 27 minutes. Total procedure time (including the time for open component) was 242 ± 75 minutes. There was no death within the first 30 days or hospital stay. MAEs occurred in 10 patients (50%). The most common MAEs were acute kidney injury (by Risk, Injury, and Failure; and Loss; and End-stage kidney disease criteria) in 6 patients (30%), estimated blood loss >1 L, respiratory failure requiring reintubation in 2 patients (10%) each, and paraplegia and ischemic colitis in 1 patient (5%) each. One patient (5%) required temporary, new-onset dialysis. CONCLUSIONS: PMEG using low-profile Zenith Alpha thoracic stent graft was safe with no early mortality and acceptable early morbidity.

Predictors of Major Adverse Limb Events after Open Forefoot Amputation in Patients with Chronic Limb-Threatening Ischemia.

BACKGROUND: Management of patients with chronic limb-threatening ischemia (CLTI) and extensive foot necrosis presents a challenge for limb salvage. Our study evaluates preoperative risk factors that contributed to durability and efficacy of limb salvage after open transmetatarsal amputation (TMA) in patients with critical limb-threatening ischemia. METHODS: We abstracted data from patients who underwent open TMA at Los Angeles County-University of Southern California Medical Center and Keck Hospital of University of Southern California from 2009 to 2018. Multivariable logistic regression analysis, adjusting for preoperative risk factors, was used to examine predictors of major adverse limb events (MALE). The aim was to evaluate outcomes following open TMA with MALE as the primary outcome. Our hypotheses were that outcomes would be worse for patients with foot infections and renal failure. RESULTS: Forty-three open TMAs were done in 39 patients during the study period. The cohort had a mean age of 63 ± 11.6 years, 89% had a history of diabetes, 95% hypertension (HTN), 54% had end-stage renal disease (ESRD), and 26% were current smokers. MALE occurred in 39% of the cohort. Sex, race, indication, HTN, smoking status, and history of prior ipsilateral revascularization or minor amputations were not associated with MALE (P > 0.05). Multivariate logistic regression found ESRD to be an independent predictor of MALE (odds ratio 7.43, 95% confidence interval 1.12-49.17, P = 0.038) after adjusting for clinically significant covariates. CONCLUSIONS: Open TMA provides acceptable rates of limb salvage for complex patients with CLTI. ESRD is an independent risk factor for MALE following open TMA in these patients. Vigilant follow-up is essential for this morbid patient population given poorer outcomes after forefoot amputation.