THE IMPORTANCE OF DYNAMIC EFFECTS ON THE ENZYME ACTIVITY: X-RAY STRUCTURE AND MOLECULAR DYNAMICS OF ONCONASE MUTANTS.

Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme

Deamidation of Proteins: The crystal structure of bovine pancreatic ribonuclease with an isoaspartyl residue at position 67

The non-enzymatic deamidation of asparagine residues in proteins is a widely occurring reaction, both in vivo and in vitro. Although the importance of this process is commonly recognised, only little structural information is available on it. In order to evaluate the structural effects of this reaction in proteins, we have determined the crystal structure of a ribonuclease A derivative in which asparagine 67 has been replaced by an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. The overall structure of the model, refined to a crystallographic R-factor of 0.159 at a resolution of 1.9 Å, is very similar to that of the native protein, but considerable deviations are observed in the region delimited by the disulphide bridge 65–72. In particular, the insertion of an extra methylene group in the main chain at residue 67 breaks up the hydrogen bond network that makes this region rather rigid in ribonuclease A. On the basis of the structure observed, some of the slightly but significantly different properties of this deamidated derivative, with respect to the native enzyme, can be explained

Through-and-through femorofemoral wire retrograde technique for the E-nside precannulated inner branch thoracoabdominal stent graft

The E-nside multibranched stent graft (Jotec GmbH, Hechingen, Germany) is the first and only off-the-shelf thoracoabdominal prosthesis with precannulated inner branches. Usually, after the device deployment, target vessel stenting will be facilitated by antegrade inner branch cannulation to retrieve the precannulated guide. In the literature, the use of antegrade access has been associated with the potential risk of cerebral and systemic embolization. Therefore, other retrograde techniques have been described. We have reported a new retrograde approach using a precannulated through-and-through femorofemoral wire technique for target vessel catheterization

“Redo” 2D–3D Fusion Technique during Endovascular Redo Aortic Repair

Purpose: The present study aims to describe a new 2D–3D fusion registration method in the case of endovascular redo aortic repair and compare the accuracy of the registration using the previously implanted devices vs. bones as landmarks. Materials and Methods: This single-center study prospectively analyzed all the patients that underwent elective endovascular re-interventions using the Redo Fusion technique between January 2016 and December 2021 at the Vascular Surgery Unit of the Fondazione Policlinico Universitario A. Gemelli (FPUG)—IRCCS in Rome, Italy. The fusion overlay was performed twice, first using bone landmarks (bone fusion) and the second using radiopaque markers of a previous endovascular device (redo fusion). The pre-operative 3D model was fused with live fluoroscopy to create a roadmap. Longitudinal distances between the inferior margin of the target vessel in live fluoroscopy and the inferior margin of the target vessel in bone fusion and redo fusion were measured. Results: This single-center study prospectively analyzed 20 patients. There were 15 men and five women, with a median age of 69.7 (IQR 42) years. The median distance between the inferior margin of the target vessel ostium in digital subtraction angiography and the inferior margin of the target vessel ostium in bone fusion and redo fusion was 5.35 mm and 1.35 mm, respectively (p ≤ 0.0001). Conclusions: The redo fusion technique is accurate and allows the optimization of X-ray working views, supporting the endovascular navigation and vessel catheterization in case of endovascular redo aortic repair

FISSIT (Fistula Surgery in Italy) study: A retrospective survey on the surgical management of anal fistulas in Italy over the last 15 years

Background: Surgical treatment of anal fistulas is still a challenge. The aims of this study were to evaluate the adoption and healing rates for the different surgical techniques used in Italy over the past 15 years. Methods: This was a multicenter retrospective observational study of patients affected by simple and complex anal fistulas of cryptoglandular origin who were surgically treated in the period 2003-2017. Surgical techniques were grouped as sphincter-cutting or sphincter-sparing and as technology-assisted or techno-free. All patients included in the study were followed for at least 12 months. Results: A total of 9,536 patients (5,520 simple; 4,016 complex fistulas) entered the study. For simple fistulas, fistulotomy was the most frequently used procedure, although its adoption significantly decreased over the years (P < .0005), with an increase in sphincter-sparing approaches; the overall healing rate in simple fistulas was 81.1%, with a significant difference between sphincter-cutting (91.9%) and sphincter-sparing (65.1%) techniques (P = .001). For complex fistulas, the adoption of sphincter-cutting approaches decreased, while sphincter-sparing techniques were mildly preferred (P < .0005). Moreover, there was a significant trend toward the use of technology-assisted procedures. The overall healing rate for complex fistulas was 69.0%, with a measurable difference between sphincter-cutting (81.1%) and sphincter-sparing (61.4%; P = .001) techniques and between techno-free and technology-assisted techniques (72.5% and 55.0%, respectively; P = .001). Conclusion: Surgical treatment of anal fistulas has changed, with a trend toward the use of sphincter-sparing techniques. The overall cure rate has remained stable, even if the most innovative procedures have achieved a lower success rate

Dynamic Prediction of Rectal Cancer Relapse and Mortality Using a Landmarking-Based Machine Learning Model: A Multicenter Retrospective Study from the Italian Society of Surgical Oncology—Colorectal Cancer Network Collaborative Group

Background: Almost 30% of patients with rectal cancer (RC) who submit to comprehensive treatment experience relapse. Surveillance plays a leading role in early detection. The landmark approach provides a more flexible and dynamic framework for survival prediction. Objective: This large retrospective study aims to develop a machine learning algorithm to profile the patient prognosis, especially the risk and the onset of RC relapse after curative resection. Methods: A cohort of 2450 RC patients were analyzed using landmark analysis. Model A applied a classical cause-specific Cox approach with a landmarking approach, while Model B implemented a landmarking-based RSF (random survival forest) competing risk algorithm. The two models were compared in terms of predictive and interpretative ability. A bootstrapped validation strategy was employed to validate the model’s performance and prevent overfitting. The best-performing hyperparameters were selected systematically, ensuring the model’s robustness within the landmark approach. The study assessed these factors’ importance and interactions using RSF and compared the predictive accuracy to that of the classical Cox model. Results: Model B outperformed Model A (mean C-index 0.95 vs. 0.78), capturing complex interactions and providing dynamic, individualized relapse predictions. Clinical factors influencing survival outcomes were identified across time with the landmark approach allowing for more accurate and timely predictions. Conclusions: The landmark approach offers an improvement over traditional methods in survival analysis. By accommodating time-dependent variables and the evolving nature of patient data, this approach provides a precise tool for profiling RC survival, thereby supporting more informed and dynamic clinical decision-making

RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis

Patients with inflammatory bowel diseases are at increased risk for colorectal cancer, but the molecular mechanisms linking inflammation and cancer are not well defined. We earlier showed that carboxylated N-glycans expressed on receptor for advanced glycation end products (RAGE) and other glycoproteins mediate colitis through activation of nuclear factor kappa B (NF-κB). Because NF-κB signaling plays a critical role in the molecular pathogenesis of colitis-associated cancer (CAC), we reasoned that carboxylated glycans, RAGE and its ligands might promote CAC. Carboxylated glycans are expressed on a subpopulation of RAGE on colon cancer cells and mediate S100A8/A9 binding to RAGE. Colon tumor cells express binding sites for S100A8/A9 and binding leads to activation of NF-κB and tumor cell proliferation. Binding, downstream signaling and tumor cell proliferation are blocked by mAbGB3.1, an anti-carboxylate glycan antibody, and by anti-RAGE. In human colon tumor tissues and in a mouse model of CAC, we found that myeloid progenitors expressing S100A8 and S100A9 infiltrate regions of dysplasia and adenoma. mAbGB3.1 administration markedly reduces chronic inflammation and tumorigenesis in the mouse model of CAC and RAGE-deficient mice are resistant to the onset of CAC. These findings show that RAGE, carboxylated glycans and S100A8/A9 play essential roles in tumor–stromal interactions, leading to inflammation-associated colon carcinogenesis

Early Outcomes of a Novel Off the Shelf Preloaded Inner Branch Endograft for the Treatment of Complex Aortic Pathologies in the ItaliaN Branched Registry of E-nside EnDograft (INBREED)

Objective: The aim of this study was to investigate the early outcomes of a novel off the shelf pre-loaded inner branched thoraco-abdominal endograft (E-nside) in the treatment of aortic pathologies.Methods: Data from a physician initiated national multicentre registry on patients treated with the E-nside endograft, were prospectively collected and analysed. Pre-operative clinical and anatomical characteristics, procedural data, and early outcomes (90 days) were recorded in a dedicated electronic data capture system. The primary endpoint was technical success. Secondary endpoints were early mortality (90 days), procedural metrics, target vessel patency, endoleak rate, and major adverse events (MAEs) at 90 days.Results: In total, 116 patients from 31 Italian centres were included. Mean + standard deviation (SD) patient age was 73 + 8 years and 76 (65.5%) were male. Aortic pathologies included degenerative aneurysm in 98 (84.5%), post-dissection aneurysm in five (4.3%), pseudoaneurysm in six (5.2%), penetrating aortic ulcer or intramural haematoma in four (3.4%), and subacute dissection in three (2.6%). Mean + SD aneurysm diameter was 66 + 17 mm; aneurysm extent was Crawford I -III in 55 (50.4%), IV in 21 (19.2%), pararenal in 29 (26.7%), and juxtarenal in four (3.7%). The procedure setting was urgent in 25 (21.5%) patients. Median procedural time was 240 minutes (interquartile range [IQR] 195, 303), with a median contrast volume of 175 mL (IQR 120, 235). The endograft's technical success rate was 98.2% and the 90 day mortality rate was 5.2% (n = 6; 2.1% for elective repair and 16% for urgent repair). The 90-days cumulative MAE rate was 24.1% (n = 28). At 90 days, there were 10 (2.3%) target vessel related events (nine occlusions and one type IC endoleak) and one type 1A endoleak requiring re-intervention.Conclusion: In this real life, non-sponsored registry, the E-nside endograft was used for the treatment of a broad spectrum of aortic pathologies, including urgent cases and different anatomies. The results showed excellent technical implantation safety and efficacy, as well as early outcomes. Longer term follow up is needed to better define the clinical role of this novel endograft

Use of an Off the Shelf Inner Branch Thoraco-abdominal Endograft for the Treatment of Juxtarenal and Pararenal Aortic Aneurysms

Objective: To investigate outcomes of an off the shelf pre-loaded inner branched endograft (E-nside) for the treatment of juxtarenal and pararenal abdominal aortic aneurysms (JP-AAAs). Methods: Data from a multicentre registry (INBREED), including patients treated with the E-nside endograft, were collected and analysed prospectively. Patients treated for JP-AAA were included. Pre-operative clinical and anatomical characteristics, procedural data, and 30 day and one year outcomes were recorded. Endpoints were technical success, 30 day death, major adverse events (MAEs), and one year freedom from target vessel instability. Results: Of 185 consecutively treated patients, 47 (25.4%) had a JP-AAA (juxtarenal n = 10, 21%; pararenal n = 37, 79%) and were included in the study; 183 target vessels were incorporated through an inner branch. Procedural setting was emergency or urgent in 18 patients (38%) owing to a contained aortic rupture (n = 2, 4%), symptomatic aneurysm (n = 4, 9%), or aneurysm > 70 mm (n = 12, 26%). The mean length of aortic coverage above the coeliac trunk was 116 ± 7 mm. Technical success was 100% and 30 day mortality rate 4% (n = 2 urgent cases). The 30 day cumulative MAE rate was 26% (n = 12): two stroke (4%); and seven spinal cord ischaemia (15%), with six in an elective setting (21%) and one in an urgent setting (6%), and five leading to permanent paraplegia or paraparesis (10%). Freedom from target vessel instability was 99% after 30 days and 97 ± 3% after one year. Conclusion: Use of an off the shelf inner branched device for treating JP-AAA was feasible in urgent and elective settings, with high technical success and satisfactory target vessel stability at one year. In the treatment of JP-AAA, stroke and spinal cord ischaemia may be associated with arm access and the increased aortic coverage that the design brings

Outcomes of off-the-shelf preloaded inner branch device for urgent endovascular thoraco-abdominal aortic repair in the ItaliaN Branched Registry of E-nside EnDograft

Objective: The aim of this study was to report the outcomes of endovascular urgent thoracoabdominal aortic (TAAA) repair, using an off-the-shelf preloaded inner branch device (E-nside; Artivion). Methods: Data from a physician-initiated national multicenter registry, including patients treated with E-nside endograft (INBREED) were prospectively collected (2020-2024); only urgent cases were included in this study. Primary outcomes were technical success and mortality at 30 days. Secondary outcomes were spinal cord ischemia rate, stroke rate, major adverse events (MAE) as also branch instability at 12 months. Results: Of 185 patients enrolled in the INBREED, 64 (34.5%) were treated in a urgent setting and were included in the study. Reason for urgent repair was presence of aneurysm-related symptoms in 31 patients (48.4%), a contained rupture in eight (12.5%), and a large aneurysm >80 mm in 25 (39.1%). Extent of repair was I to III in 32 patients (50%) and IV in 32 (50%); 18 (28%) had a narrow (<25 mm) paravisceral aortic lumen. An adjunctive proximal thoracic endograft was deployed in 29 patients (45.3%); a distal bifurcated abdominal endograft was used in 33 (51.5%). Two hundred forty-nine target vessels (97.2%) were successfully incorporated through an inner branch from an upper arm (81.2%) or femoral (18.8%) access. A balloon expandable stent was used in 184 (75.7%) target vessels, a self-expandable stent in 59 (24.3%). Mean time for target vessel bridging was 39.9 ± 28.4 minutes per target vessel. Thirty-day cumulative major adverse event (MAE) rate was 28%, and mortality occurred in five patients (9.1%). There was one postoperative stroke (1.6%), and the spinal cord ischemia (SCI) rate was 8% (n = 5). For the 249 target vessels successfully incorporated through an inner branch, 1-year freedom from target vessel instability was 93% ± 3% after 1 year. Conclusions: The E-nside represents a valid solution for the urgent treatment of TAAAs, including symptomatic and ruptured TAAAs, as well as large asymptomatic TAAAs that cannot wait for a custom-made device. The preloaded inner branches and available proximal and distal graft diameters might be useful in urgent settings and provided satisfactory early and 1-year results, in terms of both endograft and target vessel stability. Further studies are required to assess the clinical role of E-nside for urgent TAAA repair