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Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as β-galactoside binding lectins. However, certain members of this family have shown selective affinity toward specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7, and -12) toward a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I versus type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity toward poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance experiments. Particularly, we identified oligosaccharide blood group A antigen tetraose 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited interleukin-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to the low temperature, the Antarctic marine environment is challenging for protein functioning. Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologs, resulting in enhanced reaction rates at low temperatures. The Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) genome is one of the few examples of coexistence of multiple hemoglobin genes encoding, among others, two constitutively transcribed 2/2 hemoglobins (2/2Hbs), also named truncated Hbs (TrHbs), belonging to the Group II (or O), annotated as PSHAa0030 and PSHAa2217. In this work, we describe the ligand binding kinetics and their interrelationship with the dynamical properties of globin Ph-2/2HbO-2217 by combining experimental and computational approaches and implementing a new computational method to retrieve information from molecular dynamic trajectories. We show that our approach allows us to identify docking sites within the protein matrix that are potentially able to transiently accommodate ligands and migration pathways connecting them. Consistently with ligand rebinding studies, our modeling suggests that the distal heme pocket is connected to the solvent through a low energy barrier, while inner cavities play only a minor role in modulating rebinding kinetics., (© 2024 The Protein Society.)

Introduction: Duplex ultrasound (DUS) is the modality of choice for surveillance of popliteal artery aneurysms (PAAs). However, noninvasive vascular laboratories have no standard guidelines for reporting results. This study assessed reports of PAA DUS for inclusion of information pertinent to operative decision-making and timing of surveillance., Methods: This study was a retrospective review of a multi-institutional repository that was queried for all patients with a PAA from 2008 to 2022 and confirmed via manual chart review. DUS reports were abstracted and images were individually annotated for features of interest including dimensions, flow abnormalities, and percent thrombus burden., Results: A total of 166 PAAs in 130 patients had at least one DUS available for viewing. Postoperative surveillance of PAAs was performed at several intervals: the first at 30 months (IQR 3.7-113, n = 44), the second at 64 months (IQR 20-172, n = 31), and the third at 152 months (IQR 46-217, n = 16) after the operation. The largest diameter of operative PAAs (median 27.5 mm, IQR 21.8-38.0) was significantly greater than nonoperative PAAs (median 20.9 mm, IQR 16.7-27.3); p < 0.01. Fewer than 33 (21%) reports commented on patency of distal runoff. We calculated an average percent thrombus of 60% (IQR 19-81) in nonoperative PAAs, which is significantly smaller than 75% (IQR 58-89) in operative PAAs; p < 0.01., Conclusion: In this multi-institutional retrospective study, PAAs are often not followed at intervals recommended by the Society for Vascular Surgery guidelines and do not include all measurements necessary for clinical decision-making in the multi-institutional repository studied. There should be standardization of PAA DUS protocols performed by all noninvasive vascular laboratories to ensure completeness of PAA DUS images and inclusion of characteristics pertinent to clinical decision-making in radiology reports., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration., (© 2023 Avolio et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Given the relative rarity of ruptured and symptomatic type I-III thoracoabdominal aortic aneurysms (TAAA), data is scarce with regard the outcomes of those who survive to repair. The goal of this study was to determine short and long-term outcomes after open repair of type I-III TAAA surgery for ruptured and symptomatic TAAA and compare the results to elective TAAA repairs., Methods: All open type I-III TAAA repairs performed from 1987 to 2015 were evaluated using an institutional database. Charts were retrospectively evaluated for perioperative outcomes: major adverse event (MAE), in-hospital death, spinal cord ischemia (SCI) and long-term survival. Ruptured, symptomatic and elective repair cohorts were created for comparison. Univariate analysis was performed using the Fisher's exact test for categorical variables and analysis of variance (ANOVA) for continuous variables. Logistic regression was used for in-hospital endpoints; survival analysis was performed with Cox proportional hazards modelling and Kaplan-Meier techniques., Results: Five hundred-sixteen patients had an open type I-III TAAA repair during the study period. Fifty-nine (11.4%) were performed for rupture and 51 (9.9%) were performed for symptomatic aneurysms (RAs). Ruptured and symptomatic groups were more likely to be older, female, and have larger presenting aortic diameters. Most of the ruptured and symptomatic cases were transferred from an outside facility (59.3% and 54.9%, respectively). Intraoperatively, the elective cohort was more likely to receive left heart bypass as an operative adjunct; ruptures were less likely to receive a renal bypass, and operative time was highest for the elective cohort. Perioperative mortality was 18.6% for ruptured, 2.0% for symptomatic, and 7.4% for elective indications. Ruptures were most likely to require new hemodialysis after repair (20.3% vs. 10.3% for elective, P = 0.02). On adjusted analysis, ruptures were more likely to suffer from perioperative death (adjusted odds ratio [AOR]: 4.5, 95% confidence interval (CI): 1.7-11.4) and MAEs (AOR: 2.8, 95% CI: 1.4-5.4). Ruptured and symptomatic aneurysms were not independently associated with SCI; however, preoperative hemodynamic instability was predictive (AOR: 8.7, 95% CI: 1.7-44.2). Both rupture and symptomatic cases were associated with decreased survival on Kaplan-Meier analysis with 5-year survival for ruptures at 35%, symptomatic at 47.7% and elective at 63.7%, P < 0.001. Adjusted hazards of death were 1.2 (95% CI: 0.9-1.8) in the symptomatic cohort and 2.3 (95% CI: 1.5-3.7) in the ruptured cohort., Conclusions: Open ruptured and symptomatic type I-III TAAA repairs can be performed with acceptable morbidity and mortality. Most symptomatic and rupture repairs were performed after transfer from another institution. Postoperative SCI is most strongly related to the preoperative hemodynamic status of the patient., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Antiplatelet therapy has been a pillar of management for peripheral artery disease (PAD). However, a significant subset of patients with PAD will be resistant to certain antiplatelet medications and, therefore, have an increased risk of graft and/or stent thrombosis unknown to the surgeon. At present, no point-of-care testing to identity which patients will experience benefit from these medications has been incorporated into the treatment guidelines. Thromboelastography with platelet mapping affords an opportunity to evaluate real-time coagulation dynamics and platelet function. In the present prospective, observational study, we aimed to delineate the variation in response to antiplatelet therapy in patients with PAD undergoing revascularization., Methods: All patients who were undergoing named vessel revascularization during December 2020 through April 2022 were prospectively enrolled. Platelet mapping assays were performed in three clinical phases: preoperative, postoperative inpatient, and postoperative outpatient. The distribution of platelet reactivity within patients receiving mono- vs dual antiplatelet therapy was assessed, and a between-group inferential analysis was performed. The effect of comorbidities and intervention subtype on platelet inhibition was also analyzed., Results: A total of 521 platelet mapping samples from 143 individual patients were analyzed using thromboelastography with platelet mapping. We found wide variability in the distribution of platelet inhibition, with a range of 0 to 100 and an interquartile range of 37.6. Although platelet inhibition with clopidogrel 75 mg was higher on average (44.8 ± 30.2) than that with aspirin 81 mg (24.6 ± 23.7) or aspirin 325 mg (27.1 ± 26.4; P = .001), clopidogrel at 75 mg demonstrated the highest variability in response., Conclusions: These data have demonstrated significant variability in the response to both mono- and dual antiplatelet therapy in PAD patients undergoing lower extremity revascularization. Future research on the effect of this variability in response on the clinical outcomes could provide invaluable understanding of the perioperative thrombotic risk., (Copyright © 2022 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Background: Clinical outcomes in women with peripheral artery disease (PAD) after revascularization procedures are worse compared to men, yet there is little in the existing literature as why this may be the case. Platelet Mapping is an emerging point-of-care viscoelastic technology that measures the comprehensive properties of a blood clot, including fibrin-platelet interactions. This prospective observational study aimed to characterize the clinical and Platelet Mapping profiles of female and male patients undergoing lower extremity revascularization, and to correlate Platelet Mapping distribution to thrombotic potential., Methods: All patients with a diagnosis of PAD undergoing named vessel open or endovascular revascularization to re-establish inflow, outflow, or both, during December 2020 and January 2022 were prospectively included. Patients were followed clinically for thrombosis for up to 1 year. Platelet Mapping assays were performed in 3 clinical phases: preoperative, postoperative inpatient, and postoperative outpatient. Inferential analysis between female and male patient was performed. The quartile distribution of Platelet Mapping metrics associated with thrombosis was used to infer to thrombotic potential., Results: One hundred seven patients were enrolled, of which 37 (34.6%) were female. Female patients had significantly lower rates of uncontrolled diabetes (2.7% vs. 18.6%), hypertension requiring combination therapy (37.8% vs. 58.6%), chronic kidney disease (27.0% vs. 51.4%), coronary artery disease (29.7% vs. 57.1%), and myocardial infarction (16.2% vs. 35.7%) (all P < 0.05). Platelet reactivity was significantly higher in female patients with greater platelet aggregation (75.9 ± 23.3 vs. 63.5 ± 28.8) and lower platelet inhibition (23.8 ± 23.4 vs. 36.8 ± 28.9) (all P < 0.01). This trend was consistent over time when stratified by the postoperative inpatient and postoperative outpatient clinical phases. There was no statistically discernible difference in the use of antiplatelet therapy between groups, yet female patients continued to exhibit greater platelet reactivity when analyzed by the type of pharmacologic regimen (platelet aggregation on mono-antiplatelet therapy: 80.6 ± 21.0 in women versus 69.4 ± 25.0 in men; platelet aggregation on dual antiplatelet therapy: 67.9 ± 23.8 in women versus 44.8 ± 31.8 in men) (all P < 0.01). Twenty-one patients experienced postoperative graft/stent thrombosis within the study period. In relation to the overall study population, patients with thrombosis had Platelet Mapping metrics above the 50th percentile of overall platelet aggregation distribution., Conclusions: There is a growing appreciation for the differences in etiology, disease progression, and outcomes of cardiovascular conditions as they relate to sex. In this cohort, traditional cardiovascular risk factors were in lower prevalence in female patients. Platelet reactivity was found to be higher across clinical phases and antiplatelet regimens. High platelet reactivity was also associated with an increased incidence of thrombosis after lower extremity revascularization. These hypothesis-generating findings provide the basis for further exploration of sex-specific coagulation profiling in PAD patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Spring blossom honey from regions with many rape fields tends to crystalize rapidly after harvesting. The crystallization process needs to be controlled by stirring in order to avoid the formation of coarse crystals and to ensure the creaminess of honey. The aim of this study was to investigate how various parameters of the stirring process influence the creaminess of spring blossom honey in order to give recommendations for beekeeping practices. The creaminess was quantified by measuring the crystal size by microscopic analysis, measuring the whiteness index by color analysis using CIE Lab and by sensory analysis. We investigated the influence of five stirring parameters, including the type of stirring device, honey pretreatment, stirring temperature (14 °C to room temperature), stirring interval (1 to 24 times) and stirring time (1-15 min) on the creaminess of honey. We found that the stirring temperature is the most important factor for honey creaminess. At the optimal temperature of 14 °C, other factors like seed honey, stirring time and stirring interval have only a neglectable effect. If the optimal temperature of 14 °C cannot be maintained, as it may happen in beekeepers' practice, sieving the honey with a mesh size of 200 µm before stirring, the addition of seed honey prepared with a kitchen food processor, and using a stirring screw and stirring several times per day is recommended.

Background: Postoperative infection and wound dehiscence rates are higher than expected in peripheral artery disease and contribute significantly to limb loss and mortality. Microvascular pathology characterized by microthrombi and increased platelet aggregation have been cited as contributing factors to poor wound healing and infection. The emergence of viscoelastic assays, such as thromboelastography with platelet mapping (TEG-PM), have been utilized to identify prothrombotic states and may provide insight into a patient's microvascular coagulation profile. This prospective, observational study aimed to determine if TEG-PM could predict poor wound healing or infection following lower extremity revascularization., Methods: All patients undergoing revascularization between December 2020 and January 2022 were prospectively included and followed for wound complications or non-surgical site infections of the index limb. TEG-PM metrics at the first postoperative follow-up in the nonevent group was compared to the TEG-PM sample preceding the diagnosis of infection/dehiscence in the event group. Cox proportional hazards (PH) regression was used to model the predictive value of viscoelastic parameters. Cut-point analysis to determine high-risk groups was determined by performing receiver operating characteristic curve analysis., Results: Of the 102 patients, 18.6% experienced infection/dehiscence. The TEG-PM sample analyzed in the event group was, on average, 19.5 days prior to the diagnosis of an event. The event group had significantly higher maximum clot amplitude (MA) (47.3 mm ± 16.0 vs. 30.6 mm ± 15.3, P < 0.01), higher platelet aggregation (71.3% ± 27.7 vs. 31.2% ± 24.0, P < 0.01), and lower platelet inhibition (28.7% ± 27.7 vs. 68.7% ± 24.1, P < 0.01). Cox PH analysis identified platelet aggregation as an independent and consistent predictor of infection (hazard ratio = 1.04, 95% confidence interval 1.03-1.06, P < 0.01). An optimal cut-point of > 33.2 mm MA, > 46.6% platelet aggregation, or < 55.8% platelet inhibition identifies those with infection/dehiscence with 79.0-89.5% sensitivity., Conclusions: These are the first data to provide a quantitative link between prothrombotic viscoelastic coagulation profiles with the development of infection/dehiscence. Based on the cut-points of > 33.2 mm MA, > 46.6% platelet aggregation, or < 55.8% platelet inhibition, we recommend consideration of an enhanced antimicrobial or antithrombotic approach for these high risk groups., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The structure and dynamics of membranes are crucial to ensure the proper functioning of cells. There are some compounds used in therapeutics that show nonspecific interactions with membranes in addition to their specific molecular target. Among them, two compounds recently used in therapeutics against COVID-19, remdesivir and favipiravir, were subjected to molecular dynamics simulation assays. In these, we demonstrated that the compounds can spontaneously bind to model lipid membranes in the presence or absence of cholesterol. These findings correlate with the corresponding experimental results recently reported by our group. In conclusion, insertion of the compounds into the membrane is observed, with a mean position close to the phospholipid head groups.

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.

Background: There is a paucity of data regarding the conversion rate from dry gangrene to wet gangrene after lower extremity revascularization. This study aimed to determine the rate of conversion from dry to wet gangrene within 30 days post-procedure in patients who underwent endovascular or open revascularization for critical limb ischemia. Secondary aims included determining the time to conversion and associated risk factors with conversion., Methods: A multicenter, retrospective review was performed utilizing the MGH/Brigham Healthcare System's Research Patient Data Registry (RPDR). All adult patients who had lower extremity dry gangrene that underwent a revascularization procedure (endo, open, hybrid) from April 2002 to March 2020 were included. Patients who had no lower extremity gangrene, a concurrent amputation with the revascularization procedure, or wet gangrene on initial presentation were excluded. Univariate analysis was performed using the Fisher's exact test and Wilcoxon rank-sum test., Results: There were 1,518 patients identified who underwent revascularization; 194 (12.8%) patients met inclusion criteria and served as our study cohort. There were 15 (7.7%) conversions from dry to wet gangrene within 30 days post-procedure. The mean time to conversion was 13.5 ± 8.6 days. Univariate analysis did not identify any associated risk factors for conversion., Conclusions: The rate of dry to wet gangrene conversion post revascularization is 7.7% within 30 days. The mean time of conversion is 13.5 ± 8.6 days., (Copyright © 2022. Published by Elsevier Inc.)

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.

The pathogenic agent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters into human cells through the interaction between the receptor binding domain (RBD) of its spike glycoprotein and the angiotensin-converting enzyme 2 (ACE2) receptor. Efforts have been made towards finding antivirals that block this interaction, therefore preventing infection. Here, we determined the binding affinity of ACE2-derived peptides to the RBD of SARS-CoV-2 experimentally and performed MD simulations in order to understand key characteristics of their interaction. One of the peptides, p6, binds to the RBD of SARS-CoV-2 with nM affinity. Although the ACE2-derived peptides retain conformational flexibility when bound to SARS-CoV-2 RBD, we identified residues T27 and K353 as critical anchors mediating the interaction. New ACE2-derived peptides were developed based on the p6-RBD interface analysis and expecting the native conformation of the ACE2 to be maintained. Furthermore, we found a correlation between the helicity in trifluoroethanol and the binding affinity to RBD of the new peptides. Under the hypothesis that the conservation of peptide secondary structure is decisive to the binding affinity, we developed a cyclized version of p6 which had more helicity than p6 and approximately half of its K D value., (© 2022 Carolina Sarto et al., published by De Gruyter, Berlin/Boston.)

Introduction: Silver fir (Abies alba Mill.) is one of the most valuable conifer wood species in Europe. Among the main opportunistic pathogens that cause root and butt rot on silver fir are Armillaria ostoyae and Heterobasidion abietinum. Due to the different enzymatic pools of these wood-decay fungi, different strategies in metabolizing the phenols were available., Objective: This work explores the changes in phenolic compounds during silver fir wood degradation., Methodology: Phenols were analyzed before and after fungus inoculation in silver fir macerated wood after 2, 4 and 6 months. All samples were analyzed using high-performance liquid chromatography coupled to a hybrid quadrupole-orbitrap mass spectrometer., Results: Thirteen compounds, including simple phenols, alkylphenyl alcohols, hydroxybenzoketones, hydroxycinnamaldehydes, hydroxybenzaldehydes, hydroxyphenylacetic acids, hydroxycinnamic acids, hydroxybenzoic acids and hydroxycoumarins, were detected. Pyrocatechol, coniferyl alcohol, acetovanillone, vanillin, benzoic acid, 4-hydroxybenzoic acid and vanillic acid contents decreased during the degradation process. Methyl vanillate, ferulic acid and p-coumaric were initially produced and then degraded. Scopoletin was accumulated. Pyrocatechol, acetovanillone and methyl vanillate were found for the first time in both degrading and non-degrading wood of silver fir., Conclusions: Despite differences in the enzymatic pool, both fungi caused a significant decrease in the amounts of phenolic compounds with the accumulation of the only scopoletin. Principal component analysis revealed an initial differentiation between the degradation activity of the two fungal species during degradation, but similar phenolic contents at the end of wood degradation., (© 2021 The Authors. Phytochemical Analysis published by John Wiley & Sons Ltd.)

Objective: Splenectomy is often performed during open thoracoabdominal aortic aneurysm (TAAA) repair, because capsular tears are common and can be associated with significant bleeding. It is unknown whether splenectomy affects the short- or long-term outcomes after TAAA repair., Methods: All open type I to IV TAAA repairs performed from 1987 to June 2015 were evaluated using a single institutional database. The primary endpoints were in-hospital death, major adverse events (MAE) and long-term survival. The secondary endpoint was hospital length of stay (LOS). All repairs performed for aneurysm rupture were excluded. Univariate analysis was conducted using the Fisher's exact test for categorical variables and the Wilcoxon rank sum test for continuous variables. Logistic and linear multivariable regression were used for the in-hospital endpoints, and survival analyses were performed using Cox proportional hazards modeling and Kaplan-Meier techniques., Results: A total of 649 patients met the study inclusion criteria. Of the 649 patients, 150 (23%) underwent concurrent splenectomy (CS) and six required emergency splenectomy secondary to bleeding postoperatively, leading to 156 cases of total in-hospital splenectomy. The perioperative mortality rate was 5.2% in the CS group and 5.2% in the non-CS group (P = 1.0). MAE were experienced by 48% of the CS patients compared with 34% of the non-CS patients (P = .003). Multivariable analysis revealed splenectomy was not independently predictive of perioperative death (adjusted odds ratio, 0.95; 95% confidence interval [CI], 0.41-2.23; P = .9). However, splenectomy was independently associated with any MAE (adjusted odds ratio, 1.78; 95% CI, 1.19-2.65; P = .005). Splenectomy was also associated with a longer length of stay (+5.39 days; 95% CI, 1.86-8.92; P = .003). No survival difference was found between the cohorts in the total splenectomy cohort in the unadjusted (log-rank P = 1.0) or adjusted (splenectomy adjusted hazard ratio, 1.02; 95% confidence interval, 0.78-1.35; P = .9)., Conclusions: CS during open TAAA repair did not lead to increased perioperative mortality but did lead to significantly increased perioperative morbidity and longer hospital lengths of stay. We found no difference in long-term survival outcomes when CS was performed. Splenectomy during TAAA repair did not affect long-term survival., (Copyright © 2021 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Lapatinib and tofacitinib are small-molecule kinase inhibitors approved for the treatment of advanced or metastatic breast cancer and rheumatoid arthritis, respectively. So far, the mechanisms which are responsible for their activities are not entirely understood. Here, we focus on the interaction of these drug molecules with phospholipid membranes, which has not yet been investigated before in molecular detail. Owing to their lipophilic characteristics, quantitatively reflected by large differences of the partition equilibrium between water and octanol phases (expressed by logP values), rather drastic differences in the membrane interaction of both molecules have to be expected. Applying experimental (nuclear magnetic resonance, fluorescence and ESR spectroscopy) and theoretical (molecular dynamics simulations) approaches, we found that lapatinib and tofacitinib bind to lipid membranes and insert into the lipid-water interface of the bilayer. For lapatinib, a deeper embedding into the membrane bilayer was observed than for tofacitinib implying different impacts of the molecules on the bilayer structure. While for tofacitinib, no influence to the membrane structure was found, lapatinib causes a membrane disturbance, as concluded from an increased permeability of the membrane for polar molecules. These data may contribute to a better understanding of the cellular uptake mechanism(s) and the side effects of the drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12 -/- ) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation., (© 2019 Federation of American Societies for Experimental Biology.)

Galectins (formerly known as "S-type lectins") are a subfamily of soluble proteins that typically bind β-galactoside carbohydrates with high specificity. They are present in many forms of life, from nematodes and fungi to animals, where they perform a wide range of functions. Particularly in humans, different types of galectins have been described differing not only in their tissue expression but also in their cellular location, oligomerization, fold architecture and carbohydrate-binding affinity. This distinct yet sometimes overlapping distributions and physicochemical attributes make them responsible for a wide variety of both intra- and extracellular functions, including tremendous importance in immunity and disease. In this review, we aim to provide a general description of galectins most important structural features, with a special focus on the molecular determinants of their carbohydrate-recognition ability. For that purpose, we structurally compare the human galectins, in light of recent mutagenesis studies and novel X-ray structures. We also offer a detailed description on how to use the solvent structure surrounding the protein as a tool to get better predictions of galectin-carbohydrate complexes, with a potential application to the rational design of glycomimetic inhibitory compounds. Finally, using Gal-1 and Gal-3 as paramount examples, we review a series of recent advances in the development of engineered galectins and galectin inhibitors, aiming to dissect the structure-activity relationship through the description of their interaction at the molecular level., (Copyright © 2019 Modenutti, Capurro, Di Lella and Martí.)

Background: Antioxidative enzymes contribute to a parasite's ability to counteract the host's intracellular killing mechanisms. The facultative intracellular oyster parasite, Perkinsus marinus, a sister taxon to dinoflagellates and apicomplexans, is responsible for mortalities of oysters along the Atlantic coast of North America. Parasite trophozoites enter molluscan hemocytes by subverting the phagocytic response while inhibiting the typical respiratory burst. Because P. marinus lacks catalase, the mechanism(s) by which the parasite evade the toxic effects of hydrogen peroxide had remained unclear. We previously found that P. marinus displays an ascorbate-dependent peroxidase (APX) activity typical of photosynthetic eukaryotes. Like other alveolates, the evolutionary history of P. marinus includes multiple endosymbiotic events. The discovery of APX in P. marinus raised the questions: From which ancestral lineage is this APX derived, and what role does it play in the parasite's life history?, Results: Purification of P. marinus cytosolic APX activity identified a 32 kDa protein. Amplification of parasite cDNA with oligonucleotides corresponding to peptides of the purified protein revealed two putative APX-encoding genes, designated PmAPX1 and PmAPX2. The predicted proteins are 93% identical, and PmAPX2 carries a 30 amino acid N-terminal extension relative to PmAPX1. The P. marinus APX proteins are similar to predicted APX proteins of dinoflagellates, and they more closely resemble chloroplastic than cytosolic APX enzymes of plants. Immunofluorescence for PmAPX1 and PmAPX2 shows that PmAPX1 is cytoplasmic, while PmAPX2 is localized to the periphery of the central vacuole. Three-dimensional modeling of the predicted proteins shows pronounced differences in surface charge of PmAPX1 and PmAPX2 in the vicinity of the aperture that provides access to the heme and active site., Conclusions: PmAPX1 and PmAPX2 phylogenetic analysis suggests that they are derived from a plant ancestor. Plant ancestry is further supported by the presence of ascorbate synthesis genes in the P. marinus genome that are similar to those in plants. The localizations and 3D structures of the two APX isoforms suggest that APX fulfills multiple functions in P. marinus within two compartments. The possible role of APX in free-living and parasitic stages of the life history of P. marinus is discussed.

Endogenous lectins can control critical biological responses, including cell communication, signaling, angiogenesis and immunity by decoding glycan-containing information on a variety of cellular receptors and the extracellular matrix. Galectin-1 (Gal-1), a prototype member of the galectin family, displays only one carbohydrate recognition domain and occurs in a subtle homodimerization equilibrium at physiologic concentrations. Such equilibrium critically governs the function of this lectin signaling by allowing tunable interactions with a preferential set of glycosylated receptors. Here, we used a combination of experimental and computational approaches to analyze the kinetics and mechanisms connecting Gal-1 ligand unbinding and dimer dissociation processes. Kinetic constants of both processes were found to differ by an order of magnitude. By means of steered molecular dynamics simulations, the ligand unbinding process was followed monitoring water occupancy changes. By determining the water sites in a carbohydrate binding place during the unbinding process, we found that rupture of ligand-protein interactions induces an increase in energy barrier while ligand unbinding process takes place, whereas the entry of water molecules to the binding groove and further occupation of their corresponding water sites contributes to lowering of the energy barrier. Moreover, our findings suggested local asymmetries between the two subunits in the dimer structure detected at a nanosecond timescale. Thus, integration of experimental and computational data allowed a more complete understanding of lectin ligand binding and dimerization processes, suggesting new insights into the relationship between Gal-1 structure and function and renewing the discussion on the biophysics and biochemistry of lectin-ligand lattices., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Antibodies against spike proteins of influenza are used as a tool for characterization of viruses and therapeutic approaches. However, development, production and quality control of antibodies is expensive and time consuming. To circumvent these difficulties, three peptides were derived from complementarity determining regions of an antibody heavy chain against influenza A spike glycoprotein. Their binding properties were studied experimentally, and by molecular dynamics simulations. Two peptide candidates showed binding to influenza A/Aichi/2/68 H3N2. One of them, termed PeB, with the highest affinity prevented binding to and infection of target cells in the micromolar region without any cytotoxic effect. PeB matches best the conserved receptor binding site of hemagglutinin. PeB bound also to other medical relevant influenza strains, such as human-pathogenic A/California/7/2009 H1N1, and avian-pathogenic A/Mute Swan/Rostock/R901/2006 H7N1. Strategies to improve the affinity and to adapt specificity are discussed and exemplified by a double amino acid substituted peptide, obtained by substitutional analysis. The peptides and their derivatives are of great potential for drug development as well as biosensing.

Proteins undergo dynamic structural changes to function within the range of physical and chemical conditions of their microenvironments. Changes in these environments affect their activity unless the respective mutations preserve their proper function. Here, we examine the influenza A virus spike protein hemagglutinin (HA), which undergoes a dynamic conformational change that is essential to the viral life cycle and is dependent on endosomal pH. Since the cells of different potential hosts exhibit different levels of pH, the virus can only cross species barriers if HA undergoes mutations that still permit the structural change to occur. This key event occurs after influenza A enters the host cell via the endocytic route, during its intracellular transport inside endosomes. The acidic pH inside these vesicles triggers a major structural transition of HA that induces fusion of the viral envelope and the endosomal membrane, and permits the release of the viral genome. HA experiences specific mutations that alter its pH stability and allow the conformational changes required for fusion in different hosts, despite the differences in the degree of acidification of their endosomes. Experimental and theoretical studies over the past few years have provided detailed insights into the structural aspects of the mutational changes that alter its susceptibility to different pH thresholds. We will illustrate how such mutations modify the protein's structure and consequently its pH stability. These changes make HA an excellent model of the way subtle structural modifications affect a protein's stability and enable it to function in diverse environments., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recognition and complex formation between proteins and carbohydrates is a key issue in many important biological processes. Determination of the three-dimensional structure of such complexes is thus most relevant, but particularly challenging because of their usually low binding affinity. In silico docking methods have a long-standing tradition in predicting protein-ligand complexes, and allow a potentially fast exploration of a number of possible protein-carbohydrate complex structures. However, determining which of these predicted complexes represents the correct structure is not always straightforward. In this work, we present a modification of the scoring function provided by AutoDock4, a widely used docking software, on the basis of analysis of the solvent structure adjacent to the protein surface, as derived from molecular dynamics simulations, that allows the definition and characterization of regions with higher water occupancy than the bulk solvent, called water sites. They mimic the interaction held between the carbohydrate -OH groups and the protein. We used this information for an improved docking method in relation to its capacity to correctly predict the protein-carbohydrate complexes for a number of tested proteins, whose ligands range in size from mono- to tetrasaccharide. Our results show that the presented method significantly improves the docking predictions. The resulting solvent-structure-biased docking protocol, therefore, appears as a powerful tool for the design and optimization of development of glycomimetic drugs, while providing new insights into protein-carbohydrate interactions. Moreover, the achieved improvement also underscores the relevance of the solvent structure to the protein carbohydrate recognition process.

The hybridization performance of a set of 12-mer RNA:RNA duplexes containing 2'-C-methyluridine, 5-bromo-2'-C-methyluridine, or (2'S)-2'-deoxy-2'-C-methyluridine was analyzed. Melting point temperatures of the modified duplexes showed an important ΔT(m) decrease (-8.9 to -12.5 °C), while circular dichroism experiments indicated that the helix was still A-type, suggesting a localized disturbance disorder. Molecular dynamics simulations using AMBER were carried out in order to gain structural knowledge about the effect of the 2'-C-methyl modification in double stranded environments. On the other hand, in an attempt to explain the behavior of the 2'-deoxy-2'-C-methyl nucleosides in single stranded environments, like the 10-23 DNAzyme core, molecular dynamic simulations were performed, incorporating the modified analogues into single stranded reported stem-loop structures, studding the sugar conformations along the MD trajectories. It was observed that, despite their preferential conformational states, the 2'-C-methyl analogues are flexible enough to adopt a different puckering in single stranded environments.

In the past decade, increasing efforts have been devoted to the study of galectins, a family of evolutionarily conserved glycan-binding proteins with multifunctional properties. Galectins function, either intracellularly or extracellularly, as key biological mediators capable of monitoring changes occurring on the cell surface during fundamental biological processes such as cellular communication, inflammation, development, and differentiation. Their highly conserved structures, exquisite carbohydrate specificity, and ability to modulate a broad spectrum of biological processes have captivated a wide range of scientists from a wide spectrum of disciplines, including biochemistry, biophysics, cell biology, and physiology. However, in spite of enormous efforts to dissect the functions and properties of these glycan-binding proteins, limited information about how structural and biochemical aspects of these proteins can influence biological functions is available. In this review, we aim to integrate structural, biochemical, and functional aspects of this bewildering and ancient family of glycan-binding proteins and discuss their implications in physiologic and pathologic settings., (© 2011 American Chemical Society)

Galectins, a family of evolutionarily conserved animal lectins, have been shown to modulate signaling processes leading to inflammation, apoptosis, immunoregulation, and angiogenesis through their ability to interact with poly-N-acetyllactosamine-enriched glycoconjugates. To date 16 human galectin carbohydrate recognition domains have been established by sequence analysis and found to be expressed in several tissues. Given the divergent functions of these lectins, it is of vital importance to understand common and differential features in order to search for specific inhibitors of individual members of the human galectin family. In this work we performed an integrated computational analysis of all individual members of the human galectin family. In the first place, we have built homology-based models for galectin-4 and -12 N-terminus, placental protein 13 (PP13) and PP13-like protein for which no experimental structural information is available. We have then performed classical molecular dynamics simulations of the whole 15 members family in free and ligand-bound states to analyze protein and protein-ligand interaction dynamics. Our results show that all galectins adopt the same fold, and the carbohydrate recognition domains are very similar with structural differences located in specific loops. These differences are reflected in the dynamics characteristics, where mobility differences translate into entropy values which significantly influence their ligand affinity. Thus, ligand selectivity appears to be modulated by subtle differences in the monosaccharide binding sites. Taken together, our results may contribute to the understanding, at a molecular level, of the structural and dynamical determinants that distinguish individual human galectins.

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Lectins are able to recognize specific carbohydrate structures through their carbohydrate recognition domain (CRD). The lectin from the mushroom Agaricus bisporus (ABL) has the remarkable ability of selectively recognizing the TF-antigen, composed of Galβ1-3GalNAc, Ser/Thr linked to proteins, specifically exposed in neoplastic tissues. Strikingly, the recently solved crystal structure of tetrameric ABL in the presence of TF-antigen and other carbohydrates showed that each monomer has two CRDs, each being able to bind specifically to different monosaccharides that differ only in the configuration of a single hydroxyl, like N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc). Understanding how lectin CRDs bind and discriminate mono and/or (poly)-saccharides is an important issue in glycobiology, with potential impact in the design of better and selective lectin inhibitors with potential therapeutic properties. In this work, and based on the unusual monosaccharide epimeric specificity of the ABL CRDs, we have performed molecular dynamics simulations of the natural (crystallographic) and inverted (changing GalNAc for GlcNAc and vice-versa) ABL-monosaccharide complexes in order to understand the selective ligand recognition properties of each CRD. We also performed a detailed analysis of the CRD local solvent structure, using previously developed methodology, and related it with the recognition mechanism. Our results provide a detailed picture of each ABL CRD specificity, allowing a better understanding of the carbohydrate selective recognition process in this particular lectin., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The stability of proteins involves a critical balance of interactions of different orders of magnitude. In this work, we present experimental evidence of an increased thermal stability of galectin-1, a multifunctional beta-galactoside-binding protein, upon binding to the disaccharide lactose. Analysis of structural changes occurring upon binding of lectin to its specific glycans and thermal denaturation of the protein and the complex were analyzed by circular dichroism. On the other hand, we studied dimerization as another factor that may induce structural and thermal stability changes. The results were then complemented with molecular dynamics simulations followed by a detailed computation of thermodynamic properties, including the internal energy, solvation free energy, and conformational entropy. In addition, an energetic profile of the binding and dimerization processes is also presented. Whereas binding and cross-linking of lactose do not alter galectin-1 structure, this interaction leads to substantial changes in the flexibility and internal energy of the protein which confers increased thermal stability to this endogenous lectin. Given that an improved understanding of the physicochemical properties of galectin-glycan lattices may contribute to the dissection of their biological functions and prediction of their therapeutic applications, our study suggests that galectin binding to specific disaccharide ligands may increase the thermal stability of this glycan-binding protein, an effect that could influence its critical biological functions.

Formation of protein ligand complexes is a fundamental phenomenon in biochemistry. During the process, significant solvent reorganization is produced along the contact surface and many water molecules strongly bound to the protein's ligand binding site must be displaced. Both the thermodynamics and kinetics of this process are complex and a clear understanding at the microscopic level has been not achieved so far. Special attention has been paid to the structure of water molecules on carbohydrate recognition sites of various proteins, and many studies support the idea that displacement of these water molecules should have a crucial effect on the binding free energy. Molecular dynamics (MD) simulations in explicit water solvent is a very promising approach for this type of studies. Using MD simulations combined with statistical mechanics analysis, thermodynamic properties of these water molecules can be computed and analyzed in a comparative view. Using this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hGal-1). Specifically, we defined water sites (WS) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. In the present work, we selected a group of proteins whose ligand bound complexes have been already structurally characterized in order to extend the analysis of the role of the surface associated water molecules in the ligand binding and recognition process. The selected proteins are concanavalin-A (Con-A), galectin-3 (Gal-3), cyclophilin-A (Cyp-A), and two modules CBM40 and CBM32 of the multimodular bacterial sialidase. Our results show that the probability of finding water molecules inside the WS, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an hydroxyl group of the ligand in the protein-ligand complex. This information can be used to analyze in detail the solvation structure of the carbohydrate recognition domain (CRD) and its relation to the possible protein ligand complexes and suggests addition of OH-containing functional groups to displace water from high p(v) WS to enhance drugs, specially glycomimetic-drugs, protein affinity, and/or specificity.

Galectin-1 (Gal-1), a member of a family of evolutionarily conserved glycan-binding proteins, binds specifically to poly-N-acetyllactosamine-enriched glycoconjugates. Through interactions with these glycoconjugates, this protein modulates inflammatory responses and contributes to tumor progression and immune cell homeostasis. The carbohydrate recognition domain includes the single protein tryptophan (Trp68). UV resonance Raman spectroscopy and molecular dynamic simulation were used to examine the change in the environment of the Trp on ligand binding. The UV Raman spectra and the calculated water radial distribution functions show that, while no large structural changes in the protein follow lactose binding, substantial solvent reorganization occurs. These new insights into the microscopic role of water molecules in Gal-1 binding to its specific carbohydrate ligands provides a better understanding of the physicochemical properties of Gal-1-saccharide interactions, which will be useful for the design of synthetic inhibitors for therapeutic purposes.

Human galectin-1, a galactosil-terminal sugar binding soluble protein, is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein interactions. Recent studies revealed that it plays a key role as a modulator of cellular differentiation and immunological response. In this work, we have investigated the solvation properties of the carbohydrate recognition domain of Gal-1 by means of molecular dynamics simulations. Water sites (ws) were identified in terms of radial and angular distribution functions, and properties such as water residence times, interaction energies, and free-energy contributions were evaluated for those sites. Our results allowed us to correlate the thermodynamic properties of the ws and their binding pattern with the N-acetilgalactoside ligand. These results let us further infer that the water molecules located at the ws, which exhibit much more favorable binding, are the ones replaced by -OH groups of the sugar.

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.