System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

Histological and histomorphometric analysis of bone tissue using customized titanium meshes with or without resorbable membranes: A randomized clinical trial.

OBJECTIVES: To date, no clinical studies have investigated the effect of using resorbable collagen membrane in conjunction with customized titanium mesh to promote bone formation in guided bone regeneration. Therefore, a non-inferiority analysis (one-sided 95% CI approach) was designed to compare the augmented bone gained using meshes with and without collagen membranes, through histological and histomorphometric investigations. MATERIALS AND METHODS: Thirty patients undergoing bone augmentation procedures at both maxillary and mandible sites were randomly treated with customized titanium meshes alone (M-, n = 15) or covered with resorbable membrane (M+, n = 15), in both cases filled with autogenous bone and xenograft. After 6 months of healing, bone tissue biopsies were taken from the augmented region. The bone tissue (B.Ar), grafting material (G.Ar), and non-mineralized tissue (NMT.Ar) areas were quantified through histomorphometric analysis, as were the osteoid area (O.Ar) and its width. RESULTS: Collagen membrane did not appear to significantly influence the investigated parameters: B.Ar, G.Ar, NMT.Ar, and O.Ar were similar between Group M- (34.3%, 11.5%, 54.1%, 1.95 µm2 , respectively) and Group M+ (35.3%, 14.6%, 50.2%, and 1.75 µm2 , respectively). Considering the overall population, significantly higher rates of newly formed bone were obtained in mandibular sites, while non-mineralized and dense connective tissue rates were higher in the maxilla (p < .05). CONCLUSIONS: The application of collagen membrane over titanium mesh did not lead to significant results. Bone formation appeared significantly different in the maxilla compared with the mandible. Additional studies are required to further investigate the issues observed.

A 3D micro-CT assessment of composition and structure of bone tissue after vertical and horizontal alveolar ridge augmentation using CAD/CAM-customized titanium mesh.

OBJECTIVES: To date, no studies have exploited micro-CT in humans to evaluate bone morphology and structure after bone augmentation with CAD/CAM-customized titanium mesh, in mandible and maxilla. The aim of this study was to assess the composition and microstructure of bone biopsy through micro-CT analysis. MATERIALS AND METHODS: Bone augmentation at both maxillary and mandible sites was performed on 30 patients randomly treated with customized mesh, either alone (M-) or covered with resorbable membrane (M+), in both cases filled 50:50 with autogenous bone and xenograft. After 6 months, biopsies were taken and micro-CT was performed on consecutive 1-mm-thick VOIs from coronal to apical side, measuring tissue volumes, trabecular thickness, spacing, and number. RESULTS: In both groups, irrespective of membrane use, bone tissue (M-: 29.76% vs. M+: 30.84%) and residual graft material (M-: 14.87% vs. M+: 13.11%) values were similar. Differences were site-related (maxillary vs. mandibular) with higher percentage of bone tissue and trabecular density of low-mineralized bone and overall bone in the mandible. CONCLUSIONS: The composition and structure of bone tissue, as assessed by micro-CT after alveolar ridge augmentation using CAD/CAM-customized titanium meshes, showed similar features regardless of whether a collagen membrane was applied.

An Advanced Human Bone Tissue Culture Model for the Assessment of Implant Osteointegration In Vitro.

In the field of biomaterials for prosthetic reconstructive surgery, there is the lack of advanced innovative methods to investigate the potentialities of smart biomaterials before in vivo tests. Despite the complex osteointegration process being difficult to recreate in vitro, this study proposes an advanced in vitro tissue culture model of osteointegration using human bone. Cubic samples of trabecular bone were harvested, as waste material, from hip arthroplasty; inner cylindrical defects were created and assigned to the following groups: (1) empty defects (CTRneg); (2) defects implanted with a cytotoxic copper pin (CTRpos); (3) defects implanted with standard titanium pins (Ti). Tissues were dynamically cultured in mini rotating bioreactors and assessed weekly for viability and sterility. After 8 weeks, immunoenzymatic, microtomographic, histological, and histomorphometric analyses were performed. The model was able to simulate the effects of implantation of the materials, showing a drop in viability in CTR+, while Ti appears to have a trophic effect on bone. MicroCT and a histological analysis supported the results, with signs of matrix and bone deposition at the Ti implant site. Data suggest the reliability of the tested model in recreating the osteointegration process in vitro with the aim of reducing and refining in vivo preclinical models.

Vertebral Bone Marrow Clot towards the Routine Clinical Scenario in Spine Surgeries: What about the Antimicrobial Properties?

Exploring innovative techniques and treatments to improve spinal fusion procedures is a global challenge. Here, we provide a scientific opinion on the ability of a vertebral bone marrow (vBM) clot to provide a local combined delivery system not only of stem cells, signaling biomolecules and anti-inflammatory factors but also of molecules and proteins endowed with antimicrobial properties. This opinion is based on the evaluation of the intrinsic basic properties of the vBM, that contains mesenchymal stem cells (MSCs), and on the coagulation process that led to the conversion of fibrinogen into fibrin fibers that enmesh cells, plasma but above all platelets, to form the clot. We emphasize that vBM clot, being a powerful source of MSCs and platelets, would allow the release of antimicrobial proteins and molecules, mainly cathelicidin LL- 37, hepcidin, kinocidins and cationic host defense peptides, that are per se gifted with direct and/or indirect antimicrobial effects. We additionally highlight that further studies are needed to deepen this knowledge and to propose vBM clot as multifunctional bioscaffold able to target all the main key challenges for spinal fusion surgery.

Magnesium Alloys in Orthopedics: A Systematic Review on Approaches, Coatings and Strategies to Improve Biocompatibility, Osteogenic Properties and Osteointegration Capabilities.

There is increasing interest in using magnesium (Mg) alloy orthopedic devices because of their mechanical properties and bioresorption potential. Concerns related to their rapid degradation have been issued by developing biodegradable micro- and nanostructured coatings to enhance corrosion resistance and limit the release of hydrogen during degradation. This systematic review based on four databases (PubMed®, Embase, Web of Science™ and ScienceDirect®) aims to present state-of-the-art strategies, approaches and materials used to address the critical factors currently impeding the utilization of Mg alloy devices. Forty studies were selected according to PRISMA guidelines and specific PECO criteria. Risk of bias assessment was conducted using OHAT and SYRCLE tools for in vitro and in vivo studies, respectively. Despite limitations associated with identified bias, the review provides a comprehensive analysis of preclinical in vitro and in vivo studies focused on manufacturing and application of Mg alloys in orthopedics. This attests to the continuous evolution of research related to Mg alloy modifications (e.g., AZ91, LAE442 and WE43) and micro- and nanocoatings (e.g., MAO and MgF2), which are developed to improve the degradation rate required for long-term mechanical resistance to loading and excellent osseointegration with bone tissue, thereby promoting functional bone regeneration. Further research is required to deeply verify the safety and efficacy of Mg alloys.

Bone Regeneration Guided by a Magnetized Scaffold in an Ovine Defect Model.

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

Gender-Specific Differences in Human Vertebral Bone Marrow Clot.

Recently, our group described the application of vertebral bone marrow (vBMA) clot as a cell therapy strategy for spinal fusion. Its beneficial effects were confirmed in aging-associated processes, but the influence of gender is unknown. In this study, we compared the biological properties of vBMA clots and derived vertebral mesenchymal stem cells (MSCs) from female and male patients undergoing spinal fusion procedures and treated with vBMA clot. We analyzed the expression of growth factors (GFs) in vBMA clots and MSCs as well as morphology, viability, doubling time, markers expression, clonogenicity, differentiation ability, senescence factors, Klotho expression, and HOX and TALE gene profiles from female and male donors. Our findings indicate that vBMA clots and derived MSCs from males had higher expression of GFs and greater osteogenic and chondrogenic potential compared to female patients. Additionally, vBMA-clot-derived MSCs from female and male donors exhibited distinct levels of HOX and TALE gene expression. Specifically, HOXA1, HOXB8, HOXD9, HOXA11, and PBX1 genes were upregulated in MSCs derived from clotted vBMA from male donors. These results demonstrate that vBMA clots can be effectively used for spinal fusion procedures; however, gender-related differences should be taken into consideration when utilizing vBMA-clot-based studies to optimize the design and implementation of this cell therapy strategy in clinical trials.

Effects of dietary copper and zinc hydroxychloride supplementation on bone development, skin quality and hematological parameters of broilers chickens.

This study was carried out to evaluate the effects of supplementation with different levels of copper (Cu) and zinc (Zn), using two mineral sources (sulphate and hydroxy forms), on the bone characteristics, skin strength/elasticity, and haematological parameters of broilers. A total of 1792 1-day-old male Cobb-500 broiler chickens were randomly distributed among eight dietary treatments, using Cu sulphate (CSM) or hydroxychloride (CHC), and Zn sulphate (ZSM) or hydroxychloride (ZHC). The dietary treatments were as follows: (1) low-CSM/high-ZSM, (2) high-CSM/high-ZSM, (3) low-CHC/low-ZHC, (4) low-CHC/medium-ZHC, (5) low-CHC/high-ZHC, (6) high-CHC/low-ZHC, (7) high-CHC/medium-ZHC, and (8) high-CHC/high-ZHC. On Day 42, blood samples were collected from one bird/pen to analyze the haematological parameters. Finally, two birds/pen were slaughtered, and the tibia and femur were collected to analyze the quality of bone and skin. The means were subjected to ANOVA and, when significant, compared by Tukey's test (p < 0.05) or Dunnett's (p < 0.05) test. The haematological parameters were not influenced by mineral supplementation. However, the inclusion of low ZHC enhanced the skin strength compared to high ZHC (p = 0.046). Furthermore, the bone mineral density of the tibia proximal epiphysis, tibia ash and tibia mineral content were positively improved with supplementation of low-CHC/medium-ZHC compared to high-CHC/medium-ZHC. This study demonstrated that hydroxy compounds are potential alternatives for replacing sulphate supplements in broiler diets. Moreover, among the Cu and Zn levels, the low CHC (15 mg/kg) and medium ZHC (100 mg/kg) improved bone development and skin integrity, suggesting that the combination of Cu and Zn can be a nutritional strategy to prevent the incidence of leg disorders in broilers.

Intake and digestibility of two diets for nine-banded armadillos (Dasypus novemcinctus: Linnaeus, 1758) kept under human care.

The study aimed to evaluate metabolic parameters, nutrient intake, and absorption of two diets formulated for Dasypus novemcinctus armadillos under human care. Were studied two diets: D1-a diet with dry dog food, ground beef, and boiled chicken egg with shells; D2-a diet with the same ingredients as D1, with added banana and papaya. Both are mixed in water. The parameters analyzed were body weight (BW), weight gain (WG), maintenance energy requirement (MER), Lee index, biometrics, body condition score, glucose, triglycerides, total cholesterol, and cholesterol fractions for eight male armadillos. Dietary intake (DI) and efficiency, nutrient intake, metabolizable energy, and digestibility coefficient for the diets were evaluated in six male armadillos. The diet that included fruits showed higher BW, WG, MER, Lee index, and better glucose metabolism. Both diets promoted increases in WG and Lee Index, as well as improvements in glucose metabolism. The diet without fruit improved the lipid profile of the animals. D2 presented the highest DI, better dietary efficiency, and higher energy intake but also a lower crude fiber intake. However, it showed the best utilization of gross fiber and all other nutrients. In conclusion, the diets constituted an adequate nutritional option for captive armadillos and can be used in malnutrition and pathological processes recovery. Although both diets were adequate, the nonfruit diet was preferable due to the improved lipid profile.

Antiosteoporotic Nanohydroxyapatite Zoledronate Scaffold Seeded with Bone Marrow Mesenchymal Stromal Cells for Bone Regeneration: A 3D In Vitro Model.

BACKGROUND: Bisphosphonates are widely employed drugs for the treatment of pathologies with high bone resorption, such as osteoporosis, and display a great affinity for calcium ions and apatitic substrates. Here, we aimed to investigate the potentiality of zoledronate functionalized hydroxyapatite nanocrystals (HAZOL) to promote bone regeneration by stimulating adhesion, viability, metabolic activity and osteogenic commitment of human bone marrow derived mesenchymal stromal cells (hMSCs). METHODS: we adopted an advanced three-dimensional (3D) in vitro fracture healing model to study porous scaffolds: hMSCs were seeded onto the scaffolds that, after three days, were cut in halves and unseeded scaffolds were placed between the two halves. Scaffold characterization by X-ray diffraction, transmission and scanning electron microscopy analyses and cell morphology, viability, osteogenic differentiation and extracellular matrix deposition were evaluated after 3, 7 and 10 days of culture. RESULTS: Electron microscopy showed a porous and interconnected structure and a uniform cell layer spread onto scaffolds. Scaffolds were able to support cell growth and cells progressively colonized the whole inserts in absence of cytotoxic effects. Osteogenic commitment and gene expression of hMSCs were enhanced with higher expressions of ALPL, COL1A1, BGLAP, RUNX2 and Osterix genes. CONCLUSION: Although some limitations affect the present study (e.g., the lack of longer experimental times, of mechanical stimulus or pathological microenvironment), the obtained results with the adopted experimental setup suggested that zoledronate functionalized scaffolds (GHAZOL) might sustain not only cell proliferation, but positively influence osteogenic differentiation and activity if employed in bone fracture healing.

Use of low-molecular weight heparin, transfusion and mortality in COVID-19 patients not requiring ventilation.

It is still debated whether prophylactic doses of low-molecular- weight heparin (LMWH) are always effective in preventing Venous Thromboembolism (VTE) and mortality in COVID-19. Furthermore, there is paucity of data for those patients not requiring ventilation. We explored mortality and the safety/efficacy profile of LMWH in a cohort of Italian patients with COVID-19 who did not undergo ventilation. From the initial cohort of 422 patients, 264 were enrolled. Most (n = 156, 87.7%) received standard LMWH prophylaxis during hospitalization, with no significant difference between medical wards and Intensive Care Unit (ICU). Major or not major but clinically relevant hemorrhages were recorded in 13 (4.9%) patients: twelve in those taking prophylactic LMWH and one in a patient taking oral anticoagulants (p: n.s.). Thirty-nine patients (14.8%) with median age 75 years. were transfused. Hemoglobin (Hb) at admission was significantly lower in transfused patients and Hb at admission inversely correlated with the number of red blood cells units transfused (p < 0.001). In-hospital mortality occurred in 76 (28.8%) patients, 46 (24.3%) of whom admitted to medical wards. Furthermore, Hb levels at admittance were significantly lower in fatalities (g/dl 12.3; IQR 2.4 vs. 13.3; IQR 2.8; Mann-Whitney U-test; p = 0.001). After the exclusion of patients treated by LMWH intermediate or therapeutic doses (n = 32), the logistic regression showed that prophylaxis significantly and independently reduced mortality (OR 0.31, 95% CI 0.13-0.85). Present data show that COVID-19 patients who do not require ventilation benefit from prophylactic doses of LMWH.

Histological examination of a retrieved custom-made 3D-printed titanium vertebra : Do the fine details obtained by additive manufacturing really promote osteointegration?

PURPOSE: In the present report it is described the design, the manufacturing and the successful surgical implant of one of the first 3D custom titanium vertebra realized with Additive Manufacturing technique and its use for the spinal reconstruction after en-bloc resection for primary osteogenic sarcoma. METHODS: Clinical case presentation and the design of the 3D custom titanium vertebra was reported. It was also described the complex procedures adopted to evaluate the retrieved device from the histological point of view, as a tumor relapse hit the patient, one year after the reconstruction procedure. RESULTS: The histological evaluation confirmed that the resection technique exerts an important role in promoting bone formation: vertebral body osteotomies favored the reconstruction procedure and maximized the contact area between host bone/vertebral prosthesis thus favoring the bone tissue penetration and device colonization. CONCLUSION: The sharing of these results is very important as they represent the starting point for improving the knowledge starting from the evidence obtained in a challenging clinical condition and with post-operative treatments that could be never reproduced in preclinical model.

Genetic variability in Brazilian castor (Ricinus communis) germplasm assessed by morphoagronomic traits and gray mold reaction.

The characterization and conservation of castor accessions in germplasm bank are essential in order to breeding programs achieve its goals. Despite Brazil having the 4th largest castor germplasm bank in the world, castor diversity in Brazil remains little explored. Thus, this study aimed at characterize castor accessions collected in different Brazilian regions by means of 31 morphoagronomic traits and gray mold reaction. Forty accessions of the Universidade do Estado de São Paulo (UNESP), Botucatu, SP, Brazil, germplasm bank were evaluated. Genetic parameters were estimated for the quantitative traits, and the accessions were grouped by Ward method using the standardized Euclidean distance and the simple coincidence index for quantitative and qualitative data, respectively. Qualitative and quantitative traits were important to understand and differentiate castor accessions. The accessions showed a high variation regarding the castor gray mold reaction. The accessions assessed in this study have been preserved and can be used as a source for genetic variability in the development of new castor varieties in breeding programs.

Vitamin D and Platelets: A Menacing Duo in COVID-19 and Potential Relation to Bone Remodeling.

Global data correlate severe vitamin D deficiency with COVID-19-associated coagulopathy, further suggesting the presence of a hypercoagulable state in severe COVID-19 patients, which could promote thrombosis in the lungs and in other organs. The feedback loop between COVID-19-associated coagulopathy and vitamin D also involves platelets (PLTs), since vitamin D deficiency stimulates PLT activation and aggregation and increases fibrinolysis and thrombosis. Vitamin D and PLTs share and play specific roles not only in coagulation and thrombosis but also during inflammation, endothelial dysfunction, and immune response. Additionally, another 'fil rouge' between vitamin D and PLTs is represented by their role in mineral metabolism and bone health, since vitamin D deficiency, low PLT count, and altered PLT-related parameters are linked to abnormal bone remodeling in certain pathological conditions, such as osteoporosis (OP). Hence, it is possible to speculate that severe COVID-19 patients are characterized by the presence of several predisposing factors to bone fragility and OP that may be monitored to avoid potential complications. Here, we hypothesize different pervasive actions of vitamin D and PLT association in COVID-19, also allowing for potential preliminary information on bone health status during COVID-19 infection.

Evidence from systematic reviews on photobiomodulation of human bone and stromal cells: Where do we stand?

This study summarizes the available evidence from systematic reviews on the in vitro effects of photobiomodulation on the proliferation and differentiation of human bone and stromal cells by appraising their methodological quality. Improvements for future studies are also highlighted, with particular emphasis on in vitro protocols and cell-related characteristics. Six reviews using explicit eligibility criteria and methods selected in order to minimize bias were included. There was no compelling evidence on the cellular mechanisms of action or treatment parameters of photobiomodulation; compliance with quality assessment was poor. A rigorous description of laser parameters (wavelength, power, beam spot size, power density, energy density, repetition rate, pulse duration or duty cycle, exposure duration, frequency of treatments, and total radiant energy), exposure conditions (methods to ensure a uniform irradiation and to avoid cross-irradiation, laser-cell culture surface distance, lid presence during irradiation) and cell-related characteristics (cell type or line, isolation and culture conditions, donor-related factors where applicable, tissue source, cell phenotype, cell density, number of cell passages in culture) should be included among eligibility criteria for study inclusion. These methodological improvements will maximize the contribution of in vitro studies on the effects of photobiomodulation on human bone and stromal cells to evidence-based translational research.

Platelet Features and Derivatives in Osteoporosis: A Rational and Systematic Review on the Best Evidence.

Background: With the increase in aging population, the rising prevalence of osteoporosis (OP) has become an important medical issue. Accumulating evidence showed a close relationship between OP and hematopoiesis and emerging proofs revealed that platelets (PLTs), unique blood elements, rich in growth factors (GFs), play a critical role in bone remodeling. The aim of this review was to evaluate how PLT features, size, volume, bioactive GFs released, existing GFs in PLTs and PLT derivatives change and behave during OP. Methods: A systematic search was carried out in PubMed, Scopus, Web of Science Core Collection and Cochrane Central Register of Controlled Trials databases to identify preclinical and clinical studies in the last 10 years on PLT function/features and growth factor in PLTs and on PLT derivatives during OP. The methodological quality of included studies was assessed by QUIPS tool for assessing risk of bias in the clinical studies and by the SYRCLE tool for assessing risk of bias in animal studies. Results: In the initial search, 2761 studies were obtained, only 47 articles were submitted to complete reading, and 23 articles were selected for the analysis, 13 on PLT function/features and growth factor in PLTs and 10 on PLT derivatives. Risk of bias of almost all animal studies was high, while the in the clinical studies risk of bias was prevalently moderate/low for the most of the studies. The majority of the evaluated studies highlighted a positive correlation between PLT size/volume and bone mineralization and an improvement in bone regeneration ability by using PLTs bioactive GFs and PLT derivatives. Conclusions: The application of PLT features as OP markers and of PLT-derived compounds as therapeutic approach to promote bone healing during OP need to be further confirmed to provide clear evidence for the real efficacy of these interventions and to contribute to the clinical translation.

Prophylactic protocol for dental care in ligneous gingivitis due to severe plasminogen deficiency: Case report and review of literature.

INTRODUCTION: Severe plasminogen (PLG) deficiency causes ligneous conjunctivitis, a rare disease characterized by the growth of fibrin-rich pseudomembranes on mucosal surfaces; gums involvement leads to ligneous gingivitis (LG). Specific therapy for LG is not available yet. We report a prophylactic treatment with enoxaparin and fresh frozen plasma (FFP) for invasive dental procedures in a patient with LG, and a review of literature on LG treatment. METHODS: A 43-year-old female with LG was studied. In order to prevent LG recurrence after dental care, FFP before and the day after the procedure, and enoxaparin were administered in addition to proper minimally invasive dentistry techniques and implant surgery. RESULTS: Plasminogen deficiency was confirmed by reduced PLG antigen (25 µg/mL) and activity (20%) levels, and genetic analysis. PLG levels rose to 46% after FFP transfusion and returned to baseline after 48 hours. Minimally invasive dental procedures and implants were performed. Small gingival pseudomembranes developed soon thereafter in some cases but disappeared within a few weeks; no bleeding complications were observed. CONCLUSIONS: In our patient with LG, the adoption of combined haematological and dentistry protocols appeared to be safe and effective in preventing abnormal gingival pseudomembranes growth after dental interventions, maintaining a healthy periodontal condition.

PAI-1 and protein C as early markers of veno-occlusive disease in patients treated for Wilms tumor.

BACKGROUND: Hepatic veno-occlusive (VOD) disease has been described in hematopoietic stem cell transplantation (HSCT), solid tumors, and acute lymphoblastic leukemia. The incidence of VOD in Wilms tumor (WT) ranges from 1.2% to 8%. The diagnosis of VOD is clinical, and there are no validated laboratory biomarkers. PROCEDURE: We prospectively evaluated the specificity and sensitivity of plasminogen-activator inhibitor-1 (PAI-1) and protein C as diagnostic markers of VOD in WT patients. Fifty patients treated from 2008 to 2016 for WT were eligible. VOD was diagnosed according to modified Seattle criteria and retrospectively reclassified according to the recently published criteria for VOD in pediatric HSCT patients. RESULTS: VOD occurred in 6 of 50 patients (12%) after 20 to 97 days from starting chemotherapy. The average duration of VOD was 10 days (range, 4-13 days). PAI-1 levels were elevated in all VOD patients, while a decrease in protein C levels was observed in 33% of patients with VOD. PAI-1 antigen (Ag) values ≥ 26.4 ng/mL demonstrated high sensitivity and specificity for the clinical diagnosis of VOD with sensitivity 100%, specificity 93%; whereas protein C levels below 34.5% had sensitivity 67%, specificity 100%. Both PAI-1 and protein C had an high negative predictive value: PAI-1 Ag 100%; protein C 95%. CONCLUSIONS: PAI-1 Ag and protein C have good sensitivity and specificity for the diagnosis of VOD in WT patients. Their high negative predictive value can be used in the differential diagnosis of liver toxicity, especially in VOD episodes with absent or delayed hyperbilirubinemia.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.