The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.

Type Vicenza variant of von Willebrand disease (VWD) is characterized by a low plasma von Willebrand factor (VWF) level and supranormal VWF multimers. Two candidate mutations, G2470A and G3864A at exons 17 and 27, respectively, of the VWF gene were recently reported to be present in this disorder. Four additional families, originating from northeast Italy, with both mutations of type Vicenza VWD are now described. Like the original type Vicenza subjects, they showed a mild bleeding tendency and a significant decrease in plasma VWF antigen level and ristocetin cofactor activity but normal platelet VWF content. Unlike the original patients, ristocetin-induced platelet aggregation was found to be normal. Larger than normal VWF multimers were also demonstrated in the plasma. Desmopressin (DDAVP) administration increased factor VIII (FVIII) and VWF plasma levels, with the appearance of even larger multimers. However, these forms, and all VWF oligomers, disappeared rapidly from the circulation. The half-life of VWF antigen release and of elimination was significantly shorter than that in healthy counterparts, so that at 4 hours after DDAVP administration, VWF antigen levels were close to baseline. Similar behavior was demonstrated by VWF ristocetin cofactor activity and FVIII. According to these findings, it is presumed that the low plasma VWF levels of type Vicenza VWD are mainly attributed to reduced survival of the VWF molecule, which, on the other hand, is normally synthesized. In addition, because normal VWF-platelet GPIb interaction was observed before or after DDAVP administration, it is proposed that type Vicenza VWD not be considered a 2M subtype. (Blood. 2002;99:180-184)

SUMMARY 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.

SummaryA deletion/insertion polymorphism (4G or 5G) in the promoter of the plasminogen activator inhibitor type 1 gene has been suggested to be involved in regulation of the synthesis of the inhibitor, the 4G allele being associated with enhanced gene expression. A relationship between 4G/5G polymorphism and PAI-1 levels was found in patients with cardiovascular and metabolic diseases, but not in healthy subjects. In the present work we studied the distribution of PAI-1 4G/5G geno-type and its relation to fibrinolytic capacity in 70 unrelated patients with deep vein thrombosis. Each patient was assayed before and after 20 min. Venous occlusion for euglobulin lysis time, t-PA antigen and activity, and PAI-1 antigen and activity. The prevalence of 5G homozygous carriers was significantly lower in patients than in controls (10% vs. 26%, p = 0.009). The 5G allele frequency was reduced, even though not significantly, in DVT patients compared to healthy subjects (0.40 vs. 0.51, respectively). In the patient group, the mean PAI-1 antigen and activity levels were significantly higher than among controls and related to the 4G/5G polymorphism. In patients with 4G/5G and 4G/4G genotype a significant correlation was found between PAI-1 levels and the global fibrinolytic activity as evaluated by euglobulin lysis time. The prevalence of a reduced fibrinolytic potential due to PAI-1 excess was 45.7% among DVT patients. Moreover, the prevalence of PAI-1 induced hypofibrinolysis was strongly related to PAI-1 polymorphism, since it was significantly lower in 5G homo-zygous patients (28.6%) than in both 4G/5G carriers (55.3%, p <0.001) and 4G homozygous patients (57.9%, p <0.001).In conclusion, in patients with deep vein thrombosis the 4G polymorphism of PAI-1 gene promoter may influence the expression of PAI-1 and it should be taken into consideration as a facilitating condition for pathological fibrinolysis together with other environmental and genetic factors. Whether this has any significance in regard to the pathogenesis of venous thrombosis remains to be proven.

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.

PurposeThe purpose of this study was to test the ability of DAC®, a fast resorbable, antibacterial-loaded hydrogel coating, to prevent acute bacterial colonization in an in vivo model of an intra-operatively highly contaminated implant.MethodsA histocompatibility study was performed in 10 adult New Zealand rabbits. Then, methicillin-resistant Staph. aureus were inoculated in the femur of 30 adult New Zealand rabbits at the time of intra-medullary nailing; vancomycin-loaded DAC® coated nails were compared to controls regarding local and systemic infection development.ResultsHistocompatibility study showed no detrimental effect of DAC® hydrogel on bone tissue after 12 weeks from implant. After seven days from implant, none of the rabbits receiving vancomycin-loaded DAC® nail showed positive blood cultures, compared to all the controls; vancomycin-loaded DAC® coating was associated with local bacterial load reduction ranging from 72 to 99 %, compared to controls.ConclusionsVancomycin-loaded DAC® coating is able to significantly reduce bacterial colonization in an animal model of an intra-operatively highly contaminated implant, without local or general side effect.

The relationship between venous thromboembolism (VTE) and cancer is supported by several pathogenetic factors, including circulating microparticles (MP) originating from different cells and often bearing tissue factor. Since VTE often complicates the clinical course of patients with glioblastoma multiforme (GBM; WHO grade IV astrocytoma) and the role of MPs in these patients population is still not clear, this prospective study was conducted to evaluate the procoagulant activity of circulating MP (MP activity) in GBM patients. We enrolled 61 GBM patients undergoing gross-total or subtotal surgical resection followed by combined radio-chemotherapy; 20 healthy volunteers were tested as controls. Blood samples for MP activity and hemostatic profiles were obtained before and then 1 week and 1, 4, and 7 months after surgery. GBM patients had significantly higher mean MP activity levels than healthy controls before and 7 days after surgery. During the follow-up, MP activity levels became significantly lower 1 and 4 months after surgery (P = 0.007 and P = 0.018, respectively) than prior to surgery, but this decrease was only seen in the subgroup achieving complete tumor resection. MP activity levels increased in 7 (63.6%) of 11 patients who developed VTE. The different incidence of the increase in MP activity levels between patients with and without VTE was statistically significant (χ (2) = 4.93, P = 0.026; relative risk 1.38, 95% CI 1.03-1.86). GBM patients may have an increase in MP-associated procoagulant activity that could contribute to any prothrombotic states and increases the likelihood of VTE complications; this procoagulant activity drops during control of disease.

Genetic and acquired factors may influence phenotypic expression of inherited thrombophilia. Hypofibrinolysis due to excess PAI-1 can be found in patients with deep vein thrombosis (DVT) and 4G/5G polymorphism of the PAI-1 gene may modulate the inhibitor's synthesis. In 149 patients with inherited thrombophilia, the possible thrombotic contribution of both 4G/5G polymorphism and PAI-1 plasma levels was evaluated. Sixty-seven patients with idiopathic DVT and 98 normal subjects were also studied. By comparison with controls, a significantly higher prevalence of 4G/4G genotype was seen in idiopathic DVT and in thrombophilia patients, although in this latter group the difference only remained significant in cases symptomatic for thrombosis (p = 0.01). The 4G/4G genotype was associated with a greater risk of thrombosis both in symptomatic thrombophilia patients (OR 2.85, 95% CI 1.26-6.46) and in idiopathic DVT patients (OR 3.1, 95% CI 1.26-7.59). The greater frequency of 4G allele in symptomatic thrombophilia patients with respect to controls was statistically significant (p = 0.04). Compared to healthy subjects, PAI-1:Ag levels were higher in symptomatic thrombophilia patients and related to the 4G/5G polymorphism, with significantly higher values in the 4G/4G carriers. In conclusion, PAI-1 4G/5G polymorphism may influence PAI-1 expression and thrombotic risk in patients with inherited thrombophilia.