Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with ...associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.
Research on ubiquitin (Ub) signaling has focused primarily on homogeneously linked polyUb. Although polyUb containing different linkages within the same chain exist, their structures and signaling ...properties are unknown. These mixed-linkage chains could be unbranched (i.e., no more than one lysine or methionine linkage per Ub) or branched. Here, we examined the structure, dynamics, receptor selectivity, and disassembly of branched and unbranched tri-Ub containing both K48 and K63 linkages. Each linkage was virtually indistinguishable from its counterpart in homogeneously linked polyUb. Linkage-selective receptors from hHR23A and Rap80 preferentially bound to the K48 or K63 linkages in the branched trimer. Linkage-selective deubiquitinases specifically cleaved their cognate Ub-Ub linkages in mixed-linkage chains, and the 26S proteasome recognized and processed branched tri-Ub. We conclude that mixed-linkage chains retain the distinctive signaling properties of their K48 and K63 components and that these multiple signals can be recognized by multiple linkage-specific receptors. Finally, we propose a new, comprehensive notation for Ub and Ub-like polymers.
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► K48 and K63 linkages in branched and unbranched tri-Ub retain their structural features ► Linkage-selective receptors recognize K48 and K63 linkages in the branched tri-Ub ► Linkage-specific deubiquitinases cleave their cognate Ub-Ub linkages in the mixed-linkage chains ► The 26S proteasome recognizes and processes branched K48- and K63-linked tri-Ub
The outcomes of linkage mixing in polyubiquitin are poorly understood. Nakasone et al. show that Lys48 and Lys63 linkages retain their distinctive structural and signaling properties in mixed-linkage branched and unbranched chains, suggesting a possibly of encoding multiple signals in the same polyubiquitin chain.
Sex-mismatched hematopoietic cell transplantation is linked to increased graft-versus-host disease and mortality in myeloablative conditioning. Here we evaluated outcomes of 1,041 adult transplant ...recipients at two centers between 2006 and 2013 and investigated how the effect of sex-mismatching differed in myeloablative, reduced-intensity, and non-myeloablative total lymphoid irradiation with anti-thymocyte globulin conditioning. Among patients who underwent myeloablative conditioning, male recipients with female donors had increased chronic graft-versus-host disease (hazard ratio 1.83, P<0.01), increased non-relapse mortality (hazard ratio 1.84, P=0.022) and inferior overall survival (hazard ratio 1.59, P=0.018). In contrast, among patients who received reduced-intensity conditioning, male recipients with female donors had increased acute graft-versus-host disease (hazard ratio 1.96, P<0.01) but no difference in non-relapse mortality or overall survival. Among the patients who underwent total lymphoid irradiation with anti-thymocyte globulin, male recipients with female donors showed no increase in graft-versus-host disease or non-relapse mortality. Notably, only in the cohort receiving total lymphoid irradiation with anti-thymocyte globulin were male recipients with female donors significantly associated with reduced relapse (hazard ratio 0.64, P<0.01), and allo-antibody responses against H-Y antigens were predictive of reduced relapse. In the cohort given total lymphoid irradiation with anti-thymocyte globulin, the graft-versus-leukemia effect resulted in superior overall survival in recipients of sex-mismatched grafts (HR 0.69, P=0.037). In addition, only in the cohort treated with total lymphoid irradiation with anti-thymocyte globulin were female recipients with male donors associated with reduced relapse (hazard ratio 0.59, P<0.01) and superior survival (hazard ratio 0.61, P=0.014) compared with sex-matched pairs. We conclude that the risks and benefits of sex-mismatched transplants appear to differ according to conditioning strategy and this could affect donor selection.
Abstract Background Controversy continues regarding the use of powerful anticoagulants for venous thromboembolism prophylaxis in patients undergoing Total Knee Arthroplasty (TKA). In order to comply ...with institution-mandated guidelines and pressure from hospitalist intent on complying with conventionally recommended anticoagulation guidelines, we singularly changed our chemoprophylaxis practice from using aspirin to Lovenox and noted that transfusion rates increased substantially. Methods A retrospective case review was performed to evaluate transfusion requirement differences in primary TKA patients receiving Lovenox (Unilateral TKA: n=135, Bilateral TKA: n=44) or aspirin (Unilateral TKA: n=153, Bilateral TKA: n=45) for venous thromboembolism prophylaxis. Pearsons chi-square tests were used to evaluate surgical complications and the rate of transfusions between aspirin and Lovenox groups. Independent t-tests were used to evaluate the units of packed red blood cells transfused, hemoglobin drop, and hematocrit drop between aspirin and Lovenox groups. Results Lovenox was found to significantly increase ( P < 0.01) the rate of transfusion, units of packed red blood cells, hemoglobin drop, and hematocrit drop compared to aspirin in both unilateral and bilateral TKA patients, without significantly decreasing venous thromboembolism events (aspirin: 3 pulmonary embolisms and 4 deep venous thrombosis; Lovenox: 3 pulmonary embolisms and 2 deep venous thrombosis). Conclusion Our findings suggest that aspirin is as effective as Lovenox in preventing venous thromboembolism, and that the use of Lovenox significantly increases the likelihood of requiring transfusions following surgery.
In Brazil, the main strategy adopted to contain Visceral Leishmaniasis (VL) is the controversial culling of dogs with reagent serology for Canine VL (CVL). Despite there are studies showing that ...significant reduction of human cases has not been observed, as well as there are works demonstrating the occurrence of false-positive results in the confirmatory test, the protocol has been maintained. Researches that can reinforce the existence and persistence of this problem, as well as bring concrete alternatives are pivotal. In this context, the aim of this work was to evaluate and compare the serological, molecular and parasitological methods employed for CVL detection in Brazil, in dogs with diverse clinical profiles, from two endemic areas of Pernambuco state. Comparisons among TR-DPP®, EIE-LVC and qPCR (animals from Goiana-PE: 91) demonstrated that agreements varied from ‘poor’ to ‘moderate’ (kappa = 0.162–0.442), and a triple agreement occurred in 61.36% (54/88) of the samples. The highest percentage of agreement was obtained between TR-DPP® and EIE-LVC within the polysymptomatic group (93.33%; 14/15). Of the 34 dogs with reagent serology from Caruaru-PE, 17 (50%) and 29 dogs (85.29%) were positive for qPCR and parasitological exam, respectively. By comparing serology, qPCR and parasitological exam, the lowest percentage of agreements were obtained within the asymptomatic group (40%–72.72%). It was possible to observe that the percentage of agreement tended to decrease according to the absence of clinical manifestations in the dogs. Thus, from the fact that all diagnostic tools evaluated have their limitations, it is very important to be careful before to propose an alternative set of diagnostic criteria. Besides, the answer for better results in the control of CVL may not be in the choose of the best set of diagnostic tools, but it may be in the strategy of culling itself. In this context, it is very important to invest in alternative control measures, such as mass vaccination and treatment of dogs, thus reducing the transmission to the vector and helping to avoid new canine and human cases.
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•There was no good or excellent agreement between none of the tools analyzed for CVL.•The percentage agreement between the methods tended to decrease in asymptomatic dogs.•False-positive and false-negative results may be occurring in the control programs.•The strategy of culling dogs with CVL must be rigorously reanalyzed and discussed.•It is pivotal to invest in alternative measures for CVL control in Brazil.
Sensors and actuators based on piezoelectric plates have shown increasing demand in the field of smart structures, including the development of actuators for cooling and fluid-pumping applications ...and transducers for novel energy-harvesting devices. This project involves the development of a topology optimization formulation for dynamic design of piezoelectric laminated plates aiming at piezoelectric sensors, actuators and energy-harvesting applications. It distributes piezoelectric material over a metallic plate in order to achieve a desired dynamic behavior with specified resonance frequencies, modes, and enhanced electromechanical coupling factor (EMCC). The finite element employs a piezoelectric plate based on the MITC formulation, which is reliable, efficient and avoids the shear locking problem. The topology optimization formulation is based on the PEMAP-P model combined with the RAMP model, where the design variables are the pseudo-densities that describe the amount of piezoelectric material at each finite element and its polarization sign. The design problem formulated aims at designing simultaneously an eigenshape, i.e., maximizing and minimizing vibration amplitudes at certain points of the structure in a given eigenmode, while tuning the eigenvalue to a desired value and also maximizing its EMCC, so that the energy conversion is maximized for that mode. The optimization problem is solved by using sequential linear programming. Through this formulation, a design with enhancing energy conversion in the low-frequency spectrum is obtained, by minimizing a set of first eigenvalues, enhancing their corresponding eigenshapes while maximizing their EMCCs, which can be considered an approach to the design of energy-harvesting devices. The implementation of the topology optimization algorithm and some results are presented to illustrate the method.
UK Black African/Black Caribbean women remain disproportionately affected by HIV. Although oral pre-exposure prophylaxis (PrEP) could offer them an effective HIV prevention method, uptake remains ...limited. This study examined barriers and facilitators to PrEP awareness and candidacy perceptions for Black African/Black Caribbean women to help inform PrEP programmes and service development.
Using purposive sampling through community organisations, 32 in-depth, semi-structured interviews were conducted with Black African/Black Caribbean women living in London and Glasgow between June and August 2018. Participants (aged 19-63) included women of varied HIV statuses to explore perceptions of sexual risk and safer sex, sexual health knowledge and PrEP attitudes. A thematic analysis guided by the Social Ecological Model was used to explore how PrEP perceptions intersected with wider safer sex understandings and practices.
Four key levels of influence shaping safer sex notions and PrEP candidacy perceptions emerged: personal, interpersonal, perceived environment and policy. PrEP-specific knowledge was low and some expressed distrust in PrEP. Many women were enthusiastic about PrEP for others but did not situate PrEP within their own safer sex understandings, sometimes due to difficulty assessing their own HIV risk. Many felt that PrEP could undermine intimacy in their relationships by disrupting the shared responsibility implicit within other HIV prevention methods. Women described extensive interpersonal networks that supported their sexual health knowledge and shaped their interactions with health services, though these networks were influenced by prevailing community stigmas.
Difficulty situating PrEP within existing safer sex beliefs contributes to limited perceptions of personal PrEP candidacy. To increase PrEP uptake in UK Black African/Black Caribbean women, interventions will need to enable women to advance their knowledge of PrEP within the broader context of their sexual health and relationships. PrEP service models will need to include trusted 'non-sexual health-specific' community services such as general practice.
Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub ...signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of UbPT, and we expect that its future uses will help define and investigate the ubiquitin interactome.
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•Photoleucine was successfully incorporated into fully synthetic ubiquitin monomers•Embedded photoleucine permitted binding to the hydrophobic patch of ubiquitin•Enzymatically polymerized ubiquitin phototrap captured Ub-binding receptors•The first PC region of Rpn1, either isolated or proteasome-incorporated, bound polyUb
Application of polyubiquitin-phototrap (polyUbPT), a novel set of chain-specific inducible photo-crosslinking probes, enables trapping of transient partners through the hydrophobic patch of ubiquitin. PolyUbPT captured Rpn1 from intact proteasome complexes. Rpn1 joins Rpn10 and Rpn13 as a proteasome subunit with affinity for polyUb and Ub-like domains.
Focal lesions within the subchondral bone, termed subchondral bone cysts (SBCs), are clinically accepted radiographic markers of advanced osteoarthritis (OA), but their etiology in the hip is not ...well understood.
This study used micro-computed tomography (μCT), and histological and immunocytological analysis to examine the prevalence, size, location, and morphological and cellular features of SBCs found within 34 femoral heads (14 male, 20 female; age range = 43–80 years) obtained from total hip arthroplasty procedures.
SBCs were common—present in 91% of the femoral heads examined—and frequently commuted with the surface of the femoral head, but otherwise showed no preferred anatomical location. Few associations were found between SBC features and patient characteristics such as BMI, age and sex. SBCs were also heterogenous in composition, ranging from fibrous (most common) to predominantly fatty (least common) and often containing vasculature, nerve fibers, cartilage islands, and bony spicules. Despite this heterogeneity, focal abnormalities in bone density and cartilage thickness were consistently observed. Bone adjacent to SBCs was denser than that in the primary compressive group, and cartilage thickness in regions overlying SBCs was lower than in non-overlying regions. In contrast to these local bony changes, μCT-based finite element analyses indicated that the stiffness of the primary compressive group was only mildly affected by SBCs.
These findings indicate that SBCs in the femoral head involve extensive perturbations in cellular activity, culminating in myriad skeletal tissue types and spatially heterogenous changes in bone and cartilage morphology that are likely to affect OA progression.
Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ...ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles.
•Di-Ubs of all possible lysine linkages were made and studied by NMR and DUB assays•K27-Ub2 has unique NMR characteristics, dynamics, and resistance to DUB cleavage•K27-Ub2 adopts open conformations in solution capable of bidentate binding to receptors•K27-Ub2 binds UBA2 domain of hHR23A through bidentate interactions similar to K48-Ub2
Castañeda et al. assembled diubiquitin chains of every lysine linkage and found that K27 linkage exhibits unique structural and dynamic characteristics. It is the most resistant to deubiquitinases and adopts open conformations capable of bidentate interactions with receptors. Similar to K48-diubiquitin, K27-diubiquitin forms a sandwich-like complex with hHR23A-UBA2.
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