Local Flaps to Cover Skin Necrosis after Skin-sparing Mastectomy and Prepectoral Reconstruction from PreQ-20 Trial.

Background: In recent years, mastectomy has increasingly been indicated for women at high risk and those with breast cancer. Prepectoral reconstruction with polyurethane implant is an option for these patients. Nevertheless, this procedure can become complicated with exposure of the implant. The aim of this article is to describe the feasibility of local flaps to treat skin necrosis and dehiscence after prepectoral reconstruction and its impact on implant loss. Methods: This study includes the women who met the inclusion/exclusion criteria of the PreQ-20 protocol (12), which assessed patients with exposed implant who required a local flap for its coverage. Three types of flaps were used: thoracoepigastric, lateral thoracic, and batwing. Results: The study included 226 skin-sparing mastectomies and immediate reconstruction using prepectoral implants (52.7% bilateral mastectomies). Some 20.9% of the patients showed complications, with wound dehiscence the most frequent. Thirteen local flaps to cover the implant were performed. All flaps presented appropriate perfusion; however, the implant cover failed in six patients (46.2%). Conclusions: The use of local flaps can be a low-morbidity option for preventing implant loss when skin dehiscence or necrosis occurs and delays in oncology treatments.

Outcomes of initially nonoperative management of diverticulitis with abscess formation in immunosuppressed patients. DIPLICAB study COLLABORATIVE group.

AIM: Management of diverticulitis with abscess formation in immunosuppressed patients (IMS) remains unclear. The main objective of the study was to assess short- and long-term outcomes between IMS and immunocompetent patients (IC). The secondary aim was to identify risk factors for emergency surgery. METHODS: A nationwide retrospective cohort study was performed at 29 Spanish referral centres between 2015-2019 including consecutive patients with first episode of diverticulitis classified as modified Hinchey Ib or II. IMS included immunosuppressive therapy, biologic therapy, malignant neoplasm with active chemotherapy and chronic steroid therapy. A multivariate analysis was performed to identify independent risk factors to emergency surgery in IMS. RESULTS: A total of 1395 patients were included; 118 IMS and 1277 IC. There were no significant differences in emergency surgery between IMS and IC (19.5% and 13.5%, p = 0.075) but IMS was associated with higher mortality (15.1% vs. 0.6%, p < 0.001). Similar recurrent episodes were found between IMS and IC (28% vs. 28.2%, p = 0.963). Following multivariate analysis, immunosuppressive treatment, p = 0.002; OR: 3.35 (1.57-7.15), free gas bubbles, p < 0.001; OR: 2.91 (2.01-4.21), Hinchey II, p = 0.002; OR: 1.88 (1.26-2.83), use of morphine, p < 0.001; OR: 3.08 (1.98-4.80), abscess size ≥5 cm, p = 0.001; OR: 1.97 (1.33-2.93) and leucocytosis at third day, p < 0.001; OR: 1.001 (1.001-1.002) were independently associated with emergency surgery in IMS. CONCLUSION: Nonoperative management in IMS has been shown to be safe with similar treatment failure than IC. IMS presented higher mortality in emergency surgery and similar rate of recurrent diverticulitis than IC. Identifying risk factors to emergency surgery may anticipate emergency surgery.

Probing Changes in the Local Structure of Active Bimetallic Mn/Ru Oxides during Oxygen Evolution.

Identifying the active site of catalysts for the oxygen evolution reaction (OER) is critical for the design of electrode materials that will outperform the current, expensive state-of-the-art catalyst, RuO2. Previous work shows that mixed Mn/Ru oxides show comparable performances in the OER, while reducing reliance on this expensive and scarce Pt-group metal. Herein, X-ray photoelectron spectroscopy and X-ray absorption spectroscopy (XAS) are performed on mixed Mn/Ru oxide materials for the OER to understand structural and chemical changes at both metal sites during oxygen evolution. The results show that the Mn-content affects both the oxidation state and local coordination environment of Ru sites. Operando XAS experiments suggest that the presence of MnOx might be essential to achieve high activity likely by facilitating changes in the O-coordination sphere of Ru centers.

Diverticulitis with abscess formation: Outcomes of non-operative management and nomogram for predicting emergency surgery: The Diplicab Study Collaborative Group.

BACKGROUND: To assess short- and long-term outcomes from non-surgical management of diverticulitis with abscess formation and to develop a nomogram to predict emergency surgery. METHODS: This nationwide retrospective cohort study was performed in 29 Spanish referral centers, including patients with a first episode of a diverticular abscess (modified Hinchey Ib-II) from 2015 to 2019. Emergency surgery, complications, and recurrent episodes were analyzed. Regression analysis was used to assess risk factors, and a nomogram for emergency surgery was designed. RESULTS: Overall, 1,395 patients were included (1,078 Hinchey Ib and 317 Hinchey II). Most (1,184, 84.9%) patients were treated with antibiotics without percutaneous drainage, and 194 (13.90%) patients required emergency surgery during admission. Percutaneous drainage (208 patients) was associated with a lower risk of emergency surgery in patients with abscesses of ≥5 cm (19.9% vs 29.3%, P = .035; odds ratio 0.59 [0.37-0.96]). The multivariate analysis showed that immunosuppression treatment, C-reactive protein (odds ratio: 1.003; 1.001-1.005), free pneumoperitoneum (odds ratio: 3.01; 2.04-4.44), Hinchey II (odds ratio: 2.15; 1.42-3.26), abscess size 3 to 4.9 cm (odds ratio: 1.87; 1.06-3.29), abscess size ≥5 cm (odds ratio: 3.62; 2.08-6.32), and use of morphine (odds ratio: 3.68; 2.29-5.92) were associated with emergency surgery. A nomogram was developed with an area under the receiver operating characteristic curve of 0.81 (95% confidence interval: 0.77-0.85). CONCLUSION: Percutaneous drainage must be considered in abscesses ≥5 cm to reduce emergency surgery rates; however, there are insufficient data to recommend it in smaller abscesses. The use of the nomogram could help the surgeon develop a targeted approach.

Nanoscaffold effects on the performance of air-cathodes for microbial fuel cells: Sustainable Fe/N-carbon electrocatalysts for the oxygen reduction reaction under neutral pH conditions.

Nanostructured electrocatalysts for microbial fuel cell air-cathodes were obtained via use of conductive carbon blacks for the synthesis of high performing 3D conductive networks. We used two commercially available nanocarbons, Black Pearls 2000 and multiwalled carbon nanotubes, as conductive scaffolds for the synthesis of nanocomposite electrodes by combining: a hydrothermally carbonized resin, a sacrificial polymeric template, a nitrogenated organic precursor and iron centers. The resulting materials are micro-mesoporous, possess high specific surface area and display N-sites (N/C of 3-5 at%) and Fe-centers (Fe/C < 1.5at.%) at the carbon surface as evidenced from characterization methods. Voltammetry studies of oxygen reduction reaction activity were carried out at neutral pH, which is relevant to microbial fuel cell applications, and activity trends are discussed in light of catalyst morphology and composition. Tests of the electrocatalyst using microbial fuel cell devices indicate that optimization of the nanocarbon scaffold for the Pt-free carbon-based electrocatalysts results in maximum power densities that are 25% better than those of Pt/C cathodes, at a fraction of the materials costs. Therefore, the proposed Fe/N-carbon catalysts are promising and sustainable high-performance cathodic materials for microbial fuel cells.

Untangling Cooperative Effects of Pyridinic and Graphitic Nitrogen Sites at Metal-Free N-Doped Carbon Electrocatalysts for the Oxygen Reduction Reaction.

Metal-free carbon electrodes with well-defined composition and smooth topography are prepared via sputter deposition followed by thermal treatment with inert and reactive gases. X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy show that three carbons of similar N/C content that differ in N-site composition are thus prepared: an electrode consisting of almost exclusively graphitic-N (NG ), an electrode with predominantly pyridinic-N (NP ), and one with ≈1:1 NG :NP composition. These materials are used as model systems to investigate the activity of N-doped carbons in the oxygen reduction reaction (ORR) using voltammetry. Results show that selectivity toward 4e-reduction of O2 is strongly influenced by the NG /NP site composition, with the material possessing nearly uniform NG /NP composition being the only one yielding a 4e-reduction. Computational studies on model graphene clusters are carried out to elucidate the effect of N-site homogeneity on the reaction pathway. Calculations show that for pure NG -doping or NP -doping of model graphene clusters, adsorption of hydroperoxide and hydroperoxyl radical intermediates, respectively, is weak, thus favoring desorption prior to complete 4e-reduction to hydroxide. Clusters with mixed NG /NP sites display synergistic effects, suggesting that co-presence of these sites improves activity and selectivity by achieving high theoretical reduction potentials while facilitating retention of intermediates.

PKCθ and HIV-1 Transcriptional Regulator Tat Co-exist at the LTR Promoter in CD4(+) T Cells.

PKCθ is essential for the activation of CD4(+) T cells. Upon TCR/CD28 stimulation, PKCθ is phosphorylated and migrates to the immunological synapse, inducing the activation of cellular transcription factors such as NF-κB and kinases as ERK that are critical for HIV-1 replication. We previously demonstrated that PKCθ is also necessary for HIV-1 replication but the precise mechanism is unknown. Efficient HIV-1 transcription and elongation are absolutely dependent on the synergy between NF-κB and the viral regulator Tat. Tat exerts its function by binding a RNA stem-loop structure proximal to the viral mRNA cap site termed TAR. Besides, due to its effect on cellular metabolic pathways, Tat causes profound changes in infected CD4(+) T cells such as the activation of NF-κB and ERK. We hypothesized that the aberrant upregulation of Tat-mediated activation of NF-κB and ERK occurred through PKCθ signaling. In fact, Jurkat TetOff cells with stable and doxycycline-repressible expression of Tat (Jurkat-Tat) expressed high levels of mRNA for PKCθ. In these cells, PKCθ located at the plasma membrane was phosphorylated at T(538) residue in undivided cells, in the absence of stimulation. Treatment with doxycycline inhibited PKCθ phosphorylation in Jurkat-Tat, suggesting that Tat expression was directly related to the activation of PKCθ. Both NF-κB and Ras/Raf/MEK/ERK signaling pathway were significantly activated in Jurkat-Tat cells, and this correlated with high transactivation of HIV-1 LTR promoter. RNA interference for PKCθ inhibited NF-κB and ERK activity, as well as LTR-mediated transactivation even in the presence of Tat. In addition to Tat-mediated activation of PKCθ in the cytosol, we demonstrated by sequential ChIP that Tat and PKCθ coexisted in the same complex bound at the HIV-1 LTR promoter, specifically at the region containing TAR loop. In conclusion, PKCθ-Tat interaction seemed to be essential for HIV-1 replication in CD4(+) T cells and could be used as a therapeutic target.

Shoc2/Sur8 protein regulates neurite outgrowth.

The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction.

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids.

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.

The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.

Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein.

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.

Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction.

Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59-64 and 303-307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor-ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.

Modification and activation of Ras proteins by electrophilic prostanoids with different structure are site-selective.

Cyclopentenone prostanoids (cyP) arise as important modulators of inflammation and cell proliferation. Although their physiological significance has not been fully elucidated, their potent biological effects have spurred their study as leads for the development of therapeutic agents. A key determinant of cyP action is their ability to bind to thiol groups in proteins or in glutathione through Michael addition. Even though several protein targets for cyP addition have been identified, little is known about the structural determinants from the protein or the cyP that drive this modification. The results herein presented provide the first evidence that cyP with different structures target distinct thiol sites in a protein molecule, namely, H-Ras. Whereas 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and Delta12-PGJ2 preferentially target the C-terminal region containing cysteines 181 and 184, PGA1 and 8-iso-PGA1 bind mainly to cysteine 118, located in the GTP-binding motif. The biological counterparts of this specificity are the site-selective modification and activation of H-Ras in cells and the differential interaction of cyP with H, N, and K-Ras proteins. Cysteine 184 is unique to H-Ras, whereas cysteine 118 is present in the three Ras homologues. Consistent with this, PGA1 binds to and activates H-, N-, and K-Ras, thus differing from the preferential interaction of 15d-PGJ2 with H-Ras. These results put forward the possibility of influencing the selectivity of cyP-protein addition by modifying cyP structure. Furthermore, they may open new avenues for the development of cyP-based drugs.