Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.

The binding of different model membranes with PKCε C2 domain is not dependent on membrane curvature but affects the sequence of events during unfolding.

The C2 domain of novel protein kinases C (nPKC) binds to membranes in a Ca2+-independent way contributing to the activation of these enzymes. We have studied the C2 domain of one of these nPKCs, namely PKCε, and confirmed that it establishes a strong interaction with POPA, which is clearly visible through changes in chemical shifts detected through 31P-MAS-NMR and the protection that it exerts on the domain against thermal denaturation seen through DSC and FT-IR. In this study, using two-dimensional correlation analysis (2D-COS) applied to infrared spectra, we determined the sequence of events that occur during the thermal unfolding of the domain and highlighted some differences when phosphatidic acid or cardiolipin are present. Finally, by means of FRET and DLS experiments, we wanted to determine the effect of membrane curvature on the domain/membrane interaction by using lysophosphatidylcholine to introduce positive curvature as a control and we observed that the effect of these phospholipids on the protein binding is not exerted through the change of membrane curvature.

Interaction of Vitamin K1 and Vitamin K2 with Dimyristoylphosphatidylcholine and Their Location in the Membrane.

Vitamin K1 and vitamin K2 play very important biological roles as members of chains of electron transport as antioxidants in membranes and as cofactors for the posttranslational modification of proteins that participate in a number of physiological functions such as coagulation. The interaction of these vitamins with dimyristoylphosphatidylcholine (DMPC) model membranes has been studied by using a biophysical approach. It was observed by using differential scanning calorimetry that both vitamins have a very limited miscibility with DMPC and they form domains rich in the vitamins at high concentrations. Experiments using X-ray diffraction also showed the formation of different phases as a consequence of the inclusion of either vitamin K at temperatures below the phase transition. However, in the fluid state, a homogeneous phase was detected, and a decrease in the thickness of the membrane was accompanied by an increase in the water layer thickness. 2H NMR spectroscopy showed that both vitamins K induced a decrease in the onset of the phase transition, which was bigger for vitamin K1, and both vitamins decreased the order of the membrane as seen through the first moment (M1). 1H NOESY MAS-NMR showed that protons located at the rings or at the beginning of the lateral chain of both vitamins K interacted with a clear preference with protons located in the polar part of DMPC. On the other hand, protons located on the lateral chain have a nearer proximity with the methyl end of the myristoyl chains of DMPC. In agreement with the 2H NMR, ATR-FTIR (attenuated total reflectance Fourier transform infrared spectroscopy) indicated that both vitamins decreased the order parameters of DMPC. It was additionally deduced that the lateral chains of both vitamins were oriented almost in parallel to the myristoyl chains of the phospholipid.

Mechanical ventilator as a shared resource for the COVID-19 pandemic.

COVID-19, the causative agent of which is a new type of coronavirus called SARS-CoV-2, has caused the most severe pandemic in the last 100 years. The condition is mainly respiratory, and up to 5% of patients develop critical illness, a situation that has put enormous pressure on the health systems of affected countries. A high demand for care has mainly been observed in intensive care units and critical care resources, which is why the need to redistribute resources in critical medicine emerged, with an emphasis on distributive justice, which establishes the provision of care to the largest number of people and saving the largest number of lives. One principle lies in allocating resources to patients with higher life expectancy. Mechanical ventilator has been assumed to be an indivisible asset; however, simultaneous mechanical ventilation to more than one patient with COVID-19 is technically possible. Ventilator sharing is not without risks, but the principles of beneficence, non-maleficence and justice prevail. According to distributive justice, being a divisible resource, mechanical ventilator can be shared; however, we should ask ourselves if this action is ethically correct.

COVID-19, cuyo agente causal es un nuevo tipo de coronavirus denominado SARS-CoV-2, ha provocado la pandemia más grave en los últimos 100 años. La afección es principalmente respiratoria y hasta 5 % de los pacientes desarrolla enfermedad crítica, lo cual ha producido una enorme presión sobre los sistemas de salud de los países afectados. Principalmente se ha observado alta demanda de atención en las unidades de cuidados intensivos y de recursos de atención vital. De ahí la necesidad de redistribuir los recursos en medicina crítica, con énfasis en la justicia distributiva, la cual establece atender al mayor número de personas y salvar el mayor número de vidas. Un principio estriba en asignar los recursos a pacientes con mayores expectativas de vida. Se ha dado por hecho que el ventilador mecánico es un bien indivisible; sin embargo, técnicamente es posible la ventilación mecánica simultánea a más de un paciente con COVID-19. La acción de compartir el ventilador no está exenta de riesgos, pero prevalecen los principios de beneficencia, no maleficencia y justicia. Conforme la justicia distributiva, al ser un bien divisible, el ventilador mecánico puede ser compartido, sin embargo, cabe preguntarse si esta acción es éticamente correcta.

Anticancer Agent Edelfosine Exhibits a High Affinity for Cholesterol and Disorganizes Liquid-Ordered Membrane Structures.

Edelfosine is an anticancer drug with an asymmetric structure because, being a derivative of glycerol, it possesses two hydrophobic substituents of very different lengths. We showed that edelfosine destabilizes liquid-ordered membranes formed by either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine, sphingomyelin (SM), and cholesterol (1:1:1 molar ratio) or SM and cholesterol (2:1 molar ratio). This was observed by differential scanning calorimetry in which phase transition arises from either of these membrane systems after the addition of edelfosine. The alteration in the liquid-ordered domains was characterized by using a small-angle X-ray diffraction that revealed the formation of gel phases as a consequence of the addition of edelfosine at low temperatures and by a wide-angle X-ray diffraction that confirmed changes in the membranes, indicating the formation of these gel phases. The increase in phase transition derived by the edelfosine addition was further confirmed by Fourier-transform infrared spectroscopy. The effect of edelfosine was compared with that of structurally analogue lipids: platelet-activating factor and 1-palmitoyl-2-acetyl- sn-glycero-3-phosphocholine, which also have the capacity of destabilizing liquid-ordered domains, although they are less potent than edelfosine for this activity, and lysophosphatidylcholine, which lacks this capacity. It was concluded that edelfosine may be associated with cholesterol favorably competing with sphingomyelin, and that this sets sphingomyelin free to undergo a phase transition. Finally, the experimental observations can be described by molecular dynamics calculations in terms of intermolecular interaction energies in phospholipid-cholesterol membranes. Higher interaction energies between asymmetric phospholipids and cholesterol than between sphingomyelin and cholesterol were obtained. These results are interesting because they biophysically characterize one of the main molecular mechanisms to trigger apoptosis of the cancer cells.

Phenolic Group of α-Tocopherol Anchors at the Lipid-Water Interface of Fully Saturated Membranes.

α-Tocopherol is considered to carry on a very important role as an antioxidant for membranes and lipoproteins and other biological roles as membrane stabilizers and bioactive lipids. Given its essential role, it is very important to fully understand its location in the membrane. In this work, the vertical location of vitamin E in saturated membranes has been studied using biophysical techniques. Small- and wide-angle X-ray diffraction experiments show that α-tocopherol alters the water layer between bilayers in both 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), indicating its proximity to this surface. The quenching of the intrinsic fluorescence of α-tocopherol indicates a low quenching efficiency by acrylamide and a higher quenching by 5-doxyl-PC than by 9- and 16-doxyl-PC. These results suggest that in both DMPC and DPPC membranes, the chromanol ring is not far away from the surface of the membrane but within the bilayer. 1H nuclear Overhauser enhancement spectroscopy magic-angle spinning-nuclear magnetic resonance studies showed that α-tocopherol is localized in a similar manner in DMPC and DPPC membranes, with the chromanol ring embedded in the upper part of the hydrophobic bilayer. Using attenuated total reflection-Fourier transform infrared spectroscopy, it was observed that the tail chain of α-tocopherol lies nearly parallel to the acyl chains of DMPC and DPPC. Taking these results together, it was concluded that in both DMPC and DPPC, the hydroxyl group of the chromanol ring will establish hydrogen bonding with water on the membrane surface, and the main axis of the α-tocopherol molecule will be perpendicular to the bilayer plane.

Both idebenone and idebenol are localized near the lipid-water interface of the membrane and increase its fluidity.

Idebenone is a synthetic analog of coenzyme Q; both share a quinone moiety but idebenone has a shorter lipophilic tail ending with a hydroxyl group. Differential scanning calorimetry experiments showed that both idebenone and idebenol widened and shifted the phase transition of 1,2-dipalmitoylphosphatidylcholine (DPPC) to a lower temperature and a phase separation with different concentrations of these molecules was observed. Also small angle X-ray diffraction and wide angle X-ray diffraction revealed that both, idebenone and idebenol, induced laterally separated phases in fluid membranes when included in DPPC membranes. Electronic profiles showed that both forms, idebenone and idebenol, reduced the thickness of the fluid membrane. (2)H NMR measurements showed that the order of the membrane decreased at all temperatures in the presence of idebenone or idebenol, the greatest disorder being observed in the segments of the acyl chains close to the lipid-water interface. (1)H NOESY MAS NMR spectra were obtained using 1-palmitoyl-2-oleoyl-phosphatidylcholine membranes and results pointed to a similar location in the membrane for both forms, with the benzoquinone or benzoquinol rings and their terminal hydroxyl group of the hydrophobic chain located near the lipid/water interface of the phospholipid bilayer and the terminal hydroxyl group of the hydrophobic chain of both compounds located at the lipid/water interface. Taken together, all these different locations might explain the different physiological behavior shown by the idebenone/idebenol compared with the ubiquinone-10/ubiquinol-10 pair in which both compounds are differently localized in the membrane.

The increase in positively charged residues in cecropin D-like Galleria mellonella favors its interaction with membrane models that imitate bacterial membranes.

A comparative study of three synthetic peptides, namely neutral Cecropin D-like G. mellonella (WT) and two cationic peptides derived from its sequence, ΔM1 (+5) and ΔM2 (+9) is reported in this work. The influence of charge on the interactions between peptides and membranes and its effect on phase were studied by calorimetric assays. Differential scanning calorimetry (DSC) showed that ΔM2 peptide showed the strongest effect when the membrane contained phosphatidylcholine (PC) and phosphatidylglycerol (PG), increasing membrane fluidization. Fourier transform infrared spectroscopy (FTIR) was used to determine lipid segregation in the presence of peptides. When WT and ΔM1 bound to model membrane containing PG and PC (1:1 molar ratio) a separation of both lipids was observed. Meanwhile, ΔM2 peptide also induced a demixing of PG-peptide rich domains separated from PC. FTIR experiments also suggested that the presence of ΔM1 and ΔM2 peptides increased lipid carbonyl group hydration in DMPG membrane fluid phase, However, hydration at the interface level in fluid phase was notably increased in the presence of WT and ΔM1 peptides in DMPC/DMPG. Overall the increase in positively charged residues favors the interaction of the peptides with the negatively charged membrane and its perturbation.

The vertical location of α-tocopherol in phosphatidylcholine membranes is not altered as a function of the degree of unsaturation of the fatty acyl chains.

α-Tocopherol is a natural preservative that prevents free radical chain oxidations in biomembranes. We have studied the location of α-tocopherol in model membranes formed by different unsaturated phosphatidylcholines, namely 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC). Small angle X-ray diffraction revealed that α-tocopherol was well mixed with all the phospholipids. In all the cases only one lamellar phase was detected. Very modest changes occasioned by α-tocopherol were observed in the electron density profiles. The results obtained from quenching of α-tocopherol intrinsic fluorescence by acrylamide showed that this vitamin was inefficiently quenched in the four types of membranes, indicating that the fluorescent chromanol ring was poorly accessible for this hydrophilic quencher. Compatible with that, quenching by doxyl derivatives of phosphatidylcholines indicated that the chromanol ring was close in the four membranes to the nitroxide probe located at position 5. Quenching by doxyl-phosphatidylcholines also indicated that the efficiency of quenching was higher in POPC than in the other unsaturated phospholipids. 1H-MAS-NMR showed that α-tocopherol induced chemical shifts of protons from the phospholipids, especially of those bonded to carbons 2 and 3 of the acyl chains of the four phospholipids studied. The 1H-MAS-NMR NOESY results suggested that the lower part of the chromanol ring was located between the C3 of the fatty acyl chains and the centre of the hydrophobic monolayer for the four phospholipid membranes studied. Taken together, these results suggest that α-tocopherol is located, in all the membranes studied, with the chromanol ring within the hydrophobic palisade but not far away from the lipid-water interface.

Methylene blue prevents retinal damage in an experimental model of ischemic proliferative retinopathy.

Perinatal asphyxia induces retinal lesions, generating ischemic proliferative retinopathy, which may result in blindness. Previously, we showed that the nitrergic system was involved in the physiopathology of perinatal asphyxia. Here we analyze the application of methylene blue, a well-known soluble guanylate cyclase inhibitor, as a therapeutic strategy to prevent retinopathy. Male rats (n = 28 per group) were treated in different ways: 1) control group comprised born-to-term animals; 2) methylene blue group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery; 3) perinatal asphyxia (PA) group comprised rats exposed to perinatal asphyxia (20 min at 37°C); and 4) methylene blue-PA group comprised animals born from pregnant rats treated with methylene blue (2 mg/kg) 30 and 5 min before delivery, and then the pups were subjected to PA as above. For molecular studies, mRNA was obtained at different times after asphyxia, and tissue was collected at 30 days for morphological and biochemical analysis. Perinatal asphyxia produced significant gliosis, angiogenesis, and thickening of the inner retina. Methylene blue treatment reduced these parameters. Perinatal asphyxia resulted in a significant elevation of the nitrergic system as shown by NO synthase (NOS) activity assays, Western blotting, and (immuno)histochemistry for the neuronal isoform of NOS and NADPH-diaphorase activity. All these parameters were also normalized by the treatment. In addition, methylene blue induced the upregulation of the anti-angiogenic peptide, pigment epithelium-derived factor. Application of methylene blue reduced morphological and biochemical parameters of retinopathy. This finding suggests the use of methylene blue as a new treatment to prevent or decrease retinal damage in the context of ischemic proliferative retinopathy.

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1.

Proteins containing C2 domains are the sensors for Ca(2+) and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca(2+) binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands ß3-ß4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca(2+)-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

Signaling through C2 domains: more than one lipid target.

C2 domains are membrane-binding modules that share a common overall fold: a single compact Greek-key motif organized as an eight-stranded anti-parallel ß-sandwich consisting of a pair of four-stranded ß-sheets. A myriad of studies have demonstrated that in spite of sharing the common structural ß-sandwich core, slight variations in the residues located in the interconnecting loops confer C2 domains with functional abilities to respond to different Ca(2+) concentrations and lipids, and to signal through protein-protein interactions as well. This review summarizes the main structural and functional findings on Ca(2+) and lipid interactions by C2 domains, including the discovery of the phosphoinositide-binding site located in the ß3-ß4 strands. The wide variety of functions, together with the different Ca(2+) and lipid affinities of these domains, converts this superfamily into a crucial player in many functions in the cell and more to be discovered. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

The C1B domains of novel PKCε and PKCη have a higher membrane binding affinity than those of the also novel PKCδ and PKCθ.

The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of these enzymes. The four different C1B domains of novel PKCs (δ, ε, θ and η) were studied, together with different lipid mixtures containing acidic phospholipids and diacylglycerol or phorbol ester. The results show that either in the presence or in the absence of diacylglycerol, C1Bε and C1Bη exhibit a substantially higher propensity to bind to vesicles containing negatively charged phospholipids than C1Bδ and C1Bθ. The observed differences between the C1B domains of novel PKCs (in two groups of two each) were also evident in RBL-2H3 cells and it was found that, as with model membranes, in which C1Bε and C1Bη could be translocated to membranes by the addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1Bδ and C1Bθ were not translocated when soluble phosphatidic acid was added, and diacylglycerol was required to achieve a detectable binding to cell membranes. It is concluded that two different subfamilies of novel PKCs can be established with respect to their propensity to bind to the cell membrane and that these peculiarities in recognizing lipids may explain why these isoenzymes are specialized in responding to different triggering signals and bind to different cell membranes.

Visual outcomes after bilateral trifocal diffractive intraocular lens implantation.

BACKGROUND: In recent years new models of intraocular lenses are appearing on the market to reduce requirements for additional optical correction. The purpose of this study is to assess visual outcomes following bilateral cataract surgery and the implant of a FineVision® trifocal intraocular lens (IOL). METHODS: Prospective, nonrandomized, observational study. Vision was assessed in 44 eyes of 22 patients (mean age 68.4 ± 5.5 years) before and 3 months after surgery. Aberrations were determined using the Topcon KR-1 W wave-front analyzer. LogMAR visual acuity was measured at distance (corrected distance visual acuity, CDVA 4 m), intermediate (distance corrected intermediate visual acuity, DCIVA 60 cm) and near (distance corrected near visual acuity, DCNVA 40 cm). The Pelli-Robson letter chart and the CSV-1000 test were used to estimate contrast sensitivity (CS). Defocus curve testing was performed in photopic and mesopic conditions. Adverse photic phenomena were assessed using the Halo v1.0 program. RESULTS: Mean aberration values for a mesopic pupil diameter were: total HOA RMS: 0.41 ± 0.30 µm, coma: 0.32 ± 0.22 µm and spherical aberration: 0.21 ± 0.20 µm. Binocular logMAR measurements were: CDVA -0.05 ± 0.05, DCIVA 0.15 ± 0.10, and DCNVA 0.06 ± 0.10. Mean Pelli-Robson CS was 1.40 ± 0.14 log units. Mean CSV100 CS for the 4 frequencies examined (A: 3 cycles/degree (cpd), B: 6 cpd, C: 12 cpd, D: 18 cpd) were 1.64 ± 0.14, 1.77 ± 0.18, 1.44 ± 0.24 and 0.98 ± 0.24 log units, respectively. Significant differences were observed in defocus curves for photopic and mesopic conditions (p < 0.0001). A mean disturbance index of 0.28 ± 0.22 was obtained. CONCLUSIONS: Bilateral FineVision IOL implant achieved a full range of adequate vision, satisfactory contrast sensitivity, and a lack of significant adverse photic phenomena. TRIAL REGISTRATION: Eudract Clinical Trials Registry Number: 2014-003266-2.

Lamellar gel (lß) phases of ternary lipid composition containing ceramide and cholesterol.

Lipid lateral segregation into specific domains in cellular membranes is associated with cell signaling and metabolic regulation. This phenomenon partially arises as a consequence of the very distinct bilayer-associated lipid physico-chemical properties that give rise to defined phase states at a given temperature. Until now lamellar gel (Lß) phases have been described in detail in single or two-lipid systems. Using x-ray scattering, differential scanning calorimetry, confocal fluorescence microscopy, and atomic force microscopy, we have characterized phases of ternary lipid compositions in the presence of saturated phospholipids, cholesterol, and palmitoyl ceramide mixtures. These phases stabilized by direct cholesterol-ceramide interaction can exist either with palmitoyl sphingomyelin or with dipalmitoyl phosphatidylcholine and present intermediate properties between raft-associated phospholipid-cholesterol liquid-ordered and phospholipid-ceramide Lß phases. The present data provide novel, to our knowledge, evidence of a chemically defined, multicomponent lipid system that could cooperate in building heterogeneous segregated platforms in cell membranes.

Membrane docking mode of the C2 domain of PKCε: an infrared spectroscopy and FRET study.

The C2 domain of PKCε binds to negatively charged phospholipids but little is known so far about the docking orientation of this domain when it is bound. By using a FRET assay we have studied the binding of this domain to model membranes. We have also used ATR-Fourier transform infrared spectroscopy with polarized light (ATR-FTIR) to determine the docking mode by calculating the ß-sandwich orientation when the domain is bound to different types of model membranes. The vesicle lipid compositions were: POPC/POPE/POPA (22:36:42) imitating the inner leaflet of a plasma membrane, POPC/POPA (50:50) in which POPE has been eliminated with respect to the former composition and POPC/POPE/CL (43:36:21) imitating the inner mitochondrial membrane. Results show that the ß-sandwich of the PKCα-C2 domain is inclined at an angle α close to 45° to the membrane normal. Some differences were found with respect to the extent of binding as a function of phospholipid composition and small changes on secondary structure were only evident when the domain was bound to model membranes of POPC/POPA: in this case, the percentage of ß-sheet of the C2 domain increases if compared with the secondary structure of the domain in the absence of vesicles. With respect to the ß-sandwich orientation, when the domain is bound to POPC/POPE/CL membranes it forms an angle with the normal to the surface of the lipid bilayer (39°) smaller than that one observed when the domain interacts with vesicles of POPC/POPA (49°).

A memetic dynamic coral reef optimisation algorithm for simultaneous training, design, and optimisation of artificial neural networks.

Artificial Neural Networks (ANNs) have been used in a multitude of real-world applications given their predictive capabilities, and algorithms based on gradient descent, such as Backpropagation (BP) and variants, are usually considered for their optimisation. However, these algorithms have been shown to get stuck at local optima, and they require a cautious design of the architecture of the model. This paper proposes a novel memetic training method for simultaneously learning the ANNs structure and weights based on the Coral Reef Optimisation algorithms (CROs), a global-search metaheuristic based on corals' biology and coral reef formation. Three versions based on the original CRO combined with a Local Search procedure are developed: (1) the basic one, called Memetic CRO; (2) a statistically guided version called Memetic SCRO (M-SCRO) that adjusts the algorithm parameters based on the population fitness; (3) and, finally, an improved Dynamic Statistically-driven version called Memetic Dynamic SCRO (M-DSCRO). M-DSCRO is designed with the idea of improving the M-SCRO version in the evolutionary process, evaluating whether the fitness distribution of the population of ANNs is normal to automatically decide the statistic to be used for assigning the algorithm parameters. Furthermore, all algorithms are adapted to the design of ANNs by means of the most suitable operators. The performance of the different algorithms is evaluated with 40 classification datasets, showing that the proposed M-DSCRO algorithm outperforms the other two versions on most of the datasets. In the final analysis, M-DSCRO is compared against four state-of-the-art methods, demonstrating its superior efficacy in terms of overall accuracy and minority class performance.

The membrane binding kinetics of full-length PKCα is determined by membrane lipid composition.

Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands.

Drug-eluting vs. conventional balloon for side branch dilation in coronary bifurcations treated by provisional T stenting.

OBJECTIVE: The origin of the side branch (SB) is the most common site for restenosis in coronary bifurcations. The end-point is to compare the results of SB dilation with drug-eluting balloon (DEB group) versus conventional balloon (BAL group) in bifurcations treated with provisional T stenting. METHODS AND RESULTS: Each group included 50 patients. In DEB, the origin of SB was dilated with a Sequent(®) Please balloon. In both groups, a Taxus Liberté(®) stent was implanted in the main vessel, with kissing balloon postdilation. If the outcome for the SB was suboptimal, a Taxus stent was implanted in BAL and a bare stent in DEB group. An angiographic follow-up and IVUS were scheduled for 12 months later. Adverse events (MACE) were 24% in BAL versus 11% in DEB (P = 0.11), with greater revascularization (TLR) in the BAL group (22% vs. 12%, P = 0.16). At angiographic follow-up, there was a lower percentage of SB restenosis in the DEB group (20% vs. 7%, P = 0.08), with less late loss (0.40 mm vs. 0.09 mm, P = 0.01). CONCLUSION: Side branch dilation with a drug-eluting balloon resulted in better angiographic outcomes than with a conventional balloon, with less late loss and restenosis at the 12-month follow-up.