Rotation of the c-Ring Promotes the Curvature Sorting of Monomeric ATP Synthases.

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane-spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high-curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts. The formation of transient protein dimers relies on the membrane-mediated attractive interaction of the order of 1.5 kB T produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic-like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in biological membranes.

Atomic crystal structure and sugar specificity of a ß-trefoil lectin domain from the ectomycorrhizal basidiomycete Laccaria bicolor.

Lectins from fruiting bodies are a diverse group of sugar-binding proteins from mushrooms that face the biologically relevant challenge of discriminating self- from non-self carbohydrate structures, therefore providing a basis for an innate defence system. Such a system entails both detection and destruction of invaders and/or feeders, and in contrast to more complex organisms with immense immune systems, these two functions normally rely on multitasking lectins, namely, lectins with different functional modules. Here, we present a novel fungal lectin, LBL, from the basidiomycete Laccaria bicolor. Using a diverse set of biophysical techniques, we unveil the fine details of the sugar-binding specificity of the N-terminal ß-trefoil of LBL (LBL152), whose structure has been determined at the highest resolution so far reported for such a fold. LBL152 binds complex poly-N-Acetyllactosamine polysaccharides and also robust LBL152 binding to Caenorhabditis elegans and Drosophila melanogaster cellular extracts was detected in microarray assays, with a seeming preference for the fruit fly adult and pupa stages over the larva stage. Prediction of the structure of the C-terminal part of LBL with AlphaFold reveals a tandem repeat of two structurally almost identical domains of around 110 amino acids each, despite sharing low sequence conservation.

Overexpression of the White Opaque Switching Master Regulator Wor1 Alters Lipid Metabolism and Mitochondrial Function in Candida albicans.

Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans; increased colonization of this yeast in this niche has implicated the master regulator of the white-opaque transition, Wor1, by mechanisms not completely understood. We have addressed the role that this transcription factor has on commensalism by the characterization of strains overexpressing this gene. We show that WOR1 overexpression causes an alteration of the total lipid content of the fungal cell and significantly alters the composition of structural and reserve molecular species lipids as determined by lipidomic analysis. These cells are hypersensitive to membrane-disturbing agents such as SDS, have increased tolerance to azoles, an augmented number of peroxisomes, and increased phospholipase activity. WOR1 overexpression also decreases mitochondrial activity and results in altered susceptibility to certain oxidants. All together, these changes reflect drastic alterations in the cellular physiology that facilitate adaptation to the gastrointestinal tract environment.

Calmodulin-dependent KCNE4 dimerization controls membrane targeting.

The voltage-dependent potassium channel Kv1.3 participates in the immune response. Kv1.3 is essential in different cellular functions, such as proliferation, activation and apoptosis. Because aberrant expression of Kv1.3 is linked to autoimmune diseases, fine-tuning its function is crucial for leukocyte physiology. Regulatory KCNE subunits are expressed in the immune system, and KCNE4 specifically tightly regulates Kv1.3. KCNE4 modulates Kv1.3 currents slowing activation, accelerating inactivation and retaining the channel at the endoplasmic reticulum (ER), thereby altering its membrane localization. In addition, KCNE4 genomic variants are associated with immune pathologies. Therefore, an in-depth knowledge of KCNE4 function is extremely relevant for understanding immune system physiology. We demonstrate that KCNE4 dimerizes, which is unique among KCNE regulatory peptide family members. Furthermore, the juxtamembrane tetraleucine carboxyl-terminal domain of KCNE4 is a structural platform in which Kv1.3, Ca2+/calmodulin (CaM) and dimerizing KCNE4 compete for multiple interaction partners. CaM-dependent KCNE4 dimerization controls KCNE4 membrane targeting and modulates its interaction with Kv1.3. KCNE4, which is highly retained at the ER, contains an important ER retention motif near the tetraleucine motif. Upon escaping the ER in a CaM-dependent pattern, KCNE4 follows a COP-II-dependent forward trafficking mechanism. Therefore, CaM, an essential signaling molecule that controls the dimerization and membrane targeting of KCNE4, modulates the KCNE4-dependent regulation of Kv1.3, which in turn fine-tunes leukocyte physiology.

How rotating ATP synthases can modulate membrane structure.

F1Fo-ATP synthase (ATP synthase) is a central membrane protein that synthetizes most of the ATP in the cell through a rotational movement driven by a proton gradient across the hosting membrane. In mitochondria, ATP synthases can form dimers through specific interactions between some subunits of the protein. The dimeric form of ATP synthase provides the protein with a spontaneous curvature that sustain their arrangement at the rim of the high-curvature edges of mitochondrial membrane (cristae). Also, a direct interaction with cardiolipin, a lipid present in the inner mitochondrial membrane, induces the dimerization of ATP synthase molecules along cristae. The deletion of those biochemical interactions abolishes the protein dimerization producing an altered mitochondrial function and morphology. Mechanically, membrane bending is one of the key deformation modes by which mitochondrial membranes can be shaped. In particular, bending rigidity and spontaneous curvature are important physical factors for membrane remodelling. Here, we discuss a complementary mechanism whereby the rotatory movement of the ATP synthase might modify the mechanical properties of lipid bilayers and contribute to the formation and regulation of the membrane invaginations.

Cartas a mis pacientes: Homeopatía, ciencia y arte de la curación ¿Por qué? / Letters to my patients: Homeopathy, science and art of healing Why?

María llegó a la consulta con un cabestrillo hecho de trapos y palos. Era una mujer indígena mixteca de la montaña, proveniente de la Sierra Azul de Oaxaca, en México. La traían cargada sus dos hijos. Cuando la encontré, ella tenía unos 60 años. Era una mujer de campo y muy avejentada, pequeña y encogida, con el pelo corto y blanco. No podía valerse por sí misma porque tenía todas las articulaciones muy hinchadas, tanto, que eran del doble de su tamaño normal. Además, cuando las mostraba brillaban y tenían una tonalidad entre rosada y nacarada por la enorme cantidad de líquido que había dentro y por la intensa inflamación crónica; era una especie de artritis serosa y un cuadro de reumatismo articular crónico con intensa agudización de carácter deformante. Ya tenía todos los dedos de las manos y los pies desviados en modo evidente. No obstante la situación, la mujer era dulce y amable, incluso para quejarse, y tenía la actitud de extrema humildad de la gente sencilla indígena, característica del pueblo mexicano. La mujer lloraba fácilmente y estaba abatida y desesperada dentro de su silencio. Sus hijos decían que había cambiado y estaba muy irritable, muy diferente a como era antes.

Overcoming challenges to support us resumption of high specific activity cobalt-60.

Domestic production of high specific activity 60Co was halted after a target rupture in 2012 at the Advanced Test Reactor (ATR). The Isotope Program (IP) within the US Department of Energy (DOE) Office of Science tasked a multilaboratory team of researchers and managers from Oak Ridge and Idaho National Laboratories with the redesign the radioisotope capsule. The objective of this effort was to create a more robust and reliable design, compared to the pre-2012 target. The team successfully completed this task to produce the DOE-IP cobalt (Co) production capsule design. Furthermore, 66 capsules were successfully fabricated by Oak Ridge National Laboratory (ORNL) and delivered to Idaho National Laboratory (INL) for irradiation in the ATR between January 2014 and October 2016. This paper describes the efforts of the team to prepare and disposition the two initial DOE-IP Co production capsules that were processed in March 2020. These efforts include performing accurate production predictions, experimentally validating predictions with assay measurements, shipping with the Orano-furnished Battelle Energy Alliance Research Reactor shipping package, and disassembling capsules at the isotope vendor site.

Evidencia del mensaje de sufrimiento hereditario / Evidence for the hereditary suffering message

En este artículo pondremos un ejemplo de la vida real para demostrar con claridad todo lo escrito. Se trata del caso de una mujer de 35 años que llega a la consulta con un solo síntoma importante para ella. No le viene la menstruación desde hace años. Y si le viene, es solo esporádicamente. Refiere que la última vez que le ha venido regularmente fue cuando se separó de su novio; después se sintió libre y contenta, segura de sí misma. Años después, se ha vuelto a enamorar y han vuelto a desaparecer las menstruaciones. Cuando se configura la historia familiar nos encontramos (sin dar demasiados detalles) una historia con ciertas características que han constituido el lenguaje primordial, inconsciente y consciente, de la vida de la paciente. Resulta ser que, durante tres generaciones, mujeres dominantes y severas con los demás han tenido hijos naturales a escondidas, antes de casarse con sus maridos respectivos, que también han tenido otros hijos naturales fuera del matrimonio con anterioridad. Al final, acabaron casándose de manera oficial con sus respectivas mujeres (abuelas y madre de la paciente), ya que éstas habían quedado embarazadas. Ellos, hombres sin responsabilidad amorosa, vivieron ajenos a las obligaciones como padres; el condicionamiento social los forzó a mantener la casa y los hijos, pero sin que ello implicara la capacidad para proteger y educar a los hijos que, por tanto, siempre dependieron solo de ellas.

¿Qué Significa la Enfermedad, mi Enfermedad Personal, la Enfermedad que me Pertenece? / What does the disease mean, my Personal Illness, Illness that belongs to me?

Después de las reflexiones expuestas en los artículos anteriores podemos hacer una síntesis que nos permita responder a preguntas concretas que todos queremos conocer para comprender el sufrimiento personal. Recordemos que todo ser viviente, como elemento de una especie que naturalmente está sometida a todos los procesos de selección para dar lo mejor o extinguirse en el tiempo, nace constitucionalmente sano, aunque incompleto y limitado. (AU)

Vascular endothelial growth factor expression as a biomarker of prognosis in patients with chondrosarcoma, Ewing's sarcoma and osteosarcoma. Current concepts.

With the advent of the molecularly targeted therapies, identifying molecular therapeutic targets and molecolar marker is increasingly important, especially in neoplastic diseases. Several studies show VEGF is involved in neo-angiogenesis in many solid cancers, as breast, lung, renal, gastric carcinomas, through promoting endothelial cell growth and migration. Conversely the relationship between VEFG and tumours of the musculoskeletal system is yet unclear, in particular the role of VEGF has not yet been completely understood in these tumours. Chondrosarcoma, Ewing's Sarcoma and Osteosarcoma are the tumours of the musculoskeletal system in which the activity of VEGF has been closely studied. The present study aims to give an overview focused on the relationship between VEGF and these three cancers.

Infections in primary intramedullary nailing of open tibial fractures: a review article.

OBJECTIVE: Open fractures are insidious and life-threatening injuries. They frequently involve the leg. Our aim is to evaluate the efficacy of primary intramedullary nailing as a definitive treatment of open tibial fractures, compared to other fixation tools in terms of deep infections, healing fracture time and bony repair. PATIENTS AND METHODS: We reviewed the available literature concerning treatment and management of open tibial fractures. RESULTS: Primary intramedullary nailing is similar to delayed intramedullary nailing after external fixation in terms of union, malunion and nonunion rate. Furthermore, primary intramedullary nailing is superior compared to all other fixation tools, in terms of development of deep infections, provided that a good debridement of soft tissue, lavage of the fracture site and adequate antibiotic prophylaxis should be performed before nailing. CONCLUSIONS: Primary intramedullary nailing should be considered for the definitive treatment of open tibial fractures, providing many advantages in terms of rehabilitation, time of hospitalization and costs.

Enhanced Cytotoxic Activity of Mitochondrial Mechanical Effectors in Human Lung Carcinoma H520 Cells: Pharmaceutical Implications for Cancer Therapy.

Cancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10-N-nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating from opposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation of cancer cells.

Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death.

BACKGROUND: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail. METHODS: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic. RESULTS: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact region. The membrane remodeling effect of NAO, as well as the formation of H-dimers, was also confirmed in cultured fibroblasts, as shown by the ultrastructure alteration of the mitochondrial cristae. CONCLUSIONS: We conclude that membrane adhesion induced by NAO stacking accounts for the supramolecular basis of its cytotoxicity. GENERAL SIGNIFICANCE: Mitochondria are a potential target for cancer and gene therapies. The alteration of the mitochondrial structure by membrane remodeling agents able to form supramolecular assemblies via adhesion properties could be envisaged as a new therapeutic strategy.

Qualitative Research Process Applied to Organ Donation.

The family interview for organ donation continues to be one of the main concerns of transplant coordinators. We approach the family because we need to know the opinion of the person who has just died about the donation of his or her organs. The objective of this article is to describe how the application of the qualitative methodology has been followed to perceive what the relatives live in the moment in which they are informed of the death of their relative and they are offered the option of organ donation. Phenomenology is a philosophy that deals with the granting of meanings. It can simply describe or try to interpret them. Our informants were selected from among donor families until the 11 families were obtained. We use the Giorgi method because it allows us to identify the essence of a phenomenon and transform the lived experience into a textual expression of its essence. The unit of analysis was, each of the statements of the discourse that describes and interprets a reality, what we call "essence" or "meaning units." It must be the minimum expression of an episode and the experience in relation to it. We have detected 24 themes of the meaning units are most important for families in our area. Families agree on talking about donation, mourning, death, consolation from the donation, and the importance of the support received. Phenomenology is adequate to deepen into the feelings and interests of families in the process of donation.

Correction: The natural compound Jatrophone interferes with Wnt/ß-catenin signaling and inhibits proliferation and EMT in human triple-negative breast cancer.

[This corrects the article DOI: 10.1371/journal.pone.0189864.].

Dynamic cellular maps of molecular species: Application to drug-target interactions.

The design of living cell studies aimed at deciphering the mechanism of action of drugs targeting proteins with multiple functions, expressed in a wide range of concentrations and cellular locations, is a real challenge. We recently showed that the antitumor drug plitidepsin (APL) localizes sufficiently close to the elongation factor eEF1A2 so as to suggest the formation of drug-protein complexes in living cells. Here we present an extension of our previous micro-spectroscopy study, that combines Generalized Polarization (GP) images, with the phasor approach and fluorescence lifetime imaging microscopy (FLIM), using a 7-aminocoumarin drug analog (APL*) as fluorescence tracer. Using the proposed methodology, we were able to follow in real time the formation and relative distribution of two sets of APL-target complexes in live cells, revealing two distinct patterns of behavior for HeLa-wt and APL resistant HeLa-APL-R cells. The information obtained may complement and facilitate the design of new experiments and the global interpretation of the results obtained with other biochemical and cell biology methods, as well as possibly opening new avenues of study to decipher the mechanism of action of new drugs.

Single dose cefazolin is safe and effective for pre-operative prophylaxis in orthopaedic oncology.

Surgical site infections (SSI) are a common potentially preventable complication after surgical procedures. A standardized antibiotic prophylaxis in elective orthopaedic surgery plays a major role in lowering SSI. At present, there is little published evidence regarding standardized antibiotic prophylaxis in orthopaedic oncological surgery. We introduced a prophylactic antibiotic protocol for orthopaedic oncological surgery in our hospital. The proposed protocol consists in "one-shot" intravenous administration of Cefazolin 2g, 30 min before surgery. In our setting, this preoperative antibiotic prophylaxis regimen was associated with a markedly lower rate of SSI's. There is no current evidence in favour of greater effectiveness of prophylaxis beyond 24/48 h after surgery compared to our pre-surgical "one-shot" administration; by contrast, prolonged post-surgical prophylaxis is likely to undermine the patient's bacterial flora and select resistant pathogens. These results are preliminary and should be used to start planning a standardised prophylactic protocol to prevent SSI's after orthopaedic oncological surgery.

Study of rabbit erythrocytes membrane solubilization by sucrose monomyristate using laurdan and phasor analysis.

The study of surfactant and bio membranes interaction is particularly complex due to the diversity in lipid composition and the presence of proteins in natural membranes. Even more difficult is the study of this interaction in vivo since cellular damage may complicate the interpretation of the results, therefore for most of the studies in this field either artificial or model systems are used. One of the model system most used to study biomembranes are erythrocytes due to their relatively simple structure (they lack nuclei and organelles having only the plasma membrane), their convenient experimental manipulation and availability. In this context, we used rabbit erythrocytes as a model membrane and Laurdan (6-lauroyl-2-dimethylaminonaphthalene) as the fluorescent probe to study changes promoted in the membrane by the interaction with the sucrose monoester of myristic acid, ß-d-fructofuranosyl-6-O-myristoyl-α-d-glucopyranoside (MMS). Surfactant and erythrocytes interaction was studied by measuring hemoglobin release and the changes in water content in the membrane sensed by Laurdan. Using two-photon excitation, three types of measurements were performed: Generalized Polarization (analyzed as average GP values), Fluorescence Lifetime Imaging, FLIM (analyzed using phasor plots) and Spectral imaging (analyzed using spectral phasor). Our data indicate that at sublytical concentration of surfactant (20µM MMS), there is a decrease of about 35% in erythrocytes size, without changes in Laurdan lifetime or emission spectra. We also demonstrate that as hemolysis progress, Laurdan lifetime increased due to the decrease in hemoglobin (strong quencher of Laurdan emission) content inside the erythrocytes. Under these conditions, Laurdan spectral phasor analyses can extract the information on the water content in the membrane in the presence of hemoglobin. Our results indicate an increase in membrane fluidity in presence of MMS.

The natural compound Jatrophone interferes with Wnt/ß-catenin signaling and inhibits proliferation and EMT in human triple-negative breast cancer.

Metastatic breast cancer is the leading cause of worldwide cancer-related deaths among women. Triple negative breast cancers (TNBC) are highly metastatic and are devoid of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) amplification. TNBCs are unresponsive to Herceptin and/or anti-estrogen therapies and too often become highly chemoresistant when exposed to standard chemotherapy. TNBCs frequently metastasize to the lung and brain. We have previously shown that TNBCs are active for oncogenic Wnt10b/ß-catenin signaling and that WNT10B ligand and its downstream target HMGA2 are predictive of poorer outcomes and are strongly associated with chemoresistant TNBC metastatic disease. In search of new chemicals to target the oncogenic WNT10B/ß-CATENIN/HMGA2 signaling axis, the anti-proliferative activity of the diterpene Jatrophone (JA), derived from the plant Jatropha isabelli, was tested on TNBC cells. JA interfered with the WNT TOPFLASH reporter at the level between receptor complex and ß-catenin activation. JA efficacy was determined in various subtypes of TNBC conventional cell lines or in TNBC cell lines derived from TNBC PDX tumors. The differential IC50 (DCI50) responsiveness was compared among the TNBC models based on etiological-subtype and their cellular chemoresistance status. Elevated WNT10B expression also coincided with increased resistance to JA exposure in several metastatic cell lines. JA interfered with cell cycle progression, and induced loss of expression of the canonical Wnt-direct targets genes AXIN2, HMGA2, MYC, PCNA and CCND1. Mechanistically, JA reduced steady-state, non-phosphorylated (activated) ß-catenin protein levels, but not total ß-catenin levels. JA also caused the loss of expression of key EMT markers and significantly impaired wound healing in scratch assays, suggesting a direct role for JA inhibiting migration of TNBC cells. These results indicate that Jatrophone could be a powerful new chemotherapeutic agent against highly chemoresistant triple negative breast cancers by targeting the oncogenic Wnt10b/ß-catenin signaling pathway.

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts.

Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.