Proteomic analysis of the effect of hemin in breast cancer.

Heme, an iron-containing prosthetic group found in many proteins, carries out diverse biological functions such as electron transfer, oxygen storage and enzymatic reactions. Hemin, the oxidised form of heme, is used to treat porphyria and also to activate heme-oxygenase (HO) which catalyses the rate-limiting step in heme degradation. Our group has previously demonstrated that hemin displays antitumor activity in breast cancer (BC). The aim of this work has been to study the effect of hemin on protein expression modifications in a BC cell line to gain insight into the molecular mechanisms of hemin antitumor activity. For this purpose, we carried out proteome analysis by Mass Spectrometry (MS) which showed that 1309 proteins were significantly increased in hemin-treated cells, including HO-1 and the proteases that regulate HO-1 function, and 921 proteins were significantly decreased. Furthermore, the MS-data analysis showed that hemin regulates the expression of heme- and iron-related proteins, adhesion and cytoskeletal proteins, cancer signal transduction proteins and enzymes involved in lipid metabolism. By biochemical and cellular studies, we further corroborated the most relevant in-silico results. Altogether, these results show the multiple physiological effects that hemin treatment displays in BC and demonstrate its potential as anticancer agent.

Iron cycle disruption by heme oxygenase-1 activation leads to a reduced breast cancer cell survival.

Heme oxygenase-1 (HO-1), which catalyzes heme degradation releasing iron, regulates several processes related to breast cancer. Iron metabolism deregulation is also connected with several tumor processes. However the regulatory relationship between HO-1 and iron proteins in breast cancer remains unclear. Using human breast cancer biopsies, we found that high HO-1 levels significantly correlated with low DMT1 levels. Contrariwise, high HO-1 levels significantly correlated with high ZIP14 and prohepcidin expression, as well as hemosiderin storage. At mRNA level, we found that high HO-1 expression significantly correlated with low DMT1 expression but high ZIP14, L-ferritin and hepcidin expression. In in vivo experiments in mice with genetic overexpression or pharmacological activation of HO-1, we detected the same expression pattern observed in human biopsies. In in vitro experiments, HO-1 activation induced changes in iron proteins expression leading to an increase of hemosiderin, ROS levels, lipid peroxidation and a decrease of the growth rate. Such low growth rate induced by HO-1 activation was reversed when iron levels or ROS levels were reduced. Our findings demonstrate an important role of HO-1 on iron homeostasis in breast cancer. The changes in iron proteins expression when HO-1 is modulated led to the iron accumulation deregulating the iron cell cycle, and consequently, generating oxidative stress and low viability, all contributing to impair breast cancer progression.

Transcriptomic response in thermally challenged seahorses Hippocampus erectus: The effect of magnitude and rate of temperature change.

Hippocampus erectus inhabiting the shallow coastal waters of the southern Gulf of Mexico are naturally exposed to marked temperature variations occurring in different temporal scales. Under such heterogeneous conditions, a series of physiological and biochemical adjustments take place to restore and maintain homeostasis. This study investigated the molecular mechanisms involved in the response of H. erectus to increased temperature using transcriptome analysis based on RNA-Seq technology. Data was obtained from seahorses after 0.5-h exposure to combinations of different target temperatures (26 °C: control, and increased to 30 and 33 °C) and rates of thermal increase (abrupt: < 5 min; gradual: 1-1.5 °C every 3 h). The transcriptome of seahorses was assembled de novo using Trinity software to obtain 29,211 genes and 30,479 transcripts comprising 27,520,965 assembled bases. Seahorse exposure to both 30 and 33 °C triggered characteristic processes of the cellular stress response, regardless of the rate of thermal change. The transcriptomic profiles of H. erectus suggest an arrest of muscle development processes, the activation of heat shock proteins, and a switch to anaerobic metabolism within the first 0.5 h of exposure to target temperatures to ensure energy supply. Interestingly, apoptotic processes involving caspase were activated principally in gradual treatments, suggesting that prolonged exposure to even sublethal temperatures results in the accumulation of deleterious effects that may eventually terminate in cellular death. Results herein validate 30 °C and 33 °C as potential upper limits of thermal tolerance for H. erectus at the southernmost boundary of its geographic distribution.

Distribution patterns, carbon sources and niche partitioning in cave shrimps (Atyidae: Typhlatya).

Cave shrimps of the Typhlatya genus are common and widespread in fresh, brackish and marine groundwater throughout the Yucatan Peninsula (Mexico). These species are ideal models to test niche partitioning within sympatric species in oligotrophic systems. Nevertheless, their food sources remain unidentified, and despite their frequency and functional importance, distribution and abundance patterns of these species within caves have not been fully recognized. Here, we describe the abundance of three Typhlatya species in different temporal and spatial scales, investigate changes in water conditions, and potential sources of carbon as an indication of food origin. Species composition and abundance varied markedly in space and time revealing patterns that differed from one system to another and in relation to environmental parameters. Isotope analysis showed that each species reflects a particular δ13C and Δ14C fingerprint, suggesting they feed in different proportions from the available carbon sources. Overall, our findings suggest a niche partitioning of habitat and feeding sources amongst the three Typhlatya species investigated, where environmental characteristics and physiological differences could play an important role governing their distribution patterns.

Effect of a gradually increasing temperature on the behavioural and physiological response of juvenile Hippocampus erectus: Thermal preference, tolerance, energy balance and growth.

The physiological and behavioural responses of ectotherms to temperature is strongly dependent on the individuals' previous thermal history. Laboratory based studies investigating the mechanisms of thermoregulation in marine ectotherms, however, rarely consider key temporal elements of thermal exposure, such as the rate at which temperature changes. We tested the hypothesis that juvenile seahorses, Hippocampus erectus, from a tropical coastal lagoon in Yucatan, Mexico, would exhibit variations in physiological and behavioural descriptors of thermoregulation when submitted to contrasting regimes during 30 days: temperature constant at 25 °C (C 25); gradually increasing 1 °C every 5 days from 25 to 30 °C (GI 25-30); and constant at 30 °C (C 30). Immediately after exposure, critical maximum temperature, thermal preference, oxygen consumption, partial energy balance, growth rate and survival of seahorses were measured. Seahorses exposed to GI 25-30 showed a significantly higher critical thermal maxima (37.8 ±â€¯0.9 °C), preference (28.7 ±â€¯0.4 °C), growth (1.10 ±â€¯0.49%) and survival (97.6%) than those exposed to C 30 (36.5 ±â€¯1, 29.4 ±â€¯0.3 °C, 0.48 ±â€¯0.32%, 73.8%, respectively). Both high temperature regimes induced metabolic depression, but ramping resulted in a greater amount of energy assimilated (278.9 ±â€¯175.4 J g-1 day-1) and higher energy efficiency for growth (89.8%) than constant exposure to 30 °C (115.4 ±â€¯63.4 J g-1 day-1, 65.3%, respectively). Gradually increasing temperature allowed physiological mechanisms of thermal adjustment to take place, reflecting the capacity of juvenile H. erectus to respond to environmental change. Despite its advantage, this capacity is limited in time, since the cumulative effect of thermal exposure affected metabolic performance, eventually compromising survival. The study of seahorse response to thermal variations in the context of ocean warming needs to consider the temporal elements of thermal exposure to foresee its vulnerability under future scenarios.

Estudio genético de la fractura femoral atípica mediante la secuenciación del exoma en tres hermanas afectas y tres pacientes no relacionadas / Genetic study of atypical femoral fractures using exome sequencing in three affected sisters and three unrelated patients

Objetivos: Las fracturas atípicas de fémur (FAF) son un tipo de fracturas poco frecuentes, a menudo relacionadas con un tratamiento prolongado con bisfosfonatos (BPs). Actualmente no se conocen con exactitud sus mecanismos patogénicos y no hay pruebas para identificar aquellos pacientes con un alto riesgo de sufrir una FAF. El objetivo de este trabajo es investigar las bases genéticas de las FAFs. Material y métodos: Se secuenció el exoma completo de 3 hermanas y de 3 pacientes adicionales no relacionadas, todas tratadas con BPs durante más de 5 años. Se seleccionaron variantes compartidas por las hermanas, de baja frecuencia y potencialmente patogénicas, y se construyó una red de interacciones de genes y proteínas con los datos hallados. Resultados: Identificamos 37 variantes raras (en 34 genes) compartidas por las 3 hermanas, algunas de ellas no descritas anteriormente. La variante más llamativa fue la mutación p.Asp188Tyr en el enzima geranilgeranil pirofosfato sintasa (codificada por el gen GGPS1), de la vía del mevalonato y esencial para la función del osteoclasto. Otro hallazgo interesante fueron dos mutaciones (una en las 3 hermanas y una en una paciente no relacionada) en el gen CYP1A1, implicado en el metabolismo de los esteroides. Identificamos otras variantes que también podrían estar involucradas en la susceptibilidad a las FAFs o en el fenotipo osteoporótico subyacente, tales como las presentes en los genes SYDE2, NGEF, COG4 y la FN1. Conclusiones: Nuestros datos son compatibles con un modelo donde la acumulación de variantes de susceptibilidad podría participar en la base genética de las FAF

Objectives: Atypical femoral fractures (AFF) are rare, often related to long-term bisphosphonate (BPs) treatment. Their pathogenic mechanisms are not precisely known and there is no evidence to identify patients with a high risk of AFF. The aim of this work is to study the genetic bases of AFFs. Material and methods: Whole-exome sequencing was carried out on 3 sisters and 3 unrelated additional patients, all treated with BPs for more than 5 years. Low frequency, potentially pathogenic variants shared by the 3 sisters, were selected, were selected and a network of gene and protein interactions was constructed with the data found. Results: We identified 37 rare variants (in 34 genes) shared by the 3 sisters, some not previously described. The most striking variant was the p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase (encoded by the GGPS1 gene), from the mevalonate pathway and essential for osteoclast function. Another noteworthy finding was two mutations (one in the 3 sisters and one in an unrelated patient) in the CYP1A1 gene, involved in the metabolism of steroids. We identified other variants that could also be involved in the susceptibility to AFFs or in the underlying osteoporotic phenotype, such as those present in the SYDE2, NGEF, COG4 and FN1 genes. Conclusions: Our data are compatible with a model where the accumulation of susceptibility variants could participate in the genetic basis of AFFs

Anatomical variation of the anterior belly of the digastric muscle: case report and clinical implications

The digastric muscle is a suprahyoid muscle composed of two bellies connected by an intermediate tendon.This muscle participates in deglutition and mandibular movements. The anterior belly of the digastric muscleis localized superficially to the mylohyoid and deeply to the platysma muscle. During dissection of this regionof an embedded cadaver, an accessory anterior belly of digastric muscle was observed bilaterally. The accessorybellies were similar but not symmetrical. They were composed of two segments, one long and one short, onboth sides, and when observed together these appeared to form the letter “X”. The accessory fibers, on bothsides, originated from the anterior digastric muscle and inserted medially to the digastric fossa. Anatomicvariations of the digastric muscle may influence mastication and deglutition. Moreover, the accessory digastricmuscle affects diagnostic imaging and therapeutic procedures in head and neck surgery and must be consideredin procedures involving this area.

Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size.

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Visuotopic mapping through a multichannel stimulating implant in primate V1.

We report on our efforts to establish an animal model for the development and testing of a cortical visual prostheses. One-hundred-fifty-two electrodes were implanted in the primary visual cortex of a rhesus monkey. The electrodes were made from iridium with an activated iridium oxide film, which has a large charge capacity for a given surface area, and insulated with parylene-C. One-hundred-fourteen electrodes were functional after implantation. The activity of small (2-3) neuronal clusters was first recorded to map the visually responsive region corresponding to each electrode. The animal was then trained in a memory (delayed) saccade task, first with a visual target, then to a target defined by direct cortical stimulation with coordinates specified by the stimulating electrode's mapped receptive field. The SD of saccade endpoints was approximately 2.5 larger for electrically stimulated versus visual saccades; nevertheless, when trial-to-trial scatter was averaged out, the correlation between saccade end points and receptive field locations was highly significant and approached unity after several months of training. Five electrodes were left unused until the monkey was fully trained; when these were introduced, the receptive field-saccade correlations were high on the first day of use (R = 0.85, P = 0.03 for angle, R = 0.98, P < 0.001 for eccentricity), indicating that the monkey had not learned to perform the task empirically by memorizing reward zones. The results of this experiment suggest the potential for rigorous behavioral testing of cortical visual prostheses in the macaque.

Attractor networks for shape recognition.

We describe a system of thousands of binary perceptrons with coarse-oriented edges as input that is able to recognize shapes, even in a context with hundreds of classes. The perceptrons have randomized feedforward connections from the input layer and form a recurrent network among themselves. Each class is represented by a prelearned attractor (serving as an associative hook) in the recurrent net corresponding to a randomly selected subpopulation of the perceptrons. In training, first the attractor of the correct class is activated among the perceptrons; then the visual stimulus is presented at the input layer. The feedforward connections are modified using field-dependent Hebbian learning with positive synapses, which we show to be stable with respect to large variations in feature statistics and coding levels and allows the use of the same threshold on all perceptrons. Recognition is based on only the visual stimuli. These activate the recurrent network, which is then driven by the dynamics to a sustained attractor state, concentrated in the correct class subset and providing a form of working memory. We believe this architecture is more transparent than standard feedforward two-layer networks and has stronger biological analogies.

Tumor ovárico virilizante oculto: "un problema diagnóstico". Estudio de 2 casos / Occult virilizing ovarian tumor: a diagnostic problem. Study of 2 cases

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Biocompatibility of the polyurethane resin of the castor bean inserted into the alveolar bone of the dog.

The biocompability of the polyurethane resin of the castor bean (Ricinus vulgaris) was studied following its insertion into the alveolar bone of dogs, after extraction of their premolar teeth. The resin was left to polymerise in the dental alveolus. Excess of material due to polymerisation was removed and polishing was employed to smooth and adapt the occlusal surface to the margins of the alveolar bone. This allowed a perfect suture of the mucosa together with the periosteum. The resin remained in the dental alveolus for 90 days. It was observed that the polyurethane was replaced by osteoid and bone tissues and no immune or inflammatory reactions were detected. There has been work on and discussion about the use of the polyurethane in grafts, prostheses and orthoses. Attention was paid to all the surgical steps, in particular the preservation of the periosteal integrity. Further research is being followed in our Department in order to test the biocompatibility of the material presented in this paper when used together with metallic or ceramic implants.

Effective neural response function for collective population states.

Collective behaviour of neural networks often divides the ensemble of neurons into sub-classes by neuron type; by selective synaptic potentiation; or by mode of stimulation. When the number of classes becomes larger than two, the analysis, even in a mean-field theory, loses its intuitive aspect because of the number of dimensions of the space of dynamical variables. Often one is interested in the behaviour of a reduced set of sub-populations (in focus) and in their dependence on the system's parameters, as in searching for coexistence of spontaneous activity and working memory; in the competition between different working memories; in the competition between working memory and a new stimulus; or in the interaction between selective activity in two different neural modules. For such cases we present a method for reducing the dimensionality of the system to one or two dimensions, even when the total number of populations involved is higher. In the reduced system the familiar intuitive tools apply and the analysis of the dependence of different network states on ambient parameters becomes transparent. Moreover, when the coding of states in focus is sparse, the computational complexity is much reduced. Beyond the analysis, we present a set of detailed examples. We conclude with a discussion of questions of stability in the reduced system.

Cryopreservation of hematopoietic progenitor cells with 5-percent dimethyl sulfoxide at -80 degrees C without rate-controlled freezing.

BACKGROUND: Cryopreservation of hematopoietic cells with the rate-controlled method is used in the majority of centers. In recent years, there has been a trend toward the simplification of the process. STUDY DESIGN AND METHODS: A simplified method for cryopreservation was developed with 5-percent dimethyl sulfoxide (DMSO) as the sole cryoprotectant without rate-controlled freezing. Experiments were done with progressive concentrations of DMSO, ranging from 0 to 10 percent. With DMSO concentrations from 5- to 10-percent, the best recovery and viability for hematopoietic progenitor cells were observed. Hematopoietic progenitor cells with plasma and 5-percent DMSO were frozen and stored in a -80 degrees C mechanical freezer. Ten patients with solid and hematologic malignancies underwent transplantation with autologous hematopoietic progenitor cells. RESULTS: The median number of transfused mononuclear cells and CD34+ cells was 3.70 (3.1-8.2) x 10(8) per kg and 1.70 (0.8-6.5) x 10(6) per kg, respectively. The median number of transfused colony-forming units-granulocyte-macrophage was 12.45 (3.4-55.3) x 10(4) per kg. All patients showed rapid and sustained engraftment. The mean times to reach a neutrophil count of 0.5 x 10(9) per L and a platelet count of 50 x 10(9) per L were 11.50 +/- 1.70 and 13.90 +/- 3.98 days, respectively. All patients are alive and without transfusion requirements in complete remission 2 to 8 months after transplantation. CONCLUSION: This simplified cryopreservation technique will be useful for institutions without rate-controlled freezing facilities. Moreover, this method diminishes the amount of DMSO infused to patients, as well as its toxicity.

A protein secreted by Trypanosoma cruzi capable of inducing the entry of inert particles into HeLa cells.

Trypanosoma cruzi requires an intracellular environment to multiply within its mammalian host. We describe the purification and some properties of a protein secreted exclusively by the metacyclic (infective) forms of the parasite. This permeabilizing protein (relative molecular mass 64,000) was secreted under our experimental conditions only when the parasites interacted with HeLa cells, HeLa membranes, or wheat-germ lectin. The protein is thermostable, and its biological activity is inhibited by formaldehyde but not by ethanol or acetone. At low concentrations and over short treatment times, this protein acts as a permeabilizer and induces endocytosis. No significant protease or neuraminidase activity was found. When adsorbed onto bentonite particles and incubated in the presence of non-phagocytic cells the protein facilitated the penetration of the particles into the cells. Immune serum directed against the protein neutralized its cytotoxic action and reduced the rate of penetration of metacyclic forms into both macrophages and non-phagocytic cells. Our results suggest that the protein secreted by the parasite plays a key role in the penetration of its infective form into the host cell.

Preconception tort liability: recognizing a strict liability cause of action for DES grandchildren.

Over the past decade more than 1,000 "DES daughters" have filed lawsuits against the manufacturers of DES, alleging that their in utero exposure to the drug caused various reproductive tract abnormalities, including cancer. Plaintiffs now allege that their grandmothers' use of DES during pregnancy caused genetic damage leading to cancer in third generations. This Note addresses the validity of preconception tort liability in the context of third-generation DES cases. Plaintiffs in preconception tort liability cases have sought recovery under both negligence and strict liability causes of action. Courts should recognize the validity of preconception tort liability and allow a strict liability cause of action in third-generation cases.

[Antibiotic prophylaxis with piperacillin in vaginal hysterectomy. Study of 1 dose versus 3 doses]. / Profilaxis antibiótica con piperacilina en la histerectomiá vaginal. Estudio de una dosis frente a tres.

A homogeneous and consecutive series of 93 patients treated with vaginal hysterectomy is studied in a controlled, randomized clinical essay. The patients were divided in two groups. One of the groups received one single dose of 49 of piperacillin 30 minutes before surgery; the other, three doses of 4 g of the same antibiotic 30 minutes before surgery and 6 & 12 hours after it. No significant difference was found between both groups and therefore it is fair to suppose that one single preoperative dose of piperacillin shows similar efficacy to the usually used three doses, one preoperative and two postoperative.