GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells.

In this study, we investigated the inter-organelle communication between the Golgi apparatus (GA) and mitochondria. Previous observations suggest that GA-derived vesicles containing phosphatidylinositol 4-phosphate (PI(4)P) play a role in mitochondrial fission, colocalizing with DRP1, a key protein in this process. However, the functions of these vesicles and potentially associated proteins remain unknown. GOLPH3, a PI(4)P-interacting GA protein, is elevated in various types of solid tumors, including breast cancer, yet its precise role is unclear. Interestingly, GOLPH3 levels influence mitochondrial mass by affecting cardiolipin synthesis, an exclusive mitochondrial lipid. However, the mechanism by which GOLPH3 influences mitochondria is not fully understood. Our live-cell imaging analysis showed GFP-GOLPH3 associating with PI(4)P vesicles colocalizing with YFP-DRP1 at mitochondrial fission sites. We tested the functional significance of these observations with GOLPH3 knockout in MDA-MB-231 cells of breast cancer, resulting in a fragmented mitochondrial network and reduced bioenergetic function, including decreased mitochondrial ATP production, mitochondrial membrane potential, and oxygen consumption. Our findings suggest a potential negative regulatory role for GOLPH3 in mitochondrial fission, impacting mitochondrial function and providing insights into GA-mitochondria communication.

Accelerating sexual maturation of male Anastrepha ludens (Diptera: Tephritidae) fruit flies by adding two juvenile hormone analogues.

BACKGROUND: Improving the mating competitiveness and survival of sterile males are direct means to increase the effectiveness of the sterile insect technique (SIT). Some insecticide growth regulators, such as the juvenile hormone analogue (JHA) methoprene, have been used to improve the mating competitiveness of male tephritid flies by reducing their sexual maturation period. However, the application of methoprene reduces fly resistance to stress and decreases survival. Here, we compared the effects of methoprene and pyriproxyfen (PPF), another JHA, in Anastrepha ludens males. PPF is an insect growth regulator that exhibits higher negative effects on the larval molting process than methoprene or natural juvenile hormone. Both compounds were administered at two doses (0.05% and 0.10%) via the male diet immediately after emergence. RESULTS: Our results show that both PPF and methoprene reduced male sexual maturation. However, PPF-treated males exhibited a shorter maturation period and obtained more matings at a given age than methoprene-treated males. No significant differences were observed between the two PPF doses tested (0.05% and 0.10%). Male survival was equally reduced by the two compounds. CONCLUSION: Our results demonstrate that PPF accelerated sexual development without reducing the mating propensity of sterile male flies and can be used as a suitable alternative for methoprene. © 2023 Society of Chemical Industry.

Biological role of fructose in the male reproductive system: Potential implications for prostate cancer.

BACKGROUND: Over the last 20 years, fructose has gradually emerged as a potential metabolic substrate capable of promoting the growth and progression of various cancers, including prostate cancer (PCa). The biological and molecular mechanisms that underlie the effects of fructose on cancer are beginning to be elucidated. METHODS: This review summarizes the biological function of fructose as a potential carbon source for PCa cells and its role in the functionality of the male reproductive tract under normal conditions. RESULTS: The most recent biological advances related to fructose transport and metabolism as well as their implications in PCa growth and progression suggest that fructose represent a potential carbon source for PCa cells. Consequently, fructose derivatives may represent efficient radiotracers for obtaining PCa images via positron emission tomography and fructose transporters/fructose-metabolizing enzymes could be utilized as potential diagnostic and/or predictive biomarkers for PCa. CONCLUSION: The existing data suggest that restriction of fructose from the diet could be a useful therapeutic strategy for patients with PCa.

Immature stages of Utetes anastrephae (Hymenoptera: Braconidae) developed in Anastrepha fruit fly larvae (Diptera: Tephritidae).

The morphology of the immature stages of Utetes anastrephae (Hymenoptera: Braconidae), a native parasitoid of larvae of flies of the Neotropical genus Anastrepha (Diptera: Tephritidae), is shown. This study aimed to characterize the immature stages and morphological changes in the development of the koinobiont endoparasitoid in two species of larval hosts, Anastrepha obliqua and Anastrepha ludens. The definition of structures and morphological changes during development was made through daily microscopic observations and photographs of dissected hosts. The immature development of the parasitoid corresponds to a holometabolous insect with three well-defined stages: egg (two days), larva with three larval instars (approximately eight days), and pupa (six days). Similar development times were obtained in the two host species. Males and females completed their cycle in 17 and 18 days, respectively. During egg-first instar development, host antagonistic activity through melanization and encapsulation as mortality factors was evident and frequent only in A. obliqua. These results serve as basic knowledge for the use of this parasitoid in the biological control of fruit flies.

Effect of the Sterile Insect Technique and Augmentative Parasitoid Releases in a Fruit Fly Suppression Program in Mango-Producing Areas of Southeast Mexico.

The Sterile Insect Technique (SIT), by means of sterile male releases of Anastrepha ludens (Loew), coupled with Augmentative Biological Control (ABC), by releasing the parasitoid Diachasmimorpha longicaudata (Ashmead), was evaluated in a commercial mango production area for one year. The obtained results were compared with mean fruit fly population values from two previous years without the combined use of both techniques. The treatments were: SIT + ABC, SIT, ABC, and Control, and each treatment was established in blocks of 5000 Ha separated by distances of 5-10 km. The evaluations were carried out through fruit sampling to assess percent parasitism and trapping of adult flies to obtain Flies per Trap per Day (FTD) values. The mean percentage of parasitism increased from 0.59% in the control treatment to 19.38% in the block with ABC. The FTD values decreased from ~0.129 and ~0.012 in the control block to 0.0021 in the block with SIT and ABC, representing a 98% suppression. The difference between the two periods in the control block was not significant. We conclude that the integration of both techniques resulted in an additive suppression of the pest population, supporting the use of both control techniques in an area-wide pest management context.

Impact of interorganelle coordination between the conventional early secretory pathway and autophagy in cellular homeostasis and stress response.

The conventional early secretory pathway and autophagy are two essential interconnected cellular processes that are crucial for maintaining cellular homeostasis. The conventional secretory pathway is an anabolic cellular process synthesizing and delivering proteins to distinct locations, including different organelles, the plasma membrane, and the extracellular media. On the other hand, autophagy is a catabolic cellular process that engulfs damaged organelles and aberrant cytosolic constituents into the double autophagosome membrane. After fusion with the lysosome and autolysosome formation, this process triggers digestion and recycling. A growing list of evidence indicates that these anabolic and catabolic processes are mutually regulated. While knowledge about the molecular actors involved in the coordination and functional cooperation between these two processes has increased over time, the mechanisms are still poorly understood. This review article summarized and discussed the most relevant evidence about the key molecular players implicated in the interorganelle crosstalk between the early secretory pathway and autophagy under normal and stressful conditions.

Additive Effect of Releasing Sterile Insects Plus Biocontrol Agents against Fruit Fly Pests (Diptera: Tephritidae) under Confined Conditions.

Pest control models integrating the use of the sterile insect technique (SIT) and augmentative biological control (ABC) have postulated that it is possible to obtain a synergistic effect from the joint use of these technologies. This synergistic effect is attributed to the simultaneous attack on two different biological stages of the pest (immature and adult flies), which would produce higher suppression on the pest populations. Here we evaluated the effect of the joint application of sterile males of A. ludens of the genetic sexing strain Tap-7 along with two parasitoid species at the field cage level. The parasitoids D. longicaudata and C. haywardi were used separately to determine their effect on the suppression of the fly populations. Our results showed that egg hatching percentage was different between treatments, with the highest percentage in the control treatment and a gradual reduction in the treatments with only parasitoids or only sterile males. The greatest induction of sterility (i.e., the lowest egg hatching percentage) occurred with the joint use of ABC and SIT, demonstrating that the earlier parasitism caused by each parasitoid species was important reaching high levels of sterility. Gross fertility rate decreased up to 15 and 6 times when sterile flies were combined with D. longicaudata and C. haywardi, respectively. The higher parasitism by D. longicaudata was determinant in the decrease of this parameter and had a stronger effect when combined with the SIT. We conclude that the joint use of ABC and SIT on the A. ludens population had a direct additive effect, but a synergistic effect was observed in the parameters of population dynamics throughout the periodic releases of both types of insects. This effect can be of crucial importance in the suppression or eradication of fruit fly populations, with the added advantage of the low ecological impact that characterizes both techniques.

Medfly Population Suppression through Augmentative Release of an Introduced Parasitoid in an Irrigated Multi-Fruit Orchard of Central-Western Argentina.

Biological control through the augmentative release of parasitoids is an important complementary tool that may be incorporated into other strategies for the eradication/eco-friendly control of pest fruit flies. However, not much information is available on the effectiveness of fruit fly parasitoids as biocontrol agents in semi-arid and temperate fruit-growing regions. Therefore, this study evaluated the effect of augmentative releases of the larval parasitoid Diachasmimorpha longicaudata (Ashmead) on Ceratitis capitata (Wiedemann) (medfly) populations over two fruit seasons (2013 and 2014) on a 10 ha irrigated fruit farm in San Juan province, central-western Argentina. The parasitoids were mass reared on irradiated medfly larvae of the Vienna-8 temperature-sensitive lethal genetic sexing strain. About 1692 (±108) parasitoids/ha were released per each of the 13 periods throughout each fruit season. Another similar farm was chosen as a control of non-parasitoid release. The numbers of captured adult flies in food-baited traps and of recovered fly puparia from sentinel fruits were considered the main variables to analyze the effect of parasitoid release on fly population suppression using a generalized least squares model. The results showed a significant decrease (p < 0.05) in the medfly population on the parasitoid release farm when compared to the Control farm, demonstrating the effectiveness of augmentative biological control using this exotic parasitoid. Thus, D. longicaudata could be used in combination with other medfly suppression strategies in the fruit production valleys of San Juan.

Prescription of High-intensity Aerobic Interval Training Based on Oxygen Uptake Kinetics.

Endurance training results in diverse adaptations that lead to increased performance and health benefits. A commonly measured training response is the analysis of oxygen uptake kinetics, representing the demand of a determined load (speed/work) on the cardiovascular, respiratory, and metabolic systems, providing useful information for the prescription of constant load or interval-type aerobic exercise. There is evidence that during high-intensity aerobic exercise some interventions prescribe brief interval times (<1-min), which may lead to a dissociation between the load prescribed and the oxygen uptake demanded, potentially affecting training outcomes. Therefore, this review explored the time to achieve a close association between the speed/work prescribed and the oxygen uptake demanded after the onset of high-intensity aerobic exercise. The evidence assessed revealed that at least 80% of the oxygen uptake amplitude is reached when phase II of oxygen uptake kinetics is completed (1 to 2 minutes after the onset of exercise, depending on the training status). We propose that the minimum work-time during high-intensity aerobic interval training sessions should be at least 1 minute for athletes and 2 minutes for non-athletes. This suggestion could be used by coaches, physical trainers, clinicians and sports or health scientists for the prescription of high-intensity aerobic interval training.

Irradiation and parasitism affect the ability of larval hemocytes of Anastrepha obliqua for phagocytosis and the production of reactive oxygen species.

The development of the parasitoid Doryctobracon crawfordi (Viereck) (Hymenoptera: Braconidae) in Anastrepha obliqua (McQuart) (Diptera: Tephritidae) larvae is unviable in nature; however, if the host larva is irradiated at 160 Gy, the parasitoid develops and emerges successfully. This suggests that radiation affects the immune responses of A. obliqua larvae, while the underlying mechanisms remain to be revealed. Using optical and electronic microscopies we determined the number and type of hemocyte populations found inside the A. obliqua larvae, either nonirradiated, irradiated at 160 Gy, parasitized by D. crawfordi, or irradiated and parasitized. Based on flow cytometry, the capacity to produce reactive oxygen species (ROS) was determined by the 123-dihydrorhodamine method in those hemocyte cells. Five cell populations were found in the hemolymph of A. obliqua larvae, two of which (granulocytes and plasmatocytes) can phagocytize and produce ROS. A reduction in the number of cells, mainly of the phagocytic type, was observed, as well as the capacity of these cells to produce ROS, when A. obliqua larvae were irradiated. Both radiation and parasitization decreased the ROS production, and when A. obliqua larvae were irradiated followed by parasitization by D. crawfordi, the reduction of the ROS level was even greater. In contrast, a slight increase in the size of these cells was observed in the hemolymph of the parasitized larvae compared to those in nonparasitized larvae. These results suggest that radiation significantly affects the phagocytic cells of A. obliqua and thus permits the development of the parasitoid D. crawfordi.

Increasing radiation doses in Anastrepha obliqua (Diptera: Tephritidae) larvae improve parasitoid mass-rearing attributes.

Doses of 40, 80, 120, and 160 Gy were applied to 5-, 6-, 7-, and 8-day-old Anastrepha obliqua larvae, which were exposed to the Neotropical-native braconids Doryctobracon crawfordi and Utetes anastrephae and the Asian braconid Diachasmimorpha longicaudata. These tests were performed to know the effect of the increase in host radiation on the emergence of the aforementioned parasitoids and the related consequences of oviposition on the host. The study was based on the fact that higher radiation doses may cause a decrease in the host immune activity. There was a direct relationship between the increase in radiation dose and the parasitoid emergence. Both, the weight and the mortality of the host larvae were not affected by radiation. Although the larval weight of the larvae was lower and the mortality was higher in the younger larvae. Both, the number of scars and immature stages per host puparium originated from the younger larvae were lower than those from older larvae. Only U. anastrephae superparasitized more at lower radiation. Superparasitism by D. longicaudata was more frequent at 160 Gy. Qualitative measurements of melanin in the larvae parasitized showed that the levels were lower with increasing radiation. As radiation doses increased, the antagonistic response of the A. obliqua larva was reduced. Host larvae aged 5- and 6-day-old irradiated at 120-160 Gy significantly improve parasitoid emergence. This evidence is relevant for the mass production of the three tested parasitoid species.

Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates.

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

[Participation in the community after the rehabilitation: multi-center study in Argentina] / Participación en la comunidad en personas con patologías neurológicas luego de la rehabilitación: estudio multicéntrico en Argentina

Introduction: Introduction: Participation is the dynamic and complex interaction between the individual's health condition, bodily functions, activities that can be carried out and environmental factors. Measuring it helps to understand the impact of disability. Objectives: Describe the activities and participation in subjects with neurological pathologies, discharged from hospitalization for rehabilitation. Secondly, to compare the clinical-demographic characteristics and the participation among wheelchair users with respect to non-users. Material and method: Observational, prospective, cross-sectional, multicenter study. Based on a survey of people over 18 years of age with pathologies of neurological origin discharged from rehabilitation from 6 centers in Argentina. Results: 282 people responded, 69% men with an average age of 50 years and discharged 22 months ago. The most common diagnosis was cerebrovascular accident. The self-perception of participation was 49 out of 90, and those who do not use a wheelchair report a higher level of participation. The greatest satisfaction was in areas of interpersonal relationships. 50% require assistance to use transportation in the community. 61% neither work nor study, nor do they engage in sports activities (65%). 61% of wheelchair users cannot go to places in the community because they are inaccessible. Conclusion: Less participation in community activities was observed, mainly due to architectural barriers and difficulties in using transport in wheelchair users. The family occupies a central place so that they can integrate into the community.

Introducción: La participación es la interacción dinámica y compleja entre la condición de salud del individuo, las funciones corporales, las actividades que puede realizar y los factores ambientales. Medirla ayuda a comprender el impacto de la discapacidad. Objetivos: Describir las actividades y participación en sujetos con patologías neurológicas, dados de alta de internación para rehabilitación. Secundariamente comparar las características clínico-demográficas y la participación entre usuarios de silla de ruedas respecto a personas no usuarias. Material y método: Estudio observacional, prospectivo, transversal, multicéntrico. Basado en una encuesta a mayores de 18 años con patologías de origen neurológico dados de alta de rehabilitacion de 6 centros de Argentina. Resultados: Respondieron 282 personas, 69% hombres con una media de edad de 50 años y dados de alta hace 22 meses. El diagnóstico más frecuente fue el accidente cerebrovascular. La autopercepción de la participación fue de 49 sobre 90, y los que no utilizan silla de ruedas refieren mayor nivel de participación. La mayor satisfacción fue en áreas de relaciones interpersonales. El 50% requiere de asistencia para utilizar los transportes en la comunidad. El 61% no trabaja ni estudia, así como tampoco realizan actividades deportivas (65%). Al 61% de los usuarios de silla de ruedas no pueden ir a lugares de la comunidad por ser inaccesibles. Conclusión: Se observó menor participación en actividades comunitarias, principalmente por barreras arquitectónicas y por dificultades para usar el transporte en usuarios de silla de ruedas. La familia ocupa un lugar central para que puedan integrarse en la comunidad.

Rehabilitación física en pacientes con obesidad post COVID19: Una revisión narrativa

El COVID19 ha afectado a millones de personas a nivel mundial. Entre los pacientes contagiados que se agravan y requieren de cuidados intensivos avanzados; además de largas estadías de hospitalización, se encuentran quienes tienen obesidad. Debido a la gran prevalencia de personas con obesidad, tanto en países desarrollados como en en vías de desarrollo, y a las distintas secuelas que experimentan debido al efecto directo del virus como al tratamiento que reciben, es necesario comprender la fisiopatología asociada a la severidad del contagio. Otro aspecto importante a considerar es ¿cómo las secuelas del tratamiento en las unidades de pacientes críticos pueden afectar el estado de salud de estas personas? El propósito de esta revisión fue indagar en la literatura sobre la rehabilitación fisica en pacientes con obesidad que han padecido COVID19 con el objetivo de tener una mirada integral que apunte a potenciar los resultados de la rehabilitación durante todo el curso de la enfermedad. Se revisaron antecedentes en bases de datos como Pubmed, la literatura y ante la escasa evidencia sobre el proceso de rehabilitación en las personas con obesidad se realizó una revisión narrativa del paciente con obesidad que enferma de COVID19 y que luego de una hospitalización prolongada debe ser reintegrado a sus actividades habituales. Se enfatiza en la fisiopatología asociada a la inmovilización prolongada de un paciente con comorbilidades previas y se proponen estrategias de rehabilitación basadas en el entrenamiento físico adaptado a su nueva condición de salud.

ABSTRACT COVID19 is a pandemic that has affected all of humanity and is still far from being eradicated, despite efforts to vaccinate the population. Among infected patients whose symptoms worsen and require advanced intensive care; in addition to long hospital stays, there are people with obesity. Due to the high prevalence of people with obesity, both in developed and developing countries, and the different sequelae they experience due to the direct effect of the virus and the treatment they receive, it is necessary to understand the pathophysiology associated with the severity of the contagion, as well as treatment sequelae among intensive care patients with the goal of having a comprehensive view that aims to enhance the results of rehabilitation throughout the course of the disease. Post-discharge sequelae depend on the severity of the disease, previous comorbidities, and length of hospitalization. This review presents a global panorama of obese patients who become ill with COVID19 and who, after a prolonged hospitalization, return to normal daily routines. Emphasis is placed on the pathophysiology associated with prolonged immobilization of a patient with previous comorbidities and on rehabilitation strategies based on physical training adapted to the new health condition.

Tracheostomy Decannulation and Disorders of Consciousness Evolution.

BACKGROUND: Tracheostomy is a frequent surgical procedure in subjects with chronic disorders of consciousness (DOC). There is no consensus about safety of tracheostomy decannulation in this population.The aim of our study was to estimate if DOC improvement is a predictor for tracheostomy decannulation. Secondary outcomes include mortality rate and discharge destination. METHODS: We conducted an observational, retrospective, case-control study at a weaning and rehabilitation center (WRC). We included tracheostomized subjects with DOC admitted between August 2015 and December 2017. We matched groups based on the consciousness level at admission assessed withthe coma recovery scale revised (CRS-R). Subjects who were later decannulated formed the cases, while those that remained tracheostomized at the end of follow-up formed the controls. Improvement of DOC was defined as a progress in the categories of the CRS-R. RESULTS: 22 subjects were included in each group. No significant differences were found in clinical and demographic variables, except that controls had longer neurologic injury evolution (65.5 vs 51 days, P = .047), more tracheostomy days at admission to ourinstitution (53 vs 33.5, P = .02), and higher prevalence of neurological comorbidities (12 vs 4, P = .03). Subjects who improved their DOC had more chances of being decannulated (OR 11.28, 95% CI 1.96-123.08). Tracheostomy decannulation could not be achieved in most subjects who did not improve from vegetative state (VS) (OR 0.13, 95% CI 0.02-0.60). 8 subjects, however, could be decannulated in VS, with only one decannulation failure and no deaths. Mortality was higher in controls (0 vs 6, P = .02), especially among VS (0 vs 5, P = .049). No significant differences were found in discharge destination between groups. CONCLUSIONS: Subjects who improve their DOC are more likely to achieve tracheostomy decannulation. Some subjects in VS were decannulated, with lower mortality than those who remained tracheostomized.

TNF-α-activated eNOS signaling increases leukocyte adhesion through the S-nitrosylation pathway.

Nitric oxide (NO) is a key factor in inflammation. Endothelial nitric oxide synthase (eNOS), whose activity increases after stimulation with proinflammatory cytokines, produces NO in endothelium. NO activates two pathways: 1) soluble guanylate cyclase-protein kinase G and 2) S-nitrosylation (NO-induced modification of free-thiol cysteines in proteins). S-nitrosylation affects phosphorylation, localization, and protein interactions. NO is classically described as a negative regulator of leukocyte adhesion to endothelial cells. However, agonists activating NO production induce a fast leukocyte adhesion, which suggests that NO might positively regulate leukocyte adhesion. We tested the hypothesis that eNOS-induced NO promotes leukocyte adhesion through the S-nitrosylation pathway. We stimulated leukocyte adhesion to endothelium in vitro and in vivo using tumor necrosis factor-α (TNF-α) as proinflammatory agonist. ICAM-1 changes were evaluated by immunofluorescence, subcellular fractionation, immunoprecipitation, and fluorescence recovery after photobleaching (FRAP). Protein kinase Cζ (PKCζ) activity and S-nitrosylation were evaluated by Western blot analysis and biotin switch method, respectively. TNF-α, at short times of stimulation, activated the eNOS S-nitrosylation pathway and caused leukocyte adhesion to endothelial cells in vivo and in vitro. TNF-α-induced NO led to changes in ICAM-1 at the cell surface, which are characteristic of clustering. TNF-α-induced NO also produced S-nitrosylation and phosphorylation of PKCζ, association of PKCζ with ICAM-1, and ICAM-1 phosphorylation. The inhibition of PKCζ blocked leukocyte adhesion induced by TNF-α. Mass spectrometry analysis of purified PKCζ identified cysteine 503 as the only S-nitrosylated residue in the kinase domain of the protein. Our results reveal a new eNOS S-nitrosylation-dependent mechanism that induces leukocyte adhesion and suggests that S-nitrosylation of PKCζ may be an important regulatory step in early leukocyte adhesion in inflammation.NEW & NOTEWORTHY Contrary to the well-established inhibitory role of NO in leukocyte adhesion, we demonstrate a positive role of nitric oxide in this process. We demonstrate that NO induced by eNOS after TNF-α treatment induces early leukocyte adhesion activating the S-nitrosylation pathway. Our data suggest that PKCζ S-nitrosylation may be a key step in this process.

Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis.

Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.

D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy.

Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

High-Speed Bodyweight Resistance Training Improves Functional Performance Through Maximal Velocity in Older Females.

The authors compared the effects of bodyweight resistance training at moderate- or high-speed conditions on muscle power, velocity of movement, and functional performance in older females. In a randomized, single-blinded noncontrolled trial, participants completed 12 weeks (three sessions/week) of bodyweight resistance training at high (n = 14; age = 70.6 ± 4.3 years) or moderate (n = 12; age = 72.8 ± 4.2 years) speeds. Data were analyzed with an analysis of variance (Group × Time) with α level set at <.05. After the intervention, timed up and go test performance (p < .05) and the rising from a chair test mean (22.4%) and maximal velocity (28.5%), mean (24.4%) and maximal power (27.7%), normalized mean (25.1%), and normalized maximal power (28.5%) increased in the high-speed group (p < .05). However, the moderate-speed group achieved no improvements (Δ6.7-14.4%; p > .2). The authors conclude that high-speed bodyweight resistance training is an effective and economically practical strategy to improve the functional capacity of older women relevant to daily life activities.