Nitration-mediated activation of the small GTPase RhoA stimulates cellular glycolysis through enhanced mitochondrial fission.

Mitochondrial fission and a Warburg phenotype of increased cellular glycolysis are involved in the pathogenesis of pulmonary hypertension (PH). The purpose of this study was to determine whether increases in mitochondrial fission are involved in a glycolytic switch in pulmonary arterial endothelial cells (PAECs). Mitochondrial fission is increased in PAEC isolated from a sheep model of PH induced by pulmonary overcirculation (Shunt PAEC). In Shunt PAEC we identified increases in the S616 phosphorylation responsible for dynamin-related protein 1 (Drp1) activation, the mitochondrial redistribution of Drp1, and increased cellular glycolysis. Reducing mitochondrial fission attenuated cellular glycolysis in Shunt PAEC. In addition, we observed nitration-mediated activation of the small GTPase RhoA in Shunt PAEC, and utilizing a nitration-shielding peptide, NipR1 attenuated RhoA nitration and reversed the Warburg phenotype. Thus, our data identify a novel link between RhoA, mitochondrial fission, and cellular glycolysis and suggest that targeting RhoA nitration could have therapeutic benefits for treating PH.

Endothelin-1 acutely increases nitric oxide production via the calcineurin mediated dephosphorylation of Caveolin-1.

Endothelin (ET)-1 is an endothelial-derived peptide that exerts biphasic effects on nitric oxide (NO) levels in endothelial cells such that acute exposure stimulates-while sustained exposure attenuates-NO production. Although the mechanism involved in the decrease in NO generation has been identified but the signaling involved in the acute increase in NO is still unresolved. This was the focus of this study. Our data indicate that exposing pulmonary arterial endothelial cells (PAEC) to ET-1 led to an increase in NO for up to 30min after which levels declined. These effects were attenuated by ET receptor antagonists. The increase in NO correlated with significant increases in pp60Src activity and increases in eNOS phosphorylation at Tyr83 and Ser1177. The ET-1 mediated increase in phosphorylation and NO generation were attenuated by the over-expression of a pp60Src dominant negative mutant. The increase in pp60Src activity correlated with a reduction in the interaction of Caveolin-1 with pp60Src and the calcineurin-mediated dephosphorylation of caveolin-1 at three previously unidentified sites: Thr91, Thr93, and Thr95. The calcineurin inhibitor, Tacrolimus, attenuated the acute increase in pp60Src activity induced by ET-1 and a calcineurin siRNA attenuated the ET-1 mediated increase in eNOS phosphorylation at Tyr83 and Ser1177 as well as the increase in NO. By using a Caveolin-1 celluSpot peptide array, we identified a peptide targeting a sequence located between aa 41-56 as the pp60Src binding region. This peptide fused to the TAT sequence was found to decrease caveolin-pp60Src interaction, increased pp60Src activity, increased eNOS pSer1177 and NO levels in PAEC and induce vasodilation in isolated aortic rings in wildtype but not eNOS knockout mice. Together, our data identify a novel mechanism by which ET-1 acutely increases NO via a calcineurin-mediated dephosphorylation of caveolin-1 and the subsequent stimulation of pp60Src activity, leading to increases in phosphorylation of eNOS at Tyr83 and Ser1177.

Mitochondrial network dynamics in pulmonary disease: Bridging the gap between inflammation, oxidative stress, and bioenergetics.

Once thought of in terms of bioenergetics, mitochondria are now widely accepted as both the orchestrator of cellular health and the gatekeeper of cell death. The pulmonary disease field has performed extensive efforts to explore the role of mitochondria in regulating inflammation, cellular metabolism, apoptosis, and oxidative stress. However, a critical component of these processes needs to be more studied: mitochondrial network dynamics. Mitochondria morphologically change in response to their environment to regulate these processes through fusion, fission, and mitophagy. This allows mitochondria to adapt their function to respond to cellular requirements, a critical component in maintaining cellular homeostasis. For that reason, mitochondrial network dynamics can be considered a bridge that brings multiple cellular processes together, revealing a potential pathway for therapeutic intervention. In this review, we discuss the critical modulators of mitochondrial dynamics and how they are affected in pulmonary diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), and pulmonary arterial hypertension (PAH). A dysregulated mitochondrial network plays a crucial role in lung disease pathobiology, and aberrant fission/fusion/mitophagy pathways are druggable processes that warrant further exploration. Thus, we also discuss the candidates for lung disease therapeutics that regulate mitochondrial network dynamics.

Inflammatory lung injury is associated with endothelial cell mitochondrial fission and requires the nitration of RhoA and cytoskeletal remodeling.

Higher levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a TLR4 agonist, are associated with poor clinical outcomes in sepsis-induced acute lung injury (ALI). Little is known regarding the mechanisms by which eNAMPT is involved in ALI. Our recent work has identified a crucial role for mitochondrial dysfunction in ALI. Thus, this study aimed to determine if eNAMPT-mediated inflammatory injury is associated with the loss of mitochondrial function. Our data show that eNAMPT disrupted mitochondrial bioenergetics. This was associated with cytoskeleton remodeling and the loss of endothelial barrier integrity. These changes were associated with enhanced mitochondrial fission and blocked when Rho-kinase (ROCK) was inhibited. The increases in mitochondrial fission were also associated with the nitration-mediated activation of the small GTPase activator of ROCK, RhoA. Blocking RhoA nitration decreased eNAMPT-mediated mitochondrial fission and endothelial barrier dysfunction. The increase in fission was linked to a RhoA-ROCK mediated increase in Drp1 (dynamin-related protein 1) at serine(S)616. Another TLR4 agonist, lipopolysaccharide (LPS), also increased mitochondrial fission in a Drp1 and RhoA-ROCK-dependent manner. To validate our findings in vivo, we challenged C57BL/6 mice with eNAMPT in the presence and absence of the Drp1 inhibitor, Mdivi-1. Mdivi-1 treatment protected against eNAMPT-induced lung inflammation, edema, and lung injury. These studies demonstrate that mitochondrial fission-dependent disruption of mitochondrial function is essential in TLR4-mediated inflammatory lung injury and identify a key role for RhoA-ROCK signaling. Reducing mitochondrial fission could be a potential therapeutic strategy to improve ARDS outcomes.

In vitro nematicidal and acaricidal effect of biosurfactants produced by Bacillus against the root-knot nematode Nacobbus aberrans and the dust mite Tyrophagus putrescentiae.

In the present study, the nematicidal and acaricidal activity of three biosurfactants (BS) produced by strains of the Bacillus genus was evaluated. The BS produced by the Bacillus ROSS2 strain presented a mortality of 39.29% in juveniles (J2) of Nacobbus aberrans at a concentration of 30 mg/mL, this same strain is the one that presented the highest mortality in Tyrophagus putrescentiae, which was 57.97% at a concentration of 39 mg/mL. The BS were qualitatively identified by thin layer chromatography and are lipid in nature based on the retention factor (Rf). While the GC-MS analysis identified two main compounds that are 4,7-Methano-1H-indene-2,6-dicarboxylic acid, 3a,4,7,7a-tetrahydro-1, and Methyl 4-(pyrrol-1-yl)-1,2,5-oxadiazole-3-carboxylate1, which is the polar part indicated by the presence of dicarboxylic acid and carboxylate groups; while the non-polar portion can be interpreted as a hydrocarbon chain of variable length. Based on the present results, BS can be an alternative for the biocontrol of the root-knot nematode N. aberrans and the mite T. putrescentiae.

Loss of SOX18/CLAUDIN5 disrupts the pulmonary endothelial barrier in ventilator-induced lung injury.

Mechanical strain contributes to ventilator-induced lung injury (VILI) through multi-factorial and complex mechanisms that remain unresolved. Prevailing evidence suggests that the loss of pulmonary endothelial tight junctions (TJs) plays a critical role. TJs are dynamically regulated by physiologic and hemodynamic forces to stabilize the endothelial barrier. The transcription factor sex-determining region Y-box (SOX)-18 is important in regulating blood vessel development and vascular permeability through its ability to regulate the transcription of Claudin-5, an endothelial TJ protein. Previously, we demonstrated that SOX18 expression is increased by shear stress in the pulmonary endothelium. Therefore, in this study, we investigated how mechanical strain mediated through cyclic stretch affects the SOX18/Claudin-5 regulatory axis. Our data demonstrate that SOX18 and Claudin-5 are downregulated in human lung microvascular endothelial cells (HLMVEC) exposed to cyclic stretch and the mouse lung exposed to high tidal mechanical ventilation. Overexpression of SOX18 reduced the loss of Claudin-5 expression in HLMVEC with cyclic stretch and preserved endothelial barrier function. Additionally, overexpression of Claudin-5 in HLMVEC ameliorated barrier dysfunction in HLMVEC exposed to cyclic stretch, although SOX18 expression was not enhanced. Finally, we found that the targeted overexpression of SOX18 in the pulmonary vasculature preserved Claudin-5 expression in the lungs of mice exposed to HTV. This, in turn reduced lung vascular leak, attenuated inflammatory lung injury, and preserved lung function. Together, these data suggest that enhancing SOX18 expression may prove a useful therapy to treat patients with ventilator-induced lung injury.

Bio-directed Chemical Study of Pleurotus ostreatus Spent Substrate and Its Nematicidal Activity.

PURPOSE: In the present study, the nematicidal activity of the chemical fractionation of the spent substrate of the edible mushroom Pleurotus ostreatus against eggs and L3 larvae of Haemonchus contortus was evaluated. METHODS: The hydroalcoholic extract of the spent substrate was subjected to a bipartition with ethyl acetate giving two fractions: one aqueous (F. Ac) and one organic (F. AcOET). Both fractions were evaluated against eggs and L3 larvae at different concentrations (5, 2.5, 1.25, 0.625 and 0.3125 mg/mL) and 2% methanol, PBS and thiabendazole (5 mg/mL) as controls. Chemical fractionation of F. AcOET was performed in open column chromatography where 76 fractions were obtained and when analyzed by thin layer chromatography (TLC) were grouped into 11 mixtures (R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R11). These mixtures were evaluated at 10 mg/mL against eggs and L3 larvae. The data obtained were analyzed by ANOVA and a Tukey test in the SAS V9 program. RESULTS: The results showed that, in the evaluation of F. AcOET and F. Ac, for the inhibition of egg hatching, the highest percentages were 78.80 and 76.89% at 5 mg/mL, respectively. As for the percentage of larval mortality, F. AcOET obtained 60.91 and F. Ac 29.77% at 5 mg/mL. The results of the evaluations of the mixtures showed that mixtures R4, R5 obtained 100 and 95.41% larval mortality and mixtures R6 and R7 presented 100% inhibitory activity of egg hatching, so these were analyzed by gas chromatography mass spectrometry finding compounds such as vanillin, ß-sitosterol, ρ-methyl ρ-hydroxycinnamate and ρ-hydroxybenzaldehyde. CONCLUSION: The results of the present study demonstrate that the spent substrate of P. ostreatus has potential anthelmintic activity against H. contortus. Moreover, by reusing and taking advantage of this substrate, its environmental pollution effects can be reduced.

Activation of the mechanosensitive Ca2+ channel TRPV4 induces endothelial barrier permeability via the disruption of mitochondrial bioenergetics.

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.

HER2/HER3 regulates lactate secretion and expression of lactate receptor mRNA through the MAP3K4 associated protein GIT1.

One of the major features of cancer is Otto Warburg's observation that many tumors have increased extracellular acidification compared to healthy tissues. Since Warburg's observation, the importance of extracellular acidification in cancer is now considered a hallmark of cancer. Human MAP3K4 functions upstream of the p38 and JNK mitogen activated protein kinases (MAPKs). Additionally, MAP3K4 is required for cell migration and extracellular acidification of breast cancer cells in response to HER2/HER3 signaling. Here, we demonstrate that GIT1 interacts with MAP3K4 by immunoprecipitation, while cellular lactate production and the capacity of MCF-7 cells for anchorage independent growth in soft agar were dependent on GIT1. Additionally, we show that activation of HER2/HER3 signaling leads to reduced expression of lactate receptor (GPR81) mRNA and that both, GIT1 and MAP3K4, are necessary for constitutive expression of GPR81 mRNA. Our study suggests that targeting downstream proteins in the HER2/HER3-induced extracellular lactate signaling pathway may be a way to inhibit the Warburg Effect to disrupt tumor growth.

Hunting in a peasant community of the Sierra de Huautia, Morelos, Mexico / Cacería en una comunidad campesina de la sierra de huautla, morelos, méxico

RESUMEN La Reserva de la Biosfera Sierra de Huautla es un reservorio de biodiversidad del trópico seco mexicano. Las comunidades campesinas que la habitan históricamente han aprovechado los recursos naturales para su subsistencia, entre ellos la fauna silvestre. Con el objetivo de caracterizar las prácticas de cacería en la comunidad de Pitzotlán, Morelos, se aplicaron entrevistas a diez campesinos locales, así como observación participante, recorridos guiados y registro de la caza durante siete meses. Se registraron 37 especies de fauna con valor de uso, principalmente para alimento (57 %), disminuir daños agropecuarios (49 %) y en la medicina tradicional (27 %). De septiembre de 2018 a marzo de 2019 se contabilizó la captura de 70 individuos pertenecientes a 17 especies en 41 eventos de cacería. Las especies con mayor porcentaje de individuos capturados fueron iguana negra Ctenosaurapectinata (19 %), huilota Zenaida spp. (19 %), tejon Nasua narica (14 %), venado Odocoileus virginianus (10 %) y tortola Columbina inca (9 %). Se estimó un total de 278,93 kg de biomasa consumida, el 80 % aportada por el venado cola blanca. La cacería se realizó en un radio de 3.5 km del entorno comunitario, por medio de "espiadas", "arreadas", "acorralamiento", "lampareadas", el uso de perros y en eventos oportunistas. La cacería es una actividad complementaria a las estrategias de subsistencia de la comunidad estudiada, cuyo arraigo cultural permite obtener bienes materiales, el esparcimiento y la socialización.

ABSTRACT The Sierra de Huautla Biosphere Reserve is a biodiversity reservoir in the Mexican dry tropics. The peasant communities historically inhabiting it have been used its natural resources for their subsistence, including wildlife. In order to characterize the hunting practices in the community of Pitzotlán, Morelos, interviews were applied to ten local farmers, as well as participant observation, guided tours and hunting recording for seven months. 37 species of fauna with use values were registered, most of them for food (57 %), to reduce agricultural damage (49 %) and to use in traditional medicine (27 %). From September 2018 to March 2019, the capture of 70 individuals belonging to 17 species was counted in 41 hunting events. The species with the highest percentage of captured individuals were iguana negra Ctenosaura pectinata (19 %), huilota Zenaida spp. (19 %), tejon Nasua narica (14 %), Odocoileus virginianus (10 %) and tortola Columbina inca (9 %). It was estimated that 278.93 kg of biomass were consumed, 80 % was contributed by the white-tailed deer. The hunting was carried out within a 3.5 km ratio from the community environment, through "spied on", "herded", "cornered" and, "lampareadas", with the use of dogs and in opportunistic events. Hunting is a complementary activity in the subsistence strategies of the studied community, whose cultural roots allow obtaining material goods, recreation and socialization.

Fauna silvestre con valor de uso en la Reserva de la Biosfera Sierra de Huautla, Morelos, México / Wild fauna with use value in the Biosphere Reserve Sierra de Huautla, Morelos, Mexico

Resumen El objetivo de este trabajo fue sistematizar las especies apropiadas, formas de apropiación y valores de uso asignados a la fauna silvestre en comunidades de la Reserva de la Biosfera Sierra de Huautla, México, mediante un análisis documental y trabajo de campo. Se registraron 46 especies con valor de uso pertenecientes a 31 familias. La clase con mayor número de especies apropiadas son las aves (42.5%), seguidas de los mamíferos (34%). El 64% de las especies se apropian con fines alimentarios y el 38.3% se utilizan para el tratamiento de enfermedades en la medicina tradicional. Otros usos son adornos, amuletos, elaboración de herramientas y como animal de compañía. La apropiación de fauna silvestre se realiza por medio de la cacería, la recolección y la pesca. Las armas de fuego como la escopeta es el principal instrumento para la cacería. También se utilizan perros, machetes, resorteras, tarrayas, anzuelos, nudos corredizos y se capturan manualmente. La apropiación puede ser una actividad oportunista u organizada por medio de las arreadas, espiadas, lampareadas, campeadas, acorralamiento y recolección manual. La apropiación de fauna silvestre es una actividad culturalmente arraigada en las comunidades de la reserva. Sin embargo, el análisis de la relación sociedad-fauna en la región es limitado, por lo que es necesario generar información que promueva el manejo sostenible del recurso faunístico.

Abstract The aims of this paper were the systematization of appropriated species, appropriation forms and assigned use values of wildlife species in the Biosphere Reserve of the Sierra de Huautla, Mexico, by means of documentary analysis and field work. We recorded 46 species with use value pertaining to 31 families. Birds accounted for the highest number of appropriated species (42.5%), followed by mammals (34%). Of the total species, 64% are appropriated for alimentary purposes and are used in the treatment of diseases in traditional medicine. Others are used as ornaments, talismans, in tool manufacture and as pet animals. Wildlife appropriation is achieved by hunting, collection and fishing. Fire arms such as shotguns are the main instrument used in hunting. Dogs, machetes, slingshots, nets, hooks, slipknots, are also used, or they are captured by hand. Appropriation can be an opportunistic or organized activity by means of "arreadas" (herding), "espiadas" (opportunistic observation), "lampareadas" (spotlighting), "campeadas" (scouring), "acorralamiento" (cornering) and manual collection. Wildlife appropriation is a culturally rooted activity in the reserve communities. Nevertheless, the analysis of the relationship society-fauna in the region is limited and this is why the generation of information that may enable a sustainable management of the faunistic resource is necessary.

Estudio etnozoológico de los vertebrados silvestresde la comunidad Bonifacio García, Morelos, México / Ethnozoological study of wild vertebrates of the Bonifacio Garcia community, Morelos, Mexico

Resumen El objetivo de la investigación fue analizar el conocimiento etnozoológico que conservan de los vertebrados silvestres los habitantes de la comunidad Bonifacio García en Morelos, México. Se aplicaron entrevistas semiestructuradas a 30 informantes clave, mediante la observación participante y recorridos guiados se buscaron rastros como huellas, excretas, madrigueras, pieles y observación in vivo para la identificación de las especies citadas y se calcularon los siguientes índices: valor de diversidad de uso para cada especie (VDE) y de uso medicinal (VDM), así como el índice de diversidad de las enfermedades tratadas (IVDE). Se reportan 41 especies de vertebrados que los entrevistados reconocen, de los cuales 28 son aprovechados: mamíferos 39.3%, aves 32%, reptiles 18%, peces 7.1% y anfibios 3.6%. Los principales valores de uso fueron alimentario (0.54), medicinal (0.46) y ornamental (0.43). El venado cola blanca, la iguana negra y la víbora de cascabel registraron 0.57 de VDE y un VDM de 0.19. Las principales enfermedades tratadas son: cáncer, afecciones respiratorias, enfermedades de la piel y reumatismo con un IVDE de 0.17. La apropiación de los vertebrados silvestres se lleva a cabo por medio de la cacería de subsistencia en el campo (50%), terrenos de cultivo (43%) y el traspatio (7%) utilizando con mayor frecuencia escopeta como arma de captura. Se concluye que la apropiación de vertebrados permite complementar necesidades básicas de alimentación y salud; por lo que los conocimientos etnozoológicos deben de integrarse a políticas públicas que permitan la conservación y manejo de la fauna silvestre en la comunidad.

Abstract The aim of this study was to analyze the ethnozoological knowledge that the inhabitants of the Bonifacio García community, Morelos, Mexico, conserve about wild vertebrate. Semi-structured interviews were applied to 30 key informants, through participant observation and guided tours, traces such as footprints, excreta, burrows and pelts and in vivo observation were researched for the identification of the mentioned species and the following indexes were calculated: value of diversity of use for each species (VDS) and of medicinal uses (DVM), as well as the diversity index of the treated diseases (DITD). The study recorded a total of 42 wild vertebrate species, of these 28 are used: mammals 39.3%, birds 32%, reptiles 18%, fish 7.1% and amphibia 3.6%. The main use values were alimentary (0.54), medicinal (0.46) and ornamental (0.43). The white-tailed deer, the black iguana and the rattlesnake registered 0.57 of VDS and a DVM of 0.19. The main diseases treated are cancer, respiratory and skin diseases and rheumatism with an DITD of 0.17. The animals are obtained by means of subsistence hunting in the fields (50%), croplands (43%) and backyards (7%), using most frequently shotguns as arms for capture. We conclude that the appropriation of vertebrates complements basic needs for food and health; therefore, ethnozoological knowledge must be integrated into public policies that enable the conservation and management of wild fauna in the community.